NADP-malic Enzyme Species Research Articles

Stomatal response to VPD in C4 plants with different biochemical sub-pathways.

C4 NAD-malic enzyme (NAD-ME) species occurs in drier regions and exhibit different drought responses compared to C4 NADP-malic enzyme (NADP-ME) species. However, a physiological mechanism explaining the geographical discrepancies remains uncertain. This study examined gas exchange patterns that might explain different distributions observed between two subtypes of C4 photosynthesis. We measured the response of leaf gas exchange to vapour pressure deficit (VPD) and CO2 in plants from six distinct C4 clades having closely related NAD-ME and NADP-ME species using a Li-Cor 6400 gas exchange system. We found that NAD-ME species exhibited greater relative reductions in stomatal conductance with increases in VPD than NADP-ME species but observed no consistent subtype differences in C4 cycle activity as indicated by the initial slope of the A response to intercellular CO2 concentration. Based on these results, we hypothesise the greater response of gs to increasing VPD may enable NAD-ME plants to outperform NADP-ME plants in hot, dry environments where VPD is normally high.

Shade compromises the photosynthetic efficiency of NADP-ME less than that of PEP-CK and NAD-ME C4 grasses.

The high energy cost and apparently low plasticity of C4 photosynthesis compared with C3 photosynthesis may limit the productivity and distribution of C4 plants in low light (LL) environments. C4 photosynthesis evolved numerous times, but it remains unclear how different biochemical subtypes perform under LL. We grew eight C4 grasses belonging to three biochemical subtypes [NADP-malic enzyme (NADP-ME), NAD-malic enzyme (NAD-ME), and phosphoenolpyruvate carboxykinase (PEP-CK)] under shade (16% sunlight) or control (full sunlight) conditions and measured their photosynthetic characteristics at both low and high light. We show for the first time that LL (during measurement or growth) compromised the CO2-concentrating mechanism (CCM) to a greater extent in NAD-ME than in PEP-CK or NADP-ME C4 grasses by virtue of a greater increase in carbon isotope discrimination (∆P) and bundle sheath CO2 leakiness (ϕ), and a greater reduction in photosynthetic quantum yield (Φmax). These responses were partly explained by changes in the ratios of phosphoenolpyruvate carboxylase (PEPC)/initial Rubisco activity and dark respiration/photosynthesis (Rd/A). Shade induced a greater photosynthetic acclimation in NAD-ME than in NADP-ME and PEP-CK species due to a greater Rubisco deactivation. Shade also reduced plant dry mass to a greater extent in NAD-ME and PEP-CK relative to NADP-ME grasses. In conclusion, LL compromised the co-ordination of the C4 and C3 cycles and, hence, the efficiency of the CCM to a greater extent in NAD-ME than in PEP-CK species, while CCM efficiency was less impacted by LL in NADP-ME species. Consequently, NADP-ME species are more efficient at LL, which could explain their agronomic and ecological dominance relative to other C4 grasses.

Photosynthetic Diversity in Indian Grass Flora

Plants following C3 and C4 pathways of photosynthesis respond differently to environmental variables such as temperature and precipitation. The C3 and C4 plants therefore, comprise specific groups which can help predict species response to climatic changes. Grasses alone account for nearly half of the known C4 species and half of the grass species are estimated to follow C4 photosynthesis. Indian grass flora has about 250 genera and 1,250 odd species, 55 % of which are of C4 type. The C4 tribe Andropogoneae, exclusively of NADP-malic enzyme (NADP-ME) sub-type, alone represents 50 % of the Indian C4 grasses. NADP-ME grasses exhibit high moisture affinity and constitute more than three-fourth of the Indian C4 grasses. The greater representation of NADP-ME species in the Indian grass flora could be attributed to the onset of Indian monsoons during the late Miocene, when the main adaptive radiation in grasses took place, the world over.

Kinetic analyses of recombinant isoforms of phospho enolpyruvate carboxylase from Hydrilla verticillata leaves and the impact of substituting a C 4-signature serine

Hydrilla verticillata (L.f.) Royle is a single-cell C 4 NADP-malic enzyme species in which the C 4 and Calvin cycles operate in the same cell; phospho enolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) is the initial carboxylase. The cDNAs of two Hydrilla PEPC isoforms were sequenced; one encodes the photosynthetic form, HVPEPC4, and the other an anaplerotic, HVPEPC3. Both lack the C 4-signature Ser. Recombinant proteins were generated and the C 3-Ala was replaced by the C 4-Ser using site-directed mutagenesis. Kinetic analyses of the recombinant isoforms indicated that rHVPEPC4 had a higher K 0 .5 (PEP) (0.3 mM), more typical of C 4 PEPC, despite the lack of a signature Ser. Replacing Ala with Ser in rHVPEPC4 further increased the K 0.5(PEP) to 0.78 mM, but reduced the V max by 25%. However, a similar Ser substitution in rHVPEPC3 not only increased the K 0.5(PEP) from 0.15 to 0.92 mM but also increased the I 50(MAL), V max and k cat. Thus, with a single amino acid substitution rHVPEPC3 kinetically became similar to a C 4 PEPC. In all cases, glucose-6-phosphate lowered the K 0.5(PEP) values and reduced malate inhibition. Although, the C 4-Ser had an impact, overall the C 4 characteristics of HVPEPC4 are conferred by other parts of its primary structure.

Analysis of donors of electrons to photosystem I and cyclic electron flow by redox kinetics of P700 in chloroplasts of isolated bundle sheath strands of maize

Bundle sheath chloroplasts of NADP-malic enzyme (NADP-ME) type C4 species have a high demand for ATP, while being deficient in linear electron flow and oxidation of water by photosystem II (PSII). To evaluate electron donors to photosystem I (PSI) and possible pathways of cyclic electron flow (CEF1) in isolated bundle sheath strands of maize (Zea mays L.), an NADP-ME species, light-induced redox kinetics of the reaction center chlorophyll of PSI (P700) were followed under aerobic conditions. Donors of electrons to CEF1 are needed to compensate for electrons lost from the cycle. When stromal electron donors to CEF1 are generated during pre-illumination with actinic light (AL), they retard the subsequent rate of oxidation of P700 by far-red light. Ascorbate was more effective than malate in generating stromal electron donors by AL. The generation of stromal donors by ascorbate was inhibited by DCMU, showing ascorbate donates electrons to the oxidizing side of PSII. The inhibitors of NADPH dehydrogenase (NDH), amytal and rotenone, accelerated the oxidation rate of P700 by far-red light after AL, indicating donation of electrons to the intersystem from stromal donors via NDH. These inhibitors, however, did not affect the steady-state level of P700+ under AL, which represents a balance of input and output of electrons in P700. In contrast, antimycin A, the inhibitor of the ferredoxin-plastoquinone reductase-dependent CEF1, substantially lowered the level of P700+ under AL. Thus, the primary pathway of ATP generation by CEF1 may be through ferredoxin-plastoquinone, while function of CEF1 via NDH may be restricted by low levels of ferredoxin-NADP reductase. NDH may contribute to redox poising of CEF1, or function to generate ATP in linear electron flow to O2 via PSI, utilizing NADPH generated from malate by chloroplastic NADP-ME.

Identification of C4 responsive genes in the facultative C4 plant Hydrilla verticillata

The aquatic monocot Hydrilla verticillata (L.f.) Royle is a well-documented facultative C4 NADP-malic enzyme species in which the C4 and Calvin cycles operate in the same cell with the specific carboxylases confined to the cytosol and chloroplast, respectively. Several key components had already been characterized at the molecular level, thus the purpose of this study was to begin to identify other, less obvious, elements that may be necessary for a functional single-cell C4 system. Using differential display, mRNA populations from C3 and C4 H. verticillata leaves were screened and expression profiles compared. From this study, 65 clones were isolated and subjected to a customized macroarray analysis; 25 clones were found to be upregulated in C4 leaves. Northern and semi-quantitative RT-PCR analyses were used for confirmation. From these screenings, 13 C4 upregulated genes were identified. Among these one encoded a previously recognized C4 phosphoenolpyruvate carboxylase, and two encoded distinct pyruvate orthophosphate dikinase isoforms, new findings for H. verticillata. Genes that encode a transporter, an aminotransferase and two chaperonins were also upregulated. Twelve false positives, mostly housekeeping genes, were determined from the Northern/semi-quantitative RT-PCR analyses. Sequence data obtained in this study are listed in the dbEST database (DV216698 to DV216767). As a single-cell C4 system that lacks Kranz anatomy, a better understanding of how H. verticillata operates may facilitate the design of a transgenic C4 system in a C3 crop species.

Photosynthesizing Tissue Development in C4 Cotyledons of Two Salsola Species (Chenopodiaceae)

The quantitative anatomy of developing cotyledons of NAD-malic enzyme species Salsola incanescens and NADP-malic enzyme species S. paulsenii (Chenopodiaceae) was studied. S. incanescens belongs to the group of species with foliar type of seedling development characterized by slowly growing cotyledons and a rosette form at juvenility. The rosette is the consequence of fast leaf formation, which was correlated with a low rate of leaf growth. S. paulsenii belongs to the group with the cotyledonous type of seedling development. A high growth rate of cotyledons, slow leaf formation, and absence of the rosette characterize this type. Slow leaf formation was correlated with a high rate of leaf growth. The Kranz–anatomy in cotyledons of S. incanescens (atriplicoid type) and S. paulsenii (salsoloid type) determines the duration of cotyledon development proceeding for 15 days after seed germination. The rate of growth changes during the developmental period was correlated with the type of seedling development. Cotyledons of a foliar species S. incanescens exhibit 2 to 5 times slower growth changes in cotyledon area, width, thickness, volume of mesophyll and bundle sheath cells, and number of chloroplasts per bundle sheath cell than the cotyledons of a cotyledonous species S. paulsenii. During cotyledon development in both species, the number of chloroplasts per mesophyll cell remained unchanged, and developmental changes in the bundle sheath occurred at higher rate than in mesophyll cells. Thus, these two indices seem to be independent of the type of Kranz–anatomy. The presence of atriplicoid type cotyledons in the species with salsoloid structure of true leaves might indicate a close genetic relationship between these two patterns of Kranz-anatomy.

Analyzing the Light Energy Distribution in the Photosynthetic Apparatus of C4 Plants Using Highly Purified Mesophyll and Bundle-Sheath Thylakoids.

The chlorophyll fluorescence characteristics of mesophyll and bundle-sheath thylakoids from plant species with the C4 dicarboxylic acid pathway of photosynthesis were investigated using flow cytometry. Ten species with the NADP-malic enzyme (NADP-ME) biochemical type of C4 photosynthesis were tested: Digitaria sanguinalis (L.) Scop., Euphorbia maculata L., Portulaca grandiflora Hooker, Saccharum officinarum L., Setaria viridis (L.) Beauv., Zea mays L., and four species of the genus Flaveria. This study also included three species with NAD-ME biochemistry (Atriplex rosea L., Atriplex spongiosa F. Muell., and Portulaca oleracea L.). Two C4 species of unknown biochemical type were investigated: Cyperus papyrus L. and Atriplex tatarica L. Pure mesophyll and bundle-sheath thylakoids were prepared by flow cytometry and characterized by low-temperature fluorescence spectroscopy. In pure bundle-sheath thylakoids from many species with C4 photosynthesis of the NADP-ME type, significant amounts of photosystem II (PSII) emission can be detected by fluorescence spectroscopy. Simulation of fluorescence excitation spectra of these thylakoids showed that PSII light absorption contributes significantly to the apparent excitation spectrum of photosystem I. Model calculations indicated that the excitation energy of PSII is efficiently transferred to photosystem I in bundle-sheath thylakoids of many NADP-ME species.

Geographical Distribution of C4 Acid Decarboxylation Types and Associated Structural Variants in Native Australian C4 Grasses (Poaceae)

The native Australian C4 grass (Poaceae) flora is estimated to comprise 347 (57%) NADP-ME (NADP- malic enzyme), 193 (32%) NAD-ME (NAD-malic enzyme), and 65 (11%) PCK (PEP carboxykinase) type species. All C4 types are best represented in northern tropical Queensland, within the megatherm seasonal (summer) rainfall bioclimate of Nix. NADP-ME species are numerically dominant in 48 out of 73 State and Territory subdivisions, including 23 wholly or partly within the megathermlmesotherm arid bioclimate which closely corresponds to the arid and semiarid zones covering c. 80% of Australia. NAD-ME species numbers are proportionately at their highest in this bioclimate; PCK species may be the most dependent there on high soil moisture availability. The extent of the megatherm seasonal bioclimate is parallelled by the distribution of most PCK species and of many species of all C4 types with a suberised lamella in the cell walls of their 'photosynthetic carbon reduction' (or Kranz) tissue. Whilst the physiological reasons for these correlations are unknown, it is clear that C4 type alone is neither the sole determinant of geographical distribution nor necessarily always an adaptation to a particular bioclimatic regime. Taxonomic, ecological and historical factors in relation to C4 type distribution are discussed.

Photosynthesis in Mesophyll Protoplasts and Bundle Sheath Cells of Various Types of C4 Plants IV. Enzymes of kespiratory Metabolism and Energy Utilizing Enzymes of Photosynthetic Pathways')

Summary A study was made of the distribution of energy utilizing enzymes of carbon assimilation and key enzymes of respiratory metabolism between mesophyll and bundle sheath cells of 3 groups of C4 plants: NADP-malic enzyme species, NAD-malic enzyme species, and phosphoenolpyruvate (PEP) carboxykinase species. A number of enzymes of photosynthetic metabolism including some energy utilizing enzymes of carbon assimilation and photorespiratory enzymes showed preferential distribution between mesophyll and bundle sheath cells. Pyruvate Pi dikinase, NADP-malate dehydrogenase and carbonic anhydrase were predominantly localized in mesophyll preparations of all 3 groups of C4 species. NADP-malic enzyme species, as compared with NAD-malic enzyme and PEP carboxykinase species, showed much higher activity of NADP-malate dehydrogenase in mesophyll cells. Phosphoribulokinase of the regenerative phase of the reductive pentose phosphate pathway was localized in bundle sheath cells of all 3 C4 groups. Enzymes of the reductive phase of the reductive pentose phosphate pathway, glycerate-3-phosphate kinase, NADP)-glyceraldehyde-3-phosphate dehydrogenase, and triose-phosphate isomerase were high in activity in both photosynthetic cell types of the various species examined. In a qualitative evaluation of energy requirements in carbon assimilation in C photosynthesis ATP would be required in mesophyll cells for pyruvate Pi dikinase; in bundle sheath cells for phosphoribulokinase; NADPH would be required in mesophyll cells for NADP-malate dehydrogenase; and ATP and NADPH would be required in both mesophyll and bundle sheath cells for the reductive phase of carbon assimilation. Aspartate- and alanine aminotransferase, suggested to have a role in C4 photosynthesis in some C4 species, were high in activity in both cell types in NAD-malic enzyme and PEP carboxykinase species with relatively low activity in NADP-malic enzyme species. Enzymes apparently associated with dark respiratory metabolism including glucose-6phosphate dehydrogenase and 6-phosphogluconic dehydrogenase of the oxidative pentose phosphate pathway, cytochrome c oxidase of the mitochondria, 3-phosphoglycerate mutase and enolase of glycolysis were relatively active in both mesophyll and bundle sheath cells in species of all 3 groups. However, on a chlorophyll basis the cytochrome c oxidase activity was 2 to 3 fold higher in activity in bundle sheath than in mesophyll cells. In particular with Zea mays the cytochrome c oxidase was about 4 fold more active in bundle sheath than in mesophyll cells. Enzymes which may be involved in photo respiratory metabolism were primarily associated with bundle sheath cells. The results are discussed with respect to sites of energy utilization in C4 metabolism and differences in carbon assimilation in 3 C4 groups.

Photosynthesis in mesophyll protoplasts and bundle sheath cells of various type of C4 plants. III. Fluorescence emission spectra, delayed light emission, and P700 content

Summary Mesophyll protoplasts from C 3 and C 4 plants and bundle sheath cells from C 4 plants were enzymatically isolated and purified from representative species of Gramineae. Chloroplast particles prepared by breaking protoplasts and cells with a French press were used for comparative photochemical studies. In C 4 grasses having high concentrations of NADP-malic enzyme, the ratio of fluorescence at 685 nm/730 nm, at liquid nitrogen temperature, and the delayed light emission was from 3 to 15 fold higher in mesophyll protoplast extracts than in bundle sheath cell extracts. This variation was species dependent. Based on delayed light emission and liquid nitrogen fluorescence emission spectra ratio 685 nm/730 nm, the relative level of photosystem II in bundle sheath chloroplast particles of NADP-malic enzyme species is Setaria lutescens, Setaria faberii > Digitaria sanguinalis > Zea mays > sugarcane, Sorghum bicolor , and Sorghum halepense . In Sorghum bicolor and Sorghum halepense at least 94 % of the photosystem II activity of the leaf is in mesophyll chloroplasts. The P700 content is 1.4 to 2.2 fold higher in bundle sheath extracts compared to mesophyll extracts of species having high NADP-malic enzyme. In C 4 grasses having high NAD-malic enzyme, such as Panicum capillare and Panicum miliaceum , the delayed light emission and liquid nitrogen fluorescence emission spectra at 685 nm/730 nm was higher in bundle sheath extracts than in mesophyll extracts. On the other hand, the P700 activity in these species was about 1.4 fold higher in mesophyll protoplasts extracts than in bundle sheath extracts. In C4 grasses having high phosphopyruvate carboxy-kinase, the liquid nitrogen fluorescence emission spectra were similar in mesophyll protoplast and bundle sheath extracts, the delayed light emission was higher in mesophyll extracts than in bundle sheath extracts, while the P700 activity was higher in bundle sheath than in mesophyll extracts. The results are discussed in relation to proposed energy requirements for carbon metabolism in mesophyll and bundle sheath cells of various C 4 species.

Biochemical and cytological relationships in C4 plants

C4 plants can be divided into three groups based on differences in activities of three decarboxylating enzymes: NADP-malic enzyme, NAD-malic enzyme, and phosphopyruvate carboxykinase.In the Gramineae the three C4 groups are distinguished by anatomical and ultrastructural characteristics of bundle-sheath chloroplasts. NADP-malic enzyme species lack well-developed grana in bundle-sheath chloroplasts (grana reduced) and the bundle-sheath chloroplasts are in the centrifugal position. NAD-malic enzyme species have bundle-sheath chloroplasts in the centripetal position and contain grana. Phosphopyruvate carboxykinase species have bundle-sheath chloroplasts in the centrifugal position and they contain grana. NADP-malic enzyme species of the Gramineae have only been found in the subfamilies Aristidoideae and Panicoideae. With the exception of the genera Panicum, and Urochloa, NAD-malic enzyme species and phosphopyruvate carboxykinase species have only been found in the subfamily Eragrostoideae. C4 species of the genus Panicum are found among all three of the C4 groups.The dicotyledonous C4 species examined fall into two groups: those having high NADP-malic enzyme and those having high NAD-malic enzyme. No phosphopyruvate carboxykinase C4 species have been found among the dicotyledons. The NADP-malic enzyme C4 species of the dicotyledons like NADP-malic enzyme species of the Gramineae have bundle-sheath chloroplasts with reduced grana but in contrast to NADP-malic enzyme species of the Gramineae the bundle-sheath chloroplasts are in the centripetal position. The NAD-malic enzyme species of the dicotyledons like the NAD-malic enzyme species of the Gramineae have bundlesheath chloroplasts in the centripetal position with well developed grana.The results are discussed in terms of evolutionary and functional diversification of C4 plants.