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Effects of CO₂ enrichment and potassium supply on growth and inorganic nutrition of chrysanthemum (Dendranthema… Hoyos, Juan C. 1992

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EFFECTS OF CO2 ENRICHMENT AND POTASSIUM SUPPLY ONGROWTH AND INORGANIC NUTRITION OF CHRYSANTHEMUM(Dendranthema grandiflora Tzvelev)byJUAN CAMILO HOYOS C.Agronomo, Escuela Agricola PanamericanaB. S. Fruit Crops, University of FloridaA THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDepartment of Plant ScienceWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIADecember 1992© Juan C. Hoyos, COPYRIGHT 1992RELEASE FORMIn presenting this thesis in partial fulfillment of the requirements for an advanceddegree at The University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission forextensive copying of this thesis for scholarly purposes may be granted by the Headof my Department or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of Plant ScienceThe University of British Columbia2357 Main MallVancouver, B. C.Canada V6T 1Z4Date: December 10, 1992iiABSTRACTBeyond the effects of photosynthesis, plant responses to CO2 enrichment aremodified by secondary environmental factors and internal processes, includingmineral relations and carbon partitioning. Leaf abnormalities sometimes developunder long-term CO2 enrichment, and previous research has correlated them withaltered nutrient requirements and distribution.To explore the roles of CO2 enrichment on the development of leafabnormalities, three experiments were performed on chrysanthemum(Dendranthema grandiflora Tzvelev) plants cv 'Envy'. CO2 enrichment and K+supply effects on growth, carbon partitioning, and the inorganic nutrient contents inplants were studied.CO2 enrichment increased plant dry mass, mainly due to increased leaf drymass. In CO2 enriched plants, increased leaf area of branch leaves resulted in anincrease in total leaf area. There was no significant further increase above 1200A L L-1 CO2. Leaf starch concentration was increased by CO2 enrichment, althoughthere was no evidence of excessive starch accumulation. CO2 enrichment did nothave a significant effect on the leaf concentrations of sucrose, glucose, fructose orprotein. Total leaf content of inorganic nutrients was not changed by CO2enrichment. CO2 enrichment decreased the starch-corrected concentrations ofsome nutrients in leaves borne on main stems. That change was significant for K,Ca and Mn, and also for the ratio of Mn to Zn. In leaves borne on the lower part ofbranches, CO2 enrichment induced a slight reduction in N concentration.Interactions between CO2 enrichment and K supply on the nutrient relations ofplants were seldom detected.Increasing K limited leaf dry mass accumulation and leaf area production,reduced nutrient concentrations, and induced leaf chlorosis. The interpretation ofK effects was restricted by the possible detrimental effect of acetic acid. Thisnegative effect was probably enhanced by daily drifts in the pH of nutrient solutions.Both acetic acid additions and the size of pH drifts increased with increasing Ksupply. A possible beneficial effect of Na, when K supply was low, furtherconfounded the interpretation of K supply effects.Interveinal chlorosis appeared on both the uppermost leaves of main stemsand the lower leaves borne on branches, after one week of CO2 enrichment. It wasmore pronounced in the latter. Leaf chlorosis decreased toward the bottom ofmain stems, and toward the top of branches. Increasing K supply enhancedchlorosis in both CO2 enriched and unenriched plants. No relationship wasdetected between leaf chlorosis and leaf starch concentration or leaf temperature.Reduced leaf concentration of Mn coupled with alterations in the Mn to Zn ratio,and the appearance and distribution of symptoms, suggest that Mn deficiency playeda role in the induction of chlorosis by CO2 enrichment. However, other nutrients,such as K or N, or non-nutritional factors could be involved in the disorder.These studies confirmed that CO2 enrichment reduces leaf nutrientconcentration and makes plants more susceptible to nutrient stresses. Theimportance of inorganic nutrition in the regulation of plant responses to CO2enrichment was also verified.ivTABLE OF CONTENTSAbstract^ iiTable of Contents^ ivList of Tables viiiList of Figures ixList of Symbols^ xiAcknowledgements )diDedication xiiiChapter 1 Introduction^ 1Chapter 2 Literature Review 62.1 Importance of CO2 Enrichment Studies on Plants^ 62.2 History of CO2 Enrichment^ 92.3 Effects of CO2 Enrichment on Stomatal Conductance^ 112.4 Effects of CO2 Enrichment on Photosynthesis 132.5 Effects of CO2 Enrichment on Carbon Metabolism^ 152.5.1 Effects of CO2 Enrichment on Photosynthates 162.5.2 Effects of Starch Accumulation on Photosynthesis ^ 182.5.3 Effects of CO2 Enrichment on Protein Concentration 182.6 Effects of CO2 Enrichment on Plant Growth^ 192.7 Leaf Abnormalities Induced by CO2 Enrichment 222.7.1 History^ 222.7.2 Foliar Abnormalities ^ 222.8 Interactions Between CO2 and Plant Growth Regulators^ 252.9 Physiological Roles of K 262.9.1 Cation-Anion Balance^ 262.9.2 Osmoregulation and Cell Expansion^ 272.9.3 Stomatal Movement 272.9.4 Photosynthesis^ 28V2.9.5 Enzyme Activation and Carbon Metabolism^ 292.9.6 Phloem Transport and Sucrose Movement 302.9.7 Protein Synthesis^ 312.9.8 Substitution of K by Na 312.10 Interactions Between CO2 Enrichment and Mineral Nutrition^322.10.1 Responses of CO2 Enriched Plants to Mineral Nutrition ^332.10.2 Changes in the Mineral Nutrition of CO2 Enriched Plants ^332.10.3 Mineral Nutrition and Leaf Injury of CO2 Enriched Plants^35Chapter 3 Effects of CO2 Enrichment and K Nutrition on Growth ofChrysanthemum Plants^ 363.1 Introduction.^ 363.2 Materials and methods 393.2.1 First Experiment, Summer 1990^ 393.2.1.1 Plant culture^ 393.2.1.2 Experimental design 413.2.1.3 Statistical analysis 423.2.2 Second Experiment, Summer 1991^ 433.2.2.1 Plant Culture^ 433.2.2.2 Experimental Design and Statistical Analysis^463.3 Results^ 473.3.1 Growth Assessment, Summer 1990 Experiment^473.3.1.1 Effects of CO2 Enrichment on Plant Growth (1990)^473.3.1.2 Effects of CO2 Enrichment on Growth of Plant Parts (1990) ^ 473.3.1.3 Effects of CO2 Enrichment on Growth Indices (1990)^ 553.3.2 Growth Assessment, Summer 1991 Experiment^573.3.2.1 Effects of CO2 Enrichment and K Supply on Plant Growth(1991)^ 573.3.2.2 Effects of CO2 Enrichment and K Supply on Growth of PlantParts (1991) 633.3.2.3 Effects of CO2 Enrichment and K Supply on Growth Indices(1991)^ 703.4 Discussion^ 743.4.1 General Effects of CO2 Enrichment and K Supply on Plant Growth ^ 74vi3.4.2 Effects of CO2 Enrichment and K Nutrition on Growth ofPlant Parts ^ 773.4.3 Effects of CO2 Enrichment and K Nutrition on Growth Indices ^ 80Chapter 4 Effects of CO2 Enrichment and K Supply on Carbon MetaboliteConcentration and Inorganic Nutrient Status in Leaves^ 834.1 Introduction^ 834.2 Materials and Methods^ 874.2.1 Plant Culture 874.2.2 Determination of Photosynthate and Chlorophyll Concentration inLeaves^ 874.2.2.1 Extraction and Separation of Starch and Soluble Sugars^ 884.2.2.2 Determination of Starch ^ 884.2.2.3 Determination of Sucrose, D-Glucose and D-Fructose^ 894.2.2.4 Determination of Total Protein 904.2.2.5 Determination of Chlorophyll ^ 914.2.3 Determination of Inorganic Nutrient Concentration in Leaves ^ 924.2.3.1 Sampling Procedures^ 924.2.3.2 Leaf Tissue Analysis 934.2.3.3 Expression of the Inorganic Nutrient Concentration^ 934.2.3.4 Statistical Analysis^ 954.2.3.5 Relationships of Leaf Chlorosis with the Effects of CO2Enrichment on Plant Nutrition.^ 964.3 Results^ 974.3.1 Effects of CO2 Enrichment and K Supply on the Concentrations ofCarbon Metabolites in Leaves^ 974.3.2 Effects of CO2 Enrichment and K supply on the Inorganic NutrientStatus of Main Stem Leaves 1014.3.2.1 Total Leaf Contents of Nutrients^ 1014.3.2.2 Starch-Corrected Concentrations of Nutrients ^ 1054.3.2.3 Nutrient Concentrations in Main Stem Leaves (Dry MassBasis)^ 1124.3.3 Effects of CO2 Enrichment and K supply on the Inorganic NutrientStatus of Leaves Borne on the Lower Part of Branches^ 1124.3.3.1 Total Leaf Contents of Nutrients^ 1124.3.3.2 Starch-Corrected Concentrations of Nutrients in Leaves ^ 116vii4.3.4 Effects of CO2 Enrichment and K Supply on Leaf Chlorosis^ 1214.3.5 Nutritional Status in Relation to CO2 Induced Leaf Chlorosis^ 1274.4 Discussion^ 1294.4.1 Effects of CO2 Enrichment and K Supply on the Concentration ofPhotosynthates in Leaves^ 1294.4.1.1 Concentrations of Sugars 1294.4.1.2 Concentration of Starch 1294.4.1.3 Total Protein Concentration^ 1314.4.2 Effects of CO2 Enrichment on the Nutrient Status in Leaves^ 1324.4.2.1 Effects of CO2 Enrichment on Leaf Content of Nutrients ^ 1324.4.2.2 Effects of CO2 Enrichment on Leaf Concentration of InorganicNutrients^ 1324.4.3 Effects of K Supply on the Leaf Concentration of Nutrients^ 1364.4.4 Effects of CO2 Enrichment and K Nutrition on Leaf Chlorosis ^ 1374.4.5 Nutritional Status in Relation to CO2 induced Leaf Chlorosis ^ 139Chapter 5 General Discussion^ 142Chapter 6 Conclusions 150Literature Cited^ 153Appendix 1 166Appendix 2 167Appendix 3^ 169Appendix 4 171Appendix 5 174Appendix 6^ 182Appendix 7 183Appendix 8 184viiiLIST OF TABLESSummary of statistical significance of growth measures, summer 1990 ^ 48Regression coefficients for growth measures, summer 1990^49Summary of statistical significance, of growth measures, summer 1991 ^58Regression coefficients for growth measures, summer 1991^59Summary of statistical significance, concentration of metabolites in leavesborne on main stems and on the lower part of branches, summer 1991. 98Summary of statistical significance, total content of elements in mainstem leaves^ 102Summary of statistical significance, ratios between total content of cationsin main stem leaves.^ 104Summary of statistical significance, growth measures (loge) of main stemleaves.^ 106Summary of statistical significance, starch-corrected concentration ofnutrients in main stem leaves.^ 106Summary of statistical significance, ratios between starch-correctedconcentration of cations in main stem leaves.^ 110Summary of statistical significance, concentration (wn/WL) of elementsin main stem leaves.^ 113Summary of statistical significance, total content of elements in leavesborne on the lower part of branches.^ 114Summary of statistical significance, ratios between total contents ofcations in leaves borne on the lower part of branches.^ 114Summary of statistical significance growth measures (loge) of leavesborne on the lower part of branches, summer 1991.^ 117Summary of statistical significance, concentration (wn/WLNs) ofelements in leaves borne on the lower part of branches^ 117Summary of statistical significance of ratios between concentration(wn/VVI,Ns) of cations in leaves borne on the lower part of branches ^ 120Summary of statistical significance, concentration (wn/WL) of elementsin leaves borne on the lower part of branches^ 122Summary of statistical significance for leaf chlorophyll concentration,summer and fall 1991 ^ 122Table 3.1Table 3.2Table 3.3Table 3.4Table 4.1Table 4.2Table 4.3Table 4.4Table 4.5Table 4.6Table 4.7Table 4.8Table 4.9Table 4.10Table 4.11Table 4.12Table 4.13Table 4.14ixLIST OF FIGURESFigure 3.1 Diagram of the recirculating hydroponic system used for the 1991experiments.^ 45Figure 3.2 Effects of CO2 enrichment on W (a), LA (b) and WL (c),summer 1990. 52Figure 3.3 Effects of CO2 enrichment on LAb (a) WLb (b) Ws (c), Wb (d) and Wr(e), summer 1990.^ 54Figure 3.4 Effects of CO2 enrichment on AGR (a), R (b), E (c), F (d), LWR (e)and SLA (f), summer 1990.^ 56Figure 3.5 Effects of CO2 enrichment and K supply on W (a, b), LA (c, d), and WL(e, f), summer 1991 64Figure 3.6 Effects of CO2 enrichment and K supply on LAb (a, b) and LAu (c, d),summer 1991.^ 65Figure 3.7 Effects of CO 2 enrichment and K supply on WLm (a, b), WL1 (c, d), andWLu (e, f), summer 1991.^ 67Figure 3.8 Effects of CO2 enrichment and K supply on Ws (a, b) and Wb (c, d),summer 1991.^ 68Figure 3.9 Effects of CO2 enrichment and K supply on Wr (a, b) and Wy (c, d),summer 1991. 69Figure 3.10 Effects of CO2 enrichment and K supply on AGR (a, b), R (c, d),summer 1991.^ 72Figure 3.11 Effects of CO2 enrichment and K supply on F (a, b) and SLA (c, d),summer 1991. 73Figure 4.1 Effects of CO2 enrichment and K supply on starch concentration inleaves borne on main stems (a, b), leaves borne on the lower part ofbranches (c, d), and leaves borne on the upper part of branches (e, f) ofchrysanthemum plants, summer^ 99Figure 4.2 Effects of CO2 enrichment and K supply on total contents of P (a), Mn(b) in main stem leaves 103Figure 4.3 Effects of CO2 enrichment and K supply on the ratio between totalcontents of Mn and Zn in main stem leaves. Legend as on figure 4.2.103Figure 4.4 Effects of CO2 enrichment and K supply on the starch-correctedconcentration of N (a), P (b), K (c) and Ca (d) in main stem leaves^ 108xFigure 4.5 Effects of CO2 enrichment and K supply on the starch-correctedconcentrations of Mn (a), Cu (b), Zn (c) and Al (d)in main stem leaves.^ 109Figure 4.6 Effects of CO2 enrichment and K supply on the ratio between thestarch-corrected concentrations of Mn and Zn in main stem leaves.....111Figure 4.7 Effects of CO2 enrichment and K supply on leaf contents of N (a), P (b),Zn (c), Fe (d), B (e) and Na (f) in leaves borne on the lower part ofbranches.^ 115Figure 4.8 Effects of CO2 enrichment and K supply on the starch-correctedconcentrations of N (a), K (b), Mg (c) and Na (d) in leaves borne on thelower part of branches.^ 118Figure 4.9 Effects of CO2 enrichment and K supply on the starch-correctedconcentrations of Mn (a), Fe (b), Zn (c), Cu (d) and B (e) in leavesborne on the lower part of branches.^ 119Figure 4.10 Effects of CO2 enrichment and K supply on chlorophyll concentrationin leaves of main stems (a, b), the lower part of branches (c, d), and theupper part of branches (e, f), summer 1991. 124Figure 4.11 Effects of CO2 enrichment and K supply on chlorophyll concentrationin leaves of main stems (a, b), the lower (c, d) and the upper part ofbranches (e, f), fall 1991 ^ 126xiLIST OF SYMBOLSSymbol^Description (units)AGR Absolute growth rate (g day-1)F^Leaf area ratio (cm-2 g-1)H Harvest indexLA^Plant leaf area (loge cm-2)LAb Leaf area of leaves borne on branches (loge cm-2)LAI^Leaf area of leaves borne on the lower part of branches (loge cm-2)LAm Leaf area of leaves borne on main stems (loge cm-2)LAu^Leaf area of leaves borne on the upper part of branches (loge cm-2)LWR Leaf weight ratioPPFD^Photosynthetic photon flux density (mMol s-1 cm-2)R Relative growth rate (dayl)SLA^Specific leaf area (cm-2 g-1)SRR Shoot to root ratioE Unit leaf rate (g-2 day-1)wn^Inorganic nutrient mass (loge g)W Plant dry mass (loge g)Wb^Dry mass of branches (loge g)WL Leaf dry mass (loge g)WI+^Dry mass of leaves borne on branches (loge g)WLb Dry mass of leaves borne on the upper part of branches (loge g)WLI^Dry mass of leaves borne on the lower part of branches (loge g)VVLm Dry mass of leaves borne on main stems (loge g)WLNs^Leaf dry mass minus starch (loge g)Wr Dry mass of roots (loge g)Ws^Dry mass of main stems (loge g)Wstarch^Starch mass (loge g)WY Dry mass of flowers (loge g)xiiACKNOWLEDGEMENTSI am grateful to the many people and institutions who have helped me duringthese three years. In particular I thank Dr. Peter Jolliffe for his advice during thedevelopment of the project, and for his financial support during the last stage of mystudies. Financial support was also provided by the University of British Columbiathrough a University Graduate Fellowship. The advice of other members of mysupervisory committee, Dr. Brian Ellis, Dr. George Eaton and Dr. Robert Guy, atdifferent times and in many aspects is acknowledged with gratitude. I am verythankful to Angela Moloney for her invaluable help in plant management and datacollection during the summer 1991 experiment. I thank Derek White for histechnical support to keep the equipment and chambers running, Dr. Thomas Vogtand Leroy Scrubb for their assistance and advice in performing the analysis ofcarbon metabolites, and Yoder Canada for providing the chrysanthemum cuttingsfor the experiments. My appreciation is due also to Angela Moloney, NancyFurness and Murali Srinivasan for their helpful comments during the developmentof this manuscript.My especial thanks go to my wife Beatriz for her constant spiritual andphysical support during these years in Canada.DEDICATIONTo the memory of my father and to my mother.To my wife.1CHAPTER 1 INTRODUCTIONInterest in CO2 relations of plants arises from the significance of CO2 to plantfunction and from the increasing levels of CO2 in the earth's atmosphere during thepast century. Atmospheric CO2 increases are predicted to double the presentconcentrations in the next 50 to 100 years (Gates 1983). Despite such dramaticincreases, projected global CO2 concentrations would still be below those necessaryto sustain maximum growth rates in most plants (Hicklenton 1988). Also, CO2enrichment has been utilized in protected plant culture for many years, and this hasprovided the basic understanding of plant responses to high CO2 (Goldsberry 1986,Hicklenton 1988, Mortensen 1987, Nelson 1991). Although a great body ofinformation on the benefits of CO2 enrichment under protected cultivation hasexisted for many years (Wittwer 1970), research is still needed on methods forobtaining maximum benefits from CO2 enrichment (Hicklenton 1988).Photosynthesis is the predominant source of chemical energy and carboncompounds for growth processes, setting the upper limit for primary productivity ofgreen plants. Atmospheric CO2 is the substrate for this process in higher plants,and is therefore the source of carbon upon which plant growth depends (Huber et al.1985). The rate at which photosynthesis proceeds via the C3 pathway is oftenlimited by the present atmospheric CO2 concentrations, of about 350 A L L-1.Increasing CO2 above such levels induces higher net photosynthetic rates in manyplant species, particularly in C3 plants (Pearcy and Bjorkman 1983). Mostinvestigated plant species have the C3 pathway (Salisbury and Ross 1985).Plant responses to CO2 enrichment, however, are not solely derived fromphotosynthetic changes. They are also influenced by the effects of high CO2 on the2partitioning of carbon (Ehret and Jolliffe 1985b, Liu 1990, Madore and Grodzinski1985) and inorganic nutrients (Liu 1990, Tripp et al. 1991). Morphologicalresponses to high CO2 include increased branching (Rogers et al. 1984a), increasedstem elongation (Madore and Grodzinski 1985, Mortensen 1987), thicker leaveswith more densely packed palisade cells (Hofstra and Hesketh 1975, Thomas andHarvey 1983), reduced stomatal density (Gislerod and Nelson 1989), modified shootto root ratio (Sionit et al. 1981b, Tripp et at 1991), and earlier reproductivedevelopment (Wittwer 1970). Most studies also report increased leaf area (Fordand Thorne 1967, Hofstra and Hesketh 1975, Sionit et al. 1981b), and when leaf areais increased in the seedling stage subsequent growth may increase as well(Kriedemann and Wong 1984).Responses of plants to high CO2 are also modified by interactions with otherenvironmental factors including the supply of photosynthetically active radiation(Ehret and Jolliffe 1985a), temperature (Ehret and Jolliffe 1985a, Yelle et al. 1987),relative humidity (Gislerod and Nelson 1989, Mortensen 1987) and the supply ofinorganic nutrients (Goudriaan and Ruiter 1985, Mortensen 1987, Patterson andFlint 1982).Studies of the interaction between high CO2 and inorganic nutrition of plantsare scarce (Porter and Grodzinski 1989). High CO 2 levels usually increase plantdry matter accumulation, and thus may increase the demand for inorganic nutrients(Kimball 1986). Lack of sufficient nutrient uptake and translocation may limit thestimulation of plant growth by high CO2 (Mortensen 1987). Uptake of N and P insoybean plants was increased by CO2 enrichment (Cure et al. 1988a, 1988b, Israel1990). In tomato plants, NO3 uptake and translocation was increased by CO23enrichment, but the effect was dependent on root zone temperature (Yelle et al.1987). Nutrient uptake was not increased in CO2-enriched cotton (Wong 1979) orcocklebur (Hocking and Meyer 1985).Effects of elevated CO2 on plant functions may interact with inorganicnutrient uptake and translocation. Transpiration reduction, due to reducedstomatal aperture under high CO2, may alter the uptake and translocation ofnutrients (Glass 1989, Madsen 1975), thereby affecting plant growth (Gislerod andNelson 1989, Mortensen 1987). Decreased tissue N concentrations as a result ofCO2 enrichment were correlated with reduced photosynthetic rates (Larigauderieet al. 1988). Increased translocation of NO3" from roots to shoots was correlatedwith increased growth of CO2 enriched tomato plants (Yelle et at. 1987). Leafconcentrations of K and Na also affect transpiration rates and the water useefficiency by plants (Marschner 1987), and their abundance may influence plantresponses to high CO2.Leaf abnormalities, including chlorosis, necrosis and curling, have beenreported in connection with prolonged exposure of plants to CO2 enrichment. Suchleaf abnormalities have been observed on leaves of dwarf bean (Ehret and Jolliffe1985a, Liu 1990), cotton (Hesketh et at. 1971), tomato (Madsen 1974, Tripp et al.1991), monoecious cucumber (Peet 1986), chrysanthemum and many othergreenhouse ornamental plants (Van Berkel 1984, Goldsberry 1986, Mortensen1987). Leaf abnormalities induced by CO2 enrichment may be widespread, butrecognition of this problem was delayed due to the variety of symptoms observed(Van Berkel 1984, Ehret and Jolliffe 1985a). Symptoms vary according toenvironmental conditions (Mortensen 1987), plant species or even cultivar (Van4Berkel 1984). Leaf abnormalities may affect photosynthetic and plant growthresponses to CO2 enrichment (Ehret and Jolliffe 1985a), thus affecting yield, qualityand aesthetic value of ornamental plants.Studies on dwarf bean plants found limited growth response to CO2enrichment (Jolliffe and Ehret 1985). Dry mass was increased, but there was noincrease in leaf area. Leaf abnormalities occurred on primary leaves followingapproximately three weeks of CO2 enrichment (Ehret and Jolliffe 1985a, Liu 1990).The onset of leaf abnormalities was correlated with increased remobilization of Nand K from affected leaves to younger tissues (Liu 1990).The cause of CO2 induced leaf abnormalities is not fully understood.Injurious effects of CO2 enrichment on leaves have been attributed to four possiblereasons: (1) high leaf temperatures induced by limited transpiration at highirradiance, particularly when there are sudden changes in the water balance withinthe plant (Van Berkel 1984), (2) high starch content in leaves (Ehret and Jolliffe1985a, Hesketh et at. 1971, Madsen 1975), which may impair chloroplast function(Cave et al. 1981, Wulff and Strain 1982) or affect other processes by modifying theC:N ratio (Kuehny et at. 1991); (3) reduced leaf nutrient concentration, as aconsequence of limited nutrient supply (Liu 1990), uptake or translocation fromroot to shoots induced by lower transpiration rates (Mortensen 1987); and (4)reduced leaf nutrient concentration due to increased nutrient remobilization fromolder leaves to new or stronger sinks (Liu 1990, Tripp eta!. 1991).The present research was conducted to explore the interacting effects of CO2enrichment and K nutrition on growth, and on the development of leafabnormalities in chrysanthemum plants cv. 'Envy'. The first study (Chapter 3)5examined the progress of growth under CO2 enrichment alone, and under CO2enrichment with different levels of K supply. The distribution of growth amongvarious plant parts was assessed in order to determine the timing and location ofresponses. The second study (Chapter 4) considered the impact of CO2 enrichmentand different levels of K supply on leaf nutrient status and the appearance of leafabnormalities.6CHAPTER 2 LITERATURE REVIEWThis review focuses on secondary factors affecting function and growth ofplants grown under CO2 enrichment, with the main emphasis being placed oninteractions between CO 2 enrichment and K nutrition. Comprehensive reviews onCO2 enrichment were published by Enoch and Kimball (1986a, 1986b) and Lemon(1983). As well, the metabolic functions of K have been reviewed (Huber 1984b,Lauchli and Pfluger 1978, Marschner 1986).2.1 IMPORTANCE OF CO2 ENRICHMENT STUDIES ON PLANTSThe study of effects of carbon dioxide (CO2) enrichment on plants is a topicof intensive research which is supported by three main interests. Due to theimportance of CO2 in plant function, physiological changes induced by high CO2have major effects on plant growth. CO2 enrichment has been extensively used formore than three decades to enhance productivity of commercial greenhouse crops.Concern about rising global levels of CO2 increased the interest in CO2 researchduring the last decade.When other conditions are not limiting, plant growth and agricultural yieldare enhanced by elevated CO2 concentrations (Kimball 1983, Wittwer 1983). CO2enrichment liberates plants from the common limitation of carbon supply forphotosynthesis (Mortensen 1987). The fate of the additional carbon provided andother secondary effects, however, are also important in determining plant responsesto high CO2 (Ehret and Jolliffe 1985b). In addition to its role in photosynthesisCO2 has important roles in several metabolic processes. CO2 is the product ofrespiration; CO2 also interacts with abscisic acid (ABA) to regulate stomatal7aperture by affecting K movement to or from guard cells; ionic forms of CO2 (i.e.HCO3-) participate in membrane transport systems; and CO2 interacts in the growthregulating action of ethylene. As a consequence of the involvement of CO2 in suchdiverse physiological processes, plant responses to high CO2 cannot be explainedsimply on the basis of its photosynthetic effects (Ehret and Jolliffe 1985a). CO2enrichment alters carbon partitioning in leaves (Geiger et al. 1983, Ho et al. 1977,Hoddinott and Jolliffe 1988, Madore and Grodzinski 1985). In addition, CO2enrichment induces changes in secondary factors within the plant which alsoinfluence plant growth responses (Ehret and Jolliffe 1985b). These changes includeincreases in leaf area, altered dry matter partitioning (Ehret and Jolliffe 1985b,Jolliffe and Hoddinott 1988, Liu 1990), the induction of leaf injury (Liu 1990, Trippet al. 1991, Van Berkel 1984), and altered nutrient relations (Goudriaan and deRuiter 1985, Kuehny et at. 1991, Liu 1990, Patterson and Flint 1982, Tripp eta!.1991). Whereas much research has been performed on photosynthetic responses toCO2 enrichment, less is known of non-photosynthetic responses to CO2 enrichment,and the interactions of CO2 enrichment with secondary environmental factors,particularly the availability of inorganic nutrients (Porter and Grodzinski 1989).Chrysanthemum (Dertdranthema grandiflora Tzvelev) is an importantgreenhouse crop worldwide in economic terms (Eng et al. 1985). As with mostgreenhouse crops, chrysanthemum benefits from high CO2 atmospheres (Eng et al.1985, Goldsberry 1986, Nelson 1991). CO2 is often a limiting factor in commercialproduction of greenhouse crops, particularly during winter months, when solarradiation is available for photosynthesis but low outdoor temperatures restrictgreenhouse ventilation (Eng et at. 1985, Hicklenton 1988, Mastalerz 1977). Under8such conditions CO2 levels inside the greenhouse may drop to 100 A L L-1 five to sixhours after sunrise (Wittwer and Robb 1964), a concentration which is only slightlyabove the compensation point for most C3 species (Eng et al. 1985, Mastalerz 1977).Therefore, in northern latitudes (north of the 40th parallel), CO2 enrichment hasbeen widely practised to improve productivity and quality of greenhouse crops(Goldsberry 1986, Hanan 1986, Hicklenton 1988, Mastalerz 1977, Mortensen 1987,Wittwer 1983). In southern climates where more ventilation is needed due to hightemperatures, CO2 enrichment is possible only for a comparatively short season.Yet the high temperatures and irradiances prevalent under such conditions wouldresult in the greatest use efficiency of CO2. This implies that CO2 enrichmentwould be significantly more beneficial at low latitudes, if only supplemental CO2could be applied for longer periods of time (Hicklenton 1988).Detrimental effects of high CO2 on plants have occasionally been reported.Most of these reports discuss a variety of leaf injuries observed under CO2enrichment (Van Berkel 1984, Goldsberry 1986, Jolliffe and Ehret 1985a,Mortensen 1987, Peet et al. 1986). Nevertheless, even before any visible damagewas apparent on CO2 enriched bean leaves, photosynthesis was reduced, indicatingthat adverse effects may sometimes reduce the benefits of CO2 enrichment withoutbeing noticeable (Ehret and Ehret 1985a). The nature of CO2-induced leaf injuriesis unclear, yet few studies have been done to explore them.Immediate benefits of CO2 enrichment on greenhouse crops haveencouraged research on this topic for more than three decades (Goldsberry 1986).The basic scientific knowledge provided by such studies is valuable now, in view ofthe rapid increases in the CO2 levels taking place in the earth's atmosphere.9Current increases in atmospheric CO2 are estimated at about 2 ,u L L-1 per year, andit is predicted that present atmospheric CO2 concentration may double in the nextcentury (Kimball 1983). The primary effect of such a change on agriculturalproduction is expected to be due to the increased CO2 supply per se, rather than dueto the climate change induced by higher CO2 concentrations (Kimball 1983). Thishas stimulated further studies on the potential direct and indirect effects of CO2enrichment on plant growth and crop productivity (Enoch and Kimball 1986, Lemon1983, Mortensen 1987).2.2 HISTORY OF CO2 ENRICHMENTIncorporation of organic materials (e.g. straw, animal manure) in the growingmedium is the oldest and simplest method of CO2 enrichment. As microbialoxidation of organic waste proceeds, heat is generated and CO2 is released to theatmosphere. Such a technique was successfully used in France during the 19thcentury. In this century, CO2 enrichment has been applied in the same way togreenhouse crops, often unconsciously, through the use of organic materials asamendments to improve soil characteristics (Hicklenton 1988). Experiments ondirect use of CO2 enrichment performed at the beginning of the century in Englandproduced adverse results, due to gas contaminants as was discovered later. Later in1904, the benefits of CO2 enrichment on plant growth were demonstrated by theFrench scientist Demoussy. By 1918, Cummings and Jones established thebeneficial effects of CO2 on growth and yield of plants in greenhouses in the USA(Hicklenton 1988). Despite these pioneering studies, commercial adoption of thetechnique was slow both in Europe and North America. It was not until the late101950's that new studies reawakened interest in CO2 enrichment and instigatedworldwide commercial application. Such change was probably induced by theavailability of relatively inexpensive sources of uncontaminated CO2 (i.e. pure CO2,combustion of propane, combustion of natural gas). In the early 1960's, researchdealt with the effects of elevated CO2 concentrations on vegetative growth of lettuceand fruit yield of tomato, leading to the adoption of CO2 enrichment for productionof such crops (Hicklenton 1988). About the same time, research began on theeffects of CO2 enrichment on greenhouse flower crops (Goldsberry 1986).Researchers focused their attention on commercially important species such as rose,carnation and poinsettia. Increases in vegetative growth, flower size and shorteningof the time to blooming were induced by CO2 concentrations up to 600 AL L-1.Benefits of CO2 enrichment on flower crops were harder to quantify economicallythan the yield improvements recorded on vegetable crops (Goldsberry 1986,Hicklenton 1988). Subsequent studies confirmed the benefits of CO2 enrichment inimproving flower quality and yield on many ornamental plant species (Goldsberry1986, Hicklenton 1988) including chrysanthemums (Eng et al. 1983, 1985,Goldsberry 1986, Hughes and Cockshull 1971a, 1971b, 1972, Kuehny eta!. 1991,Mortensen and Moe 1983). Nowadays, CO2 enrichment is widely practised innorthern latitudes where infrequent ventilation during the winter months allows aneasy and economical maintenance of a 1000 il L L-1 CO2 concentration (Eng eta!.1985, Hicklenton 1988). Even in southern regions, new control systems andproduction methods (Kimball and Mitchell 1979, Willits and Peet 1981) are makingCO2 enrichment an economically attractive option during part of the year(Hicklenton 1988).11CO2 enrichment accompanied with proper environmental conditionsfacilitates the accelerated production of higher quality crops with greater yields(Hicklenton 1988). While abundant information is available supporting thebenefits of CO2 enrichment (Kimball 1983, Wittwer 1970), research today is focusedon the development of methods for maximizing these benefits (Hicklenton 1988).2.3 EFFECTS OF CO2 ENRICHMENT ON STOMA TAL CONDUCTANCERemoval of intercellular CO2 by mesophyll cells induces stomates of mostspecies to open during photosynthesis (Salisbury and Ross 1985). Conversely, highCO2 concentrations cause partial closure of stomates (Gislerod and Nelson 1989,Morison 1985, Salisbury and Ross 1985), and as a consequence stomatalconductance is usually decreased under CO2 enrichment (Bravdo 1986, Ehret andJolliffe 1985b, Sionit and Kramer 1986). Changes in stomatal aperture modifyseveral functions, including photosynthesis, transpiration, leaf temperature, wateruse efficiency and plant water status (Pearcy and Bjorkman 1983). Water useefficiency of CO2 enriched plants is increased because water consumption is reducedand at the same time photosynthesis is increased (Kimball 1986b, Morison 1985,Mortensen 1987, Rogers et at. 1984, Sionit and Kramer 1986).Decreased stomatal conductance was suggested in some studies as the causeof the reduced photosynthesis sometimes observed during prolonged exposure toelevated CO2 (Aoki and Yabuki 1977, Imai and Murata 1979, Peet et al. 1986). Inmost reports, however, decreased stomatal conductance only had a small effect onthe inhibition of photosynthesis caused by long term exposure to CO2 enrichment(Ehret and Jolliffe 1985b, Spencer and Bowes 1986).12The sensitivity of stomates to CO2 varies with plant species, leaf age andenvironmental conditions (Gislerod and Nelson 1989, Morison 1985). Also, CO2interacts with abscisic acid in controlling stomatal opening (Radin and Ackerson1982). High irradiance enhances the closing effect of high CO2 (Sharkey andRaschke 1981, Wong et al. 1978). Increased stomatal aperture of CO2 enrichedplants grown under high relative humidity was suggested as the cause for greatergrowth of chrysanthemum (Gislerod and Nelson 1989). CO2 enrichment reducedthe leaf osmotic potential of wheat plants. The pattern of decline of leaf osmoticpotential as water stress increased in CO2 enriched plants suggested superioradaptation to water stress and turgor maintenance in such plants (Sionit et at.1981b). Similarly, CO2 enrichment delayed or prevented the onset of severe waterstress on soybean plants (Rogers et al. 1984).Rising CO2 concentrations may reduce transpiration rates by 20% to 40%(Morison 1985, Rogers et at. 1984). Lower rates of transpiration coupled withincreased net photosynthesis and growth, suggest that the supply of inorganicnutrients for CO2 enriched greenhouse crops should be higher (Kimball 1986b).For example growth responses of CO2 enriched Begonia plants under high relativehumidity and scarce nutrient supply were reduced, and leaf chlorosis was observed.However, when nutrient supply was increased such effects were prevented. Thisindicates that decreased transpiration may affect the inorganic relations of CO2enriched plants (Mortensen 1987).132.4 EFFECTS OF CO2 ENRICHMENT ON PHOTOSYNTHESISCO2 enrichment affects the CO2 exchange characteristics of plants, andexerts a major influence on the carbon assimilation process (Ehret and Jolliffe1985b). CO2 enrichment has a positive effect on photosynthesis mainly byincreasing the ratio of CO2 to 02 at RuBP carboxylase (rubisco), decreasing thephotorespiratory activity (Enoch et al. 1978; Vu et al. 1989). Additional CO2 alsoincreases the photosynthetic substrate thus enhancing net photosynthesis(Mortensen 1987), particularly under greenhouse conditions. A decline inphotosynthetic capacity, however, has been observed on plants under long termexposure to CO2 enrichment (Ehret and Jolliffe 1985b).Diffusion governs the transport of CO2 from the external atmosphere tofixation sites inside the chloroplasts. High atmospheric CO2 concentrationsincrease the gradient between the atmosphere and fixation sites, directly promotingCO2 diffusion (Bravdo 1986). In such a way, high CO2 atmospheres increase CO2fixation by rubisco, leading to higher net photosynthesis (Mortensen 1987; Salisburyand Ross 1985, Wong 1980).Increased photosynthesis rates are a common initial response to CO2enrichment in non-woody species such as tomato (Hicklenton and Jolliffe 1980,Yelle et al. 1987), cucumber (Peet et al. 1986), chrysanthemums (Mortensen 1984,Mortensen and Moe 1983a), and water hyacinth (Spencer and Bowes 1986).However, a decline in photosynthetic capacity has been observed in plants exposedfor long terms (days or weeks) to CO2 enrichment (Aoki and Yabuki 1977, Deluciaet al. 1978, Ehret and Jolliffe 1985b, Imai and Murata 1978, Mortensen and Moe1983b, Peet et a 1986). This acclimation response is influenced by the previous14growing environment and growing conditions at the time of measurement (Peet et al.1986). Hence, when leaves from CO2 enriched plants and leaves from unenrichedplants were compared at the same CO2 level, leaves grown under CO2 enrichmenthad lower photosynthetic rates. If tested at the same CO2 concentrations at whichthe plants were grown, however, the CO2 enriched leaves had higher photosynthesisrates (Ehret and Jolliffe 1985b, Hicklenton and Jolliffe 1978, Huber et al. 1984,Poorter et al. 1988, Vu et al. 1989). Photosynthetic rates of chrysanthemum plantsdecreased after two weeks of CO2 enrichment, compared to those of unenrichedplants (Mortensen 1986). It is suggested that concentrations of about 900 A L L-1CO2 saturate the photosynthetic capacity of chrysanthemum plants (Hughes andCockshull 1972a, 1972b, Mortensen and Moe 1983b, Skoye and Toop 1973).Hence, concentrations up to 1000 A L L-1 CO2 are recommended for chrysanthemumgrowing (Mortensen 1987, Hicklenton 1988).This decline in photosynthetic capacity has been associated with decreasedactivities of rubisco (Hicklenton and Jolliffe 1980, Peet et al. 1986, Porter andGrodzinski 1984, Sage et al. 1988, Yelle et al. 1989) and carbonic anhydrase (CA)(Peet et al. 1986, Porter and Grodzinski 1984), excessive starch accumulation(Nafziger and Koller 1976), lower stomatal conductance (Imai and Murata 1978,Spencer and Bowes 1986), indirect growth effects (Poorter et al. 1988), and leafinjury (Ehret and Jolliffe 1985a). Photosynthetic rates in plants can be reduced byCO2 enrichment without visible leaf injury (Ehret and Jolliffe 1985a). Althoughchanges in photosynthetic activity due to high CO2 have been correlated withchanges in rubisco activity (Hicklenton and Jolliffe 1980, Peet et al. 1986, Porter andGrodzinski 1984), a cause and effect relationship has yet to be demonstrated.15Studies on five plant species indicated that the measured rubisco content was higherthan that required to support the observed photosynthetic rates (Sage et al. 1989).In rose plants, neither rubisco nor CA activities were affected by CO2 enrichment(Beeson and Graham 1991). The maintenance of normal levels of rubisco and CAactivities, which allowed the maintenance of high levels of carbon assimilation, wassuggested as the reason for long term yield increases under long term CO2enrichment of roses (Beeson and Graham 1991).The demand by sink tissues may also influence photosynthetic capacity(Zeevart 1979, Gifford and Evans 1981). Secondary factors, such leaf age andirradiance level, which can affect the source-sink relationships within the plant, mayalso modulate the photosynthetic responses of plants (Beeson and Graham 1991,Ehret and Jolliffe 1985b, Woltz 1969). High sink demand induced the maintenanceof higher photosynthetic rates in plants under CO2 enrichment (Beeson and Graham1991, Mauney et al. 1978, Mor and Halevy 1979, Peet et al. 1976), and CO2enrichment reduced the photosynthetic rates of sink-limited plants (Clough et al.1981, Peet 1984). It appears that continuous demand for the extra photosynthatesproduced under CO2 enrichment prevents feedback inhibition that would decreasethe activity of nibisco, and thus photosynthesis (Beeson and Graham 1991).2.5 EFFECTS OF CO2 ENRICHMENT ON CARBON METABOLISMBeyond the impact of CO2 enrichment on photosynthesis, plant responses toCO2 enrichment also depend on internal and external secondary factors. Amongthose, the fate of the additional photosynthates within the plant is critical indetermining plant responses to CO2 enrichment (Ehret and Jolliffe 1985b). Starch16accumulation in leaves is a characteristic response to high CO2 (Chatterton et al.1972, Ehret and Jolliffe 1985b, Liu 1990, Madore and Grodzinski 1985). In CO2enriched plants carbon partitioning is altered, and this may lead to photosynthateretention in the leaves (Hoddinott and Jolliffe 1988, Liu 1990, Madore andGrodzinski 1985). Restrictions in carbon partitioning may be induced by excessphotosynthetic activity over export due to limited capacity of the transport systemor, more probably, by a lower sink demand (Ehret and Jolliffe 1985b).2.5.1 Effects of CO2 Enrichment on PhotosynthatesCO2 enrichment may affect both synthesis of sucrose and subsequent exportof sugars from leaves, as shown by its influence on the content and diurnal variationof starch and sugars in leaves of cotton (Delucia et at. 1978) and white clover(Scheidegger and Nosberger 1984). While both starch and sucrose synthesisincreased with increasing CO2 availability, the increase in starch was always greaterthan in sucrose (Sharkey et al. 1985). Starch was mainly accumulated in the firsthalf of the light period, and sugar in the second half. Accumulation of both starchand sugar during the photoperiod was followed by degradation and export of bothduring the dark period (Scheidegger and Nosberger 1984). In CO2 enriched plants,however, leaf starch was not totally depleted in the dark, leading to starchaccumulation (Delucia et al. 1978, Nafziger and Koller 1976, Madore andGrodzinski 1985). Sugar accumulation in leaves appeared to be caused by source-sink imbalances (Scheidegger and Nosberger 1984).The rate of sucrose synthesis increases as photosynthesis rate is increased,but it does not increase in parallel with the net carbon exchange rate. This suggests17that biochemical factors within the cell influence the capacity for sucrose synthesis(Huber et al. 1985). The rate of sucrose synthesis in leaves is regulated mainly bychanges in the activity of existing enzymes, particularly sucrose phosphate synthase(SPS), and fructose-1,6-bisphosphate-l-phosphatase, which are not increased byCO2 enrichment (Huber et al. 1984a).The direction and extent of sucrose transport is mainly determined by thesucrose gradient between the source leaf and sinks (Beevers 1985). CO2 maydirectly or indirectly affect H+ -coupled transfer mechanisms such as phloemloading, sucrose storage, and solute transport into expanding cells (Tolbert 1983).CO2 enrichment increased photoassimilate export from leaves of dwarf cucumber,and also altered the nature of the metabolites exported (Madore and Grodzinski1985). Similarly, CO2 enrichment increased carbon transport in tomato (Ho et al.1977) and sugar beet (Geiger et al. 1983) leaves. In contrast, proportionally less ofthe newly fixed carbon was exported from primary leaves of bean plants (Hoddinottand Jolliffe 1988) or soybean (Huber et al. 1984a).Leaf concentrations of sucrose were increased in CO2 enriched sugar beet.In such plants, sucrose is stored mainly in the vacuole, which may be less readilyavailable for transport than sucrose found in the cytoplasm or in the phloem ofminor veins (Geiger et al. 1983). Small increases in sugar concentration were alsoreported in leaves of CO2 enriched soybean (Allen et al. 1988, Havelka et al. 1984,Huber et al. 1984a, Hrubec et ai. 1985, Vu et al. 1989), and dwarf beans (Liu 1990).Other studies did not find any sugar increase in leaves of cotton, soybean, sunfloweror sorghum (Mauney et ai. 1978), soybean (Mauney et at 1978, Finn and Brun 1982,Nafziger and Koller 1976). Whereas Hofstra and Hesketh (1975) reported reduced18sugar concentration on leaves of CO2 enriched soybean. Such varied responses,even within the same species, could be related with the sampling methods ofdifferent studies, particularly age and time (of the day) of sampling.2.5.2 Effects of Starch Accumulation on PhotosynthesisStarch accumulation in CO2 enriched plants is presumed to induce a declinein the photosynthetic capacity (Azcon-Bieto 1983, Chatterton et al. 1972, Nafzigerand Koller 1976). In cotton plants, the photosynthesis reduction was reversed bytransferring plants back to low CO2 atmospheres, and the recovery of thephotosynthetic rate was correlated with starch depletion (Sasek et al. 1985). Inyoung tomato leaves, however, starch concentration was not related to the reductionin photosynthesis rate (Yelle et al. 1989a), and only a weak correlation was detectedin bean plants (Ehret and Jolliffe 1985b).Mechanisms of the inhibition of photosynthesis associated with starchaccumulation are still not well defined. Physical mechanisms of disturbance havebeen suggested. These include interference of large starch grains with theintercellular transport of CO2 (Nafziger and Koller 1976), shading of thechloroplasts by starch grains (Warren-Wilson 1966), contortion of the chloroplastlamellae, or actual disruption of the chloroplasts by large unusually shaped starchgrains (Cave et al. 1981, Wulff and Strain 1982).2.5.3 Effects of CO2 Enrichment on Protein ConcentrationCO2 enrichment did not change total protein concentration in leaves ofsoybean (Havelka et al. 1984), dwarf beans (Liu 1990, Porter and Grodzinski 1984)19or maize (Wong 1979). CO2-enriched bean leaves exhibited a greater decline intotal carbonic anhydrase activity than did unenriched ones, even though totalprotein content was unchanged, indicating that CO2 enrichment may inducequalitative changes in the protein fraction (Porter and Grodzinski 1984).2.6 EFFECTS OF CO2 ENRICHMENT ON PLANT GROWTHIn addition to its influence in the CO2 exchange characteristics and carbonmetabolism, CO2 enrichment induces changes in secondary factors which alsoinfluence growth responses of plants (Ehret and Jolliffe 1985b). Environmentalgrowing conditions (Ehret and Jolliffe 1985a, Eng et at. 1985, Hughes and Cockshull1971a, 1971b, 1972) and cultural practices affecting source sink relationships(Beeson and Graham 1991, Van Berkel 1984, Peet et al. 1986) also affect growthresponses to CO2 enrichment plants.CO2 enrichment often leads to enhanced growth and yield, can shortencropping time, and, in many instances, enhances the quality of produce (size andgrade of flowers or fruits) (Hicklenton 1988). Although benefits of CO2 enrichmentare well documented (Kimball 1983, Wittwer 1970), there are large differences inthe growth response of different species (Scheidegger and Nosberger 1984).Usually, indeterminate species show greater CO2 effects than do determinate plants(Kramer 1981). Yield effect on flower crops is often less evident than in othercrops, mainly because flower yields are based on number of blooms per plant ratherthan on bloom weight (Hicklenton 1988, Kimball 1983).Dry mass of plants is usually increased by CO2 enrichment, most of theincrease often results from the development of greater number of heavier and larger20leaves (Delucia et al. 1978, Ehret and Jolliffe 1985b, Sasek and Strain 1988, 1989).Leaf mass increase is largely due to starch accumulation in older leaves (Ehret andJolliffe 1985b, Jolliffe and Hoddinott 1988, Porter and Grodzinski 1989).Anatomical changes, such as production of thicker leaves under CO2 enrichment(Hofstra and Hesketh 1975, Thomas and Harvey 1983) may also contribute toincrease leaf mass. Although no change was observed in bean (Ehret 1983) or corn,a C4 plant (Thomas and Harvey 1983). Increases in dry mass are usually correlatedwith the leaf area response of plants (Kriedemann and Wong 1984). Most CO2studies report leaf area increases, although often such increases are smaller thanleaf mass increases (Ford and Thorne 1967, Hofstra and Hesketh 1975, Sionit et al.1981b). CO2 enrichment may enhance leaf expansion as well as leaf productionrates (Sasek and Strain 1989). A few studies, however, have reported no increase inleaf area of plants grown under CO2 enrichment (Thomas et al. 1975, Kriedemannand Wong 1984, Jolliffe and Ehret 1985, Liu 1990, Peet 1986).Growth may be more affected at certain developmental stages, and increasedleaf area production during early growth in turn may be compounded withimportant gains in future growth or development (Clough et al. 1981). Increasedgrowth under high CO2 occurs when demand for assimilates is high (Clough et al.1981); for example, during the early stages of seedling growth or early fruitdevelopment (Mauney et al. 1978). Kimball (1983) indicates that growth of youngplants appears to be stimulated more by high CO2 than does growth of matureplants. This is also supported by early increases in the relative growth rate of dwarfbean (Jolliffe and Ehret 1985), wheat (Neales and Nicholls 1978), Plantago major(Poorter et al. 1988), and tomato (Yelle et al. 1990). In monoecious cucumber, CO221enrichment did not increase fruit mass or number, nor did it increase overallbiomass, leaf area or relative growth rate beyond the first 16 days after seeding(Peet 1986).In chrysanthemum plants, effects of CO2 enrichment are highly dependent onother environmental conditions, particularly irradiance levels (Eng et al. 1985,Hughes and Cockshull 1971a, 1971a, 1972). In most cases CO2 enrichment does notincrease number of blooms per unit area (Goldsberry 1986, Hicklenton 1988), andflower initiation and maturation are only marginally affected (Hughes andCockshull 1971a). When crop yield is not increased, the main benefit of CO2enrichment for chrysanthemum is the production of thicker, heavier stems of greaterheight (Eng et al. 1985, Mortensen and Moe 1983a, Nelson 1991). Such effects canincrease the number of marketable blooms per crop, and usually make CO2enriched plants stronger and more resistant to handling and transit (Hicklenton1988). Increased plant height allows for early harvest and may increase themarketability of cut flowers, but is not a desirable feature in pot plants (Hicklenton1988, Nelson 1991). Under winter conditions, with low irradiance and low outsidetemperatures, benefits of CO2 enrichment to greenhouse grown chrysanthemumplants are more noticeable. Under such conditions, dry mass production, numberof flowers per stem, and final yield of flowers are increased (Eng et al. 1985, Hughesand Cockshull 1971a). Additional benefits accrue from increased number andquality of chrysanthemum cuttings produced during winter when CO2 enrichment isprovided at the propagation stage (Eng et al. 1983).222.7 LEAF ABNORMALITIES INDUCED BY CO2 ENRICHMENT2.7.1 HistoryDetrimental effects of CO2 enrichment were occasionally reported in earlystudies. However, the injurious effects of high CO2 were not widely recognizeduntil the mid-1980's (Ehret and Jolliffe 1985a, Van Berkel 1984). Most damageobserved earlier under CO2 enrichment was commonly attributed to contaminantsintroduced with CO2 (Ehret and Jolliffe 1985b). Pollutants associated with CO2enrichment which may cause problems to greenhouse plants fall into three generalcategories. They are, non-combustion gases with phytotoxic effects (i.e. propylene),phytotoxic products of incomplete combustion (i.e. ethylene, SO2, CO and N oxides),and gases which also have physiological effects on humans (i.e. CO) (Hicklenton1988, Nelson 1991).The recognition of CO2-induced disorders was probably delayed by theinconsistency of symptoms in different plant species, the possibility of contaminantgases, and variation in the degree of CO2-induced leaf injury, perhaps caused bysecondary environmental factors (Ehret and Jolliffe 1985a).2.7.2 Foliar AbnormalitiesReports of abnormalities in leaves of plants under long-term exposure toCO2 enrichment include, chlorosis and necrosis of older leaves of cucumber(Wittwer 1967, Heij 1984), chrysanthemums (Van Berkel 1984, Mortensen 1987),Gerbera (Van Berkel 1984), tomato (Van Berkel 1984), basil (Wallick and Zinnen1990), potato (Goudriaan and de Ruiter 1985), cotton (Hesketh et at. 1971), soybean23(Chang 1975, Hesketh et al. 1971) and dwarf beans (Ehret and Jolliffe 1985a, Liu1990). In tomato plants, rolled, deformed, and necrotic leaves increased withincreasing CO2 concentrations (Madsen 1974). As the severity of such symptomsincreased, K concentrations decreased in the affected leaves (Tripp et al. 1991).Paler foliage and marginal chlorosis on all leaves were observed on monoeciouscucumber (Peet et at. 1986). Chlorosis and necrosis of the top leaflets of newlymature leaves were observed in CO2-enriched tomato (Heij et al. 1983). Incommercial greenhouses, leaf injuries induced by CO2 enrichment have beenreported in young tomato plants (Van Berkel 1984), and were observed in severalornamental species in Burnaby Lake Greenhouses, Surrey B. C. (Jolliffe 1991,personal communication).Although the cause of CO2-induced leaf injuries is not fully understood,several physiological and environmental factors have been associated with the onsetof injury, these include irradiance (Ehret and Jolliffe 1985a, Mortensen 1987),temperature (Ehret and Jolliffe 1985a), sudden temperature changes (Van Berkel1984), and relative humidity (Mortensen 1987). In other studies the degree of leafinjury under CO2 enrichment has been correlated to increased leaf starchconcentration (Cave et al. 1981, Ehret and Jolliffe 1985a, Goudriaan and de Ruiter1983, Liu 1990, Hesketh et al. 1971, Wulff and Strain 1982).In unenriched chrysanthemum plants leaf abnormalities, similar to thoseobserved under CO2 enrichment, were associated with starch accumulation (Woltz1969, Woltz and Engelhard 1971). Symptoms observed include thickening,downward rolling of margins, chlorotic mottling, bronzing and actual necrosis ofsmall leaf areas of older leaves (Woltz 1969). Such symptoms at certain stages24closely resembled those of Mg deficiency, but the disorder occurred uniformly evenwith high levels of Mg fertilization (Woltz 1969). Conditions that favoredaccumulation of photosynthate, such as increased irradiance and heavy disbudding,also favored the appearance of the disorder (Woltz 1969). In contrast, cultivarscapable of rapid growth were less susceptible to leaf injury (Woltz and Engelhard1971).Leaf chlorosis in Gerbera plants however, was not correlated with starchconcentration. Leaf injury was correlated with the concentration of totalcarbohydrates, although in some cases, symptom severity was more closelycorrelated with the concentrations of glucose and total sugars (Van Berkel 1984).Similarly, a seasonal foliar deformation observed in CO2 enriched tomatoes hasbeen attributed to high leaf starch. However, a seasonal comparison of leaf starchand deformation severity in a later study showed no relationship between the twofactors. Instead, diminished leaf K was correlated with increased leaf deformation.The severity of damage in tomato leaves was positively correlated with increases infinal fruit yield. This effect was associated with the ability of such cultivars toremobilize K from older leaves (Tripp eta!. 1991).CO2-induced leaf injury may partly counteract beneficial responses to CO2enrichment, particularly in young plants, since it will limit the compounding ofgrowth gains, and diminish crop aesthetics (Ehret and Jolliffe 1985a). Nonetheless,even when visible damage occurred, growth under CO2 enrichment improvedoverall biomass productivity of bean plants (Jolliffe and Ehret 1984).Threshold concentrations of CO2 for damage vary considerably betweenspecies or cultivars (Hicklenton 1988), and are dependent on environmental25conditions as well (Ehret and Jolliffe 1985a, Goldsberry 1986, Mortensen 1987, VanBerkel 1984). Usually, damage has been observed with CO2 concentrationsabove 1200 A L L-1. Therefore, growers are advised to use CO2 levels between 800and 1000 A L L-1 (Hicklenton 1988, Mortensen 1987, Nelson 1991).2.8 INTERACTIONS BETWEEN CO2AND PLANT GROWTH REGULATORSCO2 interacts with plant growth regulators, particularly ethylene and abscisicacid (ABA). CO2 is a critical factor in the regulation of ethylene production inleaves, and also seems to regulate ethylene retention within plant tissues (Horton1984). High CO2 blocks some effects of ethylene. It takes about 105 A L L-1 CO2 toblock 1 it L L-1 of ethylene (Abeles 1973). CO2 is a close structural analog of alleneand CO, two compounds which can substitute for ethylene (Burg and Burg 1967).Thus, at high concentrations (5,000 to 10,000 A L L-1) CO2 inhibits many ethyleneeffects, possibly by acting as a competitive inhibitor (Burg and Burg 1967, Abeles1973, Dilley 1978). In processes where ethylene plays an intermediate role, theblocking action of CO2 may also affect the activity of other chemicals. For example,CO2 suppressed the ability of IAA and 2,4-D to inhibit growth, and the action ofABA in the induction of abscission (Abeles 1973).Interactions between CO2 and ABA provide control mechanisms for stomatalopening, providing CO2 for photosynthesis while protecting the plant againstexcessive water loss (Radin and Ackerson 1982). When CO2 decreases in theintercellular spaces, K moves into the guard cells and stomates open, allowing CO2to diffuse in. This meets the needs of photosynthesis and transpiration (in non-succulent species). If water stress develops, ABA appears in the water that moves26into the guard cells, increasing the efflux of K from the guard cells inducing stomata!closure. The degree of stomatal response to ABA depends upon the CO2concentration in guard cells, and response to CO2 depends upon the concentrationof ABA (Salisbury and Ross 1985).The induction of flowering via phytochrome requires, at least in some plantspecies, the presence of CO2 at sufficiently high concentrations. Hence, removal ofCO2 during the red light night interruptions inhibited flowering responses to theinterruptions in Xanthium pennsilvanicum (Bassi et al. 1976). CO2 may also mimicthe action of auxins, probably through its action in increasing acidity in thecytoplasm. Such an effect however, is usually temporary and not very strong(Salisbury and Ross 1985).2.9 PHYSIOLOGICAL ROLES OF K2.9.1 Cation-Anion BalanceAvailability of K may have a large impact on plant growth. K acts mainly asa highly mobile charge carrier, forming only weak complexes from which it is readilyexchangeable (Wyn Jones et al. 1979). K is the predominant cation forcounterbalancing immobile anions in the cytoplasm, and often in vacuoles, xylemand phloem (Marschner 1986). High concentrations of K are required in thecytoplasm and chloroplasts to neutralize both insoluble macromolecular anions andsoluble anions (e.g. organic acid anions and inorganic anions). K is also thepredominant counterion for NO3- in long distance transport as well as for storage in27the vacuoles. Once NO3- is reduced in leaves, the remaining K requires thestoichiometric synthesis of organic acids for charge balance (Marschner 1986).2.9.2 Osmoregulation and Cell ExpansionK makes a major contribution to the osmotic potential of the cytoplasm,vacuoles and chloroplasts. Thus, leaf water potential is closely related to Kconcentration. For maintenance of cell turgor, however, K may be replaced byother solutes such as Na and sugars (Marschner 1986), and, it seems thataccumulation of soluble sugars in K-deficient leaves is related in the maintenance ofthe leaf water potential (Huber 1984b, Pitman et at. 1971).K is required for cell expansion. Cell expansion involves the increase of theosmotic potential in vacuoles through solute accumulation, and K is the main soluteinvolved. K also interacts with IAA, cytokinins and GA, enhancing their activity ininducing cell expansion (Marschner 1986). The activation of ATPases, requiresMg + but is further stimulated by K, and in some cases to a similar extent by Na(Marschner 1986).2.9.3 Stomatal MovementIn most plant species, K content is largely responsible for turgor changes inguard cells during stomata! movement. Increased K concentrations in guard cellsresult in the uptake of water from adjacent cells, which increases guard cell turgorand leads to stomata! opening (Marschner 1986, Salisbury and Ross 1985). Eitherincreasing irradiance, or reducing CO2 concentrations can, cause a buildup of K inthe guard cells (Salisbury and Ross 1985). Light-induced accumulation of K in the28guard cells is mediated by a membrane-bound H+ efflux pump. Such accumulationof K in the guard cells has to be balanced by a counterion, which is usually malate orCt. Malate synthesis requires phosphoenol pyruvate (PEP) (as a substrate for PEPcarboxykinase) which is supplied by degradation of starch in the chloroplasts of theguard cells (Marschner 1986).2.9.4 PhotosynthesisK-deficient plants generally have reduced photosynthesis rates (Huber1984b). In the suboptimal concentration range, the K content of leaves is closelycorrelated with various parameters of CO2 exchange (Marschner 1986). Decreasedphotosynthetic rates are often associated with partial stomatal closure as a result ofthe role of K fluxes in stomata! opening (Raschke 1975). Non-stomatal limitations,however, are also involved (Huber 1984b). K reduces the resistance for CO2transfer in the mesophyll, and increasing K also promotes the synthesis of rubiscoand its activity (Mengel and Kirkby 1987). Thus, usually high leaf K concentrationsincrease CO2 assimilation (Huber 1984b). These effects of K have been related tothe high requirement for K displayed by certain regulatory enzymes inphotosynthesis, particularly pyruvate kinase and 6-phosphofructokinase (Marschner1986). K is essential for the structural integrity and function of plastids (Marschner1986). It is also the main ion mobilized to balance changes during the light-inducedH+ flux across the thylakoid membranes, thereby participating in the establishmentof the transmembrane pH gradient necessary for photophosphorylation. Increasingthe K concentration in the cytosol stimulates CO2 fixation by isolated chloroplasts(Marschner 1986).292.9.5 Enzyme Activation and Carbon MetabolismThere are more than 60 enzymes which either depend completely on, or arestimulated by K (Suelter 1970). This role of K has direct effects on the metabolismof carbon (Huber 1984b).Leaf K concentration influences the synthesis of starch and sucrose, as wellas the export rates of sugars from leaves. Starch synthesis is mediated by starchsynthase, an enzyme which is highly dependent on univalent cations, particularly K,for its activity (Marschner 1986). Na, which also promotes starch synthaseactivity, is three to four times less effective than K in that function. When a highproportion of K in leaves is replaced by Na, starch content is much lower, and thecontent of soluble carbohydrates, particularly sucrose, increases. This shift mayfavor both cell expansion in leaf tissue and phloem transport of carbon compoundsto sinks (Hawker et at. 1974).K also enhances the activity of sucrose phosphate synthase (SPS), the keyenzyme in sucrose synthesis (Huber 1984b). Decreased SPS activity in K-deficientleaves may reduce sucrose synthesis, and in turn reduce the export of hexoses,although other factors could be involved as well (Huber 1984b). Lower export ratesfrom K-deficient leaves could also result from several causes including, thereduction in photosynthesis in K-deficient leaves which thus makes less assimilateavailable for export (Huber 1984b); a greater requirement for sugars in theosmoregulation of leaves; lower rates of phloem loading; lower flow rates of sucrosein the sieve tubes; and impaired sucrose transport across the tonoplast of the storagecells in sink tissue. High levels of K and Na considerably increase the rate ofsucrose efflux from leaf cells to the apoplast (Marschner 1986).302.9.6 Phloem Transport and Sucrose MovementPhloem loading of sucrose occurs mainly in minor veins, and is similar tophloem loading of minerals. The role of K in phloem loading of sucrose, however,is not clear. Phloem loading has to be an active process, since it is directed againsta concentration gradient. It is pH dependent, and it appears that there is a directcoupling of H+ efflux and K+ influx (H+ / K+ antiport) which could account for thehigh K concentrations observed in the phloem sap (Marschner 1986). Low Kconcentrations in the apoplast might stimulate the H+ efflux pump and thereforefacilitate sucrose loading. High K concentrations are most likely inhibitory,because a high K influx could depolarize the membrane and thus decrease the H+gradient. The activation of ATPases by K might also be involved in such processes(Giaquinta 1979, Ho and Baker 1982).K enhances both phloem loading and transport of sucrose (Marschner 1986).K makes a considerable contribution to the total osmotic pressure in the sieve tubes,and thus to the flow rate of photosynthates from source to sink. In plantsadequately supplied with K, a much higher proportion of photosynthates istranslocated from leaves to storage organs such as potato tubers (Haeder et at. 1973)or storage tissues (stalk) of sugarcane (Hartt 1969). In contrast, other studies havefound that K transport of photosynthate in the phloem is not dependent onmaintenance of K at the level required for normal growth (Cho and Komor 1980,Thrower and Thrower 1976).312.9.7 Protein SynthesisK is required for protein synthesis in higher plants and for the translation ofm-RNA on wheat germ ribosomes (Marschner 1986). It also regulates themobilization of stored proteins and the degradation of nitrogenous compounds.Thus, in K-deficient plants there is an accumulation of soluble nitrogenouscompounds (Helal and Mengel 1979, Koch and Mengel 1974). In green leaves,chloroplasts account for half of both leaf RNA and leaf protein, and in C3 speciesmost of the chloroplast protein is rubisco. Accordingly, the synthesis of nthisco isparticularly impaired when K is deficient, thus affecting photosynthesis. Such aneffect may also affect other processes due to lack of enzymes (Mengel and Kirkby1987).2.9.8 Substitution of K by NaNa may replace K in less specific processes such as maintenance of theosmotic potential and cell turgor, and with less efficiency in the activation of SPSand starch synthase and some other processes. However, Na cannot replace K inprotein synthesis (Helal and Mengel 1979), or in the roles played by K in thedivision, differentiation and expansion of cells. Also, K was found to be essentialfor chlorophyll formation in expanding tissue of sugar beet, and for the induction ofnitrate reductase in spinach leaves (Marschner 1986).The extent of substitution of K by Na on nonhalophytes (glycophytes) variesamong plant species. Plant species can be classified in four groups according totheir response to Na. Most differences are related to differences in uptake ortranslocation of Na to the shoots (Marschner 1986). The effectiveness of Na32substitution in shoots is limited and differs among individual organs, and evenamong cell compartments. The extent of replacement of K by Na depends on theuptake potential for Na, which varies between species (Marschner 1971). In oldsugar beet leaves, for example, nearly all the K can be replaced by Na and the Kthus made available can be used for specific functions in meristematic andexpanding tissues.In some plant species, beneficial effects of Na on plant growth are mostmarked when K supply is inadequate (Mengel and Kirkby 1987). Na supply mayinduce increases in leaf area and the number of stomates per leaf area. Also, inplants supplied with Na, in addition to K, the stomates tend to close more rapidlythan in plants supplied exclusively with K, an effect that may improve the waterbalance of plants (Marschner 1986, Terry and Waldron 1984). Growth stimulationby Na is of practical and scientific interest, since it opens the possibility to use lowergrade potash fertilizers that contain a high proportion of Na (Marschner 1986).2.10 INTERACTIONS BETWEEN CO2 ENRICHMENT AND MINERALNUTRITIONIrradiance, temperature, relative humidity and nutrient supply may alterplant responses to CO2 enrichment. Many studies have been performed on thethree former factors, but few have investigated either the effects of CO2 enrichmenton the nutrient relations of plants, or the influence of nutrient availability on growthresponses of plants under CO2 enrichment (Porter and Grodzinski 1989). In plantsgrown under CO2 enrichment, three issues must be considered in regard to mineralnutrition. These are: 1) the effects of inorganic nutrient supply on the responses of33such plants to CO2 enrichment; 2) changes induced by CO2 enrichment in thenutrient relations of plants; and 3) the relationships of these two patterns to thedevelopment of CO2-induced leaf injury.2.10.1 Responses of CO2 Enriched Plants to Mineral NutritionAvailability of inorganic nutrients influences plant responses to CO2enrichment (Mortensen 1987). Responses to nutrient supply vary in regard to plantspecies and growing conditions. In some studies, growth of CO2 enriched nutrient-stressed plants was similar to that of adequately supplied plants under CO2enrichment (Hocking and Meyer 1985, Imai and Murata 1978, Wong 1979). Inothers, increasing availability of nutrients enhanced the effects of CO2 enrichment(Cure et al. 1988a, 1988b, Goudriaan and de Ruiter 1983, Imai and Murata 1978,Mortensen 1987, Patterson and Flint 1982, Sionit et al. 1981a, Wong 1979). CO2enrichment may increase dry matter production even at low levels of nutrient supply(Sionit et al. 1981a, Goudriaan and de Ruiter 1983, Peet and Willits 1984).However, it has been suggested that to obtain maximum benefits of CO2 enrichmentfor chrysanthemum plants, their nutritional status must be maintained at a higherlevel than normally used under non CO2-enriched conditions (Eng et al. 1985).2.10.2 Changes in the Mineral Nutrition of CO2 Enriched PlantsCO2 enrichment decreased the foliar nutrient concentration of many plantspecies including beans (Liu 1990, Porter and Grodzinski 1989), cotton (Wong1979), wheat (Hocking and Meyer 1991), monoecious cucumber (Peet et al. 1986),34celery (Tremblay 1988), lettuce (Knecht and O'Leary 1983), tomato (Tripp et al.1991) and chrysanthemum (Eng et al. 1987, Kuehny et al. 1991).Greater starch content enlarges the leaf mass of CO2 enriched leaves, thuscontributing to the relative decrease in nutrient concentration in chrysanthemum(Kuehny et al. 1991), tomato (Tripp et al. 1991), monoecious cucumber (Peet et al.1986), and bean (Porter and Grodzinski 1989). Alteration in nutrient relations inCO2 enriched plants also has been related to decreased transpiration rates (VanBerkel 1984, Madsen 1975, Mortensen 1987, Yelle et al. 1987). Changes in mineralnutrition of CO2 enriched plants may also result from inadequate supply, uptake ortranslocation of nutrients, in relation to the increased requirements of CO2 enrichedplants (Liu 1990, Tripp et al. 1991). Usually, transpiration has a greater effect ontransport than on uptake of nutrients (Marschner 1986).The effects of CO2 enrichment on nutrient uptake vary among plant species.Increased uptake of N and P was recorded in soybean (Cure et al. 1988a, 1988b,Israel et al. 1990), but no increase N uptake occurred in cotton (Wong 1979) orcocklebur (Hocking and Meyer 1985). Increased nutrient uptake of plants underCO2 enrichment was associated with growth enhancement of plants (Cure et al.1988a, 1988b, Yelle et al. 1987). Conversely, limited nutrient uptake reducedgrowth stimulation by CO2 enrichment (Porter and Grodzinski 1989). Nutrientuptake and translocation in CO2 enriched plants are affected also by environmentalconditions in the root zone. CO2 enrichment reduced movement of NO3- fromroots to shoots in tomato plants. Conditions that favored NO3- movementincreased plant growth. Therefore, growth response of plants to CO2 enrichmentwas enhanced at 30 C, the temperature at which NO3- translocation to leaves was35highest (Yelle et al. 1987). Cation uptake by Virginia pine under CO2 enrichmentwas larger than anion uptake (Luxmore et at 1986), an increase which wasassociated with acidification of the rhizosphere (Jarvis and Robson 1983).Nevertheless, due to parallel increases in leaf growth and starch accumulation,greater nutrient uptake may not always be detected as increased nutrientconcentration, as reported for CO2 enriched tomato leaves (Yelle et al. 1987).2.10.3 Mineral Nutrition and Leaf Injury of CO2 Enriched PlantsVisible symptoms of leaf abnormalities bear a superficial resemblance toeffects of nutrient imbalances (Goudriaan and de Ruiter 1983, Liu 1990). Lownutrient supply induced leaf injury in CO2 enriched Begonia (Mortensen 1987) anddwarf beans (Liu 1990). The timing of increased remobilization of N and K fromolder leaves to younger parts was also correlated with the onset of chlorosis onprimary leaves of CO2-enriched dwarf bean plants (Liu 1990) and wild radish (Kochet al. 1988). Severity of deformation in older CO2 enriched tomato leaves washighly correlated with the foliar concentration of K (Tripp eta!. 1991).36CHAPTER 3 EFFECTS OF CO2 ENRICHMENT AND K NUTRITION ONGROWM OF CHRYSANTHEMUM PLANTS3.1 INTRODUCTIONCarbon dioxide (CO2) enrichment stimulates the growth and yield of crops ingreenhouses (Goldsberry 1986; Hanan 1986) as it liberates plants from the commonlimitation of carbon supply for photosynthesis. Growth responses to supplementalCO2 differ between species, and may also change with plant age, type of carbonmetabolism (C3 vs Ca), environmental conditions, growth pattern (determinate orindeterminate), and cultural practices (particularly disbudding, pruning andharvesting). In addition to increased photoassimilate production, growth responsesto CO2 enrichment are associated with effects on physiological and biochemicalprocesses within the plant (Bravdo 1986) which may modify the partitioning andutilization of additional carbon provided (Ehret and Jolliffe 1985b). Much of theresearch so far performed on plant response to CO2 enrichment has concentrated onphotosynthetic responses. Less is known of non-photosynthetic responses to CO2enrichment, and the interactions of CO2 enrichment with secondary environmentalfactors, particularly the availability of inorganic nutrients.Research on dwarf bean plants revealed that total plant dry mass increasedin response to CO2 enrichment, although little increase in leaf area occurred (Ehretand Jolliffe 1985b). Growth analysis indicated that dry matter partitioning wasaltered by CO2 enrichment (Ehret and Jolliffe 1985b, Jolliffe and Hoddinott 1988),with the appearance of some limitation to carbon export from leaves (Hoddinottand Jolliffe 1988). In chrysanthemums, the main benefit of CO2 enrichment is the37production of thicker, heavier stems (Eng et al. 1985, Mortensen and Moe 1983a).CO2 enrichment also allows the vegetative long-day period to be reduced withoutsacrificing plant size, thus shortening the crop production cycle (Hicklenton 1988,Nelson 1991).Availability of inorganic nutrients may affect plant responses to CO2enrichment, but the effects vary between species. Increasing nutrient supplyenhanced CO2 enrichment effects in several plant species (Cure et al. 1988a, 1988b,Patterson and Flint 1982, Sionit et al. 1981). As a C4 plant, maize usually shows asmaller growth response to CO2 enrichment than do C3 species (Goudriaan deRuiter 1983) regardless of the N level (Imai and Murata 1978, Wong 1979). Manyplant species increase their dry matter accumulation in response to CO2 enrichment,even at low nutrient supply (Goudriaan de Ruiter 1983, Sionit et al. 1981a). Insome studies, growth of nutrient-stressed plants under CO2 enrichment was similarto that of adequately supplied plants (Hocking and Meyer 1985, Wong 1979), whilein other cases growth was restricted by inadequate nutrient supply (Goudriaan deRuiter 1983, Mortensen 1987, Patterson and Flint 1982, Sionit et al. 1981a).In dwarf bean, leaf chlorosis developed after three weeks of CO2 enrichment(Jolliffe and Ehret 1985a; Liu 1990). Chlorosis and other leaf abnormalities havealso been observed in a wide range of other plant species, includingchrysanthemums (Goldsberry 1986, Van Berkel 1984, Walla and Kristoffersen 1974)and other greenhouse plants (Cave et al. 1981, Goldsberry 1986, Peet 1986,Mortensen 1987, Van Berkel 1984, Tripp eta!. 1991). Visible symptoms of thesedisorders bear a superficial resemblance to the effects of nutrient deficiency orexcess. Indeed, K and N were found to become deficient in CO2 enriched primary38leaves of dwarf bean plants by the onset of the injury (Liu 1990), and leaf Kreductions were correlated with increased alterations in older tomato leaves underCO2 enrichment (Tripp et al. 1991).Hence, beyond the photosynthetic effects, growth responses to CO2enrichment may be conditioned by several factors including leaf expansion, drymatter partitioning, leaf injury, and the availability of adequate supply of inorganicnutrients. The primary aim of the studies discussed in this chapter was to evaluatethe long term effects of CO2 enrichment on growth of chrysanthemum plants underdifferent levels of K nutrition. To assess the timing and location of responseswithin plants, effects of treatments on the growth of individual plant parts andoverall growth were examined using techniques of plant growth analysis.393.2 MATERIALS AND METHODSTwo experiments were conducted. The first provided an opportunity to testexperimental systems and to observe plant responses to CO2 enrichment alone.The second experiment investigated plant responses under combinations ofatmospheric CO2 and K supply. In the first experiment pot chrysanthemum(Dendranthema grandiflora Tzvelev cv 'Envy') plants were grown for ten weeksunder three different CO2 levels, during the summer of 1990. In the secondexperiment plants of the same cultivar were grown for eight weeks under acombination of two CO2 and four K levels, during the summer of 1991.3.2.1 First Experiment, Summer 19903.2.1.1 Plant culturePlants of pot chrysanthemum cv 'Envy', were grown on a commercial peatbased soiless mix (Metromix 225, W. R. Grace, Canada) into 100 mm square plasticpots (0.67 L per pot). Cuttings came rooted on a peat based medium from thepropagator (Yoder Canada, Leamington, Ontario). Uniform cuttings wereselected, and one cutting was transplanted to each pot on May 17, 1990.During the first two weeks, the plants were grown in a controlledenvironment chamber (Percival model PG 78). Light source was a combination offive 60 W incandescent bulbs and eight 40 W cool white fluorescent tubes, providinga photosynthetic photon flux density (PPFD) of 190 p MOi nr2 s-1 at pot level, asdetermined with a quantum meter (LI-COR, model LI-185). Photoperiod was 12hours with a 4-hour night interruption to delay flower initiation using the40incandescent lamps alone (irradiance 50 Anal ilr2 S-1 PPFD at pot level), starting 4h after the beginning of the dark period. Temperatures were 22±22 C (day) and18+2 C (night). During the first week the cuttings were misted as required using ahand sprayer. Fourteen days after transplanting the plants were moved into CO2chambers to begin treatments.The CO2 chambers, operated as open gas exchange systems, as have beendescribed previously (Jolliffe and Ehret 1984; Liu 1990). The chamber inlet airsupply was obtained from outside the MacMillan Building at 12 m above groundlevel. Inlet air was driven through each chamber by an inlet fan, and the airturnover rate in each chamber was approximately two times per minute. Each inletfan had a rated output of 160 L min-1 at zero static pressure. Each CO2 chamberhad 0.33 m x 0.33 m x 0.56 m space for plant growth (volume = 60.9 L). ChamberCO2 concentrations of 350+30 iz L , 1200+60 AL.L-1 or 1800+90 L.L-' weremaintained continuously by adding 100% CO2 from compressed gas cylinders(Medigas, medical grade) to the inlet stream of ambient air. Enrichmentconcentrations were set by regulating the flow rates of CO2 via precision regulatedflowmeters. To improve spatial uniformity in gas concentration, each chambercontained four air mixing fans beneath the plants. Each mixing fan had a ratedoutput of 250 L min-1. The CO2 concentration inside each chamber was monitoreddaily by pumping air from inside the CO2 chambers through a CO2 infrared analyzer(Beckman model 864).Temperatures inside the CO2 chambers were 21±4  C (day) and 14.5 + 2.5 C(night). An irradiance of 135+15 p, MOi m-2 s-1 PPFD at pot level was provided for aphotoperiod of 9 hours. Light sources were two banks of fifteen 60 W incandescent41bulbs, each with sixteen 40 W cool white fluorescent tubes. Each bank served asthe light source to four CO2 chambers.Uniform, well rooted plants were selected to be moved into the CO2chambers. The plants were pinched leaving the eight nodes in the main stem.Dolomitic lime (4.6 g) and F-T-E fritted trace micronutrients (0.13 g) (GreenValley Fertilizer, Canada) were added to each pot, and incorporated by a thoroughirrigation. Hand watering with fertilizer solutions was applied as needed, using thenutrient concentrations shown in Appendix 1. B-nine 50 WP (N-dimethylamino-succinamic acid, Daminozide, Uniroyal) was used as a growth retardant, a practicecommonly used commercially to produce more compact pot chrysanthemumsPlants were sprayed to run-off twice, 21 and 35 days after transplanting, using ahand sprayer. Concentrations used were 3.3 mg L-1 and 2.5 mg L-1, respectively. Experimental designThe plants were arranged in a split plot design, with two replications(blocks), five ages of harvest (main plots), and three CO2 levels (subplots). Sixtyplants were assigned to each block according to their size, and then twenty plantswere randomly distributed within each of the CO2 chambers.Four randomly selected plants from each CO2 chamber were harvested ateach of five different times (t): 0, 13, 23, 40, and 54 days after transfer to the CO2chambers. At each harvest the areas of leaves borne on main stems (LA.) andthose borne on branches (LAb) were measured using a leaf area meter (LI-CORmodel LI-3000). Dry masses of leaves (WL), flowers ( Wy) and other plant partswere taken after being oven dried at 65 C for 72 hours. Roots were washed with42tap water before drying. The total dry mass of above-ground parts was summed toobtain shoot dry mass per plant (Ws), below-ground dry mass was root dry mass(WO, and the sum of all parts formed total plant dry mass (W). Statistical analysisPrimary data, i.e. the measures of leaf areas and dry masses described above,were loge-transformed, and Bartlett's test for homogeneity of group variances wasperformed, on both primary data and loge-transformed variables. Homogeneity ofvariance was improved by such transformation, and loge-transformed variables weretherefore used for analysis of variance and subsequent regression development.Forward stepwise polynomial regressions, describing the time trends of each plantmeasure per treatment, were generated using ordinary least squares. Standarderror of means were calculated as the standard deviations of the sample means,which are the standard deviation of the population divided by the square root of thesample size (Wilkinson 1990). Regressions were then used to produce indices ofgrowth rate, including absolute growth rate (AGR), [c/W/dt]; relative growth rate (R),[d(logeW)/dt]; and unit leaf rate (E), [(1/LA)(dW/c/t)]; as described by Hunt (1982).Plant proportions were assessed through the calculation of the following ratios: leafarea ratio (F), [LA/W]; specific leaf area (sLA), [LA/wq; leaf weight ratio (LwR),[WL/W]; shoot to root ratio (sRR), [ws/wd; and harvest index (H), [wilw]. Datawere analyzed using Systat 5.01 (Wilkinson 1990).3.2.2 Second Experiment, Summer 19913.2.2.1 Plant CultureThe second experiment was completed during the summer of 1991.Unrooted cuttings of pot chrysanthemum cv 'Envy' (Yoder Canada, Leamington,Ontario) were planted in 35 mm-rockwool cubes (Grodan, Denmark) for rooting,and were kept in the same controlled environment chamber described above(Percival PG 78). Environmental conditions (irradiance, photoperiod andtemperature) were the same as described above for the preliminary period of thefirst experiment. During rooting, which lasted fourteen days, the cuttings wereenclosed in clear plastic trays and were misted as often as required with a handsprayer, in order to keep relative humidity high.Once rooted, the cuttings were stored at 0 C for one or two weeks tocoordinate planting schedules. Then they were transplanted on to 80 mm rockwoolcubes (Grodan, Denmark), planting one cutting per cube. After transplanting thecuttings were placed in the same controlled chamber (Percival PG 78) with no coverfor one week, to expose them to long photoperiod. During this period the plantswere watered with the nutrient solution containing the lowest K concentration to beused later in the trial.To begin CO2 enrichment and K supply treatments, plants were pinched tothe sixth node, and sixteen uniform and well rooted plants were randomly assignedto the CO2 chambers on June 13, 1991. Temperatures in the CO2 chambers were233 C (day) and 153 C (night). Photoperiod was 9 hours, with an irradiance of190 +15 p MO1 m-2 S-1 PPFD at pot level. There was no bud removal for this crop,4344and B-nine was not applied. Atmospheric CO2 concentrations of 350+30 AL L-1 or14.00+70 AL L-1 were continuously provided. To monitor CO2 concentrations, airfrom inside each CO2 enriched chamber was pumped throughout a CO2 infraredanalyzer (ADC, model EGA 13794) for 15 minutes each hour. While in the CO2chambers, plants also were treated with nutrient solutions containing one of fourdifferent concentrations of K: 2.50, 3.75, 5.00, or 6.25 mMol L-1. Concentration ofother minerals in the solutions was held constant, and acetic acid (17.4 M) was usedto equilibrate the pH. Composition of the nutrient solutions is shown inAppendix 2.Each plant was placed into a 1 L plastic pot, which was connected by a 1.5mm (I.D.) polyethylene tube to a recirculating hydroponic system (Fig. 3.1). Theirrigation system was controlled by a microprocessor (Campbell 21x micrologger,Campbell Scientific). Three times per day the plant pots were filled with nutrientsolution, which was then allowed to drain to the supply tanks. The period ofsubmersion was between two and three minutes. The electrical conductivity (EC)was between 1.5 mmhos cm (lowest K supply) and 1 9 mmhos cm (highest Ksupply). The initial pH of the solutions was adjusted to 5.5 using acetic acid (17.4M). Subsequently, the pH and EC (with tap water) of the solutions were adjusteddaily to keep them within the initial range. Every three days the nutrient solutionswere changed to avoid large drifts in ion concentration. Samples of nutrientsolutions, fresh and three days old, were analyzed by a commercial laboratory(Pacific Soil Analysis Inc., PSAL Richmond B. C.), and the measured concentrationswere close to the theoretical range values (Appendix 3).Figure 3.1 Diagram of the recirculating hydroponic system used for the 1991experiments.463.2.2.2 Experimental Design and Statistical AnalysisThis experiment was arranged as a split-split plot, with four blocks, five agesof harvest (main plots), two CO2 levels (subplots) and four K levels (sub-subplots).Two groups of eight plants were assigned at random to each chamber, and eachgroup was to be harvested at a different age. From each age group, two plants fromevery chamber were allocated to each K level.Groups of eight plants were harvested at random on a date determinedbefore the experiment started. Plants from each replicate (i.e. each CO2enrichment by K supply combination) were harvested at five times (t): 0, 7, 14, 28 or56 days after treatments began. Until the fourth harvest, the removed plants werereplaced by another set which would be harvested on a later date.At harvest, leaves from three sections of the plant were separated formeasurement: leaves borne on main stems (LA.), the four lowest leaves borne onbranches (LAI), and leaves borne on the upper section of branches above the fourthleaf (LAu). Leaf area was measured with a leaf area meter (LI-COR, model LI-3000). Dry masses were determined after plant parts were oven dried at 65 C for72 hours. Root dry mass was determined as the difference in dry mass of therockwool cubes before and after the experiment.Data were analyzed after loge-transformation of the variables as describedfor the previous experiment (summer 1990).473.3 RESULTSBecause the experimental structure differed between the two experiments,data from the first trial, Summer 1990, and second trial, Summer 1991, will beinterpreted separately. Primary data for both trials are tabulated in Appendix Growth Assessment, Summer 1990 Experiment3.3.1.1 Effects of CO2Enrichment on Plant Growth (1990)Analysis of variance indicated that CO2 treatments had significant (p < 0.05)effects on total dry mass (W) accumulation and most other measures of growth ofthe 1990 chrysanthemum plants (Table 3.1). Interactions of the effects of CO2 withtime, or block by time, were occasionally significant. Regression coefficients and R2values generated for growth variables of this experiment are presented in table 3.2.W was significantly increased in CO2 enriched plants (Fig. 3.2a), and therewas no significant difference in W between the two CO2 enrichment levels, 1200 and1800 A L L-1. W of CO2 enriched plants was 19.5% larger than the controls after 28days, and this difference was similar by the end of the trial. Effects of CO2Enrichment on Growth of Plant Parts (1990)Total leaf area (LA) followed a similar pattern to W, although it increasedfaster at the beginning and stabilized six weeks after the initiation of treatments(Fig. 3.2b). After 28 days total leaf area of CO2 enriched plants was increased by4.5% over the control plants, and this difference was preserved until the end of thetrial.48Table 3.1 Summary of statistical significance ofgrowth measures, summer 1990.Growth Data, Summer 1990Source^df W^LA^LAm LAb WL VVLm VVLbBlock 1^ns^ns^*^ns^*^*t^4^* ns^*^*^*^*Block*t^4^ns^ns^ns^ns^ns^nsCO2 2^* ns^*^*^ns^*t*CO2^8^ns^ns^ns^ns^ns^ns^nsBlock*CO2st 10^ns^ns^ns^*^ns^ns^ns* means significant (p <0.05)ns means non significant (p > 0.05)Table 3.1 (continued) Summary of analysis of variance ofgrowth measures summer 1990.Growth data, summer 1990Source^Wb Ws Wr WY H^F LWR SLA SRRBlock *^ns^ns^*t^ *^ns^ns^* *^*Block*t^ns^ns^*^*^*^ns^ns^*CO2 * ns^ns^* *^*t*CO2^ns^ns^ns^ns^ns^*^ns^*Block*CO2*t^ns ns^* ns^ns^ns^ns* means significant (p < 0.05)ns means non significant (p > 0.05)49Table 3.2 Regression coefficients for growth measures,summer 1990CO2: 350 p L L-1Variable Constant t t2 t3 R2W 0.38 0.04 -3.31E-06 0.92LA 5.40 0.08 -9.24E-04 0.96VVL -0.23 0.05 -6.91E-06 0.93LAm 5.35LAb 3.37 0.20 -3.00E-03 0.95WLm -0.35 5.79E-05 0.11WLb -2.55 0.19 -2.00E-03 0.95Ws -1.47 0.02 0.77Wb -3.21 0.10 -1.05E-05 0.96Wr -0.90 0.03 0.78Wy -0.41 0.00F 5.07 0.02 -8.11E-06 0.62LWR -0.61 0.01 -3.60E-06 0.69SLA 5.82 0.00SRR 0.91 0.03 -3.89E-05 0.62H 0.68 -0.11 1.98E-05 0.5050Table 3.2 (continued) Regression coefficients for growthmeasures, summer 1990CO2: 1200 /IL L-1Variable Constant t t2 t3 R2W 0.33 0.06 -5.09E-06 0.98LA 5.39 0.09 -9.81E-04 0.12VVL -0.32 0.08 -7.18E-04 0.96LAm 5.28 0.00 0.97LAb 3.45 0.20 -3.00E-03 0.96WLm -0.44 0.01 0.19WI,b -2.40 0.19 -2.00E-03 0.97Ws -1.50 0.03 0.87Wb -3.14 0.12 -1.37E-05 0.00Wr -0.95 0.05 -2.99E-06 0.75Wy 0.11 -6.00E-03 1.12E-04 0.61F 5.12 0.01 -6.79E-06 0.47LWR -0.62 0.00 -3.79E-06 0.41SLA 5.83 0.98SRR 0.93 0.02 -2.77E-03 0.91H 0.68 -0.17 4.19E-05 0.4851Table 3.2 (continued) Regression coefficients for growthmeasures, summer 1990CO2: 1800 p L L-1Variable Constant t2 t3 R2W 0.40 0.06 -6.14E-06 0.95LA 5.45 0.09 -1.00E-03 0.97WL -0.19 0.08 -7.76E-04 0.95LAm 5.44LAb 3.33 0.21 -3.00E-03 0.96WLm -0.26 0.00 0.06WLb -2.46 0.20 -2.00E-03 0.96Ws -1.49 0.04 -3.87E-06 0.85Wb -3.14 0.14 -1.00E-03 0.97Wr 4.00 0.05 -3.18E-06 0.87Wy 0.18 -5.00E-03 1.02E-04 0.47F 5.11 0.01 -6.13E-06 0.58LWR -0.61 0.00 -2.92E-06 0.77SLA 5.76 0.00S RR 1.07 0.02 -3.64E-04 0.26H 0.67 -0.15 3.39E-05 0.46312^18^24^30 36^42^48^54Mae (days)12^18^24^30 36^42^48^54Time (days)Figure 3.2a52Plant Dry MassSummer 1990Figure 3.2bLeaf AreaSummer 1990Figure 3.2eLeaf Dry MassSummer 1990Time (days)Figure 32 Effects of CO2 enrichment on W (a), LA (b) and WL (c),summer 1990.53The area of leaves borne on branches (LAb) significantly increased with timeand CO2 enrichment (Fig. 3.3a). Thus, most of the variation in total leaf areaobserved amongst CO2 levels came from the effect on LAb.Changes in total leaf mass, WL (Fig. 3.2c) followed a similar pattern to totaldry mass. Leaves borne on main stems (WL„,) significantly increased their massover time, but no significant effect of CO2 enrichment was detected. It was noticed,however, that plants under 1800 A L L-1 CO2 tended to produce heavier leaves onmain stems than the other treatments. Initial variability in mass and area of leavesborne on main stems was large. Thus, small changes in these leaves could not bedetected as significant.The dry mass of leaves borne on branches (WLb) was significantly increasedby CO2 enrichment (Fig. 3.3b). Such leaves began to appear on branches after oneweek of CO2 enrichment (i.e. after one week of pinching) and their mass increaseduntil the sixth week of treatment. These leaves were the main cause of the increasein total leaf mass induced by CO2 enrichment.Thy mass of main stems (Ws), branches (Wb) and roots (Wr) significantlyincreased with time from the beginning of the experiment, and were alsosignificantly increased by CO2 enrichment (Fig. 3.3c, 3.3d, 3.3e). The rate of changeof these variables, however was about ten times smaller than the rate of mass gainof leaves. No statistically significant effects of CO2 were detected on flower mass(Wy), or on the timing of flower production.3.512^18^24^30^36^42Thus (days)12^18^24^30^36^42Mae (days)312^18^24^30^36^42Tune (days)54Figure 33aLeaf Area of BranchesSummer 1990Figure 3.3eStem Dry MassSummer 1990Figure 33bBranch Leaf Dry MassSummer 1990Figure 3.3dBranch Dry MassSummer 1990Figure 3.3eRoots Dry MassSummer 1990Figure 3.3 Effects of CO2 enrichment on LAb (a) WLb (b) % (c), Wb (d)and % (e), summer 1990. CO2 levels (p L L-1), 350 (^); 1200 (^);1800 (^).553.3.1.3 Effects of CO2Enrichment on Growth Indices (1990)Absolute growth rate (AGR) increased with time, and differences increasedtowards the end of the experiment (Fig. 3.5a). AGR was higher in CO2 enrichedplants, although no differences were observed between the two CO2 enrichmentlevels.Relative growth rate (R) decreased from the beginning of the experiment atall CO2 levels (Fig. 3.5b). Plants under CO2 enrichment had higher R, but the rateof decrease was much more pronounced than in the control plants. Hencetreatment differences in R were small at the end of the study.Unit leaf rate (E) was higher in plants under CO2 enrichment (Fig. 3.5c). Inall treatments there was a slight decrease in E up to about 24 days of CO2enrichment, after which a rapid increase over time was observed. The rate ofincrease in E was much higher on CO2 enriched plants than on the controls, butlittle difference was observed between the two CO2 enrichment treatments.Leaf area ratio (F) increased early in the study, and higher values wereobserved in the control plants than the CO2 enriched plants for most of the study(Fig. 3.5d). Leaf weight ratio (LWR) had a significant response to CO2 and theresponse changed over time (Fig. 3.5e). LWR showed a slight increase at thebeginning followed by a large decrease after 4 weeks. LWR decreased morerapidly on plants grown at 1200 A L L-1 CO2. Hence differences between the 1200/A L  L-1 CO2 grown plants and the other treatments developed towards the end of thestudy. Specific leaf area (SLA) was decreased by the highest CO2 level (Fig. 3.50.The ratio of shoot to root dry mass (SRR) was significantly reduced by CO2enrichment.6^12^18 24 30 36 42 48 54Time (days)6^12^18^24^30^36 42^48^54Time (days) -0.00080.00070^6^12^18^24^30^36^42^48^54Time (days)0.00060.0005-0.00040.00030.00020.0001-6^12 18 24 30 36 42 48 54Time (days)8.0i7.0-16.0:-4.0-3.0:2.00.0-10 6^12^18^24^30^36Time (days)42^48 5456Figure 3.4a^ Figure 3.4bAbsolute Growth Rate^Relative Growth RateSummer 1990^ Summer 1990Figure 3.4c^ Figure 3.4dUnit Leaf Rate (E)^ Leaf Area Ratio (F)Summer 1990 Summer 1990Figure 3.4eLeaf Weight RatioSummer 19900^6^12 18^24 10 36 42 48 54Time (days)Figure 3.4fSpecific Leaf AreaSummer 1990Figure 3.4 Effects of CO2 enrichment on AGR (a), R (b), E (c), F (d), LWR(e) and SLA (f), summer 1990. CO2 levels (AL L-1), 350 (^ ),1200(^); 1800 (^).573.3.2 Growth Assessment, Summer 1991 Experiment3.3.2.1 Effects of CO2Enrichment and K Supply on Plant Growth (1991)The analysis of variance growth variables from the summer of 1991 (Table3.3) indicated that CO2 enrichment had significant (p < 0.05) effects on total drymatter accumulation, on most of the dry mass variables, and on the SLA and F ofthe plants. K supply had significant effects on most of the primary variables, butthe effect of K supply was not significant on most of the growth indices evaluated.Interactions of the effects of CO2 enrichment with time were significant for nearlyhalf of the dependent variables. Interactions of the effects of K supply and CO2enrichment were significant only in a few cases. Interactions of the effects of Ksupply with time were significant for about half of the dependent variables.Interactions of the effects of K supply with CO2 enrichment and time weresignificant mostly for measurements from leaves borne on the lower part ofbranches. Block effects were significant for most of the primary variables, whilstthe interaction of the effects of block and time were significant in most cases.Regression coefficients and R2 values for growth variables of this experiment arepresented in table 3.4.58Table 3.3 Summary of statistical significance, ofgrowth measures, summer 1991.Growth Data, Summer 1991Source df W LA LAm LAI LAu WL WLm VVLI VVLuBlock 3 * * * * ns * * * *t 3 * * * * * * * * *Blockst 9 * ns * * ns * * nsCO2 1 * 118 ns DS ns * * * nsCO2*t 3 * ns ns * * ns ns ns nsBlock*C 02* t 12 * * * * ns * * ns *K 3 * ns * * * ms * *Kt 9 * ns ns ns * * * nsK*CO2 3 ns * ns * ns ns ns ns IISt*K*CO2 9 ns * ns * * * ns * nsTable 3.3 (continued) Summary of statisticalsignificance of growth measures, summer 1991.Growth Data, Summer 1991Source Wb Ws Wr WY H F LVVR SLABlock ns ns * ns ns ns nst * * ns * ns *Block*t * * * * * *CO2 * ns ns ns * ns *CO2*t ns * ns ns ns * ns *Blk*CO2* t ns ns * ns * * * *K * * * * ns ns ns nsKt * * ns ns ns *K*CO2 ns ns ns ns ns ns ns nst*K*CO2 ns ns ns ns ns ns ns* means significant (p < 0.05)us means non significant (p > 0.05)59Table 3.4 Regression coefficients for growth measures,summer 1991K: 2.50 mMol L-1CO2: 350pL L-1 CO2 = 1400 II L L-1Var. Const.^t t2 t3 R2 Const. t t2 t3 R2W -1.04 0.09 -1.11E-04 0.94 -0.79 0.08 -6.89E-06 0.86LA 4.20 0.07 -6.58E-06 0.87 3.74 0.10 -1.32E-05 0.90WL -1.35 0.08 -1.20E-05 0.92 -1.10 0.07 -8.99E-06 0.75LAm 4.23 0.04 -6.66E-04 0.36 4.66 0.00 0 0.00LAI -0.17 027 -3.10E-03 0.91 -2.69 0.42 -5.09E-03 0 0.99Lk -1.08 0.21 -2.38E-05 0.85 -1.06 0.21 -2.13E-05 0.81VVLm -1.36 0.06 -9.48E-04 0.57 -0.44 0.00 4.60E-04 0 0.19VVLI -0.01 -0.07 1.00E-03 0.38 -0.80 0.00 0 0.00VVLu 0.04 -9.09E-04 2.01E-05 0.67 -0.01 6.68E-06 0.78Ws -2.28 0.07 -5.59E-04 0.82 -1.70 0.03 0 0.81Wb -0.27 -0.06 3.02E-05 0.51 -0.80 0.42Wr 46.28 0.59 -9.53E-04 0.81 46.55 0.60 -9.51E-05 0.50WY 0.12 -0.07 1.58E-05 0.25 -0.76F 5.20 -0.01 0.20 4.64 7.8E-04 -1.55-05 0.23LVVR -0.33 -0.01 -1.31E-04 0.87 -0.38 0.87SLA 5.52 0.00 1.61E-05 0.13 4.82 0.03 -4.24E-06 0.32SLAm -0.09 0.37 -4.65E-03 0.88 -2.65 0.49 -6.7E-03 0.76SLAI 5.54 0.00 5.32 0.00SLAu -0.31 0.18 -2.11E-05 0.83 -3.09 0.33 -5.6E-05 0.89S RR 0.01 0.08 4.44E-05 0.63 0.21 0.10 -1.00E-03 0.2560Table 3.4 (continued) Regression coefficients for growthmeasures, summer 19913.75 mMol L-1CO2: 350 AL L-1 CO2 = 1400 it L L-1Var. Const.^t t2 t3 R2 Const. t t2 t3 R2W -0.90 0.09 -6.67E-04 0.93 -0.61 0.07 -3.79E-06 0.93LA 3.82 0.10 -1.45E-05 0.95 3.74 0.10 -1.44E-05 0.89WL -1.34 0.07 -9.45E-06 0.93 -1.06 0.07 -8.42E-06 0.84LAm 4.62 4.94E-04 -9.46E-06 0.21 4.63LAI -2.27 0.43 -5.22E-03 0.92 -2.33 0.42 -5.06E-03 0.93LA, -1.38 0.21 -1.97E-05 0.82 -1.62 0.25 -3.2E-05 0.86VVLm -0.75 4.88E-06 0.28 -0.45 -0.01 0.23WI.4 -0.89 0.00 -0.91WI, 0.07 -1.04E-03 2.18E-05 0.70 0.05 -8.92E-04 2.19E-05 0.57Ws -1.82 0.02 0.83 -1.81 0.03 0.86Wb -0.32 -0.07 3.16E-05 0.54 -0.32 -0.05 2.86E-05 0.68Wr -9.84 0.53 -6.33E-03 0.35 -7.56 0.37 -4.06E-03 0.34Wy 1.22 -0.13 2.71E-05 0.24 -0.83F 5.01 0.00 4.82 9.01E-04 -1.87E-05 0.34LVVR -0.45 0.84 -0.34 -0.01 -2.84E-06 0.93SLA 5.37 0.01 0.37 4.63 0.05 -1.04E-05 0.60SLAm 5.45 0.08 4.78 0.03 -6.05E-06 0.33SLAI -0.01 0.30 -3.45E-03 0.38 -2.16 0.51 -6.55E-03 0.78SLAu -2.44 0.26 -3.32E-05 0.76 -3.37 0.36 -6.26E-05 0.90SRR 1.99 1.9261Table 3.4 (continued) Regression coefficients for growthmeasures, summer 1991K: 5.00 mMol L-1CO2: 35OizLL1 CO2 = 1400L L1Var. Const.^t t2 t3 R2 Const. t t2 t3 R2W -0.79 0.07 -3.78E-06 0.95 -0.46 0.05 0.83LA 4.33 0.05 0.81 3.51 0.12 -1.72E-05 0.94WL -1.31 0.06 -6.29E-06 0.91 -1.10 0.06 -6.03E-06 0.78LAm 4.64 4.17 0.04 -6.06E-04 0.47LAI -2.07 0.38 -4.39E-03 0.92 -2.48 0.43 -5.27E-03 0.96LAu -1.60 024 -3.00E-05 0.86 -1.52 0.23 -2.59E-05 0.86WLm -0.77 -1.80E-06 0.28 -0.63WL1 -1.04 -0.97WLu 0.14 -2.25E-03 4.38E-05 0.10 -1.67E-05 350E-05 0.49Ws -1.92 0.03 0.85 -1.87 0.04 0.84Wb -0.25 -0.09 3.87E-05 0.60 -0.38 -0.06 3.13E-05 0.59Wr -7.62 0.42 -4.92E-03 0.34 -4.08 2.67E-05 0.14Wy 0.83 -0.09 1.74E-05 0.18 -0.67F 4.98 -0.01 0.13 4.75 8.97E-05 -1.73E-05 0.17LVVR -0.54 0.82 -0.49 -1.95E-05 0.59SLA 5.39 0.01 0.20 4.69 0.05 -9.14E-06 0.48SLAM 5.40 0.18 5.33SLAI -2.17 0.52 -6.65E-03 0.77 -250 0.51 -6.43E-05 0.78SLAu -3.42 0.36 -6.22E-05 0.90 -3.35 0.35 -6.16E-05 0.90SRR 1.24 1.5162Table 3.4 (continued) Regression coefficients for growthmeasures, summer 1991K: 6.25 mMol L-1CO2: 350,u L L-1 CO2 = 1400/i L L-1Var. Const.^t t2 t3 R2 Const. t t2 t3 R2W -0.87 0.07 -6.28E-06 0.87 -0.44 0.07 -4.64E-04 0.84LA 3.96 0.07 -9.38E-06 0.87 3.91 0.08 -1.03E-05 0.95WL -0.79 0.03 0.64 -0.88 0.08 -8.27E-04 0.71LAm 4.54 5.17E-04 -9.74E-05 021 4.42 0.02 -6.61E-06 0.32LAI -2.31 0.40 -4.91E-03 0.96 -2.10 0.39 -4.61E-03 0.95LAu -0.75 0.13 0.78 -1.36 0.20 -2.09E-05 0.88WLm -0.63 -0.01 0.31 -0.37 0.31WLI -0.03 -0.11 1.62E-03 0.27 -1.01WL. -0.20 0.17 -2.67E-03 4.93E-05 054Ws -2.06 0.03 0.45 -1.67 0.03 0.80Wb -0.19 -0.10 3.73E-05 0.55 -0.16 -0.08 3.29E-05 0.55Wr -6.13 0.13 0.23 -7.53 0.40 -4.62E-03 0.33WY 1.21 -0.13 2.71E-05 0.29 1.56 -0.17 3.65E-05 0.35F 4.95 -1.40E-06 0.08 4.67LVVR -0.35 -0.02 0.69 -0.43 -2.46E-04 0.83SLA 3.31 0.14 -1.77E-03 0.26 4.70 0.03 -4.08E-06 0.56SLAm 3.35 0.14 4.78E-03 0.21 4.93 0.01 0.29SLAI -1.23 0.45 -5.84E-03 0.67 -2.20 0.50 -6.31E-03 0.79SLAu -2.47 0.26 -3.48E-05 0.76 -3.29 0.35 -5.87E-05 0.90SRR 1.07 1.82 -0.01 0.0763In the CO2 enriched plants, W increased steadily from the onset of treatment(Fig. 3.5a, 3.5b). Increasing K supply significantly reduced accumulation of plantmass, particularly in CO2 enriched plants. At the end of the trial, mean dry massesof CO2 enriched plants were 25% larger than the controls, although largerdifferences were observed in the first two harvests. Effects of CO2Enrichment and K Supply on Growth of Plant Parts (1991)LA increased steadily during the first six weeks (Fig. 3.5c, 3.5d). The CO2enrichment effect on LA was not significant. Plants under the highest K supplyproduced the smallest LA of any case. There was a significant interaction of CO2with K, as well as a significant interaction of these two factors over time.Area of leaves borne on main stems (LA.) was not significantly affected bytime, CO2 or K supply. The area of leaves borne on the lower part of branches(LAI) increased steadily over time until six weeks after of treatment (Fig. 3.6a, 3.6b).The CO2 enrichment effect was not significant on LAI. The effect of K supply, andthe interaction of CO2 enrichment with K supply were significant. Thus, plantsgrown under CO2 enrichment and low K supply tended to have larger LAI. Thearea of leaves borne on the upper part of branches (LAu) increased steadily overtime until the end of the experimental period (Fig. 3.6c, 3.6d). LA u showed nosignificant response to CO2. There were significant effects of K supply, and of theinteraction of CO2 enrichment with K supply over time on LAD. Plants under highCO2 produced slightly larger IA. in most of the K supply regimes.64Figure 3.5aPlant Dry MassSummer 1991 CO2 350Figure 33bPlant Dry MassSummer 1991 CO2 1400Leaf AreaSummer 1991 CO2 3503.01.014^21^28^35^42^49'lime (days)Figure 3.5eLeaf Dry MassSummer 1991 CO2 350Leaf AreaSummer 1991 CO2 1400Figure 3.5fLeaf Dry MassSummer 1991 CO2 14002.21ea 1.0-0-11Figure 3.5c Figure 3.5d14^21^28^35^42^49'lime (days)1.-1.-2. -14^21^28^35^42^49^56^ 0^1^14^21^28^35^42^49Time (days) Time (days)Figure 3.5 Effects of CO2 enrichment and K supply on W (a, b), LA (c, d),and WL (e, f), summer 1991. K levels (mMol L-1), 2.50 (^),3.75 (^), 5.00 (^), 6.25 (^).14 21 28 35 42 49 56Time (days)14 21 28 35 42 49 5665Figure 3.6aLeaf Area Lower LeavesSummer 1991 CO2 350Figure 3.6cLeaf Area Upper LeavesSummer 1991 CO2 350Figure 3.6bLeaf Area Lower LeavesSummer 1991 CO2 1400Time (days)Figure 3.6dLeaf Area Upper LeavesSummer 1991 CO2 1400Time (days)Figure 3.6 Effects of CO2 enrichment and K supply on LAb (a, b) and LA.^(c, d), summer 1991. K levels (mMol L-1), 2.50 (^ ), 3.75 (^),5.00 (^), 6.25 (^).66CO2, K, and the interaction between these factors with time producedsignificant effects on total leaf dry mass, WL (Fig. 3.5, 3.5f), resembling the patternsshown by total dry mass. WL was higher in plants grown under CO2 enrichment orwith lower levels of K supply. CO2 enrichment significantly increased dry mass ofboth leaves borne on main stems (WL,„) (Fig. 3.7a, 3.7b), and leaves borne on thelower part of branches (WLI) (Fig. 3.7c, 3.7d). The effect of K supply wassignificant on WI,' but not on WL,„. The interaction of K supply with time wassignificant on both leaf types. R2 values are low due the large variability of thesedata (Table 3.4). The effect of CO2 was not significant in leaves borne on the upperpart of branches (WL,i) (Fig. 3.7e, 3.7f). Increasing K supply reduced WL„ in mostcases.Dry mass of main stems (Ws) and branches (Wb) significantly increased overtime, and mass accumulation was greater in CO2-enriched plants (Fig. 3.8a, 3.8b).K supply influenced significantly W. Increasing K supply significantly decreasedbranch dry mass (Fig. 3.8c, 3.8d). Root dry mass (Wr) significantly increased overtime. CO2 enrichment did not have a significant effect on Wr. K supply and theinteraction of K supply with time were significant (Fig. 3.9a, 3.9b). The techniqueused for measurement caused large variability in root dry mass.Dry mass of flowers (Wy) was measured only on the last two harvests, and thetime effect was not evaluated. Nevertheless, Wy increased towards the end of thestudy (Fig. 3.9c, 3.9d). CO2 enrichment did not influence Wy. The effect of Ksupply was significant, although the ranking of the K levels varied between CO2treatments.14^21^28^35^42Time (days) 'Time (days)-1-2.14^21^28^35^42^49^26 0^1^14^21^28^35^42^49Time (days) lime (days)67Figure 3.7a^ Figure 3.7bMain Stem Leaf Dry Mass^Main Stem Leaf Dry MassSummer 1991 CO2 350 Summer 1991 CO2 1400Figure 3.7c^ Figure 3.7dLower Leaves Dry Mass^Lower Leaves Dry MassSummer 1991 CO2 350 Summer 1991 CO2 140014^21^28^35^42^49Time (days)Figure 3.7eUpper Leaves Dry MassSummer 1991 CO2 35014^21^28^35^42^49Tune (days)Figure 3.71Upper Leaves Dry MassSummer 1991 CO2 1400Figure 3.7 Effects of CO2 enrichment and K supply on WL. (a, b), WL/ (c,d), and WI,. (e, f), summer 1991. K levels (mMol L-1), 2.50 (^),3.75 (^ ), 5.00 (^), 6.25 (^).68Figure 3.8aMain Stem Dry MassSummer 1991 CO2 3500.5Figure 3.8bMain Stem Dry MassSummer 1991 CO2 14000.50.0 ^0.0-0.5 -0.5 -1.0- -1.5 -1.5-2.0-2.50 7 14 21 28 35 42 49 56Time (days)Figure 3.8cDry Mass of BranchesSummer 1991 CO2 3504:4)/------^,----- .0/1III111111111111111111111111117 14 21 28 35 42 49 56Time (days)-2.0-2.50 7 14 21 28 35 42 49 56Time (days)Figure 3.8dDry Mass of BranchesSummer 1991 CO2 14002. l^llll 1111111111111111111111107 14 21 28 35 42 49 56Time (days)llllllll lllllllll^ ll 11,111112. 0-/..........Figure 3.8 Effects of CO2 enrichment and K supply on Ws (a, b) and Wb (c,^d), summer 1991. K levels (mMol L-1), 2.50 (^), 3.75 (-),5.00 (^), 6.25 (^).••69Figure 3.9aRoots Dry MassSummer 1991 CO2 350Figure 3.9bRoots Dry MassSummer 1991 CO2 1400Figure 3.9cDry Mass of FlowersSummer 1991 CO2 350Figure 3.9dDry Mass of FlowersSummer 1991 CO2 1400Figure 3.9 Effects of CO2 enrichment and K supply on Wr (a, b) and Wy (c,d), summer 1991. K levels (mMol L-1), 2.50 (^), 3.75 (^),5.00 (^), 6.25 (^).703.3.2.3 Effects of CO2Enrichment and K Supply on Growth Indices (1991)AGR increased with time (Fig. 3.10a, 3.10b), and such increase occurredfastest in CO2 enriched plants grown with the two lowest K levels. It was slowest inplants grown with the two highest K concentrations.Relative growth rate (R) decreased from the beginning of the experiment atboth CO2 levels in most cases (Fig. 3.10c, 3.10d). Plants grown under CO2enrichment and low K supply had lower R initially, but R decreased more slowly insuch plants than in CO2 enriched plants. Plants with the highest level of K supplyhad similar initial R at both CO2 levels, but R decreased more rapidly in CO2enriched plants.Unit leaf rate (E) was higher on plants under CO2 enrichment, but theresponse of E to K supply was modified by the CO2 level. In CO2 enriched plants Edeclined during the first weeks and increased after six weeks, while on plants underlow CO2, E had a variable response to K supply.CO2 enrichment significantly affected the leaf area ratio (F), and there was asignificant interaction between the effects of CO2 enrichment and time (Fig. 3.11a,3.11b). In unenriched plants, initial F was higher than in CO2-enriched plants, and,in most cases, F decreased slowly from the beginning of the study. In plants grownunder CO2 enrichment, F increased up to the fifth week and then decreased. At theend of the experimental period, F of CO2-enriched plants was smaller than F ofunenriched plants. R2 values however, are low for these curves.LWR decreased over time in most of the treatments, but no significanteffects of time, CO2 enrichment, K supply or their interactions were detected.71Specific leaf area (SLA) was significantly affected by CO2 enrichment andthe interaction of CO2 enrichment with time (Fig. 3.11c, 3.11d). Initially, the SLAof unenriched plants was higher than the SLA of CO2 enriched plants, but the SLAof the latter increased at a faster rate. Thus, at the end of the trial the SLA of bothCO2 enriched and unenriched plants were similar. K supply did not significantlychange the SLA. The interaction of the effects of K supply and time wassignificant, hence SLA increased over time, and the rate of change was modified bythe level of K supply.Summer 19910.10-0.08-0.07-0.05-ftCO2 3500.03:0.02:0.01-0.00 I 11111^1111^1111^1111^1111^1111^1111111111^111^111^111^111^111^1110 7 14 21 28 35 42 49 56Time (days)72Figure 3.10aAbsolute Growth RateSummer 1991 CO2 3500 7 14 21 28 35 42 49 56Time (days)Figure 3.10cFigure 3.10bAbsolute Growth Rategnmmr.,- 1001 cry) 1 AAAFigure 3.10dRelative Growth Rate^Relative Growth RateFigure 3.10 Effects of CO2 enrichment and K supply on AGR (a, b), R (c,d), summer 1991. K levels (mMol L-1), 2.50 (^), 3.75 (^),5.00 (^), 6.25 (^).E 4.9-4.7• -g• 4.9-4.7-5.5-5.3-4.5-4.3^0 7 14 21 28 35 42 49 56Time (days)Figure 3.11c4.5-4.3^0 7 14 21 28 35 42 49 56Time (days)Figure 3.11d5.5-5.3-7.06.0-4.0-3.0,,,,,, WM1111111110^7^14^21 11III1128" r3imi,6"":19"" r673Figure 3.11a^ Figure 3.11bLeaf Area Ratio (F)^Leaf Area Ratio (F)Summer 1991 CO2 350 Summer 1991 CO2 1400Specific Leaf AreaSummer 1991. CO2 350.3.0 1IJLII 1,1111111,1 III 1011111111111111111111 11,4 111111t^16Time (days)Specific Leaf AreaSummer 1991. CO2 1400Time (days)Figure 3.11 Effects of CO2 enrichment and K supply on F (a, b) and SLA (c,d), summer 1991. K levels (mMol L-1), 2.50 (^), 3.75 (^),5.00 (^), 6.25 (^).3.4 DISCUSSION3.4.1 General Effects of CO2 Enrichment and K Supply on Plant GrowthThe total dry mass of chrysanthemum plants in both experiments wasenhanced by CO2 enrichment, an effect that has been reported in previous studies(Gislerod and Nelson 1989, Kimball 1986a, Mortensen 1987, Mortensen and Moe1983b). There was little effect of increasing the CO2 concentration from 1200 ii, L L-1 to 1800 AL L-1 in the first experiment, which is in agreement with previous reports(Mortensen and Moe 1983a, 1983b).Plant growth responses to CO2 enrichment were strongly affected by nutrientsupply. This was evident either when comparing responses to K supply in thesecond experiment, or by observing the differences in the resolution of growthresponses to CO2 enrichment between the first and second experiments. In the firstexperiment the plants were grown with higher levels of nutrient supply, and theabsolute growth rates were greater than in the second experiment, despite the lowerirradiance levels used in the former. Such influence of nutrient supply has beenobserved previously in CO2 enrichment studies (Goudriaan de Ruiter 1983,Mortensen 1987, Patterson and Flint 1982, Sionit et al. 1981a). Mortensen (1987)found that Begonia plants provided with high nutrient supply (4 mmhos cm-1 EC)increased growth and showed healthy foliage. By comparison, nutrient solutionswith an EC of 2 mmhos cm-1 EC increased growth over the controls but producedchlorotic leaves and elongated stems. Furthermore, no increase in growth occurredin plants supplied with nutrient solutions with an EC of 1 mmhos cm*7475In the present study, two effects interfered with the analysis of plantresponses. First, as a consequence of limited space in the CO2 growth chambers,there were complications in the experimental design and small samples had to beused, resulting in both increased variability and smaller degrees of freedom forstatistical analyses. Also, there was a direct effect of nutrient supply limiting plantresponses to CO2 enrichment.It was surprising that decreasing K supply increased plant growth, since thenormal response to K deficiency is retarded growth (Marschner 1986). It is notlikely that such an effect was caused by the concentration of K itself, sinceincreasing K from 2.5 mMol L-1 to 6.25 mMol L-1 normally would be beneficial forchrysanthemums. Indeed, for chrysanthemums in rockwool culture 5.00 mMol L -1K is recommended (Sonneveld and de Krey 1989). Also, CO2 enrichedchrysanthemums in hydroculture experiments were grown with K concentrations of6.00 to 6.25 mMol L- 1 (Gislerod and Nelson 1989, Kuehny et al. 1991); and in thefirst experiment of these studies 5.1 mMol L -1 K were used to grow plants on a peatbased medium without any toxicity.The good response of plants at low K supply appears to be related to apossible beneficial effect of Na. A similar effect was observed in alfalfa whereapplied Na increased yield when K supply was low, although no increase wasobserved when K was near the critical level (James 1988).The detrimental effects of increasing K supply were not induced by salinity,since the growth of chrysanthemum plants under CO2 enrichment was increased bynutrient concentrations up to 4 mmhos cm -1 (Mortensen 1987), and in thisexperiment EC never was over 2.5 mmhos cm -1 . Thus, other factors of the nutrient76solutions may have caused the detrimental effects. These factors include: theincreasing amounts of acetic acid and NaOH added to the nutrient solutions as Kconcentration increased, and daily drifts in pH which varied for each solution type.Nutrient concentrations, and amounts of NaOH, Na2SO4 and acetic acid added tonutrient solutions are presented in Appendix 2.In the solutions, Na concentration was the same, but the source of Nachanged. Na2SO4 was the only Na source at the lowest K level, NaOH was the Nasource at the highest level of K, and both of them were added in intermediateamounts to the other two K treatments. Since the pH of the solutions rose withincreasing NaOH, greater amounts of acetic acid were required to adjust the pH.After the initial pH adjustment, differences as large as 1.3 pH units among the twoextreme treatments were recorded between daily pH adjustments. Such differencesmay have created problems in the solubility or uptake of nutrients, and also createdincreasing requirement for acetic acid to adjust the pH after the drifts.Acetic acid, used in concentrations ranging from 0.78 mM to 3.13 mM,probably was the main detrimental factor of the nutrient solutions. This acid, andother organic acids, have been found to be phytotoxic (Harper and Lynch 1980).Organic acids applied in concentrations of 1 mM per 100 g soil inhibited rootelongation (Lynch 1978), and mass accumulation of roots and shoots(Chandrasekaran and Yoshida 1973). The undissociated species of organic acidsare readily taken up and lead to a sharp rise in membrane leakiness to inorganicions (Lee 1977). 10 mM potassium acetate induced leakiness in barley roots, as aresult of changes in the fatty acid composition of the membranes. Phytotoxicity77increased with a decreasing external pH, in an effect related to the high rates ofpermeation of the plasma membrane (Jackson and St. John 1980).3.4.2 Effects of CO2 Enrichment and K Nutrition on Growth of Plant PartsTotal plant dry mass (W), leaf dry mass (WL) and total leaf area (LA) wereincreased by CO2 enrichment in the first experiment. Changes in W and LA wereseen mainly in leaves borne on branches, as leaves borne on the main stem werealmost fully expanded when the experiments started. In the second experiment,however, when plants were grown under a combination of CO2 enrichment and Ksupply, CO2-induced changes in LA were not detected. Increases in W and LAwere consistent with many reports (Kimball 1983), including several studies onchrysanthemums (Gislerod and Nelson 1989, Hughes and Cockshull 1971a, 1971b,1972, Mortensen and Moe 1983). Response of chrysanthemum plants to CO2enrichment was increased by secondary factors, such as higher irradiance (Hughesand Cockshull 1971a, 1971b, 1972) and higher relative humidity (Gislerod andNelson 1989). In other studies, CO2 enrichment caused little or no increase on theleaf area of chrysanthemum cv 'Harim' (Mortensen 1986), dwarf beans (Jolliffe andEhret 1985, Liu 1990), tobacco (Thomas et al. 1975) and Sorghum (Mauney et al.1978).WL was increased by CO2 enrichment in both experiments. In this case also,most of the increase occurred on leaves borne on branches, although dry mass ofleaves borne on main stems also increased. These results agree with previousstudies on chrysanthemums (Gislerod and Nelson 1989, Hughes and Cockshull1971a, 1971b, 1972, Mortensen and Moe 1983).78CO2 enrichment increased the dry mass of main stems and branches, and didnot have a significant effect on flower dry mass, as reported in previous studies(Gislerod and Nelson 1989, Goldsberry 1986, Mortensen 1987, Mortensen 1986).Hughes and Cockshull (1972) found that a low irradiance delayed flowerdevelopment and reduced flower number and mass, inhibiting responses to CO2enrichment. They also reported a positive correlation between irradiance and CO2enrichment. Thus at higher irradiance levels CO2 enrichment increased flowermass (Hughes and Cockshull 1971a).Timing of flower production was not changed in this study. Although time toharvest can be reduced up to two weeks in CO2-enriched chrysanthemum plants,flower initiation is controlled by photoperiod. Thus early flower production resultsfrom an increased initial growth, which allows for both an early pinch and earlierexposure of plants to short photoperiod (Nelson 1991). Such an advantage,however, did not exist in these experiments since CO2 enrichment and shortphotoperiod to induce flowering began at the same time, and all plants werepinched at that timeRoot mass was modified by CO2 enrichment in the first experiment, but notin the second. In addition to possible biological effects of treatments, growingconditions (peat-lite vs rockwool), physical factors (pot type and size), and methodsof root growth assessment could be responsible for the inconsistency of resultsbetween studies. Such inconsistency has also been observed in previous reports.CO2 enrichment did not induce significant early changes in root dry mass of dwarfbean plants, although late changes were significant (Jolliffe and Ehret 1985). Rootmass also increased in CO2-enriched chrysanthemums grown in 20-L tanks79(Gislerod and Nelson 1989), while it was reduced in CO2-enriched tomato plants(Tripp eta!. 1991).In these studies, leaves borne on the branches were affected first, and theirchanges usually occurred faster than elsewhere in the plants. It is possible that suchchanges might subsequently have influenced changes in other plant parts. Changesin dry matter partitioning induced by CO2 enrichment have been reported inchrysanthemums (Gislerod and Nelson 1989, Mortensen and Moe 1983b), tomato(Tripp et al. 1991), cucumber (Ito 1978), dwarf bean (Jolliffe and Hoddinott 1988,Liu 1990), and tomato plants (Tripp et al. 1991). Indeed, increased fruit yield ofCO2-enriched tomato was mostly due to changes in partitioning of assimilates,rather than to increased photosynthesis (Tripp et al. 1991). Changes in dry matterpartitioning may affect the structure of the plant (Gislerod and Nelson 1989,Mortensen and Moe 1983b), and the subsequent effect on yield and morphology ofthe marketable parts is of horticultural interest (Goldsberry 1986).Increasing K supply decreased W, WL and LA in most cases but responsesvaried according to the leaf type. Increasing K supply also reduced dry mass ofstem, branches, flowers and roots. Such reductions were usually enhanced by CO2enrichment. These results are opposite to typical responses of plants to K supply(Winsor and Adams 1987). Reduction in leaf size is the first symptom of Kdeficiency (Lunt and Kofranek 1958). K deficiency also reduced plant mass (Luntand Kofranek 1958, Winsor and Adams 1987, Holcomb and White 1974), andresulted in weak spindly stems in chrysanthemums (Lunt et al. 1964). This supportthe theory, stated previously in this paper, that other factors of the nutrient solutionsbecame deleterious for plants as K supply increased.803.4.3 Effects of CO2 Enrichment and K Nutrition on Growth IndicesAbsolute growth rate (AGR) and relative growth rate (R) were enhanced byCO2 enrichment and decreasing K supply. CO2-induced increases in such indiceswere reported in chrysanthemum (Gislerod and Nelson 1989, Mortensen and Moe1983b), and other plant species (Jollife and Ehret 1985, Liu 1990, Rogers et al.1984a). In these experiments, R decreased with time from the beginning oftreatments, as reported by Gislerod and Nelson (1989) for chrysanthemums, ratherthan exhibiting the typical slight increase prior to a decline observed in other plantspecies grown from seeds (Jolliffe and Ehret 1985, Liu 1990, Rogers et al. 1984a).The steady reduction with time observed in R was due to the large initial plant mass,since the plants were grown from cuttings. R assesses the rate of change of drymass over time as a function of the total mass of the plant.Changes in the unit leaf rate (E) and leaf area ratio (F) are indicators ofmodifications in the efficiency of assimilation and partitioning of carbon in CO2-enriched plants (Hughes and Cockshull 1971a). E was increased by CO2enrichment, as reported in other studies (Jolliffe and Ehret 1985). Hughes andCockshull (1971a, 1971b) found a positive relationship of irradiance with the effectsof CO2 enrichment on the E of chrysanthemum plants. In spite of the large effect Ksupply on E in the present study, there was not a response pattern to K supply,possibly as a consequence of the confounding factors of the nutrient solutions.CO2 enrichment reduced F in the first experiment. Although F was alsoreduced in the second study, the pattern of changes in F suggests that the effects ofCO2 enrichment and K supply on the balance between LA and W varied with time.Reduction in F values indicate a smaller increase of LA relative to W. Reduced F81has been reported previously on CO2-enriched chrysanthemums (Mortensen 1986)and dwarf beans (Jolliffe and Ehret 1985, Liu 1990). Other studies on CO2-enriched chrysanthemums, however, did not find changes in F (Hughes andCockshull 1971a, 1971b).Changes in the two components of F, specific leaf area (SLA) and leaf weightratio (LWR) were also detected. In the first experiment SLA was drasticallyreduced at the highest CO2 level (1800 A L L-1), but was increased by 1200 A L L-1CO2. Changes in SLA resulted from higher WL at the 1800 A L L-1 CO2 relative toplants grown at 1200 A L L-1 CO2, since LA was similar in both treatments. In thesecond experiment, both CO2 enrichment and increasing K supply reduced SLA,while LWR was not modified. Many studies have documented CO2-inducedreductions in SLA or increases in specific leaf weight, the reciprocal of SLA (Jolliffeand Ehret 1984), although Hughes and Cockshull (1971a, 1971b) did not find suchdifferences in chrysanthemums.A small decrease or no change in the shoot to root ratio (SRR) of CO2-enriched plants was observed on in either experiment. Other studies have reportedlowered SRR in several species (Ford and Thorne 1967, Sionit et al. 1982), but nochange was observed in chrysanthemum (Hughes and Cockshull 1972) or dwarfbeans (Jolliffe and Ehret 1984). In chrysanthemums grown in hydroculture, SRRwas increased by CO2 enrichment after 4 weeks when the relative humidity was95%, but at 50% relative humidity CO2 enrichment increased SRR only after 6weeks of treatment (Gislerod and Nelson 1989). These discrepancies may be aconsequence of differences in plant species or cultivar, secondary factors (such as82relative humidity), restrictions on root growth imposed by pot size, or differenttechniques employed to assess root mass.Harvest index (H) was not changed by CO2 enrichment, indicating that theadditional growth of plants did not result in a disproportionate effect on growth offlowers. It is possible that the low irradiance of the experiment limited plantresponse to CO2 enrichment, thus preventing increases in H, as reported previouslyin chrysanthemum plants (Hughes and Cockshull 1971a, 1971b).In the second experiment, K supply influenced plant growth responses morethan did CO2 enrichment. This observation agrees with the stronger effect ofnutrient supply in relation to that of CO2 enrichment, on growth of three plantspecies reported by Patterson and Flint (1982).83CHAPTER 4 EFFECTS OF CO2 ENRICHMENT AND K SUPPLY ON CARBONMETABOLITE CONCENTRATION AND INORGANIC NUTRIENT STATUS INLEAVES4.1 INTRODUCTIONAtmospheric CO2 is the substrate for photosynthesis in higher plants,supplying the reduced forms of carbon upon which plant growth depends (Huber etal. 1985). Increasing CO2 above present atmospheric levels stimulates plant growth,when other environmental factors are not limiting (Kimball 1986b), and may do sowhen other resources are normally limiting, particularly light (Hughes andCockshull 1971a). In addition to the response of photosynthesis, the fate of fixedcarbon is critical for determining plant responses under CO2 enrichment (Tolbertand Zelitch 1983), since plant growth and development depends also on utilizationof the fixed carbon within the plant. Also, as dry matter accumulation increases inresponse to CO2 enrichment, a correspondingly greater amount of inorganicnutrients may be required by plants (Kimball 1986b), and nutrient relations appearto be changed in CO2 enriched plants (Liu 1990, Tripp et al. 1991). Availability andutilization of inorganic nutrients may, therefore, affect plant responsiveness to CO2enrichment. There is increasing evidence that long exposure to high CO2 mayinduce detrimental effects and leaf abnormalities in plants (Van Berkel 1984, Ehretand Jolliffe 1985a, Mortensen 1987, Liu 1990 Tripp et al. 1991). CO2-induced leafabnormalities resembled inorganic nutrient deficiency symptoms in many instances,for example of K in potato (Goudriaan and Ruiter 1983), K, Mg or Mn in tomato(Tripp et al. 1991), and N or K in dwarf beans (Liu 1990).84Increased leaf starch concentration is a characteristic response of many plantspecies to CO2 enrichment (Delucia et al. 1978, Ehret and Jolliffe 1985b, Porter andGrodzinski 1989). In leaves of some plant species under CO2 enrichment, sugarconcentration showed relatively small increases (Allen et al. 1988, Havelka et al.1984, Huber et al. 1984, Vu et al. 1989), although other studies did not find any sugarincrease (Mauney et al. 1978, Finn and Brun 1982, Nafziger and Koller 1976). Leafprotein concentration is usually not altered by high CO2, although qualitativechanges may occur in the protein fraction (Porter and Grodzinski 1984). K-deficient plants tend to have reduced rates of starch synthesis, while sucrosemovement from leaves increases (Marschner 1986). Substitution of up to 50% ofthe K in the nutrient medium by Na increased sucrose content in sugar beet roots.When 95% of the K was replaced by Na, however, widely varying responsesoccurred (Marschner et al. 1981).Poor mineral nutrition of plants grown under CO2 enrichment reducedgrowth responses (Goudriaan and Ruiter 1983, Liu 1990, Mortensen 1987, Sionit etal. 1981, Yelle et al. 1987, Wong 1979), and in some cases induced leaf abnormalities(Liu 1990, Mortensen 1987, Tripp et al. 1991). Low N fertilization (Wong 1979) andP shortage (Goudriaan and Ruiter 1983) rendered CO2 enrichment ineffective inseveral plant species, and CO2 enrichment adversely affected maize plants subjectedto N and K deficiency (Goudriaan and Ruiter 1983). In contrast, increasedresponse of wheat (Sionit et al. 1981) and Begonia plants (Mortensen 1987) to CO2enrichment was correlated with increasing concentrations of nutrient solutions.Enhanced NO3- translocation from roots to leaves, due to other environmentalfactors, was associated with an increased response of tomato plants to CO285enrichment (YeIle et al. 1987). It has been suggested that to obtain maximumbenefits of CO2 enrichment, nutrient supply to chrysanthemum plants must bemaintained at a higher level than normally used for unenriched plants (Eng et al.1985).Decreases in foliar nutrient concentration of CO2 enriched leaves have beenreported for many plant species (Liu 1990, Porter and Grodzinski 1989, Wong 1979,Hocking and Meyer 1991, Peet et al. 1986, Tremblay 1988, Knecht and O'Leary1983), including chrysanthemum (Eng et al. 1987, Kuehny et al. 1991). Suchreductions result at least in part from nutrient dilution by starch (Kuehny et al. 1991,Porter and Grodzinski 1989). Nevertheless, nutrient imbalances might also resultfrom reduced transpiration (Madsen 1975, Mortensen 1987), inadequate supply,uptake or translocation of nutrients (Liu 1990, Mortensen 1987, Tripp et al. 1991,Yelle et al. 1987) in relation to the increased requirements of CO2 enriched plants.Nutrient uptake and translocation in CO2 enriched Virginia pine, tomato and roses,were also affected by environmental conditions in the root zone (Luxmore et al.1986, Jarvis and Robson 1983, Yelle et al. 1987) and K supply (Woodson andBoodley 1982).The onset of leaf abnormalities has been associated with severalphysiological and environmental factors (Cave et al. 1981, Ehret and Jolliffe 1985a,Liu 1990, Mortensen 1987, Tripp eta!. 1991, Van Berkel 1984, Wulff and Strain1982), but the role of CO2 enrichment in such disorders is not fully understood.The onset and severity of leaf abnormalities have been correlated to starchaccumulation in some studies (Cave et al. 1981, Ehret and Jolliffe 1985a, Heskethet al. 1971, Liu 1990, Sharkey et al. 1985). However, leaf abnormalities were not86correlated with the starch concentrations observed in CO2 enriched Gerbera (VanBerkel 1984) and tomato plants (Tripp et al. 1991). Instead, diminished K wasassociated with increased deformation in the latter (Tripp et at. 1991). Increasedremobilization of N and K was also correlated with the onset of leaf injury in CO2enriched dwarf bean plants (Liu 1990). Low nutrient supply to CO2 enriched dwarfbean (Liu 1990) and Begonia plants (Mortensen 1987) promoted leaf injury, whilehigher nutrient supply delayed or prevented the development of such abnormalities.The present studies were done to investigate whether CO2 enrichmentaltered the presence or allocation of inorganic nutrients in chrysanthemum plants.It was intended to observe the development of CO2-induced leaf abnormalities, andto evaluate the role of both CO2 enrichment and K supply in this problem.Relationships between K supply and concentrations of starch and other carbonmetabolites in leaves were also studied in connection with leaf abnormalities.4.2 MATERIALS AND METHODS4.2.1 Plant CultureFor the study of carbon metabolites and nutrient relations, plants grownduring the summer of 1991 (Chapter 3) were utilized. For the chlorophyll analysis,results from those plants, and results from another experiment (fall 1991) wereincluded.The latter experiment was carried out during the fall of 1991, using the samelevels of both CO2 enrichment and K supply, as well as the same management andenvironmental conditions of the previous experiment (summer 1991). This studywas undertaken to study CO2 exchange characteristics of chrysanthemum plants, butonly observations on chlorophyll concentrations and leaf abnormalities will bepresented in this thesis.4.2.2 Determination of Photosynthate and Chlorophyll Concentration in LeavesSamples were harvested for analysis at five times (t): 0, 7, 14, 28 or 56 daysafter the initiation of treatments. Samples were recorded from individual plants,with separate samples being taken from leaves borne on main stems, leaves borneon the four lowest nodes of branches, and leaves borne above the fourth node onbranches. From each leaf portion, five disks (1.03 cm diameter each) were takenfrom the central, interveinal regions using a cork borer. The disks were placed inplastic vials, immediately frozen in liquid nitrogen, and stored in the freezer at -20 Cuntil analyses were performed. Starch was determined for all time points, butsugars and total protein were determined for the four initial samplings only.87884.2.2.1 Extraction and Separation of Starch and Soluble SugarsUsing a glass rod mounted on a mechanical stirrer, four frozen leaf diskswere ground in a test tube immersed in liquid nitrogen. Ground material wasstored at -20 C.Samples were suspended in 2.6 mL of a mixture of methanol: chloroform:water (M:C:W / 12:5:3 / v:v:v) and left overnight in the freezer. The next day theywere vortex mixed and centrifuged (1250 g, 10 minutes). The supernatant wasremoved and saved for the determination of soluble sugars. The pellet wasresuspended in 1 mL of M:C:W, and centrifuged (1250 g, 10 minutes). This pellet,representing the insoluble fraction, was stored in the freezer for starchdetermination.The supernatant from the second extraction was mixed with the supernatantfrom the first extraction, to form the soluble sugars extract. Then 1.4 mL doubledistilled water was added such extract, followed by vortex mixing and centrifuging(1250 g, 10 minutes). A phase separation appeared; the bottom green layer wasdiscarded while the top (aqueous) layer was saved. Such samples containing thesoluble sugars extract were stored at -20 C for analysis. Determination of StarchEnzymatic analysis of starch was performed using a -amylase andamyloglucosidase to break down the starch. The released glucose was thendetermined using the Sigma 510-A glucose kit (Sigma diagnostics), a procedure inwhich glucose is converted to glucuronic acid by glucose oxidase, releasing hydrogenperoxide, which subsequently oxidizes a color reagent (o-dianisidine) in the89presence of peroxidase resulting in color development (Sigma diagnostics,procedure 510).The pellet was suspended in 5 mL of acetate buffer (pH 4.5, 150 mM), andincubated in a shaking water bath at 55 C for 2 hours. Purified solutions of a -amylase from Aspergillus oryzae (Sigma diagnostics A-0273), 62.5 AL; andamyloglucosidase from Rhizopus mold (Sigma diagnostics A-7255), 125 AL werethen added to each tube. The mixture was incubated for two hours at roomtemperature and centrifuged (1250 g, 10 minutes).Three aliquots of the supernatant were analyzed for glucose. 50 A Lsupernatant and 150 A L acetate buffer were mixed together, suspended in two mLof combined peroxidase - glucose oxidase - color reagent solution (Sigmadiagnostics, cat. 510-A), and incubated for 30+5 minutes in a water bath at 37 C.Absorbances at 450 nm were determined in a spectrophotometer (Philips, modelP48620). Starch concentration in the extract was calculated by establishing a starchstandard curve. The standard curve was prepared by making six different dilutions(0, 5, 10, 15, 20 and 25 A g per sample) out of a starch stock solution. The stocksolution was prepared by dissolving 16.2 mg soluble starch powder (Sigma S-9765)in 100 mL acetate buffer. Starch samples were processed as any other sample tofinally get the absorbance readings for glucose. Determination of Sucrose, D-Glucose and D-FructoseThe concentration of soluble sugars in the extracts was determined using atest kit for sucrose, D-glucose and D-fructose (Boehringher Mannheim, cat. 716 260,1989). In this procedure, glucose was phosphorylated by ATP producing glucose-6-90P (G6P) in a reaction catalyzed by hexokinase. Then G6P was oxidized by NADPto form gluconate-6-P and NADPH, in the presence of glucose-6-P dehydrogenase.The content of NADPH was measured spectrophotometrically at 340 nm Theamount of NADPH formed is stoichiometric with the amount of D-glucose in thesample. Hexokinase also catalyzes the phosphorylation of D-fructose by ATP toform fructose-6-P (F6P). On completion of this reaction, F6P was converted toG6P by phosphoglucose isomerase. For determination of sucrose, the disaccharidewas hydrolyzed to D-glucose and D-fructose, in the presence fi-fructosidase(invertase) at pH 4.6. Those compounds were determined as mentioned above.Sucrose content was calculated from the difference between D-glucoseconcentrations before and after an enzymatic inversion.For determination of sugars, the extracts containing soluble sugars wereplaced in a water bath at 55 C for two hours. Then two aliquots were pipetted intodisposable cuvettes, following the procedures stated in the pipetting table of the testkit for the amount and type of enzymes to be added, and the timing for readingabsorbances (Boehringher Mannheim 1989). Absorbances at 340 nm weredetermined in a spectrophotometer (Philips, model P48620). Concentration ofeach sugar in the extracts was calculated as indicated in Appendix 10, and thenexpressed as mass of sugar per unit leaf area. Determination of Total ProteinTotal soluble protein was determined using the Bio-Rad protein microassay(Bio-Rad, cat 500-0001). This basis of this method is that when an acidic solution91of Coomassie Brilliant Blue G-250 binds to protein there is a shift in the absorbancemaximum from 465 nm to 595 nm.For determination, one frozen leaf disk (1.03 cm diameter) was ground in a2.5 mL Eppendorf tube using a glass rod mounted on a mechanical stirrer. Thetubes were kept in liquid nitrogen while grinding, and once ground, were stored at -20 C until analyzed. Ground samples were suspended in 1.5 mL Tris-HC1 (pH 7.5,25 mM) and cold centrifuged (10,000 g 10 minutes). Three 50 AL-aliquots ofsupernatant were pipetted into Eppendorf tubes and 750 A L double distilled waterplus 200 A L dye reagent concentrate (Bio-Rad, cat. 500 0006) were added.Absorbances at 595 nm were determined spectrophotometrically (Philips, modelP48620) within 30 minutes of the addition of the color reagent solution. Totalprotein concentration in samples was determined by relating their absorbanceresults to absorbances of a protein standard curve prepared with lyophilized bovinegamma globulin (Bio-Rad, cat. 500 0005). Total protein was then expressed asconcentration per unit leaf area. Determination of ChlorophyllChlorophyll concentration in leaves was determined with an electronicchlorophyll meter (Minolta, model SPAD 502). In such device chlorophyll ismeasured in SPAD values which indicate the amount of chlorophyll present in thesample leaf. SPAD values are calculated based on the amount of light transmittedby the leaf in two wavelength regions in which the absorption of chlorophyll isdifferent. The red area, approximately 650 nm, where absorption is high and92unaffected by carotene, and the infrared area, approximately 940 nm, whereabsorption is extremely low.In the summer 1991 experiment, chlorophyll concentration was measured ateach harvest time. In the fall 1991 experiment, chlorophyll concentration wasrecorded every four days, from the initiation to day 24 of treatments. In both cases,five measurements were taken from different leaves within each one of the leafsections separated for growth analysis.4.2.3 Determination of Inorganic Nutrient Concentration in Leaves4.2.3.1 Sampling ProceduresSamples for tissue analysis were taken separately from leaves borne on mainstem, and from the four lowest leaves borne on branches of each plant. Withineach of these two categories, all leaves from plants grown in the same CO2 chamber,at the same K level, and harvested at the same age were pooled for tissue analysis.Sample leaves were dried at 65 C for 72 hours, weighed, and sent for analysis.Tissue analyses of leaves borne on main stems were performed on samplesharvested after seven or 56 days from the initiation of CO 2 enrichment treatments.Leaves from both CO2 enriched and non-enriched plants grown at K concentrationsof 2.5 mMol L-1 or 6.25 mMol L-1 were analyzed.Analyses of leaves borne on branches were done only on samples harvestedafter 28 days of treatments. Leaves studied came from the unenriched and CO2enriched plants grown at K concentrations of 2.5 mMol L -1 , 3.75 mMol L-1 ,4.0 mMol L-1 or 6.25 mMol L-1.934.2.3.2 Leaf Tissue AnalysisDry leaf samples were sent for analysis to a commercial laboratory (PSAI,Richmond, B. C.). For determination of N, P, K, Ca, Mg, Mn, Al and Na, drysamples were digested with H2SO4 - LiSO4 - Se - H202 (Parkinson and Allen 1975).Subsequently, N was determined colorimetrically on an AutoAnalyzer (TechniconAutoanalyzer II Methodology, 1976). P was determined spectrophotometrically;and K, Ca, Mg, Mn, Na and Al were determined by atomic absorptionspectrophotometry. B, Fe, Zn, and Cu were determined from the dry ash ofsamples. B was determined colorimetrically using the azomethine-H method, andthe remaining elements were determined by atomic absorption spectrophotometry(PSAI, personal communication, 1991). Expression of the Inorganic Nutrient ConcentrationInorganic nutrient concentrations are often expressed as a percentage of thedry mass of tissue. This measurement provides a useful absolute measure ofnutrient status because both dry mass and nutrient content can be estimatedaccurately. However, this type of expression may sometimes be of limited value inphysiological studies of nutrient behavior (Leigh et as'. 1982). For example, in CO2enrichment studies increased leaf starch magnifies specific leaf weight of CO2enriched plants (Ehret and Jolliffe 1985a). As starch accumulates in leaves,nutrient concentrations calculated on a dry mass basis are artificially depressed(Kuehny et al. 1991), even if the absolute content is not decreased (Porter andGrodzinski 1989). Another indication of such artificial depression is that criticallevels of inorganic nutrients in CO2 enriched wheat plants were reduced along with94leaf nutrient concentrations. Thus, the use of a different criterion for interpretationof tissue analysis of CO2 enriched plants is advised (Hocking and Meyer 1991). Forassessment of tissue analysis of CO2 enriched plants, a measurement not interferedwith by starch mass should be preferred.In view of such difficulty, different approaches were used for assessment ofthe nutritional status of plants in the present study. Four different forms ofexpression of nutrient status within plants were evaluated: total content of inorganicnutrients in each leaf portion, concentration of inorganic nutrients based on leaf drymass (wn :WL), concentration of inorganic nutrients based on a starch-corrected leafdry mass (WLNs), and concentration of inorganic nutrients on a leaf area basis(wn :LA).Total content of inorganic nutrients in leaves may reveal effects oftreatments on presence or allocation of elements in plants. Such an expressionmight also give an indication of the balance between demand and supply forinorganic nutrients in plants. Total content of nutrients is particularly appropriatewhen growth limitations are imposed by growing conditions. Growth limitationscomplicate interpretation of nutrient concentrations, since small leaves with smalldry masses and less content of a given element may have similar concentrations aslarger leaves with greater content of such element.Expression of nutrient concentrations based on a starch-corrected leaf massor leaf area (if that is unaffected by CO2 enrichment) are not interfered with bystarch. The correction for starch was done by subtracting the starch mass in agiven sample from the total dry mass of such sample, and then recalculating thenutrient concentration (Kuehny et al. 1991). Expression of the nutrient95concentrations on a leaf dry mass basis (uncorrected for starch) was used only as abasis for comparison with previous studies.Ratios amongst cation concentrations were also evaluated because: (1) theeffects of treatments on nutrient status in the most affected leaves were weak whensingle elements were considered; (2) in these studies the balance of K and Na in thenutrient solutions had a large influence in plant growth; (3) CO2 enrichmentinduced changes in the starch corrected concentration of cations in other studies(Kuehny et al. 1991, Tripp et al. 1991); (4) increasing K supply induced cationaccumulation in leaves (Woodson and Boodley 1982); and (5) cations are importantenzyme activators and are affected in their biological activity by the presence andconcentration of other cations (Marschner 1986). Statistical AnalysisInorganic nutrient balance was assessed through the evaluation of treatmenteffects on single elements, and by through the evaluation of ratios amongst leafconcentration of cations. Data were analyzed using Systat 5.01 (Systat 1990).Bartlett's test for homogeneity of group variances was performed on primary data,and those values were used to perform the analysis of variance. Analyses ofvariance for leaf concentration of inorganic nutrients, and for ratios between cationconcentrations, were generated using ordinary least squares.Analyses of variance were also performed on the loge transformed values ofLA, WL, starch-corrected leaf dry mass (WLNs), and starch mass (wn) of leavesborne on main stems, and on the lower part of branches. Data used in theseanalysis comprised only the same subset corresponding to the tissue analyses.964.2.3.5 Relationships of Leaf Chlorosis with the Effects of CO2Enrichment on PlantNutrition.To further investigate links of CO2 enrichment and mineral nutrition andtheir effects on the leaf chlorosis, nutrient concentrations of the most affected leaveswere compared to those of healthier leaves. This was done because statisticalanalysis of CO2 enrichment and its effects on the nutritional status of plants wereaffected by the reciprocity between K and Na concentrations in the nutrientsolutions. Such effects made it difficult to understand the role of CO2 enrichmentenhancing the onset of leaf chlorosis. In addition, leaf nutrient concentrations werecompared with concentration ranges (i.e. "normal", "deficient", etc.) published forthe assessment of tissue analysis in chrysanthemum (Winsor and Adams 1987).4.3 RESULTS4.3.1 Effects of CO2 Enrichment and K Supply on the Concentrations of CarbonMetabolites in LeavesAnalysis of variance of the concentrations of metabolites in leaves revealedmany significant (p < 0.05) effects of CO2 enrichment, K supply and theirinteractions on starch concentration. None of the sugars which were measured,however, exhibited significant responses to CO2 enrichment, K supply or time, andtreatment effects on total protein were seldom significant. The effects of blocksand all the interactions containing blocks were often significant (Table 4.1),suggesting the existence of environmental differences between the two sets ofgrowth chambers.The effects of CO2 enrichment, K supply and time on the starchconcentration in leaves borne on main stems were significant. The interactionamong CO2 enrichment, K supply and time was also significant. CO2 enrichmentincreased starch concentration by 28% in these leaves (Fig. 4.1a, 4.1b). Differencesin starch concentrations changed during the course of the experiment, however, andit was difficult to separate out the effect of K supply.There was no significant change over time in the starch concentration ofleaves borne on the lower part of branches. CO2 enrichment significantly increasedthe starch concentration in such leaves (Fig. 4.1c, 4.1d). K supply also had asignificant effect on starch concentration, and its effect changed with CO2 level andwith time. The interactions of time with CO2 enrichment, time with K supply, andtime with CO2 and K supply were significant. Hence, increasing K supply increased9798Table 4.1 Summary of statistical significance,concentration of metabolites in leaves borne on mainstems and on the lower part of branches, summer 1991.Leaf Concentration of MetabolitesSource df Starch Protein Sucrose Glucose FructoseLeaf Type Main Lower Upper Main Lower Main Lower Main Lower Main LowerBlock 3 * * ns * * us ns ns *t 3 ns ns * us ns us ns ns nsBlock*t 9 * * us * * * * * * *CO2 1 * * * us ns ns ns ns ns us nsCO2*t 3 * ns ns ns ns ns us ns ns nsBlock*CO2*t 12 ns ns ns * * * * * * *K 3 ns * * ns ns ns ns us ns us nsK*t 9 * * ns * ns ns us ns ns us nsK*CO2 3 * * us * ns ns ns ns ns ust*K*CO2 9 * ns * us ns us ns ns ns usTable 4.1 (continued) Summary of analysis of variance,concentration of metabolites in leaves borne on theupper part of branches, summer 1991.Concentration of Metabolites, Upper Leaves of BranchesSource df Protein Sucrose Glucose FructoseBlock 2 * *CO2 1 ns us us nsBlock* CO2 2 ns us nsK 3 ns ns ns usK*CO2 3 ns ns ns ns* means significant (p <0.05)us means non significant (p > 0.05)99Figure 4.1 Effects of CO2 enrichment and K+ supplyon starch concentration in leaves borne on main stems(a, b), leaves borne on the lower part of branches (c, d),and leaves borne on the upper part of branches (e, f) ofchrysanthemum plants, summer 1991.Legend for figures a, b, c and dK+ concentration: 2.50 mMol L-1 ( )K+ concentration: 3.75 mMol L-1 ( )K+ concentration: 5.00 mMol L-1 ( )K+ concentration: 6.25 mMol L-1 ( )Legend for figures e and fK+ concentration:^2.50 mMol L-1K+ concentration:^3.75 mMol L-1K+ concentration:^5.00 mMol L-1K+ concentration:^6.25 mMol L-11^1 :7***t/ / 15629100Figure 4.1aStarch Main Stem LeavesSummer 1991. CO2 350.....................14^21^28^35^42^49rune (days)Figure 4.1c5Starch Lower LeavesSummer 1991. CO2 35014^21^28^35^42^49Tune (days)Figure 4.1eStarch Upper Leaves of BranchesSummer 1991.  CO2 350Dais after treatmentFigure 4.1 bStarch Main Stem LeavesSummer 1991. CO2 1400Tyne (days)Figure 4.1dStarch Lower LeavesSummer 1991. CO2 1400'rune (days)Figure 4.1fStarch Upper Leaves of BranchesSummer 1 991. CO2 1 40028^ 56D90 after treatment101the starch concentration in CO2 enriched plants, while in unenriched plants,increasing K supply tended to produce larger starch concentrations at the beginningbut reduced starch later on. CO2 enrichment significantly increased the starchconcentration in leaves borne on the upper part of branches (Fig. 4.1e, 4.1f). Ksupply or time did not have a significant effect on these leaves.4.3.2 Effects of CO2 Enrichment and K supply on the Inorganic Nutrient Status ofMain Stem Leaves4.3.2.1 Total Leaf Contents of NutrientsAnalysis of variance of the total content of inorganic nutrients in leavesborne on main stems (Table 4.2) did not indicate significant effects (p < 0.05) ofCO2 enrichment or any interaction of CO2 enrichment effects with time.Increasing K supply significantly diminished total leaf contents of P (-17%) and Mn(-35%) (Fig. 4.2a, 4.2b). The effects of time were occasionally significant. Therewas one significant effect of blocks and occasional significance of the effects ofblock interactions. The effects of the interactions of CO2 enrichment with K supply,K supply with time, and CO2 with K and time were seldom significant.Analysis of variance of nine different ratios amongst total content of cationsin leaves borne on main stems (Table 4.3) determined significant effects (p < 0.05)of CO2 treatments on the ratio of Mn to Zn, and significant effects of K supply ontwo ratios. Interactions of the effects of CO2 with time, or K supply with time, orblocks with time were occasionally significant. The effects of time, and the102Table 4.2 Summary of statistical significance, totalcontent of elements in main stem leaves.Total Content of Inorganic Nutrients, Main Stem LeavesSource df N P K Ca Mg Mn Fe B Zn Cu AlBlock 3 ns ns ns ns ns ns ns ns ast 1 ns as us ns ns ns ns ns ns nsBlock*t 3 as ns ns as ns ns * ITS * nsCO2 1 ITS ITS ns ns ns ns ns ns ns ns nsCO2*t 1 ns as ns ns ns ITS as ns ns ns ITSBlock*CO2*t 6 * ITS ITS ns ITS ns ns * ns *K 1 ns * ns ns ns * ns ns ITS ns ITSK*t 1 ns ns ns * ITS ns ns ITS * nsK*CO2 1 ns ns ns ITS ns ns as ITS ns nst*K*CO2 1 ns as ns ns ns ns as * ITS ITS ns* means significant (p <0.05)ns means non significant (p > 0.05)Figure 4.2aPhosphorusMain Stem LeavesDays d trearnertFigure 4.2bManganeseMain Stem LeavesDais or reamersFigure 4.2 Effects of CO2 enrichment and K supply on total contents of P(a), Mn (b) in main stem leaves.K( mMol L-1) \ CO2(AL L-1)2.506.25This legend will be used in figures 4.2 to 4.6.Mn:ZnMain Stem Leaves 34ao-2 6-1 81.4-1 00.6 7 56Deis al seamenFigure 4.3 Effects of CO2 enrichment and K supply on the ratio betweentotal contents of Mn and Zn in main stem leaves. Legend as on figure 4.2.350 1400.2cc104Table 43 Summary of statistical significance, ratiosbetween total content of cations in main stem leaves.Ratios: Total Content of Cations, Main Stem LeavesSourceK:CaK:MgCa:MgMn:ZnMn:FeMn:CuZn:FeZn:CuFe:CuBlock 3 ns ns ns ns ns ns nst 1 ns ns ns ns ns ns ns ns nsBlock*t 3 * ns * ns ns * ns * *CO2 1 ns ns ns * ns ns ns ns nsCO2*t 1 ns us ns ns ns ns ns ns nsBlock*CO2* t 6 ns ns ns ns ns ns ns * *K 1 ns ns ns * ns * us ns nsK*t 1 ns ns ns ns ns ns ns ns nsK*CO2 1 ns ns ns ns ns ns ns ns nst*K*CO2 1 us ns ns ns ns ns ns ns ns* means significant (p < 0.05)ns means non significant (p > 0.05)105interactions between CO2 enrichment and K supply, and among CO2 enrichmentand K supply with time were not significant.CO2 enrichment significantly decreased the ratio between total leaf contentsof Mn and Zn (Fig. 4.3). Increasing K supply decreased the ratios between totalleaf contents of Mn to Zn, and Mn to Cu. Starch-Corrected Concentrations of NutrientsOnly one significant (p < 0.05) effect was detected on the starch-correcteddry mass of leaves borne on main stems, WLNs (Table 4.4). Comparison of thisvariation with LA and WL indicated that WLNs is the more appropriate forassessment of nutrient concentration in those leaves; i.e. WLNs provides a stablebasis for expressing nutritional status because WLNs was seldom affected byexperimental sources of variation.Analysis of variance of the starch-corrected concentration of inorganic nutrients inleaves borne on main stems revealed significant (p < 0.05) effects of CO2enrichment on four elements (Table 4.5). K supply had significant effects on fiveelements, including P and Mn which also had their total contents affected. Therewas one significant effect of the interaction of CO2 with time, and four of theinteraction of K supply with time. Occasional changes were induced by time andthe interaction of CO2 enrichment with K supply over time, while block effects andblock interactions were significant in many cases.Block variability can be attributed mainly to three factors: (1) differencesbetween CO2 enrichment chambers; (2) differences between plant groups which106Table 4.4 Summary of statistical significance, growthmeasures (loge) of main stem leaves.loge Growth Measures, Main Stem LeavesSource df LA VVL WI-NS WstarchBlock 3 ns ns *t 1 ns ns ns nsBlock*t 3 ns * * nsCO2 1 ns ns ns *CO2*t 1 ns ns ns nsBlock*CO2*t 6 * * ns *K 1 ns ns ns *K*t 1 * * ns nsK*CO2 1 ns ns ns nst*K*CO2 1 ns ns ns ** means significant (p <0.05)ns means non significant (p > 0.05)Table 4.5 Summary of statistical significance, starch-corrected concentration of nutrients in main stem leaves.Starch-Corrected Concentration, Main Stem LeavesSource df N P K Ca Mg Mn Fe B Zn Cu AlBlock 3 * ns ns * * *t 1 ns ns ns ns ns ns ns ns ns nsBlock*t 3 * * * ns * ns * * * * *CO2 1 ns ns * * ns * ns ns ns ns *CO2*t 1 ns ns ns ns ns * ns ns ns ns nsBlock*CO2*t6 * * * * * ns ns ns ns * nsK 1 * ns ns us * ns ns * * nsK*t 1 * * ns us ns * us ns ns ns *K*CO2 1 ns ns ns us ns ns ns ns ns ns nst*K*CO2 1 ns ns ns ns * ns ns ns ns ns ns* means significant (p < 0.05)ns means non significant (p > 0.05)107started treatments at different times and possibly had differences in the initial plantnutritional status; and (3) variability in the tissue analysis.CO2 enrichment significantly reduced the starch-corrected concentration ofK (-25%), Ca (-22%), Mn (-30%) and Al (-26%) (Fig. 4.4, 4.5). CO2 enrichmentproduced a consistent pattern of reduction in the starch-corrected concentrations ofall other nutrients, the only exception being Zn at the first time. Mn concentrationdecreased under CO2 enrichment, and such reduction was particularly large after 56days of treatments (Fig. 4.5a).Increasing K supply significantly diminished leaf concentrations of N (-14%),P (-25%), Mn (-35%), Zn (-3%) and Cu (-5%). Incremental K supply also reducedstarch-corrected concentration of most elements with two exceptions: Zn at low CO2the second time of measurement, and Cu in most treatments. Starch-correctedconcentrations of N, P, Mn and Al increased over time in plants grown with low Ksupply, but decreased with increasing K supply. Mn concentration increased overtime in plants under low K supply but decreased in plants grown under high Ksupply.Analysis of variance of 9 different ratios amongst the starch-correctedconcentration of cations in leaves borne on main stems determined one significant(p < 0.05) effect of CO2 enrichment (on the Mn to Zn ratio), and two significanteffects of K supply (Table 4.6). There was no significant interaction between CO2enrichment and K supply. Other effects and interactions were seldom significant.CO2 enrichment significantly decreased the ratio of Mn to Zn (Fig. 4.6). IncreasingK supply decreased the ratios Mn to Zn, and Mn to Cu.° 3.0132.1 2.6• 20--.., 0.6.c7^56Days of treatment4.64.0- 3.6,-,.ce 3.0co0• 4.4aNitrogenMain Stem LeavesFigure 4.4bPhosphorusMain Stem Leaves7^66Days of treatmentFigure 4.4cPotassiumMain Stem Leaves2al 0.53 0.40.33ae 4.4dCalciumMain Stem Leaves7^56Days of treatment7^56Days of treatmentFigure 4.4 Effects of CO2 enrichment and K supply on the starch-correctedconcentration of N (a), P (b), K (c) and Ca (d) in main stem leaves. Legendas on figure 4.2.109Figure 4.5aManganeseMain Stem Leaves7^56Days of treatmentFigure 4.5cZincMain Stem LeavesFigure 4.5bCopperMain Stem Leaves7^56Days of treatmentFigure 4.5dAluminumMain Stem Leaves7^56^ 7^56Days of treatment Days of treatmentFigure 4.5 Effects of CO2 enrichment and K supply on the starch-correctedconcentrations of Mn (a), Cu (b), Zn (c) and Al (d) in main stem leaves.Legend as on figure 4.2.110Table 4.6 Summary of statistical significance, ratiosbetween starch-corrected concentration of cations inmain stem leaves.Ratios: Starch-Corrected Concentration of Cations, Main Stem LeavesSource dfK:CaK:MgMg:CaMn:ZnMn:FeMn:CuZn:FeZn:CuFe:CuBlock 3 ns ns * ns ns ns ns nst 1 ns ns ns ns ns ns ns ns nsBlock*t 3 * ns ns ns ns * ns * *CO2 1 ns ns ns * ns ns ns ns nsCO2*t 1 * ns ns ns ns ns ns ns nsBlock*CO2st 6 ns ns ns ns ns ns ns * *K 1 ns ns ns ns * ns ns nsK*t 1 ns ns ns ns ns ns ns ns nsK*CO2 1 ns ns ns ns ns ns ns ns nst*K*CO2 1 ns ns ns ns ns ns ns ns ns* means significant (p <0.05)ns means non significant (p > 0.05)111Figure 4.6 Effects of CO2 enrichment and K supply on the ratio between thestarch-corrected concentrations of Mn and Zn in main stem leaves. Legendas on figure 4.2.1124.3.2.3 Nutrient Concentrations in Main Stem Leaves (Thy Mass Basis)Analyses of variance of the concentration of inorganic nutrients on a drymass basis in leaves borne on main stems revealed significant (p < 0.05) effects ofCO2 enrichment on seven elements, out of 11 tested. K supply had significanteffects on four elements (Table 4.7). There was no significant effects of theinteraction of CO2 enrichment with K supply.4.3.3 Effects of CO2 Enrichment and K supply on the Inorganic Nutrient Status ofLeaves Borne on the Lower Part of Branches4.3.3.1 Total Leaf Contents of NutrientsAnalyses of variance indicated that CO2 enrichment did not induce anysignificant (p < 0.05) changes on either leaf content of nutrients (Table 4.8), or onratios between the leaf content of cations in leaves borne on the lower part ofbranches (Table 4.9). K supply had significant effects on the leaf content of sixnutrients and on most ratios between total leaf content of cations. The interactionbetween the effects of CO 2 enrichment and K supply was not significant in any case.No time response was tested because only leaves harvested at one time (28 days)were analyzed.Increasing K supply significantly reduced total leaf contents of N, P, Na, Feand B (Fig. 4.7). Increases in K supply up to the second level decreased total leafcontent of Zn, but further increases increased leaf content of Zn (Fig. 4.7c) Effectsof CO2 enrichment were variable: of 44 cases evaluated (11 elements at 4 times)CO2 enrichment increased leaf content of elements 21 times and reduced their leaf113Table 4.7 Summary of statistical significance,concentration (wfi/WL) of elements in main stemleaves.Concentration (wn:VVL), Main Stem LeavesSource df N P K Ca Mg Mn Fe B Zn Cu AlBlock 3 * * * ns ns * * *t 1 ns ns ns * ns ns ns ns ns ns nsBlock*t 3 * * * ns * ns * * ns *CO2 1 * * * * ns ns ns *CO2*t 1 ns ns ns ns ns * ns ns ns *B1ock*CO2*t6 * * * * * ns ns ns ns ns nsK 1 * * ns ns ns * ns ns * ns nsK*t 1 ns ns ns ns ns ns ns nsK*CO2 1 ns ns ns ns ns ns ns ns ns ns nst*K*CO2 1 ns * ns ns ns ns ns ns ns ns ns* means significant (p <0.05)ns means non significant (p > 0.05)114Table 4.8 Summary of statistical significance, totalcontent of elements in leaves borne on the lower part ofbranches.Total Content, Leaves of BranchesSource df N P K Ca Mg Mn B Zn Cu NaBlock 3 * ns ns * ns ns *CO2 1 ns ns ns ns ns ns ns ns ns ns nsBlock*CO2 3 ns ns * ns ns ns ns * ns us nsK 3 * * ns ns ns ns * * ns *K*CO2 3 ns ns ns ns ns us ns ns ns us ns* means significant (p <0.05)ns means non significant (p > 0.05)Table 4.9 Summary of statistical significance, ratiosbetween total contents of cations in leaves borne on thelower part of branches.Ratios: Total Content of Cations, Leaves of BranchesSourcedlK:CaK:MgK:NaCa:MgCa:NaMg:NoMn:Zn^Mn:CuMn:Fe^Zn:FeZn:CuFe:CuBlock 3 * ns * ns * * us us * * *CO2 1 ns ns us ns ns us us us ns us us usBlock*CO2 3 * * us ns ns us us ns ns us * *K 3 ns * * * * * * ns ns *K*CO2 3 ns ns us ns us us ns ns ns us us us* means significant (p <0.05)us means non significant (p > 0.05)FigSodiumLower Leaves of Branches0. 4.7a^ Figure 4.7bNitrogen^ PhosphorusLower Leaves of Branches Lower Leaves of Branches250^375^&CO^6.25^ 250^375^500^6.25K supply (mMol L-I) K sum./ (mMol L-1)Figure 4.7c^ Figure 4.7dFigure 4.7eBoronLower Leaves of Branches250^3.75^508^6.25K stwiy (rnMol L-1)250^3.75^5.00^6.25K suFIN (rnMcd L-1)Figure 4.7 Effects of CO2 enrichment and K supply on leaf contents of N (a),P (b), Zn (c), Fe (d), B (e) and Na (f) in leaves borne on the lower part ofbranches. CO2 concentrations, 350AL L-1^1400AL L-1 (^).116content 23 times With micronutrients a pattern of lower leaf content in CO2enriched plants with low K supply, and higher leaf content in unenriched plants withhigh K supply (except for B at 5 mMol L-1 K) was observed. Starch-Corrected Concentrations of Nutrients in LeavesAnalysis of variance of the loge transformed values of growth measures ofleaves borne on the lower part of branches (Table 4.10) indicated that WLNs was assuitable as WL for the evaluation of nutrient concentration in these leaves.Therefore, WLNs was used again for assessment of concentrations.Analysis of variance of the starch-corrected nutrient concentrations in leavesborne on the lower part of branches (Table 4.11) indicated only one significant (p <0.05) effect of CO2 enrichment, while K supply induced significant changes onseveral elements.The starch-corrected concentration of N was reduced by CO2 enrichment,although the effect was not large quantitatively (P = 0.041) (Fig. 4.8a). Increasing Ksupply significantly reduced the starch-corrected concentrations of N (-18%) and Na(-46%) and increased concentrations of K (28%) and Mg (11%) (Fig. 4.8).Increasing K supply up to the second level decreased the concentrations of Mn andZn (Fig. 4.9), but further increments in K supply increased their leaf concentrations.Analyses of variance of 12 different ratios amongst starch-correctedconcentrations of cations in leaves borne on the lower part of branches (Table 4.12),did not detected significant (p < 0.05) effects of CO2 enrichment. K supply inducedsignificant changes in several ratios. There was no significant interaction betweenthe effects of CO2 enrichment and K supply.117Table 4.10 Summary of statistical significance growthmeasures (loge) of leaves borne on the lower part ofbranches, summer 1991.Growth Measures (loge), Leaves of BranchesSource^df^LA^VVL^Virl,Ns^WstarchBlock^3^ns^* nsCO2 1 ns ns^ns^nsB1ock*CO2^3^*^ns ns nsK^3 *^*^*K*CO2^3 ns ns ** means significant (p < 0.05)ns means non significant (p > 0.05)Table 4.11 Summary of statistical significance,concentration (wn/VVLNs) of elements in leaves borne onthe lower part of branches.Starch-Corrected Concentration, Leaves of BranchesSource df^N P^K^Ca^Mg Fe^B^Cu^Mn Zn NaBlock^3^ns^ns^*^ns^*^ns^*^*CO2^1^*^ns^ns^ns^ns^ns^ns^ns^ns^ns^nsB1ock*CO2 3^ns^as^*^ns^ns^ns^ns^ns^ns^*^nsK^3^*^ns^*^ns^*^*^*^ns^* *K*CO2^3^ns^ns^ns^ns^ns^ns^ns^ns^*^ns^ns* means significant (p < 0.05)ns means non significant (p > 0.05)MagnesiumLower Leaves of Branches0.380.36 -0.34-0.32-0.30-0.28-0.26-0.24-0.22^,50^3.75^5.02. 0^6.25K supply (mMol L-1)Figure 4.8d118Figure 4.8aNitrogenLower Leaves of Branches2.50^3.75^5.00^6.25K supply (mMol L-1)Figure 4.8cPotassiumLower Leaves of Branches3.75^5.00^6.25K supply (mMol L-1)Figure 4.8bSodiumLower Leaves of Branches4000- 3500.cwoo-1-0§i 2500-c2000-E.a 1 500-1 000250^3.75^5.00^6.25K supply (mMol L-1)^5.80^5.60-- 5.40-,--.c 5.20 -•C1-35.00-§I4.60-4.40-o 4.20-4.00-^3. 80 ^Figure 4.8 Effects of CO2 enrichment and K supply on the starch-correctedconcentrations of N (a), K (b), Mg (c) and Na (d) in leaves borne on thelower part Of branches.CO2 concentrations, 350 ,uL Li (^1400 AL L-1 ( ^ ')-ManganeseLower Leaves of Branches1 8016060-119Figure 4.9a40 2.50^3.75^5.00^6.25K supply (mMol L-1)Figure 4.9cZincLower Leaves of BranchesFigure 4.9b2.50^3;5^5.60^6.25K supply (mMol L-1)Figure 4.9dBoronLower Leaves of Branches135 1 30-1 25-1 20-11 5-11 0-1 05-1 00-95-90-85IronLower Leaves of Branches2.50^3.75^5.00^6.25^2.50^3;5^5.00^6.25K supply (mMol L-1) K supply (mMol L-1)Figure 4.9 Effects of CO2 enrichment and K supply on the starch-correctedconcentrations of Mn (a), Fe (b), Zn (c), Cu (d) and B (e) in leaves borne onthe lower part of branches.CO2 concentrations, 350 p L L-1 ( • ), 1400AL L-1 ( ----+---- ).120Table 4.12 Summary of statistical significance of ratiosbetween concentration (wn/VVLNs) of cations in leavesborne on the lower part of branches.Ratios, Starch-Corrected Concentrations, Leaves of BranchesSource dlK:CaK:MgK:NaCa:MgCa:NaMg:No Mn:Zn^Zn:CuMn:Fe^Mn:Cu Zn:FeFe:CuBlock 3 ns * * * ns ns *Block* 1 ns ns ns ns ns ns ns ns ns ns ns nsCO2*t 3 * * ns ns ns ns ns ns ns * ns *K 3 ns * * * * * ns * ns * *K*CO2 3 ns ns ns us ns ns ns ns ns ns ns ns* means significant (p < 0.05)ns means non significant (p > 0.05)121Analyses of variance of nutrient concentrations expressed on a dry mass basis(wn:WL) revealed significant (p < 0.05) effects of CO2 enrichment only on N(Table 4.13). K supply had significant effects on six elements: N, P, Ca, Cu, Zn andNa. The interaction of CO2 enrichment with K supply had a significant effect on Zn.4.3.4 Effects of CO2 Enrichment and K Supply on Leaf ChlorosisDuring the 1990 experiment, only a slight leaf chlorosis in a few leaves borneon main stems was observed after three weeks of CO2 enrichment. Such chlorosissoon disappeared.In the experiments performed in 1991, leaf chlorosis was observed after oneweek of CO2 enrichment. Paler foliage and interveinal chlorosis on the uppermostleaves of main stems was noticed after one week of treatment. Chlorosis increasedwith increasing K supply and CO2 enrichment, and there were no symptoms underthe lowest K levels of unenriched plants. New leaves sprouting on branches ofaffected plants were chlorotic and had greener tips. Leaf chlorosis receded towardsthe bottom of the main stem. It also decreased towards the top of branches ofaffected plants, until it was not present in the uppermost leaves of branches. Themost affected leaves were on plants grown at the highest level of K supply, whichwere yellowish in color with greener tips and veins. However, leaves borne on thelower part of branches of affected plants remained chlorotic, and the foliage of theupper part was of a paler green color.Chlorophyll concentrations of leaves reflected visual observations of plants.Analysis of variance of leaf chlorophyll concentrations of the two experimentscarried out during 1991 (Table 4.14), revealed significant (p < 0.05) effects of time122Table 4.13 Summary of statistical significance,concentration (wn/WL) of elements in leaves borne onthe lower part of branches.Concentration (wn:WL), Leaves of BranchesSource df N P K Ca Mg Fe B Cu Mn Zn NaBlock 3 * ns ns ns ns *CO2 1 ns ns ns ns ns ns ns ns ns nsBlock*CO2 3 ns * ns ns ns ns ns ns ns nsK 3 * * ns * ns ns ns ns * *K* CO2 3 ns ns ns ns ns ns ns ns ns * ns* means significant (p < 0.05)ns means non significant (p > 0.05)Table 4.14 Summary of statistical significance for leafchlorophyll concentration, summer and fall 1991.Chlorophyll ConcentrationsSummer FallLeaf type df Main df Lower df Upper dl Main df Lower df UpperBlock 3 * 3 ns 3 ns 2 2 ns 2t 4 ns 2 * 1 * 6 * 3 * 1 *Bloclot 12 * 6 3 * 12 ns 6 ns 2 nsCO2 1 ns 1 * 1 ns 1 * 1 * 1 *t*CO2 4 * 2 ns 1 * 6 ns 3 * 1 nsBlock*CO2*t 15 * 9 * 6 ns 14 ns 8 ns 4 nsK 3 * 3 * 3 * 3 * 3 * 3 *t*K 12 6 * 3 18 9 3 nsCO2*K 3 ns 3 3 * 3 ns 3 * 3 *t*CO2*K 12 ns 6 3 ns 18 ns 9 ns 3 ns* means significant (p < 0.05)ns means non significant (p > 0.05)123and K supply, as well as significant effects of the interactions of time with K supply,and CO2 enrichment with K supply in most leaf types. There were some effects ofCO2 enrichment, and some effects of the CO2 interaction with time.Chlorophyll concentration in leaves borne on main stems of plants grownduring the summer of 1991 was not significantly affected by either CO2 enrichmentor the interaction of CO2 enrichment with K supply. Increasing K significantlyreduced chlorophyll concentration of such leaves (Fig. 4.10a, 4.10b), and differencesincreased over time, as the chlorophyll concentration in CO2-enriched plantsdecreased. In leaves borne on the lower part of branches, the chlorophyllconcentration increased slightly over time (Fig. 4.10c, 4.10d). The effects of CO2enrichment and K supply were both significant. Interactions of the effects of CO2enrichment with K supply, CO2 enrichment with time, and time with K supply werealso significant. There was a significant decrease in chlorophyll concentration withincreasing K supply. CO2 enrichment initially reduced chlorophyll concentration,thus increasing differences induced by K supply, but such differences decreasedtowards the end of the experiment. Chlorophyll concentration in leaves borne onthe upper portions of branches followed similar trends to leaves borne on the lowerpart of branches (Fig. 4.10e, 4.10f), although the effect of CO2 enrichment was notsignificant. Chlorophyll concentration of these leaves was higher than that of theleaves borne on the lower part of branches, but lower than the concentration inleaves borne on the main stems.In plants grown during the fall of 1991, CO2 enrichment reduced chlorophyllconcentration in all types of leaves, with the exception of leaves borne on mainstems (Fig. 4.11). Increasing K supply reduced chlorophyll concentration of leaves.124Figure 4.10 Effects of CO2 enrichment and K supplyon chlorophyll concentration in leaves of main stems (a,b), the lower part of branches (c, d), and the upper partof branches (e, f), summer 1991.Legend for figures a, b, c and dK concentration:K concentration:K concentration:K concentration:2.50 mMol L-13.75 mMol L-15.00 mMol L-16.25 mMol L-1Legend for figures e and fK concentration:^2.50 mMol L-1K concentration:^3.75 mMol L-1K concentration:^5.00 mMol L-1K concentration:^6.25 mMol L-1This legend will be used for figures 4.10 and 4.11.'0125Figure 4.10aChlorophyll Main StemSummer 1991. CO2 35014^21^28^35^42Time (days)Figure 4.10oChlorophyll Lower LeavesSummer 1991. CO2 350Time (days)Figure 4.10eChlorophyll Upper LeavesSummer 1991.   CO2 35028^56Dap after treatmentFigure 4.10bChlorophyll Main StemSummer 1991. CO2 140024^35^42^4911me (days)Figure 4.10dChlorophyll Lower LeavesSummer 1991. CO2 1400in14^21^28^35^42^49Time (days)Figure 4.10fChlorophyll Upper LeavesSummer 1991. CO2 1400Days atter treadmerta_126Figure 4.11aChlorophyll Main Stem LeavesFall 1991. CO2 35014Ilme (days)Figure 4.11cChlorophyll Lower LeavesFall 1991. CO2350Figure 4.11bChlorophyll Main Stem LeavesFall 1991. CO2 1400Figure 4.11dChlorophyll Lower LeavesFa111991. CO2 1400Figure 4.11eChlorophyll Upper LeavesFall 1991. CO2 350Figure 4.11fChlorophyll Upper LeavesFall 1991. CO2 140020^24Dais atter treatment20^ 24Dais atter treatmentFigure 4.11 Effects of CO2 enrichment and K supply on chlorophyllconcentration in leaves of main stems (a, b), the lower (c, d) and the upperpart of branches (e, f), fall 1991. Legend as on figure 4.10.127Leaf chlorophyll concentration significantly increased over time, excepting on leavesborne on the lower part of branches, in which chlorophyll remained constant.Leaves borne on main stems of plants grown with low K supply became particularlydark green, and in some cases presented necrotic tips. On leaves borne on theupper part of branches, the increase in chlorophyll concentration over time wasgreater on unenriched than on CO2-enriched plants, particularly at higher levels ofK supply.It was also noticed that plants grown under CO2 enrichment and lower levelsof K supply had thicker stems, and developed a purplish coloration.4.3.5 Nutritional Status in Relation to CO2-Induced Leaf ChlorosisIncreasing K supply induced leaf chlorosis. Plants grown at the higher levelsof K supply (5.0 and 6.25 mMol L-1) presented paler foliage and had slightinterveinal chlorosis on the uppermost leaves of their main stems after one week oftreatment. CO2 enrichment increased the symptoms. In leaves of CO2 enrichedplants grown with high K supply, the starch-corrected concentrations of N, K, Ca,Mn and Fe were below "normal" recommended levels (Winsor and Adams 1987).Furthermore, at that time Fe concentration was reduced below the critical level inthe worst case; CO2 enrichment increased Zn concentrations; and reduced the ratiosK to Ca and Mn to Zn.The four lowest leaves of branches were the most affected by thecombination of high K supply and CO2 enrichment. Starch-corrected concentrationof N decreased with CO2 enrichment and increasing K supply, but even the lowest Nlevel was within the "normal" range. Leaf K was over the maximum recommended128on all samples, and it increased with greater K supply, although CO2 enrichmenttended to reduce leaf K concentration. Leaf Na concentration increased with CO2enrichment, particularly at the highest and lowest levels of K supply. The ratio of Kto Na decreased with CO2 enrichment particularly at the highest level of K supply,indicating that reductions in Na uptake were not linearly related with increases in Ksupply. In addition, Zn and Mn concentrations were 38% and 20% higher in theseleaves than in unenriched leaves at the same K levels, although they were below the"excessive" range. Such concentrations, however, were 308% and % higher thanconcentrations of those elements in the healthier (greener and larger) leavesobserved in the experiment.4.4 DISCUSSION4.4.1 Effects of CO2 Enrichment and K Supply on the Concentration ofPhotosynthates in Leaves4.4.1.1 Concentrations of SugarsThe significant effect of blocks on the sugar concentrations in leaves mayhave been a consequence of the variability due to differences in the time ofsampling during the day, since whole blocks were sampled at the time of harvest.Sugar concentration in leaves is dynamic, and large changes occur during the day(Huber et al. 1985). Such variability might have been the cause of conflicting resultsin the sugar concentration in leaves reported in other CO2 enrichment studies.Some reports indicate no change in soluble sugar concentrations (Mauney et al.1978, Finn and Brun 1982; Nafziger and Koller 1976). Others report a slightincrease in sucrose concentration in soybean leaves caused by CO2 enrichment(Huber et al. 1984, Hrubec et al. 1985). Still others found lower sugar concentrationin enriched soybean leaves (Hofstra and Hesketh 1975). Van Berkel (1984) founda close correlation between the concentration of sugars and the expression of leafchlorosis in Gerbera, but this was not observed in the present study. Concentration of StarchIt appears that main stem leaves served initially as a source of carbohydratesfor development, as indicated by the reduction of starch concentration early in thisexperiment (Summer 1991, results). Towards the end of the experiment, however,129130starch concentration in such leaves increased in CO2 enriched plants. In leavesborne on the lower or upper parts of branches, starch concentrations usually wereslightly increased by CO2 enrichment, although concentrations were lower than inleaves borne on main stems. CO2 enrichment has been found to increase leafstarch concentration in many plant species (Dons 1988, Ehret and Jolliffe 1985b, Liu1990, Peet et cii. 1986, Spencer and Bowes 1986, Sasek et al. 1985). In plants underCO2 enrichment the export of increased levels of metabolite may be limited by thecapacity of the transport system (Ehret and Jolliffe 1985a, Liu 1990) or by reducedsink demand (Ehret and Jolliffe 1985a).Previous studies have found correlations of starch accumulation with theonset of leaf abnormalities under CO2 enrichment (Cave et cii. 1981, Ehret andJolliffe 1985a, Hesketh et al. 1971, Liu 1990, Madsen 1984). In this study, the onsetof leaf chlorosis, and the lowest chlorophyll content recorded, coincided with higherstarch concentrations in leaves borne on the lower part of branches. However,starch concentrations found in such leaves were too low to be presumed as theparticular cause of chlorosis. Furthermore, starch concentrations in these leaveswere less than half of those concentrations found in leaves borne on main stems,which were less chlorotic.Starch accumulation has been correlated with photosynthetic dysfunction andleaf chlorosis induced by CO2 enrichment (Cave et al. 1981, Ehret and Jolliffe 1985a,Goudriaan and Ruiter 1985, Madsen 1974). It was suggested that the formation oflarge and unusually shaped starch grains within the chloroplasts may damage thegrana or its structure (Cave et al. 1981, Madsen 1974). It has also been postulated131that starch accumulation may affect the leaf by changing the C to N ratio whichseems to act as a control for many physiological plant processes (Kuehny et al. 1991).However, leaf chlorosis in Gerbera was not correlated with starchconcentration, and symptoms of starch excess (leaf roll) have not been observed intomato when chlorophyll breakdown occurs (Van Berkel 1984).The effect of K supply on leaf starch concentration was not consistent,perhaps due to the confounding factors of the nutrient solutions. It was noticed,however, that increasing K supply resulted in increased leaf starch concentration inmost cases, with the exception of leaves borne on the main stems of unenrichedplants. The largest starch concentration occurred in leaves borne on main stems ofCO2 enriched plants grown with the highest level of K supply. It is usual that as theratio of leaf K to leaf Na increases, leaf starch decreases, in part as a consequenceof the reduced efficiency of Na in the activation of starch synthase (Marschner1986). Total Protein ConcentrationNeither CO2 enrichment nor K supply significantly influenced proteinconcentrations, as reported previously by others (Havelka et al. 1984, Liu 1990,Porter and Grodzinski 1984, Wong 1979).1324.4.2 Effects of CO2 Enrichment on the Inorganic Nutrient Status in Leaves4.4.2.1 Effects of CO2Enrichment on Leaf Content of Inorganic NutrientsLeaf nutrient contents are an indication of the quantities of nutrientsassimilated into leaves through the growth process, a measure related with demand,supply and the ability of the plant to take up and mobilize nutrients. Observationsof contents of inorganic nutrients in leaves did not show major effects of CO2enrichment on the presence of nutrients within plants. There were no significantinteractions of CO2 enrichment with K supply, implying that under the conditions ofthe experiment there was no major change in nutrient uptake of the CO2 enrichedplants. This differs from reports where plants under CO2 enrichment increasedtheir nutrient uptake (Cure et al. 1988a, 1988b, Yelle et al. 1987).Changes in the ratio between leaf contents of Mn and Zn showed a subtleeffect of CO2 enrichment. This effect varied with time, leaf portion and nutritionalcondition. Effects of CO2Enrichment on Leaf Concentration of Inorganic NutrientsTissue concentrations of nutrients reflect many things, including thedeposition of nutrients into tissue and source-sink relationships. They are ameasure of the ability of the plant to maintain nutrient in the amounts required forphysiological processes. They also depend on processes of nutrient uptake andredistribution. Additional carbon fixed under CO2 enrichment usually increases thedry mass of leaf tissue relative to inorganic elements, reducing their relativeconcentration, wn:WL (Eng et al. 1985, Kuehny et al. 1991, Liu 1990, Porter and133Grodzinski 1989, Tripp et al. 1991). In this study also, the concentrations (wn:WL)of most nutrients in leaves borne on main stems were significantly reduced by CO2enrichment, with the exception of those of Fe, Zn, and B. However evaluation ofstarch-corrected concentrations (wn:WLNs) revealed significant effects only on fourelements (K, Ca, Mn and Al), although there was a consistent tendency towardsdecreased starch-corrected concentrations of nutrients under CO2 enrichment. Asimilar pattern in the differences between assessment methods was found by Kuehnyet al. (1991). A reduction of nutrient concentration not associated with starch couldresult from leaf mass increases in excess of the increase in inorganic nutrient uptake(Kuehny et al. 1991), and might be important in plant function.In dwarf beans (Liu 1990) and wild radish (Koch et at 1988), reduction in theconcentration (wn:WL) of N and K in older leaves was considered a sign ofremobilization of such elements to new tissues. CO2 enrichment also reduced thestarch-corrected concentration of K in tomato leaves (Tripp et al. 1991), and in olderleaves of chrysanthemum plants grown during winter time (830 iz MOi M-2 S-1 PPFD)(Kuehny et al. 1991). Reductions in the concentration (wn:WL) of N and K alsowere detected in the older leaves in the present study, and the starch-correctedconcentration of K in leaves borne of main stems was also significantly reduced. Kconcentrations in leaves borne on the lower part of branches were much higherrelative to concentrations in leaves borne on main stems. Kuehny et al. (1991) alsodetermined K accumulation in the uppermost leaves of CO2 enrichedchrysanthemum plants grown under winter time conditions, although no change wasobserved in plants grown during the spring.134In this study Mn, Zn and Fe were reduced by CO2 enrichment, but the effectwas only significant for Mn. Kuehny et al. (1991) also reported reduction in thestarch-corrected concentrations of Mn and Zn, increases in Fe, and no differences inCa in older chrysanthemum leaves.CO2 enrichment induced a decline in concentration of Ca, Zn and Fe inleaves of chrysanthemum plants cv. 'Dramatic'. However, leaf concentrations(wn:WL) of most elements, with the exception of P and Cu, were reduced when highpressure sodium lamps were used in conjunction with CO2 enrichment(Eng et al. 1985). In a different study on chrysanthemum plants cv. 'Fiesta', CO2enrichment reduced foliar concentrations of most nutrients on a dry mass basisduring the spring, with 1400 pMOI IM2 s-1 PPFD. With 830 A I1101 11r2 s4 PPFDduring the winter, however, only N concentration was reduced (Kuehny et al. 1991).Increasing irradiance also increased starch accumulation in older leaves andinduced leaf injury in unenriched chrysanthemum plants (Woltz 1969, Woltz andEngelhard 1971). In the present study, nutrient solution concentrations (e.g. N, P,K, Ca, Mg) were lower than those used in the studies mentioned above, andirradiance levels were much lower as well (< 200 A mol 111-2 s-1 PPFD). These twofactors, plus the confounding factors observed in the nutrient solutions, e.g. pHdrifts, increasing amounts of NaOH and Na2SO4, and the beneficial effect of Na,(Chapter 3, Section 3.4.1) perhaps limited plant responses to CO2 enrichment,particularly in younger leaves.CO2 enrichment induced large changes in ratios between major cations onlyat certain levels of K supply, suggesting links between the K supply and CO2enrichment. However, these changes were not consistent at all levels of K supply,135thus, neither CO2 enrichment nor the interaction of CO2 enrichment with K supplywere significant in most cases. This lack of consistency in response may have beencaused by the confounding factors observed in the nutrient solutions.The size and direction of changes in concentrations of inorganic nutrientsvaried according to the leaf type. Two factors probably created these differingtrends: (1) main stem leaves were mature at the initiation of treatments, while lowerleaves of branches were subjected to treatments since the time of sprouting. Thus,the former tended to be net exporters of some elements while branch leaves weremostly sinks. (2) nutrient solutions containing high levels of K restricted dry matteraccumulation and leaf area expansion of leaves borne on the lower part of branches(Chapter 3), interfering with plant responses to CO2 enrichment. Such an effectpossibly influenced CO2-induced effects on nutrient concentration in leaves.Frequent monitoring of foliar nutrient concentration is particularlyrecommended for greenhouse crops grown under CO2 enrichment (Hicklenton1988). The basis for assessment of tissue analysis is crucial, as it may lead to wrongconclusions due to changes in the leaf dry mass induced by starch accumulation.Starch-corrected concentration of nutrients in leaves is a more sensible indicator ofnutritional changes in CO2 enriched plants, although it requires a starch analysis tobe done in parallel to tissue nutrient analysis. Therefore, further studies to setcritical levels using leaves normally taken for analysis would be important, asrecommended by Hocking and Meyer (1991) for CO2 enriched wheat.1364.4.3 Effects of K Supply on the Leaf Concentration of Inorganic NutrientsK supply in the nutrient solutions affected leaf concentration of mostelements in leaves borne on both main stems and branches. On leaves borne onmain stems, increasing K supply reduced concentration of most elements. Onleaves borne on the lower part of branches, increasing K supply reduced N and Naconcentrations, and increased concentrations of the other cations. Higher cationaccumulation (K, Ca and Mg) induced by increasing K supply was also determinedin rose leaves from the upper part of shoots. That effect was associated with anincreased NO3 uptake induced by increasing K supply (Woodson and Boodley1982). In my studies, cation accumulation induced by K supply was not as large asdetermined by Woodson and Boodley (1982). The smaller increase observed in thepresent study may be a result of: inhibition of NO3- transport from roots to shoots byCO2 enrichment (Yelle et al. 1987), differences between plant species or leaf agestudied, effects of Na and acetic acid present in the nutrient solutions.Substitution capacity of K by Na is increased in plant species that havegreater ability to translocate and accumulate more Na in shoots when grown underlow-K stress (Figdore et at. 1989). Such capacity was observed in leaves borne onthe lower part of branches, where Na concentration increased with decreasing Ksupply. However, Na is also known to compete with other cations, thus causingrelated physiological disorders (Marschner 1986).Low levels of Na could be used in the nutrition of plants under CO2enrichment with several advantages. The most important is the ability of Na toincrease carbon mobilization within the plant, thus reducing starch accumulation in137leaves. In addition, Na stimulates growth, improves water use efficiency, and allowsthe use of less expensive fertilizers.4.4.4 Effects of CO2 Enrichment and K Nutrition on Leaf ChlorosisIn the first experiment, leaf chlorosis was not strong, in spite of having theplants subjected to up to 1800 A L L-1 CO2. In the 1991 experiments, CO2-inducedchlorosis was consistently observed. The difference in results between the twoyears may be a consequence of a combination of factors. (1) Irradiance was slightlyincreased by about 60 A mol m-2 s-1 PPFD in the last two experiments, and increasedirradiance has been reported to enhance leaf abnormalities under CO2 enrichment(Ehret and Jolliffe 1985a, Heij and Uffelen 1983); (2) Higher concentration in thenutrient supply was used in the first experiment compared to the 1991 experiments.Reduced nutrient concentration in the supply increased chlorosis of the lower leavesof CO2-enriched Begonia (Mortensen 1987) and bean plants (Liu 1990). (3) In thefirst experiment, the growth retardant B-nine (daminozide) was used, which delayssenescence in plant organs (Baker 1983).CO2 enrichment reduced leaf chlorophyll concentration, as has beenreported in previous studies (Patterson and Flint 1982, Wulff and Strain 1982). Onaffected plants, the uppermost leaves borne on main stems and leaves borne on thelower part of branches showed some degree of chlorosis, but new leaves sproutingon branches became progressively greener up to the top. The pattern of suchsymptoms was in some ways similar to symptoms reported by Peet eta!. (1986), whoobserved lighter green foliage, interveinal chlorosis and yellow margins in all leavesof monoecious cucumber. But many other reports indicate damage in older leaves.138Hence, chlorosis and necrosis of primary leaves, with later extension to the firsttrifoliate leaves, was observed on dwarf beans (Ehret and Jolliffe 1985a, Liu 1990).Chlorosis of older leaves was reported also on CO2-enriched Begonia (Mortensen1987) and Gerbera (Van Berkel 1984), while deformation of older leaves wasobserved in tomato (Tripp et al. 1991). The inconsistency of CO2 induced leafinjury symptoms has been recognized (Ehret and Jolliffe 1985a, Van Berkel 1984),and is probably a consequence of CO2 enrichment acting as a secondary factorenhancing leaf injury of plants.CO2 enrichment increased plant growth despite its adverse effect inincreasing the incidence of leaf abnormalities, a result obtained previously(Patterson and Flint 1982, Jolliffe and Ehret 1984, Liu 1990, Tripp et at. 1991). Inother CO2 enrichment studies, however, limited nutrient supply interfered with plantgrowth responses and also with the expression of leaf injury of Begonia (Mortensen1987).Although in 1991 CO2 enrichment increased chlorosis, the main cause ofsuch symptoms was the effect of increasing K supply. In studies done on soybeansand two weed species, nutrient effects were also greater and more consistent thanCO2 enrichment effects (Patterson and Flint 1982). In this study chrysanthemumplants were adversely affected by increasing K supply, which conflicts with reportswhere increasing nutrient concentration reduced leaf abnormalities (Mortensen1987, Liu 1990, Patterson and Flint 1982, Wulff and Strain 1982). At the sametime, older leaves borne on main stems of plants grown with low K supply becameprogressively darker in color (also confirmed by chlorophyll readings) and somenecrotic tips were observed, resembling K deficiency symptoms (Marschner 1986).139K supply reduced the leaf concentration of many nutrients, therefore it islikely that changes in more than one element were involved in the chlorosis. Itseems that acetic acid was main cause of the negative impact of increasing K supply,since acetic acid may be phytotoxic (Harper and Lynch 1980, Lynch 1978).Variable pH drifts of the nutrient solutions, which also were larger with increasingK supply, may have also cause detrimental effects. Larger pH drifts in the higher Ktreatments may have been the result of the uptake of acetic acid in solution.4.4.5 Nutritional Status in Relation to CO2 -induced Leaf ChlorosisCO2 enrichment increased leaf chlorosis on both leaves borne on main stemsand leaves borne on the lower part of branches. In spite of this, significant effectsof CO2 enrichment on concentration of inorganic nutrients in leaves borne on thelower part of branches were not detected in most cases, with the exception of aslight reduction in N. Unfortunately, tissue analysis of leaves borne on the lowerpart of branches reflect the status after 28 days of enrichment, and not that at thetime of the onset of leaf injury.CO2 enrichment reduced the starch-corrected concentrations of K, Ca andMn in leaves borne on main stems. Furthermore, in the most affected plantsstarch-corrected concentrations of N, K, Ca and Mn in leaves borne on main stemswere below the "normal" recommended levels, while Fe was below the critical level(Winsor and Adams 1987). Such changes, and reduction in the ratio Mn to Zn,were probably related to the onset of chlorosis. The observations suggest that CO2enrichment induced a sudden reduction in the concentration of several nutrients,observed as soon as seven days after treatments in leaves borne on the main stems.140Such reductions may in turn have affected development of the newer leaves,enhancing their chlorosis.Altered nutrient relations induced by CO2 enrichment appear to beassociated with the onset of leaf chlorosis. Consistent reductions in the nutrientconcentrations were observed in leaves borne on the main stems of CO2 enrichedplants, although no nutritional changes were detected in the most chlorotic leaves ofbranches. It seems that the main role of CO2 in enhancing chlorosis was via thereduction of Mn concentration. This conclusion is supported by: the CO2-induceddecrease in Mn concentration and the alteration of the ratio of Mn to Zn; theappearance of interveinal chlorosis symptoms in leaves; the pattern of distributionof symptoms in plants, in uppermost leaves of main stem and lower leaves ofbranches, with paler green foliage on the other leaves of branches, which are similarto Mn-deficiency symptoms described for chrysanthemums (Winsor and Adams1987, Roorda et al. 1980); and the role of Mn as a semimobile element in plants(Marschner 1986), which is also related to the distribution of symptoms in the plant.Symptoms resembling Mn deficiency have been described under CO2 enrichment(Tripp et al. 1991). In other studies, CO2 enrichment decreased Mn concentrationin chrysanthemums, particularly under high irradiance (Eng et al. 1985, Kuehny et al.1991).It is also possible that other nutrients were involved in the onset of leafchlorosis. For example, leaf K and N were also decreased by CO2 enrichment.Reduce leaf K has been associated with leaf injury in CO2 enriched tomato (Trippet al. 1991), and dwarf bean (Liu 1990) plants. In the latter study, N reductionswere also associated with leaf chlorosis. Fe deficiency could also be involved in the141chlorosis, since it was found at very low levels at the highest K levels, although at theintermediate levels it was not low. In other studies, however, CO2 enrichmentincreased Fe concentration (Kuehny et at. 1991). It is possible that, in this study, thereduction of Fe concentration at the highest level of K supply was related to Feprecipitation induced by shifts in the pH of the nutrient solution.Limitations in interpretation of this experiment arose from several sourcesincluding: the toxic effect of acetic acid, the inability to control more closely pHchanges in the nutrient supply; the pooling chlorotic leaves with healthier ones fortissue analysis; differences in the timing of sampling the different leaf portions;small sample size; large variability of analytical results; and possible non-nutritionalactions of treatments which were not monitored. Furthermore, in this experiment,irradiance and nutrient supply concentrations were low, and the expression of CO2-induced leaf abnormalities is enhanced by high irradiance (Eng et al. 1985, Ehretand Jolliffe 1985a, Kuehny et al. 1991) and high concentration of the nutrient supply(Mortensen 1987).142CHAPTER 5 GENERAL DISCUSSIONThese studies have indicated that CO2 treatment effects on the mineralrelations of plants are important in regulating plant responses to high CO2,confirming results of previous studies (Liu 1990, Sionit et al. 1981, Tripp et al. 1991,Wong 1979). It was also shown that the supply of inorganic nutrients influencedgrowth responses and nutrient relations of CO2 enriched plants, as reportedpreviously by others (Goudriaan and Ruiter 1985, Liu 1990, Mortensen 1987,Patterson and Flint 1982).CO2 enrichment increased plant dry mass, a common response ofchrysanthemum plants (Eng et al. 1985, Gislerod and Nelson, Hughes and Cockshull1971a, Mortensen 1986), although CO2 concentration above 1200 A L L-1 did notproduce a significant additional increase in dry mass, as indicated by Mortensen andMoe (1983a). Increases in leaf dry mass, particularly of leaves borne on branches,accounted for most of the increased growth in CO2 enriched plants. Thus, most ofthe increase in leaf mass was associated with leaf area increases, as found byKriedemann and Wong (1978). Increasing CO2 did not change flower mass, asreported by most authors (Goldsberry 1986, Hicklenton 1988, Nelson 1991),although others have found increased flower mass when CO2 enrichment wasapplied with supplemental irradiance (Eng et al. 1985, Hughes and Cockshull1971a). Time to flowering was little affected, as reported previously by others(Hicklenton 1988, Mortensen 1986, Nelson 1991). This probably occurred becauseCO2 enrichment was started after the plants were pinched, since earliness to flowerin chrysanthemums is related to increases in early vegetative growth (Eng et al. 1985,Mortensen 1986, Nelson 1991).143Plant growth responses to CO2 enrichment were influenced by nutrientsupply. Absolute growth rates were higher in the first experiment than in thesecond one, probably as a result of higher nutrient supply, since N, P, and Caconcentrations were 35% to 50% higher than in the second experiment. In thesecond experiment plant growth, responses to CO2 enrichment were also affected byK supply. These observations support the idea that nutrient supply may affect plantgrowth responses more strongly than does CO2 enrichment, as established byPatterson and Flint (1982).Increasing K supply tended to be detrimental for plants. Other componentsof the nutrient solutions may have been responsible for the detrimental effects.Acetic acid added for pH adjustment was probably the main adverse factor, since ithas been found toxic to plants (Chandrasekaran and Yoshida 1973, Harper andLynch 1980). In addition, differences as large as 1.3 pH units among the twoextreme K treatments were recorded between daily pH adjustments, with thehighest K treatment exhibiting the greatest daily drift. Such pH differences mayhave created dissimilarities in the solubility or uptake of nutrients.In contrast, plants supplied with low K (50% the recommended) grew well,introducing another confounding factor for the K supply treatments. This probablywas a consequence of a beneficial effect of Na, induced by a relatively high Na to Kratio. The substitution of K by Na, and the growth stimulation by Na in plants havebeen documented (Figdore et cd. 1989, James 1988, Marschner 1986, Marschner1971). Na can replace K in older leaves thus releasing K for functions where it isessential. Growth stimulation by Na is induced mainly by its effect on cellexpansion which may increase leaf area production. Na also improves the water144balance and water use efficiency of plants as compared to those supplied only withK (Marschner 1986). Further understanding of the interactions between K, Na andCO2 enrichment is necessary, particularly dealing with the effects of Na supply ondifferent plant species or cultivars.Starch concentrations increased in leaves of CO2 enriched plants, as has beenwidely reported (Ehret and Jolliffe 1985a, Havelka et al. 1984a, Peet et al. 1986,Scheidegger and Nosberger). In contrast, increasing the Na to K ratio in thenutrient supply decreased leaf starch concentrations, such an effec could result, atleast in part, of the lower efficiency of Na in activating starch synthase (Marschner1986). This response of plants to Na supply could be beneficial for CO2 enrichedplants, because low levels of Na supply would allow an increase in growth byreducing the possible harmful effects of starch accumulation under CO2 enrichment.CO2 enrichment modified the nutrient relations of chrysanthemum plants.Although the total content of inorganic nutrients in leaves borne on main stems wasnot altered, CO2 enrichment decreased the starch-corrected concentrations ofnutrients in leaves borne on main stems. The lack of change in total leaf nutrientcontents implies that neither uptake nor translocation of inorganic nutrients weremodified by CO2 enrichment, contrasting with a previous study which recordedincreased uptake of NO3" in CO2 enriched tomato (Yelle et at. 1987), although theydid not detect changes in leaf concentrations.Low nutrient concentrations have been reported previously in leaves of CO2enriched chrysanthemums (Eng et at. 1985, Kuehny et al. 1991), and have beenobserved in other plant species (Goudriaan and Ruiter 1985, Koch et at. 1988, Liu1990, Porter and Grodzinski 1989, Tripp et at. 1991). The present study, and the145studies mentioned above, indicate that although CO2 enrichment induces a generalreduction in the tissue concentration of nutrients, its effect is greater on somenutrients than on others. Such variation seems to depend on other factors such assink strength (Tripp et at 1991), nutrient supply (Liu 1990), or other environmentalconditions (Eng et at. 1985, Kuehny et at. 1991). Based on this general reduction intissue concentrations, several researchers have suggested a 'nutrient dilution' effectin CO2 enriched plants, mainly due to increased leaf starch (Eng et al. 1985, Kuehnyet at. 1991, Porter and Grodzinski 1989). Since starch is essentially a storageproduct, such a dilution would not be as important in plant function as alterations inleaf nutrient concentrations independent from the leaf starch mass. In addition toincreased starch, lower tissue concentrations of nutrients could result fromincreased shoot growth in excess of increases in nutrient uptake (Porter andGrodzinski 1984), limited translocation (Mortensen 1987, Tripp et at. 1991), orincreased remobilization of nutrients (Koch et at. 1988, Liu 1990, Tripp et at. 1991).The two former explanations may be closer to the evolution of symptoms observedin these studies.In the present studies, the depression in the leaf concentrations of K, Ca, Mnand Al were not removed when the data was expressed on a starch-free dry massbasis. In CO2 enriched chrysanthemums, Kuehny et at. (1991) reported reductionsin the starch-corrected concentrations of Mn, Zn and Cu, and Eng et at. (1985)found reductions in most nutrients (not corrected for starch), but Mn was reducedonly when lighting was applied for long photoperiods. Lower starch-correctedconcentration of K was reported in tomato, due to increased remobilization fromolder leaves to fruits (Tripp et at. 1991). Also, increased remobilization of N and K146from older leaves to young tissues was reported in dwarf beans (Liu 1990) and wildradish (Koch et al. 1988). In this study, higher concentrations of N, K, P and Mn inleaves borne on branches and decreased concentrations of such elements in leavesborne on main stems, suggest that they were remobilized. Due to differences in thetiming of sampling for leaves borne on main stems and leaves borne on the lowerpart of branches, however, it is not possible to confirm such a statement.K supply induced larger changes than CO2 enrichment in the starch-corrected concentrations of most nutrients. Increasing K supply reduced the leafconcentration of many nutrients, but only the total leaf content of P and Mn werechanged by K supply. No interactions were detected between K supply and CO2enrichment in either case. The effect of K supply and the lack of interaction foundin this experiment are not conclusive, however, due to the confounding factorsmentioned above.Increasing K supply induced leaf chlorosis. Exposure of plants to CO2enrichment enhanced the leaf chlorosis. CO2-induced leaf injury in several plantspecies has been associated with leaf starch increases (Cave et al. 1981, Ehret andJolliffe 1985a, Wulff and Strain 1982). In this study, however, leaf starch did notappear to be related with leaf abnormalities, which agrees with reports on tomatoand Gerbera (Van Berkel 1984) and tomato (Tripp et al. 1991). Chlorophyllbreakdown induced by increased leaf temperature, which could be enhanced byCO2-induced stomatal closure and transpiration reductions, was suggested as thecause of chlorosis in tomato and Gerbera. High irradiance increased chlorosis,particularly when it occurred after dull weather and sources of water within theplant were limited (Van Berkel 1984). Primary bean leaves that developed injury147during CO2 enrichment also exhibited the largest increase in stomatal diffusiveresistance. However, such a change was observed at the same time as the onset ofleaf injury, and it was difficult to judge whether stomatal changes were the cause orthe consequence of leaf injury (Ehret and Jolliffe 1985a). In the presentexperiments, however, excessive leaf temperatures were unlikely to have occurredbecause of the low irradiance levels used.In contrast, altered nutrient relations appear to be associated with the onsetof leaf chlorosis. Consistent reductions in the nutrient concentrations wereobserved in leaves borne on the main stems of CO2 enriched plants, although nonutritional changes were detected in the most chlorotic leaves of branches. Itseems that the main role of CO2 in enhancing chlorosis was via the reduction in Mnconcentration. This conclusion is supported by several factors: the decrease in Mnconcentration and the alteration of the ratio of Mn to Zn induced by CO2enrichment; the appearance of symptoms in leaves (interveinal chlorosis); thepattern of distribution of symptoms in plants (uppermost leaves of main stem andlower leaves of branches, with somewhat paler green foliage on the other leaves ofbranches) which are similar to Mn-deficiency symptoms in chrysanthemum plants(Winsor and Adams 1987, Roorda et al. 1980); and the role of Mn as a semimobileelement in plants (Marschner 1986), which is also related to symptom distribution inthe plant. It is also possible, however, that other nutrients were involved in theobserved disorder. For example, K and N were also decreased by CO2 enrichment,and have been associated with leaf injury in other studies (Tripp et at. 1991, Liu1990). Non-nutritional effects of treatments, which were not studied, are anotherpossible cause of chlorosis.148It appears that CO2 enrichment has a secondary role in the nutrient balancein plants. Thus, nutrient relations of CO2 enriched plants are modified by thecombination of effects of CO2 enrichment, nutrient supply, internal factors such assource sink relationships, and other environmental factors (Kuehny et at. 1991, Liu1990, Luxmore et ca 1986, Mortensen 1987, Yelle et at. 1987). According to theoverall growing conditions, a wide variety of symptoms and growth responses mayoccur on CO2 enriched plants. It seems that during the first days of CO2enrichment the nutrient requirements of young tissues were increased. Plantsunder certain conditions were not able to fulfill such needs, thus causing the onset ofleaf chlorosis. Later, leaf chlorosis faded towards the top of the branches as plantsappeared to reach a balance.Limitations in the nutrient supply may reduce growth responses to CO2enrichment, and under certain conditions may cause leaf injuries reducing qualityand yield of greenhouse crops (Van Berkel 1984, Hicklenton 1988). If the CO2levels in the earth's atmosphere continue to rise, reference values for interpretationof tissue analysis must be adjusted for other crops as well (Hocking and Meyer1991). To take full advantage of CO2 enrichment, inorganic nutrition has to beadjusted appropriately to plant species or cultivars, growing conditions and culturalpractices. Tissue analysis is advised for monitoring the nutrient status of CO2enriched plants (Hicklenton 1988, Nelson 1991). However, reference values usedfor evaluation of tissue concentrations must be adjusted properly in respect to thoseof unenriched plants (Hocking and Meyer 1991) otherwise they may be misleading.CO2 enrichment affects the mineral relations of plants, and responses of CO2enriched plants depend on nutrient supply. However, relatively few studies have149explored such topics. To explain CO2-induced leaf injuries, several hypotheses havebeen advanced, but their cause remains unclear. Further studies are needed tounderstand plant function under CO2 enrichment, to define the causes of leaf injury,and thus accordingly define parameters for management of plant nutrition underCO2 enrichment and different environmental conditions.150CHAPTER 6 CONCLUSIONS1. Growth responses observed in these studies were similar to thosereported in other CO2 enriched plant species, including chrysanthemum CO2enrichment increased plant dry mass, mainly as a consequence of greater leaf mass.However, CO2 concentrations above 1200 A I., L-1 did not induce significantadditional growth. Only minor changes occurred in flower mass, and time toblooming was not shortened by increasing CO2 levels.2. Growth responses to CO2 enrichment were associated with nutrientsupply. Differences in the absolute growth rates of the first and second experimentsindicate that under CO2 enrichment greater growth increases occurred with highernutrient supply, when no other limiting factors were present.^In the latterexperiment increasing K supply tended to reduce plant growth. Such an effect mayhave limited the response of plants to CO2 enrichment and interfered with theinteractions of CO2 enrichment with K supply. The detrimental effects caused byincreasing K supply were perhaps a consequence of increasing acetic acid, which isknown to have phytotoxic effects. However, variable drifts in the pH of the nutrientsolutions may have also added to the problem. There was also a beneficialresponse to Na when K supply was low, further confounding the analysis of growthresponses to K supply.3. CO2 enrichment increased leaf concentrations of starch, as has beenreported in previous studies. In contrast, leaf starch concentration decreased as theNa to K ratio in the nutrient supply increased. This effect could be beneficial forplants grown under high CO2 to lessen the harmful effects of starch accumulation inleaves.1514. CO2 enrichment did not alter significantly the total content of nutrients inleaves, implying that nutrient demand did not change. However, CO2 enrichmentreduced the concentrations (we: WL) of most nutrients in leaves borne on mainstems, reflecting changes in the allocation of nutrients within the plant. Leafnutrient concentrations were reduced in part by greater leaf starch content, an effectthat has been called 'nutrient dilution' (Kuehny et al. 1991). Beyond such dilution,CO2 enrichment significantly reduced the starch-corrected concentrations of K, Caand Mn, and altered the Mn to Zn ratio in leaves borne on main stems. CO2enrichment also caused a slight decrease in the starch-corrected concentration of Nin leaves borne on the lower part of branches.5. K supply induced stronger effects than CO2 enrichment on leaf nutrientconcentrations. This effect influenced the onset of leaf chlorosis and plant growth.Hence, increasing K supply induced leaf chlorosis on the uppermost leaves of mainstems and on leaves borne on the lower part of branches.6. Exposure of plants to CO2 enrichment significantly enhanced leafchlorosis. Leaf abnormalities under CO2 enrichment have been associatedpreviously with leaf starch accumulation, increased leaf temperatures, or nutrientimbalances. In these studies, however, leaf chlorosis did not appear to be relatedwith leaf starch accumulation or with increased leaf temperatures. Instead,alterations in the nutrient relations of the plant appeared to be the cause. CO2enrichment decreased starch-corrected concentrations of most nutrients in leavesborne on main stems, but only minor effects were detected in leaves borne on thelower part of branches, although these leaves were more chlorotic at the time ofmeasurement. However, the appearance of symptoms and the pattern of chlorosis152distribution in the plant, coupled with decreased Mn concentration and an alteredMn to Zn ratio, suggest that Mn deficiency played a role in the onset of leafchlorosis. Nevertheless, other nutrients, such as K and N were also decreased byCO2 enrichment, and could have contributed to leaf chlorosis. Non-nutritionaleffects of treatments, which were not studied, are another possible cause ofchlorosis.7. It appears that during the first days of CO2 enrichment the nutrientrequirements of young tissues increased. Plants under certain conditions were notable to fulfill such needs, thus causing the onset of leaf chlorosis. Later, leafchlorosis faded towards the top of the branches as plants appeared to reach abalance.8. These results support the conclusion that CO2 enrichment alters thenutrient relations of plants. They also indicate that supply of inorganic nutrientsdetermines plant responses to CO2 enrichment. Higher nutrient supply may favorgrowth of CO2 enriched plants, as long as the plant is able to balance uptake,demand, and source sink relationships.153LITERATURE CITEDAbeles, F. B., 1973. Ethylene in plant biology. 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J., 1987. Effect of atmospheric CO2concentration and root zone temperature on growth, mineral nutrition, andnitrate reductase activity of greenhouse tomato. J. Amer. Soc. Hort. Sci.112:1036-1040.Zeevart, J. A. D., 1979. Regulation of assimilate partitioning. In: Partitioning ofassimilates. Summary reports of a workshop held at Michigan StateUniversity, East Lansing, MI. Am. Soc. Plant Physiol. Rockville, MD.pp. 14-17.166APPENDIX 1Appendix 1. Composition of the nutrient solutions,used during the summer of 1990,Nutrient Solutions, Summer 1990,ug L-1 lig L-1Week N P K Ca Mg Mn Fe Zn Cu B Mo1 to 3 200 77 200 50 25.0^0.03 0.40 0.02 0.03 0.06 0.0084 190 89 206 50 25.0^0.02 0.40 0.02 0.03 0.05 0.0075 to 6 200 39 200 50 25.0^0.01 3.00 0.01 0.02 0.03 0.0047 to 8 100 16 178 40 25.0^1.00 3.00 0.25 0.10 0.10 0.0109 NO FERTILIZERFertilizer Used as sources:20- 20 - 2013 - 0 - 4411 - 15 - 29Ca(NO3)MgSO4Iron chelate (Sequestrene 138)167APPENDIX 2Appendix 2. Composition of nutrient solutions,summer and fall of 1991. Nutrient Solutions, Summer and Fall 1991Macroelements Microelementsmg L-1 mmol L-1 pg L-1 ,u mol L-1NO3 - N 136.6 9.8 Fe 3.40 0.7NH4 - N 25.1 1.8 Mn 1.00 18.2P 31.0 1.0 B 0.22 20.0Ca 111.0 2.8 Zn 0.20 3.0Mg 24.0 1.0 Cu 0.03 0.5S 95.2 3.0 Mo 0.05 0.5Na 913 4.0K concentration in nutrient solutions:mMol L-1^: 2.50 3.75^5.00 6.25mg L-1^:^98 147 196 245Na sources,^g I:1 of nutrient solution:NaOH 0.00 2.64^5.28 7.94Na2S 04^14.10 9.42 4.70 0.00Acetic acid concentration (added to adjust the initial pH of solutions):mM *^ 0.0 0.78^1.57 3.13* (Approximate initial concentration. Variable amounts were used for daily adjustments.)168Appendix 2.^(continued)^Fertilizers used to preparenutrient solutions, summer and fall 1991.Fertilizers added to nutrient solutions (g L -1)K Supply (Mmol L -1) 2.50 3.75 5.00 6.25FertilizerAmmonium nitrate 0.1476 0.1476 0.1476 0.1476Calcium nitrate 0.5840 0.5840 0.5840 0.5840Potassium nitrate 0.1609 0.1609 0.1609 0.1609Monopotassium sulphate 0.1360 0.1360 0.1360 0.1360Potassium sulphate 0.0000 0.1176 0.2352 03528Magnesium sulfate 0.2436 0.2436 0.2436 0.2436Sodium sulphate 0.2819 0.1882 0.0941 0.0000Sodium hydroxide 0.0000 0.0528 0.1057 0.1588Iron chelate (Sequestrene 138) 0.0340 0.0340 0.0340 0.0340Manganese chelate 0.0083 0.0083 0.0083 0.0083Solubor 0.0011 0.0011 0.0011 0.0011Zinc chelate 0.0014 0.0014 0.0014 0.0014Copper sulphate 0.0001 0.0001 0.0001 0.0001Ammonium molybdate 0.0001 0.0001 0.0001 0.0001169APPENDIX 3Appendix 3.1 Concentration of major elements(mg L-1) in the nutrient solution just after preparation(new), and three days old (old). Results of analysisperformed at PSM, Richmond, B. C.pH, EC (mmhos cm4) and Mean Concentration (mg L-1) of Major Elements in Nutrient Solution,Summer 1991K (mg L-1) Sol. Type pH EC NH4-N NO3-N P K Ca Mg Na98 new 5.5 1.84 30 140 33.8 90 93.5 23.5 93.5147 new 5.2 1.94 31 141 34.2 139 92.8 23.17 88.8196 new 5.4 1.77 30 136 34.2 185 98.5 22.3 81.8245 new 5.4 2.14 30 135 33.5 236 95.5 22.3 78.298 old 6.1 1.82 27 141 31.5 87 100.0 22.0 116.5147 old 6.8 1.82 24 107 30.5 135 90.0 21.5 79.5196 old 6.3 1.88 19 83 30.5 186 85.0 23.0 54.0245 old 7.2 1.81 12 20 20.0 255 75.0 19.0 34.0170Appendix 3.2 Concentration of minor elements (mg L -1)in the nutrient solution just after preparation (new),and three days old (old). Results of analysis performedat PSM, Richmond, B. C.Mean Concentration (mg L-1) of Minor Elements in Nutrient Solution, Summer 1991 K (mg L-1) Sol. Type Cu Zn Fe Mn B98 new 0.24 0.74 3.37 1.06 <0.05147 new 0.24 0.72 3.18 1.03 <0.05196 new 0.25 0.80 2.97 1.03 <0.05245 new 0.26 0.80 2.85 1.02 <0.0598 old 0.27 2.05 2.15 0.99 <0.05147 old 0.22 2.50 1.50 0.46 <0.05196 old 0.24 3.78 1.75 0.92 <0.05245 old 0.23 2.71 1.05 0.66 <0.05171APPENDIX 4Appendix 4 Means and standard errors of the logetransformed growth variables, summer 1990.CO2 350 p L L-1Var.*Meant (days) 0SE Mean12SE Mean22SE Mean39SE Mean53SE0.44 0.05 0.84 0.07 1.26 0.06 2.02 0.11 2.20 0.06LA 5.39 0.05 6.28 0.06 6.73 0.05 7.19 0.06 7.14 0.04VVL -023 0.05 0.36 0.07 0.78 0.06 131 0.08 1.38 0.04LAm 5.27 0.05 5.31 0.06 5.40 0.04 5.41 0.06 5.35 0.06LAb 3.17 0.12 5.79 0.07 6.42 0.06 7.00 0.07 6.95 0.05VVLm -0.33 0.06 -0.39 0.07 -0.30 0.06 -0.23 0.07 -0.20 0.07WLb -2.72 0.13 -0.29 0.08 036 0.08 1.07 0.09 1.15 0.054.96 0.03 5.43 0.03 5.47 0.03 5.17 0.05 4.93 0.05SLA 5.63 0.03 5.91 0.02 5.95 0.03 5.88 0.02 5.76 0.04LWR -0.67 0.02 -0.48 0.02 -0.48 0.01 -0.70 0.03 -0.82 0.02Ws -1.48 0.05 -1.23 0.11 -1.01 0.04 -0.61 0.11 -0.42 0.06Wb -3.19 0.10 -2.02 0.10 -129 0.08 0.09 0.15 0.38 0.08Wr -0.74 0.06 -0.79 0.07 -0.38 0.09 0.42 0.11 0.45 0.07Wy -0.49 0.49 -1.86 0.24 0.31 0.09* Units of measurement are in the list of symbols172Appendix 4 (continued) Means and standard errors ofthe loge transformed growth variables, summer 1990.CO2 1200 p L L-1MeanVar.* \ t (days) 0SE Mean12SE Mean22SE Mean39SE Mean53SE0.38 0.07 0.90 0.05 1.62 0.04 2.28 0.06 2.63 0.04LA 5.37 0.05 6.34 0.04 6.98 0.03 7.32 0.04 7.46 0.03WL -0.30 0.07 0.39 0.05 1.10 0.04 1.47 0.04 1.67 0.02LAm 5.24 0.06 5.32 0.04 5.45 0.05 5.35 0.09 5.46 0.04LAb 3.25 0.08 5.88 0.05 6.74 0.04 7.16 0.04 7.31 0.03WLm -0.42 0.09 -0.42 0.04 -0.26 0.06 -0.31 0.10 -0.14 0.05VVLb -2.55 0.11 -0.20 0.07 0.79 0.05 1.28 0.05 1.49 0.034.99 0.04 5.44 0.02 5.36 0.03 5.04 0.04 4.83 0.03SLA 5.68 0.06 5.94 0.02 5.89 0.03 5.85 0.02 5.79 0.02LVVR -0.68 0.03 -0.51 0.02 -0.53 0.02 -0.81 0.03 -0.96 0.01Ws -1.42 0.08 -1.34 0.05 -0.84 0.08 -0.37 0.04 -0.11 0.06Wb -3.14 0.12 -1.82 0.07 -0.65 0.08 0.51 0.08 0.99 0.05Wr -0.85 0.11 -0.60 0.08 0.09 0.07 0.77 0.07 1.04 0.03WY -2.17 0.83 -1.67 0.13 0.73 0.09* Units of measurement are in the list of symbols173Appendix 4 (continued) Means and standard errors ofthe loge transformed growth variables, summer 1990.CO2 1800 p L I:1MeanVar.* \ t (days) 0SE Mean12SE Mean22SE Mean39SE Mean53SEW 0.43 0.10 1.00 0.03 1.72 0.07 2.29 0.06 2.60 0.04LA 5.43 0.07 6.40 0.04 7.00 0.04 7.31 0.04 7.39 0.02WL -0.18 0.08 0.52 0.04 1.20 0.05 1.52 0.05 1.65 0.02LAm 5.32 0.07 5.43 0.05 5.55 0.04 5.42 0.08 5.48 0.05LAb 3.11 0.09 5.91 0.03 6.74 0.05 7.14 0.03 7.23 0.02WLm -0.28 0.08 -0.26 0.06 -0.10 0.05 -0.20 0.09 -0.13 0.05WI,b -2.63 0.17 -0.10 0.04 0.89 0.06 1.31 0.04 1.47 0.03F 5.00 0.04 5.40 0.03 5.28 0.05 5.02 0.05 4.79 0.03SLA 5.60 0.02 5.88 0.02 5.80 0.05 5.79 0.03 5.74 0.02LWR -0.61 0.03 -0.47 0.02 -0.52 0.03 -0.78 0.03 -0.95 0.03Ws -1.47 0.13 -1.14 0.06 -0.69 0.06 -0.32 0.07 -0.16 0.04Wb -3.13 0.09 -1.66 0.05 -0.63 0.16 051 0.08 0.92 0.06Wr -0.88 0.15 -0.70 0.06 0.16 0.10 0.72 0.08 1.06 0.06WY -1.55 0.76 -1.62 0.07 0.68 0.06* Units of measurement are in the list of symbols174APPENDIX 5Appendix 5 Means and standard errors of the logetransformed growth variables, summer 1991.CO2: 350 pL L-1K:^2.50 mMol L-1Var.*^t (days) 0Mean SE7Mean SE14Mean SE28Mean SE56Mean SE-1.10 0.12 -0.32 0.11 -0.15 0.03 0.94 0.08 1.53 0.05LA 4.17 0.04 4.70 0.08 5.04 0.04 6.44 0.05 6.96 0.04WL -1.43 0.06 -0.66 0.10 -0.44 0.03 0.63 0.09 1.06 0.03LAm 4.17 0.04 4.70 0.08 459 0.09 4.83 0.19 4.48 0.08LAI 3.98 0.11 5.34 0.07 5.23 0.06LAu 5.74 0.13VVLm 4.43 0.06 -0.66 0.21 0.81 0.03 -0.55 0.10 -1.04 0.12WI4 -1.63 0.03 -0.75 0.04 -0.82 0.04VVLi, -0.24 0.055.27 0.02 5.02 0.06 5.19 0.09 5.50 0.05 5.44 0.15LVVR -0.33 0.06 -0.34 0.06 -0.29 0.01 -0.31 0.03 -0.47 0.02SLA 5.60 0.03 5.37 0.05 5.48 0.08 5.82 0.04 5.91 0.04SLAm 5.60 0.03 5.37 0.05 5.40 0.12 5.38 0.21 5.51 0.09SLAI 5.62 0.09 6.09 0.07 6.05 0.05SLAu 5.98 0.08Ws -2.38 0.09 -1.57 0.10 -1.55 0.04 -0.91 0.09 -0.29 0.12Wb -2.89 0.20 -0.54 0.05 1.32 0.13Wr -12.92 1.85 -9.00 2.93 -1.27 0.14 -0.20 0.16SRR 12.60 1.82 8.84 2.89 2.21 0.18 1.73 0.14-2.94 1.11Units of measurement are in the list of symbols175Appendix 5 (continued) Means and standard errors ofthe loge transformed growth variables, summer 1991.CO2K:1400p L L-12.50 mMol L-1Var.* \ t (days) 0Mean SE7Mean SE14Mean SE28Mean SE56Mean SEW -1.10 0.19 -0.10 0.09 0.03 0.12 1.05 0.03 1.81 0.13LA 4.17 0.04 4.64 0.05 4.67 0.10 6.37 0.05 6.99 0.08VVL -1.43 0.12 -0.42 0.10 -0.23 0.08 0.73 0.06 1.42 0.08LAm 4.17 0.04 4.64 0.05 4.57 0.11 4.88 0.15 4.53 0.14LAI 2.3 0.05 5.17 0.02 5.1 0.12LAu 5.70 0.04 6.67 0.04 6.75 0.04VVLm 4.43 0.06 -0.42 0.10 -0.51 0.06 -0.54 0.06 -0.94 0.12WL1 -1.74 0.16 -0.73 0.06 -0.66 0.11W14, 0.22 0.07 0.62 0.07 1.11 0.11F 5.27 0.02 4.74 0.14 4.64 0.18 5.31 0.14 5.18 0.04LVVR -0.33 0.09 -0.32 0.01 -0.26 0.05 -0.33 0.07 -0.39 0.15SLA 5.60 0.03 5.06 0.19 4.90 0.21 5.64 0.13 5.57 0.04SLAm 5.60 0.03 5.06 0.19 5.08 0.26 5.42 0.13 5.47 0.08SLAI 4.03 0.15 5.91 0.06 5.76 0.06SLAu 5.48 0.04 6.05 0.05 5.63 0.12Ws -2.38 0.09 -1.42 0.08 -1.46 0.16 -0.64 0.36 0.10Wb -2.90 0.51 0.12 0.12 1.77 0.03Wr 40.89 3.32 40.83 3.38 -0.83 0.05 0.22 0.12SRR 10.80 3.17 10.86 3.19 1.88 0.08 1.58 0.14H -4.08 0.44 -2.62 0.87 -2.09 0.62* Units of measurement are in the list of symbols176Appendix 5 (continued) Means and standard errors ofthe loge transformed growth variables, summer 1991.CO2K:350 iu L 1113.75 mMol L-1Var.* \ t (days)0Mean SE7Mean SE14Mean SE28Mean SE56Mean SEW -1.10 0.05 -0.47 0.11 -0.15 0.03 0.91 0.07 1.39 0.18LA 4.17 0.04 4.64 0.09 4.96 0.06 6.42 0.06 6.97 0.02WL -1.43 0.05 -0.83 0.02 -0.47 0.03 030 0.05 0.97 0.10LAm 4.17 0.04 4.64 0.09 4.70 0.04 4.80 0.10 4.51 0.03LAI 3.44 0.24 5.15 0.09 5.21 0.12'Au 5.68 0.14WLm -1.43 0.07 -0.83 0.06 -0.70 0.03 -0.76 0.06 4.07 0.04W14 -2.07 0.07 -0.97 0.1 -0.84 0.04VVLI, -0.49 0.13F 5.27 0.02 5.11 0.06 5.11 0.06 5.51 0.12 5.59 0.11LVVR -0.33 0.01 -0.36 0.12 -0.32 0.01 -0.41 0.11 -0.41 0.11SLA 5.60 0.03 5.47 0.07 5.44 0.06 5.92 0.03 6.00 0.05SLAm 5.60 0.03 5.47 0.07 5.40 0.07 5.56 0.10 5.58 0.06SLAI 531 0.24 6.11 0.05 6.06 0.09SLA, 6.17 0.04Ws -2.38 0.06 -1.68 0.03 -1.44 0.04 -1.12 0.14 -0.46 0.15Wb -2.87 0.19 -0.81 0.08 1.25 0.21Wr -7.86 3.23 -1.27 0.43 -1.29 0.47 -0.05 0.12SRR 2.19 0.48 1.44 0.20H -2.19 0.71* Units of measurement are in the list of symbols177Appendix 5 (continued) Means and standard errors ofthe loge transformed growth variables, summer 1991.CO2K:1400pL L'i3.75 mMol L-1Var.* \ t (days)0Mean SE7Mean SE14Mean SE28Mean SE56Mean SEW -1.10 0.05 -0.14 0.12 -0.06 0.04 0.98 0.08 1.87 0.20LA 4.17 0.04 4.54 0.02 4.85 0.05 6.37 0.05 7.05 0.04VVL -1.43 0.12 -0.46 0.07 -035 0.02 0.78 0.11 138 0.17lAm 4.17 0.04 4.54 0.02 4.59 0.05 4.84 0.08 4.57 0.04LAI 334 0.19 5.08 0.08 534 0.09LAu 5.59 0.02 6.61 0.05 6.74 0.06VVLm -2.38 0.15 -1.47 0.05 -1.45 0.08 -0.88 0.08 0.12 0.08VVLI -1.87 0.15 -0.92 0.1 -0.71 0.14WLu -0.11 0.19 0.76 0.16 1.01 0.09F 5.27 0.02 4.68 0.12 4.91 0.04 5.40 0.07 5.18 0.16LWR -0.33 0.14 -0.32 0.13 -0.29 0.01 -0.20 0.04 -0.49 0.09SLA 5.60 0.03 5.00 0.16 5.20 0.05 5.60 0.06 5.68 0.11SLAm 5.60 0.03 5.00 0.16 5.19 0.07 5.51 0.18 5.43 0.04SLAI 522 021 6.00 0.10 6.05 0.15SLAu 5.70 0.19 5.84 0.12 5.72 0.05Ws -2.38 0.04 -1.12 0.14 -0.88 0.12 -0.46 0.05 0.12 0.15Wb -234 0.08 -0.36 0.07 1.95 0.03Wr -5.55 2.12 -2.47 0.26 -0.69 0.38 0.56 0.16SRR 5.41 2.00 2.41 0.25 1.66 0.35 1.31 0.05H -3.29 0.94 -2.55 0.79 -2.17 0.65* Units of measurement are in the list of symbols178Appendix 5 (continued) Means and standard errors ofthe loge transformed growth variables, summer 1991.CO2K:350 p L L-15.00 mMol L-1Var.* \ t (days)0Mean SE7Mean SE14Mean SE28Mean SE56Mean SEW -1.10 0.09 -0.46 025 -023 0.03 0.78 0.03 1.52 0.13LA 4.17 0.04 4.64 0.04 4.91 0.09 6.27 0.13 6.95 0.06VVL -1.43 0.09 -0.83 0.28 -0.53 0.03 0.29 0.03 1.08 0.17LAm 4.17 0.04 4.64 0.04 4.63 0.09 4.77 0.13 4.52 0.16LAI 3.46 0.16 4.75 0.13 5.35 0.08LAo 5.50 0.04VVLm -1.43 0.09 -0.83 0.34 -0.69 0.03 -0.83 0.03 -1.08 0.17VVI.4 -2.49 0.07 -1.29 0.1 -0.81 0.1WLu -0.46 0.08F 5.27 0.02 5.10 0.06 5.14 0.07 5.49 0.14 5.43 0.14LVVR -0.33 0.02 -0.37 0.03 -031 0.01 -0.49 0.01 -0.44 0.04SLA 5.60 0.03 5.47 0.07 5.44 0.08 5.98 0.03 5.87 0.07SLAm 5.60 0.03 5.47 0.07 5.32 0.09 5.59 0.06 5.60 0.14SLAI 5.95 0.11 6.04 0.04 6.16 0.10SLAt, 5.97 0.04Ws -2.38 0.11 -1.64 0.15 -1.57 0.04 -1.05 0.04 -0.19 0.06Wb -3.09 0.31 -1.13 0.14 1.46 0.13Wr -5.81 3.44 -1.10 0.37 -0.45 0.13 0.69 0.14SRR 5.35 3.44 0.87 0.37 1.23 0.15 0.83 0.15H -2.75 1.08* Units of measurement are in the list of symbols179Appendix 5 (continued) Means and standard errors ofthe loge transformed growth variables, summer 1991.CO2K:1400p L L-15.00 mMol L-1Var.* \ t (days)0Mean SE7Mean SE14Mean SE28Mean SE56Mean SEW -1.10 0.03 -0.30 0.05 -0.01 0.12 0.96 0.03 1.82 0.11LA 4.17 0.04 4.44 0.06 4.85 0.09 6.36 0.03 7.01 0.05WL -1.43 0.03 -0.63 0.09 -0.31 0.07 0.64 0.03 1.30 0.13LAm 4.17 0.04 4.44 0.06 4.69 0.09 4.92 0.06 4.74 0.08LAI 2.87 0.18 5.16 0.12 5.01 0.1LAo 5.09 0.08 6.06 0.06 6.32 0.02WLm -1.43 0.03 -0.63 0.09 -0.45 0.07 -0.73 0.03 -0.71 0.12VVLI -2.35 0.15 -0.73 0.06 -0.9 0.08WL, -0.92 0.43 0.12 0.08 0.45 0.06F 5.27 0.02 4.74 0.08 4.86 0.14 5.41 0.12 5.20 0.11LWR -0.33 0.01 -0.33 0.04 -0.30 0.09 -0.32 0.01 -0.51 0.03SLA 5.60 0.03 5.06 0.12 5.16 0.17 5.72 0.07 5.71 0.03SLAm 5.60 0.03 5.06 0.12 5.15 0.17 5.65 0.07 5.45 0.06SLAI 5.22 0.10 5.89 0.09 5.91 0.05SLAu 6.00 0.36 5.94 0.04 5.87 0.06Ws -2.38 0.04 -1.60 0.06 -1.39 0.09 -0.86 0.04 0.17 0.07Wb -2.84 0.10 -0.54 0.06 1.73 0.13Wr -5.79 3.46 4.58 0.36 -4.25 3.96 0.67 0.13SRR 5.49 3.44 1.57 0.37 5.20 3.89 1.14 0.17H -3.27 0.83 -2.22 0.60 -2.62 0.82* Units of measurement are in the list of symbols180Appendix 5 (continued) Means and standard errors ofthe loge transformed growth variables, summer 1991.CO2K:350 pL L-16.25 mMol L-1Var.* \ t (days)0Mean SE7Mean SE14Mean SE28Mean SE56Mean SEW -1.10 0.16 -0.48 0.09 -0.18 0.03 0.74 0.06 1.01 0.09LA 4.17 0.04 4.57 0.08 4.75 0.06 6.18 0.04 6.43 0.05WL -1.43 0.21 -0.76 0.03 -0.49 0.03 0.28 0.07 0.54 0.05"Am 4.17 0.04 4.57 0.08 4.61 0.06 4.74 0.12 4.46 0.06LAI 2.73 0.15 4.79 0.06 4.66 0.08I-Au 5.65 0.11 5.60 0.07VVLm -1.43 0.21 -0.76 0.02 -0.62 0.04 -0.89 0.07 -1.08 0.09WI.4 -2.65 0.25 -1.26 0.1 -1.4 0.16WLu -0.20 0.07 -0.40 0.62F 5.27 0.02 5.06 0.08 4.93 0.07 5.44 0.15 5.42 0.10LVVR -0.33 0.05 -0.28 0.08 -0.31 -0.45 0.01 -0.47 0.10SLA 5.60 0.03 5.34 0.05 5.24 0.10 5.90 0.06 5.89 0.04SLAm 5.60 0.03 534 0.05 522 0.09 5.63 0.07 5.53 0.03SLAI 5.38 0.22 6.05 0.12 6.06 0.11SLAG 5.85 0.08 6.00 0.62Ws -2.38 0.07 -2.06 0.10 -1.52 0.02 -1.24 0.07 -0.64 0.05Wb -3.06 0.25 -1.38 0.17 0.83 0.06Wr -5.34 3.60 -5.46 3.56 -0.44 0.25 0.54 0.08SRR 4.86 3.58 5.28 3.58 1.18 0.15 0.47 0.15H -2.98 0.66 -3.54 0.83* Units of measurement are in the list of symbols181Appendix 5 (continued) Means and standard errors ofthe loge transformed growth variables, summer 1991.CO2K:1400p L L-16.25 mMol L-1Var.* \ t (days)0Mean SE7Mean SE14Mean SE28Mean SE56Mean SEW -1.10 0.03 -0.23 0.07 0.40 0.10 0.70 0.03 1.33 0.12LA 4.17 0.04 4.50 0.11 4.99 0.07 6.02 0.07 6.71 0.03VVL -1.43 0.03 -0.56 0.05 0.19 0.07 0.34 0.03 0.83 0.13lAin 4.17 0.04 4.50 0.11 4.82 0.06 4.85 0.04 4.52 0.11LAI 3.07 0.12 4.77 0.15 5.12 0.08LAu 6.66 0.07 6.72 0.07 0.91 0.07WLm -1.43 0.03 -0.56 0.05 0.10 0.12 -0.52 0.03 -1.03 0.13W14 -2.34 0.22 -1.21 0.12 -1.01 0.19VVLu 0.73 0.02 1.17 0.05 0.50 0.05F 5.27 0.02 4.73 0.14 4.58 0.07 5.32 0.17 5.38 0.08LVVR -0.33 0.01 -0.33 0.03 -0.22 0.08 -0.36 0.01 -0.50 0.02SLA 5.60 0.03 5.07 0.17 4.80 0.08 5.68 0.16 5.87 0.05SLAm 5.60 0.03 5.07 0.17 4.73 0.09 5.37 0.22 5.54 0.01SLAI 5.41 0.14 5.99 0.13 6.13 0.13SLA, 5.93 0.05 5.55 0.06 -0.76 0.06Ws -2.38 0.04 -1.51 0.15 -1.24 0.14 -0.99 0.04 -0.19 0.13Wb -2.55 0.18 -1.10 0.45 1.03 0.17Wr -5.60 3.51 4.70 0.32 -0.52 0.30 0.46 0.13SRR 5.37 3.43 2.10 0.27 1.22 0.13 0.87 0.21H -2.37 0.79 -2.37 0.61 -1.12 0.14* Units of measurement are in the list of symbols182APPENDIX 6Appendix 6.1 Summer 1991: Summary of analysis ofvariance, concentration of nutrients (wn:LA) in leavesborne on main stemsInorganic Nutrient Concentration (wn:LA), Main Stem LeavesSource df N P K Ca Mg Mn Fe B Zn Cu AlBlock 3 ns * 'is * 'is ns 'is ns 'is 'is 'ist 1 'is ns * ns 'is 'is ns ns 'is ns nsBlock*t 3 * * ns * ns ns * * ns * *CO2 1 ns 'is ns ns ns 'is 'is ns 'is ns nsCO2*t 1 'is 'is 'is 'is ns ns 'is ns ns 'is 'isBlock*CO2*t 6 ns ns ns 'is ns ns ns 'is 'is * nsK 1 'is * ns ns ns * ns ns 'is ns 'isKt 1 ns ns ns 'is 'is ns ns 'is ns ns 'isK*CO2 1 ns ns ns ns ns ns ns ns 'is 'is 'ist*K*CO2 1 'is 'is ns 'is 'is ns 'is ns ns ns 'isAppendix 6.2. Summer 1991: Summary of analysis ofvariance, concentrationof inorganic nutrients (wn:LA)in leaves borne on the lower part of branches.Inorganic Nutrient Concentration (wn:LA), Leaves of BranchesSource df N P K Ca Mg Fe B Cu Mn Zn NaBlock 3 * ns 'is ns 'is 'is *CO2 1 ns ns ns ns ns 'is ns ns ns nsBlock*CO2 3 'is ns ns 'is 'is ns 'is ns ns ns 'isK 3 ns 'is ns ns ns ns ns * ns * *K*CO2 3 ns ns ns 'is 'is ns ns ns 'is ns ns* means significant (p < 0.05)ns means non significant (p > 0.05)183APPENDIX 7Standard curve utilized for determination of starch concentration in extracts.Starch Standard Curve0.`•-•••V.0 0.300.20.10.0•I^fCx = (y -1.55) 1 40.99R2= 0.990^5^10^15 70^75 3(mg starch per 200 mL sample184APPENDIX 8The concentration of sugars in leaf extracts was determined according to thefollowing equation:c=AA*{(V*MW)/(e*d*v*1000)}concentration (g L-1)change in absorbance (340 nm)final volumesample volumeMW: molecular mass of sugare^: absorption coefficient of NADPHd^: light path (cm)where:c :A :V :v :


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