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Some morphological and physiological differences between a normal Sitka spruce and a yellow mutant Scott, G.R. Apr 30, 1969

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SOME MORPHOLOGICAL AND PHYSIOLOGICAL DIFFERENCES BETWEEN A NORMAL SITKA SPRUCE AND A YELLOW MUTANT A Thesis submitted in Partial Fulfillment of the Degree of Bachelor of Science in Forestry We accept this thesis as conforming to the required standard The University of British Columbia April, 1969 by G. R. SCOTT ABSTRACT Sun and shade foliage was collected from a normal and mutant Sitka spruce (Picea Sltchensis Bong. Carr) and analyzed as to morpho-logical and physiological differences. The amount of chlorophyll a, cnbrophyll b, and carotene pigments was determined; needle length and width, length of last year's growth, and number of needles on last year's were measured. Pigment content was measured on top and bottom half of a golden scion to indicate within scion variation. Some mutant needles were cut in half, and pigment content measured in both halves to indi-cate within needle variation. Results indicate that the sun foliage of the mutant contains about five times less chlorophylls and slightly more carotene than ntSrmal sun foliage. In shade foliages, the mutant contained about 1.5 times more of the three pigments. The morphological studies show that the mutation causes a stunting of some needle characteristics in the mutant foliage. Previous studies indicate that this genetic mutation either disrupts the production of normal proteinous grana within the chloro-4 plasts or slows the rate of production of the chlorophylls. In the mutant either mechanism is activated by direct sunlight. TABLE OF CONTENTS Page I INTRODUCTION 1 II THE GOLDEN SPRUCE 2 Location of the mutant 2 Description of the mutant 2 III LITERATURE REVIEW 10 IV MATERIALS AND METHODS 18 Material Collection 18 Pigment extraction and analysis 18 Morphological measurements 20 Electron microscopy 21 V RESULTS 22 Pigment Analysis 22 Analysis of Variance - pigment content 22 Duncan's New Multiple Range test - pigment content ? ? ? 25 Needle dimension analysis 26 Analysis of Variance and Duncan's New Multiple Range test 26 VI DISCUSSION 2 9 Pigment analysis Needle morphology analysis 30 VII SUMMARY 3 3 N \ LIST OF ILLUSTRATIONS Figure Page 1 Location of Golden Spruce on Graham Island 3 2 Location of Graham Island 4 3 The Golden Spruce - View from across Yakoun River . . . . 5 3a The Golden Spruce - Aerial View 5 4 Golden Spruce Sun Foliage 6 5 Sun Foliage of Normal and Mutant Spruce 7 6 Underside of Mutant Sun Foliage 7 7 Side View of Mutant Sun Foliage 8 8 Within Needle Variation 9 9 Cross Section of Leaf Primordial 9 10 Chloroplasts of Nicotiana tabacum 12 11 Diagrammatic section through a chloroplast 13 12 The effect of light intensity, darkness and mutant genes on the development of a chloroplast 16 13 The region of the crowns where the five samples of foliage (A-E) were collected 19 14 Amount of plant pigment present in the five sampled foliages 24 LIST OF TABLES Table Page 1 Genetic Systems Controlling Chloroplast Structure and Function 15 2 Pigment Contents in (mg/g.) 23 ACKNOWLEDGEMENT / The author wishes to make grateful acknowledgement for the assistance of Dr. J. Worrall of the Faculty of Forestry, U.B.C. during the course of this study. Without his moral as well as scientific support, this thesis would never have been completed. I - INTRODUCTION Graham Island of the Queen Charlottes is one of the few areas in Canada which partially escaped glaciation during the Pleistocene. On the island, botanists have discovered eleven species of flowering plants previously unknown. Their ancestry is believed to go back beyond the ice age when similar plants on the mainland were wiped out. Any connection between the origin of these exotics and the origin of the !fgolden spruce11, Picea sitchensis(Bong.) Carr. is open to conjecture. The mystery of these Islands and the fact that there is only one of these "golden spruce11 once worshipped by the Haida Indians adds a little spice to the investigation. A scientist would prefer to call the golden individual a nmutant,! meaning !lone that has changed by some genetic variation51. In this thesis the less romantic viewpoint is assumed to be valid, bringing the investigation within the realm of science. This allows the author to make the assumption that previous investigation of mutation in other species may apply to the golden spruce. Mutation causes many visible as well as microscopic functions of plants and animals to change. The golden spruce has a mantle of bright yellow foliage on the outer portions of the crown. In this thesis, the cause and effect of this phenomenon will be investigated as to needle morphology, pigment content and chloroplast structure. II - THE GOLDEN SPRUCE Location of the Mutant T h e Picea sitchensis (Bong.) Carr. ,!aureaK or "Golden Spruce11 is found on Graham Island of the Queen Charlotte Group. (Figure I) Graham Island is approximately 550 air miles NNW of Vancouver, B.C. and 50 miles from the mainland coast (Figure 2). The climate is humid, with approximately 60 inches of precipitation per year. Powell (1965) classifies the island within the "outer coastn climatological region of British Columbia. Rowe (1959) classifies it as nCoast Forest Region, Queen Charlotte Islands section.1' The micro-site is a well-drained silt loam soil (Day, 1957) \tfhich is common on the Yakoun River lowlands. The site productivity is high, around site index 180 at 100 years. The tree is growing in as-sociation with other old growth sitka spruce of approximately the same age. Some young spruce are coming in as understory. Description of the Mutant The !tgolden spruce11 has recently been measured and found to be 160 feet in total height and approximately seven feet in diameter at breast height (Figures 3 and 3a). Increment borings have not been taken but age is estimated at about 400 years. The tree has the shape of a near perfect cone and the foliage of the outer "surf1 leaves (Kramer and Kozlowslci, 1960) is a bright golden yellow (Figure 4). FIGURE 2 - LOCATION OF GRAHAM ISLAND V/JA/Coc/I \?NLMQiO /V FlGUXE i. 5 Figure 3- The Golden Spruce - View from across Yakoun River Figure 3a - The Golden Spruce - Aerial View Figure 4 - Golden spruce Sun Foliage The inner shade foliage of the golden sprufce is green but not as dark as normal shade foliage. Figure 5 shows the difference between mutant sun foliage and normal spruce sun foliage. In this Figure, the outer scions are normal foliage and the three inner scions are from the mutant. The middle scion shows the underside of mutant sun foliage. / Figure 5 - Sun Foliage of Normal and Mutant Spruce Figure 6 shows the underside of the mutant sun foliage in Figure 4. Figure 6 - Underside of Mutant Sun Foliage 8 Figures 4 and 6 show within scion variation in colour of sun leaves from a top view. Figure 7 shows the same variation in the scion of Figures 4 and 6 from a side view. Figure 7 - Side View of Mutant Sun Foliage The previous figures show within scion and between scion variation in colour. Figure 8 shows that there is also within needle variation. The four larger green needles are from normal spruce ana the six yellow to yellow-green needles are from the mutant. Note that mutant needles A show colour variation between longitudinal halves and needles B show variation between top and bottom halves. Figure 8 also indicates a difference in needle dimension. This phenomenon will be discussed later in the thesis. Figure 8 - Within Needle Variation Leaf priniordia xdlthin the next yearTs buds were dissected to see if there was any difference between the mutant and normal. In Figure 9 the bud with the long stalk is from the normal spruce and the two with short stalks are from the mutant. All three show the presence of chlorophyll by their green colour. Figure 9 - Cross Section of Leaf Primordia Ill - LITERATURE REVIEW Photosynthesis in higher plants depends on the presence of the green pigment chlorophyll (Lindstrom, 1920). Curtis (1940) stated that chlorophylls are always found within chloroplasts (plastids) in normal photosynthetic plant cells. Robbins, Weier, and Stocking, (1957) basically agreed but qualified the statement, excepting the blue-green algae and certain bacteria. Von Wettstein (1961) reported that cnloroplast defects have been found in every species of lower and higher plants closely studied. These defects are caused by alteration, destruc-tion, or inactivation of hereditary factors. As many as 60 distinct genetic factors influence chloroplast formation (Curtis, 1940). Srb and Owen (1955) stated that more than twenty different dominant genes in corn affect chlorophyll formation and the absence of any one or a combination of these genes will cause chlorophyll deficiency. Three phenomena can result in different phenotypical expression of physiological or morphological traits. These are mutation, recombina-tion, and environmental modification (Burley, 1965). According to Demerec (1935), chlorophyll deficiency can manifest itself in the seedling stage, the mature stage, or in both. Four plants forms are distinguished by their chlorophyll content. These are albino, virescent, pale-green and variegated. Albinos contain no chlorophyll and die in the seedling stage. Virescents are albinos in the seedling but form chlorophyll and subse-quently develop normally. Pale-green have a reduced quantity of pigment at certain stages of development and variegated forms contain varying amounts of chlorophyll in different parts of the leaf. Some of these forms occur naturally within species while others occur rarely, and are due to mutation. The rate at which chlorophyll is produced may be controlled by genetic or environmental factors. Light and air temperature affects this rate naturally (Kramer and Kozlowski, 1960). Bright light causes the decomposition of chlorophyll (Galston, 1959, Gustafson 1940, Shirley, 1929). Therefore, shade leaves usually have a higher concentration of chlorophyll than sun leaves. Galston (1955) and Walles (1967) suggest that this mechanism is caused by a photo-oxidation reaction. Another effect of light is to orient the plastids within the photosynthetic cells (Zurzychi, 1953). The plastids become arranged so that at low light intensity the broad surface of the plastid will be perpendicular to the direction of light and at hight intensity the broad surface of the plastid will be parallel to the direction of light. This effect maximizes the absorption of light energy at low intensities and avoids the destruct-ive effect of high intensities. Granick, (1955) said that light intensity affects the size and number of palisade cells compared to mesophyli cells. Whileside (1934) mentioned that although the presence of caro-tenoid pigments is an inherent characteristic, environmental conditions have an appreciable effect on the amount of them present. Curtis (1940) found a close parallel between the effects of iron, light, and oxygen and the amount of carotene present. With the aid of the electron microscope the structure of plastids has been studied extensively (von Wettstein 1958, 1959, I960), (see Figure 10). 12 Figure 10 Chloroplasts of j^ icotiana tab a cum, var. Samsun, mesophyll cells. Section through a normal mature chloroplast showing the lamellar structure and the stacks of lamellar disks forming the grana (compare with Fig.19.11 in text). Below. Section through a plastid of a white shoot of an albomaculata variegated plant. The lamellar system has degenerated into vesicles. Magnification: top figure 31,200 x, bottom figure 19,200 x.(After von Wettstein and Ericksson, 1963.) The lamellar structure in plastids acts to concentrate chlorophyll. The molecules of pigment are attached to lipoproteins stabilizing the pigment within the grana. These grana are laminated green structures composed of compartments and connected to other grana by a system of lamellae (see Figure 11). Figure 11 - Diagrammatic section through a chloroplast of a higher plant, according to von Wettstein. The double membrane encloses a lamellar system embedded in a granular stroma. The grana are formed by closely packed stacks of disks, the disks being linked by larger lamellae. The black circles are drops of chromolipoids, called globuli. From D. von Wettstein Presumably, the purpose of this structural arrangement is to facilitate the trapping and transfering of energy during photosynthesis. It has been suggested by MacLachlan and Zalick (1963) that mutation alters the lipoprotein within the grana resulting in the reduction in amount of chlorophyll. These authors further stated that the slow rate of accumu-lation in the mutant may be due to a high rate of chlorophyll destruction 14 or to a partial inhibition of chlorophyll synthesis. A plant does not necessarily die or even show a reduction in vigour when the rate of pigment formation is slow. Studies with virescent cotton mutants by Benedict and Kohel (1968) have revealed that at saturating light intensity the chloroplasts in yellow mutants may be eight times more efficient in C02 fixation than normal green chloroplasts. Schmid and Gaffron (1967a and 1967b) revealed that tobacco mutants containing 1/8 to 1/30 the normal chlorophyll could not compensate their respiration at nor-mal photosynthetic light saturation (5,000-12,000 ergs/sec.?cm2). How-ever, the mutants could assimilate two to three times more C02 at their light saturation of approximately 50,000 ergs/sec#cm2. They further suggested that the aurea variety contains more chlorophyll action centres in relation to total light absorbing pigment, this being due to either smaller units, higher enzyme content, or both. Genes influence the production of enzymes in the chloroplast which trigger a chain of chemical processes. The genes also influence the formation of enzyme inhibitors which control the rate of chemical reaction. Any alteration, destruction, or inactivation of processes or structures by hereditary factors are controlled by the genome, the plastome or the plasmone. Table I shows the genetic systems controlling chloroplast structure and function (from von Wettstein, 1961). 15 Table I - Genetic Systems Controlling Chloroplast Structure and Function Localization Studied With Origin Example GENOME PLASTOME Chromosomes Extranuclear PLASMONE Extranuclear Gene-mutation Deficiencies Mutations Hybrids Gene dependent plastome mut. Variegated forms Spontaneous Barley Induced Corn Spontaneous Nicotiana Spontaneous Oenethera Induced Corn, Barley Spontaneous Humulus According to Benedict and Kohel (1968)the nuclear genes affect the plastid formation by producing activators or inhibitors which affect the activity of the plastid RNA and DNA. It is the plastid RNA and DNA that ultimately controls plastid structure and function and pigment quan-tity and function. Benedict and Kohel also found that the organelles are controlled by an adaptive mechanism which is responsive to light. A great number of genes control the formation of the chloro-plasts (von Wettstein, 1959). If these genes are changed by mutation, the development in the chloroplast structure may cease at a certain stage of differentiation. Figure 12 illustrates the effect of light intensity, darkness and mutation on the development of the chloroplast. Xantha 3, Xantha 10 and Albina are mutants and indicate where differentiation may be stopped. A normal plastid will develop according to the left side of the Figure and a mutant according to the right. 16 Figure 12 - The effect of light intensity, darkness and mutant genes on the development of the chloroplast. (from D. von Wettstein, 1958) In some cases the structural blocks can be associated with the failure of pigment synthesis or the failure to incorporate the pig-ments into the plastid structure. Von Wettstein further found that the plastids in a barley mutant could form the vesicles in a normal manner but could not aggregate the lamellar discs to form the normal grana. He suggests that chlorophyll is not a structural component of the grana, since the grana can form when the amount of chlorophyll has been reduced. Plastids may first develop normally and later be destroyed by cytoplasmic influences. Walles (1967) worked with an aurea mutant of Picea ables (L.) (Karst.) and found that the mutant contained 40% of the amount of chlorophyll found in wild type normal seedlings. This was due to a reduction in the grana-lamellae system. These abnormal chloroplasts were found in needles only to the point at which they turned normal green. Old, dark green needles contained normal chloroplasts. The aurea factor affected plastid development either by suppressing chlorophyll synthesis or by interfering with the grana formation. IV - MATERIALS AND METHODS a - MATERIAL COLLECTION Five samples of foliage x^ ere taken from different regions of the crowns of two Sitka spruce (note Figure 13). The normal spruce selected is about 50 feet from the mutant, is somewhat younger, and slightly taller. The two trees are as close as possible so as to reduce any difference due to environmental variation. The foliage was stored in dark green plastic bags to reduce the effect of light on pigment breakdown. At U.B.C. the foliage was kept in the dark and under refrigeration at 35?F. until used. b PIGMENT EXTRACTION AND ANALYSIS The pigments were extracted and analysed according to Methods of Analysis of the A.0.A.C.(see Reference). Needles were harvested, chopped with a razor blade, and weighed to 1/100 gram. The material was then ground thoroughly in a cold mortar with a small amount of clean sand and CaC03. The extractor used was 100% acetone. Needles were then ground in approximately 10 ml. of the acetone. This was repeated until 50 ml. of acetone was used and the needle material was white. The solution was then centrifuged at 10,000 R.P.M. for five minutes to remove suspended material from the solution. The optical density of the solution was determined using a Unieam scan spectrophotometer. The machine was set to scan the spectrum from 40Qmu to 850mu. This procedure was repeated giving two replications for each of the five samples A, B, C, D, and E. The concentration in mg./g freshweight of chlorophyll a and b were Figure 13 - Golden Spruce sun leaves shade leaves E - Golden middle outer C - Golden lower outer B - Golden lower inner Normal sun leaves .shade leaves A - Green outer D Green inner Figure 13: The region of the crowns where the five samples of foliage (A~E) were collected. calculated using the specific absorption coefficients of MacKinney (1940). ca = (12.3D663 - 0.86D645)V ' lOOOdw Cb = (19.3D645 - 3.6D663)V lOOOdw where: C = concentration in mg/g fresch weight, a = chlorophyll a b = chlorophyll b D = optical density at wavelength indicated. V = final volume of extract d 85 length of light path in cm. W = fresh wt. of needles extracted. The concentration of the carotenoids was calculated using the equation of von Wettstein (1957). Cc - 4.G95D44Q - 0-2680^ where: c = carotenoids MORPHOLOGICAL MEASUREMENTS Certain needle measurements were taken to ascertain if there were any morphological differences as well as pigmentation differences. The variables measured in the five samples (A-E) were: length of last year's growth (cm.), number of needles in last year's growth, needle length, and needle width. From this information number of needles per unit (cm.) length of branch and length-width ratio of needles was determined and compared for the five samples. Microslides of longitudinal sections of buds were taken to see any difference in colour of the needle primordia of the mutant and normal spruce. ELECTRON MICROSCOPY An attempt was made by Dr. R.W. Meyer of the Forest Products Laboratory, U.B.C. to analyse mutant and normal chloroplasts with the aid of an electron microscope. The resulting photographs were blurred and detail was indiscernible, probably due to poor fixation of the chloro-plasts. Time was not available to repeat the experiment. V - RESULTS Pigment Analysis Appendix I, Figure I, (Trial I) and Figure 2 (Trial 2) show the variation of pigment content between the five foliages (A-E) as ana-lysed with the Unicam scan spectrophotometer. The peak at around wave-length 650 millimicrons indicates the presence of the chlorophylls and the peak 400-500 indicates the carotenoids. Appendix I, Figure 3 indicates within needle variation of pigment content between the top and bottom halves of golden outer foliage. Appendix I, Figure 4 indicates between needle variation in pigment content between the top and bottom half of a golden outer scion. Values of absorptance were taken from the graphs at wavelength 440 mu (carotene), 645 mu (chlorophyll b) , and 663 mu (chlorophyll a). The absorptance values were then converted to pigment content (milligrams of pigment per gram of fresh needles) using the formulae of MacKinney (1940) and von Wettstein (1957). Table 2 lists these values for the five foliages and Figure 14 shows these results graphically. The figures for within needle and within scion variation in pigment content are listed in Table 2. Analysis of Variance-pigment content An analysis of variance performed on the data of pigment content of the five foliages indicated that at the 1% and 5% levels, there were significant differences between chlorophyll a, chlorophyll b, ana carotene. The analysis results are: TABLE 2 - PIGMENT CONTENT IN MILLIGRAMS PER GRAM OF THE FIVE FOLIAGES ( A-E ), AND PIGMENT VARIATION WITHIN THE GOLDEN MUTANT FOLIAGES ( F-I ). CHL. a CHL. b CAROTENE A - GREEN OUTER Tl* l.k2k 0.726 2.9^5 T2** 1.53^ O.832 2.93b B - GOLDEN INNER Tl 0.950 O.WK 1.826 T2 1.0^6 O . W 2.169 C - GOLDEN LOWER OUTER Tl 0.257 0.109 ^.316 T2 0.265 0.110 k.koo D - GREEN INNER Tl 1.9^5 1.030 3.938 T2 1.^ 2*+ 0.726 3.200 E - GOLDEN MID. OUTER Tl 0.118 0.06^ 2.862 T2 0.08^ - 0.07^ 2.580 F - UPPER i SCION Tl 0.11k O.Ohb 3.057 G - LOWER i SCION Tl 0.367 O.lMf ^.793 H - LOWER i NEEDLE Tl 0.057 0.181+ 7.779 I - UPPER i NEEDLE Tl 0.278 0.086 W4-09 * T1 is the abbreviation of TRIAL 1 ** T2 is the abbreviation of TRIAL 2 ) Y ) F igure 14 -AMOUNT OF PLANT PIGMENT PRESENT IN M ILLIGRAMS PER G ;RAM OF " ["HE F I VF AMPLED FOI 1 ! t W3ES. 1 V L 0 L_ 1 r / 5 A. U) (D ~o CD C.) 3 M? o E rn L. CD L. <D a 2 i U) E CD I? rr r i i 1 ? 1 2 1 ? 3 O 2 J 1 2 3 5 1 I i 1 1 /J & C D E < Green Outer Golden Inner Goldsa ' Lovse8 Quick Green /nnsp. OOLOFN MlOOUOurfK 1 CHLOROPHYLL A 2 CHLOROPHYLL B 3 n A TDAmiPW B> 25 Chlorophyll a df ss ms F Treatments 4 4,0138 1,0035 34** Error 5 0.1476 o.02952 Chlorophyll b df ss ms F 4 0.963 0.241 7.4* 4 0.130 0.0325 Carotene df ss ms F Treatment 4 2.634 0.658 12.07** Error 5 0.3725 0.0545 Treatment Error Duncan's New Multiple Range test-pigment content The ANOVA showed that there were differences, and the Duncan's test was used to show which means were significantly different. The results of the test are: Chlorophyll a E C B A D 0.101 0.261 0.998 1.479 1-685 Chlorophyll b E C B A D 0.069 0.IO8 0.489 0.726 o.878 Carotene B E A D C 1.997 2.725 2.939 3.569 4.385 * - indicates significance at the 1% level;**: 5% level. 26 Needle dimension analysis The parameters measured were: number of needles on one year's growth, length of one year's growth, needle length ana needle width. The data was analysed by an IBM 360-67 computer which was programmed to cal-culate the number of needles per cm. of growth and the length-width ratio of the needles. The computer then performed an analysis of variance and a Duncan's New Multiple Range test. This was done for each of the five foliages. The needle measurement data is listed in Tables I and 5 of Appendix 2. The results of the tests are: Analysis of variance and Duncan's New Multiple Range test: 1 - Needle Length ANOVA Source df ss ms F Foliage 4 0.93926 0.23482 54.9** Error 141 0.60303 0.42768 Total 145 0.15423 Duncan's New Multiple Range Test Foliage Sample C E B D A 15.874 16.65 20.66 21.643 21.787 2 - Needle Width Source Foliage Error Total df 4 141 145 ANOVA ss 0.1907 0.1645 0.3552 ms 0.4767 0.1166 F 40.86** 27 Duncan's New Multiple Range Test Foliage Sample B C D E A 1.03000 1.1527 1.2063 1.2735 1.3667 Length-width Ratio ANOVA Source df ss ms F Foliage 4 0.9896 0, ,2474 54. 63** Error 141 0.6417 0, ,4551 Total 145 0.1631 Duncan's New Multiple Range Test Foliage Sample E C A D B 13.1511 13.826 16.058 18.077 20.182 Number of Needles in One Year's Growth ANOVA Source df ss ms F Foliage 4 0.1434 0.3586 11.62** Error 25 0.7718 0.3087 Total 29 0.2206 Duncan's New Multiple Range Test Foliage Sample D C A B E 86.33 114.71 116.50 118.83 158.60 Length of One Year's Growth Source Foliage Error Total df 4 25 29 ANOVA ss 0.359 0.2199 0.5791 ms 0.898 0.8797 F 10.2** C 4.757 Duncan's New Multiple Range Test Foliage Sample E D A 5.8 6.267 7.467 B 7.583 6 - Number of Needles per Centimeter of Length ANOVA Source df ss ms F Foliage 4 0.8885 0.2222 43.86** Error 25 0.1267 0.5067 Total 29 0.1015 Duncan's New Multiple Range Test Foliage Sample D A B C E 13.73 15.45 15.72 24.63 27.52 This data is found on the computer sheets in Appendix 3. VI - DISCUSSION a ~ Pigment analysis From the data of pigment analysis it is obvious that there is some appreciable difference in pigment content between the golden and normal trees. The green sun foliage (A), contained about double the amount of carotene (2.939 mg/g) than chlorophyll a (1.479 mg/g). In correspond-ing mutant foliage (C), the ratio of carotene to chlorophyll a was about 16-1 (4.358-0.261 mg/g). The normal sun foliage (A) had about six times more chlorophyll a than the mutant (C) (1.479-0.261 mg/g). The mutant (C) contained more carotene than the corresponding normal foliage (A) (4.358-2.939 mg/g. A further indication of this light induced destruction of the chlorophyll pigments was found in the analysis of within scion and within needle variation in pigment content of golden sun foliage. The lower or somewhat shaded needles of a golden sun scion (E) was found to contain three times more chlorophyll a than upper more sun directed needles (0.367-0.144 mg/g). The carotene content indicated more in the shaded needles (4.793-3.057 mg/g) but carotene reduction was not as dramatic as chlorophyll a reduction. Needles from the top, sun-oriented part of the scion contained less of both chlorophylls and carotene in the top one half of the needles than the bottom one half of the needles. As was to be expected, the shade foliage (D) of the normal tree contained slightly more of the three pigments than normal sun foliage (A). This was also evident when comparing golden sun (C) and normal shade (B) foliages, but here the chlorophylls have been reduced to a much greater extent. k ~ Needle morphology analysis The needle length analysis indicates that golden sun and shade needles are shorter than corresponding normal needles. It is interesting to note from the Duncan's test that the mean golden shade needle length was not significantly different from the normal shade needle length but that golden sun needles are highly significantly different from normal sun needle lengths. This indicates that under shaded conditions the growth in needle length closely approximates the growth rate of normal needles. The fact that normal shade and sun needles show no significant difference in needle length indicates the slow growth rate is caused by the mutation and probably by light action. Needle width showed no trend in dimension as was found in length measurement. Normal sun needles were found to be both longest and widest. The number of needles shows no particular trend. This character is probably controlled by genes not associated with metabolism, so would be unaffected by the mutation. Length of one yearfs growth is a growth function and shows some-what the same trend as length-x^ ide ratio. The data indicates that the greater the length of one year's growth, the greater the length-width ratio. It is difficult to say whether this phenomenon is a result of genet-ic or environmental factors or a combination of both. It may be that chlorophyll deficiency causes a stunting of growth. There is mention in the literature that growth hormones (auxins) may be broken down by direct sunlight. (Calston & Hand, 1949, Gustaffson, 1909). Golden sun foliage (C and E) had the shortest needles and golden shade (B) the longest but (B) was not significantly different from normal sun needles (A). The analysis further indicated that there are more needles per centimetre of scion length in golden foliage than in golden shade or nor-mal foliage. This results as mutant sun scions are stunted with the same number of needles per scion. This stunting probably results from a sec-ondary action of the mutation. That is, stunted length results from chlorophyll deficiency or possibly the inactivation of certain growth hormones. The length-width ratio showed that golden sun needles (E and C) were stubbiest (short and wide), while golden shade needles were more elongated. This, like stunting of needle length, could be brought about by auxin inactivation. The investigation seems to indicate that the change in pigment content and change in growth rate are associated with relative light intensity. Under normal conditions, sun foliage usually contains less chlorophyll and carotene than shade foliage. In the mutant spruce, this photo-oxidation reduction reaction is greatly increased on the outer sun foliage. Generally, this may be caused in one of two ways, both of which may be induced by high light intensity. A normal photosynthetic cell contains enzymes which control the rate of pigment formation and destruc-tion. Presumably, genes control the rate of formation of enzyme inhibi-tors which control enzyme activity and ultimately control the rate of photosynthesis. A gene mutation could, by changing the amount of light activated enzyme inhibitor normally present, increase the rate of chlorophyll destruction. The second way in which mutation aided by the energy of light could result is in a decrease in chlorophyll content, involves a structural rather than a metabolic change. If the genetic character of a phenotype x^ ere changed so that the chemical composition of the plastid were altered to encourage light induced reduction of the lipoproteins,there would be a reduction in the "packing" of chlorophyll within the grana and a reduction in total pigment content. From the pigment and foliage dimension analysis there is evidence of a correlation between reduced growth and chlorophyll defi-ciency. It is impossible from this investigation to say whether the same mutation is causing both phenomen or whether chlorophyll deficiency results in stunted growth. Further studies would be necessary to establish a definite conclusion. Chlorophylls are always found within plastids, specifically chloroplasts. Any alteration of the structure or function of the chloroplasts may result in a change in the amount of pigments and change the rate of photosynthesis. The quantity of plant pigment found in golden Sitka spruce foliage was significantly different from the quantity found in normal 5itka spruce foliage. The difference is not the same between all types of foliage studied. Golden shade foliage was found to contain about 50% less of all three pigments measured as compared to corresponding normal foliage. At the same time, golden sun foliage contains 40% more carotene, but five tines less chlorophyll a. It is this change in the carotene to chlorophyll ratio in the golden foliage that results in its golden colour on the outer sun branches. Golden sun needles are shorter than normal shade needles, but there is no significant correlation between needle widths. Shade foliage of both trees showed higher calculated values of length-width ratio than did golden sun foliage. There seems to .be no relationship between pigment content and number of needles per one yearcf growth. This probably results as these two functions are not related as are pigment content and needle length. The length of one year of growth shox*7s that golden sun scions are shorter than either golden shade or normal sun scions. This further indicates that the mutation causes a stunting of growth as well as a reduction in chlorophyll synthesis. As golden sun scions are shorter and number of needles is unaffected by the mutation, it seems logical that these scions should have more needles per unit of length. This probably causes the appearance of the golden tree foliage to be 34 more compact than a normal sitka spruce. The analysis generally indicates that the amount of chlorophylls present is directly proportional, in a rough way, to needle length and scion length. Further statistical analysis would be necessary to establish this correlation. Without further investigation into chloroplast structure by electron microscopy this thesis, as far as understanding more fully the cause of chlorophyll reduction, is inconclusive. The prepared electron micrographs were useless in this investigation. Bibliography Association of Official Agricultural Chemists, 1965. Published by A.O.A.C. Washington, D.C., pages 114-115. Benedict, C.R. and R.J. Kohel, 1968. Characteristics of a virescent cotton mutant. Plant Physiology 43: 1611-15. Burley, J., 1965. Genetic variation in Picea Sitchensis. Commonx^ ealth Forestry Review 44 (1) 119: 47-59. Curtis, O.F., 1940. A study of certain factors affecting the formation of carotene in leaves. Cornell University Ph.D. thesis. 126 pp. Day, W.R., 1957. Sitka spruce in British Columbia. Gt. Britain Forestry Commission, Bull. 28: 110 pages. Demerec, M., 1935. Behavior of chlorophyll in inheritance. Cold Spring Harbor Symp. Quantitative Biology 3: 80-86. Galston, A.W., 1955. Some metabolic consequences of the administration of indolacetic acid to plant cells. In: The Chemistry and Mode of Action of Plant Growth by R.L. Wain and F. Wightman. Published by U. of London, 652 pp. Galston, A.W., 1959. Phototropism of stems, roots and coleoptiles. Hand-buchder Pflanzenphysiologie XVII Is 429-492. Galston, A.W. and M.E. Hand, 1949. The physiology of light action. Am. Jour. Bot. 36: 85-94. Granich, S., 1955. Plastid structure, development and inheritance. Encyclopedia of Plant Phys. 1: 507-564. Gustafson, A. 1940. The mutation system of the chlorophyll apparatus. Lunds Univ. Arssks. N.F. Avd. 2, 36: 1-40. Kramer, P. and T. Kozlowski, 1960. The Physiology of Trees. McGraw Hill Book Co. pp. 62-63 Lindstrom, E.W., 1920. Chlorophyll factors in maize. Jour. Heredity 11: 269-277. MacKinney, G. , 1940. Criteria for purity of chlorophyll preparations. J. Niol. Chem. 132: 91-109. MacLachlan, S. and S. Zalick, 1963. Plastid structure, chlorophyll con-centration, and free amino acid composition of a chlorophyll mutant of barley. Can. Jour. Botany. 41: 1053-1062. Powell, J.M., 1965. Annual and seasonal temperature and precipitation trends in B.C. since 1890. D.O.T. Canada Dept. of Forestry, pp. 43-60. Robbins, W. Weier, T. and C. Stocking, 1957. Botany. John Wiley and Sons, page 31. Rowe, J.S., 1959. Forest Regions of Canada, Canada Dept. of Forestry, Bull. 123. pp. 39-42. Schmid, G. and H. Gaffron, 1967a. Light metabolism and chlorophyll struc-ture in chlorophyll deficient tobacco mutants. J. Gen.Physiol. 50: 563-82. Schmid, G. and H. Gaffron, 1967b. Quantum requirements for photosynthesis in chlorophyll deficient plants with unusual lamellar structures. J. Gen. Physiol. 50: 2131-44. Shirley, H.L., 1929. The influence of light intensity and light quality upon the growth of plants. Am.Jour.Bot. 16: 354-390. Srb, A.M. and R.D. Owen., 1955. General Genetics. Chap. 5. The impact of environment. W.H.Freeman and Co., San Francisco, Cal. von Wettstein, D., 1957. Chlorophyll-Letale und der submikroskopioche formwechsel der plastiden. Enptl.Cell Res. 12: 427-506. von Wettstein, D., 1958. The formation of plastid structures. Broolchaven Symposia in Biology 11: 138-159. von Wettstein, D., 1959. The effect of genetic factors on submicroscopic structures of the chloroplast. T. Ultrastruct. Res. 3: 234. von Wettstein, D. , 1960. Multiple allelism in induced chlorophyll mutants. II. Error in the aggregation of the lamellar discs in the chloroplast. Hereditas 46: 700-08. von Wettstein, D., 1961. Nuclear and cytoplasmic factors in development of chloroplast structure and function. Can. Jour. Bot. 39: 1537-45. von Wettstein, D. and A. Ericksson, 1963. The genetics of chloroplast. Proc. Eleventh Int. Congr. Genetics, The Hague Netherlands, 640 pages. Walles, B., 1967. The homozygous and heterozygous effects of an aurea mutation on plastid development in spruce. Studia Forestalia Suecica. 60: 20 pages. Whiteside, A.G.O., 1934. The influence of environment on the carotenoid content of hard red spring wheat. Cereal Chem. 11: 615-625. Zurzychi, J., 1953. Arrangement of chloroplasts and light absorption in plant cells. Acta Soc. Bot. Poloniae 22: 299-320. 44 APPENDIX I r-UNICAM SP.800 """"" Sill f ' ? ?><>, ? , i. ? .A*? JL I, ULTRAVIOLET SPECTROPHOTOMETER U N I C A M SP.800 KM wavelength millimicrons FIGURE 3 oLoert Folmgi? COUTGG SAMPLE AND FORMULA ALIGN WITH INDEX ON THE RECORDER H Bottom N?volets N I S & Q L & 5 Top f " CONCENTRATION REFERENCE PATH LENGTH t & - O SCAN SPEED FAST | [ / f SLOW [ | DATE o r e * A 7 C R S c o t t* 20 ^  u> ? CO 3 40 r+ P 3 eo2 C?U!o|u? N?. 600678 1 1 r ULTRAVIOLET SPECTROPHOTOMETER f II U NIC AM S P. 800 I* i ! flip 1CAM INSTRUH?*T! 2-0 wavelength millimicrons FIGURE 4 Boo 8 10 - f u> ? 3 40?* 60g 80 100 CaUlo|w? N?. 600878 SAMPLE AND FORMULA G O C D ? M O ^ T / f ^ F O L i P l G t Z -F- UPP&4 2 Sc/o/V CONCENTRATION REFERENCE PATH LENGTH SCAN SPEED FAST [ ~ p f SLOW { { DATE OPERATOR ( j . S O O T K i r* v APPENDIX II "A" NORMAL SUN FOLIAGE No./cm. Number Length of Needle Dimensions Needles 1 yr.Growth L. cm. W.mm. 13.3 101 7.6 2.24 1.3 17.7 129 7.3 2.325 1.14 12.6 72 5.7 2.345 1.41 16.6 136 8.2 1.855 1.71 16.6 143 8.6 2.25 1.41 15.9 118 7.4 2.38 1.60 2.30 1.42 2.345 1.38 2.06 1.25 2.13 1.54 1.97 1.50 2.18 1.15 2.04 1.30 2.16 1.30 2.17 1.33 2.27 1.29 2.265 1.35 2.25 1.37 2.18 1.29 2.13 1.18 2.11 1.28 2.23 1.18 2.24 1.25 2.11 1.48 2.24 1.15 1.94 1.36 2.22 1.25 2.29 1.47 1.83 1.30 2.31 1.41 1 SHADE FOLIAGE "Bf! GOLDEN Number L.of Needle Needles Annual L. cm. W.mm. 134 8.7 2.085 0.98 127 8.3 2.12 1.09 103 7.2 1.89 0.99 137 8.6 2.145 1.08 97 6.5 1.88 1.12 115 6.2 2.095 1.10 2.135 1.03 1.97 1.05 2.04 1.1 1.89 0.98 1.925 .92 2.22 1.15 2.17 0.98 2.16 1.19 2.1 0.92 2.04 1.1 2.12 1.05 1.93 1.08 2.04 0.99 2.02 1.00 2.16 1.12 , 1.935 0.89 2.15 1.00 2.11 1.05 2.095 0.89 2.02 0.99 2.14 0.81 2.09 1.09 2.16 1.07 2.15 1.09 SUN FOLIAGE (Lcwer) "C" GOLDEN Number Length Needles Needles Growth L .mm. W. mm. 99 3.6 13.7 1.21 103 3.5 10.5 1.2 123 5.7 10.9 1.0 113 5.2 15.3 1.1 112 4.5 10.15 1.1 143 6.4 15.6 1.15 110 4.4 12.3 1.11 12.1 1.0 11.7 1.03 12.2 1.38 18.1 1.19 17.4 1.11 14.25 1.0 12.95 1.25 18.7 1.05 17.35 1.13 17.55 1.02 18.75 1.14 20.0 1.09 19.45 1.15 16.0 1.08 19.45 1.35 17.3 1.28 18.4 1.1 17.15 1.12 19.2 1.32 17.2 1.2 20.0' 1.28 17.65 1.19 15.015 1.25 "D" NORMAL SHADE FOLIAGE No./cm. Number Length of L W Needles Growth 11.6 57 4.9 cm. 23.1 ,1 1.04 1 12.9 90 7.0 23.2 1.20 14.7 88 6.0 19.6 1.33 15.1 98 6.5 22.3 1.05 15.1 98 6.5 22.6 1.19 13.0 87 6.7 21.85 1.28 19.9 1.26 23.85 1.20 19.9 1.3 23.2 1.25 22.0 1.20 21.3 1.30 19.95 0.89 21.9 1.21 22.2 1.30 21.55 1.39 22.4 1.30 20.3 1.30 17.6 1.0 18.9 1.19 23.2 1.1 22.4 1.18 23.6 1.27 23.15 1.23 20.25 1.2 22.0 1.21 22.25 1.23 22.0 1.29 21.0 1.2 22.2 1.1 "E" GOLDEN SUN FOLIAGE (Middle) -No ? / cm ? Number Leng th of L ? mm ? W ? mm? Needles Growth 23.3 156 6.7 cn. 18.9 1.15 27.0 143 5.3 19.9 1.31 28.1 157 5.6 20.15 1.11 30.9 167 5.4 20.2 1.25 28.3 170 6.0 18.85 1.09 17.9 1.35 13.65 1.05 17.6 1.30 19.75 1.41 15.45 1.19 18.4 1.48 17.0 1.38 18.15 1.39 16.6 1.40 16.65 1.48 15.9 1.25 15.3 1.43 17.65 1.3 16.5 1.18 16.5 1.40 12.6 1.25 12.7 1.18 17.8 1.2 17.7 1.2 11.45 1.22 10.1 1.16 n APPENDIX III N E E D L E L E N G T H A N A L Y S I S OF V A R I A N C E S O U R C E DF SUM SQ MEAN SQ F ERROR T R E E 4 0 . 9 3 9 2 6 E 0 3 0 . 2 3 4 8 2 E 0 3 5 4 ^ 9 0 ERROR 1 4 1 0 . 6 0 3 0 3 E 0 3 0 . 4 2 7 6 8 E 01 T O T A L 1 4 5 0 . 1 5 4 2 3 E 0 4 MEANS T R E E 1 2 3 4 5 2 1 . 7 8 6 7 2 0 . 6 6 0 0 1 5 . 8 7 3 3 2 1 . 6 4 3 3 1 6 . 6 5 0 0 A N A L Y S I S C O M P L E T E . N E E D L E WIDTH A N A L Y S I S OF V A R I A N C E S O U R C E DF SUM SQ MEAN SQ F ERROR T R E E 4 0 . 1 9 0 6 9 E 0 1 0 . 4 7 6 7 2 E 0 0 4 0 . 8 6 ERROR 141 0 . 1 6 4 4 9 E 0 1 0 . 1 1 6 6 6 E - 0 1 T O T A L 145 0 . 3 5 5 1 8 E 01 MEANS T R E E 1 2 3 4 5 1 . 3 6 6 7 1 . 0 3 0 0 1 . 1 5 2 7 1 . 2 0 6 3 1 . 2 7 3 5 A N A L Y S I S C O M P L E T E . L - W R A T I O A N A L Y S I S OF V A R I A N C E S O U R C E DF SUM SQ MEAN SQ F ERROR T R E E 4 0.98963E C3 0.24741E 03 54.36 ERROR 141 0.64172E 03 0.45512E 01 T O T A L 145 0.16314E 04 MEANS T R E E 1 2 3 4 5 16.0576 20.1816 13.8263 18.0772 13.1511 A N A L Y S I S C O M P L E T E . NO OF NEEDLES ANALYSIS OF V \ R IA N C E SOURCE DF SUM SQ MEAN SQ F ERROR TREE 4 0 . 1 4 3 4 5 E 05 0 . 3 5 8 6 3 E 04 1 1 . 6 2 ERROR 25 0 . 7 T 1 8 3 E 0 4 0 . 3 0 8 7 3 E 0 3 TOTAL 2 9 0 . 2 2 0 6 3 E 0 5 MEANS TREE 1 2 3 4 5 1 1 6 . 5 0 0 0 1 1 8 . 8 3 3 3 1 1 4 . 7 1 4 3 8 6 . 3 3 3 3 1 5 8 . 6 0 0 0 ANALYSIS COMPLETE. > L E N G T H OF 1 Y E A R GROWTH A M A L Y S I S OF V A R I A N C E S O U R : E DF SUM SQ MEAN SQ F ERROR T R E E 4 0 . 3 5 9 2 0 E 02 0 . 8 9 8 0 0 E 01 1 0 . 2 1 ERROR 2 5 0 . 2 1 9 9 2 E 02 0 . 8 7 9 6 9 E 0 0 T O T A L 2 9 0 . 5 7 9 1 2 E 0 2 M E A M S R<EE 1 2 3 4 5 7 . 4 6 6 7 7 . 5 3 3 3 4 . 7 5 7 1 6 . 2 6 6 7 5 . 8 0 0 0 V V A M A L Y S I S C O M P L E T E . J NEEDLES PER L. l ANALYSIS OF V A R I A N C E SOURCE DF SUM SQ MEAN SQ F ERROR TREE 4 0.88850E 03 0.22212E 03 43.84 ERROR 25 0.12668E 03 0.5067IE 01 TOTAL 29 0.10152E 04 MEANS TREE 1 2 3 4 5 15.4500 15.7167 24.6286 13.7233 27.5200 \ \ A N A L Y S I S C O M P L E T E . Y ) N E E D L E L E N G T H ERROR MEAN SQUARE = 4 . 2 7 6 8 0 D E G R E E S OF FREEDOM = 1 2 1 . L E V E L OF S I G N I F I C A N C E = 5 P E R C E N T 3 1 5 . 8 7 3 3 0 0 3CK 5 1 6 . 6 4 9 9 9 4 2 6 . 2 2 0 . 6 5 9 9 8 8 3 0 . 4 2 1 . 6 4 3 2 9 5 3 0 . 1 2 1 . 7 8 6 6 9 7 3 0 . > } N E E D L E WIDTH ERROR MiEAN SQUARE = 0 . 0 1 1 6 7 D E G R E E S OF FREEDOM = 1 2 1 . L E V E L OF S I G N I F I C A N C E = 5 P E R C E N T 2 1 . 0 3 0 0 0 0 3_0_. 3 1 . 1 5 2 6 9 9 3 0 . 4 1 . 2 0 6 3 0 0 3 0 . 5 1 . 2 7 3 4 9 9 2 6 . 1 1 . 3 6 6 6 9 9 3 0 . 2 3 4 5 1 ) L?W R A T I O ERROR MEAN SQUARE = 4 . 5 5 1 2 0 D E G R E E S OF FREEDOM = 1 2 1 . L E V E L OF S I G N I F I C A N C E = 5 P E R C E N T 1 3 . 1 5 1 0 99 2 6 . 3 1 3 . 8 2 6 3 0 0 3 0 . 1 1 6 . 0 5 7 5 8 7 3 0 . 4 1 8 . 0 7 7 1 9 4 3 0 . 2 2 0 . 1 8 1 5 9 5 3 0 . 1 4 STOP 0 E X E C U T I O N T E R M I N A T E D $ S I G N O F F \ NO OF N E E D L E S ERROR MEAN SQUARE = 3 0 8 . 7 2 9 9 8 D E G R E E S OF FREEDOM = 2 5 . L E V E L OF S I G N I F I C A N C E = 5 P E R C E N T 4 8 6 . 3 3 3 2 9 8 3 1 1 4 . 7 1 4 2 9 4 7 . 1 1 1 6 . 5 0 0 0 0 0 6 . 2 1 1 8 . 8 3 3 2 98 6 . 5 1 5 8 . 5 9 9 9 9 1 5 . 1 4 3 1 2 ? ? M U M B?JI BS3 Jtiia E l ksj ts?3 EM bsss sm MS> KM KR BSi K& 5 \ \ L E N G T H OF 1 YEAR GROWTH ERROR MEAN SQUARE = 0 . 8 7 9 6 9 D E G R E E S OF FREEDOM = 2 5 . L E V E L OF S I G N I F I C A N C E = 5 P E R C E N T 4 . 7 5 70 99 7 . 5 5 . 7 9 9 9 9 9 5 . 4 6 . 2 6 6 7 0 0 6 . 1 7 . 4 6 6 7 0 0 6 . 2 7 . 5 8 3 3 0 0 6 . \ 5 ?> E X E C U T I O N T E R M I N A T E D $RUN DUNCANS E X E C U T I O N B E G I N S DUNCAN NEW M U L T I P L E RANGE T E S T N E E D L E S PER L . ERROR MEAN SQUARE = 5 . 0 6 7 1 0 D E G R E E S OF FREEDOM = 2 5 . L E V E L OF S I G N I F I C A N C E = 5 P E R C E N T 4 1 3 . 7 3 3 2 9 9 6 . 1 1 5 . 4 5 0 0 0 0 6 . 2 1 5 . 7 1 6 7 0 0 ? 6 . 3 2 4 . 6 2 8 5 8 6 7 . 5 2 7 . 5 1 9 9 8 9 5 . 4 1 2 3 5 } 

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