Flaveria pringlei (C3) andFlaveria trinervia (C4) under NaCl stress

The C₄ speciesFlaveria trinervia is obviously better adapted to saline environments than the C₃ speciesF. pringlei. Treatment with 100 mM NaCl diminished crop growth rate inF. pringlei by 38% but not inF. trinervia. Under saline conditions, more assimilates were invested in leaf growth inF. trinervia but not inF. pringlei. Electrolyte concentration inF. trinervia in control and salt treated plants is lower than inF. pringlei. Fluorescence data do not indicate a damage of PS 2 charge separation in both species. Whether the C₄ photosynthetic pathway inF. trinervia is responsible for the improved salt tolerance compared toF. pringlei remains an open question.     

Evolution of C4 phosphoenolpyruvate carboxylase in the genus Alternanthera: gene families and the enzymatic characteristics of the C4 isozyme and its orthologues in C3 and C3/C4 Alternantheras

Phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.3) is a key enzyme of C₄ photosynthesis. It has evolved from ancestral non-photosynthetic (C₃) isoforms and thereby changed its kinetic and regulatory properties. We are interested in understanding the molecular changes, as the C₄ PEPCases were adapted to their new function in C₄ photosynthesis and have therefore analysed the PEPCase genes of various Alternanthera species. We isolated PEPCase cDNAs from the C₄ plant Alternanthera pungens H.B.K., the C₃/C₄ intermediate plant A. tenella Colla, and the C₃ plant A. sessilis (L.) R.Br. and investigated the kinetic properties of the corresponding recombinant PEPCase proteins and their phylogenetic relationships. The three PEPCases are most likely derived from orthologous gene classes named ppcA. The affinity constant for the substrate phosphoenolpyruvate (K _0.5 PEP) and the degree of activation by glucose-6-phosphate classified the enzyme from A. pungens (C₄) as a C₄ PEPCase isoform. In contrast, both the PEPCases from A. sessilis (C₃) and A. tenella (C₃/C₄) were found to be typical C₃ PEPCase isozymes. The C₄ characteristics of the PEPCase of A. pungens were accompanied by the presence of the C₄-invariant serine residue at position 775 reinforcing that a serine at this position is essential for being a C₄ PEPCase (Svensson et al. 2003). Genomic Southern blot experiments and sequence analysis of the 3′ untranslated regions of these genes indicated the existence of PEPCase multigene family in all three plants which can be grouped into three classes named ppcA, ppcB and ppcC.     

Transfer of C(4) Photosynthetic Characters through Hybridization of Flaveria Species

Transfer of C₄ photosynthetic traits was studied through hybridization of Flaveria trinervia (Spreng.) Mohr (C₄) and Flaveria brownii A.M. Powell (C₄-like) with Flaveria linearis Lag. (C₃-C₄) and the C₃ species Flaveria pringlei Gandoger (C₃). Fertility was low, based on irregular chromosome pairing and low pollen stainability, except in F. brownii × F. linearis which had bivalent pairing and 76% stainable pollen. Hybrids had apparent photosynthesis values of 71 to 148% of the midparental means, while the CO₂ compensation concentration was similar to the C₄ or C₄-like parent, except in hybrids having the C₃ species F. pringlei as a parent. Inhibition of apparent photosynthesis by O₂, and phosphoenolpyruvate carboxylase and NADP-malic enzyme activities and subunit levels in the hybrids were closer to the C₃ or C₃-C₄ parent. The species F. brownii and F. trinervia were equal in their capacity to transfer reduced O₂ inhibition of AP and CO₂ compensation concentration values to hybrids with F. linearis (C₃-C₄), although hybrids with F. trinervia had higher PEPC activity. The O₂ inhibition of AP was correlated with the logarithm of activities of phosphoenolpyruvate carboxylase (r = −0.95) and NADP-malic enzyme (r = −0.87). These results confirm that C₄ traits can be transferred by hybridization of C₃-C₄ and C₄ or C₄-like species, with a higher degree of C₄ photosynthesis than exists in C₃-C₄ species, and at least in F. brownii × F. linearis, fertile progeny are obtained.     

Analysis of Promoter Activity for the Gene Encoding Pyruvate Orthophosphate Dikinase in Stably Transformed C4 Flaveria Species

The C₄ enzyme pyruvate orthophosphate dikinase is encoded by a single gene, Pdk, in the C₄ plant Flaveria trinervia. This gene also encodes enzyme isoforms located in the chloroplast and in the cytosol that do not have a function in C₄ photosynthesis. Our goal is to identify cis-acting DNA sequences that regulate the expression of the gene that is active in the C₄ cycle. We fused 1.5 kb of a 5′ flanking region from the Pdk gene, including the entire 5′ untranslated region, to the uidA reporter gene and stably transformed the closely related C₄ species Flaveria bidentis. β-Glucuronidase (GUS) activity was detected at high levels in leaf mesophyll cells. GUS activity was detected at lower levels in bundle-sheath cells and stems and at very low levels in roots. This lower-level GUS expression was similar to the distribution of mRNA encoding the nonphotosynthetic form of the enzyme. We conclude that cis-acting DNA sequences controlling the expression of the C₄ form in mesophyll cells and the chloroplast form in other cells and organs are co-located within the same 5′ region of the Pdk gene.     

Enzymic and Photosynthetic Characteristics of Reciprocal F(1) Hybrids of Flaveria pringlei (C(3)) and Flaveria brownii (C(4)-Like Species)

The activities of key C₄ enzymes in gel-filtered, whole-leaf extracts and the photosynthetic characteristics for reciprocal F₁ hybrids of Flaveria pringlei (C₃) and F. brownii (C₄-like species) were measured to determine whether any inherited C₄-photosynthetic traits are responsible for their reduced CO₂ compensation concentration values (AS Holaday, S Talkmitt, ME Doohan Plant Sci 41: 31-39). The activities of phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, and NADP-malic enzyme (ME) for the reciprocal hybrids are only about 7 to 17% of those for F. brownii, but are three- to fivefold greater than the activities for F. pringlei. The low activities of these enzymes in the hybrids appear to be the result of a partial dominance of F. pringlei genes over certain F. brownii genes. However, no such dominance occurs with respect to the expression of genes for NADP-malate dehydrogenase, which is as active in the hybrids as in F. brownii. In contrast to the situation with the enzymes above, cytoplasmic factors appear to determine the inheritance of NAD-ME. The NAD-ME activity in each hybrid is comparable to that in the respective maternal parent. Pulse-chase ¹⁴CO₂ incorporation analyses at ambient CO₂ levels indicate that the hybrids initially assimilate 7 to 9% of the total assimilated CO₂ into C₄ acids as compared to 3.5% for F. pringlei. In the hybrids, the percentage of ¹⁴C in malate decreases from an average of 6.5 to 2.1% after a 60-second chase in ¹²CO₂/air. However, this apparent C₄-cycle activity is too limited or inefficient to substantially alter CO₂ exchange from that in F. pringlei, since the values of net photosynthesis and O₂ inhibition of photosynthesis are similar for the hybrids and F. pringlei. Also, the ratio of the internal to the external CO₂ concentration and the initial slopes of the plot of CO₂ concentration versus net photosynthesis are essentially the same for the hybrids and F. pringlei. At 45 micromoles CO₂ per mole and 0.21 mole O₂ per mole, the hybrids assimilate nearly fivefold more CO₂ into C₄ acids than does F. pringlei. Some turnover of the malate pool occurs in the hybrids, but the labelling of the photorespiratory metabolites, glycine and serine, is the same in these plants as it is in F. pringlei. Thus, although limited C₄-acid metabolism may operate in the hybrids, we conclude that it is not effective in altering O₂ inhibition of CO₂ assimilation. The ability of the hybrids to assimilate more CO₂ via phosphoenolpyruvate carboxylase at low levels of CO₂ than does F. pringlei may result in an increased rate of reassimilation of photorespiratory CO₂ and CO₂ compensation concentrations below that of their C₃ parent. If the hybrids do possess a limited C₄ cycle, it must operate intracellularly. They are not likely to have inherited an intercellular compartmentation of C₄ enzymes, since F. brownii has incomplete compartmentation of key C₃ and C₄ enzymes.     

Loss of the Transit Peptide and an Increase in Gene Expression of an Ancestral Chloroplastic Carbonic Anhydrase Were Instrumental in the Evolution of the Cytosolic C4 Carbonic Anhydrase in Flaveria

C₄ photosynthesis has evolved multiple times from ancestral C₃ species. Carbonic anhydrase (CA) catalyzes the reversible hydration of CO₂ and is involved in both C₃ and C₄ photosynthesis; however, its roles and the intercellular and intracellular locations of the majority of its activity differ between C₃ and C₄ plants. To understand the molecular changes underlying the evolution of the C₄ pathway, three cDNAs encoding distinct β-CAs (CA1, CA2, and CA3) were isolated from the leaves of the C₃ plant Flaveria pringlei. The phylogenetic relationship of the F. pringlei proteins with other embryophyte β-CAs was reconstructed. Gene expression and protein localization patterns showed that CA1 and CA3 demonstrate high expression in leaves and their products localize to the chloroplast, while CA2 expression is low in all organs examined and encodes a cytosolic enzyme. The roles of the F. pringlei enzymes were considered in light of these results, other angiosperm β-CAs, and Arabidopsis (Arabidopsis thaliana) “omics” data. All three F. pringlei CAs have orthologs in the closely related C₄ plant Flaveria bidentis, and comparisons of ortholog sequences, expression patterns, and intracellular locations of their products indicated that CA1 and CA2 have maintained their ancestral role in C₄ plants, whereas modifications to the C₃ CA3 gene led to the evolution of the CA isoform that catalyzes the first step in the C₄ photosynthetic pathway. These changes included the loss of the chloroplast transit peptide and an increase in gene expression, which resulted in the high levels of CA activity seen in the cytosol of C₄ mesophyll cells.     

Degree of C(4) Photosynthesis in C(4) and C(3)-C(4)Flaveria Species and Their Hybrids : II. Inhibition of Apparent Photosynthesis by a Phosphoenolpyruvate Carboxylase Inhibitor

Hybrids have been made between species of Flaveria exhibiting varying levels of C₄ photosynthesis. The degree of C₄ photosynthesis expressed in four interspecific hybrids (Flaveria trinervia [C₄] × F. linearis [C₃-C₄], F. brownii [C₄-like] × F. linearis, and two three-species hybrids from F. trinervia × [F. brownii × F. linearis]) was estimated by inhibiting phosphoenolpyruvate carboxylase in vivo with 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate (DCDP). The inhibitor was fed to detached leaves at a concentration of 4 mm, and apparent photosynthesis was measured at atmospheric levels of CO₂ and at 20 and 210 mL L⁻¹ of O₂. Photosynthesis at 210 mL L⁻¹ of O₂ was inhibited 32% by DCDP in F. linearis, by 60% in F. brownii, and by 87% in F. trinervia. Inhibition in the hybrids ranged from 38 to 52%. The inhibition of photosynthesis by 210 mL L⁻¹ of O₂ was increased when DCDP was used, except in the C₄ species, F. trinervia, in which photosynthesis was insensitive to O₂. Except for F. trinervia, control plants with less O₂ sensitivity (more C₄-like) exhibited a progressively greater change in O₂ inhibition of photosynthesis when treated with DCDP. This increased O₂ inhibition probably resulted from decreased CO₂ concentrations in bundle sheath cells due to inhibition of phosphoenolpyruvate carboxylase. The inhibition of photosynthesis by DCDP is concluded to underestimate the degree of C₄ photosynthesis in the interspecific hybrids because increased direct assimilation of atmospheric CO₂ by ribulose bisphosphate carboxylase may compensate for inhibition of phosphoenolpyruvate carboxylase.     

Environmental Effects on Photorespiration of C(3)-C(4) Species : I. Influence of CO(2) and O(2) during Growth on Photorespiratory Characteristics and Leaf Anatomy

The possibility of altering CO₂ exchange of C₃-C₄ species by growing them under various CO₂ and O₂ concentrations was examined. Growth under CO₂ concentrations of 100, 350, and 750 micromoles per mole had no significant effect on CO₂ exchange characteristics or leaf anatomy of Flaveria pringlei (C₃), Flaveria floridana (C₃-C₄), or Flaveria trinervia (C₄). Carboxylation efficiency and CO₂ compensation concentrations in leaves of F. floridana developed under the different CO₂ concentrations were intermediate to F. pringlei and F. trinervia. When grown for 12 days at an O₂ concentration of 20 millimoles per mole, apparent photosynthesis was strongly inhibited in Panicum milioides (C₃-C₄) and to a lesser degree in Panicum laxum (C₃). In P. milioides, acute starch buildup was observed microscopically in both mesophyll and bundle sheath cells. Even after only 4 days exposure to 20 millimoles per mole O₂, the presence of starch was more pronounced in leaf cross-sections of P. milioides compared to those at 100 and 210 millimoles per mole. Even though this observation suggests that P. milioides has a different response to low O₂ with respect to translocation of photosynthate or sink activity than C₃ species, the concentration of total available carbohydrate increased in shoots of all species by 33% or more when grown at low O₂. This accumulation occurred even though relative growth rates of Festuca arundinacea (C₃) and P. milioides grown for 4 days at 210 millimoles per mole O₂, were inhibited 83 and 37%, respectively, when compared to plants grown at 20 millimoles per mole O₂.     

Evolution of the C<Subscript>4</Subscript> phosphoenolpyruvate carboxylase promoter of the C<Subscript>4</Subscript> species <Emphasis Type="Italic">Flaveria trinervia</Emphasis>: the role of the proximal promoter region

Background  

The key enzymes of photosynthetic carbon assimilation in C₄ plants have evolved independently several times from C₃ isoforms that were present in the C₃ ancestral species. The C₄ isoform of phosphoenolpyruvate carboxylase (PEPC), the primary CO₂-fixing enzyme of the C₄ cycle, is specifically expressed at high levels in mesophyll cells of the leaves of C₄ species. We are interested in understanding the molecular changes that are responsible for the evolution of this C₄-characteristic PEPC expression pattern, and we are using the genus Flaveria (Asteraceae) as a model system. It is known that cis-regulatory sequences for mesophyll-specific expression of the ppcA1 gene of F. trinervia (C₄) are located within a distal promoter region (DR).     

Photosynthetic Characteristics of C(3)-C(4) Intermediate Flaveria Species : III. Reduction of Photorespiration by a Limited C(4) Pathway of Photosynthesis in Flaveria ramosissima

The initial products of photosynthesis by the C₃ species Flaveria cronquistii, the C₄ species F. trinervia, and the C₃-C₄ intermediate species F. ramosissima were determined using a pulse-chase technique with ¹⁴CO₂-¹²CO₂. The intermediate species F. ramosissima incorporated at least 42% of the total soluble ¹⁴C fixed into malate and aspartate after 10 seconds of photosynthesis in ¹⁴CO₂, as compared with 90% for the C₄ species F. trinervia and 5% for the C₃ species F. cronquistii. In both F. ramosissima and F. trinervia, turnover of labeled malate and aspartate occurred during a chase period in ¹²CO₂, although the rate of turnover was slower in the intermediate species. Relative to F. cronquistii, F. ramosissima showed a reduced incorporation of radioactivity into serine and glycine during the pulse period. These results indicate that a functional C₄ pathway of photosynthesis is operating in F. ramosissima which can account for its reduced level of photorespiration, and that this species is a true biochemical intermediate between C₃ and C₄ plants.     

Anatomical and enzymic studies of leaves of a C3 × C4Flaveria F1 hybrid exhibiting reduced photorespiration

Flaveria pringlei exhibits C₃ CO₂ compensation concentration (Г) values averaging 53 μl CO₂/l at 21% (v/v) O₂ and 25 ± 2°C. When this species is hybridized with the C₄ species, F. brownii (male) ($\text{Г = 6 μ}\text{l CO}{}_2\text{/}\text{l}$), the F₁ hybrid plants exhibit an average Г value of 31 μl CO₂/l at 21% O₂.Although light micrographs of leaf cross-sections show that the leaves of the hybrid plants possess the mesophyll arrangement characteristic of F. pringlei leaves, the hybrid plants have some bundle-sheath chloroplasts. However, the numbers of these organelles do not appear to be intermediate with respect to the numbers in the parents and are closest to the small number present in the bundle-sheath cells of F. pringlei leaves. The activities of key C₄ enzymes (in μmol · mg Chl^?1 · h^?1) are: phosphoenolpyruvate (PEP) carboxylase, 121; pyruvate, orthophosphate (P_i) dikinase, 26; NADP-malate dehydrogenase, 2529; and NADP-malic enzyme, 82. All of these activities are substantially higher than in F. pringlei, but are only 7–10% of those in F. brownii (with the exception of the NADP-malate dehydrogenase activity). These data suggest that a C₄ cycle might be operating to a limited extent in the hybrid plants resulting in reduced photorespiration.Whether or not C₄ photosynthesis occurs in these hybrid plants, they represent the first reported C₃ × C₄ F₁ hybrids to exhibit reduced Γ-values. This cross and its reciprocal should be useful models for studying the anatomical and biochemical factors determining the development of limited C₄ photosynthesis in C₃ species.     

Molecular properties of phosphoenolpyruvate carboxylase from C3, C3−C4 intermediate,and C4 Flaveria species

Phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) from Flaveria trinervia Mohr (C₄), F. floridana Johnston (C₃–C₄), and F. cronquistii Powell (C₃) leaves were compared by electrotransfer blotting/enzyme-linked immunoassay (Western-blot analysis), mobility of the native enzyme in polyacrylamide gels and in isoelectric focusing (IEF) gels, peptide mapping, and in-vitro translation of RNA isolated from each plant. The PEPCases from the C₃ and C₃–C₄ plants were very similar to each other in terms of electrophoretic mobilities on gels and isoenzyme patterns on IEF gels, and identical in peptide mapping. Quantitative differences were noted, however, in that the C₃–C₄ intermediate plant contained more PEPCase overall and that the relative activity of individual isoenzymes shifted between the C₃ and C₃–C₄ intermediate PEPCases. The PEPCase from the C₄ plant had a different isoenzyme pattern, a different peptide map, and was far more abundant than the other two enzymes. Western blot analysis demonstrated the cross-reactivity of PEPCases from all three Flaveria species with antibody raised against maize PEPCase. The results provide evidence, at the molecular level, that supports the view of C₃–C₄ intermediate species as C₃-like plants with some C₄-like photosynthetic characteristics, but there are differences from the C₃ plant in the quantity and properties of the PEPCase from the C₃–C₄ intermediate plant.Abbreviations IEF isoelectric focusing - kDa kilodalton - PEPCase phosphoenolpyruvate carboxylase - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase     

Photosynthesis in Flaveria brownii A.M. Powell : A C(4)-Like C(3)-C(4) Intermediate

Leaves of Flaveria brownii exhibited slightly higher amounts of oxygen inhibition of photosynthesis than the C₄ species, Flaveria trinervia, but considerably less than the C₃ species, Flaveria cronquistii. The photosynthetic responses to intercellular CO₂, light and leaf temperature were much more C₄-like than C₃-like, although 21% oxygen inhibited the photosynthetic rate, depending on conditions, up to 17% of the photosynthesis rate observed in 2% O₂. The quantum yield for CO₂ uptake in F. brownii was slightly higher than that for the C₄ species F. trinervia in 2% O₂, but not significantly different in 21% O₂. The quantum yield was inhibited 10% in the presence of 21% O₂ in F. brownii, yet no significant inhibition was observed in F. trinervia. An inhibition of 27% was observed for the quantum yield of F. cronquistii in the presence of 21% O₂. The photosynthetic response to very low intercellular CO₂ partial pressures exhibited a unique pattern in F. brownii, with a break in the linear slope observed at intercellular CO₂ partial pressure values between 15 and 20 μbar when analyzed in 21% O₂. No significant break was observed when analyzed in 2% O₂. When taken collectively, the gas-exchange results reported here are consistent with previous biochemical studies that report incomplete intercellular compartmentation of the C₃ and C₄ enzymes in this species, and suggest that F. brownii is an advanced, C₄-like C₃-C₄ intermediate.     

Characterization of a novel class of plant homeodomain proteins that bind to the C4 phosphoenolpyruvate carboxylase gene of Flaveria trinervia

We are interested in the regulatory mechanisms responsible for the mesophyll-specific expression of C₄ phosphoenolpyruvate carboxylase (PEPCase). A one-hybrid screen resulted in the cloning of four different members of a novel class of plant homeodomain proteins, which are most likely involved in the mesophyll-specific expression of the C₄ PEPCase gene in C₄ species of the genus Flaveria. Inspection of the homeodomains of the four proteins reveals that they share many common features with homeodomains described so far, but there are also significant differences. Interestingly, this class of homeodomain proteins occurs also in Arabidopsis thaliana and other C₃ plants. One-hybrid experiments as well as in vitro DNA binding studies confirmed that these novel homeodomain proteins specifically interact with the proximal region of the C₄ PEPCase gene. The N-terminal domains of the homeodomain proteins contain highly conserved sequence motifs. Two-hybrid experiments show that these motifs are sufficient to confer homo- or heterodimer formation between the proteins. Mutagenesis of conserved cysteine residues within the dimerization domain indicates that these residues are essential for dimer formation. Therefore, we designate this novel class of homeobox proteins ZF-HD, for zinc finger homeodomain protein. Our data suggest that the ZF-HD class of homeodomain proteins may be involved in the establishment of the characteristic expression pattern of the C₄ PEPCase gene.     

Photosynthetic Characteristics of C(3)-C(4) Intermediate Flaveria Species : I. Leaf Anatomy, Photosynthetic Responses to O(2) and CO(2), and Activities of Key Enzymes in the C(3) and C(4) Pathways

Four species of the genus Flaveria, namely F. anomala, F. linearis, F. pubescens, and F. ramosissima, were identified as intermediate C₃-C₄ plants based on leaf anatomy, photosynthetic CO₂ compensation point, O₂ inhibition of photosynthesis, and activities of C₄ enzymes. F. anomala and F. ramosissima exhibit a distinct Kranz-like leaf anatomy, similar to that of the C₄ species F. trinervia, while the other C₃-C₄ intermediate Flaveria species possess a less differentiated Kranz-like leaf anatomy. Photosynthetic CO₂ compensation points of these intermediates at 30°C were very low relative to those of C₃ plants, ranging from 7 to 14 microliters per liter. In contrast to C₃ plants, net photosynthesis by the intermediates was not sensitive to O₂ concentrations below 5% and decreased relatively slowly with increasing O₂ concentration. Under similar conditions, the percentage inhibition of photosynthesis by 21% O₂ varied from 20% to 25% in the intermediates compared with 28% in Lycopersicon esculentum, a typical C₃ species. The inhibition of carboxylation efficiency by 21% O₂ varied from 17% for F. ramosissima to 46% for F. anomala and were intermediate between the C₄ (2% for F. trinervia) and C₃ (53% for L. esculentum) values. The intermediate Flaveria species, especially F. ramosissima, have substantial activities of the C₄ enzymes, phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, NADP-malic enzyme, and NADP-malate dehydrogenase, indicating potential for C₄ photosynthesis. It appears that these Flaveria species may be true biochemical C₃-C₄ intermediates.