▪ Abstract The efficiency of photosynthetic carbon assimilation in higher plants faces significant limitations due to the oxygenase activity of the enzyme Rubisco, particularly under warmer temperatures or water stress. A drop in atmospheric CO₂ and rise in O₂ as early as 300 mya provided selective pressure for the evolution of mechanisms to concentrate CO₂ around Rubisco in order to minimize oxygenase activity and the resultant loss of carbon through photorespiration. It is well established that a carbon-concentrating mechanism occurs in some terrestrial plants through the process of C₄ photosynthesis. These plants are characterized as having Kranz-type leaf anatomy, with two structurally and biochemically specialized photosynthetic cell types, mesophyll and bundle sheath, that function coordinately in carbon assimilation. C₄ photosynthesis has evolved independently many times with great diversity in forms of Kranz anatomy, structure of dimorphic chloroplasts, and biochemistry of the C₄ cycle. The most dramatic variants of C₄ terrestrial plants were discovered recently in two species, Bienertia cycloptera and Borszczowia aralocaspica (family Chenopodiaceae); each has novel compartmentation to accomplish C₄ photosynthesis within a single chlorenchyma cell. This review discusses the amazing diversity in C₄ systems, how the essential features of C₄ are accomplished in single-cell versus Kranz-type C₄ plants, and speculates on why single-cell C₄ plants evolved.

The genus Oryza, which includes rice (Oryza sativa and Oryza glaberrima) and wild relatives, is a useful genus to study leaf properties in order to identify structural features that control CO₂ access to chloroplasts, photosynthesis, water use efficiency, and drought tolerance. Traits, 26 structural and 17 functional, associated with photosynthesis and transpiration were quantified on 24 accessions (representatives of 17 species and eight genomes). Hypotheses of associations within, and between, structure, photosynthesis, and transpiration were tested. Two main clusters of positively interrelated leaf traits were identified: in the first cluster were structural features, leaf thickness (Thick_leaf), mesophyll (M) cell surface area exposed to intercellular air space per unit of leaf surface area (S_mes), and M cell size; a second group included functional traits, net photosynthetic rate, transpiration rate, M conductance to CO₂ diffusion (g_m), stomatal conductance to gas diffusion (g_s), and the g_m/g_s ratio. While net photosynthetic rate was positively correlated with g_m, neither was significantly linked with any individual structural traits. The results suggest that changes in g_m depend on covariations of multiple leaf (S_mes) and M cell (including cell wall thickness) structural traits. There was an inverse relationship between Thick_leaf and transpiration rate and a significant positive association between Thick_leaf and leaf transpiration efficiency. Interestingly, high g_m together with high g_m/g_s and a low S_mes/g_m ratio (M resistance to CO₂ diffusion per unit of cell surface area exposed to intercellular air space) appear to be ideal for supporting leaf photosynthesis while preserving water; in addition, thick M cell walls may be beneficial for plant drought tolerance.

Summary: Kranz anatomy, with its separation of elements of the C₄ pathway between two cells, has been an accepted criterion for function of C₄ photosynthesis in terrestrial plants. However, Bienertia cycloptera (Chenopodiaceae), which grows in salty depressions of Central Asian semi‐deserts, has unusual chlorenchyma, lacks Kranz anatomy, but has photosynthetic features of C₄ plants. Its photosynthetic response to varying CO₂ and O₂ is typical of C₄ plants having Kranz anatomy. Lack of night‐time CO₂ fixation indicates it is not acquiring carbon by Crassulacean acid metabolism. This species exhibits an independent, novel solution to function of the C₄ mechanism through spatial compartmentation of dimorphic chloroplasts, other organelles and photosynthetic enzymes in distinct positions within a single chlorenchyma cell. The chlorenchyma cells have a large, spherical central cytoplasmic compartment interconnected by cytoplasmic channels through the vacuole to the peripheral cytoplasm. This compartment is filled with mitochondria and granal chloroplasts, while the peripheral cytoplasm apparently lacks mitochondria and has grana‐deficient chloroplasts. Immunolocalization studies show enzymes compartmentalized selectively in the CC compartment, including Rubisco in chloroplasts, and NAD‐malic enzyme and glycine decarboxylase in mitochondria, whereas pyruvate, Pi dikinase of the C₄ cycle is localized selectively in peripheral chloroplasts. Phosphoenolpyruvate carboxylase, a cytosolic C₄ cycle enzyme, is enriched in the peripheral cytoplasm. Our results show Bienertia utilizes strict compartmentation of organelles and enzymes within a single cell to effectively mimic the spatial separation of Kranz anatomy, allowing it to function as a C₄ plant having suppressed photorespiration; this raises interesting questions about evolution of C₄ mechanisms.

While malate and fumarate participate in a multiplicity of pathways in plant metabolism, the function of these organic acids as carbon stores in C₃ plants has not been deeply addressed. Here, Arabidopsis (Arabidopsis thaliana) plants overexpressing a maize (Zea mays) plastidic NADP-malic enzyme (MEm plants) were used to analyze the consequences of sustained low malate and fumarate levels on the physiology of this C₃ plant. When grown in short days (sd), MEm plants developed a pale-green phenotype with decreased biomass and increased specific leaf area, with thin leaves having lower photosynthetic performance. These features were absent in plants growing in long days. The analysis of metabolite levels of rosettes from transgenic plants indicated similar disturbances in both sd and long days, with very low levels of malate and fumarate. Determinations of the respiratory quotient by the end of the night indicated a shift from carbohydrates to organic acids as the main substrates for respiration in the wild type, while MEm plants use more reduced compounds, like fatty acids and proteins, to fuel respiration. It is concluded that the alterations observed in sd MEm plants are a consequence of impairment in the supply of carbon skeletons during a long dark period. This carbon starvation phenotype observed at the end of the night demonstrates a physiological role of the C₄ acids, which may be a constitutive function in plants.

• Leaf anatomy, stomatal density, and leaf conductance were studied in 10 species of Spartina (Poaceae) from low versus high salt marsh, and freshwater habitats. • Internal structure, external morphology, cuticle structure, and stomatal densities were studied with light and electron microscopy. Functional significance of leaf structure was examined by measures of CO₂ uptake and stomatal distributions. • All species have Kranz anatomy and C₄δ¹³C values. Freshwater species have thin leaves with small ridges on adaxial sides and stomata on both adaxial and abaxial sides. By contrast, salt marsh species have thick leaves with very pronounced ridges on the adaxial side and stomata located almost exclusively on adaxial leaf surfaces. Salt marsh species also have a thicker cuticle on the abaxial than on the adaxial side of leaves, and CO₂ uptake during photosynthesis is restricted to the adaxial leaf surface. • Salt marsh species are adapted to controlling water loss by having stomata in leaf furrows on the adaxial side, which increases the boundary layer, and by having large leaf ridges that fit together as the leaf rolls during water stress. Differences in structural–functional features of photosynthesis in Spartina species are suggested to be related to adaptations to saline environments.