The 'hydrology' of leaves: co-ordination of structure and function in temperate woody species

The hydraulic conductance of the leaf lamina (K_lamina) substantially constrains whole‐plant water transport, but little is known of its association with leaf structure and function. K_lamina was measured for sun and shade leaves of six woody temperate species growing in moist soil, and tested for correlation with the prevailing leaf irradiance, and with 22 other leaf traits. K_lamina varied from 7.40 × 10^?5 kg m^?2 s^?1 MPa^?1 for Acer saccharum shade leaves to 2.89 × 10^?4 kg m^?2 s^?1 MPa^?1 for Vitis labrusca sun leaves. Tree sun leaves had 15–67% higher K_lamina than shade leaves. K_lamina was co‐ordinated with traits associated with high water flux, including leaf irradiance, petiole hydraulic conductance, guard cell length, and stomatal pore area per lamina area. K_lamina was also co‐ordinated with lamina thickness, water storage capacitance, 1/mesophyll water transfer resistance, and, in five of the six species, with lamina perimeter/area. However, for the six species, K_lamina was independent of inter‐related leaf traits including leaf dry mass per area, density, modulus of elasticity, osmotic potential, and cuticular conductance. K_lamina was thus co‐ordinated with structural and functional traits relating to liquid‐phase water transport and to maximum rates of gas exchange, but independent of other traits relating to drought tolerance and to aspects of carbon economy.     

Hydraulic architecture of leaf blades: where is the main resistance?

The hydraulic architecture of Laurus nobilis L. and Juglans regia L. leaves was studied using three different approaches: (1) hydraulic measurements of both intact leaves and of leaves subjected to treatments aimed at removing the extra‐vascular resistance; (2) direct measurements of the vascular pressure with a pressure probe; and (3) modelling the hydraulic architecture of leaf venation system on the basis of measurements of vein densities and conductivities. The hydraulic resistance of leaves (R_leaf) either cut, boiled or frozen–thawed was reduced by about 60 and 85% with respect to control leaves for laurel and walnut, respectively. Direct pressure drop measurements suggested that 88% of the resistance resided outside the vascular system in walnut. Model simulations were in agreement with these results provided vein hydraulic conductance was 0.12–0.28 that of the conductance predicted by Poiseuille's law. The results suggest that R_leaf is dominated by substantial extra‐vascular resistances and therefore contrast with the conclusions of recent studies dealing with the hydraulic architecture of the leaf. The present study confirms the ‘classical’ view of the hydraulic architecture of leaves as composed by a low‐resistance component (the venation) and a high‐resistance component (the mesophyll).     

Bundle sheath lignification mediates the linkage of leaf hydraulics and venation

The lignification of the leaf vein bundle sheath (BS) has been observed in many species and would reduce conductance from xylem to mesophyll. We hypothesized that lignification of the BS in lower‐order veins would provide benefits for water delivery through the vein hierarchy but that the lignification of higher‐order veins would limit transport capacity from xylem to mesophyll and leaf hydraulic conductance (K_leaf). We further hypothesized that BS lignification would mediate the relationship of K_leaf to vein length per area. We analysed the dependence of K_leaf, and its light response, on the lignification of the BS across vein orders for 11 angiosperm tree species. Eight of 11 species had lignin deposits in the BS of the midrib, and two species additionally only in their secondary veins, and for six species up to their minor veins. Species with lignification of minor veins had a lower hydraulic conductance of xylem and outside‐xylem pathways and lower K_leaf. K_leaf could be strongly predicted by vein length per area and highest lignified vein order (R² = .69). The light‐response of K_leaf was statistically independent of BS lignification. The lignification of the BS is an important determinant of species variation in leaf and thus whole plant water transport.     

The Péclet effect on leaf water enrichment correlates with leaf hydraulic conductance and mesophyll conductance for CO(2)

Leaf water gets isotopically enriched through transpiration, and diffusion of enriched water through the leaf depends on transpiration flow and the effective path length (L). The aim of this work was to relate L with physiological variables likely to respond to similar processes. We studied the response to drought and vein severing of leaf lamina hydraulic conductance (K_lamina), mesophyll conductance for CO₂ (g_m) and leaf water isotope enrichment in Vitis vinifera L cv. Grenache. We hypothesized that restrictions in water pathways would reduce K_lamina and increase L. As a secondary hypothesis, we proposed that, given the common pathways for water and CO₂ involved, a similar response should be found in g_m. Our results showed that L was strongly related to mesophyll variables, such as K_lamina or g_m across experimental drought and vein‐cutting treatments, showing stronger relationships than with variables included as input parameters for the models, such as transpiration. Our findings were further supported by a literature survey showing a close link between L and leaf hydraulic conductance (K_leaf = 31.5 × L^?0.43, r² = 0.60, n = 24). The strong correlation found between L, K_lamina and g_m supports the idea that water and CO₂ share an important part of their diffusion pathways through the mesophyll.     

Leaf morphology of 40 evergreen and deciduous broadleaved subtropical tree species and relationships to functional ecophysiological traits

We explored potential of morphological and anatomical leaf traits for predicting ecophysiological key functions in subtropical trees. We asked whether the ecophysiological parameters stomatal conductance and xylem cavitation vulnerability could be predicted from microscopy leaf traits. We investigated 21 deciduous and 19 evergreen subtropical tree species, using individuals of the same age and from the same environment in the Biodiversity‐Ecosystem Functioning experiment at Jiangxi (BEF‐China). Information‐theoretic linear model selection was used to identify the best combination of morphological and anatomical predictors for ecophysiological functions. Leaf anatomy and morphology strongly depended on leaf habit. Evergreen species tended to have thicker leaves, thicker spongy and palisade mesophyll, more palisade mesophyll layers and a thicker subepidermis. Over 50% of all evergreen species had leaves with multi‐layered palisade parenchyma, while only one deciduous species (Koelreuteria bipinnata) had this. Interactions with leaf habit were also included in best multi‐predictor models for stomatal conductance (g_s) and xylem cavitation vulnerability. In addition, maximum g_s was positively related to log ratio of palisade to spongy mesophyll thickness. Vapour pressure deficit (vpd) for maximum g_s increased with the log ratio of palisade to spongy mesophyll thickness in species having leaves with papillae. In contrast, maximum specific hydraulic conductivity and xylem pressure at which 50% loss of maximum specific xylem hydraulic conductivity occurred (Ψ₅₀) were best predicted by leaf habit and density of spongy parenchyma. Evergreen species had lower Ψ₅₀ values and lower maximum xylem hydraulic conductivities. As hydraulic leaf and wood characteristics were reflected in structural leaf traits, there is high potential for identifying further linkages between morphological and anatomical leaf traits and ecophysiological responses.     

Mechanisms for minimizing height‐related stomatal conductance declines in tall vines

The ability to transport water through tall stems hydraulically limits stomatal conductance (g_s), thereby constraining photosynthesis and growth. However, some plants are able to minimize this height‐related decrease in g_s, regardless of path length. We hypothesized that kudzu (Pueraria lobata) prevents strong declines in g_s with height through appreciable structural and hydraulic compensative alterations. We observed only a 12% decline in maximum g_s along 15‐m‐long stems and were able to model this empirical trend. Increasing resistance with transport distance was not compensated by increasing sapwood‐to‐leaf‐area ratio. Compensating for increasing leaf area by adjusting the driving force would require water potential reaching ?1.9 MPa, far below the wilting point (?1.2 MPa). The negative effect of stem length was compensated for by decreasing petiole hydraulic resistance and by increasing stem sapwood area and water storage, with capacitive discharge representing 8–12% of the water flux. In addition, large lateral (petiole, leaves) relative to axial hydraulic resistance helped improve water flow distribution to top leaves. These results indicate that g_s of distal leaves can be similar to that of basal leaves, provided that resistance is highest in petioles, and sufficient amounts of water storage can be used to subsidize the transpiration stream.     

Potassium mediates coordination of leaf photosynthesis and hydraulic conductance by modifications of leaf anatomy

Typical symptoms of potassium deficiency, characterized as chlorosis or withered necrosis, occur concomitantly with downregulated photosynthesis and impaired leaf water transport. However, the prominent limitations and mechanisms underlying the concerted decreases of leaf photosynthesis and hydraulic conductance are poorly understood. Monocots and dicots were investigated based on responses of photosynthesis and hydraulic conductance and their components and the correlated anatomical determinants to potassium deficiency. We found a conserved pattern in which leaf photosynthesis and hydraulic conductance concurrently decreased under potassium starvation. However, monocots and dicots showed two different hydraulic‐redesign strategies: Dicots tended to show a decreased minor vein density, whereas monocots reduced the size of the bundle sheath and its extensions, rather than the minor vein density; both of these strategies may restrain xylem and outside‐xylem hydraulic conductance. Additionally, potassium‐deprived leaves developed with fewer mesophyll cell‐to‐cell connections, leading to a reduced area being available for liquid‐phase flow. Further quantitative analysis revealed that mesophyll conductance to CO₂ and outside‐xylem hydraulic resistance were the major contributors to photosynthetic limitation and increased hydraulic resistance, at more than 50% and 60%, respectively. These results emphasize the importance of potassium in the coordinated regulation of leaf photosynthesis and hydraulic conductance through modifications of leaf anatomy.     

EFFECTS OF 2,3,5-TRIIODOBENZOIC ACID ON THE STRUCTURE OF SOYBEAN LEAVES

The effects of 2,3,5-triiodobenzoic acid (TIBA) on soybean leaves (Glycine max. [L.] Merrill ‘Harosoy‘) include thickening with intensification of color and some raised intercostal regions, giving a wrinkled appearance. These effects are not restricted to early stages of leaf development but are pronounced during and after unfolding of the leaf. Proliferation of tracheary elements, increased procambial activity, and hypertrophy of bundle sheath extension cells occurred in the leaflet midvein of the youngest expanded leaf treated with 50 ppm or 100 ppm of TIBA. The youngest treated leaves exhibited differential growth rates and expansion within the palisade and spongy layers. Hypertrophy of spongy cells in these leaves occurred independently or simultaneously with elongation of the upper and lower palisade layers. The palisade and spongy tissues had undergone cell division and expansion at a greater pace than the epidermal layers. This, along with hypertrophy in the bundle sheath extension cells, would explain the wrinkled appearance of the lamina. The treated leaf became thicker than the control as a result of the increased number of cells in the spongy layer and elongation in the palisade layer. The observed aberrations in leaf structure suggest that TIBA interferes with some auxin-translocating system within the plant.     

Leaf hydraulic conductance varies with vein anatomy across Arabidopsis thaliana wild‐type and leaf vein mutants

Leaf venation is diverse across plant species and has practical applications from paleobotany to modern agriculture. However, the impact of vein traits on plant performance has not yet been tested in a model system such as Arabidopsis thaliana. Previous studies analysed cotyledons of A. thaliana vein mutants and identified visible differences in their vein systems from the wild type (WT). We measured leaf hydraulic conductance (K_leaf), vein traits, and xylem and mesophyll anatomy for A. thaliana WT (Col‐0) and four vein mutants (dot3‐111 and dot3‐134, and cvp1‐3 and cvp2‐1). Mutant true leaves did not possess the qualitative venation anomalies previously shown in the cotyledons, but varied quantitatively in vein traits and leaf anatomy across genotypes. The WT had significantly higher mean K_leaf. Across all genotypes, there was a strong correlation of K_leaf with traits related to hydraulic conductance across the bundle sheath, as influenced by the number and radial diameter of bundle sheath cells and vein length per area. These findings support the hypothesis that vein traits influence K_leaf, indicating the usefulness of this mutant system for testing theory that was primarily established comparatively across species, and supports a strong role for the bundle sheath in influencing K_leaf.     

Bundle sheath extensions affect leaf structural and physiological plasticity in response to irradiance

Coordination between structural and physiological traits is key to plants' responses to environmental fluctuations. In heterobaric leaves, bundle sheath extensions (BSEs) increase photosynthetic performance (light‐saturated rates of photosynthesis, A_max) and water transport capacity (leaf hydraulic conductance, K_leaf). However, it is not clear how BSEs affect these and other leaf developmental and physiological parameters in response to environmental conditions. The obscuravenosa (obv) mutation, found in many commercial tomato varieties, leads to absence of BSEs. We examined structural and physiological traits of tomato heterobaric and homobaric (obv) near‐isogenic lines grown at two different irradiance levels. K_leaf, minor vein density, and stomatal pore area index decreased with shading in heterobaric but not in homobaric leaves, which show similarly lower values in both conditions. Homobaric plants, on the other hand, showed increased A_max, leaf intercellular air spaces, and mesophyll surface area exposed to intercellular airspace (S_mes) in comparison with heterobaric plants when both were grown in the shade. BSEs further affected carbon isotope discrimination, a proxy for long‐term water‐use efficiency. BSEs confer plasticity in traits related to leaf structure and function in response to irradiance levels and might act as a hub integrating leaf structure, photosynthetic function, and water supply and demand.     

Arabidopsis leaf hydraulic conductance is regulated by xylem sap pH,controlled, in turn,by a P-type H+-ATPase of vascular bundle sheath cells

The leaf vascular bundle sheath cells (BSCs) that tightly envelop the leaf veins, are a selective and dynamic barrier to xylem sap water and solutes radially entering the mesophyll cells. Under normal conditions, xylem sap pH below 6 is presumably important for driving and regulating the transmembranal solute transport. Having discovered recently a differentially high expression of a BSC proton pump, AHA2, we now test the hypothesis that it regulates the xylem sap pH and leaf radial water fluxes. We monitored the xylem sap pH in the veins of detached leaves of wild-type Arabidopsis, AHA mutants and aha2 mutants complemented with AHA2 gene solely in BSCs. We tested an AHA inhibitor (vanadate) and stimulator (fusicoccin), and different pH buffers. We monitored their impact on the xylem sap pH and the leaf hydraulic conductance (K_leaf), and the effect of pH on the water osmotic permeability (P_f) of isolated BSCs protoplasts. We found that AHA2 is necessary for xylem sap acidification, and in turn, for elevating K_leaf. Conversely, AHA2 knockdown, which alkalinized the xylem sap, or, buffering its pH to 7.5, reduced K_leaf, and elevating external pH to 7.5 decreased the BSCs P_f. All these showed a causative link between AHA2 activity in BSCs and leaf radial hydraulic water conductance.     

Photosynthetic Characteristics of Portulaca grandiflora, a Succulent C(4) Dicot : CELLULAR COMPARTMENTATION OF ENZYMES AND ACID METABOLISM

The succulent, cylindrical leaves of the C₄ dicot Portulaca grandiflora possess three distinct green cell types: bundle sheath cells (BSC) in radial arrangement around the vascular bundles; mesophyll cells (MC) in an outer layer adjacent to the BSC; and water storage cells (WSC) in the leaf center. Unlike typical Kranz leaf anatomy, the MC do not surround the bundle sheath tissue but occur only in the area between the bundle sheath and the epidermis. Intercellular localization of photosynthetic enzymes was characterized using protoplasts isolated enzymatically from all three green cell types.     

Photosystem II efficiency of the palisade and spongy mesophyll in <Emphasis Type="Italic">Quercus coccifera</Emphasis> using adaxial/abaxial illumination and excitation light sources with wavelengths varying in penetration into the leaf tissue

The existence of major vertical gradients within the leaf is often overlooked in studies of photosynthesis. These gradients, which involve light heterogeneity, cell composition, and CO₂ concentration across the mesophyll, can generate differences in the maximum potential PSII efficiency (F _V/F _M or F _V/F _P) of the different cell layers. Evidence is presented for a step gradient of F _V/F _P ratios across the mesophyll, from the adaxial (palisade parenchyma, optimal efficiencies) to the abaxial (spongy parenchyma, sub-optimal efficiencies) side of Quercus coccifera leaves. For this purpose, light sources with different wavelengths that penetrate more or less deep within the leaf were employed, and measurements from the adaxial and abaxial sides were performed. To our knowledge, this is the first report where a low photosynthetic performance in the abaxial side of leaves is accompanied by impaired F _V/F _P ratios. This low photosynthetic efficiency of the abaxial side could be related to the occurrence of bundle sheath extensions, which facilitates the penetration of high light intensities deep within the mesophyll. Also, leaf morphology (twisted in shape) and orientation (with a marked angle from the horizontal plane) imply direct sunlight illumination of the abaxial side. The existence of cell layers within leaves with different photosynthetic efficiencies makes appropriate the evaluation of how light penetrates within the mesophyll when using Chl fluorescence or gas exchange techniques that use different wavelengths for excitation and/or for driving photosynthesis.     

Reporter‐based forward genetic screen to identify bundle sheath anatomy mutants in A. thaliana

The evolution of C₄ photosynthesis proceeded stepwise with each small step increasing the fitness of the plant. An important pre‐condition for the introduction of a functional C₄ cycle is the photosynthetic activation of the C₃ bundle sheath by increasing its volume and organelle number. Therefore, to engineer C₄ photosynthesis into existing C₃ crops, information about genes that control the bundle sheath cell size and organelle content is needed. However, very little information is known about the genes that could be manipulated to create a more C₄–like bundle sheath. To this end, an ethylmethanesulfonate (EMS)‐based forward genetic screen was established in the Brassicaceae C₃ species Arabidopsis thaliana. To ensure a high‐throughput primary screen, the bundle sheath cells of A. thaliana were labeled using a luciferase (LUC68) or by a chloroplast‐targeted green fluorescent protein (sGFP) reporter using a bundle sheath specific promoter. The signal strengths of the reporter genes were used as a proxy to search for mutants with altered bundle sheath anatomy. Here, we show that our genetic screen predominantly identified mutants that were primarily affected in the architecture of the vascular bundle, and led to an increase in bundle sheath volume. By using a mapping‐by‐sequencing approach the genomic segments that contained mutated candidate genes were identified.     

Stem hydraulic supply is linked to leaf photosynthetic capacity: evidence from New Caledonian and Tasmanian rainforests

A strong relationship between hydraulic supply of water to leaves and maximum photosynthetic capacity was found in a group of seven conifers and 16 angiosperm species, including two vessel‐less taxa, from similar rainforest communities in New Caledonia and Tasmania (Australia). Stem hydraulic supply was expressed as the hydraulic conductivity of branches in terms of leaf area supplied (K_L) and leaf photosynthetic capacity was measured as the mean quantum yield of PSII (Ø_PSII) in leaves exposed to full sun, as determined by chlorophyll fluorescence analysis. A single, highly significant linear regression (r² = 0·74) described the relationship between hydraulic conductivity and quantum yield in all species. This suggests that the maximum photosynthetic rate of leaves is constrained by their vascular supply. In both rainforest locations, the K_L of conifer wood overlapped broadly with that of associated vessel‐bearing and vessel‐less angiosperms indicating a degree of hydraulic convergence in these forests.     

Stem and leaf hydraulic properties are finely coordinated in three tropical rain forest tree species

Coordination of stem and leaf hydraulic traits allows terrestrial plants to maintain safe water status under limited water supply. Tropical rain forests, one of the world's most productive biomes, are vulnerable to drought and potentially threatened by increased aridity due to global climate change. However, the relationship of stem and leaf traits within the plant hydraulic continuum remains understudied, particularly in tropical species. We studied within‐plant hydraulic coordination between stems and leaves in three tropical lowland rain forest tree species by analyses of hydraulic vulnerability [hydraulic methods and ultrasonic emission (UE) analysis], pressure‐volume relations and in situ pre‐dawn and midday water potentials (Ψ). We found finely coordinated stem and leaf hydraulic features, with a strategy of sacrificing leaves in favour of stems. Fifty percent of hydraulic conductivity (P₅₀) was lost at ?2.1 to ?3.1 MPa in stems and at ?1.7 to ?2.2 MPa in leaves. UE analysis corresponded to hydraulic measurements. Safety margins (leaf P₅₀ – stem P₅₀) were very narrow at ?0.4 to ?1.4 MPa. Pressure‐volume analysis and in situ Ψ indicated safe water status in stems but risk of hydraulic failure in leaves. Our study shows that stem and leaf hydraulics were finely tuned to avoid embolism formation in the xylem.     

A possible optical role of the bundle sheath extensions of the heterobaric leaves of Vitis vinifera and Quercus coccifera

Heterobaric leaves are characterized by transparent regions in their lamina, due to the occurrence of bundle sheath extensions. Fused silica fibre‐optic microprobes were used to monitor light gradients and part of the spectral regime along the bundle sheath extensions, as well as along the mesophyll in the heterobaric leaves of two representative plants, one mesomorphic (Vitis vinifera L.) and one xeromorphic (Quercus coccifera L.). It was found that the attenuation of collimated visible light by the bundle sheath extensions of both plants was weaker than the attenuation by the photosynthetic parenchyma layers. However, only a small portion of the amount of light that strikes the leaf surface is transmitted through these structures. The adaxial epidermis covering the bundle sheath extensions, as well as the mesophyll, afforded similar effective protection against UV radiation in both tissues. The relative amount of the forward‐scattered visible light inside the bundle sheath extensions approached that detected by the microprobe at the adaxial illuminated leaf surface. Moreover, light transmitted through the bundle sheath extensions was enriched mainly in the blue and red regions, compared to light transmitted through the photosynthetic tissue. The time course of photosynthetic starch formation in the leaves of V. vinifera detected by iodine staining showed that the accumulation of starch during the first minutes of illumination was high within photosynthetic parenchyma cells adjacent to the bundle sheath extensions. The data showed that bundle sheath extensions act as transparent ‘windows’ which enrich the neighbouring mesophyll areas with high levels of photosynthetically active radiation (400–700 nm). The phenomenon was more pronounced in the thick and compact sclerophyllous leaves of Q. coccifera by virtue of the greater abundance of bundle sheath extensions as compared to that in V. vinifera. The enhancement of the light micro‐environment within the deep internal layers of the mesophyll may affect the photosynthetic performance of such leaves, giving adaptive advantages.     

Photochemical Characteristics of Mesophyll and Bundle Sheath Chloroplasts from C4 Plants

Mesophyll and bundle sheath chloroplasts were isolated by differential grinding from the leaves of two NADP-ME C₄ plants, Setaria italica Beauv. cv. H-1, Pennisetum typhoides S & H. cv. AKP-2, and a NAD-ME C₄ species Amaranthus paniculatus L. The mesophyll chloroplasts of C₄ plants possessed slightly lower K_m for ADP and Pi than those of bundle sheath chloroplasts. The Hill reaction activities and noncyclic photophosphorylation rates of the bundle sheath chloropiasts from S. italica and P. typhoides were less than one-fifth of those by the mesophyll chloroplasts. But the bundle sheath chloroplasts of A. paniculatus exhibited high rates of Hill reaction, cyclic as well as noncyclic photophosphorylation. The pigment- and eyiochrome composition suggested a relative enrichment of PS 1 in bundle sheath chloroplasts of S. italica and P. typhoides. The chain exists in both mesophyll and bundle sheath chloroplasts. As much as 35–52% of leaf chlorophyll was located in the bundle sheath chloroplasts. The photochemical activities of bundle sheath chloroplasts are significant though a major part of leaf photochemical potential is associated with the mesophyll chloroplasts.     

Distribution of Photosynthetic Enzymes between Mesophyll, Specialized Parenchyma and Bundle Sheath Cells of Arundinella hirta

Arundinella hirta L. is a C₄ plant having an unusual C₄ leaf anatomy. Besides mesophyll and bundle sheath cells, A. hirta leaves have specialized parenchyma cells which look morphologically like bundle sheath cells but which lack vascular connections and are located between veins, running parallel to them. Activities of phosphoenolpyruvate and ribulose-1,5-bisphosphate carboxylases and phosphoenolpyruvate carboxykinase, NADP-and NAD-malic enzymes were determined for whole leaf extracts and isolated mesophyll protoplasts, specialized parenchyma cells, and bundle sheath cells. The data indicate that A. hirta is a NADP-malic enzyme type C₄ species. In addition, specialized parenchyma cells and bundle sheath cells are enzymatically alike. Compartmentation of enzymes followed the C₄ pattern with phosphoenolpyruvate carboxylase being restricted to mesophyll cells while ribulose-1,5-bisphosphate carboxylase and decarboxylating enzymes were restricted to bundle sheath and specialized parenchyma cells.     

Anatomy, chloroplast structure and compartmentation of enzymes relative to photosynthetic mechanisms in leaves and cotyledons of species in the tribe Salsoleae (Chenopodiaceae)

Certain members of the family Chenopodiaceae are the dominant species of the deserts of Central Asia; many of them are succulent halophytes which exhibit C4-type CO2 fixation of the NAD- or NADP-ME (malic enzyme) subgroup. In four C4 species of the tribe Salsoleae, the Salsoloid-type Kranz anatomy in leaves or stems was studied in relation to the diversity in anatomy which was found in cotyledons. Halocharis gossypina, has C4 NAD-ME Salsoloid-type photosynthesis in leaves and C3 photosynthesis in dorsoventral non-Kranz cotyledons; Salsola laricina has C4 NAD-ME Salsoloid-type leaves and C4 NAD-ME Atriplicoid-type cotyledons; Haloxylon persicum, has C4 NADP-ME Salsoloid-type green stems and C3 isopalisade non-Kranz cotyledons; and S. richteri has C4 NADP-ME Salsoloid-type leaves and cotyledons. Immunolocalization studies on Rubisco showed strong labelling in bundle sheath cells of leaves and cotyledons of organs having Kranz anatomy. The C4 pathway enzyme phosphoenolpyruvate carboxylase was localized in mesophyll cells, while the malic enzymes were localized in bundle sheath cells of Kranz-type tissue. Immunolocalization by electron microscopy showed NAD-ME is in mitochondria while NADP-ME is in chloroplasts of bundle sheath cells in the respective C4 types. In some C4 organs, it was apparent that subepidermal cells and water storage cells also contain some chloroplasts which have Rubisco, store starch, and thus perform C3 photosynthesis. In non-Kranz cotyledons of Halocharis gossypina and Haloxylon persicum, Rubisco was found in chloroplasts of both palisade and spongy mesophyll cells with the heaviest labelling in the layers of palisade cells, whereas C4 pathway proteins were low or undetectable. The pattern of starch accumulation correlated with the localization of Rubisco, being highest in the bundle sheath cells and lowest in the mesophyll cells of organs having Kranz anatomy. In NAD-ME-type Kranz organs (leaves and cotyledons of S. laricina and leaves of H. gossypina the granal index (length of appressed membranes as a percentage of total length of all membranes) of bundle sheath chloroplasts is 1.5 to 2.5 times higher than that of mesophyll chloroplasts. In contrast, in the NADP-ME-type Kranz organs (S. richteri leaves and cotyledons and H. persicum stems) the granal index of mesophyll chloroplasts is 1.5 to 2.2 times that of the bundle sheath chloroplasts. The mechanism of photosynthesis in these species is discussed in relation to structural differences.