Use of inhibitors to distinguish between C4 acid decarboxylation mechanisms in bundle sheath cells of C4 plants

Both malate and aspartate were decarboxylated at the 4-carbonposition by isolated bundle sheath strands of C₄ plants butto different extents depending upon the species. In Digitariasanguinalis, an NADP-malic enzyme (NADP-ME) species, 100 µMoxalic acid blocked malate decarboxylation through NADP-ME withoutaffecting aspartate decarboxylation which apparently occursthrough NAD-ME. In several phosphoenolpyruvate carboxykinase(PEP-CK) type C₄ species, 200 µM 3-mercaptopicolinic acid(3-MPA), an inhibitor of PEP-CK, specifically inhibited themalate decarboxylation and partially inhibited aspartate decarboxylation.The aspartate decarboxylation insensitive to 3-MPA may occurthrough NAD-ME. Neither inhibitor prevented C₄ acid decarboxylationin bundle sheath cells of NAD-ME species. The inhibitors thusserved to differentiate between the decarboxylation of C₄ acidsin PEP-CK and NADP-ME type C₄ species through their major decarboxylasefrom that of their less active decarboxylation through NAD-ME. ¹ Present address: Department of Biochemistry and Microbiology,Rutgers University, New Brunswick, NJ 08903, U. S. A. (Received January 28, 1977; )     

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.     

Leaf Anatomy and Carbon Discrimination in NAD-malic Enzyme Panicum Species and their Hybrids Differing in Bundle Sheath Cell Ultrastructure

The relationship between leaf anatomy, ultrastructure and carbondiscrimination was investigated in leaves of two F₁hybrids (F₁-1and F₁-2) between two different types of the grassPanicum [anNAD-malic enzyme (ME) C₄species], which differ in bundle sheathultrastructure. The female parent was Kabulabula grass, whichhas centrifugal chloroplasts in bundle sheath cells and is designatedan NAD-ME(F) species, while the male parent was Makarikari grass,which has centripetal chloroplasts in the bundle sheath cellsand is designated an NAD-ME(P) species. Suberin lamellae arepresent in Kabulabula grass but are lacking in Makarikari grass.Both F₁hybrids had the same chromosome number (2n =36) as theparents but exhibited both univalent (about 45%) and bivalent(about 55%) chromosome pairing which was the major basis forthe identification of F₁hybrids. In F₁-1, elongated bundle sheathcell chloroplasts are arranged mainly in a centripetal position,similar to those in the male parent, Makarikari grass. In contrast,most of the bundle sheath cells in F₁-2 are packed with starch-containingchloroplasts, although in some cells chloroplasts tended tobe centripetally arranged. In both F₁hybrids, suberin lamellaewere found in the bundle sheath cell walls, similar to the femaleparent, Kabulabula grass. The ¹³C values of both F₁hybrids were-11.4 to -11.7, almost the same as those of Kabulabula grass(-11.4), but significantly higher than those of Makarikari grass(-12.7). These results indicate that the chloroplast orientationin the bundle sheath cells and the presence of suberin lamellaeare not obligatorily linked in their expression and suggestthat suberin lamellae may play an important role in discriminationagainst¹³C. Panicum ; NAD-malic enzyme species; hybrid; chloroplast position; ¹³C discrimination; suberin lamellae     

Maximum Quantum Yields of O2 Evolution in C4 Plants under High CO2

The quantum yields of photosynthetic O₂ evolution were measuredin 15 species of C₄ plants belonging to three different decarboxylationtypes (NADP-ME type, NAD-ME type and PEP-CK type) and 5 speciesof C₃ plants and evaluated relative to the maximum theoreticalvalue of 0.125 mol oxygen quanta^-1. At 25°C and 1% CO₂,the quantum yield in C₄ plants averaged 0.079 (differences betweensubgroups not significant) which was significantly lower thanthe quantum yield in C₃ plants (average of 0.105 for 5 species).This lower quantum yield in C₄ plants is thought to reflectthe requirement of energy in the C₄ cycle. For the C₄ NADP-MEtype plant Z. mays and NAD-ME type plant P. miliaceum, quantumyields were also measured over a range of CO₂ levels between1 and 20%. In both species maximum quantum yields were obtainedunder 10% CO₂ (0.105 O₂ quanta_-1 in Z. mays and 0.097 O₂ quanta_-1in P. miliaceum) indicating that at this CO₂ concentration thequantum yields are similar to those obtained in C₃ plants underCO₂ saturation. The high quantum yield values in C₄ plants undervery high CO₂ may be accomplished by direct diffusion of atmosphericCO₂ to bundle sheath cells, its fixation in the C₃ pathway,and feedback inhibition of the C₄ cycle by inorganic carbon. (Received June 6, 1995; Accepted August 15, 1995)     

C4 syndrome of the species in theDichotomiflora group of the genusPanicum (Gramineae)

Leaf anatomy, pattern of post-illumination CO₂ burst (PIB) and activity of three C₄-acid decarboxylating enzymes in C₄ photosynthesis were investigated with the leaves of five species in theDichotomiflora group of the genusPanicum. All species had mestome sheaths, exhibited the sharp pattern of PIB in less than 30 sec of darkness and were classified as NAD-malie enzyme species biochemically. However, they clearly fell into two groups according to the difference in chloroplast location in bundle sheath cells (BSC).P. coloratum var.makarikariense, P. lanipes andP. stapfianum had centripetal chloroplasts, whereasP. laevifolium andP. longijubatum had centrifugal chloroplasts, whereas cv. Kabulabula and cv. Solai had centrifugal chlorplasts. The results indicate that theDichotomiflora group had the two leaf anatomical variations of NAD-malic enzyme species. In addition, the results onP. coloratum suggest that this species may be divided into two separate species by chloroplast location in BSC. The ultrastructural features of leaves ofP. dichtomiflorum, NAD-malic enzyme species with centrifugal chloroplasts, were also investigated. Chloroplasts in BSC had well-developed grana, and numerous large mitochondria with extensively developed internal membrane structure were restricted to the area between the chloroplsts and the vacuole in BSC.     

C4-Dicarboxylic acid metabolism in bundle-sheath chloroplasts,mitochondria and strands of Eriochloa borumensis Hack., a phosphoenolpyruvate-carboxykinase type C4 species

C₄-acid metabolism by isolated bundlesheath chloroplasts, mitochondria and strands of Eriochloa borumensis Hack., a phosphoennolpyruvate-carboxykinase (PEP-CK) species, was investigated. Aspartate, oxaloacetate (OAA) and malate were decarboxylated by strands with several-fold stimulation upon illumination. There was strictly light-dependent decarboxylation of OAA and malate by the chloroplasts, but the chloroplasts did not decarboxylate aspartate in light or dark. PEP was a primary product of OAA or malate decarboxylation by the chloroplasts and its formation was inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea or NH₄Cl. There was very little conversion of PEP to pyruvate by bundle-sheath chloroplasts, mitochondria or strands. Decarboxylation of the three C₄-acids by mitochondria was light-independent. Pyruvate was the only product of mitochondrial metabolism of C₄-acids, and was apparently transaminated in the cytoplasm since PEP and alanine were primarily exported out of the bundle-sheath strands. Light-dependent C₄-acid decarboxylation by the chloroplasts is suggested to be through the PEP-CK, while the mitochondrial C₄-acid decarboxylation may proceed through the NAD-malic enzyme (NAD-ME) system. In vivo both aspartate and malate are considered as transport metobolites from mesophyll to bundle-sheath cells in PEP-CK species. Aspartate would be metabolized by the mitochondria to OAA. Part of the OAA may be converted to malate and decarboxylated through NAD-ME, and part may be transported to the chloroplasts for decarboxylation through PEP-CK localized in the chloroplasts. Malate transported from mesophyll cells may serve as carboxyl donor to chloroplasts through the chloroplastic NAD-malate dehydrogenase and PEP-CK. Bundle-sheath strands and chloroplasts fixed ¹⁴CO₂ at high rates and exhibited C₄-acid-dependent O₂ evolution in the light. Studies with 3-mercaptopicolinic acid, a specific inhibitor of PEP-CK, have indicated that most (about 70%) of the OAA formed from aspartate is decarboxylated through the chloroplastic PEP-CK and the remaining (about 30%) OAA through the mitochondrial NAD-ME. Pyruvate stimulation of aspartate decarboxylation is discussed; a pyruvate-alanine shuttle and an aspartate-alanine shuttle are proposed between the mesophyll and bundle-sheath cells during aspartate decarboxylation through the PEP-CK and NAD-ME system respectively.Abbreviations CK carboxykinase - -Kg -ketoglutarate - ME malic enzyme - 3-MPA 3-mercaptopicolinic acid - OAA oxaloacetate - PEP phosphoenolpyruvate - R5P ribose-5-phosphate     

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     

Leaf anatomy, post-illumination CO2 burst and NAD-malic enzyme activity of Panicum dichotomiflorum

Light microscopic observation of leaf blades of Panicum dichotomiflorumshowed that a mestome sheath was present and chloroplasts inbundle sheath cells were in the centrifugal position. However,a sharp pattern of post-illumination CO₂ burst was observedin less than 30 sec after the extinction of light. Among threeC₄-acid decarboxylating enzymes, only the activity of NAD-malicenzyme was high. These results indicate that P. dichotomiflorumis a NAD-malic enzyme type species having centrifugal chloroplastsin bundle sheath cells and the sharp pattern of post-illuminationCO₂ burst is closely correlated with the C₄-acid decarboxylationsystem through NAD-malic enzyme ¹This research was supported by a grant from the Ministry ofAgriculture, Forestry and Fishery (GEP55-II-1-7). (Received August 18, 1980; )     

Leaf anatomy, post-illumination CO2 burst and NAD-malic enzyme activity of Panicum dichotomiflorum

Light microscopic observation of leaf blades of Panicum dichotomiflorumshowed that a mestome sheath was present and chloroplasts inbundle sheath cells were in the centrifugal position. However,a sharp pattern of post-illumination CO₂ burst was observedin less than 30 sec after the extinction of light. Among threeC₄-acid decarboxylating enzymes, only the activity of NAD-malicenzyme was high. These results indicate that P. dichotomiflorumis a NAD-malic enzyme type species having centrifugal chloroplastsin bundle sheath cells and the sharp pattern of post-illuminationCO₂ burst is closely correlated with the C₄-acid decarboxylationsystem through NAD-malic enzyme ¹This research was supported by a grant from the Ministry ofAgriculture, Forestry and Fishery (GEP55-II-1-7). (Received August 18, 1980; )     

Structural features of NAD-malic enzyme type C4 Eleocharis: An additional report of C4 acid decarboxylation types of the cyperaceae

The genusEleocharis, a blade-less sedge group, has been very recently recorded to include NAD-malic enzyme type C₄ species. The ultrastructural features of culms of two C₄ representatives in the genus were examined in relation to the C₄ acid decarboxylation type. They possessed non-chlorophyllous mestome sheath cells between mesophyll cells and Kranz cells, and were confirmed biochemically to be NAD-malic enzyme type. The oval or lenticular chloroplasts with well-developed grana are scattered in the Kranz cells with abundant large mitochondria, and do not show such centripetal position as is known in the “classical NAD-malic enzyme type”. The suberized lamellae occur in the mestome sheath cells internally surrounding the Kranz sheath and may contribute to maintaining high CO₂ concentration in the Kranz cells. These new structural features of the NAD-malic enzyme type found inEleocharis are added to the structural and functional relationships of the C₄ types in the Cyperaceae reported previously     

Functional characterization of phosphoenolpyruvate carboxykinase-type C4 leaf anatomy: immuno-, cytochemical and ultrastructural analyses

BACKGROUND AND AIMS: Species having C4 photosynthesis belonging to the phosphoenolpyruvate carboxykinase (PEP-CK) subtype, which are found only in family Poaceae, have the most complex biochemistry among the three C4 subtypes. In this study, biochemical (western blots and immunolocalization of some key photosynthetic enzymes) and structural analyses were made on several species to further understand the PEP-CK system. This included PEP-CK-type C4 species Urochloa texana (subfamily Panicoideae), Spartina alterniflora and S. anglica (subfamily Chloridoideae), and an NADP-ME-type C4 species, Echinochloa frumentacea, which has substantial levels of PEP-CK. KEY RESULTS: Urochloa texana has typical Kranz anatomy with granal chloroplasts scattered around the cytoplasm in bundle sheath (BS) cells, while the Spartina spp. have BS forming long adaxial extensions above the vascular tissue and with chloroplasts in a strictly centrifugal position. Despite some structural and size differences, in all three PEP-CK species the chloroplasts in mesophyll and BS cells have a similar granal index (% appressed thylakoids). Immunolocalization studies show PEP-CK (which catalyses ATP-dependent decarboxylation) is located in the cytosol, and NAD-ME in the mitochondria, in BS cells, and in the BS extensions of Spartina. In the NADP-ME species E. frumentacea, PEP-CK is also located in the cytosol of BS cells, NAD-ME is very low, and the source of ATP to support PEP-CK is not established. CONCLUSIONS: Representative PEP-CK species from two subfamilies of polyphyletic origin have very similar biochemistry, compartmentation and chloroplast grana structure. Based on the results with PEP-CK species, schemes are presented with mesophyll and BS chloroplasts providing equivalent reductive power which show bioenergetics of carbon assimilation involving C4 cycles (PEP-CK and NAD-ME, the latter functioning to generate ATP to support the PEP-CK reaction), and the consequences of any photorespiration.     

Panicum milioides, a Gramineae plant having Kranz leaf anatomy without C4-photosynthesis

Light and electron microscopic observations of the leaf tissueof Panicum milioides showed that the bundle sheath cells containeda substantial number of chloroplasts and other organelles. Theradial arrangement of chlorenchymatous bundle sheath cells,designated as Kranz leaf anatomy, has been considered to bespecific to C₄ plants. However, photosynthetic ¹⁴CO₂ fixationand ¹⁴CO₂ pulse-and-chase experiments revealed that the reductivepentosephosphate pathway was the main route operating in leavesof P. milioides. The interveinal distance of the leaves wasintermediate between C₃and C₄Gramineae species. These resultsindicate that P. milioides is a natural plant species havingchracteristics intermediate between C₃ and C₄ types. (Received March 6, 1975; )     

Properties of Leaf NAD-Malic Enzyme from the Inducible Crassulacean Acid Metabolism Species Mesembryanthemum crystallinum

NAD-malic enzyme (NAD-ME) functions to decarboxylate malatein the light in leaves of certain species displaying Crassulaceanacid metabolism (CAM). The properties of NAD-ME in desaltedextracts from the inducible CAM species, Mesembryanthemum crystallinumwere examined. The shapes of the malate saturation curve andthe activity versus pH curve at 10 mM malate were dependenton the presence of the activator CoA. The malate saturationcurve was sigmoidal in the absence of an activator and hyperbolicin the presence of CoA. The pH optimum with 10mM malate andMn²⁺ as cofactor was as low as 6.5 without an activator, andincreased to 7.2 in the presence of CoA. Fumarate activationwas synergistic with CoA above pH 7.2. The enzyme displayedhysteretic behavior under suboptimal assay conditions. Rapid extraction and desalting of the enzyme (<1.5 mim) followedimmediately by assay did not reveal any difference in the propertiesof the enzyme on a day/night basis. It is proposed that diurnalregulation of the enzyme in vivo is mediated by pH and malatelevel without a change in the oligomeric form of the enzyme.The molecular weight of the enzyme was approximately 350,000at pH 6.5 or 7.8. The enzyme obtained from M. crystallinum inthe C₃ mode was very similar to the CAM enzyme except that itdisplayed a lower V_max. ³ Current address: MSU-DOE Plant Research Lab, Michigan StateUniversity, E. Lansing, Michigan, U.S.A. 48824. (Received October 2, 1984; Accepted December 20, 1984)     

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     

Salsola arbusculiformis, a C3-C4Intermediate in Salsoleae (Chenopodiaceae)

Salsola arbusculiformis is identified as a C₃–C₄intermediatespecies based on anatomical, biochemical and physiological characteristics.This is the first report of a naturally occurring intermediatespecies in the Chenopodiaceae, the family with the largest numberof C₄species amongst the dicots. In the genus Salsola, mostspecies have Salsoloid anatomy with Kranz type bundle sheathcells and C₄photosynthesis, while a few species have Sympegmoidanatomy and were found to have non-Kranz type bundle sheathcells and C₃photosynthesis. In the cylindrical leaves of C₄Salsolawith Salsoloid type anatomy, there is a continuous layer ofdistinct, chlorenchymatous Kranz type bundle sheath cells surroundedby a single layer of mesophyll cells; whereas species with Sympegmoidtype anatomy have an indistinct bundle sheath with few chloroplastsand multiple layers of chlorenchymatous mesophyll cells. However,S. arbusculiformis has intermediate anatomical features. Whileit has two-to-three layers of mesophyll cells, characteristicof Sympegmoid anatomy, it has distinctive, Kranz-like bundlesheath cells with numerous chloroplasts and mitochondria. Measurementsof its CO₂compensation point and CO₂response of photosynthesisshow S. arbusculiformis functions as an intermediate specieswith reduced levels of photorespiration. The primary means ofreducing photorespiration is suggested to be by refixing photorespiredCO₂in bundle sheath cells, since analysis of photosyntheticenzymes (activity and immunolocalization) and¹⁴CO₂labellingof initial fixation products suggests minimal operation of aC₄cycle. Copyright 2001 Annals of Botany Company Immunolocalization, photosynthetic enzymes, C₃–C₄intermediate, C₄-plants, leaf anatomy, Chenopodiaceae, Salsola arbusculiformis     

Occurrence of C3 and C4 photosynthesis in cotyledons and leaves of Salsola species (Chenopodiaceae)

Most species of the genus Salsola (Chenopodiaceae) that have been examined exhibit C₄ photosynthesis in leaves. Four Salsola species from Central Asia were investigated in this study to determine the structural and functional relationships in photosynthesis of cotyledons compared to leaves, using anatomical (Kranz versus non-Kranz anatomy, chloroplast ultrastructure) and biochemical (activities of photosynthetic enzymes of the C₃ and C₄ pathways, ¹⁴C labeling of primary photosynthesis products and ¹³C/¹²C carbon isotope fractionation) criteria. The species included S. paulsenii from section Salsola, S. richteri from section Coccosalsola, S. laricina from section Caroxylon, and S. gemmascens from section Malpigipila. The results show that all four species have a C₄ type of photosynthesis in leaves with a Salsoloid type Kranz anatomy, whereas both C₃ and C₄ types of photosynthesis were found in cotyledons. S. paulsenii and S. richteri have NADP- (NADP-ME) C₄ type biochemistry with Salsoloid Kranz anatomy in both leaves and cotyledons. In S. laricina, both cotyledons and leaves have NAD-malic enzyme (NAD-ME) C₄ type photosynthesis; however, while the leaves have Salsoloid type Kranz anatomy, cotyledons have Atriplicoid type Kranz anatomy. In S. gemmascens, cotyledons exhibit C₃ type photosynthesis, while leaves perform NAD-ME type photosynthesis. Since the four species studied belong to different Salsola sections, this suggests that differences in photosynthetic types of leaves and cotyledons may be used as a basis or studies of the origin and evolution of C₄ photosynthesis in the family Chenopodiaceae.This revised version was published online in October 2005 with corrections to the Cover Date.     

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.     

The Identification of Photosynthetic Subpathways of Some C4 and CAM Plants

The photosynthetic subpathways of five C4 plants and one CAM plant were distinguished according to their chemical, physiological and cytological characteristics. Based on C4 acid decarboxylation enzymes, four C4 plants of Setaria glauca, Sporobolus indicus, Zoysia tenuifolia and Leptochloa chinensis all exhibited the functional high activities of PEP carboxykinase and aspartate aminotransferase as seen in the known PEP-CK subtype. The δ13C value of –12.43% in leaves of L. chinensis was also consistent with that range among PEP-CK subtype. So, these species were classified into PEP-CK subtype. However, their chloroplasts in bundle sheath cells were evenly distributed, not as that displayed centrifugally or centripetally in three typical subtypes. The even arrangement of chloroplasts in bundle sheath cells was likely to be an evolutional intermediate from centripetal (NAD ME type) to centrifugal types (NADP-ME and most PEP-CK types). The high activities of NAD-malic enzyme and aspartate aminotransferase, accompanied with the centripetally located chloroplasts, 0.057 of quantum yield and tile δ13C value of –15.3% in leaves of C4 dicot Euphobia hirta indicated characteristics of NAD-ME subtype. Moreover, CAM plant Aloe vera clearly fell into PEP-CK sybtype because of its high activity of PEP-CK both in whole leaf and green tissue.     

Structural, biochemical, and physiological characterization of C4 photosynthesis in species having two vastly different types of kranz anatomy in genus Suaeda (Chenopodiaceae)

C (4) species of family Chenopodiaceae, subfamily Suaedoideae have two types of Kranz anatomy in genus Suaeda, sections Salsina and Schoberia, both of which have an outer (palisade mesophyll) and an inner (Kranz) layer of chlorenchyma cells in usually semi-terete leaves. Features of Salsina (S. AEGYPTIACA, S. arcuata, S. taxifolia) and Schoberia type (S. acuminata, S. Eltonica, S. cochlearifoliA) were compared to C (3) type S. Heterophylla. In Salsina type, two layers of chlorenchyma at the leaf periphery surround water-storage tissue in which the vascular bundles are embedded. In leaves of the Schoberia type, enlarged water-storage hypodermal cells surround two layers of chlorenchyma tissue, with the latter surrounding the vascular bundles. The chloroplasts in Kranz cells are located in the centripetal position in Salsina type and in the centrifugal position in the Schoberia type. Western blots on C (4) acid decarboxylases show that both Kranz forms are NAD-malic enzyme (NAD-ME) type C (4) species. Transmission electron microscopy shows that mesophyll cells have chloroplasts with reduced grana, while Kranz cells have chloroplasts with well-developed grana and large, specialized mitochondria, characteristic of NAD-ME type C (4) chenopods. In both C (4) types, phosphoenolpyruvate carboxylase is localized in the palisade mesophyll, and Rubisco and mitochondrial NAD-ME are localized in Kranz cells, where starch is mainly stored. The C (3) species S. heterophylla has Brezia type isolateral leaf structure, with several layers of Rubisco-containing chlorenchyma. Photosynthetic response curves to varying CO (2) and light in the Schoberia Type and Salsina type species were similar, and typical of C (4) plants. The results indicate that two structural forms of Kranz anatomy evolved in parallel in species of subfamily Suaedoideae having NAD-ME type C (4) photosynthesis.     

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

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