Comparative Study: Photosynthesis & Transpiration in Acacia Koa Leaves & Phyllodes, Exercises of Literature

Download Comparative Study: Photosynthesis & Transpiration in Acacia Koa Leaves & Phyllodes and more Exercises Literature in PDF only on Docsity! PHOTOSYNTHESIS, RESPIRATION, TRANSPIRATION, AND GROWTH OF ACACIA KOA SEEDLINGS AS AFFECTED BY PHOTOSYNTHETIC PHOTON FLUX DENSITY A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAII IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN AGRONOMY AND SOIL SCIENCE DECEMBER 1981 By Gerald Alan Walters Dissertation Committee: Duane P. Bartholomew, Chairman James A. Silva Peter P. Rotar Paul C. Ekern Douglas J. C. Friend We certify that we have read this dissertation and that in our opinion it is satisfactory in scope and quality as a dissertation for the degree of Doctor of Philosophy in Agronomy and Soil Science. DISSERTATION COMMITTEE decreased with time, while the percentage of stem dry weight increased. Phyllodes developed only on seedlings exposed to light of at least light-saturating levels. The data indicated that koa seedlings can survive and grow only at light levels equal to or greater than 25 percent of full sunlight. The vigor of koa seedlings grown at less than 25 percent full sunlight declined with time and it appeared that they would eventually die. This minimum light requirement accounts for the scarcity of natural koa reproduction in an undisturbed koa forest. Koa leaves and phyllodes readily adapted to changes in available light. Leaves and phyllodes grown in full sunlight developed the characteristics of sun leaves. Conversely, at 27 percent of full sunlight, both leaf forms developed the characteristics of shade leaves. Fully-developed leaves and phyllodes only adapted physiologically to changes in available light. Partially-developed leaves and phyllodes adapted both physiologically and anatomically to changes in available light. Seedlings with phyllodes grown in 27 percent of full sunlight for 6 to 8 weeks developed leaves at the terminals. Seedlings again produced phyllodes when placed in full sunlight. V TABLE OF CONTENTS Page ACKNOWLEDGMENTS................................................... iii A B S TRACT ............................................................ iv LIST OF T A B L E S ..................................................... viii LIST OF I L L U STRATIONS ............................................ x CHAPTER I. GENERAL INTRODUCTION ................................ 1 CHAPTER II. GENERAL LITERATURE REVIEW Carbon Dioxide Exchange of Leaves ................ 6 Transpiration by Leaves ........................... 12 Growth and Development of P l a n t s .................. 15 CHAPTER III. PHOTOSYNTHESIS, RESPIRATION, AND TRANSPIRATION OF JUVENILE AND MATURE LEAF FORMS OF ACACIA KOA Introduction ........................................ 19 Literature Review ................................. 19 Materials and Methods ............................. 23 R e s u l t s ............................................. 32 Discussion.......................................... 42 CHAPTER IV. PHOTOSYNTHESIS, RESPIRATION, AND GROWTH OF ACACIA KOA SEEDLINGS EXPOSED TO DIFFERENT LIGHT LEVELS Introduction ........................................ 53 Literature Review ................................. 53 Materials and Methods ............................. 58 R e s u l t s ............................................. 61 Discussion.......................................... 72 CHAPTER V. ADAPTATION OF ACACIA KOA LEAVES AND PHYLLODES TO CHANGES IN AVAILABLE LIGHT Introduction 79 . Literature Review ................................. 80 Materials and Methods ............................. 82 R e s u l t s ............................................. 85 Discussion.......................................... 93 CHAPTER VI. GENERAL SUMMARY AND CONCLUSIONS ................... 101 vi TABLE OF CONTENTS (Continued) Page APPENDIX A Solar Radiation, Cumulative Rainfall, and Temperatures at Waimanalo, Hawaii for the Period October 1, 1980 to April 28, 1981 ......... 104 LITERATURE CITED ................................................... 105 vii LIST OF ILLUSTRATIONS Figure Page 1 Acacia Koa Seedling with a) Leaves and b) Phyllodes........................................ 4 2 An Acacia Koa Phyllode in the Leaf Assimilation Chamber for Gas Exchange Determinations ............................... 26 3 Diagram of the Semi-closed System for Leaf Gas Exchange Determinations, Department of Agronomy and Soil S c i e n c e ............. 27 4 Semi-closed system for leaf gas exchange determinations, Department of Agronomy and Soil Scie n c e........................... 28 5 Cross Section of a) Leaflet (210X) and b) Phyllode (105X) of Acacia K o a ....................... 34 X 6 Rate of (^-exchange as a Function of Incident Photosynthetic Photon Flux Density of Leaves and Phyllodes of Acacia K o a .......................................... 36 7 Rate of CC^-exchange as a Function of Incident Photosynthetic Photon Flux Density of Acacia Koa Phyllodes in Vertical and Flat Positions......................... 39 8 Dry Weight Increments of Stem (A ), Leaves (*), Roots (□), and Total (+) of Acacia Koa Seedlings Grown Under Different Light Treatments ............................. 65 CHAPTER I GENERAL INTRODUCTION Koa (Acacia koa Gray) has been called the Monarch of Hawaiian forests. This is a fitting description of this native leguminous species that occurs on all the larger islands, forming a part of the forest cover on about 500,000 acres (Nelson and Wheeler 1963), or about 25 percent of the forested areas of the state. Koa, one of the most common native tree species of the Hawaiian Island ecosystem, provides a habitat for several species of native birds (Goodwin and Aldrich 1966, Munro 1960), mollusks (Kondo 1970), and insects (Gagne et al. 1970, Gressitt and Davis 1969, Swezey 1925). Many native plant species grow in association with koa. Koa is also a renewable economic resource and is Hawaii's most valuable timber tree. Skolmen (1970) estimated that the koa industry generates more than $1.3 million per year. Koa grows to heights of more than 45 m. The circumference of one tree measured 8.4 m (Littlecott 1969). The technical wood properties of koa are almost identical to those of black walnut (Juglans nigra L.) (Skolmen 1968) and the beauty of finished koa and black walnut are comparable. Koa has many uses, but because of its high value, it is used mainly for cabinets, furniture, gun stocks, veneer, and craft pieces. Koa forests are not as extensive today as they once were (Hall 1904, Whitesell 1964) and the area continues to dwindle for a number of reasons. In the past 50 years, an estimated 100,000 acres of koa forest have been cleared for pasture. Koa is relished by cattle who prevent the regeneration of koa forests by eating all seedlings and \ 1 sprouts. Koa forest regeneration is also hampered by competition from numerous introduced and native plant species and by insects and diseases that attack young and old trees. Fire has also taken its toll on koa forests. One fire burned for more than 2 months through the koa forests on the island of Hawaii. Logging has not reduced the area of koa forests because koa regenerates rapidly in openings created by logging. If koa forests are to be perpetuated at their present extent, or perhaps increased, both natural and artificial regeneration must be accomplished. Natural koa regeneration is scarce in undisturbed forests. Approximately 200 to 300 seedlings and root suckers per acre are present in an established forest and most are of recent origin. The majority of seedlings present are less than 3 inches tall; most . root suckers are less than 2 feet tall. Few seedlings survive for more than 1 year (Sccwcroft and Nelson 1976). However, if the forest canopy is removed and the mineral soil is exposed, seeds which may have lain in the soil for 25 years (Judd 1920), germinate rapidly. Estimates of up to 143,500 koa seedlings per acre have been tallied in a plot after removal of the forest canopy (Judd 1925). Whether the high rate of germination after the disturbance resulted from increased soil temperature or light, or to an altered soil moisture or oxygen regime, or combinations of these factors, is not known. When natural regeneration occurs in adequate numbers after removal of the upper story vegetation, spatial distribution of seedlings is generally very uneven. Artificial regeneration is therefore necessary to ensure fully-stocked forests. Artificial 2 develop near the wound have true leaves (Rock 1913). Photosynthetic photon flux density (PPFD) is one of the most important environmental factors because it supplies the energy required for plant photosynthesis, growth, and development. Photosynthetic photon flux density can be readily manipulated in the nursery and forest so that seedlings are exposed to optimum levels during ontogeny, thus promoting rapid growth and development. In the nursery, PPFD can be controlled by the use of lamps or shade cloth. In the forest, it can be controlled by manipulating species composition and spacing. However, the present lack of data on the relationship between PPFD and photosynthesis and growth of koa, makes it impossible to develop management strategies for promoting koa growth by manipulating the light regime. This research was conducted to accomplish the following objectives: Objective 1— Determine the photosynthetic, respiration, and transpiration characteristics of leaves and phyllodes of Acacia koa. Objective 2— Determine the photosynthetic, respiration, and growth characteristics of Acacia koa seedlings exposed to different PPFD. Objective 3— Determine how Acacia koa leaves and phyllodes adapt to changes in PPFD. 5 CHAPTER II GENERAL LITERATURE REVIEW Despite the economic and ecological importance of koa, data describing its growth and development in the nursery and field and the physiological biochemical data necessary to explain that growth and development, are lacking. Because 90 to 95 percent of the dry weight of plants are derived from photosynthetic C02 assimilation (Zelitch 1975a), an understanding of the partitioning of photosynthate into various plant parts is necessary. Some of the CC>2 fixed during photosynthesis is utilized by respiration in the light and darkness to produce energy and products that are used in the maintenance and construction of plant tissue (Ledig et al. 1976). The rate of photosynthesis is often related to water deficits in the plant. As deficits occur because moisture loss by transpiration exceeds uptake, cell elongation rates decline and eventually photosynthetic rates decrease because of increased stomatal and mesophyll diffusive resistances (Begg and Turner 1976, Zelitch 1975b). This general literature review will focus on the processes of photosynthesis, respiration, transpiration, and their subsequent effects on plant growth and development. Because of the lack of information on Acacia species in general, and koa specifically, the review includes information on other tree and plant species. Carbon Dioxide Exchange of Leaves Photosynthesis is a photochemical reaction in which water plus carbon dioxide (C02) in the presence of light and chlorophyll yields sugar. The overall reaction of photosynthesis is given by the 6 simplified equation: nCO + 2nH 0* + light chloroplasts (CH 0) + nO * + nfl 0 ̂ ̂ n z ^ The initial phase of photosynthesis is the trapping of light energy by chlorophyll contained in plant organs, principally the leaves. During this phase, electrons are removed from water, oxygen is released, and high energy pyridine nuceleotide and adenosine triphosphate molecules are produced. These energy-rich molecules are used to reduce carbon dioxide during the second phase of photosynthesis as well as to provide energy for other physiological processes (Zelitch 1971). This energy-trapping process of the initial phase is apparently the same for all higher plants, but the manner in which the plant reduces carbon dioxide varies with species (Govindjee and Govindjee 1974). All woody plants and trees examined thus far, with the exception of mangrove (Joshi et al. 1974) and larch (Fry and Phillips 1976), have characteristics of plants (Downton 1971). Classification as a plant implies the absence of a highly photosynthetically active vascular bundle sheath in the leaf tissue, the potential of high rates of photorespiration, the predominance of ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBPcase) as the major photosynthetic C02 fixation enzyme, and a minimum CO^ compensation concentration of about 50 ppm C02 (Black 1973, Zelitch 1971). Photorespiration reduces net photosynthesis by releasing part of the C02 fixed in photosynthesis with a loss of energy associated with its fixation (Black et al. 1976, Chollet and Ogren 1975, Goldsworthy 1970, Schrader 1976, Zelitch 1975a). The high CO^ compensation concentration 7 physiological, and biochemical events. The pattern of leaf i development is similar for different species (Schaedle 1975). During leaf expansion, increases in photosynthetic CO^-uptake can be related to development of internal leaf structure and stomates (Homann 1975, Isebrands and Larson 1973), a decrease in diffusion resistance (Homann 1975) , synthesis of chlorophyll (Dickmann 1971b), development of physiological and structural integrity of the membrane-bound phosphorylation system of chloroplasts (Dickmann 1971b, Hernadez-Gil and Schaedle 1973), Fraction I protein synthesis, increases in RuBPcase activity (Dickmann 1971b), and a sharp decline in mitochondrial respiration (Dickmann et al. 1975). A leaf is mature when leaf expansion ceases and leaf anatomy has essentially stabilized. Maturation does not proceed at a uniform rate throughout the leaf. The lamina tip matures first, both structurally and functionally, and maturation then proceeds basipetally, the leaf base and margins maturing last (Isebrands and Larson 1973). At maturity, mesophyll cells and intercellular spaces are fully developed, stomatal formation is complete, and the leaf vascular system is fully functional (Isebrands and Larson 1973). A leaf that has reached anatomical maturity is functionally mature as well (Dickmann et al. 1975). The potential capacity for net photosynthesis is maximum and COj-compensation concentration and dark respiration reach a minimum (Dickmann 1971a, Dickmann and Gjerstad 1973, Larson et al. 1969, Loach and Little 1973). The Hill reaction and RuBPcase activities are maximum (Dickmann et al. 1975, Ghosh 1973). A mature leaf functions only as an exporter of photosynthate (Larson et al. 10 1969), although an expanding leaf may be exporting simultaneously from mature regions and importing to immature regions (Larson et al. 1972, Schaedle 1975). Following the completion of leaf expansion, a more or less steady state condition of photosynthetic performance persists for a duration of 10 to 40 days, depending on species, time of year, and environmental conditions (Schaedle 1975). Subsequently, with increasing leaf age, net photosynthesis declines (Bormann 1956, Bormann 1958, Clark 1961, Dickmann 1971a, Freeman 1952, Furukawa 1973, Kozlowski and Keller 1966, Logan 1970, Thrower 1967, Wardlaw 1968). Associated with this decline is a reduction in the chlorophyll, \ protein, and nucleic acid content of the leaf (Schaedle 1975), and diffusive resistance to water and CC^ increase (Davis and McCree 1978). The photosynthetic activity of the whole plant is greatly affected by the proportion and arrangement of photosvnthetically active leaves. The maintenance of high photosynthetic activity is dependent on the continued adding of and the rapid maturation of leaves (Dickmann 1971b). Many tree species progress through several distinct leaf stages during their first year; cotyledons are followed by primary leaves, and eventually, secondary leaves. Each leaf type is morphologically and anatomically distinct (Ledig et al. 1976). Marshall and Kozlowski (1976) found that cotyledons of woody angiosperms contributed significantly to the plants' chances for survival and growth if care was taken to avoid injury and to provide them with favorable conditions. Primary leaves have higher rates of 11 CO^-uptake than secondary leaves (Ledig et al. 1976). -2 -1The CER or net photosynthesis (mg CO^ dm h ) is the algebraic sum of CO^ fixed photosynthetically and CO^ released by respiration. Under near optimal conditions, trees have CER that range -2 -1from 10 to 30 mg CC^ dm h (Brix 1967, Krueger and Ferrell 1965, Krueger and Ruth 1969, Schaedle 1975, Verduin 1953). Jarvis and Jarvis (1964) found that maximum rates of CO^-uptake for temperate -2 -1zone evergreen conifers ranged from 5 to 10 mg CO^dm h compared -2 -1to rates of 10 to 20 mg CO^ dm h for deciduous broad-leaved trees and shrubs. Larcher (1969b) reported that photosynthetic rates of evergreen broad-leaved species from temperate and warm temperate -2 -1regions ranged from 14 to 17 mg CO^ dm h . However, many values of net photosynthesis found in the literature are thought to be unreliable because of peculiarities in the method of determination (Brittain and Cameron 1973, Jarvis and Jarvis 1964). Differences in environmental factors— light intensity and quality, water, nutrition, CO^ content— and differences in plant material— attached or detached leaves, sun leaves or shade leaves, seedlings or larger plants— used by researchers are factors which could have affected ohotosynthetic rates (Zelawski and Walker 1976). Transpiration by Leaves Transpiration is the loss of water by evaporation from plants. It differs from the general process of evaporation, because the water vapor does not evaporate from a free surface but must pass through the epidermis and cuticle or through pores; i.e., the stomata or lenticels. Transpiration may take place from any exposed part of the 12 decreases transpiration, but relative humidity is a function of temperature, and this in turn is influenced by light. Wind may cause an increase or decrease in transpiration. Wind increases the water vapor pressure gradient and reduces r by blowing away the vapor, cl causing an increase in evaporation. If the leaf is warmed by sunlight, wind will lower the temperature, causing a decrease in transpiration (Salisbury and Ross 1978). Growth and Development of Plants Growth, in terms of dry matter production, results mainly from net photosynthetic fixation of carbon dioxide (Schrader 1976, Zelitch 1975b). However, correlations between growth and photosynthesis of single leaf trees and crop plants have been weak, or even zero (Carter 1972, Elmore 1980, Gordon and Gatherum 1968, Ledig and Perry 1969, Moss and Musgrave 1971). The poor correlations result because measurements on a single leaf do not account for partitioning, for the effects of mutual shading, or for senescence of older leaves. Some of the photosynthate is utilized for maintenance respiration, as well as for growth (Baker et al. 1972, Ledig et al. 1976, McCree 1974). Maintenance respiration produces the energy to maintain tissue integrity and function. Growth respiration provides the energy and carbon skeletons for the synthesis of new tissue (Baker et al. 1972). The respiration components change with ontogeny. As plants grow, more dry weight is put in nonproductive tissues like stems and branches, whose integrity and function must be maintained. Growth respiration has been found to be proportional to photosynthesis (Ledig et al. 1976). Maintenance and growth respiration rates are substantially 15 higher for shoots than for roots (Ledig et al. 1976). Dry matter production per unit of soil area, often expressed as the crop growth -2 -1rate (g m day ), is the cumulative result of net photosynthesis by a unit area of an average leaf in the canopy multiplied by the leaf area index after deduction of photosynthate used in respiration, and thus represents accumulation of real increments of organic matter for the entire plant. Stem growth of tree seedlings over time is usually sigmoid. During the logarithmic phase, establishment of the seedling begins as the radical penetrates the soil and the cotyledons, if they are epigeal, begin to carry on photosynthesis. Growth rate (the increase in size per unit of time) is slow during this phase, apparently because the germinating seed has fewer cells capable of growth. The growth rate continuously increases during this phase as more cells are formed. During the second or linear phase, growth continues at a constant, usually maximum rate for some time. The third phase is the senescence phase during which dry matter continues to increase, but more slowly, so the rate decreases (Salisbury and Ross 1978). Growth of the stem and roots has been found to be episodic and alternating (Krueger and Trappe 1967). In a study with pitch pine (Pinus rigida Mill.), Ledig et al. (1976) found that for about the first 2 1/2 months, root growth was rapid while leaves were slowly increasing in dry weight. For the next 3 1/2 months, root growth was nil while leaf growth was rapid. Then the phase shifted so that root growth was again rapid. Although growth of the stem and roots alternated, the rate of dry weight gain for the whole plant was nearly 16 linear over the entire period of measurement. Growth can be defined in terms of increased dimension, mass, or combinations of these for the whole plant or plant organ. For growth analysis, the primary values are usually the dry weight of whole plants and/or parts (stems, leaves, roots), and the dimensions of assimilatory apparatus (leaf area, weight of chlorophyll, etc.). These values are determined at specified intervals, and from them calculations are made that describe the growth of the plants and their various parts as well as the relationship between assimilatory apparatus, generally leaves, and dry matter production. Relative growth rate (RGR), milligrams dry weight produced per gram of dry matter per unit of time, indicates the efficiency of dry matter production under specific environmental conditions (Brix 1967). The RGR can be calculated by multiplying the net assimilation rate (NAR) by the leaf area ratio (LAR). The NAR describes the net production efficiency of the assimilatory apparatus and can be calculated using one of several formulas. The formula used in this study was: NAR = w 2~w l . In A2 - In A . a 2-a l t2 - t, where and are the total plant dry weight at initiation (t^) and at some later time (t^), A 2 and A^ are the total projected plant leaf area at times t_ and t2 and t2 - t^ is the sampling interval. This formula assumes that A and W are linearly related. 17 Muell., A. koa does not develop phyllodes until some months after germination. The development of phyllodes by koa is associated with increasing physiological age and a reduced rate of shoot growth. For example, formation of phyllodes is initiated on slow-growing lateral shoots at a time when the rapidly-growing terminal leader is still forming juvenile compound leaves. Reversion from adult to juvenile foliage occurs if slow-growing shoots are forced into rapid growth by environmental manipulation such as increased supplies of water or fertilizer (Borchert 1976). Genetic variability of heterophylla development in seedling populations of A. melanoxylon R. Br. has been shown by Borchert (1976). The shoot elongation rate of some plants was less than one-half the average rate and phyllodes formed very early on these plants while others grew rapidly and retained juvenile leaves for a long time. The modification of phenotype that occurs with the conversion from leaves to phyllodes is an apparent adaption to increased temperature and aridity (Allsop 1965, Coaldrake 1971). The relative size of the bipinnate leaves of A. melanoxylon and the rate of change from leaves to phyllodes were related by Farrell and Ashton (1978) to site rainfall. Leaves were larger and were retained longer on seedlings growing on sheltered sites than on seedlings growing on exposed sites. Phyllode shape and size of A. melanoxylon were highly correlated with the distribution of annual rainfall. Phyllodes in drier areas were smaller and more symmetric than phyllodes in wetter areas (Farrell and Ashton 1978). Leaves and phyllodes of Acacia species are inclined (measured 20 from the horizontal) at different angles. Leaves are displayed horizontally. Phyllodes are displayed almost vertically. The mean phyllode inclination for A. harpophylla was 83° (Connor, et al. 1971). Leaf inclination influences irradiance penetration through the leaf canopy, and therefore determines the energy available to leaves and/or phyllodes for photosynthesis. The greater the leaf inclination, the smaller will be the direct irradiance, therefore, steeply-inclined leaves have lower CER than horizontal leaves. However, a canopy of steeply-inclined leaves can have a greater CER than an equivalent canopy having horizontal leaves (Trenbath and Angus 1975). The higher rate is due to enhanced efficiency of light utilization. The effect of leaf inclination on CER is strongly related to and interacts with leaf area index (LAI) and PPFD level (Gordon and Promnitz 1976, Loomis and Williams 1969, Saeki 1960). As LAI increases, more self and mutual shading occurs. Shading alters the photosynthetic light response characteristics of individual leaves. Shaded leaves have lower light-saturation and light-compensation points than do nonshaded leaves. The more LAI increases and PPFD level decreases, the more important it becomes to have steeply-inclined leaves if maximum photosynthetic rates are to be sustained. The effects of self and mutual shading can be reduced by increasing the PPFD so that light penetrates the canopy and reaches the interior and lower leaves (Hughes 1969). Under such conditions, light above the first layer of leaves exceeds that required to 21 saturate photosynthesis, and light within the canopy may approach saturation. Therefore, the greater the extent of mutual shading the higher the light intensity required for maximum photosynthetic rates (Kramer and Decker 1944). Zelawski et al. (1973), using high intensity diffuse light that penetrated the seedling canopy from all sides, increased-light saturation values of the seedlings. On cloudy or overcast days, when diffuse sky-source radiation is great, total canopy photosynthesis may be greater for some species with low photosynthetic capacity than on clear days (Allen et al. 1974). Leaf inclination also affects daily photosynthetic patterns. The rate of photosynthesis of horizontally-displayed leaves increases as the sun rises to the zenith and decreases as the sun moves toward the horizon. Because of the near vertical display of phyllodes, their daily photosynthetic pattern is characterized by a decrease as the sun rises to the zenith and an increase as the sun moves away from the zenith (Connor et al. 1971). Photosynthetic rates have been measured only for phyllodes of the heterophyllic tree A. harpophylla (Connor et al. 1971, Tunstall and Connor 1975). I could find no data for true leaves of such trees. Under optimum conditions of moisture and temperature, the photosynthetic rate of naturally-displayed phyllodes exposed to 1500 -2 -1 -2 -1pE m s light from above was 13.6 mg CO^ dm h . It was not stated whether the calculations were based on the projected leaf area or the total leaf area. No literature was found to indicate morphological, anatomical, chlorophyll, or RuBPcase characteristics of juvenile leaves or 22 transpiration, were determined for a leaf and a phyllode on each of six different plants. Well-watered plants were placed in a growth chamber. The growth chamber environment was maintained at 70 ± 5 percent relative humidity, 26 ± 2° C temperature, and 1200 ymol -2 -1m s PPFD, using a Lucalox high pressure mercury vapor lamp. The diffuse light was filtered through about 2 cm of water to reduce infrared radiation. The PPFD was modified by placing neutral density screens between the light source and the leaf assimilation chamber. An intact leaf or phyllode was sealed in a water-jacketed plexiglass leaf assimilation chamber which was connected in series to a Beckman IR 215 infrared gas analyzer (IRGA) and a C02 supply in a semi-closed system mode (Sestak et al. 1971) (Fig. 2). Temperature was maintained at 27 ± 1° C by circulating water from a constant temperature bath through the water jacket. Air and leaf temperatures within the leaf and growth chamber were monitored with 0.25 and 0.13 mm copper-constantin thermocuples, respectively. The thermocouple used to monitor leaf temperature was attached to the abaxial side of the leaf. For leaves and phyllodes in the horizontal position, the incident PPFD on the leaf within the leaf chamber was determined by holding horizontally a Lambda Instruments Inc. LI-190 quantum sensor. For phyllodes in the vertical position, the incident PPFD was determined by holding the quantum sensor vertically and doubling the reading. The air was circulated within the leaf chamber by a small fan. Changes in carbon dioxide (CC>2) and water vapor content of the air after passage over the leaves were determined using the IRGA and a General Eastern 1100 AP dew point hygrometer (Figs. 3 25 26 Figure 2. An Acacia koa phyllode in the leaf assimilation chamber for gas exchange determinations. r n CLC - DP- - R - - □ II - - -humidity control WD- - -water bath IR- - - -infrared gas analyzer F - - - -flow meter p - - - -pump - -controlled environment chamber - -assimilation chamber - -light source - -dew point hygrometer - -recorder Figure 3. Diagram of the semi-closed system for leaf gas exchange determinations, Department of Agronomy and Soil Science. N> ' • j Transpiration (T) rate was calculated by the equation T = (Co-Cj)F A where Cq and equal the water vapor density of the outgoing and incoming air streams (gem , 3 -1F equals the flow rate (cm s ), and 2A equals the projected leaf area (cm ). The water vapor density of the air streams was calculated from the dew point temperature of the air streams measured with a dew point hygrometer and from air and leaf temperatures. Total diffusion resistance in the leaf was calculated by the equation R = Cc - [(CQ + Cj)/2] 2 T where C equals the saturation water vapor density in the leaf -3(gem ) at the leaf temperature and the other parameters are as given in equation 1. Leaf conductance was calculated as the reciprocal of R. Chlorophyll Determination The amounts of chlorophyll (chi) a, b, total chi (a + b), and their ratio (a/b) were determined using the methodology of Arnon (1949). One-half gram fresh weight of leaf material taken from the middle part of the leaf or phyllode, was ground for 15 seconds with a Brinkman Polytron homogenizer in about 15 ml of 80 percent acetone and about 0.1 g of magnesium carbonate. Magnesium carbonate was added to prevent chlorophyll breakdown. The leaf material was further extracted with acetone until colorless and the extract was filtered through Whatman No. 1 filter paper to remove plant debris. The / 30 extract was brought to 100 ml with acetone. Optical densities of the extracts were measured in a Beckman Spectronic 100 at wavelengths of 663 and 645 nm. The chlorophyll content of the extract was determined from a nomogram of optical density vs milligrams chlorophyll (S^stak 1971). Total Soluble Protein and Ribulose-1,5-bisphosphate Carboxylase (RuBPcase) Determinations Koa leaves and phyllodes were destemmed and the surface areas of 1.0-g samples were determined. The leaf sample was ground in a Brinkman Polytron homogenizer for about 10 seconds with 20 ml of an extraction solution consisting of 50 mM Tris buffer, 10 mM MgCl^/ and 0.2 mM EDTA, adjusted to pH 7.2 with HC1 (Blenkinsop and Dale 1974). The pH of the resulting suspension was adjusted to 7.0 with NH^OH. The suspension was centrifuged for 30 minutes at 7500 g. The TSP content was assayed according to the method of Bradford (1976). A 0.025-ml sample was mixed with 5 ml dye-reagent solution (1 ml Bio-Rad protein assay dye reagent concentrate + 4 ml extraction solution). After about 10 minutes, the optical density of the sample was determined in a Bausch and Lomb Spectronic 20 (A595)• The weight of protein in the sample was determined from a calibration curve made using different amounts of bovine serum albumin in the dye reagent. The RuBPcase content of the supernate was determined by ammonium sulfate fractionation (Paulsen and Lane 1966, Wilson and McCalla 1968) and dye-reagent techniques (Bradford 1976). The initial extract was brought to 50 percent saturation with solid ammonium sulfate (320 g/2, 31 of extract). After standing for 30 minutes, the solution was centrifuged at 13,000 g for 30 minutes. The precipitate was resuspended in 2 ml of extraction solution. A 0.012-ml sample was mixed in 5-ml dye-reagent solution (1 ml dye-reagent concentrate + 4 ml extraction solution). After about 10 minutes, the optical density of the sample was determined in a Bausch and Lomb Spectronic 20 (A 5g5) • The of RuBPcase was determined from the same calibration curve as was used for TSP. Data Analysis The means and standard deviations were determined for each morphological characteristic, CER, chi a and b, TSP, RuBPcase, transpiration, and total leaf conductance for leaves and phyllodes. The degrees of freedom used in the Student's t-test for comparing the different means from samples of equal size were 18 for the morphological characteristics and 10 for all others. Unless otherwise noted, all differences reported as significant, are significant at least at the 0.05 level of probability. Results Morphological Characteristics Leaves and phyllodes differed in all observed morphological characteristics. Leaves are displayed horizontally. If the rachis bends down, the leaflets adjust so their display remain horizontal. Phyllodes are displayed vertically. Average length and width of the leaves are greater than that of the phyllodes (Table 2). Average area of the leaves (one side) is almost 3 times greater than the area of phyllodes. Phyllodes, however, are more than twice as thick as the 32 35 Table 3 Stomatal Frequency and Pore Length in Acacia Koa Leaves and Phyllodes Leaf Stomatal frequency Upper (left) Lower (right) Epidermis Epidermis Pore length - - - - mm”2 _ _ _ _ mm Leaves Phyllodes + 200 230 230 .020 .018 +Stomata are found only close to the major veins. anisolateral, having greater stomatal frequency on the abaxial surface than on the adaxial surface. Leaflets have about 200 stomates per square millimeter on the lower surface and just a few stomates along the major veins on the upper surface. Average stomate pore length of the leaflet and the phyllode was similar. The total stomatal pore area per square millimeter of leaf surface (1 side of leaflets and 2 2sides of phyllodes) was estimated to be 0.117 mm for phyllodes 2compared to 0.063 mm for leaflets. CO ̂ .-exchange Rates of Leaves and Phyllodes Leaves and phyllodes have similar CER at a given PPFD when calculations were based on the projected unit leaf area (Fig. 6). Light saturation occurs for both leaf forms at about 1200 ymol -2 -1m s (about 60 percent of full sunlight). Mean maximum rates of -2 -1 -2 -1photosynthesis were about 24 mg CO^ <3m h (15 ymol m s ) for both leaves and phyllodes. The photosynthetic rates declined with Phyllode (horizontal) o ' Figure 6. Rate of C02~exchange as a function of incident photosynthetic photon flux density of leaves and phyllodes of Acacia koa. decreasing PPFD at about the same rate for both leaf forms. The light - 2 - 1compensation point was about 25 ymol m s and was similar for both leaves and phyllodes. The COcompensation concentration was about 55 ppm for both leaves and phyllodes. Although the leaves and phyllodes had similar CER when determined on a projected unit leaf area basis, they had different CER when determined on the projected organ area basis. Leaves displayed about 3 times more projected surface area than phyllodes so that on an organ basis, the CER for leaves was greater than for phyllodes (Table 4). 37 Table 4 C02~exchange Rate by a Single Fully-expanded Acacia koa Leaf and Phyllode, Based on Projected Leaf Area Leaf CER at measurement PPFD (ymol m"2s_l) form 1200 1000 375 165 Dark Leaves 28.9+ mg CO 2 leaf 28.2 ■lh-1 17.8 7.2 -2.2 Phyllodes 9.6 9.3 5.4 1.8 -0.9 +Each value is a mean of six observations. -2 -1At PPFD of 1200, 1000, and 375 ymol m s the CER of leaves was -2 -1about 3 times that of phyllodes. At 165 ymol m s the CER of leaves was 4 times that of phyllodes, while in the dark the difference was about 2.4 times. Phyllodes are displayed vertically, so in nature the PPFD is 40 Table 5 Chlorophyll in Leaves and Phyllodes of Acacia Koa Leaf formr Chlorophyll a b Total (a + b) a/b mg g”^+ mg dm-^ mg g”l mg dm-2 mg g~l mg dm-2 Leaves 1.27a++ 5.39a 0.47a 2.01a 1.74a 7.40a 2.7 Phyllodes 0.86b 3.01b 0.30b 1.05b 1.16b 4.06b 2.9 +Chlorophyll is expressed on a fresh weight basis. ++Each value is a mean of six observations. Values in columns followed by the same letter do not differ significantly at the 0.05 level of probability. of chi a to b averaged about 2.7 for leaves, not significantly less than the average of 2.9 for phyllodes. Total Soluble Protein and RuBPcase in Leaves and Phyllodes On a fresh weight basis and on an area basis, leaves also had significantly greater amounts of TSP than phyllodes (Table 6). On a weight basis, leaves contained about 8.5 times more protein than the phyllodes. Total soluble protein made up about 18 percent of the dry weight of leaves and about 2 percent of phyllodes. On an area basis, leaves contained about 4 times more soluble protein than the phyllodes. Ribulose-1,5-diphosphate carboxylase (RuBPcase) levels of leaves on a fresh weight basis were about 12 times greater than those of the phyllodes (Table 6). On an area basis, leaves contained about 6 times more enzyme than the phyllodes. The percentage of the total protein complex made up of RuBPcase 41 Table 6 Total Soluble Protein and RuBPcase+ in Leaves and Phyllodes of Acacia Koa Leaf form Total soluble protein RuBPcase+ mg g‘ -1++ mg dm-^ L e a v e s P h y l l o d e 43.08a+++ 5.33b 66.34a 17.26b 19.15a 1.51b 29.49a 5.13b +Ribulose-l,5-bisphosphate carboxylase. ++Soluble protein and RuBPcase are expressed on a fresh weight basis. +++Each value is a mean of six observations. Values in columns followed by the same letter do not differ significantly at the 0.05 level of probability. also varies markedly between leaves and phyllodes. In leaves, about 44 percent of the TSP was represented by RuBPcase, whether calculated on a fresh weight or an area basis. In phyllodes, about 28 percent of the TSP was RuBPcase, regardless of the basis used for the calculation. Transpiration Rate and Total Leaf Conductance of Leaves and Phyllodes If transpiration rates are expressed on a projected unit area basis, phyllodes have significantly higher rates at 1200 and 165 ymol - 2 - 1m s than do leaves (Table 7). However, if transpiration rates are expressed on a projected organ area basis, leaves have rates 1.5 -2 -1times greater than phyllodes at 1200 umol m s and 2 times - 2 - 1 . greater at 165 ymol m s . In the dark, transpiration rate on a projected unit area basis is about the same for leaves and phyllodes, but on a projected organ area basis transpiration rate of leaves is twice that of phyllodes. 42 Transpiration by a Single Fully-expanded Acacia koa Leaf and Phyllode, Based on Projected Unit and Organ Area Table 7 I Leaf form 1200 165 Dark Unit area Organ area Unit area Organ area Unit area Organ area mg dirT^h-! mg h-l mg dirT^h--'- mg h-l mg dnT^h-! mg h-! Leaves 49.7a++ 59.6 39.6a 47.5 19.1a 22.9 Phyllodes+ 90.4b 38.0 56.5b 23.7 24.8a 10.4 +Phyllodes were oriented horizontally rather than in the natural vertical position. ++Each value is a mean of six observations. Values in a column followed by the same letter do not differ significantly at the 0.05 level of probability. In the light, the mean total leaf conductance for leaves was greater than for phyllodes, but the differences were not statistically significant (Table 8). Discussion Photosynthetic Characteristics of Leaves and Phyllodes A rate of CC^-exchange at saturating PPFD for leaves and -2 -1phyllodes of about 24 mg CO^ dm h is similar to the rates reported for eucalyptus (Brittain and Cameron 1973), apple (Malus sp.) (Heinicke and Hoffman 1933, Mika and Antoszewski 1972) and aspen (Populus sp.) (Okafo and Hanover 1978). The mean maximum CER obtained _ O _ Ifor A. koa phyllodes was about twice the rate of 13.4 mg CO^ dm h found for phyllodes of A. harpophylla (Connor et al. 1971, Tunstall and Connor 1975). No C02~exchange rates were found for leaves of carbohydrates. The quantity of RuBPcase in a leaf varies depending on the PPFD level to which the leaf is exposed. Leaves exposed to high PPFD levels generally have more RuBPcase than leaves exposed to low PPFD (Bjorkman 1968, Boardman 1977). Acacia koa leaves are adapted to full sunlight. In fact, leaflets adjust to maintain an angle perpendicular to the sun. Because of the vertical display, koa phyllodes are not subjected to the high PPFD that leaves are. The amount of RuBPcase in leaves was 6 to 12 times greater than the amount in phyllodes, depending on whether the quantity was figured on an area or fresh weight basis, respectively. Phyllodes, however, contain sufficient RuBPcase for their CERs on a leaf area basis to equal those of leaves. If CERs of leaves and phyllodes are expressed on a - 2RuBPcase (mg dm ) basis, the rate for phyllodes is about 5 times -2 -1greater than for leaves (Table 9). Data on CER (mg CO^ dm h ) -2RuBPcase (mg dm ) have not been found for other species. Data from 45 Table 9 Photosynthetic Rates of Acacia Koa Leaves and Phyllodes Expressed on the Basis of Leaf Area, Chlorophyll, and RuBPcase+ Leaf mg CO 2 mg CO 2 h“l mg CO 2 h"l form h— 1 dm” 2 (mg Chl)“l (mg RuBPcase)-^ Leaves 24.la++ 3.26a 0*8 2 s Phyllodes 22.8a 5.62b 4.44b +Ribulose-l,5-diphosphate carboxylase. ++Each value is a mean of six observations. Values in columns followed by the same letter do not differ significantly at the 0.05 level of probability. this study indicates that some of the RuBPcase in leaves may be inactive. Although leaves and phyllodes have similar CERs when expressed on a projected unit area basis, leaves have about 3 times the CER rate of phyllodes when expressed on a projected organ area (Table 4). Leaves have 3 times the projected area as do phyllodes. A leaf contributes at least 3 times more photosynthate for seedling growth and development as does a phyllode. CO^-exchange rates (CER) of both leaves and phyllodes decrease as the zenith angle of incident PPFD (0) increases. In nature, however, the decrease in CERs is not as great as would be expected because the PPFD absorbed by leaves remains approximately constant as 0 increases from 0 to 60° (Kriedeman et al. 1964). When the PPFD is in excess of or near that required to saturate photosynthesis, the small decrease in PPFD absorbed may be offset by the increase in photosynthetic efficiency at the lower PPFD levels. In addition, a reorientation of the chloroplasts with respect to the direction of incoming PPFD could result in more complete absorption of PPFD with increasing 0. CO^-exchange characteristics of leaves and phyllodes are affected by their morphology and anatomy. Leaves consist of many small leaflets with smooth surfaces while phyllodes have a large rough surface. The large, rough surface of phyllodes would be expected to result in lower boundary layer conductance than would be found for leaflets under the same wind conditions. Boundary layer conductance (g ) estimated by the formula g = 0.59 L ®*25 v°'5 where L is the cl cl 46 2 -1 leaf area (cm ) and v the wind speed (cm s ) (Milthorpe and Moorby 21974), the boundary layer conductance was calculated for a 1 cm 2leaflet and a 120 cm phyllode at different wind speeds (Table 10). 47 Table 10 Calculated Boundary Layer Conductance of a Hypothetical A. Koa Leaflet and Phyllode at Different Wind Speeds Boundary layer conductance Wind speed (v) Leaflet Phyllodes - - - - cm s-1 - - - - - 10 1.85 0.56 40 3.70 1.12 160 7.69 2.27 640 14.29 4.55 At each wind speed, the conductance of a phyllode is only one-third that of a leaflet. The roughness of the phyllode surface and the presence of hairs initially also decrease the conductance. Sunderland (1968) found that the drag on a smooth metal replica of a wheat leaf increased when a real leaf was attached to the metal surface. The increase was about 20 percent at V = 150 cm s \ growing to 50 percent at V = 50 cm s 1 because of the increasing importance of surface friction at low speeds. Phyllode conductance would be significantly less than the values shown in Table 10 because of surface roughness and area. The boundary layer conductance of a leaflet would also probably be less than the values shown in Table 10 which also have photosynthetic rates similar to those of koa (Hill et al. 1960). No transpiration rates were found for either leaves or phyllodes of any Acacia species. Leaves, as a whole, transpire about 57 percent more water than do phyllodes even when phyllodes are displayed horizontally. The change from leaves to phyllodes is an apparent adaptation to drier conditions as would occur as the plant gets larger and competition occurs within and between plants. Total leaf conductance was similar for both leaves and phyllodes (Table 8). This was unexpected because of the differences in the transpiration rate and in the morphological and anatomical characteristics between leaves and phyllodes. The boundary layer conductance for leaves would be much greater than for phyllodes because each leaflet is small, thin, and smooth, in comparison to each phyllode. The greater boundary layer conductance for leaves is apparently offset by greater stomatal conductance of phyllodes. Phyllodes have about twice the stomatal pore area per unit leaf area as leaves have. The boundary layer and stomatal conductances apparently have about equal effect as the total leaf conductance of leaves is similar to that of phyllodes. The total leaf conductances determined for A. koa phyllodes was similar to the value of 0.5 cm s ^ determined for A. harpophylla phyllodes under high PPFD and adequate leaf moisture (Tunstall and Connor 1975). At low PPFD levels and adequate moisture, total diffusive conductance for phyllodes of both species was about 0.25 cm s \ In the dark, total leaf conductance of A. harpophylla 50 phyllodes was about 0.02 cm s \ much less than the 0.07 cm s ^ obtained for A. koa phyllodes. The discrepancy could be due to a higher true conductance of A. koa phyllodes than those of A. harpophylla or to incomplete stomatal closure of the A. koa phyllodes. E c o l o g i c a l S i g n i f i c a n c e o f L e a v e s a n d P h y l l o d e s Continuous rapid plant growth is dependent upon continuous efficient absorption of PPFD. Plants which have horizontal leaves that are replaced by steeply-inclined leaves have an "ideal" foliage configuration for PPFD absorption (Trenbath and Angus 1975). The horizontal leaves of plants exposed to full sunlight intercept maximum PPFD. The larger leaves produce about 3 times more photosynthate than phyllodes. However, as the LAI increases and therefore mutual shading increases, PPFD penetration of the canopy decreases. A canopy of steeply-inclined leaves is able to intercept more PPFD under field conditions than can an equivalent canopy of horizontal leaves and, therefore, CERs are higher (Trenbath and Angus 1975). Heterophyllous plants like A. koa have this ideal foliage configuration. Horizontal leaves are replaced by steeply-inclined phyllodes. The leaves and phyllodes together allow koa and other heterophyllous species to be pioneering species. Koa makes rapid growth and therefore, is able to i compete with other species that also become established after removal of the forest canopy. The change from leaves to phyllodes is also an apparent adaptation to reduced moisture availability. Transpiration rates of phyllodes are less than leaves even when phyllodes are exposed to 51 higher than normal light levels. Organ orientation, surface, and anatomical characteristics, as previously discussed, all indicate that phyllodes are more drought resistant than leaves. Moisture loss after dark is less from phyllodes than from leaves because their stomates close after about 30 minutes in the dark, while stomates of leaves do not close until after 120 or more minutes in the dark. Tunstall and Connor (1975) found that phyllodes of A. harpophylla can carry on photosynthesis over a wider range of environmental conditions than can leaves; i.e., ambient temperatures 0 to 50° C, relative humidity 20 -2to 100 percent, and daily radiation from 5 to 20 MJm . Phyllodes are able to maintain a favorable internal moisture state even under adverse conditions. Therefore, phyllodes tend to persist even under very adverse conditions (Tunstall and Connor 1975). This persistence makes it possible for A. koa to exploit the environment when conditions again become favorable. Although no data are available for leaves of any Acacia species, it lias been observed in the nursery that A. koa plants shed their leaves when the seedlings are stressed for moisture. If leaves are lost because of moisture stress, plant recovery, even under favorable moisture conditions, is doubtful. 52 more chlorophyll per unit weight or per unit volume of leaf (Bjorkman 1975, Hoober and Stegeman 1976, Louwerse and Zweerde 1977), but the chlorophyll content per unit area of leaf surface is often lower than that of leaves grown at high PPFD (Bjorkman and Holmgren 1963, Hesketh 1968, Wild et al. 1975). The increase in size of the chloroplasts and the amount of chlorophyll per chloroplast in shade leaves is more than offset by the decrease in the number of chloroplasts per unit area of leaf surface. A change in PPFD results in changes in chloroplast ultra-structure. Grana thickness increases as PPFD decreases, but above a given PPFD no additional changes were observed (Skene 1974). The proportion of chi b relative to chi a increases as PPFD is lowered below a threshold value which is usually about the PPFD where morphological changes also occur (Bjorkman and Holmgren 1963, Boardman et al. 1972, Lewandowska et al. 1976). The higher proportion of chi b in understory species enhances their light-absorbing capacity in the wavelength region between the main blue and red bands of chi a (Boardman 1977). Leaves in the higher, more exposed part of the upperstory species have a higher proportion of chi a which allows them to utilize the blue and red wavelengths. Shade species have lower soluble leaf protein/chl ratios than sun plants. They contain smaller amounts of RuBPcase which correlate with their low levels of soluble protein (Bjorkman 1975, Boardman et al. 1972). The rate of synthesis of RuBPcase and other enzymes involved with photosynthesis is controlled by PPFD (Salisbury and Ross 1978, Tobin and Suttie 1980). Under low PPFD, the rate of synthesis is slow. As PPFD increases to the saturation level the rate of enzyme 55 synthesis also increases. Photosynthetic photon flux density levels beyond the light-saturation point may be injurious. The CO^-exchange characteristics of individual leaves vary, depending on the PPFD under which they developed. Sun leaves of trees -2 -1are light-saturated at PPFD of 600 to 900 umol m s . Shade leaves, however, are accustomed to low PPFD and become light-saturated a t P P F D o n l y o n e - f o u r t h t o o n e - t h i r d o f t h o s e r e q u i r e d t o l i g h t - saturate sun leaves. Generally, light-saturated CERs of most plants -2 -1are considerably higher for sun leaves (16 to 20 mg CO^ dm h ) than -2 -1for shade leaves (2 to 5 mg CC>2 dm h ) . The light compensation points of sun leaves are also higher than for shade leaves being 20 to -2 -1 -2 -1 30 umol m s and 2 to 10 umol m s , respectively (Berry 1975, Boardman 1977, Boehning and Burnside 1956). Shade leaves can usually photosynthesize at higher rates under low PPFD than can sun leaves (Salisbury and Ross 1978). Leaves on the same plant may have C02~exchange characteristics of sun leaves, shade leaves, or both, depending on their exposure. Leaves on the shaded side of plants growing under a high PPFD (shade leaves) have characteristics more like those of leaves on true shade plants than do the sun leaves on the sunny side. These differences result from anatomical and biochemical adaptations during leaf development as previously noted. Sun plants generally are incapable of growth or only grow slowly a t P P F D a s l o w a s t h o s e t o l e r a t e d b y e x t r e m e s h a d e p l a n t s . Conversely, species or ecotypes limited in nature to densely-shaded habitats lack the genetic ability to produce photosynthetic machinery 56 as efficient at high PPFD as that of sun plants and their leaves may even be damaged at high PPFD levels (Bjorkman 1975, Boehning 1949, Bormann 1953, Kozlowski 1949). Kozlowski (1957) found much greater inhibition of photosynthesis by PPFD over time in hardwood seedlings than in pine seedlings. He suggested that in well-exposed trees, inhibitory solarization may cause the outer leaves of tree crowns to photosynthesize less efficiently than do partially-shaded leaves. The decrease in photosynthetic efficiency was caused by photo-oxidative deactivation of some enzymes. A further decline in photosynthetic efficiency with time was caused by photo-bleaching of the chlorophyll or a combination of the two factors. Continuous exposure of plants to high PPFD can reduce their growth (Krueger and Ferrell 1965), or kill them (Ronco 1970, Ronco 1972). F o r e s t t r e e s a r e g e n e r a l l y c l a s s i f i e d a s s h a d e - t o l e r a n t o r i n t o l e r a n t , d e p e n d i n g o n t h e P P F D u n d e r w h i c h t h e y g r o w . Shade-tolerant species generally photosynthesize more efficiently at lower PPFD than intolerant species, and therefore, they are generally found as understory species or as climax types. Shade-intolerant species are commonly found in open areas such as would occur after some disturbance; i.e., fire or logging. The different photosynthetic rates of various species at different PPFD affect their ability to compete. Species composition of a forest is ultimately determined by adaptation of the different species to PPFD. Kramer and Decker (1944) found that the photosynthetic rate of shade-intolerant loblolly pine seedlings increased with PPFD up to almost full sunlight whereas the photosynthetic rate of three shade-tolerant hardwood species saturated 57 were placed on the surface of the growing media in each pot. The 90 seedlings at each light level provided a pool from which seedlings were selected for gas exchange determinations and growth analyses. The bases for seedling selection were average-sized healthy plants. Gas Exchange Determinations CO^-uptake and CO^-efflux (dark) determinations were made every 3 weeks on six seedlings from each treatment. The first determinations were made at the time the seedlings were potted; the last were made when the seedlings were 18 weeks old. The materials and methods for determining CERs were the same as described in Chapter III, except determinations were made only at PPFDs of 1250 and 165 ymol m 2s 1 and in darkness. Chlorophyll, Total Soluble Protein, and Ribulose-1,5-diphosphate Carboxylase Determinations When the seedlings were 18 weeks old, the levels of chlorophyll, TSP, and RuBPcase in the fourth or fifth leaf (LPI-4 or 5) of six seedlings from each PPFD treatment were determined. The materials and methods were as described in Chapter III. Seedling Growth Determination Every 3 weeks, after CO^ exchange determinations were made, the potting media was washed from the roots of nine seedlings from each treatment. Measurements were made of stem height and diameter, leaf area and number, and dry weight of the stem, leaves, and roots. Dry weights were determined after several days of drying at 70° C. Total biomass was determined by adding the dry weights of roots, shoot, and leaves. 60 Relative growth rate (RGR), NAR, and LAR were calculated from these data. Data Analyses The means and standard deviations for the CER, chlorophyll, TSP, and RuBPcase, were determined from the six seedlings in each treatment. Treatment means were compared using Tukey's LSD (0.05 level) value. The means and standard deviations for stem height and diameter, leaf number and area, and dry weights, and for RGR, NAR, and LAR, were determined from nine seedlings in each light level. These treatment means were also compared using Tukey's LSD (0.05 level) value. All differences reported as significant are significant at least at the 0.05 level. Results Seedling Growth and Development Stem height and diameter growth were significantly affected by light level. After 42 days, differences in stem height and diameter between the treatments were already evident (Table 11). Seedlings in 45 percent light were significantly taller than those grown in 100 percent light. Seedlings in the 77 and 27 percent treatments were also taller than those grown in the 100 percent treatment, but the differences were not significant. Stem diameter decreased as the light level was decreased, but the differences at 42 days were not significant. With each succeeding measurement, there was greater separation in stem height and diameter between the treatments. Seedlings grown in the 100 percent treatment always had significantly greater stem height and diameter than seedlings grown in the 27 61 Table 11 Stem, Leaf, and Root Development of Acacia Koa Seedlings Grown Dnder Different Light Levels Days since emergence Light level Stem Leaves Area leaf Dry weight Shoot- root ratioHeight Diameter Number Area Stem Leaves Roots Total Percent --- cm - - - - - cm ^ ---- - - - - gram - - - - --- 42 100+ 21.4a++ 0.29a 8. 3ab 100.5a 12.1 0.16a 0.36a 0.13a 0.66a 4.1a 77 22. 4ab 0.28a 8.2a 104.1a 12.7 0.21a 0.45a 0.15a 0.81a 4.5a 45 25.4b 0.27a 9.5b 113.5a 12.0 0.23a 0.47a 0.14a 0.84a 5.4a 27 23.3ab 0.23a 7.3a 94.2a 12.0 0.20a 0.38a 0.14a 0.73a 4.5a 68 100 35.4a 0.40a 11.3a 234.7a 20.8 0.54a 0.88a 0.3 lab 1.72a 4.7a 77 33.9ab 0.38a 10.lab 241.8a 23.9 0.50a 0.87a 0.34a 1.70a 4.1a 45 35 .Oab 0.37a 11.3a 246.0a 21.8 0.42a 0.75a 0.21b 1.38a 6.0a 27 28.5b 0.29b 8.9b 140.8b 15.8 0.20b 0.39b 0.10 0.70b 5.9a 93 100 60.7a 0.59a 15.4a 335.0a 21.8 1.69a 2.24a 0.89a 4.82a 4.7a 77 54.3a 0.56ab 13.3ab 374.4a 28.2 1.30ab 1.8lab 0.64ab 3.75ab 4.9a 45 54.3a 0.48b 13.0b 344.3a 26.5 1.06bc 1.36bc 0.45bc 2.87b 5.8a 27 39.8b 0.34c 12.1b 234.3a 19.4 0.61c 0.70c 0.22c 1.54c 6.1a 107 100 77.0a 0.74a 18.4a 624.6a 34.0 3.14a 4.07a 1.50a 8.70a 5.4a 77 71.4a 0.65b 17.7a 544.2a 30.8 2.52a 2.58b 1.03a 6.14b 5.2a 45 70.0a 0.61ab 17.0a 636.7a 37.4 2.36a 2.69b 0.97a 6.02b 5.3a 27 46.0b 0.41c 13.4b 224.1b 16.7 0.72b 1.01c 0.32b 2.06c 5.6a 131 100 89.7a 0.80a 18.3a 793.7a 43.4 5.54a 4.25a 2.00a 11.79a 5.0a 77 91.2a 0.8 lab 16. lab 815.5a 50.6 5.66a 4.36a 1.92ab 11.94a 5.6a 45 77.5b 0.65c 17.4a 714.7a 41.0 3.24b 3.48a 1.10b 7.81b 6.8a 27 38.7c 0.69bc 12.3b 310.4b 25.2 1.22c 1.28b 0.41c 2.92c 6.1a 161 100 111.3a 0.91a 17.7ab 728.4a 41.1 7.94a 4.41a 2.68a 15.02a 4.8a 77 102.2a 0.77b 17.8ab 641.2ab 36.0 5.40b 3.32b 1.62b 10.33b 5.6ab 45 86.1b 0.62c 19.6a 503.0b 25.6 3.12c 2.55b 0.87c 6.55c 6.8b 27 55.3c 0.49d 14.2b 217.4c 15.3 1.18d 1.12c 0.46d 2.76d 5.2ab +The light level in the fiberglass-covered greenhouse at midday is about 70 percent of that outside. ++Each value is a mean of nine observations. Values in columns for a time period followed by the same letter are not significantly different at the 0.05 level of probability. OR T H EI G H T (G ) Qf TT H EI G H T lO ) 65 >-a:o RC-E FRCM EMERGENCE iDflTSi RC-E P RCM EMERGENCE i DRTS, 15. 10 . 5. 0 -- UJx tra RC-E FROM EMERGENCE IDRTSJ RC-E FROM EMERGENCE ( O RI S ; Figure 8. Dry weight increments of stem (A), leaves {*), roots (O), and total (+) of Acacia koa seedlings grown under different light treatments. root dry weight due to treatment, differences in dry weight of stems, leaves, and roots, as a percent of total dry weight, were not significantly affected by treatment (Table 12). After 42 days, leaves, stems, and roots made up about 55, 26, and 19 percent, respectively, of the total dry weight of seedlings in each treatment. At each measurement time, the percent in stems increased. The percent of total dry weight in roots remained almost constant over time. After 161 days, stems made up about 49 percent of the total dry weight, leaves 35 percent, and roots 16 percent. Growth Analysis Components Koa relative growth rates (RGR) (or the biomass increment per unit of existing biomass) were generally similar for seedlings in each light treatment (Table 13). Relative growth rates decreased significantly with decreasing light level only during the growth period between days 42 and 68. The negative RGR for seedlings in the 27 percent light treatment during that growth period, and for seedlings in the 77, 45, and 27 percent light treatments in the growth period between days 131 and 161, was due to a decrease in mean total dry weights of the seedlings at the end of the respective growth periods (Fig. 8 and Table 11). The RGR of seedlings in the 100, 77, and 45 percent light treatments peaked during the 93- to 107-day growth period, whereas the RGR of seedlings in 27 percent light treatment peaked during the 68- to 93-day growth period. The NAR (the biomass increment per day per unit leaf area) of the seedlings in the 100 percent light treatment in each growth period, except for the period from day 107 to 131, was significantly greater 66 67 Table 12 Dry Weight Distribution in Stems, Leaves, and Roots of Acacia Koa Seedlings Grown Under Different Light Levels Days since Light Percent of total dry weight emergence level Stem Leaves Roots Percent 42 100 26 55 20 77 26 55 19 45 27 56 17 27 27 54 19 68 100 31 51 18 77 29 51 20 45 30 54 16 27 29 56 15 93 100 35 46 19 77 35 48 17 45 37 47 16 27 40 45 15 107 100 36 47 17 77 41 42 17 45 39 45 16 27 35 49 16 131 100 47 36 17 77 47 37 16 45 41 45 14 27 42 44 14 161 100 53 29 18 77 52 32 16 45 48 39 13 27 43 40 17 70 Table 14 CC^-exchange Rate by a Single Fully-expanded Acacia Koa Leaf Grown Under Different Light Levels Days since emergence Light level CER 1250 at measurement 165 PPFD Dark Percent - - - mg C02 dm~2h_1 _ _ _ 42 100 23.62a+ — -2.51a 77 17.41b — -1.49b 45 15.54bc — -1.32b 27 12.90c — -1.39b 68 100 27.47a 9.33a -2.97a 77 20.52b 8.77a -2.18b 45 21.77b 10.22a -2.13b 27 20.48b 9.30a -1.87b 93 100 27.02ab 9.22a -2.73a 77 28.34a 10.58a -2.34ab 45 26.92ab 11.14a -1.92b 27 22.96b 10.62a -1.75b 107 100 26 • 88a 9.41ab -2.13a 77 25.12ab 11.73a -1.91a 45 22.14bc 11.69a -1.65b 27 17.70c 8.71b -1.48b 131 100 30.10a 10.44a -2.43a 77 29.88a 11.66a -1.99b 45 24.39ab 10.03a -1.52b 27 18.57b 10.34a -1.68b 161 100 22.66ab 5.00a -2.89a 77 23.96ab 5.82a -2.25b 45 24.55a 5.96a -2.13b 27 19.81b 5.79a -2.04b +Each value is a mean of six observations. Values in columns for a time period followed by the same letter are not significantly different at the 0.05 level of probability. leaves were used. The CER of seedling leaves determined at 165 ymol m s was similar for each treatment. Koa seedlings grown at low light were not significantly more efficient in fixing CO^ at low PPFD than those grown at high light although the CER of seedlings grown in the 45 percent treatment had higher CERs than those grown in the 100 percent light treatment. The CER measured at 161 days in a PPFD of 165 was only about one-half the rate obtained at all other measurement times (Table 14). There is no explanation for this decrease but it was consistent for seedlings in all light treatments. The CERs determined in the dark (respiration) were consistently highest for seedlings grown in the 100 percent light treatment and decreased as the light level decreased (Table 14). The differences in dark respiration rate between seedlings in the 100 and 77 percent light were generally significant. However, dark respiration rate differences between the 77, 45, and 27 percent light treatments were not significant. There were no correlations between the dark respiration rate and CER at light-saturation for seedlings grown at any light level. Leaf contents of chi a, b, and total varied with treatment (Table 15). Leaves from plants grown in the 45 percent light treatment had significantly more chi a, b, and total than leaves from the 77 percent light treatment, which in turn had significantly more chlorophyll (a, b, total) than leaves from plants grown in the 100 percent treatment. Leaves in the 27 percent light treatment were the exception as they had chi b levels similar to those leaves from the 77 percent light ( 71 -2 -1 72 Chlorophyll Content of Acacia Koa Leaves Grown Under Different Light Levels Table 15 Light level Chlorophyll a b Total a/b mg g“l mg dnf ̂ mg g"l mg dm” ̂ mg g-1 mg dm” * 100 1.25a+ 2.15a 0.55a 0.94a 1.80a 3.09a 2.30a 77 1.61b 2.37a 0.72ab 1.05ab 2.34b 3.42a 2.24ab 45 1.87c 2.32a 1.00c 1.24b 2.87c 3.55a 1.87b 27 1.64b 2.22a 0.81bc 1.09ab 2.45b 3.32a 2.02ab +Each value is a mean of six observations. Values in columns followed by the same letter are not significantly different at the 0.05 level of probability. treatment. The ratio of chi a to b decreased as the light level decreased. The TSP and RuBPcase levels of leaves were not significantly correlated with treatment. Leaves grown in the 100 percent light treatment had significantly more TSP and RuBPcase than leaves grown at the other light treatments, but there were no significant differences between TSP and RuBPcase levels and among the other treatments (Table 16). The RuBPcase comprised about 42 percent of the TSP regardless of the light regime of the plants. Discussion Seedling Growth and Development The quantity of available light afforded by the four treatments affected the length of the establishment period and the subsequent and LAR, both changed with available light in a manner that maintained a near constant RGR. Similar results were obtained by Warrington et al. (1978) for both and species. The RGR, NAR, and LAR values obtained for koa were typical of plants in general, as they were similar to values obtained for cotton (Mauney et al. 1978), walnut (Juglans nigra L.), honeylocust (Gleditsia triacanthos L.) (Carpenter and Hanover 1974), and for northern red oak (Quercus rubra L.) (Farmer 1975). CO ̂.-Exchange Rate For plants adapted to full sunlight (nonshade plants), the CER of leaves at light saturation usually is higher for leaves that developed under full sunlight than it is for leaves that developed in deep shade. Conversely, the CER of leaves in low light is higher for leaves that developed in shade than for leaves that developed in full sunlight (Berry 1975, Bjorkman et al. 1972). The former case was true for koa leaves, but koa leaves that developed at 27 percent of the full sunlight treatment did not have a higher CER at low light (165 -2 -1Vimol m s ) than leaves that developed in full sunlight. The greater CER of koa leaves grown in the 100 percent light treatment at -2 -11250 ymol m s can probably be partially attributed to their higher RuBPcase levels (Table 14). Boardman (1977) reported that other factors that contributed to a higher CER at high light are higher levels of constituents of the electron transport chain and greater mesophyll and stomatal conductances. Though no measurements of these components were made on koa leaves, it seems likely that similar results to those reported by Boardman (1977) would have been 75 obtained. The leaf morphology of leaves grown under high light results in greater stomatal and mesophyll conductances to CO^ than is found for leaves grown under low light. The increased stomatal conductance is correlated with a greater stomatal frequency (Bjorkman et al. 1975, Charles-Edwards and Ludwig 1975, Crookston et al. 1975) and mesophyll conductance is proportional to leaf thickness and the surface area of the mesophyll cells, both of which increase when leaves develop in a high light environment (Holmgren 1968, Nobel et al. 1975). Leaves which develop in high light also have a higher overall cell metabolism than do leaves that develop under low light (Boardman 1977) and higher dark respiration rates were measured for koa leaves grown in full sunlight than for those grown under low light (Table 14). With koa, as with other species (Goodchild et al. 1972) the amount of chlorophyll increased as the available light decreased down to 45 percent of full sunlight. The higher chlorophyll in plant leaves grown under low light enhances energy absorption at low light, and therefore, the CER for shade leaves at low PPFD should be greater than for sun leaves. Koa leaves grown at 27 percent of full sunlight had significantly less chlorophyll than leaves grown at 45 percent of full sunlight. This is the opposite of what was expected. Although the light compensation point for individual koa leaves is about 25 - 2 - 1 . pmol m s (Chapter III) , perhaps the light compensation point for t h e w h o l e p l a n t i s h i g h e r . B e c a u s e o f m u t u a l s h a d i n g , m a n y o f t h e leaves may have been growing at close to the light compensation p o i n t . U n d e r s u c h c o n d i t i o n s , t h e y w o u l d n o t h a v e s u f f i c i e n t e n e r g y 76 to invest in large leaves or in light-harvesting pigments. As noted earlier, the average area of a leaf in the 27 percent light treatment was about 60 percent of the area of a leaf in the 45 percent light treatment, and only about 50 percent of the area of a leaf in the full sunlight treatment. CO^-Exchange Rate and Seedling Growth P l a n t g r o w t h i s d e p e n d e n t o n t h e s u p p l y o f p h o t o s y n t h a t e . However, the correlation between photosynthesis per unit of leaf area and growth is often poor (Evans 1975, Helms 1976, Ledig and Perry 1967, Mauney et al. 1978). In this study, correlations between CER of single leaves and stem height and diameter, and stem, leaf, and root dry weights were also poor, with r values ranging from 0.07 to 0.18. T h e p o o r c o r r e l a t i o n w a s n o t s u r p r i s i n g b e c a u s e t h e r e w e r e f e w -2 -1significant differences among CER values at 1250 ymol m s for leaves grown at the different light levels. The correlation between CER and growth probably could have been improved if the CER had been determined on whole plants instead of on single leaves. The CER varies with PPFD and PPFD varies with position in the seedling crown (Okafo and Hanover 1978). Leaves in the upper part of the crown absorb much of the incident PPFD, allowing less for the lower leaves. If the light level during growth was sufficiently great to penetrate to the lower leaves, many of these leaves would be light-saturated. However, if the growth PPFD were lower than that required for light saturation of the upper leaves, the lower leaves could be at or even below the light compensation point. The total photosynthate available for maintenance of tissue integrity and 77 To investigate the adaptive potential of koa one must examine the physiological and anatomical changes that occur in both leaves and phyllodes as the amount of available light energy changes. The objective of this study was to determine the adaptive potential of partially- and fully-developed leaves and phyllodes of koa to changes in light levels. Specifically, the changes in CER, in level of chlorophyll, TSP, RuBPcase, and specific leaf weights that resulted from changes in light levels, were determined. Literature Review The photosynthetic properties of a species can be modified by growing it under PPFDs different than normal; i.e., by growing a sun plant in the shade or conversely a shade plant in the sun (Bjorkman and Holmgren 1966, Bunce et al. 1977, Burnside and Boehning 1957). The extent of modification depends on the species or the variety within a species. The photosynthetic rate of some sun plants grown -2 -1under shade light-saturated at a PPFD at least 20 ymol m s lower than if they were grown under full sunlight, while rates of other species remained unchanged (Burnside and Boehning 1957). The light compensation point of sun plants grown in the shade decreased by -2 -1as little as 0 and as much as 20 ymol m s . When shade plants were grown under higher than normal irradiances, the photosynthetic rate became light-saturated at higher intensities in some species, but not in others (Burnside and Boehning 1957). These modifications in photosynthetic properties resulted from physiological and anatomical changes that occurred with changes in PPFD. Leaf adaptation to a new light environment results in changes in the photosynthetic rate at a 80 given PPFD, stomatal conductance, leaf anatomy, leaf-water potential, photosynthetic unit size, and glycolate oxidase and RuBPcase activity. Some changes may occur within 1 day of alteration of the light environment. Changes in chlorophyll content, numbers of photosynthetic units, specific leaf weight, and malate dehydrogenase activity are slower (Alberte et al. 1976, Bowes et al. 1972, Bunce et al. 1977, Charles-Edwards and Ludwig 1975, Patterson et al. 1977). Both anatomical and physiological changes may occur even after leaf expansion is complete (Bunce et al. 1977). Although the intrinsic photosynthetic capacity of individual leaves imposes an upper limit on a tree's potential rate of dry matter production, this potential rate is rarely approached because only part of a tree canopy is ever light-saturated. Through manipulation of canopy architecture; i.e., plant spacing, species composition, and selection for leaf orientation, the horticulturist, agronomist, or forester can increase the average PPFD on lower shaded leaves and decrease it on upper exposed leaves. The net result is a greater number of leaves assimilating CO^ at a higher average rate and with greater photosynthetic efficiency (Boardman 1977, Bunce et al. 1977). Many silvicultural systems were developed to meet the environmental requirements of different species (Smith 1962). Species such as red maple (Acer rubrum L.) and yellow-poplar (Liriodendron tulipifera L.) can be established and make rapid growth under relatively low PPFD (Kozlowski 1949). The shelterwood system was developed for such species (Smith 1962). Under this system, some large trees of the desired species are left during harvesting to 81 provide cover for the next generation. The clearcut system is used for species such as Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] which requires almost full sunlight to become established (Krueger and Ruth 1969). In the clearcut system, all stems are cut from an area. As trees grow, competition for light and other environmental factors increases. To ensure an adequate photosynthetic rate for rapid growth of the desired tree species, stand density can be regulated by periodic thinning so that sufficient PPFD is available (Smith 1962). This regulation of stand density by periodic thinning continues up to the final harvest so that the growth curve for the crop trees is as steep and smooth as is practical for a particular management system. Materials and Methods Establishment and Growth of Seedlings Seed was collected from several mature trees growing at 1600-m elevation on Mount Hualalai on the island of Hawaii. Seedlings were grown at the University of Hawaii Experiment Farm in Waimanalo, island of Oahu. The study began November 7, 1980 and was completed April 15, 1981. Seedlings were grown outside, under the solar radiation, rainfall, and temperatures shown in Appendix A. The seed was sized and scarified by the hot water treatment. One imbibed seed was sown into each plastic pot that measured 15 cm wide by 18 cm deep. The rooting medium was peat moss and vermiculite, 1 to 1 by volume. No Rhizobium bacteria was added to the media because nodules developed naturally on koa roots during earlier studies. Sixty seedlings were 82 Results Specific Leaf Weights. Fully-developed leaves in the HH treatment had a significantly higher SLW than fully-developed leaves in the LL treatment (Table 17). The leaflets of leaves in the HH treatment were smaller in area, but thicker cross section than leaflets of leaves in the LL treatment. Changing the level of available light did not result in significant differences between the SLW of leaves in the HL and HH treatments, or between leaves in the LH and LL treatments. Leaves that were only partially developed when the change in light environment occurred had SLWs that were not significantly different from leaves in the environment to which they had been moved (Table 17). Therefore, the SLW of leaves in the HL treatment was more similar to that of leaves in the LL treatment than to that of leaves in the HH treatment. Leaves in the LH treatment developed a SLW more similar to that of leaves in the HH treatment than to that of leaves in the LL treatment. C02~Exchange Rates. The CERs of fully-developed leaves in the HH treatment were not significantly different from those in the LL treatment (Table 18). Transferring fully-developed leaves to a higher or lower light did not result in significant changes in CER. Two -2 -1exceptions are the significant decrease in CER at 1250 ymol m s of leaves in the HL treatment compared to those in the HH treatment, -2 -1and the significant decrease in the CER determined at 115 ymol m s for leaves in the LH treatment compared to those in the LL treatment. There were no significant differences in dark respiration of 85 Adaptation of Leaves 86 Table 17 Changes in Specific Leaf Weight (fresh weight) of Acacia Koa Leaves and Phyllodes Exposed to Different Light Treatments Specific weight Leaves Phyllodes Stage of leaf Light treatment"1"*" Light treatment Development"*" HH HL LH LL HH HL Fully developed 1.37a+++ 1.30ab - g dm-^ - - - - 1.18bc 1.09c 3.57a 3.61a Partially developed 1.42a 1.14b 1.47a 1.18b 3.56a 3.17b "•"Stage of development when the change in light level occurred. ++Light treatments: HH— plants grown and maintained in full sunlight; HL— plants grown under full sunlight, then placed under 27 percent of full sunlight; LH— plants grown under 27 percent of full sunlight, then moved to full sunlight; LL— plants grown and maintained under 27 percent of full sunlight. +++Each value is a mean of five observations. Values in rows for a leaf form followed by the same letter are not significantly different at the 0.05 level of probability. 87 Table 18 Changes in C02~exchange Rates of Acacia Koa Leaves Exposed to Different Light Treatments Stage of leaf development"1" Light treatment"1"1" CER determined at PPFD (ymol nT^s -1) 1250 975 325 115 Dark - - - - mg CO 2 dnT^h"• 1 ____ Fully HH 26 . Sa"*”*"4" 25. 5a 16.4a 6.4ab -1.5a developed HL 21.4b 21.4a 14.6a 6 . lab -1.4a LH 23.3ab 2 2 • 6 a 14.3a 5.8a - 1 .6 a LL 23.0ab 22.5a 17.1a 8.5b - 1 .2 a Partially HH 2 0 .5ab 20.4a 1 2 .8 a 4.7a -2.4a developed HL 23.0b 2 2 .2 a 14.0a 5.2ab - 2 .0 a LH 18.7a 19.5a 14.8a 6.5b - 1 .8 ab LL 17.8a 19.5a 14.7a 6 .8 b -1.3b +Stage of development when the change in light level occurred. ++See Table 15 for an explanation of the light treatments. +++Each value is a mean of five observations. Values in columns for a leaf development stage followed by the same letter are not significantly different at the 0.05 level of probability.