Comparison of leaf structure and photosynthetic characteristics of C3 and C4 Alloteropsis semialata subspecies

Alloteropsis semialata (R. Br.) Hitchcock includes both C3 and C4 subspecies: the C3 subspecies eckloniana and the C4 subspecies semialata. We examined the leaf structural and photosynthetic characteristics of these plants. A. semialata ssp. semialata showed high activities of photosynthetic enzymes involved in phosphoenolpyruvate carboxykinase-type C4 photosynthesis and an anomalous Kranz anatomy. Phosphoenolpyruvate carboxylase; pyruvate, Pi dikinase and glycine decarboxylase (GDC) were compartmentalized between the mesophyll (M) and inner bundle sheath cells, whereas ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) occurred in both cells. A. semialata ssp. eckloniana also showed an anomalous non-Kranz anatomy, in which the mestome sheath cells included abundant chloroplasts and mitochondria. Rubisco and GDC accumulated densely in the M and mestome sheath cells, whereas the levels of C4 enzymes were low. The activity levels of photo-respiratory enzymes in both subspecies were intermediate between those in typical C3 and C4 plants. The values of CO2 compensation points in A. semialata ssp. semialata were within the C4 range, whereas those in A. semialata ssp. eckloniana were somewhat lower than the C3 range. These data suggest that the plants are C3-like and C4-like but not typical C3 and C4, and when integrated with previous findings, point to important variability in the expression of C4 physiology in this species complex. A. semialata is therefore an intriguing grass species with which to study the evolutionary linkage between C3 and C4 plants.     

Drought constraints on C4 photosynthesis: stomatal and metabolic limitations in C3 and C4 subspecies of Alloteropsis semialata

The C4 photosynthetic pathway uses water more efficiently than the C3 type, yet biogeographical analyses show a decline in C4 species relative to C3 species with decreasing rainfall. To investigate this paradox, the hypothesis that the C4 advantage over C3 photosynthesis is diminished by drought was tested, and the underlying stomatal and metabolic mechanisms of this response determined. The effects of drought and high evaporative demand on leaf gas exchange and photosynthetic electron sinks in C3 and C4 subspecies of the grass Alloteropsis semialata were examined. Plant responses to climatic variation and soil drought were investigated using a common garden experiment with well-watered and natural rainfall treatments, and underlying mechanisms analysed using controlled drying pot experiments. Photosynthetic rates were significantly higher in the C4 than the C3 subspecies in the garden experiment under well-watered conditions, but this advantage was completely lost during a rainless period when unwatered plants experienced severe drought. Controlled drying experiments showed that this loss was caused by a greater increase in metabolic, rather than stomatal, limitations in C4 than in the C3 leaves. Decreases in CO2 assimilation resulted in lower electron transport rates and decreased photochemical efficiency under drought conditions, rather than increased electron transport to alternative sinks. These findings suggest that the high metabolic sensitivity of photosynthesis to severe drought seen previously in several C4 grass species may be an inherent characteristic of the C4 pathway. The mechanism may explain the paradox of why C4 species decline in arid environments despite high water-use efficiency.     

Photosynthetic acclimation and resource use by the C3 and C4 subspecies of Alloteropsis semialata in low CO2 atmospheres

During the past 25 Myr, partial pressures of atmospheric CO₂ (C_a) imposed a greater limitation on C₃ than C₄ photosynthesis. This could have important downstream consequences for plant nitrogen economy and biomass allocation. Here, we report the first phylogenetically controlled comparison of the integrated effects of subambient C_a on photosynthesis, growth and nitrogen allocation patterns, comparing the C₃ and C₄ subspecies of Alloteropsis semialata. Plant size decreased more in the C₃ than C₄ subspecies at low C_a, but nitrogen pool sizes were unchanged, and nitrogen concentrations increased across all plant partitions. The C_3, but not C₄ subspecies, preferentially allocated biomass to leaves and increased specific leaf area at low C_a. In the C₃ subspecies, increased leaf nitrogen was linked to photosynthetic acclimation at the interglacial C_a, mediated via higher photosynthetic capacity combined with greater stomatal conductance. Glacial C_a further increased the biochemical acclimation and nitrogen concentrations in the C₃ subspecies, but these were insufficient to maintain photosynthetic rates. In contrast, the C₄ subspecies maintained photosynthetic rates, nitrogen‐ and water‐use efficiencies and plant biomass at interglacial and glacial C_a with minimal physiological adjustment. At low C_a, the C₄ carbon‐concentrating mechanism therefore offered a significant advantage over the C₃ type for carbon acquisition at the whole‐plant scale, apparently mediated via nitrogen economy and water loss. A limiting nutrient supply damped the biomass responses to C_a and increased the C₄ advantage across all C_a treatments. Findings highlight the importance of considering leaf responses in the context of the whole plant, and show that carbon limitation may be offset at the expense of greater plant demand for soil resources such as nitrogen and water. Results show that the combined effects of low CO₂ and resource limitation benefit C₄ plants over C₃ plants in glacial–interglacial environments, but that this advantage is lessened under anthropogenic conditions.     

Seasonal differences in photosynthesis between the C3 and C4 subspecies of Alloteropsis semialata are offset by frost and drought

The regional abundance of C₄ grasses is strongly controlled by temperature, however, the role of precipitation is less clear. Progress in elucidating the direct effects of photosynthetic pathway on these climate relationships is hindered by the significant genetic divergence between major C₃ and C₄ grass lineages. We addressed this problem by examining seasonal climate responses of photosynthesis in Alloteropsis semialata , a unique grass species with both C₃ and C₄ subspecies. Experimental manipulation of rainfall in a common garden in South Africa tested the hypotheses that: (1) photosynthesis is greater in the C₄ than C₃ subspecies under high summer temperatures, but this pattern is reversed at low winter temperatures; and (2) the photosynthetic advantage of C₄ plants is enhanced during drought events. Measurements of leaf gas exchange over 2 years showed a significant photosynthetic advantage for the C₄ subspecies under irrigated conditions from spring through autumn. However, the C₄ leaves were killed by winter frost, while photosynthesis continued in the C₃ plants. Unexpectedly, the C₄ subspecies also lost its photosynthetic advantage during natural drought events, despite greater water-use efficiency under irrigated conditions. This study highlights previously unrecognized roles for climatic extremes in determining the ecological success of C₃ and C₄ grasses.     

Consequences of C4 photosynthesis for the partitioning of growth: a test using C3 and C4 subspecies of Alloteropsis semialata under nitrogen-limitation

C(4) plants dominate the world's subtropical grasslands, but investigations of their ecology typically focus on climatic variation, ignoring correlated changes in soil nutrient concentration. The hypothesis that higher photosynthetic nitrogen use efficiency (PNUE) in C(4) than in C(3) species allows greater flexibility in the partitioning of growth, especially under nutrient-deficient conditions, is tested here. Our experiment applied three levels of N supply to the subtropical grass Alloteropsis semialata, a unique model system with C(3) and C(4) subspecies. Photosynthesis was significantly higher for the same investment of leaf N in the C(4) than C(3) subspecies, and was unaffected by N treatments. The C(4) plants produced more biomass than the C(3) plants at high N levels, diverting a greater fraction of growth into inflorescences and corms, but less into roots and leaves. However, N-limitation of biomass production caused size-dependent shifts in the partitioning of growth. Root production was higher in small than large plants, and associated with decreasing leaf biomass in the C(3), and inflorescence production in the C(4) plants. Higher PNUE in the C(4) than C(3) subspecies was therefore linked with greater investment in sexual reproduction and storage, and the avoidance of N-limitations on leaf growth, suggesting advantages of the C(4) pathway in disturbed and infertile ecosystems.     

Evolutionary implications of C3–C4 intermediates in the grass Alloteropsis semialata

C₄ photosynthesis is a complex trait resulting from a series of anatomical and biochemical modifications to the ancestral C₃ pathway. It is thought to evolve in a stepwise manner, creating intermediates with different combinations of C₄‐like components. Determining the adaptive value of these components is key to understanding how C₄ photosynthesis can gradually assemble through natural selection. Here, we decompose the photosynthetic phenotypes of numerous individuals of the grass Alloteropsis semialata, the only species known to include both C₃ and C₄ genotypes. Analyses of δ¹³C, physiology and leaf anatomy demonstrate for the first time the existence of physiological C₃–C₄ intermediate individuals in the species. Based on previous phylogenetic analyses, the C₃–C₄ individuals are not hybrids between the C₃ and C₄ genotypes analysed, but instead belong to a distinct genetic lineage, and might have given rise to C₄ descendants. C₃ A. semialata, present in colder climates, likely represents a reversal from a C₃–C₄ intermediate state, indicating that, unlike C₄ photosynthesis, evolution of the C₃–C₄ phenotype is not irreversible.     

Comparative cytogenetic investigation of the two subspecies of the grass Alloteropsis semialata (Poaceae)

A cytotaxonomic investigation was undertaken to assess the taxonomic status of the grasses Alloteropsis semialata subsp. eckloniana and A. semialata subsp. semialata , distinguished because of a C3 photosynthetic pathway in subsp. eckloniana and a C4 pathway in subsp. semialata. Of the 30 analysed specimens of population A, 14 (46.7%) were diploid, 14 (46.7%) hexaploid, one octoploid (3.3%) and one dodecaploid (3.3%). Of the 21 specimens of population B, 14 (66.7%) were diploid, three (14.3%) hexaploid and four (19.0%) octoploid. All the diploids belonged to subsp. eckloniana , while all the polyploids belonged to subsp. semialata. Meiosis of the diploids appeared normal, with nine bivalents and a mean metaphase I chiasma frequency of 12.7 per cell. The hexaploids displayed a large range of chromosome pairing associations, although a high percentage of bivalents was recorded (89.9%). Three of the hexaploids showed 100% bivalent pairing, but the largest multivalent found in other hexaploids was a hexavalent pairing. The three octoploids analysed had 93.5% bivalent pairing. B chromosomes were found in five diploids, two hexaploids and one octoploid. Slow-moving bivalents, two in the diploids and up to four in the polyploids segregated late at anaphase I in most specimens.     

Broadening the spectrum of photosynthesis in the grass,Alloteropsis semialata

This article comments on: Evolutionary implications of C₃–C₄ intermediates in the grass Alloteropsis semialata     

C3 and C4 Photosynthetic and Anatomical forms of Alloteropsis semialata (R.Br.) Hitchcock: I. Variability in Photosynthetic Characteristics, Water Utilization Efficiency and Leaf Anatomy

The grass Alloteropsis semialata (R.Br.) Hitchcock is uniquein that both Kranz and non-Kranz leaf anatomy has been reportedin this species. The present study investigates Kranz formsof A. semialata collected from a single ecological niche. Theseplants exhibit morphological and anatomical differences withrespect to leaf area, stomatal size and stomatal distribution.Carbon dioxide and water exchange measurements in the two formsshow the expected pattern of higher photosynthetic rate andhigher water utilization efficiency associated with Kranz anatomy.No intermediate physiological response or anatomical form wasobserved in this sample. Alloteropsis semialata (R.Br.) Hitchcock, C₃ photosynthetic, C₄ photosynthesis, water utilization, leaf anatomy, Kranz anatomy     

C3 and C4 Photosynthetic and Anatomical Forms of Alloteropsis semialata (R.Br.) Hitchcock: 2. A Comparative Investigation of Leaf Ultrastructure and Distribution of Chlorenchyma in the Two Forms

Alloteropsis semialata (R.Br.) Hitchcock is a widely distributedgrass species known to show marked morphological, anatomicaland physiological variation. An unusual feature of this grassis that it shows both C₃, and C₄ photosynthetic pathways withthe respective anatomy related to these pathways in one speciesand within a single environment. This study investigates ultrastructuraldifferences between C₃ and C₄ forms of A. semialata, and showsdistribution of chlorenchyma in the two forms, at light microscopelevel. Quantitative assessment shows marked differences in mesophylland Kranz sheath cells with regard to size and organelle content;also in distribution of starch grains, lipid droplets and organellesin the mesophyll cells of both forms. These differences arediscussed in relation to the C₃/C₄ syndrome. Measurements were made using a Digiplan electronic planimeteron transmission electron micrographs of mature leaves sectionedtransversely at mid-lamina, and on drawings of leaf sectionsshowing the distribution of chlorenchymatous tissue. Alloteropsis semialata(R.Br.) Hitchcock, leaf structure, chlorenchyma, ultrastructure, C₃-photosynthesis, C₄-photosynthesis     

Effects of irradiance and temperature on photosynthesis in C3, C4 and C3/C4 Panicum species

Species in the Laxa and Grandia groups of the genus Panicum are adapted to low, wet areas of tropical and subtropical America. Panicum milioides is a species with C₃ photosynthesis and low apparent photorespiration and has been classified as a C₃/C₄ intermediate. Other species in the Laxa group are C₃ with normal photorespiration. Panicum prionitis is a C₄ species in the Grandia group. Since P. milioides has some leaf characteristics intermediate to C₃ and C₄ species, its photosynthetic response to irradiance and temperature was compared to the closely related C₃ species, P. laxum and P. boliviense and to P. prionitis. The response of apparent photosynthesis to irradiance and temperature was similar to that of P. laxum and P. boliviense, with saturation at a photosynthetic photo flux density of about 1 mmol m^-2 s^-1 at 30°C and temperature optimum near 30°C. In contrast, P. prionitis showed no light saturation up to 2 mmol m^-2 s^-1 and an optimum temperature near 40°C. P. milioides exhibited low CO₂ loss into CO₂-free air in the light and this loss was nearly insensitive to temperature. Loss of CO₂ in the light in the C₃ species, P. laxum and P. boliviense, was several-fold higher than in P. milioides and increased 2- to 5-fold with increases in temperature from 10 to 40°C. The level of dark respiration and its response to temperature were similar in all four Panicum species examined. It is concluded that the low apparent photorespiration in P. milioides does not influence its response of apparent photosynthesis to irradiance and temperature in comparison to closely related C₃ Panicum species.Abbreviations AP apparent photosynthesis - I CO₂ compensation point - gl leaf conductance; gm, mesophyll conductance - PPFD photosynthetic photon flux density - PR apparent photorespiration rate - RuBPC sibulose bisphosphate carboxylase     

A molecular phylogeny of the genus Alloteropsis (Panicoideae, Poaceae) suggests an evolutionary reversion from C4 to C3 photosynthesis

Background and Aims: The grass Alloteropsis semialata is the only plant species withboth C₃ and C₄ subspecies. It therefore offers excellent potentialas a model system for investigating the genetics, physiologyand ecological significance of the C₄ photosynthetic pathway.Here, a molecular phylogeny of the genus Alloteropsis is constructedto: (a) confirm the close relationship between the C₃ and C₄subspecies of A. semialata; and (b) infer evolutionary relationshipsbetween species within the Alloteropsis genus. Methods: The chloroplast gene ndhF was sequenced from 12 individuals,representing both subspecies of A. semialata and all four ofthe other species in the genus. ndhF sequences were added tothose previously sequenced from the Panicoideae, and used toconstruct a phylogenetic tree. Key Results: The phylogeny confirms that the two subspecies of A. semialataare among the most recently diverging lineages of C₃ and C₄taxa currently recognized within the Panicoideae. Furthermore,the position of the C₃ subspecies of A. semialata within theAlloteropsis genus is consistent with the hypothesis that itsphysiology represents a reversion from C₄ photosynthesis. Thedata point to a similar evolutionary event in the Panicum stenodes–P.caricoides–P. mertensii clade. The Alloteropsis genusis monophyletic and occurs in a clade with remarkable diversityof photosynthetic biochemistry and leaf anatomy. Conclusions: These results confirm the utility of A. semialata as a modelsystem for investigating C₃ and C₄ physiology, and provide moleculardata that are consistent with reversions from C₄ to C₃ photosynthesisin two separate clades. It is suggested that further phylogeneticand functional investigations of the Alloteropsis genus andclosely related taxa are likely to shed new light on the mechanismsand intermediate stages underlying photosynthetic pathway evolution.     

Maintenance and growth components of dark respiration rate in leaves of C3 and C4 plants as affected by leaf temperature

The rates of maintenance and growth components of leaf dark respiration of a C₃ plant (Phaseolus vulgaris L.) and C₄ plant (Zea mays L.) as affected by temperature were studied using the McCree concept. Respiration rates were measured by means of infrared gas analysis in a closed gas exchange system. In both C₃ and C₄ species R_D and R_m increased with temperature in the temperature range (15–62 °C) studied. R_G depended on temperature with an optimum near the temperature optimum of gross photosynthetic rate, P_g. Significant correlation between R_D and R_M and between R_G and P_G was found.     

Mutual stimulation of temperature and light effects on C(4) phosphoenolpyruvate carboxylase in leaf discs and leaves of Amaranthus hypochondriacus

This article reports marked modulation of the activity and regulatory properties of phosphoenolpyruvate carboxylase (PEPC) by temperature and light in leaf discs as well as leaves of Amaranthus hypochondriacus. The activity of PEPC increased by 1.7-fold at 45 degrees C over 25 degrees C. Warm temperature also stimulated the photoactivation of PEPC. The activation by light of PEPC was 1.9-fold at 25 degrees C and increased to 2.2-fold at 45 degrees C. The sensitivity of PEPC to its inhibitor malate was less and the activation by glucose-6-phosphate (G-6-P) or inorganic phosphate (Pi) was more at 45 degrees C than that at 25 degrees C. These effects of temperature were quite pronounced in light. Similar responses were observed when detached leaves were exposed to varying ambient temperature (dry heat). The activity of PEPC increased by 1.6-fold at 45 degrees C over 25 degrees C in the dark. The activation of PEPC by light was 2.1-fold at 25 degrees C and increased to 2.6-fold at 45 degrees C. Inhibition by malate was less and activation by G-6-P or Pi was more at 45 degrees C than that at 25 degrees C. Thus, there was a marked modulation of not only the activity but also the regulatory properties of the enzyme by temperature and light, independently as well as cooperatively with each other. Further experiments suggested that PEPC was able to memorize to a significant extent the changes induced by warm temperature and that these changes were complemented by subsequent illumination. These effects were not due to changes in PEPC protein levels. We conclude that temperature and light can modulate PEPC activity and regulatory properties not only individually but also in a significantly cooperative manner with each other. As significant increases in temperature are common during daytime in tropical or subtropical conditions, we suggest that the synergistic effects of temperature and light are quite relevant in optimizing the activity of PEPC in leaves of C(4) plants.     

The effect of temperature on C(4)-type leaf photosynthesis parameters

C(4)-type photosynthesis is known to vary with growth and measurement temperatures. In an attempt to quantify its variability with measurement temperature, the photosynthetic parameters - the maximum catalytic rate of the enzyme ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco) (V(cmax)), the maximum catalytic rate of the enzyme phosphoenolpyruvate carboxylase (PEPC) (V(pmax)) and the maximum electron transport rate (J(max)) - were examined. Maize plants were grown in climatic-controlled phytotrons, and the curves of net photosynthesis (A(n)) versus intercellular air space CO(2) concentrations (C(i)), and A(n) versus photosynthetic photon flux density (PPFD) were determined over a temperature range of 15-40 degrees C. Values of V(cmax), V(pmax) and J(max) were computed by inversion of the von Caemmerer & Furbank photosynthesis model. Values of V(pmax) and J(max) obtained at 25 degrees C conform to values found in the literature. Parameters for an Arrhenius equation that best fits the calculated values of V(cmax), V(pmax) and J(max) are then proposed. These parameters should be further tested with C(4) plants for validation. Other model key parameters such as the mesophyll cell conductance to CO(2) (g(i)), the bundle sheath cells conductance to CO(2) (g(bs)) and Michaelis-Menten constants for CO(2) and O(2) (K(c), K(p) and K(o)) also vary with temperature and should be better parameterized.     

Hypersalinity effects on leaf ultrastructure and physiology in the mangrove Avicennia marina

The effects of hypersalinity on leaf ultrastructure and physiology in the mangrove, Avicennia marina, were investigated by comparing leaves of adult trees growing naturally in the field under seawater and hypersalinity conditions in Richards Bay, South Africa. We tested the hypothesis that hypersalinity has a deleterious effect on membranes and cellular organelles such as chloroplasts and mitochondria, which would impact negatively physiological processes, such as ion and water relations, and photosynthetic performance. Soil ψ and soil salinity were −2.96 ± 0.07 MPa and 35 ± 2.8 psu in the seawater salinity site, compared to −5.91 ± 0.42 MPa and 58 ± 3.6 psu respectively, in the hypersaline site. In the hypersaline site, leaves were smaller and thicker, with thicker cuticles, while chloroplasts, mitochondria and nuclei exhibited swelling and disintegration, compared to those at seawater salinity. Multivesicular structures and vesicles, observed in vacuoles, chloroplasts, mitochondria, and along cell walls and plasma membranes, were more abundant in leaves from the hypersaline than the seawater site, and were probably indicative of greater plant salt uptake in the former site. Leaf concentrations of total chlorophyll and chlorophylls a and b were lower in trees from the hypersaline site by 33%, 29%, and 45% respectively, compared to those at seawater salinity. Midday minimum xylem ψ was −3.82 ± 0.33 MPa in the seawater site and −6.47 ± 0.45 MPa in the hypersaline site. In the hypersaline site, the concentration of leaf Na⁺ was 40% higher, while those of K⁺, Ca²⁺, and Mg²⁺ were lower by 45%, 44%, and 54% respectively, than those in the seawater site. CO₂ exchange and the intrinsic photochemical efficiency of PS II were significantly lower in trees from the hypersaline site by 48 and 19% respectively. The ultrastructural evidence supported the physiological data that A. marina trees in the hypersaline site are under extreme salinity stress and that this species is growing there at the upper limit of its salt tolerance.     

Independent effects of ocean warming versus acidification on the growth,survivorship and physiology of two Acropora corals

Coral Reefs - Climate change is the greatest threat to coral reef ecosystems. Importantly, gradual changes in seawater chemistry compounds upon increasing temperatures leading to declines in...     

Low night temperature effect on photosynthate translocation of two C4 grasses

Summary Translocation of assimilates in plants of Echinochloa crus-galli, from Quebec and Mississippi, and of Eleusine indica from Mississippi was monitored, before and after night chilling, using radioactive tracing with the short-life isotope ¹¹C. Plants were grown at 28°/22°C (day/night temperatures) under either 350 or 675 l·l^-1 CO₂. Low night temperature reduced translocation mainly by increasing the turn-over times of the export pool. E. crus-galli plants from Mississippi were the most susceptible to chilling; translocation being completely inhibited by exposure for one night to 7°C at 350 l·l^-1 CO₂. Overall, plants from Quebec were the most tolerant to chilling-stress. For plants of all three populations, growth under CO₂ enrichment resulted in higher ¹¹C activity in the leaf phloem. High CO₂ concentrations also seemed to buffer the transport system against chilling injuries.     

Low humidity effects on photosynthesis in single leaves of C4 plants

Summary It was hypothesized that since sub-stomatal carbon dioxide concentrations are often saturating to photosynthesis at ambient external concentrations in C₄ plants at high light, photosynthesis might be insensitive to partial stomatal closure caused by large leaf-air water vapor pressure difference. The response of stomatal conductance and photosynthesis at high irradiance to vapor pressure difference was determined under uniform conditions in C₄ plants grown under controlled conditions, and outdoors. In several cases, photosynthesis was less sensitive to stomatal closure than it would have been if photosynthesis had a linear response to sub-stomatal carbon dioxide concentration. No change in photosynthesis at up to 25 mbar vapor pressure difference was demonstrated in the C₄ species Portulaca oleracea and Amaranthus hypochondriacus, despite reductions in stomatal conductance of 32 and 17%, respectively. Sensitivity of photosynthesis to leaf-air vapor pressure difference was found to depend on the species and on the growth conditions.     

Contrasting effects of simazine on the photosynthetic physiology and leaf morphology of two Populus clones

Differential effects of simazine (2-chloro-4,6-bis(ethylamino)- s -triazine) on the physiology of two Populus clones were investigated in a greenhouse study. Additions of 5 mg/pot simazine to young plants had no deleterious morphological or physiological effects on clone NC 5328 ( P. x euramericana cv. I 45/51; Section Aigeiros), but reduced the rate of CO₂ fixation, increased CO₂ compensation concentrations and lowered the specific leaf weight of clone NE 388 ( P. maximowiczü x P. trichocarpa cv. Kingston; section Tacamahaca). Abaxial leaf conductance to water vapor was not affected in NE 388. Deleterious effects of simazine on NE 388 were detected ca 48 h after exposure of plants to simazine and generally became more pronounced thereafter. Visual symptoms of injury were evident at ca 2 weeks after simazine application.
Toxic responses to simazine in clone NE 388 varied in different portions of the crown. Inhibition of photosynthesis and increased CO₂ compensation concentrations were more pronounced in the region of recently matured leaves, but were somewhat less in the region of expanding leaves. Older mature leaves in the lower crown region showed no visual symptoms of injury and the rate of photosynthesis and CO₂ compensation concentrations were largely unaffected.