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Influence of UV-B radiation and soil moisture stress on broccoli (Brassica oleraceae) seedings Mohan, Mercy 2003

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I N F L U E N C E OF U V - B RADIATION A N D SOIL MOISTURE STRESS O N BROCCOLI (BRASSICA OLERACEAE)  SEEDLINGS  by  MERCY MOHAN (B.Sc, 1980; M.Sc, 1982. University of Kerala, INDIA)  A THESIS SUBMITTED IN P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T FOR THE D E G R E E OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Faculty of Agricultural Sciences)  We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A August 2003 © Mercy Mohan, 2003  JUBCl THE UNIVERSITY OF BRITISH C O L U M B I A  F A C U L T Y OF G R A D U A T E STUDIES  Library Authorization  In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  M £ /Ley Name of Author ( p l e a s e  M-O print)  Date (dd/mm/yyyy)  Title of Thesis:  Year:  Degree:  2crv>3  Department of The University of British Colutfibia Vancouver, BC Canada  grad.ubc.ca/forms/?formlD=THS  p a g e 1 of 1  last updated: 2-Aug-04  11  Abstract The interaction between ultraviolet-B (UV-B; 280-320 run) radiation and soil moisture stress (SMS) on the growth of broccoli (Brassica oleracea L. var. italica cv. Purple Sprouting) seedlings was studied. The seedlings were grown under different levels of UV-B radiation and SMS and their combinations in the greenhouse. The factorial experiment consisted of sixteen treatments: four levels of SMS [normal {100% field capacity (FC)}, mild (80% FC), moderate (60% FC) and severe (40% FC)] x four levels of UV-B radiation [no UV-B (0), ambient (4 kJ m" d"), and two above ambient 2  1  levels (7 and 11 kJ m" d")]. The study showed that the responses of broccoli seedlings 2  1  grown under the combination of stresses were significantly different from those grown under individual stresses. The combination of stresses significantly increased the synthesis of epicuticular wax and UV-B absorbing compounds in the leaves and reduced leaf area compared to individual stresses. Seedlings grown under the combination of UV-B radiation and SMS had higher water potentials than the moisture stressed seedlings grown without UV-B radiation. Growth indices such as specific leaf weight (SLW), leaf area ratio (LAR), leaf weight ratio (LWR) and shoot:root ratio (SRR) indicated that the morphology and biomass allocation were strongly influenced by the interaction. UV-B irradiance with soil moisture stress also caused a reduction in growth parameters like plant height, fresh biomass and dry biomass compared to individual stresses. The results of this study suggest that UV-B radiation can minimize or mask the adverse effect of SMS on broccoli seedlings.  iii  T A B L E OF CONTENTS Page ABSTRACT LIST OF TABLES LIST OF FIGURES ACKNOWLEDGEMENTS 1.0 GENERAL INTRODUCTION 1.1 UV-B radiation 1.2 Soil moisture stress 1.3 Components of water potential  ii v vii viii 1 2 3 5  2.0 RESEARCH OBJECTIVES  6  3.0. LITERATURE REVIEW  7  3.1 Effect of UV-B radiation on the growth and development of plants 3.2 Influence of soil moisture stress on plants '. 3.3 Interaction of UV-B radiation and other environmental stresses on plants 3.4 Effect of UV-B radiation and soil moisture stress on plants 4.0 MATERIALS AND METHODS 4.1 Broccoli 4.2 UV-B radiation treatments 4.3 Soil moisture treatments 4.4 Growth measurements 4.5 Extraction and assay of UV-B absorbing compounds 4.6 Extraction and assay of epicuticular wax 4.7 Measurement of water potential 4.8 Statistical analysis  7 ..8 9 11 20 20 20 22 25 25 26 26 27  5.0 RESULTS 5.1 5.2 5.3 5.4 5.5 5.6  Effects Effects Effects Effects Effects Effects  28 of U V - B of U V - B of U V - B of U V - B of U V - B of U V - B  radiation radiation radiation radiation radiation radiation  and and and and and and  soil soil soil soil soil soil  moisture moisture moisture moisture moisture moisture  stress on primary growth parameters stress on percent water content stress on growth indices stress on epicuticular wax content stress on absorbance at 300 nm stress on water potential  6.0 DISCUSSION  48  6.1 Effects of U V - B radiation and soil moisture stress on seedling growth 6.1.1 Effects of U V - B radiation and soil moisture stress on leaf area 6.1.2 Effects of U V - B radiation and soil moisture stress on root growgth 6.1.3 Effects of U V - B radiation and soil moisture stress on plant biomass 6.2 6.3 6.4 6.5 6.6  Effects Effects Effects Effects Effects  28 38 39 42 44 46  of U V - B of U V - B of U V - B of U V - B of U V - B  radiation radiation radiation radiation radiation  and and and and and  soil soil soil soil soil  moisture moisture moisture moisture moisture  stress on growth indices stress on percent water content stress on absorbance at 300 nm stress on epicuticular wax production. stress on water potential  48 48 49 50 50 51 51 .52 54  7.0 CONCLUSIONS  56  8.0 L I T E R A T U R E CITED  63  LIST OF TABLES I. Measurement of soil moisture content using Theta Probe  22  2  The experimental design  23  3  Effects of U V - B radiation and soil moisture stress on root fresh weight  29  4. Effects of U V - B radiation and soil moisture stress on root dry weight  29  5. Effects of U V - B radiation and soil moisture stress on root length  30  6. Effects of U V - B radiation and soil moisture stress on seedling height  30  7. Effects of U V - B radiation and soil moisture stress on total fresh biomass  31  8. Effects of U V - B radiation and soil moisture on leaf number  37  9. Effects of U V - B radiation and soil moisture on fresh leaf biomass  37  10. Effects of U V - B radiation and soil moisture on leaf area  38  II. Effects of U V - B radiation and soil moisture stress percent water content  39  12. Effects of U V - B radiation and soil moisture on specific leaf weight  39  13. Effects of U V - B radiation and soil moisture on shoot:root ratio  40  14. Effects of U V - B radiation and soil moisture on leaf area ratio  41  15. Effects of U V - B radiation and soil moisture on leaf weight ratio  41  16. Effects of U V - B radiation and soil moisture on epicuticular wax/dry weight  42  17. Effects of U V - B radiation and soil moisture on absorbance at 300 nm/dry weight . . . .44 18. a. A N O V A results for the effect of U V - B radiation and SMS on broccoli seedlings root length, root fresh biomass, root dry biomass and plant height  58  18. b. A N O V A results for the effect of U V - B radiation and SMS on broccoli seedlings leaf number, leaf fresh biomass, leaf area and total fresh biomass  59  18. c. A N O V A results for the effect of U V - B radiation and SMS on broccoli seedlings growth indices  60  vi  18. d. A N O V A results for the effect of U V - B radiation and SMS on broccoli seedlings dry biomass, percent water content, absorbance at 300 nm per leaf area and absorbance at 300 nm per dry weight  61  18. e. A N O V A results for the effect of U V - B radiation and SMS on broccoli seedlings wax per leaf area, wax per dry weight and water potential  62  LIST OF FIGURES 1. Effects of UV-B radiation and soil moisture stress on dry biomass  32  2. Broccoli seedlings grown under no UV-B and different levels of soil moisture stress . . 33 3. Broccoli seedlings grown under 4 kJ m- d" UV-B and different levels of soil moisture stress for six weeks in the greenhouse  .34  4. Broccoli seedlings grown under 7 kJ m- d" UV-B and different levels of soil moisture stress for six weeks in the greenhouse  35  5. Broccoli seedlings grown under 11 kJ m- d" UV-B and different levels of 2  1  soil moisture stress for six weeks in the greenhouse 6. Effects of UV-B radiation and soil moisture stress on epicuticular wax/leaf area  36 43  7. Effects of UV-B radiation and soil moisture stress on absorbance at 300nm/leaf area .. 45 8. Effects of UV-B radiation and soil moisture stress on water potential  47  Vlll  Acknowledgements I would like to thank my supervisor Dr. Mahesh K. Upadhyaya for his guidance, patience, encouragement, and support through all these years. I would also like to thank my committee members Dr. Chris Chanway and Dr. Robert Guy for their time and valuable advice. I am especially thankful to Dr. Douglas P. Ormrod for his suggestions regarding water stress treatments. I am thankful to Dr. Rob Guy for agreeing to serve on my committee at the last stage. I would like to thank Mr. David Kaplan for his assistance in the greenhouse. I would also like to thank all my colleagues in the Faculty of Agriculture especially, Dr. Nancy Furness, Dr. Quincy Dai and Ms. Jennifer Cameron for their friendship and support. Above all, I am grateful to my husband Mr. Mohan George for being there for me in every step of the way and to my loving kids George, Ralph and Maria, for their love and patience, which gave me strength to finish up what I had started.  1  1.0 General Introduction Stress in plants is defined as an external factor that exerts a disadvantageous influence, which is usually measured in terms of survival; growth and/or crop yield (Taiz and Zeiger 1998). Under both natural and managed conditions, plants face several environmental stresses, e.g. variation in temperature, water shortage, toxic pollutants, wounding, infection, light intensity, ozone, nutrient deficiency, and insects, all of which can affect growth and productivity depending on their intensity. Factors like air temperature can cause stress just in a few minutes, but others like soil moisture stress may take several days to show effects on growth (Taiz and Zeiger, 1998). Stress plays a significant role in determining plant growth and species distribution (Taiz and Zeiger, 1998). Stress resistance, i.e. the ability to cope with unfavourable conditions, varies among plants. Generally plants show cross-resistance, i.e. resistance to cope with one stress because of acclimation to another. The mechanisms of resistance to several stresses may share many common features (Taiz and Zeiger, 1998). Understanding the physiological processes involved in the adaptation and acclimation of plants to different environmental stresses and the mechanisms behind the stress injuries is important to agriculture and ecology. Ultraviolet -B radiation (UV-B) and soil moisture stress (SMS) are two major factors that may affect plant growth under field conditions. Since the diurnal and seasonal patterns of SMS in plants overlap with periods of U V - B radiation maxima in the field (Bornman and Teramura, 1993), research into the study of combined effects of U V B radiation and SMS is important.  2  1.1 UV-B radiation Ultraviolet light is relatively high-energy radiation with wavelengths below 400 nm (i.e. U V - A = 320-400 nm; U V - B = 280-320 nm; U V - C = 100-280 nm). A l l types of U V radiation are known to damage plants. U V - C , the most energetic and damaging U V radiation, is effectively absorbed by O 3 in the stratosphere and doesn't reach the earth's surface. The U V - A region of the spectrum is not attenuated by O 3 (Stapleton, 1992), but the amount of solar U V - B radiation reaching the earth's surface is increasing, due to a decrease in the stratospheric O 3 concentration (Madronich et al. 1995) and its agricultural and ecological consequences are of concern. Even a slight increase in the total U V - B radiation is able to cause significant biological damage to plants (Jansen et al. 1998), affecting biomass and yield (Teramura, 1983; Sullivan and Teramura, 1988; Krupa and Kickert, 1989; Caldwell et al. 1989; Tevini and Teramura, 1989; Bornman and Teramura, 1993; Runeckles and Krupa, 1994; Caldwell et al. 1995). Ozone depletion is highest during winter and spring. Over the last two decades stratospheric O3 has decreased by about 12-14% in mid-latitudes in winter and by 4-6% in summer (Taiz and Zeiger, 1998). Plant species vary in sensitivity to U V - B radiation (Caldwell, 1981; Jansen et al. 1998). U V - B fluences are greater at higher elevations and the plants growing there exhibit more adaptive mechanisms than those at lower elevations (Jansen et al. 1998). Rozema et al. (1997) reported that solar U V - B radiation could induce morphogenetic effects in plants, which in turn can modify plant canopy architecture. U V - B radiation can ameliorate, aggravate or not have an effect on an environmental constraint (Bornman and Teramura, 1993). The biological impact of U V -  3  B radiation on plants depends on the balance between injury, restoration and acclimatization (Jansen et al. 1998). To improve crop growth and productivity, knowledge of how enhanced levels of U V - B radiation interact with other environmental stresses is important.  1.2 Soil moisture stress Plant water stress is common in many parts of the world. Plant productivity depends more on the availability of water than of any other environmental factor (Salisbury and Ross, 1978). Water is an important constituent of protoplasm and is essential for growth, metabolism, and movement of materials throughout the plant. Water accounts for about 80-95% of the fresh weight of herbaceous tissues and 35-75% of woody tissues (Taiz and Zeiger, 1998). Drought causes changes in the physiological and biochemical processes of plant growth and productivity (Daie, 1988). It reduces stem elongation, leaf expansion and stomatal movement (Hsiao, 1973). SMS occurs inplants when the available soil water is low and the atmospheric conditions cause continued loss of water by transpiration (Taiz and Zeiger, 1998). As the soil dries up, it becomes difficult for plants to absorb moisture, cell turgor pressure decreases and consequently plant growth is inhibited. Maintenance of high turgor is necessary for growth, since it enables the continuation of cell elongation and improves stomatal conductance at lower water potentials (Turner and Jones, 1980). When the plant is turgid, water pressure in plant tissues keeps stems upright and leaves expanded to receive sunlight (Plaster, 1992). If turgor falls below a certain minimum value [~ 0.4 MPa, (varies with species)], the cell expansion will cease, though the turgor is still positive (Taiz and Zeiger, 1998). If turgor decreases too much (i.e. to  4  0), plants will wilt. Severe water stress can cause permanent wilting. Once plants reach the permanent wilting point, turgor cannot be regained unless additional water is applied to the soil (Taiz and Zeiger, 1998). If stress is prolonged, most plants will die. Generally, plants undergo permanent wilting only when the water potential of the soil OF  w  (soil))  is less than -1.5 MPa, but this varies with species and habitat (Taiz and Zeiger,  1998). The water-limited productivity of plants is determined by the total available water and also by the water-use-efficiency of the plant (Taiz and Zeiger, 1998). A plant withstands SMS better if it is able to acquire more water or i f it has a higher water-useefficiency. Although SMS is a persistent threat to plant survival, many plants survive low soil moisture content by developing various morphological and physiological modifications (Hopkins, 1999). There are many ways by which plants can resist SMS. The most common involve either maintaining water uptake or preventing water loss. To take up water, the water potential ( ¥ ) of the plant must be lower than that of the soil (Taiz and Zeiger, 1998). When  of the soil is lower, water is held by the soil particles more tightly. SMS lowers  plant 4 . A more negative *P of leaves cause water to move into leaves increasing their /  turgor pressure (Hopkins, 1999). Water potential is a measure of the free energy of water per unit volume, and is significant since it governs the direction of water flow across membranes and through tissues (Taiz and Zeiger, 1998). Water always moves from areas of high water potential to one of lower water potential (Gil, 2002).  5  1.3 Components of ¥ Cell growth, photosynthesis and crop productivity are strongly influenced by *F (Taiz and Zeiger, 1998). *F has three major components in hydrated tissues; namely solute potential (^Ps), pressure potential Q¥p) and the gravitational potential OFg). The total  is also designated by T w .  T s is determined by the solute concentration and ^Fp is the hydrostatic pressure of the solution which can be either positive or negative (Taiz and Zeiger, 1998). Positive pressures increase the water potential while negative pressures decrease it. T g is the effect of gravity and its effect on ^Fw is only appreciable for large changes in elevation (e.g. in tall trees). Any factor that influences the free energy of water will influence and can be considered a component. Temperature Qfj) and gravity (Tg) are two such factors, but their effects are normally insignificant and can be ignored. Matric potential Q¥m) is another component of *F which is the effect of the surface interactions but is generally important only in dry soils, seeds and dry cell walls. It is negligible in hydrated cells since it only extends a few molecules of distance from the interacting surface. Therefore the only substantial components of ¥ in hydrated tissues are the solutes dissolved and hydrostatic pressure (Taiz and Zeiger, 1998). *Fw = T s + T p Pure water has a T w of 0. A l l solutions have lower ^Fw than pure water since they have fewer water molecules per volume and therefore have negative *Fw (Gil, 2002). In  6  plants *Fw is almost always negative, because pure water has a higher potential than the water inside the cell. Though the effects of individual stresses on plant growth have been investigated extensively their interactions have not been studied. In real life situations, plants have to experience and cope with several stresses simultaneously. To understand the potential impact of depletion of the stratospheric O3 layer on plants and adaptations of plants to changing environmental conditions, interactions among U V - B radiation with other environmental stresses such as drought, P A R (photosynthetically active radiation), CO2 and nutrient availability must be studied.  2.0 Research objectives The overall goal of this research was to improve our understanding of interaction between U V - B radiation and soil moisture stress on broccoli (Brassica oleracea var italica cv. Purple Sprouting) seedlings. The specific objectives were:  a) to characterise the interaction of U V - B radiation and soil moisture stress effects on broccoli seedlings  b) to determine the roles of leaf surface wax, U V - B absorbing compounds and components of *F in this interaction.  7  3.0 Literature review 3.1 Effect of UV-B radiation on the growth and development of plants U V - B radiation has many direct and indirect effects on growth and development of plants. Enhanced U V - B radiation can reduce photosynthetic rate and plant productivity, causing changes in growth, development and morphology (Teramura and Ziska, 1996; Jansen et al. 1998). U V - B radiation could affect the speed of stomatal opening and closing and thus could influence the rate of transpiration (Negash, 1987; Middleton and Teramura, 1993; Day and Voglemann, 1995). Leaf thickening, reduction in leaf area, changes in cuticular wax formation and induction and accumulation of U V - B absorbing compounds in the epidermis are major protective mechanisms against U V - B radiation damage (Taiz and Zeiger, 1998). Reduction in photosynthesis and leaf area, even to a small extent could reduce plant growth and productivity (Vu et al. 1978). U V - B radiation may alter plant biomass allocation, which may change root/shoot ratios (Biggs and Kossuth, 1978). Ormrod et al. (1997) observed decreased shoot biomass production in Arabidopsis thaliana under increased U V - B radiation. Plants accumulating less shoot biomass when exposed to U V B radiation were reported to have reduced U V - B sensitivity compared to plants with more shoot biomass (Smith et al. 2000). U V - B induced inhibition of cell expansion has been reported in cucumber cotyledons (Ballare et al. 1991), tomato hypocotyls (Ballare et al. 1995), wheat hypocotyls (Hopkins, 1997) and barley leaves (Liu et al. 1995). U V - B radiation has been reported to alter turgor pressure and cell wall extensibility, which in turn could affect cell wall expansion (Tevini and Iwanzik, 1986).  8  Leaf thickness increases with increased solar U V - B radiation (Cen and Bornman, 1990; Bornman and Voglemann, 1991) and might reduce the effect of internal U V - B radiation (Bornman and Voglemann, 1991). Cen and Bornmann (1990) observed that increased leaf thickness under elevated U V - B radiation results from increased length of pallisade parenchyma cells. Changes in leaf shape, reducing the leaf cross-sectional area exposed to U V - B radiation is also considered as a protective measure against U V - B (Barnes ef al. 1990). A n increase in anthocyanins and other U V - B absorbing compounds such as flavonoids occurs in plants after U V irradiation (Lindoo and Caldwell, 1978; Flint et al. 1985; Balakumar et al. 1993; Dunning et al. 1994; Mazza et al. 1999). These compounds are important because they help to reduce transmittance of U V - B radiation through the epidermis thereby protecting the underlying U V - B sensitive cells (Robberecht and Caldwell, 1978; Caldwell et al. 1983; Day 1993; L i et al. 1993 and Rozema et al. 1997). The direct damage to components of the photosynthetic system might be avoided by an increase in epidermal flavonoids, since they reduce U V - B penetration into the mesophyll (Caldwell et al. 1983; Tevini et al. 1991).  3.2 Influence of soil moisture stress on plants SMS is a widespread phenomenon in nature. The effects of water stress on the growth and development of plants have been studied thoroughly (Schulze and Weissenbock, 1986). SMS limits the productivity of many crops both qualitatively and quantitatively (Boyer, 1982). SMS can reduce photosynthesis, growth, protein and chlorophyll synthesis, influence enzyme activity, increase respiration and susceptibility to heat and other environmental stresses, and cause wilting (Taiz and Zeiger, 1998).  9  Increased cuticular and stomatal resistances, reduction in radiation absorption due to presence of leaf surface structures such as epicuticular wax and/or trichomes, and reduction of leaf area due to abscision, smaller leaves and leaf rolling are major acclimation mechanisms for preventing water loss from plants (Taiz and Zeiger, 1998). Epicuticular wax is very important in protecting leaves from environmental stresses and provides a significant drought tolerance mechanism to plants (Saneoka and Ogata, 1987). Increased deposition of epicuticular wax on the leaf surface reduces cuticular transpiration acting as a barrier to water diffusion and environmental pollutants (Hall and Jones, 1961; Martin and Juniper, 1970). Plants with higher deposition of epicuticular wax often show higher tissue water retention capacity. The ability to deposit more epicuticular wax is therefore a screening parameter for drought tolerance (Dedio, 1975). Many soil, climatic and environmental factors such as water, light, photoperiod, humidity, temperature, and air pollutants are also known to influence leaf epicuticular wax deposition (Baker, 1980). SMS is one of the most important factors among them. It has been reported that epicuticular wax plays a role in resistance to U V - B radiation in some plants. It acts like a hydrophobic layer and increases reflection of light from the leaf surface (Tevini and Steinmuller, 1985).  3.3 Interaction of U V - B radiation with other environmental stresses Sensitivity of crop plants to U V - B radiation is influenced by water regimes, levels of PAR; (400-700 nm), atmospheric CO2 and nutrient availability (Murali and Teramura, 1985). The interaction of U V - B radiation with other stresses depends on the specific combination of stresses (Bornman and Teramura, 1993). Caldwell and Flint (1994)  10  showed that heat resistance was increased by U V - B radiation in cucumber cotyledons. Heat, water and high light intensity stress are all related to oxidative stress (Freeman et al. 1990; Eickmeier et al. 1992), and acquired resistance to any one of them may provide cross-resistance to others. U V - B radiation is considered to be a potential cause of oxidative stress (Larson, 1988; Panagapoulos et al. 1990) and might induce increased synthesis of anti-oxidative enzymes (Balakumar et al. 1993). Several studies have reported that P A R (400-700 nm) has a direct effect on the growth responses of plants to U V - B radiation (Teramura, 1980; Warner and Caldwell, 1983; Mirecki and Teramura, 1984; Cen and Bornman, 1990). Decreased P A R and increased U V - B radiation reduce plant height and total biomass and shift biomass allocation patterns (Bornman and Teramura, 1993). These responses may vary with plant species. Combined with low levels of PAR, U V - B radiation influences photosynthesis, transpiration, respiration and stomatal conductance (Teramura et al. 1980; Mirecki and Teramura, 1984). Warner and Caldwell (1983), observed that under high P A R plants are less susceptible to U V - B radiation, and changes in the morphology and physiology of plants such as increased thickness of leaves and accumulation of flavonoids were documented. The same study indicated that high P A R along with U V - B radiation has a negative effect on photosynthesis. Plant parameters such as biomass allocation, branching, flowering, leaf thickness, emergence and senescence may be affected differentially by U V - B radiation and CO2. Generally plants respond positively to elevated CO2 and negatively to enhanced U V - B radiation. Photosynthesis, growth and primary production increase with an increase in atmospheric CO2 whereas enhanced U V - B radiation has a negative influence on plants  11  (Strain and Cure, 1985; Rozema et al. 1993; Rozema, 1995). Rozema et al. (1997) found a statistically significant interaction between the effects of CO2 and U V - B radiation on plant biomass. By increasing the flavonoid level in the epidermis, elevated CO2 might provide protection from U V - B radiation. Rozema et al. (1997) noticed a reduced sensitivity to enhanced U V - B radiation at elevated CO2. Protection from U V - B radiation could also be provided by carbohydrate accumulation as well as leaf thickening that takes place in response to CO2 enrichment (Adamse and Britz, 1992). Damage to photosynthesis by U V - B radiation could be avoided by exposure to high CO2 concentrations (Stewart and Hoddinott, 1993; Sullivan and Teramura, 1994). The interspecific variation and alteration in photosynthetic limitations might have implications for plant growth under enhanced CO2 and U V - B radiation (Sullivan, 1997).  3.4 Effect of UV-B radiation and SMS on plants The effect of U V - B radiation on plants could be influenced by other simultaneous stresses (Bornman and Teramura, 1993; Tevini, 1993). The magnitude of the effect of U V - B radiation on plants has found to be decreased or masked by SMS (Sullivan and Teramura, 1990; Tevini 1993; Nogues et al. 1998). Adaptation of plants to U V - B radiation has shown that U V - B radiation can induce multiple stress defenses (Schmidt et al. 2000). U V - B radiation might reduce tissue dehydration in the presence of SMS, by stimulating production of osmolytes which help plants maintain tissue turgor without closing stomata (Schmidt et al. 2000). It has been reported that U V - B radiation can act as an environmental signal to induce tolerance to SMS in Douglas-fir seedlings by causing changes in carbon assimilation rates and water potential (Poulson et al. 2002).  12  The responses of plants to a combination of U V - B radiation and SMS were found to be significantly different from those when the stresses were applied individually. Teramura et al. (1984); Murali and Teramura (1985 a, b) and Yang et al. (2000) reported that U V - B radiation and SMS together reduced net photosynthesis and biomass accumulation in plants compared to individual stresses. Sullivan and Teramura (1990) showed that under the combination of stresses, SMS reduced the growth and yield of soybean, but the effects of supplemental U V - B radiation were dependent on different watering regimes. Under U V - B radiation, plant biomass, leaf area and number of pods were reduced in well-watered plants whereas the plant height, specific leaf weight, seed yield and numbers were not affected. Supplemental U V - B radiation did not have any statistically significant effect on plant growth and yield under water stressed conditions. No additional plant growth reductions occurred when plants received enhanced U V - B radiation (Sullivan and Teramura, 1990). Nogues and Baker (2000) also reported that exposure to elevated levels of U V - B radiation and SMS had no significant effects on the growth or photosynthetic activities of plants like olive, lavender and rosemary. Reduced productivity in field-grown soybean exposed to U V - B radiation was moderated by soil moisture stress (Sullivan and Teramura, 1990). Under SMS (Teramura et al. 1984) or mineral deficiency (Murali and Teramura, 1985), soybeans are less susceptible to U V - B radiation but highly sensitive when the visible radiation level is low (Mirecki and Teramura, 1984; Warner and Caldwell, 1983). Reduced growth and changes including increased leaf thickness and accumulation of U V - B screening pigments were noticed in plants grown under the combination of stresses compared to control plants (Murali and Teramura, 1986; Yang et al. 2000). Well-watered plants showed significant  13  reduction in leaf area and total biomass in response to U V - B radiation but under SMS they remained unaffected by U V - B radiation (Murali and Teramura, 1986). U V - B radiation and SMS reduced total biomass in soybean during early stages of vegetative growth (Teramura et al. 1984). U V - B was more effective in changing biomass allocation to different parts compared to SMS. Soil moisture stress reduced the number of leaves and nodes and the leaf area. U V - B radiation alone lowered the rate of net photosynthesis and SMS induced an additional reduction. Growth in vegetative plants was more sensitive to the combination of stresses than the reproductive plants. The reduced sensitivity of the older plants might be due to the increased levels of flavonoids in the U V irradiated leaves (Teramura et al. 1984). Soil moisture stress can generate phosphorus deficiency in plants (Krizek et al. 1985) which may result in stunted growth and death of older leaves (Taiz and Zeiger, 1998). Reduction in plant phosphorus levels has been shown to reduce the sensitivity of soybean to U V - B radiation (Murali and Teramura, 1985). Levizou and Manetas (2001) also reported that nutrient levels could strongly modify the effects of U V - B radiation on the growth of Phlomis fruiticosa. The effect of U V - B radiation may be reduced in the presence of drought or other stresses, causing growth delay or a concurrent increase in U V - B protective mechanisms (Sullivan and Teramura, 1990). Masking of U V - B radiation effects in soil moisture stressed conditions might be due to anatomical (leaf thickening) or biochemical (accumulation of pigments) adjustments to SMS, which in turn might also provide protection from U V - B radiation (Murali and Teramura, 1986). Research to determine the mechanisms with which U V - B radiation affects water relations, leaf development and gas exchange, indicated that U V - B irradiance caused  14  significant reductions in leaf area and plant biomass, associated with a reduction in leaf cell numbers and cell division (Nogues et al. 1998). It also affected the exposed leaf surfaces by inhibiting the epidermal cell expansion. The interaction between the two stresses was significant and the effect of SMS was delayed and reduced by U V - B radiation through reductions in plant water-loss rates, stomatal conductance and leaf area (Nogues et al. 1998). Reduction in leaf area resulted in reduced plant water loss and, in turn, a slower reduction in soil water content. The slow drying of soil under U V - B irradiation might have delayed the development of SMS (Nogues et al. 1998). The interaction between U V - B radiation and SMS brought beneficial effects on the morphological and growth characteristics in cowpea seedlings (Balakumar et al. 1993). Although the combined stress inhibited some of the physiological activities of the plants, one type of stress minimized the damage by the other. The combination of stresses showed less reduction in primary growth parameters and relative water content (RWC) and a significant increase in specific leaf weight. Water stress alone reduced plant height, fresh and dry biomass and leaf area. Synthesis of anthocyanin pigments increased by 8, 36 and 25% under drought, U V - B radiation and a combination of stresses respectively in cowpea seedlings. The injurious effects were less under combined stresses compared to water stress alone (Balakumar et al. 1993). Increases in anthocyanin and phenols after U V - B exposure were also reported in Pisum sativum and Triticum aestivum (Alexieva et al. 2001). The relative water content also remained the same in pea and wheat after U V - B treatments indicating that changes in water content were not involved (Alexieva Qt al. 2001).  15  Accumulation of proline may contribute to a decline in ^ s of plants and thus a decrease in T w (Balakumar et al. 1993). This lowering in T w increases the capacity for water absorption. Compared to the control, cowpea seedlings under water stress alone showed a 3-fold increase in the proline level while the increase under the combination of stresses was only 1.5 fold. Alexieva et al. (2001) also reported increased proline levels in soil moisture stressed pea and wheat, and suggested that proline might be a droughtinduced factor which also might have a protective role in response to U V - B radiation. Phenolic and flavonoid compounds found in terrestrial plant tissues play an important part in the interaction of plants with their biotic and abiotic environments (Dakora 1995). The phenolic content of plant tissue might be critical for tolerance to U V - B radiation (Robberecht and Caldwell 1978; Wollenweber and Dietz; 1980; Caldwell et al. 1983; Karaboumiotis et al. 1992). The phenolic levels were affected positively by the light availability and increased the U V - B radiation absorbing capacity. Under field conditions, an increase in phenolic levels after herbivore attack has been reported in semi-deciduous Mediterranean shrub Phlomis fruiticosa, which may provide extra protection against enhanced U V - B radiation levels (Levizou and Manetas, 2001). While U V - B radiation lessens the effect of SMS in some plants, it has an additive, inhibitory effect on the functional processes of such plants (Balakumar et al. 1993). The protective effect of U V - B radiation in water stressed plants could be due to a direct effect of U V - B radiation on internal plant water relations by increasing the potential for osmotic adjustments in plants. U V - B induced reduction in leaf area (Vu et al. 1982) and its effect on stomatal conductance (Teramura et al. 1983) could confirm this effect. Reduction in leaf area and increase in SLW and accumulation of anthocyanins in leaf tissues were  16  noticed in water stressed plants irradiated with U V - B radiation (Sullivan and Teramura, 1990; Balakumar et al. 1993). A n increase in SLW is correlated with an increase in leaf thickness (Mirecki and Teramura 1984) which in turn could reduce U V penetration due to greater radiation absorption. Thus the anatomical and biochemical changes in leaf tissues caused by the combination of stresses provides increased protective mechanisms from damage by either stress (Murali and Teramura, 1985; Balakumar et al. 1993). A combination of stresses could reduce net carbon gain by plants and reduce biomass production, which might contribute to masking the effects of U V - B radiation (Balakumar et al. 1993). It has been suggested that when growth is restricted, other repair mechanisms help to improve U V induced damage before it becomes lethal (Beggs et al. 1986). The effect of enhanced U V - B radiation on the growth and productivity of plants was found to be dependent on plant water status (Murali and Teramura, 1986). Well-watered plants were affected deleteriously by U V - B radiation while water-stressed plants were not affected seemingly, because of the masking of U V - B radiation effects caused by growth reductions and increases in U V - B absorbing compounds and leaf thickness. Manetas et al. (1997) investigated the possible mechanisms underlying alleviation by enhanced U V - B radiation of the negative effect of SMS in Pinus pinea. Supplemental U V - B radiation had no effects in P. pinea under well-watered conditions, while under SMS, U V - B irradiated plants showed significantly less needle drop and increased photosynthetic capacity compared to plants grown under ambient U V - B radiation (Petropoulou et al. 1995). The amount of epicuticular wax increased in water stressed P. pinea plants, and a further increase was noticed with supplemental U V - B radiation  17  (Manetas et al. 1997). Under enhanced U V - B radiation, there was a large increase in cuticle thickness in water stressed plants, which could decrease cuticular transpiration. Though the water loss through the cuticle may not be that much, it has to be taken into consideration during prolonged periods of water stress (Manetas et al. 1997). The production of thicker cuticle will eventually lead to an improved water economy indirectly maintaining photosynthesis (Manetas et al. 1997). Teramura et al. (1984) investigated the combined effect of U V - B radiation and water stress on the stomatal conductance and leaf *F of greenhouse grown soybean. The diurnal pattern of leaf ¥ prior to drought showed an early morning maximum with a minimum at midday. Significantly lower midday and afternoon leaf *F and leaf conductances were noticed in water stressed, compared to well-watered plants. Under the combination of stresses U V - B radiation lowered leaf conductance but didn't have any effect on leaf v|/. The internal water relations of soybean were affected by water stress only. U V - B radiation had no effect on the turgid weight: dry weight ratio or relative water content of soybean. Faster recovery from SMS was observed in U V - B irradiated plants compared to those grown without U V - B radiation, however this fast recovery was associated with lower stomatal conductances, not with any component of internal water relations (Teramura et al. 1984). Under the combination of stresses, Poulson et al. (2002) observed about 40% higher T in U V - B irradiated Pseudotsuga menziesii seedlings compared to non-irradiated ones. Besides general growth reductions, SMS and U V - B radiation caused a reduction in the photosynthetic capacity of soybean (Sullivan and Teramura, 1990). Stomatal conductance was only minimally affected by U V - B radiation. In well-watered  18  conditions, growth and photosynthesis of soybean were affected significantly by U V - B radiation; whereas in water stressed plants these effects were masked when growth and yield were already reduced. U V - B sensitivity in soybean varied significantly with growth and developmental stage (Teramura and Sullivan, 1989). Plant height and leaf area were unaffected by U V - B radiation during vegetative stages while it had an inhibitory effect during reproductive stages. Leaf area ratio was reduced during vegetative and early reproductive stages while specific leaf weight increased only during the vegetative stage. Murali and Teramura (1985), studied the effectiveness of enhanced U V - B radiation on the growth and productivity of field grown soybean under well watered and water stressed conditions. At one stage of development (main stem with a fully developed leaf and pod 5 mm long at one of the four uppermost nodes) net photosynthesis was not affected by U V - B radiation in water stressed plants but affected negatively in wellwatered plants. Water stress during the same stage of development drastically reduced net photosynthesis, transpiration and chlorophyll concentration, but increased specific leaf weight, methanolic leaf extract absorbance and stomatal conductance. Enhanced U V - B radiation makes Mediterranean pines less sensitive to SMS providing temporary photosynthetic and growth superiority during the dry season (Petropoulou et al. 1995). Although the photosynthetic superiority under enhanced U V B radiation was temporary, it significantly affected morphological parameters and biomass at final plant harvest. U V - B radiation is also found to induce stomatal closure in some plants (Negash and Bjdrn, 1986; Grammatikopoulos et al. 1994). There are reports suggesting that photosynthesis is not affected by stomatal effects (Murali and Teramura,  19  1986; Sullivan and Teramura, 1989; Teramura et al. 1991; Ziska and Teramura 1992; Nogues etal. 1998). Although it has been shown that the combined effects of U V - B radiation and SMS result in reduced growth of plants (Teramura et al. 1983; 1984 a; 1984 b; Tevini et al. 1983; Balakumar et al. 1993), the underlying mechanisms are not understood. Since exposure to higher levels of U V - B radiation can alter the normal growth and development of economically important plants affecting their yield, an understanding of the interactions between U V - B radiation and other environmental stresses, and their physiological underpinnings, is important to improve crop growth and productivity.  20  4.0 Materials and methods 4.1 Broccoli Broccoli, a nutritionally and economically important and U V - B sensitive vegetable crop, was used to study interactions between U V - B radiation and soil moisture stress. Broccoli also has a thick layer of epicuticular wax on its leaf surface (Bitterlich and Upadhyaya, 1990, Gomez-Campo et al. 1999). Epicuticular wax plays an important role in adaptation of plants to enhanced U V - B radiation (Clark and Lister, 1975).  4.2 UV-B radiation treatment Broccoli plants were grown in U V - B chambers in a greenhouse at the University of British Columbia. Pre-burnt (lOOh) UV-B-313 40 W fluorescent tubes were used to provide ultraviolet-B radiation treatments. Each U V - B chamber had a 1.20 m long x 1.20 m wide x 1.25 m high metal frame, enclosed with Mylar film (Type D, 0.127 mm thick) (Cadillac Plastics Ltd., Burnaby, BC) to prevent U V - B radiation from escaping. U V - B fluorescent tubes were mounted at the top of each metal frame, 30 cm apart and 1.10 m above the greenhouse benches. Inside the U V - B chambers, broccoli seedlings were exposed to U V - B radiation passed through appropriate films [either a layer of Mylar film, or one, two or three layers of cellulose acetate film (diacetate type, 0.127 mm) (McMaster-Carr, New Brunswick, NJ, US)] mounted on 60 x 30 x 30 cm wooden frames. Mylar film absorbs U V - B radiation below 320 nm and cellulose acetate absorbs below 290 nm (Barnes et al. 1988). Cellulose acetate films were pre-burnt under U V - B radiation emitted from the U V - B lamps for 24 h prior to the experiment to stabilize their UV-transmission.  21  U V - B radiation (290-320 nm) was measured in single nanometer increments using an International Light IL 1700 Radiometer and an IL 782A double-slit monochromator (Harvard Apparatus, St. Lauren, PQ). Single nanometer readings were taken in a chamber covered with opaque black plastic to exclude all visible light. Biologically effective U V - B ( U V - B B E ) radiation was estimated from these readings using Caldwell's (1971) generalized plant damage action spectrum normalized to 300 nm. Integrated estimates of U V - B radiation between 290 and 315 nm were obtained under light conditions with an International Light SED240 solar blind bulk sensor (Harvard Apparatus, St. Lauren, PQ). The relationships between bulk sensor readings and U V - B B E estimates obtained using the monochromator were determined through regression analyses. Supplementary light in the greenhouse was provided by high-pressure SON-T Plus sodium lamps (P.L. light systems Inc., Beamsville, ON) spaced 1.9 m apart and 2.75 m above the greenhouse benches. Ambient temperature varied between 28 to 36 C during the day and 20 to 24 C during the night. Photosynthetically active radiation was recorded at two weeks intervals using a LI-COR LI-185 B portable light meter (LI-COR Inc., Lincoln, NE). The readings showed an average between 320 p:mol m" s" (cloudy day) and 720 pjnol m" s" (clear day). Seedlings were exposed to U V - B radiation for 8 h each day centered around the solar noon. The frames covered with a layer of Mylar film and three, two or one layer(s) of cellulose acetate (CA) film provided approximately 0 (control, because of the absorption characteristics of the greenhouse glass no natural solar U V - B B E radiation in the greenhouse), 4 (~ ambient U V - B level in Vancouver, BC), 7 ( U V - B level with ~ 18% stratospheric O 3 depletion) and 11 ( U V - B level with ~ 37% 0 depletion) kJ m" d" of 2  3  1  22  U V - B BE radiation, respectively (Dai and Upadhyaya, 2002). The ambient level of U V - B radiation for Vancouver, B C (4 kJ m" d" ) and U V - B levels corresponding to O 3 2  1  depletion scenarios of 18% (7 kJ m" d" ) and 37% (11 kJ m" d" ) were calculated using 2  1  2  1  the Bjdrn and Murphy (1985) model.  4.3 Soil moisture stress treatment Broccoli {Brassica oleracea var. italica cv. Purple Sprouting) seeds purchased from a local store (West Coast Seeds in Vancouver) were sown in 9 cm diameter plastic square pots containing 7:2:1 peat, perlite and mineral soil mixture with slow release fertilizer (Osmocote, Scotts-Sierra Horticultural Products Co., Marysville, OH, US). Initially in each pot, three plants were grown, but thinned to one plant per pot prior to the start of the treatment. Plants were subjected to four levels of soil moisture stress (SMS) treatments [SI = 100%o (no stress), S2 = 80% (mild stress), S3 = 60% (moderate stress) and S4 = 40% (severe stress) of field capacity (FC)]. The amount of soil (110 gms) used per pot was found to hold 150 ml of water at FC. Seedlings were given SMS treatments by adding the following amounts of water; SI, 150 ml water per pot; S2, 120 ml water per pot; S3, 90 ml water per pot; and S4, 60 ml water per pot. In order to spread the water stress uniformly through the soil mass (when plants were re-watered with amounts less than that required to achieve FC), capillary mats (JVK Capillary Bench Matting, Vancouver, B.C) were used for watering. The capillary mat was made of evenly compressed shredded textiles, which allowed the pot to seal itself to the mat for proper capillary action.  23  For each level of SMS treatment, pots were kept on capillary mats placed in separate plastic egg cartons (Enterprise Paper Co., Ltd. Coquitlam, BC) and water was applied onto the mat. Soil moisture content (SMC) was measured regularly using a Theta Probe Soil Moisture Sensor (Dynamax, Houston, Texas, US). The Theta Probe Soil Moisture Sensor consists of a plastic cylinder (5.25 inches long x 2.25 inches diameter) with four electrodes, each 2.25 inch long, attached to one end, and the other end connected to a moisture meter. The electrodes were pushed down into the soil and the readings were taken to determine how much water to apply to adjust SMS levels. The Theta Probe measures volumetric S M C (6 ). 6 is the ratio between the volume of water V  V  present in the sample and the total volume of the soil sample.  Table 1. Measurement of S M C using Theta Probe Soil moisture treatment  0v(V/V)  SI (100%)  39.67 ±0.145  S2 (80%)  33.72 ±0.148  S3 (60%)  27.69 ±0.108  S4 (40%)  21.73 ±0.111  Values are means ± SE of 10 replicates. S = soil moisture, SI = No SMS, S2 = mild SMS, S3 = moderate SMS, S4 = severe SMS 6 = volumetric S M C (Probe readings for different levels of SMS). V  At 100% FC, the probe measured 40% volume of water on the moisture meter, i.e. 40%» volume of water was present in 110 gms of soil at FC. For each level of SMS, the probe measured the following % volume of water per pot: 80%) SMS, 34% volume of  24  water; 60% SMS, 28% volume of water; 40% SMS, 22% volume of water, respectively (Table 1). Seedlings were watered approximately every 4 day and each time before th  watering, S M C was measured and the required amount of water was added to maintain the SMS levels. Experimental treatments were started as soon as the seedlings emerged. The seedlings were exposed to 8h of U V - B radiation each day. A split plot experimental design, with U V - B as the main factor and water stress as the subplot factor, with four blocks was used. In each block, plants were subjected to sixteen combinations of treatments as shown in Table 2.  Table 2. The Experimental Design M  CA3  CA2  CA1  SI  M SI  C A 3 SI  CA2S1  C A 1 SI  S2  MS2  CA3S2  CA2S2  C A 1 S2  S3  M S3  CA3S3  CA2S3  C A 1 S3  S4  MS4  CA3S4  CA2S4  C A 1 S4  S = soil moisture treatment, M = Mylar, C A = cellulose acetate S1 = No SMS, S2 = mild SMS, S3 = moderate SMS, S4 = severe SMS M = 0 U V - B , CA3 = 4 kJ i n d" U V - B , CA2 = 7 kJ nf d" U V - B , CA1 = 11 k J m ' d " ' U V - B . 2  1  2  1  2  Three plants were grown (one each for growth, T , and foliar U V absorbing compounds measurements) under each treatment in each block. Altogether there were 192 plants, in four blocks. The seedlings within each plastic carton, and the treatments  25  within each block, on the greenhouse bench were randomized every other day to minimize microclimate heterogeneity effects. Plants were grown for 42 days in the greenhouse. The experiment was repeated. The first experiment was started on April 24 2002, and was terminated on June th  5 2002. The repetition of the experiment was started on 26 of July 2002 and was th  th  harvested on September 5 2002. th  4.4 Growth measurements Broccoli seedlings were harvested after six weeks and plant height and leaf number recorded. Roots were washed and blotted dry on paper towels. Root, shoot and leaf fresh weights were recorded. Leaf area was measured using a LI-COR LI - 3000 portable leaf area meter (LI-COR Inc., Lincoln, N B , US). Plant parts were dried at 70 C for 72 h and dry weights recorded. Specific leaf weight (SLW) [leaf dry weight/leaf area (g cm" )], leaf weight ratio (LWR) [leaf dry weight/shoot dry weight], leaf area ratio 2  (LAR) [leaf area/shoot dry weight (cm" g" )], shoot:root dry weight ratios (SRR) [shoot 2  1  dry weight/root dry weight], and the percent water content {[(fresh weight-dry weightVfresh weight]x 100} were calculated (Hunt 1990).  4.5 Measurement of UV-B absorbing compounds To measure the U V - B absorbing compounds present in the leaves, leaf discs (1.5 cm" ) were sampled from the third true leaf of the seedlings. Samples were extracted in 2  glass tubes in 5 ml of acidified methanol mixture [MeOH:H 0:HCl (79:20:1)] (Mirecki 2  and Teramura, 1984) in a hot water bath (85 C) for 15 min, cooled to room temperature and centrifuged at 2000g for 15 min (IEC Model C L centrifuge, IntT Equip. Co. M A ) .  26  The volume of each extract was made to 25 ml in a volumetric flask by adding the mixture used for extraction. The absorbance of the extract at 300 nm (Day, 1993) was recorded using a Shimadzu U V - 160U U V / Visible recording Spectrophotometer (Kyoto, Japan).  4.6 Epicuticular Wax Measurement The fully expanded fourth true leaf of the plant was used. Leaf area was determined using LI-COR LI - 3000 portable leaf area meter. Each leaf was then dipped into 20 ml of H P L C - grade chloroform for 20 sec at room temperature. The extract containing the chloroform/wax solution was filtered through pre-rinsed (with chloroform) Whatman No. 1 filter paper and then through a pre-rinsed 0.2 um Sartorious filter using a long stemmed Buchner funnel inserted into a suction flask. The filtered chloroform was then poured into pre-weighed 25 ml glass scintillation vials. The flask and the suction filter were rinsed with a small amount of chloroform. The extracts were allowed to dry to constant weight to remove all chloroform (Barness and Brown, 1990; Chachalis et al. 2001). The vials were reweighed and the amount of wax per unit leaf area (u.g cm" ) and 2  per unit dry weight (u.g mg" ) was calculated. 1  4.7 Measurement of \|/ The pressure chamber also called 'Scholander pressure bomb' (Soil Moisture Equipment Corp. Santa Barbara, California), was used to measure ¥ of broccoli seedlings. The pressure bomb technique is a simple and rapid method for estimating plant T .  27  The required broccoli shoot was excised above the soil level and sealed into the pressure chamber with the cut end extending outside. The cut end soon turned dry since the water column was broken by excision (water was pulled into the xylem capillary by the unopposed tension). The chamber pressure was then increased slowly until the water in the xylem was brought back to the cut surface (indicated by wet/shiny appearance). At that point the chamber pressure [balance pressure (the pressure required to bring the water back to the excised surface)] was measured which is equal to the negative hydrostatic pressure (tension) that existed in the xylem of the broccoli seedling before excision.  4.8 Statistical Analysis Statistical analysis was done using SYSTAT 9.0 software (SYSTAT Inc., Chicago, IL, US). A two-way analysis of variance (ANOVA) was carried out on each plant measure to determine any significant differences in the growth characteristics of broccoli seedlings grown under the combination of U V - B radiation and water stress as well as individual treatment effects. Observations were classified according to two criteria (different levels of U V - B radiation and different levels of SMS), and data were analysed to test whether the variation in the growth characteristics was caused by different levels of U V - B radiation, different levels of water stress or by their interaction. Multirange comparisons were performed using the Bonferroni adjustment at the 5% level unless stated otherwise.  28  5.0 Results U V - B radiation, SMS and their interaction significantly influenced broccoli seedling growth. Responses of plants grown under the combination of stresses were significantly different from those grown under individual stresses (Fig. 2, 3,4 and 5). Interaction of U V - B and SMS significantly reduced plant height (Table 6), leaf area (Table 10) and dry matter production (Fig. 1) and increased SLW (Table 12), epicuticular wax content (Fig. 6) and U V - B absorbing compounds (Fig. 7) in the leaves of broccoli seedlings. Seedlings grown under the combination of stresses had higher *F than those grown under SMS without U V - B radiation (Fig. 8). The magnitude of the effects of U V B radiation on the seedlings depended on the level of SMS. Since there was no significant treatment x experiment interaction (Table 18), the results of the two experiments were pooled. The effects of the interaction were similar in both the experiments.  5.1 Effects of UV-B radiation and SMS on primary growth parameters General growth parameters like plant height (Table 6), total dry biomass (Fig. 1), leaf biomass (Table 9) and leaf area (Table 10) were significantly affected by U V - B radiation, SMS and the interaction. Root biomass accumulation was more under no U V - B and less under high U V - B (Table 3). Plants grown under high U V - B radiation with no SMS, showed more than 45% reduction in root biomass, compared to well watered plants without U V - B (Table 4). Under severe SMS, enhanced (11 kJ m" d" ) U V - B radiation 2  1  reduced root biomass accumulation by more than 36% compared to severe SMS alone and by more than 66% compared to control (Table 4). Severe SMS showed 47% reduction in root biomass accumulation compared to control. Under the combination of stresses, SMS  29  might have reduced the effect of U V - B radiation, on root biomass accumulation. SMS alone reduced seedling root length by about 18% compared to control (Table 5). Enhanced U V - B radiation with severe SMS caused only a 10% reduction compared to control. Under the combination of stresses U V - B radiation tended to reduce the effect of SMS on root length.  Table 3. Effects of U V - B radiation and SMS on root fresh weight (g) of broccoli seedlings grown in greenhouse for six weeks. ^ Soil U V - B level (kJ m"^ d" ) Moisture 0 4 7 11 1  .2.8  SI  4.1 ± 0.14a  2.6 ± 0.13a  2.1 ± 0.25a  S2  3.4 ± 0.20b  2.1±0.33b  1.4 ± 0.1 l b  2.4 ± 0.25a  S3  2.9 ± 0.15c  1.9 ± 0.38c  1.2 ± 0.12c  1.7 ± 0.13b  S4  1.8±0.32d  1.7 ± 0.05c  1.9 ± 0.1 I d  1.9 ± 0.25b  ± 0.13a  Values are means ± SE of 8 replicates pooled from two experiments. Means in each column followed by the same letter are not significantly different at P # 0.05. S I =No SMS; S 2 = mild SMS; S 3 = moderate SMS; S 4 = severe SMS.  Table 4. Effects of U V - B radiation and SMS on root dry weight (g) of broccoli seedlings grown in greenhouse for six weeks. U V - B level (kJ m d" ) Soil Moisture 11 4 7 0 l  1  SI  0.60 ± 0.01a  0.40 ± 0.03a  0.32 ± 0.02a  0.31 ± 0.04a  S2  0.44 ± 0.02b  0.30 ± 0.01b  0.29±0.09ab  0.26 ± 0.01b  S3  0.40 ± 0.03c  0.23 ± 0.03c  0.26 ± 0.07bc  0.21 ± 0.04c  S4  0.30 ±. 0.01c  0.22 ± 0.07c  0.22 ± 0 . 0 1 c  0.19±0.01c  Values are means ± SE of 8 replicates pooled from two experiments. Means in each column followed by the same letter are not significantly different at P # 0.05. S1 = No SMS; S2 = mild SMS; S3 = moderate SMS; S4 = severe SMS  30  Table 5. Effects of U V - B radiation and SMS on root length (cm) of broccoli seedlings grown in greenhouse for six weeks . U V - B level (kJ m" d" ) Soil moisture 11 7 4 0 2  1  SI  22.9 ± 0.8a  21.1 ± 0.8a  23.1  S2  19.4 ± 0.5b  20.9 ± 0.6a  21.8±0.7ab  19.5±0.7ab  S3  20.8 ± 0.9ab  20.5 ± 0.7a  23.7 ± 0.9a  20.3 ± 0.8ab  S4  18.7±0.7d  22.9 ± 0.5b  21.1  19.9 ± 0.9b  ± 0.5a  ± 0.7b  20.5 ± 0.5a  Values are means ± SE of 8 replicates pooled from two experiments. Means in each column followed by the same letter are not significantly different at P # 0.05. SI = No SMS; S2 = mild SMS; S3 = moderate SMS; S4 = severe SMS  Plant height was significantly reduced under SMS. Interaction of U V - B and SMS caused significant reduction in plant height (Table 6). Under the combination of stresses, plant height decreased with an increase in SMS and U V - B radiation. More than 6 0 % reduction in plant height was observed under severe SMS and enhanced U V - B radiation compared to control. SMS alone reduced plant height by 39% compared to control. In well watered conditions, U V - B alone didn't have much effect on plant height.  Table 6. Effects of U V - B radiation and SMS on the height (cm) of broccoli seedlings grown in greenhouse for six weeks. Soil U V - B level (kJ i n d ) moisture 0 4 7 11 2  -1  SI  22.5 ± 1.4a  21.2 ± 1.2a  21.5 ± 0.2a  S2  20.1  ± 0.8a  20.7 ± 1.3b  19.3 ± 0.9b  19.2 ± 0.8b  18.2 ± 0.2a  S3  15.2 ± 0.8c  12.3±0.7c  13.7 ± 1.3c  10.1 ± 0.1b  S4  13.1  10.2 ± 0.5d  9.6 ± 0.7d  ±0.4d  8.2 ±  Values are means ± SE of 8 replicates pooled from two experiments. Means in each column followed by the same letter are not significantly different at P # 0.05. SI = No SMS; S2 = mild SMS; S3 = moderate SMS; S4 = severe SMS  0.4c  31  U V - B radiation, SMS and their interaction induced significant reduction in total fresh (Table 7) and dry biomass accumulation (Fig. 1) compared to the control plants. The percentage of reduction was more under the combination of stresses, compared to individual stresses. However, under the combination of severe SMS and U V - B , total fresh biomass remained unaffected by U V - B radiation (Table 7). Severe SMS alone reduced dry biomass accumulation by 56% compared to well watered plants without U V - B . The percentage of reduction by elevated levels of U V - B alone was only 21 % compared to the control. Under severe SMS and enhanced U V - B , the percentage of reduction was more than 7 0 % ) compared to well watered plants without U V - B and 35% compared to severe SMS (Fig. 1).  Table 7. Effects of U V - B radiation and SMS on total fresh biomass (g) of broccoli seedlings grown in greenhouse for six weeks. . Soil U V - B level (kJ m d' ) moisture 0 4 7 1  1  11  51  16.9 ± 0.24a  14.8 ± 0.19a  13.9 ± 0.18a  13.5 ± 0.48a  52  14.7 ± 0.36b  11.9 ± 0.10b  11.2 ± 0.46b  12.1 ± 0.21b  53  8.9 ± 0.22c  6.4 ± 0.29c  6.1 ± 0.35c  7.1 ± 0.12c  54  5.5±0.44d  5.6±0.41d  5.9±0.71d  5.5 ± 0.34d  Values are means ± SE of 8 replicates pooled from two experiments. Means in each column followed by the same letter are not significantly different at P # 0.05 SI = No SMS; S2 = mild SMS; S3 = moderate SMS; S4 = severe SMS  Leaf abscission and leaf curling were noticed in plants grown under enhanced U V - B radiation (Fig. 5). Water stressed plants showed smaller leaves (Fig. 2). The combination of stresses resulted in reduced number of leaves (Table 8). Leaf abscission  32  3.0  U V - B level (kJ r r f d~ ) 2  1  Effects of U V - B radiation and SMS on the dry biomass of broccoli seedlings grown in the greenhouse for six weeks. Values are means ± SE of 8 replicates pooled from two experiments. SI = No SMS; S2 = mild SMS; S3 = moderate SMS; S4 = severe SMS.  33  2 Broccoli seedlings grown under severe (40% FC); moderate (60% FC); mild (80% FC) and no SMS (100% FC) in the greenhouse for six weeks without U V - B radiation  34  Fig. 3 Broccoli seedlings grown under 4 kJ m" d" U V - B and severe (40% FC); moderate (60% FC); mild (80% FC) and no SMS (100% FC) in the greenhouse for six weeks. 2  1  35  Fig. 4 Broccoli seedlings grown under 7 kJ m" d" U V - B and severe (40% FC); moderate (60% FC); mild (80% FC) and no SMS (100% FC) in the greenhouse for six weeks. 2  1  Fig.5 Broccoli seedlings grown under 11 kJ m" d" U V - B and severe (40% FC); moderate (60% FC); mild (80% FC) and no SMS (100% FC) in the greenhouse for six weeks 2  1  37  also contributed to reduce the number of leaves. Leaf biomass (Table 9) increased under the combination of stresses compared to SMS while leaf area (Table 10) decreased with increase in SMS and U V - B radiation. Severe SMS alone reduced leaf biomass by more than 70% compared to control. Under the combination of stresses the percentage of reduction in leaf biomass, compared to control as well as U V - B irradiated plants without SMS, was less than soil moisture stressed plants without U V - B (Table 9).  Table 8. Effects of U V - B radiation and SMS on leaf number of broccoli seedlings grown in greenhouse for six weeks. Soil U V - B level (kJ m" d" ) moisture 0 4 7 11 2  SI S2 S3 S4  9± 8± 5± 4±  0.7a 0.4b 0.7c 0.4d  9± 8± 6± 4±  0.4a 0.4b 0.4c 0.4d  8 7 6 5  ± ± ± ±  1  0.4a 0.4a 0.4b 0.4c  8± 7± 5± 4±  0.4a 0.4b 0.4c O.Od  Values are means ± SE of 8 replicates pooled from two experiments. Means in each column followed by the same letter are not significantly different at P # 0.05. SI = No SMS; S2 = mild SMS; S3 = moderate SMS; S4 = severe SMS  Table 9. Effects of U V - B radiation SMS on fresh leaf biomass (g) broccoli seedlings grown in greenhouse for six weeks. Soil U V - B level (kJ m" d" ) moisture 0 4 7 11 2  SI S2 S3 S4  6.4 ± 0.18a 4.1 ± 0.32b 2.1 ± 0.38c 1.9±0.07d  6.4 5.1 3.4 2.8  ± 1.02a ± 0.70a ±0.17b ± 0.25c  1  7.4 ± 1.15a 4.9 ± 0.57b 3.0±0.07c 2.5±0.35c  5.7 5.8 3.5 2.1  Values are means ± SE of 8 replicates pooled from two experiments. Means in each column followed by the same letter are not significantly different at P # 0.05. SI = No SMS; S2 = mild SMS; S3 = moderate SMS; S4 = severe SMS  ± 0.47a ± 0.42a ± 0.50b ±0.20c  38  Under the combination of stresses U V - B caused significant reduction in leaf area. (Table 10). U V - B alone reduced leaf area by about 25% in plants grown under enhanced U V - B radiation with no SMS. The percentage of reduction varied under different levels of SMS. Under high U V - B and severe SMS, leaf area was reduced by more than 75% compared to the control. Severe SMS alone reduced leaf area by 64% compared to control.  Table 10. Effects of U V - B radiation and SMS on total leaf area (cm" ) of broccoli seedlings grown in greenhouse for six weeks. ^ Soil U V - B level (kJ m" d"') moisture 0 4 7 2  2  11  51  2 6 0 . 9 ± 9.2a  2 0 7 . 9 ± 5.6a  2 3 0 . 6 ± 6.2a  196.6 ± 5.4a  52  1 9 9 . 9 ± 4.3b  168.3 ± 4.9b  190.5 ± 4.7b  1 4 1 . 9 ± 3.4b  53  118.6 ± 2.1c  103.7 ± 2 . 8 c  101.9 ± 0 . 8 c  98.6 ± 2.1c  54  92.7 ± 1.8d  75.4 ± 1.9d  83.2 ± 2.8d  65.9 ± 1.8d  Values are means ± SE of 8 replicates pooled from two experiments. Means in each column followed by the same letter are not significantly different at P # 0.05. SI = No SMS; S2 = mild SMS; S3 = moderate SMS; S4 = severe SMS  5.2 Effects of UV-B radiation and SMS on percent water content Soil moisture stress decreased the percent water content in broccoli seedlings (Table 11), but in the presence of U V - B radiation there was no remarkable change in the percent water content among the seedlings. Under the combination of stresses, increased U V - B increased percent water content compared to SMS. Water content was increased approximately by 10% in severely water-stressed plants with enhanced U V - B radiation compared to plants under severe SMS alone (Table 11). U V - B radiation maintained the percent water content in broccoli seedlings under water stressed conditions. The  39  percentage of water content reduction was only 1.1% under high U V - B and severe SMS compared to control, while it was reduced by about 10% under severe SMS alone.  Table 11. Effects of U V - B radiation and SMS on the percent water content of broccoli seedlings grown in greenhouse for six weeks. ^ Soil U V - B level (kJ m" d" ) moisture 0 4 7 11 2  1  SI  87.8 ± 0.54a  88.8 ± 1.3a  87.8 ± 1.2a  88.2 ± 1.4a  S2  84.5 ± 0 . 3 7 a  8 5 . 7 ± 1.1a  86.5 ± 0.78b  87.1 ± 1.1a  S3  80.3 ± 0 . 4 1 a  84.9 ± 1.3a  83.7 ± 0.89b  85.7 ± 1.4ab  S4  78.9 ± 0.59b  84.5 ± 0.86a  85.6 ± 1. l a b  86.2 ± 1.0b  Values are means ± SE of 8 replicates pooled from two experiments. Means in each column followed by the same letter are not significantly different at P # 0.05. SI = No SMS; S2 = mild SMS; S3 = moderate SMS; S4 = severe SMS  5.3 Effects of UV-B radiation and SMS on growth indices SLW (a measure of leaf thickness and/or density); was influenced by U V - B radiation, SMS and their interaction (Table 12). Plants grown under severe SMS and enhanced U V - B showed more than 45% increase in SLW compared to control (Table 12). Severe SMS alone increased SLW by 25%. Well watered plants under 4 kJ m" d'  Table 12. Effects of U V - B radiation and SMS on specific leaf weight (g m ' ) of broccoli seedlings grown in greenhouse for six weeks. U V - B level (kJ m d" ) Soil moisture (V/W) 11 4 7 0 2  SI  40.1 ± 2.1a  S2 S3 S4  1  46.4 ± 3.2a  43.2 ± 4.1a  4 1 . 2 ± 2.2a  42.4 ± 2.1b  49.3 ± 1.1a  4 0 . 7 ± 1.1a  4 7 . 6 ± 2.1b  49.2 ± 1.1c  52.5 ± 3.1a  49.3 ± 2.1a  5 6 . 4 ± 1.1c  50.5 ± 1.1c  55.1 ± 4.0a  50.7 ± 6.1b  58.1 ± 2.2c  Values are means ± SE of 8 replicates pooled from two experiments. Means in each column followed by the same letter are not significantly different at P # 0.05. S1 = No SMS; S2 = mild SMS; S3 = moderate SMS; S4 = severe SMS  40  U V - B showed about 16% increase in SLW compared to control. Under the combination of stresses, with the exception of 7 kJ m' d" U V - B , SLW increased with increase in U V 2  1  B and SMS. Under 7 kJ m" d" of U V - B , the combination of stresses didn't have much 2  1  effect on SLW compared to SMS. In water stressed conditions, U V - B increased SLW of broccoli seedlings compared to well watered plants without U V - B . U V - B radiation, SMS and their interaction all had significant effects on SRR (a measure of above vs below ground biomass). U V - B increased SRR compared to control, while SMS reduced SRR (Table 13). The combination of U V - B and mild SMS increased SRR compared to control as well as mild SMS alone. U V - B radiation with moderate and severe SMS significantly reduced SRR compared to control as well as U V B irradiated plants without SMS. Broccoli seedlings grown under enhanced U V - B and severe SMS showed more than 65% reduction in SRR compared to high U V - B treated plants without SMS, about 45% reduction compared to control and about 11% reduction compared to severe SMS alone (Table 13).  Table 13. Effects of U V - B radiation and SMS on shoot:root dry weight ratio of broccoli seedlings grown in greenhouse for six weeks. ^ Soil U V - B level (kJ nf d' ) moisture 0 4 7 11 2  51  3.5 ± 0.09a  1  5.1 ± 0.24a  5.1 ± 0.15a  5.3 ± 0.31a  52  3.1 ± 0.07a  4.9 ± 0.15a  4.4 ± 0.23b  4.4 ± 0.23a  53  2.7 ± 0.08b  2.9 ± 0.13b  2.7 ± 0.18c  2.8 ± 0.05b  54  2.2 ± 0.17c  1.9 ± 0.06b  2.1 ± 0.07c  1.9 ± 0.08c  Values are means ± SE of 8 replicates pooled from two experiments. Means in each column followed by the same letter are not significantly different at P # 0.05. SI =No SMS; S2 = mild SMS; S3 = moderate SMS; S4 = severe SMS  41  L A R (a morphological index of leafiness) was also significantly reduced by the interaction of U V - B and SMS (Table 14). SMS reduced L A R by about 11%. Under severe SMS and enhanced U V - B , the reduction was more than 20% compared to control. Enhanced U V - B radiation reduced L A R by 15% compared to control. However under 7 kJ m" d" of U V - B , the percentage of reduction was less compared to ambient and high 2  1  U V - B levels.  Table 14. Effects of U V - B radiation and water stress on leaf area ratio (cm" g" ) of broccoli seedlings grown in greenhouse for six weeks. ^ Soil moisture U V - B level (kJ m d" ) (V/W) 0 4 7 2  2  1  1  11  SI  144.9 ± 8.2a  122.3  ± 7.1a  135.6 ± 6.2a  122.9 ± 4.7a  S2  137.6 ± 3.4b  120.2 ± 5.9b  133.2 ± 2.8b  S3  131.8  ± 2.7c  126.5  ± 3.3c  127.3 ± 1.8c  S4  128.6 ± 1.7d  125.1  ± 1.9a  124.0  119.6±2.1b 117.8 ± 4.9c 115.6 ± 1.8d  ±2.7d  Values are means ± SE of 8 replicates pooled from two experiments. Means in each column followed by the same letter are not significantly different at P # 0.05. SI = No SMS; S2 = mild SMS; S3 = moderate SMS; S4 = severe SMS  Table 15. Effects of U V - B radiation and water stress on leaf weight ratio of broccoli seedlings grown in greenhouse for six weeks. U V - B level (kJ m d" ) Soil moisture (V/W) 11 4 7 0 1  SI S2 S3 S4  0.63 ± 0.04a 0.55 ± 0.02b 0.59 ± 0.02c 0.60 ± 0.03c  0.64 ± 0.65 ± 0.66 ± 0.68 ±  0.03a 0.05a 0.02b 0.02c  0.60 ± 0.69 ± 0.75 ± 0.70 ±  1  0.02a 0.02b 0.03c 0.02b  0.70 ± 0.01a 0.95 ± 0.03b 0.87 ± 0.02c 0.92 ± 0.03d  Values are means ± SE of 8 replicates pooled from two experiments. Means in each column followed by the same letter are not significantly different at P # 0.05. SI = No SMS; S2 = mild SMS; S3 = moderate SMS; S4 = severe SMS  42  Under the combination of U V - B and SMS, L W R (leaf weight, compared with the total plant dry weight) increased significantly (Table 15). Enhanced U V - B and severe water stress brought more than 46% increase in leaf weight ratio compared to well watered plants without U V - B and more than 53% compared to plants grown under severe water stress alone.  5.4 Effects of UV-B radiation and SMS on epicuticular wax content In both the experiments, U V - B radiation, SMS and their interaction had significant effects on epicuticular wax production in broccoli seedlings (Table 16). However, SMS had greater effect on epicuticular wax production compared to U V - B stress (Fig.6). The combination of stresses, significantly increased the production of epicuticular wax, compared to the control plants as well as plants stressed with U V - B alone (Table 16). Plants grown under elevated U V - B radiation and severe water stress produced three times more wax than the control.  Table 16. Effects of U V - B radiation and water stress on epicuticular wax content/dry wt (pg/mg) of broccoli seedlings grown in greenhouse for six weeks. Soil moisture U V - B level (kJ m d"') (V/W) 0 4 7 11 z  51 52 53  54  0.30  ± 0.02a  0.44 ± 0.02b 0.66 ± 0.07c 1.25 ± 0.18d  ± 0.04a  0.38 ± 0.03a  0.41  0.51 ± 0 . 1 lb 0.75 ± 0.04b 0.95 ± 0.06c  0.33  0.59 ± 0.41b 0.86 ± 0.11c 0.96 ± 0.12c  0.69 ± 0.09a 0.95 ± 0.22b 0.98 ± 0.11c  Values are means ± SE of 8 replicates pooled from two experiments. Means in each column followed by the same letter are not significantly different at P # 0.05. SI = No SMS; S2 = mild SMS; S3 = moderate SMS; S4 = severe SMS  ± 0.03a  43  Fig. 8 Effects of U V - B radiation and SMS on epicuticular wax per unit leaf area (cm") of broccoli seedlings grown in the greenhouse for six weeks. Values are means ± SE of 8 replicates pooled from two experiments. SI = no SMS; S2 = mild SMS; S3 = moderate SMS; S4 = severe SMS  44  5.5 Effects of U V - B radiation and S M S on absorbance at 300 nm In both the experiments, U V - B radiation, SMS and their interaction had significant effects on the production of U V - B absorbing compounds in broccoli seedling leaves (Fig. 7). Absorbance at 300 nm was significantly higher in U V - B irradiated plants. Enhanced U V - B radiation alone produced 15% more U V - B absorbing compounds compared to control. Enhanced U V - B irradiation with severe SMS caused a more than two fold increase in the concentration of U V - B absorbing compounds compared to plants grown under severe water stress alone, and about 65% more than the control. Severe water stress decreased the production of U V - B absorbing compounds by more than 35% compared to the control (Fig. 7). The relative concentration of U V - B absorbing compounds increased under the combination of stresses as well as with U V - B (Table 17). SMS significantly reduced the relative concentration of U V - B absorbing compounds in the leaves. Severe SMS alone decreased it by more than 35% compared to control and more than 40% compared to enhanced U V - B alone. Under severe SMS, U V B increased the concentration of U V - B absorbing compounds in the leaf.  Table 17. Effects of U V - B radiation and SMS on absorbance at 300 nm/dry wt (mg) of broccoli seedling leaves grown in greenhouse for six weeks. Soil moisture U V - B level (kJ m' d' ) (V/W) 0 4 7 11 1  51 52 53 54  0.074 ± 0.002a 0.070 ± 0.001a 0.065 ± 0.004b 0.048 ± 0.006b  0.08 ± 0.001a 0.08 ± 0.004b 0.09 ± 0.003c 0.10 ± 0.002c  1  0.08 ± 0.002a 0.09 ± 0.001b 0.09 ± 0.004c 0.10±0.004d  0.09 ± 0.002a 0.10±0.004ab 0.11 ± 0.005c 0.12±0.004d  Values are means ± SE of 8 replicates pooled from two experiments. Means in each column followed by the same letter are not significantly different at P # 0.05. SI = No SMS; S2 = mild SMS; S3 = moderate SMS; S4 = severe SMS  45  ~  CM  0.30  E CO CL)  co ro  0.25  0.20  0)  E c  o o co  +-> CO CD  0.15  0.10 A  o  c CO  .Q 1_  0.05  O  tf)  <  0.00  UV-B level (kJ m" d" ) 2  1  Fig. 7 Effects of U V - B and SMS on the absorbance at 300 nm/per unit leaf area (cm") of broccoli seedlings grown in the greenhouse for six weeks. Values are means ± SE of 8 replicates pooled from two experiments. SI = No SMS; S2 = mild SMS; S3 = moderate SMS; S4 = severe SMS  46  5.6 Effect of UV-B radiation and SMS on W Water stressed plants had considerably lower  i.e. more negative tension than  well-watered plants. Severe SMS alone decreased ¥ of broccoli seedlings by more than 2 fold compared to control (Fig. 8). Under the combination of stresses, U V - B irradiated seedlings had higher  than the non-irradiated ones. In well-watered conditions, U V - B  radiation lowered seedling 4* compared to the control. Under severe SMS, 4 and 7 kJ m d" of U V - B increased *P by 17% and 21% respectively compared to plants grown under 1  2 1 severe SMS without U V - B . When the stresses were too severe (11 kJ m" d" of U V - B and severe SMS), U V - B radiation had little or no effect on the *F of broccoli seedlings.  47  1.6  UV-B level (kJ m" d" ) 2  1  Fig. 8 Effects of U V - B radiation and SMS on plant water potential of U V - B seedlings grown in the greenhouse for six weeks. Values are means ± SE of 8 replicates pooled from two experiments. SI = no SMS; S2 = mild SMS; S3 = moderate SMS; S4 = severe SMS.  48  6.0 Discussion The effectiveness of U V - B radiation on broccoli seedlings was dependent on SMS levels. Interactions between U V - B radiation and SMS substantially affected seedling growth and induced certain beneficial as well as inhibitory effects on them. In most of the parameters measured, the combination of stresses seemed to induce considerable protective effects to prevent the damage caused by both U V - B and SMS; such as increases in epicuticular wax content, U V - B absorbing compounds and specific leaf weight, and reductions in leaf area and number. In water-stressed conditions, U V - B irradiated seedlings had higher  than the non-irradiated ones except plants grown under  7 kJm d"' U V - B and moderate SMS. Growth parameters like plant height, fresh biomass 2  and dry biomass were reduced under the combination of stresses.  6.1 Effects of UV-B and SMS on seedling growth 6.1.1 Leaf area Plant leaves are the most sensitive part to environmental stresses (Teramura, 1983). Leaf area is important to plants since it affects growth and productivity. The significant reduction in leaf area under the combination of stresses (Table 10) defends plants against both U V - B and SMS. The smaller the leaf area, the lesser will be the amount of water transpired. It has been reported that under conditions of SMS, reduced leaf expansion is beneficial to plants since it leads to reduced transpiration (Hopkins, 1999). If leaf enlargement is reduced even to a small extent, a corresponding reduction in plant growth is likely (Caldwell, 1971). The reduced leaf area under the combination of stresses could result in reduced plant water loss and consequently a slower reduction in soil water  49  content. The slow drying of soil under U V - B irradiation might cause a delay in the development of water stress. U V - B radiation has been reported to delay and reduce the severity of drought through reduction in plant water loss, stomatal conductance and leaf area (Nogues et al. 1998). Reduced leaf area, smaller leaves and leaf curling all will reduce radiation absorption. Leaf abscission observed in the seedlings is a morphogenetic response that helps to reduce the leaf area exposed to U V - B radiation. The significant reduction in the number of leaves under the combined effect (Table 8), might also be a defense against both SMS and U V - B leading to reduced leaf area causing reduced evapotranspiration and radiation absorption.  6.1.2 Root growth Root biomass is an important factor in agriculture, since it affects the growth and productivity of crop plants. U V - B radiation significantly reduced root biomass production in broccoli seedling (Table 4) indicating greater sensitivity of the roots (indirectly) to this radiation. On the other hand, root length increased under the combination of stresses (Table 5). Roots are generally less sensitive to SMS than shoots (Hopkins, 1999). The high ¥ under the combination of stresses (Fig. 8), might have enhanced relative root growth by changing the root:shoot ratio in favor of root growth and improved the capacity of the roots to absorb more water from the soil that remained moist. Root extension into deeper wetter soil is a defense against drought.  50  6.1.3 Plant biomass Total biomass accumulation is a good indicator of U V - B radiation effects on growth since it represents the plant's overall fitness to cope with an unfavourable environment (Teramura, 1983). The dry matter accumulation was significantly reduced in plants grown under the combination of U V - B radiation and SMS (Fig. 1). SMS alone reduced plant dry matter accumulation by about 56%. Under severe SMS and enhanced U V - B radiation, the percentage of reduction was more than 70% compared to the control. This substantial growth reduction might have masked the effect of U V - B radiation under SMS. When growth is restricted, other mechanisms like photoreactivation and excision and post-replication repair might help to amend the U V - B induced damage before it becomes lethal (Beggs et al. 1986)  6.2 Effect of UV-B radiation and SMS on growth indices A n increase in SLW is considered a general response to U V - B radiation (Teramura, 1983; Tevini and Teramura, 1989). Severe water stress alone increased SLW by 25% whereas severe water stress together with enhanced U V - B increased S L W by 45%o compared to control (Table 12). Leaf thickness has been suggested to reduce U V penetration, due to greater tissue absorption, increasing the length of the U V - B screening pathway (Murali and Teramura, 1986). SLW is reported to be related to drought tolerance in several crops (Crop Updates, 1998). A n increase in S L W might be related to an increase in relative water content in the leaves. Since an increase in SLW is accompanied by an increase in leaf biomass (Table 9) the increase in L W R (Table 15) and a decline in L A R (Table 14) of broccoli seedlings grown under the combination of stresses may be related to an increase in SLW.  51  Reduction in L A R accompanied by an increase in SLW has also been reported in soybean (Teramura and Sullivan, 1987). The SRR declined under the combination of stresses (Table 13). A shift in shootroot biomass allocation happened due to U V - B radiation. It has been reported that U V - B radiation may alter plant biomass partitioning which may alter shoot:root ratios (Biggs and Kossuth, 1978). A decrease in shoot:root ratio under SMS conditions is advantageous since it improves the capacity of roots to absorb more water by growing down to much deeper soil.  6.3 Effect of UV-B radiation and SMS on percent water content SMS reduced percent water content in broccoli seedlings. In well watered plants U V - B didn't have any effect on percent water content. Under the combination of stresses, U V - B radiation maintained the percent water content in broccoli seedlings, which could be considered a protective effect to prevent the damage caused by SMS. Even under severe drought conditions, U V - B reduced the effect of SMS on the seedlings by maintaining water content (Table 11).  6.4 Effect of UV-B and SMS on absorbance at 300 nm The higher concentration of U V - B absorbing compounds found in broccoli leaves grown under the combination of stresses is a typical response against both U V - B and water stress (Caldwell et al. 1983; Murali and Teramura, 1985; Balakumar et al. 1997; L i et al. 1993). The increased concentration of U V - B absorbing compounds in leaves reduces transmittance of U V - B radiation through the epidermis and protects U V - B sensitive organelles such as chloroplasts in the mesophyll tissues (Robberecht and Caldwell, 1983; Caldwell et al. 1983; Wellmann, 1982; Flint et al. 1985, Tevini et al.  52  1991, L i et al. 1993; Cen and Bornman, 1993; Day and Vogelman, 1995 and Rozema et al. 1997). It has been reported that the accumulation of U V - B absorbing compounds may provide further protection against U V - B radiation (Flint and Caldwell, 1984; Rao and Ormrod, 1995). At the seedling stage, an increase in U V - B absorbing compounds will help the plant to get acclimatized with the increasing levels of U V - B radiation. The absorbance at 300 nm in methanol extracts of leaves was expressed on the basis of leaf area (Fig. 7) as well as dry weight. (Table 17). Absorbance per unit dry weight indicates the accumulation of U V - B absorbing compounds within the tissue, and absorbance per unit leaf area indicates the capacity of these compounds to reduce the U V radiation within the leaf (Flint et al. 1985). Drought has been reported to stimulate the accumulation of U V - B absorbing compounds in some plants (Murali and Teramura, 1985; Balakumar et al. 1997). The significant increase in the accumulation of foliar U V B absorbing compounds in broccoli seedlings grown under U V - B radiation without SMS, and the significant reduction in the accumulation of U V - B absorbing compounds in the seedlings grown under SMS without U V - B radiation, strengthens the report that the significant increase in U V - B absorbing compounds under the combination of stresses is related to the plant's sensitivity to U V - B radiation (Sachs and Hio, 1986; Sullivan and Teramura, 1989; Tevini and Teramura, 1989).  6.5 Effect of UV-B radiation and SMS on epicuticular wax production Like U V - B absorbing compounds, epicuticular wax also plays an important role in reducing the deleterious effects of enhanced U V - B radiation. Since epicuticular wax constitutes an interface between the plant and its environment, it is the first recipient of  53  incident radiation (Steinmuller and Tevini, 1986). Since it increases the reflection and scatter of light away from the leaf surface (Tevini and Steinmuller, 1985), an increase in epicuticular wax content under a combination of U V - B and SMS could be a defense against U V - B radiation. It has been reported that the reflectance of leaves from which wax has been mechanically removed is much reduced (Steinmuller and Tevini, 1986, Sinclair and Thomas, 1970 and Eller and Willi, 1977). Although the leaf surface reflectance is only less than 5% of light, it will help to reflect the incident U V - B radiation away from the surface of the leaves before it reaches the mesophyll tissues (Caldwell et al. 1983). Under the combination of stresses, the amount of wax deposited per unit leaf area was significantly higher than the control as well as U V - B irradiated plants with no SMS (Fig. 6). Plants grown under elevated U V - B radiation and severe SMS produced three times more wax than the control, which could offer defense against both U V - B and SMS. The additional deposition of wax under enhanced U V - B radiation could reduce water loss from the leaf tissues (Derma, 1970). The reduced leaf area under the combination of stresses does not explain greater deposition of wax on the leaf surface because the amount of wax per unit dry weight also increased under the combination of stresses (Table 16). It is obvious that the water-stressed plants are benefited by the increase in epicuticular wax content since it reduces epicuticular permeability and therefore transpiration (Hall and Jones, 1961; Martin and Juniper, 1970; Sinclair and Thomas, 1970; Denna, 1970; Eller and Willi, 1977). While cuticular transpiration accounts for only 5-10% of the total leaf transpiration under normal conditions (Taiz and Zeiger,  54  1998), it can be critically important to plants that are under extreme stress. Reduced evapotranspiration and increased water use efficiency caused by the additional deposition of epicuticular wax under a combination of stresses might improve plant SMS tolerance and help to maintain their growth and productivity. The additional deposition of U V - B absorbing compounds and epicuticular wax may have contributed to the resistance of plants to both U V - B and SMS, by reducing absorbed radiation and transpiration (Saneka and Ogata, 1987).  6.6 Effect of UV-B radiation and SMS on *F To take up water, the 4* of the plants must be lower than the 4* of the soil. Broccoli seedlings grown under the combination of U V - B radiation and SMS had higher 4 than /  those grown under SMS without U V - B , and lower 4* than the control as well as well watered U V irradiated plants (Fig. 8). The lower the 4* of the plants, the greater the effect on drought tolerance. U V - B radiation might have increased the potential for osmotic adjustment, by lowering solute potential in broccoli seedlings. It has been reported that a relatively low 4* is a significant acclimation to SMS since it moves more s  water into leaves increasing their turgor pressure (Hopkins, 1999). Under each level of U V - B radiation, the seedling ¥ decreased with increase in SMS. When the stresses were not too severe, the interaction brought positive effects, while U V - B had a negative effect when it was extreme. Under extreme conditions of U V - B and SMS, U V - B had little or no effect on the 4* of broccoli seedlings, since it had been masked or reduced by severe SMS when growth was already reduced.  55  A l l these evidences support the fact that exposure to higher levels of U V - B radiation can alter the normal growth and development of economically important plants and that the effect of U V - B radiation can be modified by other environmental stresses like SMS. Under the combination of stresses, the effects of U V - B radiation and SMS stress were much less on broccoli seedlings compared to individual stress conditions. This decreased sensitivity might be due to reduced growth, increase in U V - B absorbing compounds and epicuticular wax in the leaves, leaf thickening and changes in the components of water potential.  During the seedling stage, individual leaflets might have received more U V - B radiation, but subsequently due to self-shading, the amount of radiation reaching the surface of the leaves could be less (Teramura and Sullivan, 1987). In order to determine the possibility of the long-term effects of increased U V - B radiation on crop plants, studies have to be conducted to maturation of the plant to know whether the sensitivity is only during the vegetative stage or persists until the reproductive stage. It has been suggested that drought can cause changes in plant development from vegetative to reproductive stages (Desclaux and Roumet, 1996).  Since agriculture depends mainly on weather and climate, it is important to know the responses of plants to changing environmental conditions. For more realistic interpretation of the hypothesis that interaction between U V - B radiation and SMS bring beneficial effects to crop plants, long term field studies are essential. Field studies would help us understand whether the yield of the crop is increased or decreased by the interaction between U V - B and SMS; or i f it causes early ripening or reduction in the quantity or quality of the fruits etc.  56  7.0 Conclusions  The conclusions drawn from this study support the hypothesis of my thesis that there is interaction between U V - B radiation and SMS and that U V - B absorbing compounds, epicuticular wax and components of 4* play an important role in their interaction. It also strengthens the suggestion that under the combination of stresses, U V B radiation and SMS contribute mutually to the welfare of plants (Cen and Bornman, 1990). This thesis is significant because it increases our knowledge about the roles of epicuticular wax, U V - B absorbing compounds and the components of 4* in this interaction which have not been investigated in a single study so far. Under the combination of stresses, production of epicuticular wax content (Table 16) and U V - B absorbing compounds (Table 17) in the leaves were significantly increased causing a reduction in U V - B transmittance as well as water-loss, which concurrently reduce the deleterious effects of U V - B and SMS. U V - B radiation might have increased the potential for osmotic adjustment in broccoli seedlings by lowering T , and might have helped s  seedlings to take up more water and maintain their turgor. Though the long-term protective function of U V - B absorbing compounds has not been studied yet, in the seedling stage an increase in U V - B absorbing compounds is important because it presumably helps the seedlings to get acclimatized to the increasing U V - B radiation.  It is important to note the limitations of this study since it was done in the greenhouse environment only. Field grown plants face several stress factors simultaneously. It has been reported that the effects of U V - B radiation can be altered by other environmental factors like PAR, C 0 , nutrient availability, etc (Teramura, 1980; 2  57  Warner and Caldwell, 1983; Mirecki and Teramura, 1984; Murali and Teramura, 1985; Strain and Cure, 1985; Cen and Bornman, 1990; Bornman and Teramura, 1993; Adamse and Britz, 1992; Rozema et al. 1993; Sullivan and Teramura, 1994; Rozema et al. 1997). Furthermore, in the greenhous, plants were grown under artificial U V - B radiation which differs from sunlight in its spectral composition. P A R level in the greenhouse was also lower than under field conditions. On the other hand, despite these limitations, this greenhouse study enabled a better understanding of the fundamental processes involved in the interaction of U V - B radiation and SMS under controlled conditions, without the interference of other stress factors.  Since my studies were focussed only on the seedling stage of broccoli, nothing is known about what will happen to the economic yield of the crop i f it is grown under the combination of U V - B radiation and SMS. It will be interesting to extend this study up to the yield stage to see the harvestable productivity of the plant.  58  Table 18. a. A N O V A results for the effect of U V - B radiation and SMS on root length, root fresh biomass, root dry biomass and plant height of broccoli seedlings grown in the green house for six weeks. Results drawn from two experiments. P value for the Parameters measured Source of variation  df RTLG  RTFB  RTDB  PH 0.544 NS  Expt  1  0.088 NS  0.935 NS  0.980 N S  UVB  3  0.000 * *  0.000 * *  0.000 * *  SMS  3  0.000**  0.000**  0.000**  0.000**  Rep  3  0.054 NS  0.249 NS  0.061 NS  0.998 NS  Rep x SMS  9  0.824 NS  0.170 NS  0.446 NS  0.827 NS  Rep x U V B  9  0.775 NS  0.588 NS  0.461 NS  0.274 NS  U V B x SMS  9  0.000 * *  0.000 * *  0.000 * *  Expt x U V B  3  0.113 NS  0.974 NS  0.193 NS  0.077 NS  Expt x SMS  3  0.268 NS  0.053 NS  0.484 NS  0.137 NS  Rep x SMS x U V B  27  0.438 NS  0.114 NS  0.821 NS .  0.720 NS  Expt x SMS x U V B  9  0.923 NS  0.068 NS  0.957 NS  0.247 NS  Error  48  R T L G = root length, RTFB = root fresh biomass, RTDB = root dry biomass and PH = plant height. Significant at P = 0.01 and NS = not significant  0.000 * *  0.000 * *  59  Table 18. b. A N O V A results for the effect of U V - B radiation and SMS on leaf number, leaf fresh biomass, leaf area and total fresh biomass of broccoli seedlings grown in the green house for six weeks. Results drawn from two experiments. P value for the Parameters measured Source of variation  df LNO  LFB  LA  FRB NS  Expt  1  0.272  UVB  3  0.040 *  0.000 * *  0.000 * *  0.000 * *  SMS  3  0.000 * *  0.000 * *  0.000 * *  0.000 * *  Rep  3  0.101  NS  0.060  NS  0.156  NS  Rep x SMS  9  0.073  NS  0.064  NS  0.586  NS  0.933  NS  Rep  9  0.610  NS  0.059  0.057  NS  0.055  NS  SMS  9  0.000  **  Expt x U V B  3  0.193  Expt x SMS  3  UVB  Rep  UVB  x  x  x  SMS  x  UVB  Expt x SMS x U V B Error  NS  NS  0.235  NS  0.000 * *  NS  0.886  0.000 * *  0.102  0.058  NS  0.000 * *  NS ,  0.169  NS  0.158  NS  0.624  NS  0.449  NS  0.966  NS  0.695  NS  0.533  NS  27  0.936  NS  0.455  NS  0.827  NS  0.709  NS  9  0.103  NS  0.191  NS  0.591  NS  48  L N O = leaf number, L F B = leaf fresh biomass, L A = leaf area and FRB = total fresh biomass * * Significant at P = 0.01, * Significant at P = 0.05 and NS = not significant  0.936  NS  60  Table 18. c. A N O V A results for the effect of U V - B radiation and SMS on growth indices of broccoli seedlings grown in the green house for six weeks. Results drawn from two experiments. P value for the Parameters measured Source of variation  df SLW  LAR  LWR  SRR  0.199 NS  0.793 NS  0.062 NS  Expt  1  0.564 NS  UVB  3  0.000**  SMS  3  0.000 * *  0.000 * *  0.000 * *  0.000 * *  Rep  3  0.178 NS  0.286 NS  0.665 NS  0.063 NS  Rep x SMS  9  0.213 NS  0.459 NS  0.485 NS  0.056 NS  Rep x U V B  9  0.391 NS  0.401 NS  0.059 NS  0.100 NS  U V B x SMS  9  0.000 **  0.001 **  0.000 * *  0.000 * *  Expt x U V B  • 3  0.1 IONS  0.540 NS  0.638 NS  0.107 NS  Expt x SMS  3  0.197 NS  0.509 NS  0.166 NS  0.310NS  Rep x SMS x U V B  27  0.066 NS  0.458 NS  0.165 NS  0.057 NS  Expt x SMS x U V B  9  0.590 NS  0.468 NS  0.102 NS  0.180 NS  Error  48  0.000 * *  0.000 * *  0.000 * *  SLW = specific leaf weight, L A R = leaf area ratio, L W R = leaf weight ratio and SRR = shoot:root ratio * * Significant at P = 0.01 and NS = not significant  61  Table 18. d. A N O V A results for the effect of U V - B radiation and SMS on dry biomass, percent water content, absorbance at 3 0 0 nm per leaf area and absorbance at 3 0 0 nm per dry weight of broccoli seedlings grown in the green house for six weeks. Results drawn from two experiments. P value for the Parameters measured Source of variation  df DRB  %WC NS  ABS/LA  ABS/DW  NS  Expt  1  0.068  UVB  3  0.000 **  0.000 * *  0.000 * *  0.030 *  SMS  3  0.000 * *  0.000**  0.033 *  0.047 *  Rep  3  0.446  NS  0.602  NS  0.452  NS  NS  0.102  0.381  NS  0.626  NS  0.309  Rep  x  SMS  9  0.720  NS  0.130  NS  0.393  NS  0.524  NS  Rep  x  UVB  9  0.240  NS  0.054  NS  0.427  NS  0.529  NS  SMS  9  0.000  **  Expt x U V B  3  0.061  NS  Expt x SMS  3  0.334  NS  27  0.196  NS  0.221  9  0.256  NS  0.202  UVB  Rep  x  x  SMS  x  UVB  Expt x SMS x U V B Error  0.000 * *  0.045 *  0.031 *  0.465  NS  0.375  NS  0.312  NS  0.070  NS  0.351  NS  0.296  NS  NS  0.478  NS  0.438  NS  0.513  NS  NS  0.409  NS  48  DRB = dry biomass, % WC = percent water content, A B S / L A = absorbance at 3 0 0 nm per leaf area and A B S / D W = absorbance at 3 0 0 nm per dry weight. Significant at P = 0.01, * Significant at P = 0.05 and NS = not significant  62  Table 18. e. A N O V A results for the effect of U V - B radiation and SMS on wax per leaf area, wax per dry weight and water potential of broccoli seedlings grown in the green house for six weeks. Results drawn from two experiments.. P value for the Parameters measured Source of variation  df WX/LA  WX/DW  ¥  Expt  1  0.118NS  0.055 NS  0.181 NS  UVB  3  0.000**  0.000 * *  0.003  SMS  3  0.000 * *  0.000 * *  0.000**  Rep  3  0.167 NS  0.115NS  0.686 NS  Rep x SMS  9  0.525 NS  0.434 NS  0.335 NS  Rep x U V B  9  0.059 NS  0.086 NS  0.321 NS  U V B x SMS  9  0.000 **  0.000 * *  Expt x U V B  3  0.207 NS  0.061 NS  0.177 NS  Expt x SMS  3  0.702 NS  0.055 NS  0.710 NS  Rep x SMS x U V B  27  0.896 NS  0.354 NS  0.617 NS  Expt x SMS x U V B  9  0.696 NS  0.730 NS  0.133 NS  Error  J  0.000 * *  48  W X / L A = wax per leaf area, W X / D W = wax per dry weight and *F = water potential. Significant at P = 0.01 and NS = not significant  63  8.0 Literature cited Adamse, P. & Britz, S.J. 1992. 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