Leaf blade structure and C4 acid decarboxylation enzymes in xCynochloris spp. (Poaceae), intergeneric hybrids between species of different C4 type

PRENDERGAST, H. D. V., STONE, N. E. & HATTERSLEY, P. W., 1988. Leaf blade structure and C₄ acid decarboxylation enzymes in x Cynochloris spp. (Poaceae), intergeneric hybrids between species of different C₄ type. x Cynochloris macivorii Clifford and Everist and x C. reynoldensis B. K. Simon (Poaceae) are intergeneric C₄ hybrids between Cynodon dactylon (L.) Pers., and NAD-malic enzyme (NAD-ME) species, and two different PEP carboxykinase (PCK) species of Chloris . Parental species of each hybrid species have the 'classical' leaf blade structure of their respective C₄ acid decarboxylation types. The outline of the photosynthetic carbon reduction (PCR or Kranz) bundle sheath and the position of the PCR cell chloroplasts in x Cynochloris are intermediate between those of the parental species, C. macivorii being more like Cynodon dactylon and x C. reynoldensis more like Chloris spp. The PCR chloroplast shape in x C. macivorii and x C. reynoldensis is like that of Cynodon dactylon and Chloris spp., respectively. Differences between the hybrids in their enzyme activities complement these structural differences: x C. macivorii has more NAD—ME and less PCK activity than x C. reynoldensis , although in both species PCK activity is the greater. Both hybrids, however, have a suberized lamella in PCR cell walls as do Chloris spp. The close taxonomic relationship between Cynodon and Chloris make these genera especially suitable for reciprocal crossing experiments aimed at increasing understanding of the genetic relationships between subtypes of the C₄ photosynthetic pathway.     

Significant involvement of PEP-CK in carbon assimilation of C4 eudicots

Background and Aims

C₄ eudicot species are classified into biochemical sub-types of C₄ photosynthesis based on the principal decarboxylating enzyme. Two sub-types are known, NADP-malic enzyme (ME) and NAD-ME; however, evidence for the occurrence or involvement of the third sub-type (phosphoenolpyruvate carboxykinase; PEP-CK) is emerging. In this study, the presence and activity of PEP-CK in C₄ eudicot species of Trianthema and Zaleya (Sesuvioideae, Aizoaceae) is clarified through analysis of key anatomical features and C₄ photosynthetic enzymes.

Methods

Three C₄ species (T. portulacastrum, T. sheilae and Z. pentandra) were examined with light and transmission electron microscopy for leaf structural properties. Activities and immunolocalizations of C₄ enzymes were measured for biochemical characteristics.

Key Results

Leaves of each species possess atriplicoid-type Kranz anatomy, but differ in ultrastructural features. Bundle sheath organelles are centripetal in T. portulacastrum and Z. pentandra, and centrifugal in T. sheilae. Bundle sheath chloroplasts in T. portulacastrum are almost agranal, whereas mesophyll counterparts have grana. Both T. sheilae and Z. pentandra are similar, where bundle sheath chloroplasts contain well-developed grana while mesophyll chloroplasts are grana deficient. Cell wall thickness is significantly greater in T. sheilae than in the other species. Biochemically, T. portulacastrum is NADP-ME, while T. sheilae and Z. pentandra are NAD-ME. Both T. portulacastrum and Z. pentandra exhibit considerable PEP-CK activity, and immunolocalization studies show dense and specific compartmentation of PEP-CK in these species, consistent with high PEP-CK enzyme activity.

Conclusions

Involvement of PEP-CK in C₄ NADP-ME T. portulacastrum and NAD-ME Z. petandra occurs irrespective of biochemical sub-type, or the position of bundle sheath chloroplasts. Ultrastructural traits, including numbers of bundle sheath peroxisomes and mesophyll chloroplasts, and degree of grana development in bundle sheath chloroplasts, coincide more directly with PEP-CK recruitment. Discovery of high PEP-CK activity in C₄ Sesuvioideae species offers a unique opportunity for evaluating PEP-CK expression and suggests the possibility that PEP-CK recruitment may exist elsewhere in C₄ eudicots.     

Assessing photosystem I and II distribution in leaves from C4 plants using confocal laser scanning microscopy

Images of chlorophyll fluorescence emitted at wavelengths above and below 700 nm were recorded from leaf sections of C₄ species using confocal laser scanning microscopy (LSM). We investigated species exhibiting both NAD-malic enzyme (NAD-ME) C₄ photosynthesis and NADP-malic enzyme (NADP-ME) C₄ photosynthesis. Comparing LSM fluorescence of leaf sections with flow-cytometrically determined fluorescence from individual chloroplasts revealed that LSM fluorescence was distorted by the optical properties of leaf sections. Leaf section fluorescence, when corrected by transmission data derived from light transmission images, agreed with flow cytometry data. The corrected LSM fluorescence yielded information on the distribution of the individual photosystems in the C₄ leaf sections: PSII concentrations in bundle sheath cells were elevated in NAD-ME species but diminished in most of the NADP-ME species investigated. The NADP-ME species, Arundinella hirta, however, showed normal PSII and increased PSI concentration in bundle sheath chloroplasts. Finally, a gradient of PSI was observed within the bundle sheath cells from Euphorbia maculata.     

Structural and biochemical bases of photorespiration in C<Subscript>4</Subscript> plants: quantification of organelles and glycine decarboxylase

In C₄ plants, photorespiration is decreased relative to C₃ plants. However, it remains unclear how much photorespiratory capacity C₄ leaf tissues actually have. We thoroughly investigated the quantitative distribution of photorespiratory organelles and the immunogold localization of the P protein of glycine decarboxylase (GDC) in mesophyll (M) and bundle sheath (BS) cells of various C₄ grass species. Specific differences occurred in the proportions of mitochondria and peroxisomes in the BS cells (relative to the M cells) in photosynthetic tissues surrounding a vein: lower in the NADP-malic enzyme (NADP-ME) species having poorly formed grana in the BS chloroplasts, and higher in the NAD-malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PCK) species having well developed grana. In all C₄ species, GDC was localized mainly in the BS mitochondria. When the total amounts of GDC in the BS mitochondria per unit leaf width were estimated from the immunogold labeling density and the quantity of mitochondria, the BSs of NADP-ME species contained less GDC than those of NAD-ME or PCK species. This trend was also verified by immunoblot analysis of leaf soluble protein. There was a high positive correlation between the degree of granal development (granal index) in the BS chloroplasts and the total amount of GDC in the BS mitochondria. The variations in the structural and biochemical features involved in photorespiration found among C₄ species might reflect differences in the O₂/CO₂ partial pressure and in the potential photorespiratory capacity of the BS cells.Abbreviations BS Bundle sheath - GDC Glycine decarboxylase - M Mesophyll - NAD-ME NAD-malic enzyme - NADP-ME NADP-malic enzyme - PCK Phosphoenolpyruvate carboxykinase     

Characterization of C4 Type Leaf Anatomy in Grasses (Poaceae). Mesophyll: Bundle Sheath Area Ratios

The cross-sectional area of ‘primary carbon assimilation’(PCA) (or mesophyll) tissue and of ‘photosynthetic carbonreduction’ (PCR) (or parenchymatous bundle sheath, PBS)tissue associated with each vein has been measured in transversesections of leaf blades of 124 grass species (Poaceae). Thespecies sample is representative of all major grass taxa, andof all photosynthetic types found in this family, viz. C₃, C₃/C₄intermediate, C₄ NADP-malic enzyme type (NADP-ME), C₄ NAD-malicenzyme type (NAD-ME) and PEP carboxykinase type (PCK). MeanPCA (or mesophyll) area per vein varies between photosynthetictypes in the order C₃ > NAD-ME > PCK = NADP-ME, mean PCR(or PBS) area per vein in the order NAD-ME > PCK = C₃ >NADP-ME, and mean PCA/PCR (or mesophyll/PBS) area ratio in theorder C₃ > NADP-ME > NAD-ME > PCK. Since grass leaveshave parallel venation, tissue areas and area ratios are directlyproportional to tissue volumes and volume ratios. Regressionanalyses of plots of PCA (or mesophyll) area per vein againstPCR (or PBS) area per vein yield characteristic slopes for photosynthetictypes. Differences between types in all these parameters arenearly always statistically significant, even within high leveltaxonomic groups (Eupanicoids and Chloridoids). However, differencesbetween major taxa (Eupanicoids, Andropogonoids, Chloridoids),within a photosynthetic type, are frequently not significant.This histometric characterization of photosynthetic types isdiscussed in relation to the co-operation of PCA and PCR tissuesin C₄ photosynthesis, to possible differences between C₄ typesin PCR spatial requirements and to the developmental originof PCR tissue. Grasses, Poaceae, C₄ photosynthesis, C₄ leaf blade anatomy, ‘Kranz’, NADP-malic enzyme, NAD-malic enzyme, PEP carboxykinase, PCA tissue, PCR tissue, taxonomy     

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.     

DIFFERING ONTOGENETIC ORIGINS OF PCR (“KRANZ”) SHEATHS IN LEAF BLADES OF C4 GRASSES (POACEAE)

The origin and early development of procambium and associated ground meristem of major and minor veins have been examined in the leaf blades of seven C₄ grass species, representing different taxonomic groups and the three recognized biochemical C₄ types (NAD-ME, PCK, and NADP-ME). Comparisons were made with the C₃ species, Festuca arundinacea. In “double sheath” (XyMS+) species (Panicum effusum, Eleusine coracana, and Sporoboìus elongatus), the procambium of major veins gives rise to xylem, phloem, and a mestome sheath; associated ground meristem differentiates into PCA (“C₄ mesophyll”) tissue and the PCR (“Kranz”) sheath. Development in the C₃ species parallels this pattern, except that associated ground meristem differentiates into mesophyll and a parenchymatous bundle sheath. In contrast, major vein procambium of “single sheath” (XyMS–) species (Panicum bulbosum, Digitaria brownii, and Cymbopogon procerus) differentiates into xylem, phloem and a PCR sheath; associated ground meristem gives rise to PCA tissue. These observations of major vein development support W. V. Brown's hypothesis that the PCR sheaths of “double sheath” (XyMS+) C₄ grasses are homologous with the parenchymatous bundle sheaths of C₃ grasses, while in “single sheath” (XyMS–) C₄ species they are homologous with the mestome sheath. Although there are some similarities in the development of the major and minor vascular bundle procambium in the C₄ species examined, the ontogeny of the smaller minor veins is characterized by a precocious delineation of the PCR sheath layer that may even precede the appearance of the distinctive cytological features of ground meristem and procambium. This contracted development in minor veins appears to be related to their close spacing in mature leaves and to their comparatively late appearance during leaf ontogeny.     

Evidence for a C4 NADP-ME photosynthetic pathway in Vetiveria zizanioides Stapf

ABSTRACT

Leaf anatomy (light and transmission electron microscopy), immunogold localization of Rubisco, photosynthetic enzyme activities, CO₂ assimilation and stomatal conductance were studied in Vetiveria zizanioides Stapf., a graminaceous plant native to tropical and subtropical areas, and cultivated in temperate climates (Northwestern Italy). Leaves possess a NADP-ME Kranz anatomy with bundle sheath cells containing chloroplasts located in a centrifugal position. Dimorphic chloroplasts were also observed; they are agranal and starchy in the bundle sheath and granal starchless in the mesophyll cells. Rubisco immunolocalization studies indicate that this enzyme occurs solely in the bundle sheath chloroplasts. Pyruvate-orthophosphate dikinase, NADP-dependent malate dehydrogenase (NADP-MDH), NADP-dependent malic enzyme (NADP-ME), PEP-carboxykinase and NAD-dependent malic enzyme (NAD-ME) activities were determined. Enzyme activity and some kinetic properties of NADP-ME and NADP-MDH as well as CO₂ compensation point and stomatal conductance values were calculated indicating a NADP-ME C₄ photosynthetic pathway. Biochemical and structural results indicate that V. zizanioides belongs to the C₄ NADP-ME variant. This plant appears to be well adapted to the varying environmental conditions typical of temperate climates, by retaining high enzyme activities and a low CO₂ compensation point.     

Variations in the Leaf Anatomy among some C4 Panicum Species

The Dichotomiflora group of Panicum contains NAD-malic enzyme(ME) species with centrifugal chloroplasts in Kranz cells, NAD-ME(F)species as well as NAD-ME species with centripetal chloroplastsin Kranz cells, NAD-ME (P) species. Many attributes of leafanatomy of 22 C₄ Panicum species were investigated to identifydifferences among four different C₄ subtypes, i.e. NADP-ME,NAD-ME(F), NAD-ME(P) and PEP-CK species grouped by the C₄-aciddecarboxylating enzymes and chloroplast location in Kranz cellsin combination. Differences were found in the number of Kranzcells surrounding a large vein, and the number surrounding asmall vein, the interveinal distances, the proportion of leafcross sectional area occupied by epidermis plus sclerenchyma,by mesophyll cells, by Kranz cells, and by vascular bundles.There were also differences in the ratios of the area of thedifferent cell types. The number of the characters significantlydifferent between a respective pair of C₄ subtypes was the largestbetween NAD-ME(F) and NAD-ME(P) species. In principal componentanalysis applied to 11 leaf anatomical characters, the differentC₄ subtypes clustered into small groups, although the rangeof variations of PEP-CK species and those of NAD-ME(F) speciesoverlapped. The results were discussed in relation to taxonomyand ecological adaptation of Panicum species in the differentC₄ subtypes. C₄ photosynthesis, NADP-malic enzyme, NAD-malic enzyme, Phosphoenolpyruvate carboxykinase, C₄ leaf anatomy, Panicum, Kranz, Dichotomiflora group     

Immunogold localization of photosynthetic enzymes in leaves of various C4 plants, with particular reference to pyruvate orthophosphate dikinase

Cellular localization of photosynthetic enzymes was investigated by immunogold electron microscopy for leaves of nine C4 grasses (three NADP-malic enzyme (NADP-ME)subtype species, three NAD-malic enzyme (NAD-ME) subtype species, and three phosphoenolpyruvate carboxykinase (PCK) subtype species), two C4 sedges (NADP-ME subtype species) and two C4 dicots (an NADP-ME and an NADP/NAD-ME subtype species). In leaves of all species, immunogold labelling was present for phosphoenolpyruvate carboxylase in the cytosol of the mesophyll cells (MC) and for ribulose-1,5-bisphosphate carboxylase/oxygenase in the chloroplasts of the bundle sheath cells (BSC). However, considerable specific variation was found in the intercellular patterns of labelling for pyruvate orthophosphate dikinase (PPDK). In the NADP-ME grasses, two NAD-ME grasses, and the dicots, significant labelling for PPDK was present in the both the BSC and the MC chloroplasts. In the other NAD-ME grass, the PCK grasses, and the sedges, labelling for PPDK was present almost exclusively in the chloroplasts of the MC. These patterns were observed in the leaves of both young seedlings and mature plants. These results indicate that the accumulation of PPDK in leaves of C4 plants is not necessarily restricted to the MC, although the chloroplasts of the MC accumulate more than those of the BSC.Key words: C4 plants, immunolocalization, phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, ribulose-1,5-bisphosphate carboxylase/oxygenase.      

Light Modulation and Localization of Sucrose Phosphate Synthase Activity between Mesophyll Cells and Bundle Sheath Cells in C(4) Species

Experiments were conducted with several Panicum species (representing the different C₄ subtypes) to examine the light modulation of sucrose phosphate synthase (SPS) activity and the effect of illumination on the distribution of SPS activity between mesophyll cells (MC) and bundle sheath cells (BSC). Activity of SPS in the light decreased in the order: C₄ > C₃-C₄ intermediate > C₃. In illuminated leaves, SPS activities were similar among the three C₄ subtypes, but SPS activity was higher for NAD-malic enzyme (NAD-ME) species with centripetal chloroplasts in BSC (NAD-ME(P) species) than for NAD-ME species with centrifugal chloroplasts in BSC (NAD-ME(F) species). Transfer of plants into darkness for 30 minutes resulted in decreased SPS activity for all species tested except Panicum bisulcatum (C₃ species) and Panicum virgatum (NAD-ME(P) species) which showed little or no change. All C₄ subtypes had some SPS activity both in MC and BSC. In the light, SPS activity was mainly in the MC for NADP-ME, NAD-ME(F) and phosphoenolpyruvate carboxykinase species, while it was mainly in the BSC for NAD-ME(P) species. In the dark, for all C₄ subtypes, SPS activity in the MC was decreased to a greater extent than that in the BSC. It is intriguing that NAD-ME(F) and NAD-ME(P) species differed in the activity and distribution of SPS activity between MC and BSC, although they are otherwise identical in the photosynthetic carbon assimilation pathway. Diurnal changes in SPS activity in the MC and BSC were also examined in maize leaves. SPS activity in the MC in maize leaves was high and relatively constant throughout the middle of the light period, dropped rapidly after sunset and increased again prior to the light period. On the other hand, SPS activity in the BSC was lower and changed more coincidently with light intensity than that in the MC. The results suggested that light activation of SPS activity located in the BSC may require higher irradiance for saturation than the SPS in the MC. We conclude that SPS may function in both MC and BSC for sucrose synthesis in the light, particularly at high light intensity, while in the dark, the major function may be in the BSC during starch degradation.     

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.     

The occurrence of C4 species in the genusRhynchospora and its significance in kranz anatomy of the cyperaceae

Rhynchospora rubra was found to have a low CO₂ compensation point, high δ¹³C value, Kranz leaf anatomy, starch present in the bundle sheath cells and narrow interveinal distance. These observations suggest thatR. rubra is a C₄ plant. A further anatomical survey revealed seven otherRhynchospora species presumably having the C₄ photosynthetic pathway. In the family Cypraceae C₄ plants therefore occur in the tribe Rhynchosporeae as well as in the Scirpeae and Cypereae. The C₄ species ofRhynchospora have a normal Kranz type of leaf anatomy, although the C₄ species ofCyperus andFimbristylis presently known have an abnormal one in which the mestome sheath without chloroplasts is interposed between the Kranz tissue and the rest of the chlorenchyma. Thus inRhynchospora the Kranz tissue is in direct contact with the rest of the chlorenchyma, and it is suggested that the Kranz tissue may be homologous with the mestome sheath.     

Km (CO2) values of ribulose-1,5-bisphosphate carboxylase in grasses of different C4 type

Statistical analysis of K_m (CO₂) values of ribulose-1,5-bisphosphate (RuBP) carboxylase from 35 C₄ grass species shows that the mean value for PEP-carboxykinase (PCK) type C₄ species (41.4±s.e. 2.2 μM CO₂) is significantly different from that of NAD-malic enzyme (NAD-ME) type species (55.3±3.1 μM CO₂) or NADP-malic enzyme (NADP-ME type species (52.5±s.e. 2.0μM CO₂). These C₄ type differences remain detectable within both the eu-panicoid and chloridoid grass subfamilies. By contrast, no between-subfamily differences were found within C₄ types. Variation in K_m (CO₂) values of RuBP carboxylase may be related to in vivo differences in CO₂ concentration at the enzyme site, mediated perhaps by differences in CO₂-leakiness of C₄ leaf ‘photosynthetic carbon reduction’ (PCR or ‘Kranz’) tissue.     

Growth, gas exchange, leaf nitrogen and carbohydrate concentrations in NAD‐ME and NADP‐ME C4 grasses grown in elevated CO2

Plants with the C₄ photosynthetic pathway have predominantly one of three decarboxylation enzymes in their bundle sheath cells. Within the grass family (Poaceae) bundle sheath leakiness to CO₂ is purported to be lowest in the nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME, EC 1.1.1.40) group, highest in the NAD-ME (EC 1.1.1.39) group and intermediate in the phosphoenolpyruvate carboxykinase (PCK, EC 4.1.1.32) group. We investigated the hypothesis that growth and photosynthesis of NAD-ME C₄ grasses would respond more to elevated CO₂ treatment than NADP-ME grasses. Plants were grown in 8-1 pots in growth chambers with ample water and fertilizer for 39 days at a continuous CO₂ concentration of either 350 or 700 µl l^?1. NAD-ME species included Bouteloua gracilis Lag. ex Steud (Blue grama), Buchloe dactyloides (Nutt.) Engelm. (Buffalo grass) and Panicum virgatum L. (Switchgrass) and the NADP-ME species were Andropogon gerardii Vittman (Big bluestem), Schizachyrium scoparium (Michx.) Nash (Little bluestem), and Sorghastrum nutans (L.) Nash (Indian grass). Contrary to our hypothesis, growth of the NADP-ME grasses was generally greater under elevated CO₂ (significant for A. gerardii and S. nutans), while none of the NAD-ME grasses had a significant growth response. Increased leaf total non-structural carbohydrate (TNC) was associated with greater growth responses of NADP-ME grasses. Decreased leaf nitrogen in NADP-ME species grown at elevated CO₂ was found to be an artifact of TNC dilution. Assimilation (A) vs intercellular CO₂ (C_i) curves revealed that leaf photosynthesis was not saturated at 350 µl l^?1 CO₂ in any of these C₄ grasses. Assimilation of elevated CO₂-grown A. gerardii was higher than in plants grown in ambient CO₂. In contrast, B. gracilis grown in elevated CO₂ displayed lower A, a trait more commonly reported in C₃ plants. Photosynthetic acclimation in B. gracilis was not related to leaf TNC or nitrogen concentrations, but A:C_i curves suggest a reduction in activity of both phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39). Some adaptation of stomatal functioning was also seen in B. gracilis and A. gerardii leaves grown in elevated CO₂. Our study shows that C₄ grasses have the capacity for increased growth and photosynthesis under elevated CO₂ even when water and nutrients are non-limiting. While it was the NADP-ME species which had significant responses in the present study, we have previously reported significant growth increases in elevated CO₂ for B. gracilis.     

Carbon isotope composition of C4 grasses is influenced by light and water supply

The carbon isotope composition of C⁴ grasses has the potential to be used as an indicator of changes in the isotopic composition and concentration of atmospheric CO2, especially for climate reconstruction. The usefulness of C₄ grasses for this purpose hinges on the assumption that their photosynthetic discrimination against ¹³C remains constant in a wide range of environmental conditions. We tested this assumption by examining the effects of light and water stress on the carbon isotope composition of C₄ grasses using different biochemical subtypes (NADP-ME, NAD-ME, PCK) in glasshouse experiments. We grew 14 different C₄ grass species in four treatments: sun-watered, sun-drought, shade-watered and shade-drought. Carbon isotope discrimination (Δ) rarely remained constant. In general, Δ values were lowest in sun-watered grasses, greater for sun-drought plants and even higher for plants of the shade-watered treatment. The highest Δ values were generally found in the most stressed grasses, the shade-drought plants. Grasses of the NADP-ME subtype were the least influenced by a change in environmental variables, followed by PCK and NAD-ME subtypes. Water availability affected the carbon isotope discrimination less than light limitation in PCK and NAD-ME subtypes, but similarly in NADP-ME subtypes. In another experiment, we studied the effect of increasing light levels (150 to 1500 μmol photons m^?2 s^?1) on the Δ values of 18 well-watered C₄ grass species. Carbon isotope discrimination remained constant until photon flux density (PFD) was less than 700 μmol photons m^?2 s^?1. Below this light level, Δ values increased with decreasing irradiance for all biochemical subtypes. The change in A was less pronounced in NADP-ME and PCK than in NAD-ME grasses. Grasses grown in the field and in the glasshouse showed a similar pattern. Thus, caution should be exercised when using C4 plants under varying environmental conditions to monitor the concentration or carbon isotopic composition of atmospheric CO2 in field/glasshouse studies or climate reconstruction.     

Comparative proteomic analysis of amaranth mesophyll and bundle sheath chloroplasts and their adaptation to salt stress

The effect of salt stress was analyzed in chloroplasts of Amaranthus cruentus var. Amaranteca, a plant NAD-malic enzyme (NAD-ME) type. Morphology of chloroplasts from bundle sheath (BSC) and mesophyll (MC) was observed by transmission electron microscopy (TEM). BSC and MC from control plants showed similar morphology, however under stress, changes in BSC were observed. The presence of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) was confirmed by immunohistochemical staining in both types of chloroplasts. Proteomic profiles of thylakoid protein complexes from BSC and MC, and their changes induced by salt stress were analyzed by blue-native polyacrylamide gel electrophoresis followed by SDS-PAGE (2-D BN/SDS-PAGE). Differentially accumulated protein spots were analyzed by LC–MS/MS. Although A. cruentus photosynthetic tissue showed the Kranz anatomy, the thylakoid proteins showed some differences at photosystem structure level. Our results suggest that A. cruentus var. Amaranteca could be better classified as a C₃–C₄ photosynthetic plant.     

Photosynthesis of Grass Species Differing in Carbon Dioxide Fixation Pathways : VIII. Ultrastructural Characteristics of Panicum Species in the Laxa Group

Ultrastructural studies of leaves of seven Panicum species in or closely related to the Laxa group and classified as C₃, C₄ or C₃-C₄ intermediate were undertaken to examine features associated with C₃ and C₄ photosynthesis. The C₃ species Panicum rivulare Trin. had few organelles in bundle sheath cell profiles (2 chloroplasts, 1.1 mitochondria, and 0.3 peroxisomes per cell section) compared to an average of 10.6 chloroplasts, 17.7 mitochondria, and 3.2 peroxisomes per bundle sheath cell profile for three C₃-C₄ species, Panicum milioides Nees ex Trin., Panicum decipiens Nees ex Trin. and Panicum schenckii Hack. However, two other C₃ species, Panicum laxum Sw. and Panicum hylaeicum Mez, contained about 0.7, 0.5, and 0.3 as many chloroplasts, mitochondria, and peroxisomes, respectively, as in bundle sheath cell profiles of the C₃-C₄ species. Chloroplasts and mitochondria in bundle sheath cells were larger than those in mesophyll cells for the C₄ species Panicum prionitis Griseb. and the C₃-C₄ species, but in C₃ species the organelles were similar in size or were smaller in the bundle sheath cells. The C₃-C₄ species and P. laxum and P. hylaeicum exhibited an unusually close association of organelles in bundle sheath cells with mitochondria frequently surrounded in profile by chloroplasts. The high concentrations in bundle sheath cells of somewhat larger organelles than in mesophyll cells correlates with the reduced photorespiration of the C₃-C₄ species.     

The C4 pathway: an efficient CO2 pump

The C₄ pathway is a complex combination of both biochemical and morphological specialisation, which provides an elevation of the CO₂ concentration at the site of Rubisco. We review the key parameters necessary to make the C₄ pathway function efficiently, focussing on the diffusion of CO₂ out of the bundle sheath compartment. Measurements of cell wall thickness show that the thickness of bundle sheath cell walls in C₄ species is similar to cell wall thickness of C₃ mesophyll cells. Furthermore, NAD-ME type C₄ species, which do not have suberin in their bundle sheath cell walls, do not appear to compensate for this with thicker bundle sheath cell walls. Uncertainties in the CO₂ diffusion properties of membranes, such as the plasmalemma, choroplast and mitochondrial membranes make it difficult to estimate bundle sheath diffusion resistance from anatomical measurements, but the cytosol itself may account for more than half of the final calculated resistance value for CO₂ leakage. We conclude that the location of the site of decarboxylation, its distance from the mesophyll interface and the physical arrangement of chloroplasts and mitochondria in the bundle sheath cell are as important to the efficiency of the process as the properties of the bundle sheath cell wall. Using a mathemathical model of C₄ photosynthesis, we also examine the relationship between bundle sheath resistance to CO₂ diffusion and the biochemical capacity of the C₄ photosynthetic pathway and conclude that bundle sheath resistance to CO₂ diffusion must vary with biochemical capacity if the efficiency of the C₄ pump is to be maintained. Finally, we construct a mathematical model of single cell C₄ photosynthesis in a C₃ mesophyll cell and examine the theoretical efficiency of such a C₄ photosynthetic CO₂ pump. This revised version was published online in August 2006 with corrections to the Cover Date.     

Suberized bundle sheaths in grasses (Poaceae) of different photosynthetic types. II. Apoplastic permeability

Summary The relative hydraulic conductivities of major and minor longitudinal veins, and the apoplastic permeability of the bundle sheaths surrounding all longitudinal and transverse veins were investigated in representatives of the C₃, C₄/NAD-ME, C₄/NAD-ME/PCK intermediate, C₄/PCK and C₄/NADP-ME photosynthetic types. Using the Hagen-Poiseuille equation and measurements of tracheary element diameters, the number of elements in each vein type and the numbers of each vein type, we calculated that 87–99% of the water flow in a longitudinal direction would be expected to occur in the major veins. The permeability of the mestome sheaths and parenchymatous bundle sheaths surrounding the veins was tested using the negatively-charged, fluorescent dye, trisodium 3-hydroxy-5,8,10-pyrenetrisulfonate (PTS). This dye proved nontoxic to plant tissue at a concentration of 0.5%, according to a deplasmolysis test with onion epidermal strips. The PTS concentration achieved in the tested grass leaves was about 0.035%, well below the toxic limit. When a solution of PTS was fed to the leaves by means of a basal cut, the dye moved into the veins of all orders. From there, it moved outward into the surrounding tissues, indicating that the sheaths surrounding the veins of all orders in all species tested were permeable. Therefore, contrary to previous predictions based on structural observations and some tracer studies, bundle sheaths with suberized cell walls do not function as endodermal layers.