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The evolution of C4 photosynthesis in many taxa involves the establishment of a two-celled photorespiratory CO2 pump, termed C2 photosynthesis. How C3 species evolved C2 metabolism is critical to understanding the initial phases of C4 plant evolution. To evaluate early events in C4 evolution, we compared leaf anatomy, ultrastructure, and gas-exchange responses of closely related C3 and C2 species of Flaveria, a model genus for C4 evolution. We hypothesized that Flaveria pringlei and Flaveria robusta, two C3 species that are most closely related to the C2 Flaveria species, would show rudimentary characteristics of C2 physiology. Compared with less-related C3 species, bundle sheath (BS) cells of F. pringlei and F. robusta had more mitochondria and chloroplasts, larger mitochondria, and proportionally more of these organelles located along the inner cell periphery. These patterns were similar, although generally less in magnitude, than those observed in the C2 species Flaveria angustifolia and Flaveria sonorensis. In F. pringlei and F. robusta, the CO2 compensation point of photosynthesis was slightly lower than in the less-related C3 species, indicating an increase in photosynthetic efficiency. This could occur because of enhanced refixation of photorespired CO2 by the centripetally positioned organelles in the BS cells. If the phylogenetic positions of F. pringlei and F. robusta reflect ancestral states, these results support a hypothesis that increased numbers of centripetally located organelles initiated a metabolic scavenging of photorespired CO2 within the BS. This could have facilitated the formation of a glycine shuttle between mesophyll and BS cells that characterizes C2 photosynthesis.

Improving global yields of important agricultural crops is a complex challenge. Enhancing yield and resource use by engineering improvements to photosynthetic carbon assimilation is one potential solution. During the last 40 million years C 4 photosynthesis has evolved multiple times, enabling plants to evade the catalytic inadequacies of the CO 2 -fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). Compared with their C 3 ancestors, C 4 plants combine a faster rubisco with a biochemical CO 2 -concentrating mechanism, enabling more efficient use of water and nitrogen and enhanced yield. Here we show the versatility of plastome manipulation in tobacco for identifying sequences in C 4 -rubisco that can be transplanted into C 3 -rubisco to improve carboxylation rate ( V C ). Using transplastomic tobacco lines expressing native and mutated rubisco large subunits (L-subunits) from Flaveria pringlei (C 3 ), Flaveria floridana (C 3 -C 4 ), and Flaveria bidentis (C 4 ), we reveal that Met-309-Ile substitutions in the L-subunit act as a catalytic switch between C 4 ( 309 Ile; faster V C , lower CO 2 affinity) and C 3 ( 309 Met; slower V C , higher CO 2 affinity) catalysis. Application of this transplastomic system permits further identification of other structural solutions selected by nature that can increase rubisco V C in C 3 crops. Coengineering a catalytically faster C 3 rubisco and a CO 2 -concentrating mechanism within C 3 crop species could enhance their efficiency in resource use and yield.

The genus Flaveria consists of 23 species with significant variation in photosynthetic physiologies. We tested whether photosynthetic pathway variation in seven co-existing Flaveria species corresponds to geographic distributions or physiological performance in C3, C4, and intermediate species growing under natural conditions in south-central Mexico. We found that Flaveria pringlei (C3) was the most widely distributed species with multiple growth habits. Numerous populations of Flaveria kochiana (C4), a recently described species with a previously unknown distribution, were located in the Mixtec region of Oaxaca. Flaveria cronquistii (C3) and Flaveria ramosissima (C3-C4) were only located in the Tehuacan Valley region while Flaveria trinervia (C4) was widely distributed. Only one population of Flaveria angustifolia (C3-C4) and Flaveria vaginata (C4-like) were located near Izucar de Matamoros. Midday leaf water potential differed significantly between Flaveria species, but did not vary according to growth habit or photosynthetic pathway. The quantum yield of photosystem II did not vary between species, despite large differences in leaf nitrogen content, leaf shape, plant size and life histories. We did not find a direct relationship between increasing C4 cycle characteristics and physiological performance in the Flaveria populations examined. Furthermore, C3 species were not found at higher elevation than C4 species as expected. Our observations indicate that life history traits and disturbance regime may be the primary controllers of Flaveria distributions in south-central Mexico.

During the evolution of angiosperms, C 4 phosphoenolpyruvate carboxylases have evolved several times independently from ancestral non-photosynthetic isoforms. They show distinct kinetic and regulatory properties when compared with the C 3 isozymes. To identify the evolutionary alterations which are responsible for C 4 -specific properties, particularly the increased tolerance towards the allosteric inhibitor L-malate, the photosynthetic phosphoenolpyruvate carboxylase of Flaveria trinervia Mohr (C 4 ) and its ortholog from the closely related C 3 plant Flaveria pringlei Gand. were examined using reciprocal enzyme chimeras. The main determinants for a high tolerance towards L-malate were located in the C-terminal region of the C 4 enzyme. The effect of interchanging the region between amino acids 296 and 437 was strongly dependent upon the activation of the enzyme by glucose-6-phosphate. This confirms earlier observations that this region is important for the regulation of the enzyme by glucose-6-phosphate and that it harbours determinants for the different response of the C 3 and the C 4 enzyme towards this allosteric activator. In addition, it was possible to demonstrate that the only C 4 -specific amino acid, a serine in the C-terminal part of the enzyme, is not involved in conferring an increased L-malate tolerance to the C 4 enzyme.

C4 photosynthesis presents a sophisticated integration of two complementary cell types, mesophyll and bundle sheath cells. It relies on the differential expression of the genes encoding the component enzymes and transporters of this pathway. The entry enzyme of C4 photosynthesis, phosphoenolpyruvate carboxylase (PEPC), is found exclusively in mesophyll cells, and the expression of the corresponding gene is regulated at the transcriptional level. In the C4 dicot Flaveria trinervia, the mesophyll-specific expression of the C4 PEPC gene (ppcA) depends on a 41-bp segment in the distal promoter region referred to as MEM1 (for mesophyll expression module1). Here, we show that a MEM1 sequence found in the orthologous ppcA gene from the C3 species Flaveria pringlei is not able to direct mesophyll-specific gene expression. The two orthologous MEM1 sequences of F. pringlei and F. trinervia differ at two positions, a G-to-A exchange and the insertion of the tetranucleotide CACT. Changes at these two positions in the C3 MEM1 sequence were necessary and sufficient to create a mesophyll-specificity element during C4 evolution. The MEM1 of F. trinervia enhances mesophyll expression and concomitantly represses expression in bundle sheath cells and vascular bundles.

C4 photosynthesis is characterized by a division of labour between two different photosynthetic cell types, mesophyll and bundle‐sheath cells. Relying on phosphoenolpyruvate carboxylase (PEPC) as the primary carboxylase in the mesophyll cells a CO2 pump is established in C4 plants that concentrates CO2 at the site of ribulose 1,5‐bisphosphate carboxylase/oxygenase in the bundle‐sheath cells. The C4 photosynthetic pathway evolved polyphyletically implying that the genes encoding the C4 PEPC originated from non‐photosynthetic PEPC progenitor genes that were already present in the C3 ancestral species. The dicot genus Flaveria (Asteraceae) is a unique system in which to investigate the molcular changes that had to occur in order to adapt a C3 ancestral PEPC gene to the special conditions of C4 photosynthesis. Flaveria contains not only C3 and C4 species but also a large number of C3–C4 intermediates which vary to the degree in which C4 photosynthetic traits are expressed. The C4 PEPC gene of Flaveria trinervia, which is encoded by the ppcA gene class, is highly expressed but only in mesophyll cells. The encoded PEPC protein possesses the typical kinetic and regulatory features of a C4‐type PEPC. The orthologous ppcA gene of the C3 species Flaveria pringlei encodes a typical non‐photosynthetic, C3‐type PEPC and is weakly expressed with no apparent cell or organ specificity. PEPCs of the ppcA type have been detected also in C3–C4 intermediate Flaveria species. These orthologous PEPCs have been used to determine the molecular basis for C4 enzyme characteristics and to understand their evolution. Comparative and functional analyses of the ppcA promoters from F. trinervia and F. pringlei make it possible to identity the cis‐regulatory sequences for mesophyll‐specific gene expression and to search for the corresponding trans‐regulatory factors.

C4 phosphoenolpyruvate carboxylases (PEPCase; EC 4.1.1.3) have evolved from ancestral non-photosynthetic (C3) isoforms during the evolution of angiosperms and thereby gained distinct kinetic and regulatory properties. In order to obtain insight into this evolutionary process we have studied the C3 isoforms, ppcB and ppcC, of the C4 dicot Flaveria trinervia (Spreng.) C. Mohr and compared them with the C4 enzyme of this species, ppcA, and its orthologue in the C3 species F. pringlei Gandoger. Phylogenetic analyses indicate that the ppcB PEPCase is the closest relative of the ppcA enzyme. In addition, the presence of ppcB also in the closely related C3 species F. pringlei suggests that this gene was present already in the ancestral C3 species and consequently that ppcA has evolved by gene duplication of ppcB. Investigation of the enzymatic properties of the ppcB and ppcC enzymes showed low and similar K 0.5-PEP values and limited activation by glucose-6-phosphate, typical of non-photosynthetic PEPCases, at pH 8.0. However, at the more physiological pH of 7.6, the ppcC enzyme displayed a substantially higher K 0.5-PEP than the ppcB counterpart, indicating their involvement in different metabolic pathways. This indication was strengthened by malate inhibition studies in which the ppcC enzyme showed 10 times higher tolerance to the inhibitor. The ppcA enzyme was, however, by far the most tolerant enzyme towards malate. Interestingly, the increased malate tolerance was correlated with a decrease in enzyme efficiency displayed by the turnover constant k cat. We therefore suggest that the increased malate tolerance, which is imperative for an efficient C4 cycle, is connected with a decreased enzyme efficiency that in turn is compensated by increased enzyme expression.

A cytosolic NADP-malic enzyme (CYTME) has been described previously in several plants, all C3 species. CYTME is distinct from the chloroplastic NADP-malic enzyme (CHLME) that is highly active in C4 species. We show that at least oneCytMe gene is present in all Flaveriaspp., including C3, C4, and C3-C4 intermediate types. Based on theCytMe expression patterns in Flaveria pringlei (C3) and Flaveria trinervia(C4), we suggest CYTME has several distinct roles, including the supplying of NADPH for cytosolic metabolism, the supporting of wound response or repair, and the balancing of cellular pH in illuminated leaves. These three roles are likely correlated withCytMe mRNAs of apparent sizes 2.0, 2.2, and 2.4 kb, respectively, which differ in the length of the 5′ untranslated regions. Various regulatory mechanisms involving RNA processing and translational efficiency are discussed.

In the most common C4 pathway for carbon fixation, an NADP-malic enzyme (NADP-ME) decarboxylates malate in the chloroplasts of bundle sheath cells. Isoforms of plastidic NADP-ME are encoded by two genes in all species of Flaveria, including C3, C3-C4 intermediate, and C4 types. However, only one of these genes,ChlMe1, encodes the enzyme that functions in the C4 pathway. We compared the expression patterns of theChlMe1 and ChlMe2 genes in developing leaves of Flaveria pringlei (C3) andFlaveria trinervia (C4) and in transgenicFlaveria bidentis (C4).ChlMe1 expression in C4 species increases in leaves with high C4 pathway activity. In the C3species F. pringlei, ChlMe1 expression is transient and limited to early leaf development. In contrast,ChlMe2 is expressed in C3 and C4species concurrent with stages in chloroplast biogenesis. Because previous studies suggest that NADP-ME activities generally reflect the level of its mRNA abundance, we discuss possible roles ofChlMe1 and ChlMe2 based on these expression patterns.

In C4 plants, phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31), a key enzyme in C4 photosynthesis, is controlled by reversible phosphorylation of a conserved Ser residue near the N-terminus. We now report the first cloning of a cDNA from a C4 plant, Flaveria trinervia, which encodes the specific protein kinase (FtPEPC-PK) involved in the phosphorylation of C4-form PEPC. Several lines of supportive evidence are: strict substrate specificity of the recombinant enzyme, prominent light/dark response of the transcript level and abundant expression in leaves of C4 plant (F. trinervia) but very low expression in a C3 plant of the same genus (Flaveria pringlei). We also discuss the possibility that the FtPEPC-PK gene has co-evolved with the PEPC gene to participate in C4 photosynthesis.

The combined effects of O2 on net rates of photosynthesis, photosystem II activity, steady-state pool size of key metabolites of photosynthetic metabolism in the C4 pathway, C3 pathway and C2 photorespiratory cycle and on growth were evaluated in the C4 species Amaranthus edulis and the C3 species Flaveria pringlei. Increasing O2 reduced net CO2 assimilation in F. pringlei due to an increased flux of C through the photorespiratory pathway. However, in A. edulis increasing O2 up to 5‐10% stimulated photosynthesis. Analysis of the pool size of key metabolites in A. edulis suggests that while there is some O2 dependent photorespiration, O2 is required for maximizing C4 cycle activity to concentrate CO2 in bundle sheath cells. Therefore, the response of net photosynthesis to O2 in C4 plants may result from the balance of these two opposing effects. Under 21 versus 5% O2, growth of A. edulis was stimulated about 30% whereas that of F. pringlei was inhibited about 40%.

biphosphate carboxylase-oxygenase (Rubisco), to oxygen and to carbon dioxide and the frequent induction Leaf diffusion conductance much increased when of anti-oxidant enzymes in plant tissues exposed to high Xanthium strumarium (C 3 ), Atriplex prostata (C 3 ) and ambient O 2 . Raven concluded that, apart from fire risks, Flaveria pringlei (C 3 ), F. sonorensis (C 3 /C 4 ), F. floridana plants would have been able to cope well. Beerling and (C 3 /C 4 ), and F. trinervia (C 4 ) plants were grown under Woodward (1997) reviewed evidence for changing fossil relatively high (28 kPa) ambient oxygen. This phenom- stomatal density over the Phanerozoic. They ascribe such enon, which increases sensitivity to water stress, changes as occurred mainly to the varying levels of could have adversely affected vegetation during the atmospheric CO 2 . However, their data show little correla- late Cretaceous, high O 2 episode. tion between CO 2 and stomatal density for CO 2 in the range of 20-80 Pa. Beerling et al. (1998a) grew plants in

Variations in length and charge of the C-terminus of the Rubisco large subunit (L) can be seen in L from different phylogenetic lineages. We examined the catalytic parameters of Rubisco from higher plants and from engineered Synechococcus rbcL in relation to differences in the C-terminus. Among three selected higher plants, spinach, wheat, and Flaveria pringlei, spinach Rubisco with the shortest C-tail extension (D-473 + 2) showed the lowest temperature response. The response of Rubisco from wheat (D-473 + 4) was intermediate, and the enzyme from F. pringlei (D-473 + 12) displayed the highest temperature response in terms of Vmax for the carboxylase reaction. This observation was further investigated in a model system: the temperature-response for carboxylation was enhanced after lengthening the C-terminus of the Synechococcus large subunit protein by two amino acid residues (DK). The results point towards the length of the C-terminus as an additional factor for controlling Rubisco activity, especially as an adaptation that widens the temperature range in which the enzyme can function. Longer C-termini, we suggest, could establish additional interactions with the protein surface.

The response curves of leaf photosynthesis to varying light, temperature and leaf-to-air vapour pressure deficit were measured in the C 3 plants Flaveria pringlei and Oryza saliva in normal air with a computerized open infrared gas analysis (IRGA) system, and the photochemical efficiency of photosystem II, described as (1-F s /F' m ) after Genty, Briantais & Baker (1989, Biochimica et Biophysica Acta 990, 87-92), was simultaneously measured with a modulated fluorometer. A model was written for rates of CO 2 fixation as a function of the true rate of O 2 evolution measured by fluorescence analysis (Jo 2 ), mesophyll conductance and intercellular CO 2 partial pressure. A second model was developed for rates of CO 2 fixation as a function of J0 2 , mesophyll conductance and stomatal conductance. In the latter case, leaf stomatal conductance was simulated using the stomatal model proposed by Leuning (1995, Plant, Cell and Environment 18, 339-355). The rates of CO 2 fixation predicted from the models were similar to rates measured by IRGA. The results indicate that there is potential to measure CO 2 fixation in C 3 plants by combining the non-invasive measurement of J0 2 by chlorophyll fluorescence analysis with the stomatal conductance model.

To gain an understanding of the molecular events underlying the evolution of C4 photosynthesis, we have undertaken a detailed study of the NADP-malic enzyme gene family in C4 and C3 species of Flaveria. Three genomic clones from the C4 species Flaveria bidentis were characterized and found to encode two highly similar chloroplastic forms of NADP-malic enzyme, termed ME1 and ME2. Genomic Southern blotting with gene-specific probes showed that both Me1 and Me2 are found in Flaveria trinervia (C4) and Flaveria pringlei (C3) as well as in F. bidentis. Northern blots demonstrated that Me1 expression in leaves parallels the degree of C4 photosynthesis in seven Flaveria species. Furthermore, whereas Me2 was expressed at a low level in both roots and leaves of F. bidentis, Me1 expression was seen only in leaves and was light-regulated. We discuss these results in the context of the evolution of C4 photosynthesis in Flaveria.

In Flaveria pringlei, a C3 plant, P protein of the glycine-cleavage system is encoded by a small gene family consisting of at least five transcriptionally active genes. We have cloned and sequenced two of these genes, gdcsPA and gdcsPB, and provide the first detailed report on the complete structure of eukaryotic gdcsP genes. Based on the lengths of exons and intervening sequences, the P-protein genes can be subdivided into two parts. In both cases the N-terminal region consists of one very long exon followed by a long intron. In contrast, the C-terminal parts show a complex mosaic structure of relatively small exons and introns. A highly conserved leucine-zipper motif was identified, which is supposed to participate in the assembly of the glycine decarboxylase multienzyme complex. The transcript derived from the gdcsPA sequence corresponds perfectly to a leaf cDNA isolated earlier. Reverse-transcriptase PCR experiments show that both genes are preferentially active in leaves. Stems contain distinctly less P protein mRNA and the relative level in roots is very low but still clearly detectable. In all three organs, but most significantly in roots, the gdcsPA transcript level is distinctly higher than that of gdcsPB. Analysis of promoter-beta-glucuronidase fusions in transgenic tobacco suggests that far-upstream elements enhance the transcriptional activity of both genes in leaves relative to stems. The analysis of distal gdcsPA promoter deletions reveals the presence of regulatory elements acting with a distinct organ preference and indicates their approximate location.

During the evolution of C4 plants from C3 plants, both the function and intracellular location of carbonic anhydrase (CA) have changed. To determine whether these changes are due to changes at the molecular level, we have studied the cDNA sequences and the expression of CA from Flaveria species demonstrating different photosynthetic pathways. In leaf extracts from F. bidentis (C4), F. brownii (C4-like), F. linearis (C3-C4) and F. pringlei (C3), two polypeptides of M(r) 31 kDa and 35 kDa cross-reacted with anti-spinach CA antibodies. However, the relative labelling intensities of the two polypeptides differed depending on the species. Northern blot analysis indicated at least two CA transcripts are present in each Flaveria species with sizes ranging from 1.1 to 1.6 kb. Carbonic anhydrase cDNAs from all four Flaveria species studied encode an open reading frame for a polypeptide of 35-36 kDa. The amino acid sequences deduced from all four Flaveria cDNAs share at least 70% homology with the sequences of other dicot CAs. The F. bidentis (C4) CA sequence was found to be the least similar of the Flaveria proteins and, as most of the sequence dissimilarity was found in the first third of the CA molecule, these differences may be involved in the intracellular targeting of CA. A neighbour-joining tree inferred from CA amino acid sequences showed that the Flaveria CAs cluster with other dicot CAs forming a group distinct from those of monocot CAs and prokaryotic and Chlamydomonas periplasmic CAs.

We have isolated and sequenced cDNA clones encoding T-protein of the glycine decarboxylase complex from three plant species, Flaveria pringlei, Solanum tuberosum and Pisum sativum. The predicted amino acid sequences of these clones are at least 87% identical and all are similar to the predicted sequences of the bovine, human, chicken and Escherichia coli T-proteins. Alignment of all these sequences revealed conserved domains, one of which showed a significant similarity to a part of the formyltetrahydrofolate synthetases from procaryotes and eucaryotes. This suggests that the T-protein sequence is not as unique as previously thought.

In Flaveria pringlei and F. cronquistii unlike other plants, H-protein of the glycine cleavage system is encoded by small multigene families. From leaf cDNA libraries and by reverse transcription of mRNA with subsequent polymerase chain reaction (PCR) amplification, we have obtained three different H-protein cDNA clones from each species. The relative levels of total H-protein mRNA, as well as of different H-protein transcripts, have been determined in leaves, stems, and roots of F. pringlei. Stems, with a total of 22% relative to leaves, contain substantial amounts of H-protein transcripts. The corresponding level in roots is relatively low (2.3 % relative to leaves) but easily detectable. One of the transcripts occurs only in leaves (HFP20) and another one (HFP13) is present exclusively in photosynthesizing organs. Only one of the H-protein transcripts (HFP4) was found in all three organs, in leaves, stems, and roots of F. pringlei.

The primary sequences of the chloroplast triose phosphate/phosphate translocator precursor proteins from C4-plants (maize mesophyll cells and Flaveria trinervia) and from the C3-type Flaveria pringlei were determined. The mature parts of these translocators possess 83-94% identical amino acid residues. The C4-translocator protein can be correctly targeted to C3-type chloroplasts and inserted into the envelope membrane. Expression of the mature parts of these chloroplast translocators (cTPT) in transformed yeast cells and subsequent reconstitution of the functional proteins reveals the difference between the recombinant translocator proteins from the two cell types with respect to the transport of phosphoenolpyruvate. Comparison of the cTPT sequences from F. pringlei and F. trinervia in combination with computer-aided molecular modelling of the substrate translocation pore leads to the suggestion, that only minor exchanges of amino acid residues between the C3- and C4-translocator proteins are sufficient to extend their substrate specificities to recognize also phosphoenolpyruvate.

C(4) photosynthesis has evolved multiple times from ancestral C(3) species. Carbonic anhydrase (CA) catalyzes the reversible hydration of CO(2) and is involved in both C(3) and C(4) photosynthesis; however, its roles and the intercellular and intracellular locations of the majority of its activity differ between C(3) and C(4) plants. To understand the molecular changes underlying the evolution of the C(4) pathway, three cDNAs encoding distinct beta-CAs (CA1, CA2, and CA3) were isolated from the leaves of the C(3) plant Flaveria pringlei. The phylogenetic relationship of the F. pringlei proteins with other embryophyte beta-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 beta-CAs, and Arabidopsis (Arabidopsis thaliana) "omics" data. All three F. pringlei CAs have orthologs in the closely related C(4) 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 C(4) plants, whereas modifications to the C(3) CA3 gene led to the evolution of the CA isoform that catalyzes the first step in the C(4) 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 C(4) mesophyll cells.

An analog of phosphoenolpyruvate, 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate (DCDP), was used to inhibit phosphoenolpyruvate carboxylase and, therefore, assess the contribution of the C4 pathway to photosynthesis in detached leaves of several C3-C4 intermediate species. There was no effect of 4 millimolar DCDP on apparent photosynthesis or on inhibition of apparent photosynthesis by 210 milliliters per liter of O2 for the C3-C4 species Panicum milioides, Moricandia arvensis, and Neurachne minor or the C3 species Flaveria pringlei. However, when leaves of Flaveria linearis (C3-C4), Flaveria brownii (C4-like), and Flaveria trinervia (C4) were fed 4 millimolar DCDP, photosynthesis was reduced 32, 60, and 90%, respectively. Photosynthetic inhibition by 210 milliliter per liter of O2 was also significantly increased in 4 millimolar DCDP-fed leaves of F. linearis and F. brownii but not in F. trinervia when compared with control values. These results with DCDP clearly demarcate C3-C4 species into species including Panicum, Moricandia, and Neurachne whose reduced photorespiration occurs without any C4 photosynthetic involvement and species of Flaveria in which C4 photosynthesis contributes to CO2 assimilation.

The genus Flaveria shows evidence of evolution in the mechanism of photosynthesis as its 21 species include C(3), C(3)-C(4), C(4)-like, and C(4) plants. In this study, several physiological and biochemical parameters of photosynthesis and photorespiration were measured in 18 Flaveria species representing all the photosynthetic types. The 10 species classified as C(3)-C(4) intermediates showed an inverse continuum in level of photorespiration and development of the C(4) syndrome. This ranges from F. sonorensis with relatively high apparent photorespiration and lacking C(4) photosynthesis to F. Among the intermediates, the photosynthetic CO(2) compensation points at 30 degrees C and 1150 micromoles quanta per square meter per second varied from 9 to 29 microbars. The values for the three C(4)-like species varied from 3 to 6 microbars, similar to those measured for the C(4) species. The activities of the photorespiratory enzymes glycolate oxidase, hydroxypyruvate reductase, and serine hydroxymethyltransferase decreased progressively from C(3) to C(3)-C(4) to C(4)-like and C(4) species. On the other hand, most intermediates had higher levels of phosphenolpyruvate carboxylase and NADP-malic enzyme than C(3) species, but generally lower activities compared to C(4)-like and C(4) species. The levels of these C(4) enzymes are correlated with the degree of C(4) photosynthesis, based on the initial products of photosynthesis. Another indication of development of the C(4) syndrome in C(3)-C(4)Flaveria species was their intermediate chlorophyll a/b ratios. The chlorophyll a/b ratios of the various Flaveria species are highly correlated with the degree of C(4) photosynthesis suggesting that the photochemical machinery is progressively altered during evolution in order to meet the specific energy requirements for operating the C(4) pathway. In the progression from C(3) to C(4) species in Flaveria, the CO(2) compensation point decreased more rapidly than did the decrease in O(2) inhibition of photosynthesis or the increase in the degree of C(4) photosynthesis. These results suggest that the reduction in photorespiration during evolution occurred initially by refixation of photorespired CO(2) and prior to substantive reduction in O(2) inhibition and development of the C(4) syndrome. However, further reduction in O(2) inhibition in some intermediates and C(4)-like species is considered primarily due to the development of the C(4) syndrome. Thus, the evolution of C(3)-C(4) intermediate photosynthesis likely occurred in response to environmental conditions which limit the intercellular CO(2) concentration first via refixation of photorespired CO(2), followed by development of the C(4) syndrome.

Phosphoenolpyruvate carboxylase (PEPCase) was shown to be encoded by a multigene family in various Flaveria species analysed. Several clones were isolated from genomic libraries of F. pringlei (C3 species) and F. trinervia (C4 species) and classified into four distinct groups according to their hybridization behaviour to a full-length cDNA clone encoding the PEPCase C4 isozyme of F. trinervia. A detailed cross-hybridization analysis demonstrated that the closest relative of most of the PEPCase genes isolated from F. trinervia and F. pringlei was not found in the same but in the other species. Northern analysis, using stringent conditions, allowed discrimination of class-specific PEPCase transcripts and revealed characteristic organ-specific expression patterns.

Summary The effect of excess copper on the expression of soluble proteins in 10-day old Phaseolus vulgaris seedlings was studied with two-dimensional electrophoresis and mass spectrometry, to find sensitive biochemical markers of exposure. Despite major differences in root Cu contents, both 15 and 50 μM Cu treatments resulted in equal enhancements of Cu in the primary leaves. Three proteins, apparently reacting in a dose-dependent manner to Cu exposure, were identified from roots. The levels of an intracellular pathogenesis-related protein and a newly identified protein homologous to PvPR1, PvPR2, were increased with increasing Cu concentration. The level of a newly identified PR-10 protein decreased in a dose-dependent manner. No significant difference was observed in the leaf protein pattern between controls and 15 μM Cu-treated plants. However, at 50 μM Cu exposure, the appearance of PvPR1 and a homologue of Arabidopsis thaliana thylakoid lumenal 17.4 kDa protein was observed. Another protein slightly enhanced by Cu treatment had sequence homology to a mitochondrial precursor of glycine cleavage system H protein of Flaveria pringlei .

C4 phosphoenolpyruvate carboxylases have evolved several times independently from non-photosynthetic C3 ancestral enzymes. To identify C4-specific determinants at the amino acid level the two orthologous ppcA PEPCases of Flaveria trinervia (C4) and Flaveria pringlei (C3) were used as a model system. In a previous publication [Bläsing et al., J. Biol. Chem. 275 (2000) 27917–27923] it was reported that the serine at position 774 is an invariant residue in all C4 phosphoenolpyruvate carboxylases. Here we show by swapping experiments and site-directed mutagenesis that the serine 774 and amino acids 296–437 explain two thirds of the C4 characteristic phosphoenolpyruvate saturation kinetics when investigated in the C3 background. In addition, the results indicate that the determinants functionally interact with each other.

The combined effects of O2 on net rates of photosynthesis, photosystem II activity, steady-state pool size of key metabolites of photosynthetic metabolism in the C4 pathway, C3 pathway and C2 photorespiratory cycle and on growth were evaluated in the C4 species Amaranthus edulis and the C3 species Flaveria pringlei. Increasing O2 reduced net CO2 assimilation in F. pringlei due to an increased flux of C through the photorespiratory pathway. However, in A. edulis increasing O2 up to 5–10% stimulated photosynthesis. Analysis of the pool size of key metabolites in A. edulis suggests that while there is some O2 dependent photorespiration, O2 is required for maximizing C4 cycle activity to concentrate CO2 in bundle sheath cells. Therefore, the response of net photosynthesis to O2 in C4 plants may result from the balance of these two opposing effects. Under 21 versus 5% O2, growth of A. edulis was stimulated about 30% whereas that of F. pringlei was inhibited about 40%.

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.

To study the molecular evolution of NADP-dependent malic enzyme (NADP-ME) in the genus Flaveria a leaf-specific cDNA library of the C3 plant F. pringlei was screened for the presence of sequences homologous to the C4 isoform gene (named modA) of the C4 plant F. trinervia. The cDNAs isolated contained varying numbers of identical restriction fragments suggesting that they were derived from a single gene. This was supported by Southern hybridisation experiments with genomic DNA from F. trinervia and F. pringlei. Nucleotide sequence analysis of a full-size clone identified the presence of a typical plastidic transit peptide and revealed that the mature modA proteins of F. trinervia (C4) and F. pringlei (C3) are 90% similar. These findings indicate that C3 plants, like C4 species, possess a plastidic isoform of NADP-ME and that the modA genes of the two species represent orthologous genes. Northern analyses showed that modA transcripts accumulate to similar levels in leaves, stems and roots of F. pringlei. The expression of this gene in F. pringlei thus appears to be rather constitutive. In contrast, the modA gene of F. trinervia is abundantly expressed in leaves, but maintains its expression in stems and roots. It has to be concluded from these data that the leaf-specific increase in the expression level was a key step which was taken during the evolution of the C4 isoform modA gene starting from a C3 ancestral gene.

Transfer of C(4) photosynthetic traits was studied through hybridization of Flaveria trinervia (Spreng.) Mohr (C(4)) and Flaveria brownii A.M. Powell (C(4)-like) with Flaveria linearis Lag. (C(3)-C(4)) and the C(3) species Flaveria pringlei Gandoger (C(3)). Fertility was low, based on irregular chromosome pairing and low pollen stainability, except in F. brownii x 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 CO(2) compensation concentration was similar to the C(4) or C(4)-like parent, except in hybrids having the C(3) species F. pringlei as a parent. Inhibition of apparent photosynthesis by O(2), and phosphoenolpyruvate carboxylase and NADP-malic enzyme activities and subunit levels in the hybrids were closer to the C(3) or C(3)-C(4) parent. The species F. brownii and F. trinervia were equal in their capacity to transfer reduced O(2) inhibition of AP and CO(2) compensation concentration values to hybrids with F. linearis (C(3)-C(4)), although hybrids with F. trinervia had higher PEPC activity. The O(2) 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 C(4) traits can be transferred by hybridization of C(3)-C(4) and C(4) or C(4)-like species, with a higher degree of C(4) photosynthesis than exists in C(3)-C(4) species, and at least in F. brownii x F. linearis, fertile progeny are obtained.

Flaveria pringlei exhibits C 3 CO 2 compensation concentration (Г) values averaging 53 μl CO 2 /l at 21% (v/v) O 2 and 25 ± 2°C. When this species is hybridized with the C 4 species, F. brownii (male) ( Г = 6 μ l CO 2 / l ), the F 1 hybrid plants exhibit an average Г value of 31 μl CO 2 /l at 21% O 2 . 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 C 4 enzymes (in μmol · mg Chl −1 · h −1 ) are: phospho enol pyruvate (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 C 4 cycle might be operating to a limited extent in the hybrid plants resulting in reduced photorespiration. Whether or not C 4 photosynthesis occurs in these hybrid plants, they represent the first reported C 3 × C 4 F 1 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 C 4 photosynthesis in C 3 species.

Short-term discrimination in assimilation of stable isotopes of carbon was measured for leaves of the C3 speciesPhaseolus vulgaris L. cv. Hawkesbury Wonder andFlaveria pringlei Gandoger, the C4 speciesAmaranthus edulis Speg., and the C3-C4 intermediate speciesPanicum milioides Nees ex. Trin,Flaveria floridana Johnson, andFlaveria anomala B.L. Robinson. Discriminations in the C3 and C4 species were similar to those expected from theoretical considerations. When ambient CO2 pressure was 330 μbar the mean discriminations in the C3 species andPanicum milioides were similar, whereas the mean discriminations inF. floridana andF. anomala were less than discrimination in C3 species andPanicum milioides. When ambient CO2 pressure was 100 μbar the mean discriminations inPanicum milioides andF. anomala were greater, and that inF. floridana was less, than that inPhaseolus vulgaris. We conclude that the pattern of discrimination inPanicum milioides is consistent with the presence of a glycine shuttle; inF. floridana andF. anomala, discrimination is consistent with the presence of a C4 pathway coupled with the operation of a glycine shuttle.

The activities of key C(4) enzymes in gel-filtered, whole-leaf extracts and the photosynthetic characteristics for reciprocal F(1) hybrids of Flaveria pringlei (C(3)) and F. brownii (C(4)-like species) were measured to determine whether any inherited C(4)-photosynthetic traits are responsible for their reduced CO(2) 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 (14)CO(2) incorporation analyses at ambient CO(2) levels indicate that the hybrids initially assimilate 7 to 9% of the total assimilated CO(2) into C(4) acids as compared to 3.5% for F. pringlei. In the hybrids, the percentage of (14)C in malate decreases from an average of 6.5 to 2.1% after a 60-second chase in (12)CO(2)/air. However, this apparent C(4)-cycle activity is too limited or inefficient to substantially alter CO(2) exchange from that in F. pringlei, since the values of net photosynthesis and O(2) inhibition of photosynthesis are similar for the hybrids and F. pringlei. Also, the ratio of the internal to the external CO(2) concentration and the initial slopes of the plot of CO(2) concentration versus net photosynthesis are essentially the same for the hybrids and F. pringlei. At 45 micromoles CO(2) per mole and 0.21 mole O(2) per mole, the hybrids assimilate nearly fivefold more CO(2) into C(4) 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 C(4)-acid metabolism may operate in the hybrids, we conclude that it is not effective in altering O(2) inhibition of CO(2) assimilation. The ability of the hybrids to assimilate more CO(2) via phosphoenolpyruvate carboxylase at low levels of CO(2) than does F. pringlei may result in an increased rate of reassimilation of photorespiratory CO(2) and CO(2) compensation concentrations below that of their C(3) parent. If the hybrids do possess a limited C(4) cycle, it must operate intracellularly. They are not likely to have inherited an intercellular compartmentation of C(4) enzymes, since F. brownii has incomplete compartmentation of key C(3) and C(4) enzymes.

The possibility of altering CO(2) exchange of C(3)-C(4) species by growing them under various CO(2) and O(2) concentrations was examined. Growth under CO(2) concentrations of 100, 350, and 750 micromoles per mole had no significant effect on CO(2) exchange characteristics or leaf anatomy of Flaveria pringlei (C(3)), Flaveria floridana (C(3)-C(4)), or Flaveria trinervia (C(4)). Carboxylation efficiency and CO(2) compensation concentrations in leaves of F. floridana developed under the different CO(2) concentrations were intermediate to F. pringlei and F. trinervia. When grown for 12 days at an O(2) concentration of 20 millimoles per mole, apparent photosynthesis was strongly inhibited in Panicum milioides (C(3)-C(4)) and to a lesser degree in Panicum laxum (C(3)). 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 O(2), 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 O(2) with respect to translocation of photosynthate or sink activity than C(3) species, the concentration of total available carbohydrate increased in shoots of all species by 33% or more when grown at low O(2). This accumulation occurred even though relative growth rates of Festuca arundinacea (C(3)) and P. milioides grown for 4 days at 210 millimoles per mole O(2), were inhibited 83 and 37%, respectively, when compared to plants grown at 20 millimoles per mole O(2).