Evolution of the C4 photosynthetic pathway: events at the cellular and molecular levels

The biochemistry and leaf anatomy of plants using C₄ photosynthesis promote the concentration of atmospheric CO₂ in leaf tissue that leads to improvements in growth and yield of C₄ plants over C₃ species in hot, dry, high light, and/or saline environments. C₄ plants like maize and sugarcane are significant food, fodder, and bioenergy crops. The C₄ photosynthetic pathway is an excellent example of convergent evolution, having evolved in multiple independent lineages of land plants from ancestors employing C₃ photosynthesis. In addition to C₃ and C₄ species, some plant lineages contain closely related C₃–C₄ intermediate species that demonstrate leaf anatomical, biochemical, and physiological characteristics between those of C₃ plants and species using C₄ photosynthesis. These groups of plants have been extremely useful in dissecting the modifications to leaf anatomy and molecular biology, which led to the evolution of C₄ photosynthesis. It is now clear that great variation exists in C₄ leaf anatomy, and diverse molecular mechanisms underlie C₄ biochemistry and physiology. However, all these different paths have led to the same destination—the expression of a C₄ CO₂ concentrating mechanism. Further identification of C₄ leaf anatomical traits and molecular biological components, and understanding how they are controlled and assembled will not only allow for additional insights into evolutionary convergence, but also contribute to sustainable food and bioenergy production strategies.     

Localization of ribulose bisphosphate carboxylase in the guard cells by an indirect, immunofluorescence technique

Ribulose bisphosphate carboxylase, a key enzyme in the photosynthetic carboxylation process, has been localized through an indirect immunofluorescent technique in the guard cells of some of the 41 species of plants examined. This sample includes 17 families of both dicotyledons and monocotyledons, one gymnosperm, and one pteridophyte. Plants were selected to represent all of the three major photosynthetic categories, namely C₃, C₄, and Crassulacean acid metabolism. Antibodies raised against tobacco (Nicotiana tabacum L.) ribulose bisphosphate carboxylase were used for this immunofluorescent study. A good degree of fluorescence was observed in the guard cells of seven out of 21 species exhibiting Crassulacean acid metabolism. C₃ plants exhibited a very low degree (almost negligible) of fluorescence, while the C₄ species did not exhibit any fluorescence.     

Phylogenetic niche conservatism in C4 grasses

Photosynthetic pathway is used widely to discriminate plant functional types in studies of global change. However, independent evolutionary lineages of C₄ grasses with different variants of C₄ photosynthesis show different biogeographical relationships with mean annual precipitation, suggesting phylogenetic niche conservatism (PNC). To investigate how phylogeny and photosynthetic type differentiate C₄ grasses, we compiled a dataset of morphological and habitat information of 185 genera belonging to two monophyletic subfamilies, Chloridoideae and Panicoideae, which together account for 90 % of the world’s C₄ grass species. We evaluated evolutionary variance and covariance of morphological and habitat traits. Strong phylogenetic signals were found in both morphological and habitat traits, arising mainly from the divergence of the two subfamilies. Genera in Chloridoideae had significantly smaller culm heights, leaf widths, 1,000-seed weights and stomata; they also appeared more in dry, open or saline habitats than those of Panicoideae. Controlling for phylogenetic structure showed significant covariation among morphological traits, supporting the hypothesis of phylogenetically independent scaling effects. However, associations between morphological and habitat traits showed limited phylogenetic covariance. Subfamily was a better explanation than photosynthetic type for the variance in most morphological traits. Morphology, habitat water availability, shading, and productivity are therefore all involved in the PNC of C₄ grass lineages. This study emphasized the importance of phylogenetic history in the ecology and biogeography of C₄ grasses, suggesting that divergent lineages need to be considered to fully understand the impacts of global change on plant distributions.     

Variations in nitrogen use efficiency reflect the biochemical subtype while variations in water use efficiency reflect the evolutionary lineage of C4 grasses at inter‐glacial CO2

C₄ photosynthesis evolved multiple times in diverse lineages. Most physiological studies comparing C₄ plants were not conducted at the low atmospheric CO₂ prevailing during their evolution. Here, 24 C₄ grasses belonging to three biochemical subtypes [nicotinamide adenine dinucleotide malic enzyme (NAD‐ME), phosphoenolpyruvate carboxykinase (PCK) and nicotinamide adenine dinucleotide phosphate malic enzyme (NADP‐ME)] and six major evolutionary lineages were grown under ambient (400 μL L^?1) and inter‐glacial (280 μL L^?1) CO₂. We hypothesized that nitrogen‐related and water‐related physiological traits are associated with subtypes and lineages, respectively. Photosynthetic rate and stomatal conductance were constrained by the shared lineage, while variation in leaf mass per area (LMA), leaf N per area, plant dry mass and plant water use efficiency were influenced by the subtype. Subtype and lineage were equally important for explaining variations in photosynthetic nitrogen use efficiency (PNUE) and photosynthetic water use efficiency (PWUE). CO₂ treatment impacted most parameters. Overall, higher LMA and leaf N distinguished the Chloridoideae/NAD‐ME group, while NADP‐ME and PCK grasses were distinguished by higher PNUE regardless of lineage. Plants were characterized by high photosynthesis and PWUE when grown at ambient CO₂ and by high conductance at inter‐glacial CO₂. In conclusion, the evolutionary and biochemical diversity among C₄ grasses was aligned with discernible leaf physiology, but it remains unknown whether these traits represent ecophysiological adaptation.     

Salt tolerance evolves more frequently in C4 grass lineages

Salt tolerance has evolved many times in the grass family, and yet few cereal crops are salt tolerant. Why has it been so difficult to develop crops tolerant of saline soils when salt tolerance has evolved so frequently in nature? One possible explanation is that some grass lineages have traits that predispose them to developing salt tolerance and that without these background traits, salt tolerance is harder to achieve. One candidate background trait is photosynthetic pathway, which has also been remarkably labile in grasses. At least 22 independent origins of the C₄ photosynthetic pathway have been suggested to occur within the grass family. It is possible that the evolution of C₄ photosynthesis aids exploitation of saline environments, because it reduces transpiration, increases water‐use efficiency and limits the uptake of toxic ions. But the observed link between the evolution of C₄ photosynthesis and salt tolerance could simply be due to biases in phylogenetic distribution of halophytes or C₄ species. Here, we use a phylogenetic analysis to investigate the association between photosynthetic pathway and salt tolerance in the grass family Poaceae. We find that salt tolerance is significantly more likely to occur in lineages with C₄ photosynthesis than in C₃ lineages. We discuss the possible links between C₄ photosynthesis and salt tolerance and consider the limitations of inferring the direction of causality of this relationship.     

Origin and evolution of C4 photosynthesis in the tribe Salsoleae (Chenopodiaceae) based on anatomical and biochemical types in leaves and cotyledons

 The Chenopodiaceae genus Salsola contains a large number of species with C₄ photosynthesis. Along with derivative genera they have a prominent position among the desert vegetation of Asia and Africa. About 130 species from Asia and Africa were investigated to determine the occurrence of C₃ versus C₄ syndrome in leaves and cotyledons, and to study specific anatomical and biochemical features of photosynthesis in both photosynthetic organs. The species studied belong to all six previously identified sections of the tribe Salsoleae based on morphological characters. Types of photosynthesis were identified using carbon ¹³C/¹²C isotope fractionation. The representatives of all systematic groups were investigated for mesophyll anatomy and biochemical subtypes by determination of enzyme activity (RUBPC, PEPC, NAD- and NADP-ME and AAT) and primary photosynthetic products. Two photosynthetic types (C₃ and C₄) and two biochemical subtypes (NAD- and NADP-ME) were identified in both leaves and cotyledons. Both Kranz and non-Kranz type anatomy were found in leaves and cotyledons, but cotyledons had more diversity in anatomical structure. Strong relationships between anatomical types and biochemical subtypes in leaves and cotyledons were shown. We found convincing evidence for a similar pattern of structural and biochemical features of photosynthesis in leaves and cotyledons within systematic groups, and evaluated their relevance at the evolutionary level. We identified six groups in tribe Salsoleae with respect to photosynthetic types and mesophyll structure in leaves and cotyledons. Two separate lineages of biochemical and anatomical evolution within Salsoleae were demonstrated based on studies of leaves and cotyledons. The sections Caroxylon, Malpighipila, Cardiandra and Belanthera have no C₃ species and only the NAD-ME C₄ subtype has been found in leaves. We suggest the C₄ species in the NADP-ME lineage evolved in Coccosalsola and Salsola sections, and originated in the subsection Arbuscula. Coccosalsola contains many species with C₃ and/or C₃-C₄ intermediate photosynthesis. Within these main evolutionary lineages, species of different taxonomic groups (sections and subsections) had differences in anatomical or/and biochemical features in leaves and cotyledons. We conclude that structural and biochemical changes in the photosynthetic apparatus in species of the tribe Salsoleae were a key factor in their evolution and broad distribution in extreme desert environments. Received January 25, 2001 Accepted July 17, 2001     

Drought limitation of photosynthesis differs between C3 and C4 grass species in a comparative experiment

Phylogenetic analyses show that C₄ grasses typically occupy drier habitats than their C₃ relatives, but recent experiments comparing the physiology of closely related C₃ and C₄ species have shown that advantages of C₄ photosynthesis can be lost under drought. We tested the generality of these paradoxical findings in grass species representing the known evolutionary diversity of C₄ NADP‐me and C₃ photosynthetic types. Our experiment investigated the effects of drought on leaf photosynthesis, water potential, nitrogen, chlorophyll content and mortality. C₄ grasses in control treatments were characterized by higher CO₂ assimilation rates and water potential, but lower stomatal conductance and nitrogen content. Under drought, stomatal conductance declined more dramatically in C₃ than C₄ species, and photosynthetic water‐use and nitrogen‐use efficiency advantages held by C₄ species under control conditions were each diminished by 40%. Leaf mortality was slightly higher in C₄ than C₃ grasses, but leaf condition under drought otherwise showed no dependence on photosynthetic‐type. This phylogenetically controlled experiment suggested that a drought‐induced reduction in the photosynthetic performance advantages of C₄ NADP‐me relative to C₃ grasses is a general phenomenon.     

Getting the most out of natural variation in C4 photosynthesis

C₄ photosynthesis is a complex trait that has a high degree of natural variation, involving anatomical and biochemical changes relative to the ancestral C₃ state. It has evolved at least 66 times across a variety of lineages and the evolutionary route from C₃ to C₄ is likely conserved but not necessarily genetically identical. As such, a variety of C₄ species are needed to identify what is fundamental to the C₄ evolutionary process in a global context. In order to identify the genetic components of C₄ form and function, a number of species are used as genetic models. These include Zea mays (maize), Sorghum bicolor (sorghum), Setaria viridis (Setaria), Flaveria bidentis, and Cleome gynandra. Each of these species has different benefits and challenges associated with its use as a model organism. Here, we propose that RNA profiling of a large sampling of C₄, C₃–C₄, and C₃ species, from as many lineages as possible, will allow identification of candidate genes necessary and sufficient to confer C₄ anatomy and/or biochemistry. Furthermore, C₄ model species will play a critical role in the functional characterization of these candidate genes and identification of their regulatory elements, by providing a platform for transformation and through the use of gene expression profiles in mesophyll and bundle sheath cells and along the leaf developmental gradient. Efforts should be made to sequence the genomes of F. bidentis and C. gynandra and to develop congeneric C₃ species as genetic models for comparative studies. In combination, such resources would facilitate discovery of common and unique C₄ regulatory mechanisms across genera.     

The Nitrogen Use Efficiency of C(3) and C(4) Plants: II. Leaf Nitrogen Effects on the Gas Exchange Characteristics of Chenopodium album (L.) and Amaranthus retroflexus (L.)

The effect of leaf nitrogen (N) on the photosynthetic capacity and the light and temperature response of photosynthesis was studied in the ecologically similar annuals Chenopodium album (C₃) and Amaranthus retroflexus (C₄). Photosynthesis was linearly dependent on leaf N per unit area (N_a) in both species. A. retroflexus exhibited a greater dependence of photosynthesis on N_a than C. album and at any given N_a, it had a greater light saturated photosynthesis rate than C. album. The difference between the species became larger as N_a increased. These results demonstrate a greater photosynthetic N use efficiency in A. retroflexus than C. album. However, at a given applied N level, C. album allocated more N to a unit of leaf area so that photosynthetic rates were similar in the two species. Leaf conductance to water vapor increased linearly with N_a in both species, but at a given photosynthetic rate, leaf conductance was higher in C. album. Thus, A. retroflexus had a greater water use efficiency than C. album. Water use efficiency was independent of leaf N in C. album, but declined with decreasing N in A. retroflexus.     

A Comparison of Dark Respiration between C(3) and C(4) Plants

Lower respiratory costs were hypothesized as providing an additional benefit in C₄ plants compared to C₃ plants due to less investment in proteins in C₄ leaves. Therefore, photosynthesis and dark respiration of mature leaves were compared between a number of C₄ and C₃ species. Although photosynthetic rates were generally greater in C₄ when compared to C₃ species, no differences were found in dark respiration rates of individual leaves at either the beginning or after 16 h of the dark period. The effects of nitrogen on photosynthesis and respiration of individual leaves and whole plants were also investigated in two species that occupy similar habitats, Amaranthus retroflexus (C₄) and Chenopodium album (C₃). For mature leaves of both species, there was no relationship between leaf nitrogen and leaf respiration, with leaves of both species exhibiting a similar rate of decline after 16 h of darkness. In contrast, leaf photosynthesis increased with increasing leaf nitrogen in both species, with the C₄ species displaying a greater photosynthetic response to leaf nitrogen. For whole plants of both species grown at different nitrogen levels, there was a clear linear relationship between net CO₂ uptake and CO₂ efflux in the dark. The dependence of nightly CO₂ efflux on CO₂ uptake was similar for both species, although the response of CO₂ uptake to leaf nitrogen was much steeper in the C₄ species, Amaranthus retroflexus. Rates of growth and maintenance respiration by whole plants of both species were similar, with both species displaying higher rates at higher leaf nitrogen. There were no significant differences in leaf or whole plant maintenance respiration between species at any temperature between 18 and 42°C. The data suggest no obvious differences in respiratory costs in C₄ and C₃ plants.     

Effects of polyploidy on photosynthesis

In polyploid plants the photosynthetic rate per cell is correlated with the amount of DNA per cell. The photosynthetic rate per unit leaf area is the product of the rate per cell times the number of photosynthetic cells per unit area. Therefore, the photosynthetic rate per unit leaf area will increase if there is a less than proportional increase in cell volume at higher ploidal levels, or if cell packing is altered to allow more cells per unit leaf area. In autopolyploids (Medicago sativa, C₃ species, and Pennisetum americanum, C₄ species) there is a doubling of photosynthesis per cell and of cell volume in the tetraploid compared to the diploid. However, there is a proportional decrease in number of cells per unit leaf area with this increase in ploidy such that the rate of photosynthesis per leaf area does not change. There is more diversity in the relationship between ploidal level (gene dosage) and photosynthetic rates per unit leaf area in allopolyploids. This is likely to reflect the effects of natural selection on leaf anatomy, and novel genetic interactions from contributed genomes which can occur with allopolyploidy. In allopolyploid wheat (C₃ species) a higher cell volume per unit DNA at the higher ploidal level is negatively correlated with photosynthesis rate per unit leaf area. Although photosynthesis per cell increases with ploidy, photosynthesis per leaf area decreases, being lowest in the allohexaploid, cultivated bread wheat (Triticum aestivum). Alternatively, doubling of photosynthetic rate per cell with doubling of DNA, with apparent natural selection for decreased cell volume per unit DNA, results in higher rates of photosynthesis per leaf area in octaploid compared to tetraploid Panicum virgatum (C₄) which may be a case of allopolyploidy. Similar responses probably occur in Festuca arundinacea. Therefore, in some systems anatomical factors affecting photosynthesis are also affected by ploidal level. It is important to evaluate that component as well as determining the effect on biochemical processes. Current information on polyploidy and photosynthesis in several species is discussed with respect to anatomy, biochemistry and bases for expressing photosynthetic rates.Abbreviations Chl chlorophyll - RuBPC ribulose-1,5-bisphosphate carboxylase     

Differential Expression of Genes in the Leaves of Sugarcane in Response to Sugar Accumulation

In C₄ sugarcane (Saccharum spp. hybrids), photosynthetic activity has been shown to be regulated by the demand for carbon from sink tissues. There is evidence, from other plant species, that sink-limitation of photosynthesis is facilitated by sugar-signaling mechanisms in the leaf that affect photosynthesis through regulation of gene expression. In this work, we manipulated leaf sugar levels by cold-girdling leaves (5°C) for 80 h to examine the mechanisms whereby leaf sugar accumulation affects photosynthetic activity and assess whether signaling mechanisms reported for other species operate in sugarcane. During this time, sucrose and hexose concentrations above the girdle increased by 77% and 81%, respectively. Conversely, leaf photosynthetic activity (A) and electron transport rates (ETR) decreased by 66% and 54%, respectively. Quantitative expression profiling by means of an Affymetrix GeneChip Sugarcane Genome Array was used to identify genes responsive to cold-girdling (56 h). A number of genes (74) involved in primary and secondary metabolic pathways were identified as being differentially expressed. Decreased expression of genes related to photosynthesis and increased expression of genes involved in assimilate partitioning, cell wall synthesis, phosphate metabolism and stress were observed. Furthermore four probe sets homologous to trehalose 6-phosphate phosphatase (TPP; EC 5.3.1.1) and trehalose 6-phosphate synthase (TPS; EC 2.4.1.15) were up- and down-regulated, respectively, indicating a possible role for trehalose 6-phosphate (T6P) as a putative sugar-sensor in sugarcane leaves.     

Effects of the Phosphoenolpyruvate Carboxylase Inhibitor 3,3-Dichloro-2-(Dihydroxyphosphinoylmethyl)propenoate on Photosynthesis: C(4) Selectivity and Studies on C(4) Photosynthesis

The effect of 3,3-dichloro-2-(dihydroxyphosphinoylmethyl)-propenoate (DCDP), an analog of phosphoenolpyruvate (PEP), on PEP carboxylase activity in crude leaf extracts and on photosynthesis of excised leaves was examined. DCDP is an effective inhibitor of PEP carboxylase from Zea mays or Panicum miliaceum; 50% inhibition was obtained at 70 or 350 micromolar, respectively, in the presence of 1 millimolar PEP and 1 millimolar HCO₃⁻. When fed to leaf sections via the transpiration stream, DCDP at 1 millimolar strongly inhibited photosynthesis in C₄ species (79-98% inhibition for a range of seven C₄ species), but only moderately in C₃ species (12-46% for four C₃ species), suggesting different mechanisms of inhibition for each photosynthetic type. The response of P. miliaceum (C₄) net photosynthesis to intercellular pCO₂ showed that carboxylation efficiency, as well as the CO₂ saturated rate, are lowered in the presence of DCDP and supported the view that carboxylation efficiency in C₄ species is directly related to PEP carboxylase activity. A fivefold increase in intercellular pCO₂ over that occurring in P. miliaceum under normal photosynthesis conditions only increased net photosynthesis rate in the presence of 1 millimolar DCDP from zero to about 5% of the maximal uninhibited rate. Therefore, it seems unlikely that direct fixation of atmospheric CO₂ by the bundle sheath cells makes any significant contribution to photosynthetic CO₂ assimilation in C₄ species. The results support the concept that C₄-selective herbicides may be developed based on inhibitors of C₄ pathway reactions.     

Temperature response of photosynthesis in C3, C4, and CAM plants: temperature acclimation and temperature adaptation

Most plants show considerable capacity to adjust their photosynthetic characteristics to their growth temperatures (temperature acclimation). The most typical case is a shift in the optimum temperature for photosynthesis, which can maximize the photosynthetic rate at the growth temperature. These plastic adjustments can allow plants to photosynthesize more efficiently at their new growth temperatures. In this review article, we summarize the basic differences in photosynthetic reactions in C₃, C₄, and CAM plants. We review the current understanding of the temperature responses of C₃, C₄, and CAM photosynthesis, and then discuss the underlying physiological and biochemical mechanisms for temperature acclimation of photosynthesis in each photosynthetic type. Finally, we use the published data to evaluate the extent of photosynthetic temperature acclimation in higher plants, and analyze which plant groups (i.e., photosynthetic types and functional types) have a greater inherent ability for photosynthetic acclimation to temperature than others, since there have been reported interspecific variations in this ability. We found that the inherent ability for temperature acclimation of photosynthesis was different: (1) among C₃, C₄, and CAM species; and (2) among functional types within C₃ plants. C₃ plants generally had a greater ability for temperature acclimation of photosynthesis across a broad temperature range, CAM plants acclimated day and night photosynthetic process differentially to temperature, and C₄ plants was adapted to warm environments. Moreover, within C₃ species, evergreen woody plants and perennial herbaceous plants showed greater temperature homeostasis of photosynthesis (i.e., the photosynthetic rate at high-growth temperature divided by that at low-growth temperature was close to 1.0) than deciduous woody plants and annual herbaceous plants, indicating that photosynthetic acclimation would be particularly important in perennial, long-lived species that would experience a rise in growing season temperatures over their lifespan. Interestingly, across growth temperatures, the extent of temperature homeostasis of photosynthesis was maintained irrespective of the extent of the change in the optimum temperature for photosynthesis (T _opt), indicating that some plants achieve greater photosynthesis at the growth temperature by shifting T _opt, whereas others can also achieve greater photosynthesis at the growth temperature by changing the shape of the photosynthesis–temperature curve without shifting T _opt. It is considered that these differences in the inherent stability of temperature acclimation of photosynthesis would be reflected by differences in the limiting steps of photosynthetic rate.     

Ecological selection pressures for C4 photosynthesis in the grasses

Grasses using the C₄ photosynthetic pathway dominate grasslands and savannahs of warm regions, and account for half of the species in this ecologically and economically important plant family. The C₄ pathway increases the potential for high rates of photosynthesis, particularly at high irradiance, and raises water-use efficiency compared with the C₃ type. It is therefore classically viewed as an adaptation to open, arid conditions. Here, we test this adaptive hypothesis using the comparative method, analysing habitat data for 117 genera of grasses, representing 15 C₄ lineages. The evidence from our three complementary analyses is consistent with the hypothesis that evolutionary selection for C₄ photosynthesis requires open environments, but we find an equal likelihood of C₄ evolutionary origins in mesic, arid and saline habitats. However, once the pathway has arisen, evolutionary transitions into arid habitats occur at higher rates in C₄ than C₃ clades. Extant C₄ genera therefore occupy a wider range of drier habitats than their C₃ counterparts because the C₄ pathway represents a pre-adaptation to arid conditions. Our analyses warn against evolutionary inferences based solely upon the high occurrence of extant C₄ species in dry habitats, and provide a novel interpretation of this classic ecological association.     

Contrasted histories of organelle and nuclear genomes underlying physiological diversification in a grass species

C₄ photosynthesis evolved multiple times independently in angiosperms, but most origins are relatively old so that the early events linked to photosynthetic diversification are blurred. The grass Alloteropsis semialata is an exception, as this species encompasses C₄ and non-C₄ populations. Using phylogenomics and population genomics, we infer the history of dispersal and secondary gene flow before, during and after photosynthetic divergence in A. semialata. We further analyse the genome composition of individuals with varied ploidy levels to establish the origins of polyploids in this species. Detailed organelle phylogenies indicate limited seed dispersal within the mountainous region of origin and the emergence of a C₄ lineage after dispersal to warmer areas of lower elevation. Nuclear genome analyses highlight repeated secondary gene flow. In particular, the nuclear genome associated with the C₄ phenotype was swept into a distantly related maternal lineage probably via unidirectional pollen flow. Multiple intraspecific allopolyploidy events mediated additional secondary genetic exchanges between photosynthetic types. Overall, our results show that limited dispersal and isolation allowed lineage divergence, with photosynthetic innovation happening after migration to new environments, and pollen-mediated gene flow led to the rapid spread of the derived C₄ physiology away from its region of origin.     

Photosynthesis of Grass Species Differing in Carbon Dioxide Fixation Pathways: V. RESPONSE OF PANICUM MAXIMUM, PANICUM MILIOIDES, AND TALL FESCUE (FESTUCA ARUNDINACEA) TO NITROGEN NUTRITION

The response of apparent photosynthesis to N nutrition was studied in the C₃ grass, tall fescue (Festuca arundinacea Schreb.), in the C₄ species Panicum maximum Jacq., and in Panicum milioides Nees ex Trin., a species with characteristics intermediate between C₃ and C₄ photosynthetic types. Plants were grown in culture solution containing 1, 5, 50, and 200 milligrams N per liter. Apparent photosynthesis was measured on the youngest fully expanded leaves at 320 microliters of CO₂ per liter of air and 21% O₂. Leaf conductance was calculated from transpiration measurements, and CO₂ compensation concentrations were also estimated. Several leaf anatomical characteristics were studied on plastic-embedded material. Leaf N content was determined on leaves which were used in photosynthesis measurements.