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Leaf Growth Development in Relation to Gas Exchange in Quercus marilandica Muenchh.
B JOUR.AL OF B
J Plant Physiol. VoL 154. pp. 302-309 (1999)
Plant Physjoloay © 1999 URBAN & FISCHER
Leaf Growth and Development in Relation to Gas Exchange in Quercus marilandica Muenchh. JoHNS. CHOINSKI Jr. 1 and RoBERT R. WisE 2 1
Department of Biology; University of Central Arkansas, Conway, AR 72035
2
Department of Biology; University ofWisconsin-Oshkosh, Oshkosh, WI 54901
Received August 5, 1997 ·Accepted April14, 1998
Summary
Photosynthesis, transpiration rates and stomatal conductances were measured using a portable infra-red gas analyzer and then correlated with structural changes occurring during the development of Quercus marilandica Muenchh. leaves. Q. marilandica was found to synthesize high levels of leaf anthocyanins (0.79 ± 0.13 gkg-1 fresh weight) during the period immediately following bud break. Carbon assimilation rates showed net respiration ( -1.3 ± 1.61..1-mol m -Z s-1) when measured in anthocyanin-containing leaves seven days after bud break (DAB), but to be near the compensation point at 17 DAB (1.1 ± 1.41..1-mol m- 2 s-1) when most of the anthocyanins were metabolized away, but the leaves not yet fully expanded. The maximum rate (8.3 ± 2.61..1-mol m _z s-1) was observed in fully expanded leaves at 37 DAB and was eight-fold higher than at 17 DAB. Transpiration rates and stomatal conductances were low at 7 DAB, but then increased 250 and 160 o/o, respectively, at 17 DAB, but only 22 and 43 o/o from 17 to 37 DAB. Ultrastructural analysis showed the leaves had small intercellular air spaces and underdeveloped chloroplasts at both 7 and 17 DAB; the leaves not appearing fully mature until expansion was complete at 37 DAB. SEM images showed 7 DAB leaves to be extensively covered with trichomes on both abaxial and adaxial surfaces. The trichomes were mostly shed by 17 DAB revealing the extensive development of stomates. It is concluded that transpiration and stomatal conductances were controlled primarily by the boundary layer resistance associated with the trichome layer at 7 DAB and the low carbon assimilation rates seen at 17 DAB were likely a consequence of sub-optimal chloroplast function and/or limitations in C0 2 uptake associated with the lack of intercellular air spaces.
Key words: Quercus marilandica Muenchh., photosynthesis, anthocyanins, gas exchange. Abbreviations: DAB = days after bud break; IRGA = infrared gas analyzer; UV (290-400nm); UV-B = ultraviolet-B radiation (290-320nm). Introduction
Flushing, young leaves from many tree species, both tropical (Lee, Brammeier, and Smith, 1987; Lee and Lowry, 1980) and temperate (Dillenburg et al., 1995; Price and Sturgess, 1938), exhibit a conspicuous red coloration caused by the presence of flavonoid anthocyanins. Typically, the anthocyanins are synthesized soon after bud break and can remain in the leaves for several weeks (Ludlow, 1991; Tuohy and Choinski, 1990). The role of these pigments is unclear,
= ultraviolet radiation
although it has been recognized that anthocyanins are synthesized as a generalized response to stress, for example, in nutrient deficient seedlings (Hodges and Nozzolillo, 1996) and might function, in some cases, as UV-B absorbers (Bate and Ludlow, 1978; Caldwell, 1971) or antifungal agents (Coley and Aide, 1989). The hypothesis that flavonoids serve as UV filters is reinforced by the observations that anthocyanins and other flavonoids are primarily located in leaf trichomes (Skaltsa et al., 1994) or in or near the adaxial leaf epidermis (Day and Vogelmann, 1995; Stapleton and Walbot, 1994; 0176-1617/99/154/302 $ 12.00/0
Leaf Development and Gas Exchange in Quercus Tevini et al., 1991) and that anthocyanin synthesis is stimulated by UV-B exposure (Gorton and Vogelmann, 1996). Alternatively, some investigators have suggested that anthocyanins may not be important UV absorbers except at high concentrations (Tevini and Teramura, 1989). For example, Liquidambar styraciflua L. seedlings grown in the field with supplemental UV-B did not show a significant increase in the levels of leaf anthocyanins or other flavonoids when compared with plants grown at ambient UV-B (Dillenburg et al.,
1995).
The potential impact of anthocyanin pigments on the photosynthetic process and the overall productivity of trees has had little investigation. Ludlow (1991) working with Ochna pulchra Hook, a southern African savanna tree, showed that young, red leaves carried out net respiration as measured by gas exchange for at least 14 days after bud break. Electron microscopic analysis revealed that many of the chloroplasts lacked fully developed grana and the red leaves had few and less developed stomata, many covered with a cuticular layer. Further studies on another African savanna tree (Brachystegia spiciformis Benth-Tuohy and Choinski, 1990) found that the presence of photosynthesis was dependent on the measurement technique used. Chloroplasts from young red leaves had measurable Hill Reaction activity and leaf discs had net positive oxygen evolution under saturating external C0 2 (Tuohy and Choinski, 1990). However, in a subsequent study done under field conditions at ambient C02> Choinski and Johnson (1993) found that Brachystegia leaves at a similar developmental stage exhibited net negative carbon assimilation rates when measured using a portable IRGA. Photosynthetic rates did increase as the leaves developed, although stomatal conductance and transpiration rates leveled off and became constant prior to the achievement of maximum carbon assimilation rate. Those data were interpreted to suggest that carbon assimilation was more likely limited by some non-stomatal factor such as chloroplast development rather than gas exchange. Radioactive tracer studies have supported the contention that young, green leaves of cottonwood (Dickson and Larson, 1981; Larson et al., 1980; Vogelmann et al., 1982) and oaks (Tomlinson et al., 1991) probably carry out little photosynthesis as they serve as sinks for carbohydrates until becoming fully mature. It is not clear, however, from these investigations if the lack of carbon export from the leaves is a consequence of the degree of chloroplast development or whether photosynthesis was being inhibited at the level of the stomate. The following report will compare rates of carbon assimilation, stomatal conductance and transpiration with leaf structure of Quercus marilandica Muenchh. (Black Jack Oak), a tree species widely distributed in southeastern North America (Little, 1971). This species was chosen because little work has been done investigating these relationships in young anthocyanin containing leaves of temperate, woody plants and because the leaves exhibited a particularly intense red coloration during the first two weeks after bud break. We will investigate whether photosynthesis in young, not fully expanded leaves is limited because of the progress of chloroplast development or other factors such as the number of or lack of development of stomata. The potential effects of anthocyanins on photosynthetic rates in young leaves will also be addressed.
303
Materials and Methods
Plant material Prior to bud break, trees growing within an approximately 1 hectare plot near Greenbrier, Arkansas were chosen as subjects for subsequent study. The possibility of previous hybridization with other oak species was a concern so a plot was selected that had been cleared for pasture land approximately 30 years earlier and therefore contained all relatively young trees, the majority of which were Quercus marilandica. The identification of these trees was provided by the University of Central Arkansas herbarium director (D. Culwell, pers. comm.). Collection of leaves was then done on a time course basis.
Gas exchange measurements Transient foliar gas exchange measurements were made in the field with an IRGA (Licor, model LI 6200) using dried air at ambient C0 2 in a closed system. Ten to fifteen readings were taken from each tree, and at each sampling time 2-3 trees were measured. For samples measured at seven days, the leaves were too small to completely cover the cuvette window and so after gas exchange measurements, leaf area was determined using a Licor (CI-202) leaf area meter. The leaves were wrapped in aluminum foil, placed in plastic bags, transferred to a cooler containing dry ice and then brought back to the laboratory where they were stored at -80 'C until pigment analyses could be done.
Leafpigment and area determinations For chlorophyll determinations, three leaf disks (or for the sevenday-old material, the whole leaf) were homogenized in a mortar and pestle containing 5 mL of 80 % (v/v) acetone, the particulates removed by centrifugation and chlorophyll content of the supernatants determined using spectrophotometry (Arnon, 1949). Anthocyanins were extracted in a similar manner using 1% HCl in methanol as the homogenizing medium and then separated from chlorophylls by solvent partitioning (Choinski and Johnson, 1993). Anthocyanin concentration in the methanolic layer was estimated using the absorptivity coefficient of Geissman (1955). All assays were performed at least five times except for the seven day material where each assay was done once for each leaf subjected to infra-red gas analysis. Leaf areas were determined as described above from at least 15 different leaves obtained from the outer branches of the tree to minimize variations caused by shading.
Microscopy For light microscopy and TEM, segments of tissue were excised and fixed in the field for 1-2h using 3% glutaraldehyde in 0.1 mol/ L potassium phosphate (pH 6.9). Fixed samples were subsequently post-fixed in 2% osmium tetroxide for 2 h, dehydrated in acetone and embedded in a low viscosity resin (Spurr, 1969). Thick sections were collected using glass knives and were stained with warm toluidine blue for light microscopy. Sections were viewed and photographed on a Nikon Optiphot light microscope. Thin sections were stained with ethanolic uranyl acetate and calcined lead acetate (Haniachi et al., 1986). Sections were viewed and photographed on a Zeiss EM lOC TEM at 60 kV. Specimens examined using the SEM were stored at -80 'C until processing. Tissues were thawed in 3% glutaraldehyde in a phosphate buffer (pH 7.2). Fixation continued for 2 h. Postfixation was for 2 h in 2% osmium tetroxide in the same buffer. Dehydration was through a graded ethanol series and critical point drying was in a Ladd CPO unit using liquid C0 2 as the tran-
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joHNS. CHOINSKI Jr. and RoBERT R. WrsE
Table 1: Pigment composition, photosynthesis', transpiration and stomatal conductance of developing leaves of Quercus marilandica Muenchh. All values are means together with their standard deviation. Leaf area
7 17 37 1 2
3
4
(cm2)
chlorophyll (gkg-')
anthocyan in (gkg-')
1.66±0.9 19.7±4.4 38.6±14.0
0.44±0.09 1.40±0.16 4.40±0.24
0.79±0.13 0.04±0.02 nd4
photosynthesis (Jlmol m- 2 s- 1) -1.29± 1.63•• 1.10± 1.4b 8.32±2.64c
transpiration (mol m- 2 s- 1)
stomatal conductance (mol m- 2 s- 1)
1.75±0.60. 6.16±3.73b 7.52± 1.4c
0.048±0.017' 0.127±0.022b 0.182±0.048c
Gas exchange measurements were done using a Licor portable IRGA; readings were taken in the mid-morning period with air temperatures between 29-36 'C and PPFD levels greater than 1200 Jlmol m- 2 s- 1• DAB = Days after bud break. Numerical values indicated with a common letter in the vertical direction are not statistically different at P = < 0.0 1. nd =Not determined.
sitional solvent. Specimens were lightly coated with gold and then viewed in a Hitachi 2460N SEM at 25 kV. Results
Leafgrowth and gas exchange studies Trees were monitored over a 37 day period; the first measurements taken on April16, 1996, seven days after bud break when anthocyanin levels appeared visually to be at their peak. The 7 day peak for leaf anthocyanin levels was also supported by an investigation of another oak species showing flush colors, Q. falcata, undertaken the previous year (Montgomery and Choinski, in preparation). Leaves expanded throughout the study, reaching a maximum value at 37 days after bud break (Table 1). Leaf expansion was paralleled by an increase in chlorophyll content showing a peak of 4.4 ± 0.24 g kg -I at 37 DAB (Table 1). Anthocyanins were at their highest level in the 7-day-old leaves (0.79 ± 0.13 g kg-1) and were barely measurable at 17 DAB. The leaves also did not appear to be visually red at this or older stages. Carbon assimilation rates showed net respiration at 7 and 17 DAB, although a very few of the leaves at 7 DAB and many of the leaves measured at 17 DAB did show some net positive carbon assimilation. The maximum photosynthetic rate was observed in the 37 DAB leaf material (8.3 ± 2.6)..lmolm- 2 s-1). The trends for stomatal conductance and transpiration rates also showed increases throughout the period studied, although the rates of increase were far lower than seen with the carbon assimilation rate data (Table 1). For example, between 17 and 37 DAB, carbon assimilation rates were eight-fold higher (650% increase), whereas stomatal conductances and transpiration rates showed less than a 50% increase.
Anatomical changes in the leaves 7DAB Figure 1 shows a LM view of a cross-section of a 7 DAB leaf. A dark staining layer can be seen adjacent to both the adaxial and abaxial surfaces, although the layer was more intense on the adaxial surface. Visual inspection of whole leaves revealed that the adaxial surface was darker red than the abaxis. Therefore, the dark layers seen in Figure 1 probably repre-
sented the anthocyanin-containing cells. Figure 1 also shows the paucity of intercellular air spaces in these young leaves; an observation confirmed when leaf cross sections were viewed in the SEM (Fig. 2). Further examination in the SEM revealed that both surfaces were completely covered with trichomes (Figs. 3, 4) making determinations of stomatal density impossible. The adaxial (Fig. 5) and abaxial (Fig. 6) external cell walls and cuticle were very thin at this early stage of development. TEM examination of? DAB leaves revealed characteristics of a young, developing leaf: i.e. small cells with numerous small vacuoles, no intercellular air spaces, and active mitosis (Fig. 7). Many dividing chloroplasts were visible (Fig. 8) while only very few (roughly <1 %) contained starch (Fig. 9). The chloroplasts all show some granal development, although the individual grana stacks appeared to be quite small, consisting of only 2 or 3 thylakoids (Fig. 8).
17DAB At 17 DAB, tissue differentiation into palisade and spongy mesophylllayers was evident (Figs. 10, 11). Trichome densities had declined markedly, allowing visualization of the adaxial and abaxial epidermises. No stomata were present on the adaxis (Fig. 12) while abaxial stomatal density was quite high (Fig. 13). The external epidermal cell walls were much thicker at this developmental stage (Figs. 14, 15) while a cuticular layer was deposited on the adaxis (Fig. 14). Vacuoles had enlarged and intercellular air spaces had developed (Fig. 16.). A few chloroplasts continued to divide (Fig. 17) and many contained starch (Fig. 18). Grana were larger than in the 7 DAB samples, but were still underdeveloped (Fig.18).
37DAB The leaves appeared to be fully mature at this stage and showed distinctive palisade and spongy mesophylllayers and intercellular air spaces (Figs. 19, 20). All of the adaxial trichomes had abscised (Fig. 21) while a few remained on the abaxis (Fig. 22). Epidermal cell walls and cuticle were thick on both the adaxial (Fig. 23) and abaxial (Fig. 24) surfaces. Cells had reached their maximum size and contained large vacuoles and numerous chloroplasts (Fig. 25). The preponderance of chloroplasts contained large starch grains (Figs. 25,
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26). Granal development was typical for a mature chloroplast (Fig. 27).
Discussion Studies with rapidly growing agronomic crops such as Phaseolus vulgaris L. or Gossypium hirsutum L. show a close corre-
lation with the stage of leaf development and increases in photosynthesis, conductance and transpiration; these increases occurring as a consequence of the development of the photosynthetic apparatus, as well as changes in the length, number and functioning of stomates (Ticha et al., 1985). Leaves of woody dicots show a more variable pattern. For example, in Brachystegia spiciformis, photosynthesis was observed to substantially increase in fully expanded leaves after maximum stomatal conductance and transpiration rates are reached; this increase seemingly triggered by the onset of the rainy season (Choinski and Johnson, 1993). Ludlow's (1991) study of Ochna pulchra Hook showed a similar pattern with net positive carbon assimilation seen when the leaves were first fully expanded, but maximum photosynthesis was not observed until approximately a month later. Ludlow suggested that the increases in photosynthetic rate were because of the degree of stomatal functioning as supported by microscopic examination, although stomatal conductances and transpiration data were not reported. This present investigation of Quercus showed that 7 DAB leaves exhibited net respi-
ration with low transpiration and stomatal conductance (Table 1). This was expected as the chloroplasts were small and still dividing with minimal granal development and a small amount of starch deposition, possibly as a consequence of carbohydrate import from photoautotrophic or storage tissues. The leaves were also completely covered with trichomes which presumably protected the leaf from desiccation during a period when the cell walls and cuticles were thin. The trichome covering also increased the boundary layer resistance and may have contributed to the low transpiration rates and stomatal conductances (Table 1). At 17 DAB, stomatal conductance and transpiration rates increased 2.7 to 3.5 fold, respectively, likely because of the loss of most of the trichomes and the concomitant decrease in boundary layer resistance. The small increases in photosynthetic rates seen at 17 DAB were likely for two reasons: the plastids were still underdeveloped, although with larger grana and more commonly observed starch deposition (Figs. 17, 18); and intercellular air spaces were small which limited mesophyll surface area and increased diffusive resistance to C0 2 (Nobel et al., 1975). Thus, by 37 DAB, a stage with completely mature chloroplasts and large intercellular air spaces, less than 50% increases in transpiration and stomatal conductance were accompanied by an 8-fold increase in photosynthesis. Another consequence of this observed delay in full photosynthetic functioning is that young leaves emerging from the bud are initially nutrient sinks (Tich:i et al., 1985). For most herbaceous plants, the maximum sink strength is achieved af-
Fig.l: Light micrograph of7 DAB leaf in cross section. Adaxial and abaxial anthocyanin-containing layers are indicated (arrows). Scale bar = 50~m. Fig. 2: SEM of a 7 DAB leaf in cross section. Scale bar= 50 ~m. Fig. 3: SEM of a 7 DAB leaf adaxial surface. Scale bar = 50 ~m. Fig. 4: SEM of a 7 DAB leaf abaxial surface. Scale bar = 50 ~m. Fig. 5: TEM of a 7 DAB leaf adaxial surface. Cuticle is apparent as a thin black line on the leaf surface (arrow). Scale bar= 1~m. Fig. 6: TEM of a 7 DAB leaf abaxial epidermis. Cuticle is apparent as a thin black line on the leaf surface (arrow). Scale bar= 1~m. Fig. 7: Low magnification TEM of a 7 DAB leaf in cross section. Note numerous small vacuoles, lack of intercellular air spaces and the cell in late anaphase (upper left). Scale bar = 10 ~m. Fig. 8: TEM of a dividing chloroplast from a 7 DAB lea£ Narrow grana are indicated (between arrow points). Scale bar= 1 ~m. Fig. 9: TEM of a starch-containing chloroplast from a 7 DAB leaf. Scale bar = 1~m.
Fig.lO: Light micrograph of a 17 DAB leaf in cross section. Intercellular air spaces are indicated (arrows). Scale bar= 50 ~m. Fig.ll: SEM of a 17 DAB leaf in cross section. Scale bar= 50 ~m. Fig.l2: SEM of 17 DAB leaf adaxial surface. Scale bar = 50 ~m. Fig.13: SEM of 17 DAB abaxial surface. Note numerous stomata. Scale bar= 50 ~m. Fig.14: TEM of 17 DAB leaf adaxial epidermis. Cuticle is distinct from the cell wall. Scale bar= 1 ~m. Fig.15: TEM of 17 DAB leaf adaxial epidermis. A distinct cuticle is lacking. Scale bar= 1~m. Fig. 16: Low magnification of 17 DAB TEM of leaf in cross section. Vacuoles and intercellular air spaces have enlarged. Scale bar= 10 ~·
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Fig.17: TEM of dividing chloroplast from 17 DAB lea£ Grana (between arrow points) are larger than in the 7 DAB chloroplasts. Intercellular air space is indicated (asterisk). Scale bar= 1 J.lm. Fig. 18: TEM of starch-containing chloroplast. Scale bar= 1J.lm. Fig. 19: Light micrograph of a 37 DAB leaf in cross section. Scale bar= 50 Jlm.
Fig. 20: SEM of a 37 DAB leaf in cross section. Scale bar= 50 Jlm. Fig. 21: SEM of 37 DAB adaxial surface. No trichomes or stomates are visible. Fungal hyphae extend across field of view. Scale bar = 50 Jlm. Fig. 22: SEM of 37 DAB abaxial surface. A few simple trichomes remain and stomata are numerous. Scale bar = 50 Jlm. Fig. 23: TEM of 37 DAB adaxial leaf epidermis. Cuticle and cell wall are both thick. Scale bar = 1 J.lm. Fig. 24: TEM of 37 DAB abaxial leaf epidermis. Cuticle and cell wall are both thick. Scale bar = 1 J.lm. Fig. 25: Low magnification TEM of 37 DAB leaf cross section. Intercellular air spaces are at maximum size. Scale bar= 10 J.lm. Fig. 26: TEM of starch-containing chloroplast from 37 DAB lea£ Scale bar = 1 J.lm. Fig. 27: TEM of well-developed grana stack. Scale bar= 1J.lm. Figure abbreviations: C = cuticle; CW = cell wall; DAB = days after bud break; IAS =intercellular air space; N =nucleus; S =starch; SEM =scanning electron micrograph; TEM =transmission electron micrograph.
308
JoHNS. CHOINSKI Jr. and ROBERT R. WisE
ter the leaf area reaches 10 to 25% of maximum with the transition to net photosynthate export beginning soon thereafter. The transition of the leaf from a photosynthate sink to a source for photosynthate has been shown to be a complex process involving a specific sequence of tissue maturation and includes a period where the leaf may act as both a sink and a source (Eschrich, 1975). It appears that leaves of woody dicots (Dickson and Larson, 1981; Larson et al., 1980; Tomlinson et al., 1991; Vogelmann et al., 1982) have a much longer period as sinks for photosynthate than do herbaceous plants (Ticha et al., 1985). Gas exchange studies with Ochna pulchra (Ludlow, 1991), Brachystegia spiciformis (Choinski and Johnson, 1993) and this present investigation support this view, showing a long period of net respiration in young leaves; however, it should be noted that the changeover to a capacity for net photosynthesis as measured by gas exchange probably precedes the point of transition from source to sink in the leaf (Larson et al., 1980). Research examining the possible role of anthocyanins in affecting the photosynthetic process is lacking. For example, it is possible that anthocyanins in the epidermis or trichomes mask or limit photosynthesis in young red leaves by reducing the amount of radiant energy received by the mesophyll tissues. Anthocyanins may also be directly associated with the chloroplast as supported by evidence that the enzymes for anthocyanin synthesis are compartmentalized in chloroplasts (Hrazida et al., 1978) and that a subcellular organelle called the anthocyanoplast identified in 13 cruciferous species (Hodges and Nozzolillo, 1996; Pecket and Small, 1980) contains a substantial proportion of extravacuolar anthocyanin. Alternatively, absorption of UV-Nblue light by anthocyanin might reduce the amount of light reaching «blue» photoreceptors responsible for chlorophyll and carotenoid synthesis, thus limiting overall photosynthetic performance (Kawallik, 1987; Rau and Schott, 1987). Ongoing research is investigating these questions. Acknowledgements
Appreciation is expressed to the University of Central Arkansas Faculty Research fund and to the University of Wisconsin-Oshkosh Faculty Development Board for financial support. Elizabeth Bass is thanked for technical assistance. References
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LEE, D. W, S. BRAMMEIER, and A. P. SMITH: The selective advantages of anthocyanins in developing leaves of Mango and cacao. Biotropica 19, 40-49 (1987). LEE, D. Wand J. B. LOWRY: Young leaf anthocyanin and solar ultraviolet. Biotropica 12, 75-76 (1980). LITTLE, E. L. Jr.: Atlas of United States Trees, Vol. 1. Conifers and Important Hardwoods. Misc. Pub. 1146, USDA Forest Service, Washington, DC (1971). LuDLOW, A. E.: Ochna pulchra Hook: Leaf growth and development related to photosynthetic activity. Ann. Bot. 68, 527-540 (1991). NoBEL, P. S., L. J. ZARAGOZA, and WK. SMITH: Relation between mesophyll surface area, photosynthetic rate, and illumination level during development for leaves of Plectranthus parviflorus Henckel. Plant Physiol. 55, 1067-1070 (1975). PECKET, R. C. and C. J. SMALL: Occurrence, location and development of anthocyanoplasts. Phytochem. 19, 2571-2576 (1980). PRICE, J. R. and V. C. STURGESS: CCXIV. A survey of anthocyanins. VI. Biochem. J. 32, 1658-1660 (1938). RAu, Wand E. I. ScHoTT: Blue light control of pigment biosynthesis. In: SENGER, H. (ed.): Blue light responses: Phenomena and Occurrence in Plants and Microorganisms, Vol. I, pp. 43-64. CRC Press, Boca Raton, Fl. (1987). SKALTSA, H., VERYKOKIDou, C. HARvALA, G. KARABOURNIOTIS, and Y. MANETAS: UV-B protective potential and flavonoid content of leaf hairs of Quercus ilex. Phytochem. 37, 987-990 (1994). SPuRR, A. R.: A low viscosity epoxy resin embedding medium for electron microscopy. J. Electron Micro. 26, 31-43 (1969). STAPLETON, A. E. and V. WALBOT: Flavonoids can protect maize DNA from the induction of ultraviolet radiation damage. Plant Physiol. 105, 881-889 (1994).
Leaf Development and Gas Exchange in Quercus TEVINI, M. and A. H. TERAMURA: UV-B effects on terrestrial plants. Photochem. and Photobiol. 50, 479-487 (1989). TEVINI, M., J. BRAUN, and G. FIESER: The protective function of the epidermal layer of rye seedlings against ultraviolet-B radiation. Photochem. Photobiol. 53, 320-333 (1991). TicHA., 1., J., CATsKY, D. HooM'IovA, J. PosPISILOVA, M. KAsE, and Z. SESTAK: Gas exchange and dry matter accumulation during leaf development. In: SESTAK, Z. (ed.): Photosynthesis during leaf development, pp. 157-216. W Junk, Dordrecht (1985).
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ToMLINSON, P. T., R. E. DICKSON, and J. G. ISEBRANDS: Acropetal leaf differentiation in Quercus rubra (Fagaceae). Amer. J. Bot. 78, 1570-1575 (1991). TuoHY, J. M. and J. S. CHOINSKI Jr.: Comparative photosynthesis in developing leaves of Brachystegia spiciformis Benth. J. Exp. Bot. 41, 919-923 (1990). VoGELMANN, T. C., P. R. LARSON, and R. E. DICKSON: Translocation pathways in the petioles and stem between source and sink leaves of Populus deltoides Bartr. Ex Marsh. Planta 156, 345-358 (1982).
J Plant Physiol. VoL 154. pp. 302-309 (1999)
Plant Physjoloay © 1999 URBAN & FISCHER
Leaf Growth and Development in Relation to Gas Exchange in Quercus marilandica Muenchh. JoHNS. CHOINSKI Jr. 1 and RoBERT R. WisE 2 1
Department of Biology; University of Central Arkansas, Conway, AR 72035
2
Department of Biology; University ofWisconsin-Oshkosh, Oshkosh, WI 54901
Received August 5, 1997 ·Accepted April14, 1998
Summary
Photosynthesis, transpiration rates and stomatal conductances were measured using a portable infra-red gas analyzer and then correlated with structural changes occurring during the development of Quercus marilandica Muenchh. leaves. Q. marilandica was found to synthesize high levels of leaf anthocyanins (0.79 ± 0.13 gkg-1 fresh weight) during the period immediately following bud break. Carbon assimilation rates showed net respiration ( -1.3 ± 1.61..1-mol m -Z s-1) when measured in anthocyanin-containing leaves seven days after bud break (DAB), but to be near the compensation point at 17 DAB (1.1 ± 1.41..1-mol m- 2 s-1) when most of the anthocyanins were metabolized away, but the leaves not yet fully expanded. The maximum rate (8.3 ± 2.61..1-mol m _z s-1) was observed in fully expanded leaves at 37 DAB and was eight-fold higher than at 17 DAB. Transpiration rates and stomatal conductances were low at 7 DAB, but then increased 250 and 160 o/o, respectively, at 17 DAB, but only 22 and 43 o/o from 17 to 37 DAB. Ultrastructural analysis showed the leaves had small intercellular air spaces and underdeveloped chloroplasts at both 7 and 17 DAB; the leaves not appearing fully mature until expansion was complete at 37 DAB. SEM images showed 7 DAB leaves to be extensively covered with trichomes on both abaxial and adaxial surfaces. The trichomes were mostly shed by 17 DAB revealing the extensive development of stomates. It is concluded that transpiration and stomatal conductances were controlled primarily by the boundary layer resistance associated with the trichome layer at 7 DAB and the low carbon assimilation rates seen at 17 DAB were likely a consequence of sub-optimal chloroplast function and/or limitations in C0 2 uptake associated with the lack of intercellular air spaces.
Key words: Quercus marilandica Muenchh., photosynthesis, anthocyanins, gas exchange. Abbreviations: DAB = days after bud break; IRGA = infrared gas analyzer; UV (290-400nm); UV-B = ultraviolet-B radiation (290-320nm). Introduction
Flushing, young leaves from many tree species, both tropical (Lee, Brammeier, and Smith, 1987; Lee and Lowry, 1980) and temperate (Dillenburg et al., 1995; Price and Sturgess, 1938), exhibit a conspicuous red coloration caused by the presence of flavonoid anthocyanins. Typically, the anthocyanins are synthesized soon after bud break and can remain in the leaves for several weeks (Ludlow, 1991; Tuohy and Choinski, 1990). The role of these pigments is unclear,
= ultraviolet radiation
although it has been recognized that anthocyanins are synthesized as a generalized response to stress, for example, in nutrient deficient seedlings (Hodges and Nozzolillo, 1996) and might function, in some cases, as UV-B absorbers (Bate and Ludlow, 1978; Caldwell, 1971) or antifungal agents (Coley and Aide, 1989). The hypothesis that flavonoids serve as UV filters is reinforced by the observations that anthocyanins and other flavonoids are primarily located in leaf trichomes (Skaltsa et al., 1994) or in or near the adaxial leaf epidermis (Day and Vogelmann, 1995; Stapleton and Walbot, 1994; 0176-1617/99/154/302 $ 12.00/0
Leaf Development and Gas Exchange in Quercus Tevini et al., 1991) and that anthocyanin synthesis is stimulated by UV-B exposure (Gorton and Vogelmann, 1996). Alternatively, some investigators have suggested that anthocyanins may not be important UV absorbers except at high concentrations (Tevini and Teramura, 1989). For example, Liquidambar styraciflua L. seedlings grown in the field with supplemental UV-B did not show a significant increase in the levels of leaf anthocyanins or other flavonoids when compared with plants grown at ambient UV-B (Dillenburg et al.,
1995).
The potential impact of anthocyanin pigments on the photosynthetic process and the overall productivity of trees has had little investigation. Ludlow (1991) working with Ochna pulchra Hook, a southern African savanna tree, showed that young, red leaves carried out net respiration as measured by gas exchange for at least 14 days after bud break. Electron microscopic analysis revealed that many of the chloroplasts lacked fully developed grana and the red leaves had few and less developed stomata, many covered with a cuticular layer. Further studies on another African savanna tree (Brachystegia spiciformis Benth-Tuohy and Choinski, 1990) found that the presence of photosynthesis was dependent on the measurement technique used. Chloroplasts from young red leaves had measurable Hill Reaction activity and leaf discs had net positive oxygen evolution under saturating external C0 2 (Tuohy and Choinski, 1990). However, in a subsequent study done under field conditions at ambient C02> Choinski and Johnson (1993) found that Brachystegia leaves at a similar developmental stage exhibited net negative carbon assimilation rates when measured using a portable IRGA. Photosynthetic rates did increase as the leaves developed, although stomatal conductance and transpiration rates leveled off and became constant prior to the achievement of maximum carbon assimilation rate. Those data were interpreted to suggest that carbon assimilation was more likely limited by some non-stomatal factor such as chloroplast development rather than gas exchange. Radioactive tracer studies have supported the contention that young, green leaves of cottonwood (Dickson and Larson, 1981; Larson et al., 1980; Vogelmann et al., 1982) and oaks (Tomlinson et al., 1991) probably carry out little photosynthesis as they serve as sinks for carbohydrates until becoming fully mature. It is not clear, however, from these investigations if the lack of carbon export from the leaves is a consequence of the degree of chloroplast development or whether photosynthesis was being inhibited at the level of the stomate. The following report will compare rates of carbon assimilation, stomatal conductance and transpiration with leaf structure of Quercus marilandica Muenchh. (Black Jack Oak), a tree species widely distributed in southeastern North America (Little, 1971). This species was chosen because little work has been done investigating these relationships in young anthocyanin containing leaves of temperate, woody plants and because the leaves exhibited a particularly intense red coloration during the first two weeks after bud break. We will investigate whether photosynthesis in young, not fully expanded leaves is limited because of the progress of chloroplast development or other factors such as the number of or lack of development of stomata. The potential effects of anthocyanins on photosynthetic rates in young leaves will also be addressed.
303
Materials and Methods
Plant material Prior to bud break, trees growing within an approximately 1 hectare plot near Greenbrier, Arkansas were chosen as subjects for subsequent study. The possibility of previous hybridization with other oak species was a concern so a plot was selected that had been cleared for pasture land approximately 30 years earlier and therefore contained all relatively young trees, the majority of which were Quercus marilandica. The identification of these trees was provided by the University of Central Arkansas herbarium director (D. Culwell, pers. comm.). Collection of leaves was then done on a time course basis.
Gas exchange measurements Transient foliar gas exchange measurements were made in the field with an IRGA (Licor, model LI 6200) using dried air at ambient C0 2 in a closed system. Ten to fifteen readings were taken from each tree, and at each sampling time 2-3 trees were measured. For samples measured at seven days, the leaves were too small to completely cover the cuvette window and so after gas exchange measurements, leaf area was determined using a Licor (CI-202) leaf area meter. The leaves were wrapped in aluminum foil, placed in plastic bags, transferred to a cooler containing dry ice and then brought back to the laboratory where they were stored at -80 'C until pigment analyses could be done.
Leafpigment and area determinations For chlorophyll determinations, three leaf disks (or for the sevenday-old material, the whole leaf) were homogenized in a mortar and pestle containing 5 mL of 80 % (v/v) acetone, the particulates removed by centrifugation and chlorophyll content of the supernatants determined using spectrophotometry (Arnon, 1949). Anthocyanins were extracted in a similar manner using 1% HCl in methanol as the homogenizing medium and then separated from chlorophylls by solvent partitioning (Choinski and Johnson, 1993). Anthocyanin concentration in the methanolic layer was estimated using the absorptivity coefficient of Geissman (1955). All assays were performed at least five times except for the seven day material where each assay was done once for each leaf subjected to infra-red gas analysis. Leaf areas were determined as described above from at least 15 different leaves obtained from the outer branches of the tree to minimize variations caused by shading.
Microscopy For light microscopy and TEM, segments of tissue were excised and fixed in the field for 1-2h using 3% glutaraldehyde in 0.1 mol/ L potassium phosphate (pH 6.9). Fixed samples were subsequently post-fixed in 2% osmium tetroxide for 2 h, dehydrated in acetone and embedded in a low viscosity resin (Spurr, 1969). Thick sections were collected using glass knives and were stained with warm toluidine blue for light microscopy. Sections were viewed and photographed on a Nikon Optiphot light microscope. Thin sections were stained with ethanolic uranyl acetate and calcined lead acetate (Haniachi et al., 1986). Sections were viewed and photographed on a Zeiss EM lOC TEM at 60 kV. Specimens examined using the SEM were stored at -80 'C until processing. Tissues were thawed in 3% glutaraldehyde in a phosphate buffer (pH 7.2). Fixation continued for 2 h. Postfixation was for 2 h in 2% osmium tetroxide in the same buffer. Dehydration was through a graded ethanol series and critical point drying was in a Ladd CPO unit using liquid C0 2 as the tran-
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joHNS. CHOINSKI Jr. and RoBERT R. WrsE
Table 1: Pigment composition, photosynthesis', transpiration and stomatal conductance of developing leaves of Quercus marilandica Muenchh. All values are means together with their standard deviation. Leaf area
7 17 37 1 2
3
4
(cm2)
chlorophyll (gkg-')
anthocyan in (gkg-')
1.66±0.9 19.7±4.4 38.6±14.0
0.44±0.09 1.40±0.16 4.40±0.24
0.79±0.13 0.04±0.02 nd4
photosynthesis (Jlmol m- 2 s- 1) -1.29± 1.63•• 1.10± 1.4b 8.32±2.64c
transpiration (mol m- 2 s- 1)
stomatal conductance (mol m- 2 s- 1)
1.75±0.60. 6.16±3.73b 7.52± 1.4c
0.048±0.017' 0.127±0.022b 0.182±0.048c
Gas exchange measurements were done using a Licor portable IRGA; readings were taken in the mid-morning period with air temperatures between 29-36 'C and PPFD levels greater than 1200 Jlmol m- 2 s- 1• DAB = Days after bud break. Numerical values indicated with a common letter in the vertical direction are not statistically different at P = < 0.0 1. nd =Not determined.
sitional solvent. Specimens were lightly coated with gold and then viewed in a Hitachi 2460N SEM at 25 kV. Results
Leafgrowth and gas exchange studies Trees were monitored over a 37 day period; the first measurements taken on April16, 1996, seven days after bud break when anthocyanin levels appeared visually to be at their peak. The 7 day peak for leaf anthocyanin levels was also supported by an investigation of another oak species showing flush colors, Q. falcata, undertaken the previous year (Montgomery and Choinski, in preparation). Leaves expanded throughout the study, reaching a maximum value at 37 days after bud break (Table 1). Leaf expansion was paralleled by an increase in chlorophyll content showing a peak of 4.4 ± 0.24 g kg -I at 37 DAB (Table 1). Anthocyanins were at their highest level in the 7-day-old leaves (0.79 ± 0.13 g kg-1) and were barely measurable at 17 DAB. The leaves also did not appear to be visually red at this or older stages. Carbon assimilation rates showed net respiration at 7 and 17 DAB, although a very few of the leaves at 7 DAB and many of the leaves measured at 17 DAB did show some net positive carbon assimilation. The maximum photosynthetic rate was observed in the 37 DAB leaf material (8.3 ± 2.6)..lmolm- 2 s-1). The trends for stomatal conductance and transpiration rates also showed increases throughout the period studied, although the rates of increase were far lower than seen with the carbon assimilation rate data (Table 1). For example, between 17 and 37 DAB, carbon assimilation rates were eight-fold higher (650% increase), whereas stomatal conductances and transpiration rates showed less than a 50% increase.
Anatomical changes in the leaves 7DAB Figure 1 shows a LM view of a cross-section of a 7 DAB leaf. A dark staining layer can be seen adjacent to both the adaxial and abaxial surfaces, although the layer was more intense on the adaxial surface. Visual inspection of whole leaves revealed that the adaxial surface was darker red than the abaxis. Therefore, the dark layers seen in Figure 1 probably repre-
sented the anthocyanin-containing cells. Figure 1 also shows the paucity of intercellular air spaces in these young leaves; an observation confirmed when leaf cross sections were viewed in the SEM (Fig. 2). Further examination in the SEM revealed that both surfaces were completely covered with trichomes (Figs. 3, 4) making determinations of stomatal density impossible. The adaxial (Fig. 5) and abaxial (Fig. 6) external cell walls and cuticle were very thin at this early stage of development. TEM examination of? DAB leaves revealed characteristics of a young, developing leaf: i.e. small cells with numerous small vacuoles, no intercellular air spaces, and active mitosis (Fig. 7). Many dividing chloroplasts were visible (Fig. 8) while only very few (roughly <1 %) contained starch (Fig. 9). The chloroplasts all show some granal development, although the individual grana stacks appeared to be quite small, consisting of only 2 or 3 thylakoids (Fig. 8).
17DAB At 17 DAB, tissue differentiation into palisade and spongy mesophylllayers was evident (Figs. 10, 11). Trichome densities had declined markedly, allowing visualization of the adaxial and abaxial epidermises. No stomata were present on the adaxis (Fig. 12) while abaxial stomatal density was quite high (Fig. 13). The external epidermal cell walls were much thicker at this developmental stage (Figs. 14, 15) while a cuticular layer was deposited on the adaxis (Fig. 14). Vacuoles had enlarged and intercellular air spaces had developed (Fig. 16.). A few chloroplasts continued to divide (Fig. 17) and many contained starch (Fig. 18). Grana were larger than in the 7 DAB samples, but were still underdeveloped (Fig.18).
37DAB The leaves appeared to be fully mature at this stage and showed distinctive palisade and spongy mesophylllayers and intercellular air spaces (Figs. 19, 20). All of the adaxial trichomes had abscised (Fig. 21) while a few remained on the abaxis (Fig. 22). Epidermal cell walls and cuticle were thick on both the adaxial (Fig. 23) and abaxial (Fig. 24) surfaces. Cells had reached their maximum size and contained large vacuoles and numerous chloroplasts (Fig. 25). The preponderance of chloroplasts contained large starch grains (Figs. 25,
305
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26). Granal development was typical for a mature chloroplast (Fig. 27).
Discussion Studies with rapidly growing agronomic crops such as Phaseolus vulgaris L. or Gossypium hirsutum L. show a close corre-
lation with the stage of leaf development and increases in photosynthesis, conductance and transpiration; these increases occurring as a consequence of the development of the photosynthetic apparatus, as well as changes in the length, number and functioning of stomates (Ticha et al., 1985). Leaves of woody dicots show a more variable pattern. For example, in Brachystegia spiciformis, photosynthesis was observed to substantially increase in fully expanded leaves after maximum stomatal conductance and transpiration rates are reached; this increase seemingly triggered by the onset of the rainy season (Choinski and Johnson, 1993). Ludlow's (1991) study of Ochna pulchra Hook showed a similar pattern with net positive carbon assimilation seen when the leaves were first fully expanded, but maximum photosynthesis was not observed until approximately a month later. Ludlow suggested that the increases in photosynthetic rate were because of the degree of stomatal functioning as supported by microscopic examination, although stomatal conductances and transpiration data were not reported. This present investigation of Quercus showed that 7 DAB leaves exhibited net respi-
ration with low transpiration and stomatal conductance (Table 1). This was expected as the chloroplasts were small and still dividing with minimal granal development and a small amount of starch deposition, possibly as a consequence of carbohydrate import from photoautotrophic or storage tissues. The leaves were also completely covered with trichomes which presumably protected the leaf from desiccation during a period when the cell walls and cuticles were thin. The trichome covering also increased the boundary layer resistance and may have contributed to the low transpiration rates and stomatal conductances (Table 1). At 17 DAB, stomatal conductance and transpiration rates increased 2.7 to 3.5 fold, respectively, likely because of the loss of most of the trichomes and the concomitant decrease in boundary layer resistance. The small increases in photosynthetic rates seen at 17 DAB were likely for two reasons: the plastids were still underdeveloped, although with larger grana and more commonly observed starch deposition (Figs. 17, 18); and intercellular air spaces were small which limited mesophyll surface area and increased diffusive resistance to C0 2 (Nobel et al., 1975). Thus, by 37 DAB, a stage with completely mature chloroplasts and large intercellular air spaces, less than 50% increases in transpiration and stomatal conductance were accompanied by an 8-fold increase in photosynthesis. Another consequence of this observed delay in full photosynthetic functioning is that young leaves emerging from the bud are initially nutrient sinks (Tich:i et al., 1985). For most herbaceous plants, the maximum sink strength is achieved af-
Fig.l: Light micrograph of7 DAB leaf in cross section. Adaxial and abaxial anthocyanin-containing layers are indicated (arrows). Scale bar = 50~m. Fig. 2: SEM of a 7 DAB leaf in cross section. Scale bar= 50 ~m. Fig. 3: SEM of a 7 DAB leaf adaxial surface. Scale bar = 50 ~m. Fig. 4: SEM of a 7 DAB leaf abaxial surface. Scale bar = 50 ~m. Fig. 5: TEM of a 7 DAB leaf adaxial surface. Cuticle is apparent as a thin black line on the leaf surface (arrow). Scale bar= 1~m. Fig. 6: TEM of a 7 DAB leaf abaxial epidermis. Cuticle is apparent as a thin black line on the leaf surface (arrow). Scale bar= 1~m. Fig. 7: Low magnification TEM of a 7 DAB leaf in cross section. Note numerous small vacuoles, lack of intercellular air spaces and the cell in late anaphase (upper left). Scale bar = 10 ~m. Fig. 8: TEM of a dividing chloroplast from a 7 DAB lea£ Narrow grana are indicated (between arrow points). Scale bar= 1 ~m. Fig. 9: TEM of a starch-containing chloroplast from a 7 DAB leaf. Scale bar = 1~m.
Fig.lO: Light micrograph of a 17 DAB leaf in cross section. Intercellular air spaces are indicated (arrows). Scale bar= 50 ~m. Fig.ll: SEM of a 17 DAB leaf in cross section. Scale bar= 50 ~m. Fig.l2: SEM of 17 DAB leaf adaxial surface. Scale bar = 50 ~m. Fig.13: SEM of 17 DAB abaxial surface. Note numerous stomata. Scale bar= 50 ~m. Fig.14: TEM of 17 DAB leaf adaxial epidermis. Cuticle is distinct from the cell wall. Scale bar= 1 ~m. Fig.15: TEM of 17 DAB leaf adaxial epidermis. A distinct cuticle is lacking. Scale bar= 1~m. Fig. 16: Low magnification of 17 DAB TEM of leaf in cross section. Vacuoles and intercellular air spaces have enlarged. Scale bar= 10 ~·
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Fig.17: TEM of dividing chloroplast from 17 DAB lea£ Grana (between arrow points) are larger than in the 7 DAB chloroplasts. Intercellular air space is indicated (asterisk). Scale bar= 1 J.lm. Fig. 18: TEM of starch-containing chloroplast. Scale bar= 1J.lm. Fig. 19: Light micrograph of a 37 DAB leaf in cross section. Scale bar= 50 Jlm.
Fig. 20: SEM of a 37 DAB leaf in cross section. Scale bar= 50 Jlm. Fig. 21: SEM of 37 DAB adaxial surface. No trichomes or stomates are visible. Fungal hyphae extend across field of view. Scale bar = 50 Jlm. Fig. 22: SEM of 37 DAB abaxial surface. A few simple trichomes remain and stomata are numerous. Scale bar = 50 Jlm. Fig. 23: TEM of 37 DAB adaxial leaf epidermis. Cuticle and cell wall are both thick. Scale bar = 1 J.lm. Fig. 24: TEM of 37 DAB abaxial leaf epidermis. Cuticle and cell wall are both thick. Scale bar = 1 J.lm. Fig. 25: Low magnification TEM of 37 DAB leaf cross section. Intercellular air spaces are at maximum size. Scale bar= 10 J.lm. Fig. 26: TEM of starch-containing chloroplast from 37 DAB lea£ Scale bar = 1 J.lm. Fig. 27: TEM of well-developed grana stack. Scale bar= 1J.lm. Figure abbreviations: C = cuticle; CW = cell wall; DAB = days after bud break; IAS =intercellular air space; N =nucleus; S =starch; SEM =scanning electron micrograph; TEM =transmission electron micrograph.
308
JoHNS. CHOINSKI Jr. and ROBERT R. WisE
ter the leaf area reaches 10 to 25% of maximum with the transition to net photosynthate export beginning soon thereafter. The transition of the leaf from a photosynthate sink to a source for photosynthate has been shown to be a complex process involving a specific sequence of tissue maturation and includes a period where the leaf may act as both a sink and a source (Eschrich, 1975). It appears that leaves of woody dicots (Dickson and Larson, 1981; Larson et al., 1980; Tomlinson et al., 1991; Vogelmann et al., 1982) have a much longer period as sinks for photosynthate than do herbaceous plants (Ticha et al., 1985). Gas exchange studies with Ochna pulchra (Ludlow, 1991), Brachystegia spiciformis (Choinski and Johnson, 1993) and this present investigation support this view, showing a long period of net respiration in young leaves; however, it should be noted that the changeover to a capacity for net photosynthesis as measured by gas exchange probably precedes the point of transition from source to sink in the leaf (Larson et al., 1980). Research examining the possible role of anthocyanins in affecting the photosynthetic process is lacking. For example, it is possible that anthocyanins in the epidermis or trichomes mask or limit photosynthesis in young red leaves by reducing the amount of radiant energy received by the mesophyll tissues. Anthocyanins may also be directly associated with the chloroplast as supported by evidence that the enzymes for anthocyanin synthesis are compartmentalized in chloroplasts (Hrazida et al., 1978) and that a subcellular organelle called the anthocyanoplast identified in 13 cruciferous species (Hodges and Nozzolillo, 1996; Pecket and Small, 1980) contains a substantial proportion of extravacuolar anthocyanin. Alternatively, absorption of UV-Nblue light by anthocyanin might reduce the amount of light reaching «blue» photoreceptors responsible for chlorophyll and carotenoid synthesis, thus limiting overall photosynthetic performance (Kawallik, 1987; Rau and Schott, 1987). Ongoing research is investigating these questions. Acknowledgements
Appreciation is expressed to the University of Central Arkansas Faculty Research fund and to the University of Wisconsin-Oshkosh Faculty Development Board for financial support. Elizabeth Bass is thanked for technical assistance. References
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ToMLINSON, P. T., R. E. DICKSON, and J. G. ISEBRANDS: Acropetal leaf differentiation in Quercus rubra (Fagaceae). Amer. J. Bot. 78, 1570-1575 (1991). TuoHY, J. M. and J. S. CHOINSKI Jr.: Comparative photosynthesis in developing leaves of Brachystegia spiciformis Benth. J. Exp. Bot. 41, 919-923 (1990). VoGELMANN, T. C., P. R. LARSON, and R. E. DICKSON: Translocation pathways in the petioles and stem between source and sink leaves of Populus deltoides Bartr. Ex Marsh. Planta 156, 345-358 (1982).