Key innovations in the evolution of Kranz anatomy and C4 vein pattern in Flaveria (Asteraceae)

Kranz anatomy and C(4) vein pattern are required for C(4) biochemical functioning in C(4) plants; however, the evolutionary timing of anatomical and biochemical adaptations is unknown. From the genus Flaveria, 16 species (C(3), C(4), intermediates [C(3)-C(4), C(4)-like]) were analyzed, novel anatomical and vein pattern characters were analyzed and key anatomical differences among photosynthetic groups were highlighted. A stepwise acquisition of anatomical and vein pattern traits prior to derived biochemistry was outlined on the basis of the phylogeny of Flaveria. Increased vein density represents a potential "precondition" contributing to lower ratios of photosynthetic tissues (mesophyll, bundle sheath) and precedes further anatomical and biochemical modifications observed in derived C(3)-C(4) intermediates. In derived Flaveria species, bundle sheath volume is modified through cell expansion, whereas mesophyll volume is altered through mesophyll cell expansion, reductions in the number of ground tissue layers, and increased vein density. Results demonstrated that key anatomical features of C(4) plants are also required for C(3)-C(4) biochemical intermediacy, and anatomical and biochemical alterations acquired during evolution of intermediacy may predispose a species for evolution of C(4) photosynthesis. C(4)-like species are similar to C(4) species, demonstrating that Kranz anatomy is fully evolved before complete C(4) biochemistry is achieved.     

Carbonic anhydrase and the molecular evolution of C4 photosynthesis

C(4) photosynthesis, a biochemical CO(2)-concentrating mechanism (CCM), evolved more than 60 times within the angiosperms from C(3) ancestors. The genus Flaveria, which contains species demonstrating C(3), C(3)-C(4), C(4)-like or C(4) photosynthesis, is a model for examining the molecular evolution of the C(4) pathway. Work with carbonic anhydrase (CA), and C(3) and C(4) Flaveria congeners has added significantly to the understanding of this process. The C(4) form of CA3, a β-CA, which catalyses the first reaction in the C(4) pathway by hydrating atmospheric CO(2) to bicarbonate in the cytosol of mesophyll cells (mcs), evolved from a chloroplastic C(3) ancestor. The molecular modifications to the ancestral CA3 gene included the loss of the sequence encoding the chloroplast transit peptide, and mutations in regulatory regions that resulted in high levels of expression in the C(4) mesophyll. Analyses of the CA3 proteins and regulatory elements from Flaveria photosynthetic intermediates indicated C(4) biochemistry very likely evolved in a specific, stepwise manner in this genus. The details of the mechanisms involved in the molecular evolution of other C(4) plant β-CAs are unknown; however, comparative genetics indicate gene duplication and neofunctionalization played significant roles as they did in Flaveria.     

The molecular evolution of β-carbonic anhydrase in Flaveria

Limited information exists regarding molecular events that occurred during the evolution of C(4) plants from their C(3) ancestors. The enzyme β-carbonic anhydrase (CA; EC 4.2.1.1), which catalyses the reversible hydration of CO(2), is present in multiple forms in C(3) and C(4) plants, and has given insights into the molecular evolution of the C(4) pathway in the genus Flaveria. cDNAs encoding three distinct isoforms of β-CA, CA1-CA3, have been isolated and examined from Flaveria C(3) and C(4) congeners. Sequence data, expression analyses of CA orthologues, and chloroplast import assays with radiolabelled CA precursor proteins from the C(3) species F. pringlei Gandoger and the C(4) species F. bidentis (L.) Kuntze have shown that both contain chloroplastic and cytosolic forms of the enzyme, and the potential roles of these isoforms are discussed. The data also identified CA3 as the cytosolic isoform important in C(4) photosynthesis and indicate that the C(4) CA3 gene evolved as a result of gene duplication and neofunctionalization, which involved mutations in coding and non-coding regions of the ancestral C(3) CA3 gene. Comparisons of the deduced CA3 amino acid sequences from Flaveria C(3), C(4), and photosynthetic intermediate species showed that all the C(3)-C(4) intermediates investigated and F. brownii, a C(4)-like species, have a C(3)-type CA3, while F. vaginata, another C(4)-like species, contains a C(4)-type CA3. These observations correlate with the photosynthetic physiologies of the intermediates, suggesting that the molecular evolution of C(4) photosynthesis in Flaveria may have resulted from a temporally dependent, stepwise modification of protein-encoding genes and their regulatory elements.     

Evolution of c4 phosphoenolpyruvate carboxylase. Genes and proteins: a case study with the genus Flaveria

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.     

Complex evolutionary transitions and the significance of c(3)-c(4) intermediate forms of photosynthesis in Molluginaceae

C(4) photosynthesis is a series of biochemical and structural modifications to C(3) photosynthesis that has evolved numerous times in flowering plants, despite requiring modification of up to hundreds of genes. To study the origin of C(4) photosynthesis, we reconstructed and dated the phylogeny of Molluginaceae, and identified C(4) taxa in the family. Two C(4) species, and three clades with traits intermediate between C(3) and C(4) plants were observed in Molluginaceae. C(3)-C(4) intermediacy evolved at least twice, and in at least one lineage was maintained for several million years. Analyses of the genes for phosphoenolpyruvate carboxylase, a key C(4) enzyme, indicate two independent origins of fully developed C(4) photosynthesis in the past 10 million years, both within what was previously classified as a single species, Mollugo cerviana. The propensity of Molluginaceae to evolve C(3)-C(4) and C(4) photosynthesis is likely due to several traits that acted as developmental enablers. Enlarged bundle sheath cells predisposed some lineages for the evolution of C(3)-C(4) intermediacy and the C(4) biochemistry emerged via co-option of photorespiratory recycling in C(3)-C(4) intermediates. These evolutionarily stable transitional stages likely increased the evolvability of C(4) photosynthesis under selection environments brought on by climate and atmospheric change in recent geological time.     

Photosynthetic pathway influences xylem structure and function in Flaveria (Asteraceae)

Higher water use efficiency (WUE) in C(4) plants may allow for greater xylem safety because transpiration rates are reduced. To evaluate this hypothesis, stem hydraulics and anatomy were compared in 16 C(3), C(3)-C(4) intermediate, C(4)-like and C(4) species in the genus Flaveria. The C(3) species had the highest leaf-specific conductivity (K(L)) compared with intermediate and C(4) species, with the perennial C(4) and C(4)-like species having the lowest K(L) values. Xylem-specific conductivity (K(S)) was generally highest in the C(3) species and lower in intermediate and C(4) species. Xylem vessels were shorter, narrower and more frequent in C(3)-C(4) intermediate, C(4)-like and C(4) species compared with C(3) species. WUE values were approximately double in the C(4)-like and C(4) species relative to the C(3)-C(4) and C(3) species. C(4)-like photosynthesis arose independently at least twice in Flaveria, and the trends in WUE and K(L) were consistent in both lineages. These correlated changes in WUE and K(L) indicate WUE increase promoted K(L) decline during C(4) evolution; however, any involvement of WUE comes late in the evolutionary sequence. C(3)-C(4) species exhibited reduced K(L) but little change in WUE compared to C(3) species, indicating that some reduction in hydraulic efficiency preceded increases in WUE.     

Assimilatory sulfate reduction in C(3), C(3)-C(4), and C(4) species of Flaveria

The activity of the enzymes catalyzing the first two steps of sulfate assimilation, ATP sulfurylase and adenosine 5'-phosphosulfate reductase (APR), are confined to bundle sheath cells in several C(4) monocot species. With the aim to analyze the molecular basis of this distribution and to determine whether it was a prerequisite or a consequence of the C(4) photosynthetic mechanism, we compared the intercellular distribution of the activity and the mRNA of APR in C(3), C(3)-C(4), C(4)-like, and C(4) species of the dicot genus Flaveria. Measurements of APR activity, mRNA level, and protein accumulation in six Flaveria species revealed that APR activity, cysteine, and glutathione levels were significantly higher in C(4)-like and C(4) species than in C(3) and C(3)-C(4) species. ATP sulfurylase and APR mRNA were present at comparable levels in both mesophyll and bundle sheath cells of C(4) species Flaveria trinervia. Immunogold electron microscopy demonstrated the presence of APR protein in chloroplasts of both cell types. These findings, taken together with results from the literature, show that the localization of assimilatory sulfate reduction in the bundle sheath cells is not ubiquitous among C(4) plants and therefore is neither a prerequisite nor a consequence of C(4) photosynthesis.     

Water-use efficiency and nitrogen-use efficiency of C(3) -C(4) intermediate species of Flaveria Juss. (Asteraceae)

Plants using the C(4) pathway of carbon metabolism are marked by greater photosynthetic water and nitrogen-use efficiencies (PWUE and PNUE, respectively) than C(3) species, but it is unclear to what extent this is the case in C(3) -C(4) intermediate species. In this study, we examined the PWUE and PNUE of 14 species of Flaveria Juss. (Asteraceae), including two C(3) , three C(4) and nine C(3) -C(4) species, the latter containing a gradient of C(4) -cycle activities (as determined by initial fixation of (14) C into C-4 acids). We found that PWUE, PNUE, leaf ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) content and intercellular CO(2) concentration in air (C(i) ) do not change gradually with C(4) -cycle activity. These traits were not significantly different between C(3) species and C(3) -C(4) species with less than 50% C(4) -cycle activity. C(4) -like intermediates with greater than 65% C(4) -cycle activity were not significantly different from plants with fully expressed C(4) photosynthesis. These results indicate that a gradual increase in C(4) -cycle activity has not resulted in a gradual change in PWUE, PNUE, intercellular CO(2) concentration and leaf Rubisco content towards C(4) levels in the intermediate species. Rather, these traits arose in a stepwise manner during the evolutionary transition to the C(4) -like intermediates, which are contained in two different clades within Flaveria.     

Changes in Rubisco kinetics during the evolution of C4 photosynthesis in Flaveria (Asteraceae) are associated with positive selection on genes encoding the enzyme

Rubisco, the primary photosynthetic carboxylase, evolved 3-4 billion years ago in an anaerobic, high CO(2) atmosphere. The combined effect of low CO(2) and high O(2) levels in the modern atmosphere, and the inability of Rubisco to distinguish completely between CO(2) and O(2), leads to the occurrence of an oxygenation reaction that reduces the efficiency of photosynthesis. Among land plants, C(4) photosynthesis largely solves this problem by facilitating a high CO(2)/O(2) ratio at the site of Rubisco that resembles the atmosphere in which the ancestral enzyme evolved. The prediction that such conditions favor Rubiscos with higher kcat(CO2) and lower CO(2)/O(2) specificity (S(C/O)) is well supported, but the structural basis for the differences between C(3) and C(4) Rubiscos is not clear. Flaveria (Asteraceae) includes C(3), C(3)-C(4) intermediate, and C(4) species with kinetically distinct Rubiscos, providing a powerful system in which to study the biochemical transition of Rubisco during the evolution from C(3) to C(4) photosynthesis. We analyzed the molecular evolution of chloroplast rbcL and nuclear rbcS genes encoding the large subunit (LSu) and small subunit (SSu) of Rubisco from 15 Flaveria species. We demonstrate positive selection on both subunits, although selection is much stronger on the LSu. In Flaveria, two positively selected LSu amino acid substitutions, M309I and D149A, distinguish C(4) Rubiscos from the ancestral C(3) species and statistically account for much of the kinetic difference between the two groups. However, although Flaveria lacks a characteristic "C(4)" SSu, our data suggest that specific residue substitutions in the SSu are correlated with the kinetic properties of Rubisco in this genus.     

Effects of low atmospheric CO2 and elevated temperature during growth on the gas exchange responses of C3, C3–C4 intermediate, and C4 species from three evolutionary lineages of C4 photosynthesis

This study evaluates acclimation of photosynthesis and stomatal conductance in three evolutionary lineages of C(3), C(3)-C(4) intermediate, and C(4) species grown in the low CO(2) and hot conditions proposed to favo r the evolution of C(4) photosynthesis. Closely related C(3), C(3)-C(4), and C(4) species in the genera Flaveria, Heliotropium, and Alternanthera were grown near 380 and 180 μmol CO(2) mol(-1) air and day/night temperatures of 37/29°C. Growth CO(2) had no effect on photosynthetic capacity or nitrogen allocation to Rubisco and electron transport in any of the species. There was also no effect of growth CO(2) on photosynthetic and stomatal responses to intercellular CO(2) concentration. These results demonstrate little ability to acclimate to low CO(2) growth conditions in closely related C(3) and C(3)-C(4) species, indicating that, during past episodes of low CO(2), individual C(3) plants had little ability to adjust their photosynthetic physiology to compensate for carbon starvation. This deficiency could have favored selection for more efficient modes of carbon assimilation, such as C(3)-C(4) intermediacy. The C(3)-C(4) species had approximately 50% greater rates of net CO(2) assimilation than the C(3) species when measured at the growth conditions of 180 μmol mol(-1) and 37°C, demonstrating the superiority of the C(3)-C(4) pathway in low atmospheric CO(2) and hot climates of recent geological time.     

The functional significance of C3-C4 intermediate traits in Heliotropium L. (Boraginaceae): gas exchange perspectives

We demonstrate for the first time the presence of species exhibiting C3-C4 intermediacy in Heliotropium (sensu lato), a genus with over 100 C3 and 150 C4 species. CO2 compensation points (Gamma) and photosynthetic water-use efficiencies (WUEs) were intermediate between C3 and C4 values in three species of Heliotropium: Heliotropium convolvulaceum (Gamma = 20 micromol CO2 mol(-1) air), Heliotropium racemosum (Gamma = 22 micromol mol(-1)) and Heliotropium greggii (Gamma = 17 micromol mol(-1)). Heliotropium procumbens may also be a weak C3-C4 intermediate based on a slight reduction in Gamma (48.5 micromol CO2 mol(-1)) compared to C3Heliotropium species (52-60 micromol mol(-1)). The intermediate species H. convolvulaceum, H. greggii and H. racemosum exhibited over 50% enhancement of net CO2 assimilation rates at low CO2 levels (200-300 micromol mol(-1)); however, no significant differences in stomatal conductance were observed between the C3 and C3-C4 species. We also assessed the response of Gamma to variation in O2 concentration for these species. Heliotropium convolvulaceum, H. greggii and H. racemosum exhibited similar responses of Gamma to O2 with response slopes that were intermediate between the responses of C3 and C4 species below 210 mmol O2 mol(-1) air. The presence of multiple species displaying C3-C4 intermediate traits indicates that Heliotropium could be a valuable new model for studying the evolutionary transition from C3 to C4 photosynthesis.     

Modification of Photosystem I Light Harvesting of Bundle-Sheath Chloroplasts Occurred during the Evolution of NADP-Malic Enzyme C4 Photosynthesis

Low-temperature emission spectra and excitation spectra for chlorophyll fluorescence were recorded from leaves of species of the genus Flaveria (Asteraceae) with C3, C3-C4-intermediate, C4-like, and C4 photosynthesis. Among the latter two groups, high chlorophyll b absorption was observed in excitation spectra for photosystem I (PSI) fluorescence. By comparing leaf data with those from isolated chloroplast fractions, the high chlorophyll b absorption was attributed to the specific properties of the bundle-sheath chloroplasts in leaves from C4 plants. The deconvolution of the PSI excitation spectra and the use of a model revealed that the contribution of photosystem II absorption to the functional antenna of PSI was markedly increased in leaves from three of the five C4-like and C4 species investigated in detail. The two other species exhibited normal, C3-like light-harvesting properties of PSI. The former species are known for efficient carbon assimilation, the latter for decreased efficiencies of carbon assimilation. It is concluded that photosystem II becomes a substantial part of the functional PSI antenna late in the evolution of C4 photosynthesis, and that the composite antenna optimizes the light-harvesting of PSI in bundle-sheath chloroplasts to meet the energy requirements of C4 photosynthesis.     

C3 photosynthesis in Aristida longifolia: Implication for photosynthetic diversification in Aristidoideae (Poaceae)

Only a small percentage of plant species undergo C(4) photosynthesis. Despite its rarity, the C(4) pathway has evolved numerous times from C(3) ancestors, with as many as 18 independent origins in grasses alone. We report non-Kranz (C(3)) anatomy in Aristida longifolia, a species in a genus of ca. 300 species previously thought to possess only Kranz (C(4)) anatomy. Leaf blade transections of A. longifolia show widely spaced vascular bundles, nonradiate chlorenchyma, and few or no chloroplasts in cells of the sheaths surrounding the vascular bundle, all features indicative of C(3) photosynthesis. Carbon isotope ratios range from -27.68 to -29.71%, likewise indicative of C(3) photosynthesis. We also reconstruct the phylogeny of Aristidoideae, comprising Aristida, Sartidia (C(3)), and Stipagrostis (C(4)), using a sample of 11 species, including A. longifolia, and DNA sequences of the nuclear ribosomal internal transcribed spacer region and the chloroplast rpl16 intron and trnL-trnF region. Sartidia and Stipagrostis resolve as sisters, and sister to this clade is Aristida. Aristida longifolia resolves as sister to the remaining species in the genus. C(3) photosynthesis is hypothesized to be ancestral in Aristidoideae, which means the C(4) pathway evolved twice in the subfamily-in Stipagrostis and early in the diversification of the Aristida clade.     

C4 isoform of NADP-malate dehydrogenase. cDNA cloning and expression in leaves of C4, C3, and C3-C4 intermediate species of Flaveria.

In C4 plants of the NADP-malic enzyme type, an abundant, mesophyll cell-localized NADP-malate dehydrogenase (MDH) acts to convert oxaloacetate, the initial product of carbon fixation, to malate before it is shuttled to the bundle sheath. Since NADP-MDH has different but important roles in leaves of C3 and C4 plants, we have cloned and characterized a nearly full-length cDNA encoding NADP-MDH from Flaveria trinervia (C4) to permit comparative structure/expression studies within the genus flaveria. The dicot genus Flaveria includes C3-C4 intermediate species, as well as C3 and C4 species. We show that the previously noted differences in NADP-MDH activity levels among C3, C4, and C3-C4 Flaveria species are in part due to interspecific differences in mRNA accumulation. We also show that the NADP-MDH gene appears to be present as a single copy among different Flaveria species, suggesting that a pre-existing gene has been reregulated during the evolution from C3 to C4 plants to accommodate the abundance and localization requirements of the C4 cycle.     

Evolution of C4 phosphoenolpyruvate carboxylase

C4 plants are known to be of polyphyletic origin and to have evolved independently several times during the evolution of angiosperms. This implies that the C4 isoform of phosphoenolpyruvate carboxylase (PEPC) originated from a nonphotosynthetic PEPC gene that was already present in the C3 ancestral species. To meet the special requirements of the C4 photosynthetic pathway the expression program of the C4 PEPC gene had to be changed to achieve a strong and selective expression in leaf mesophyll cells. In addition, the altered metabolite concentrations around C4 PEPC in the mesophyll cytoplasm necessitated changes in the enzyme's kinetic and regulatory properties. To obtain insight into the evolutionary steps involved in these altered enzyme characteristics, and even the order of these steps, the dicot genus Flaveria (Asteraceae) appears to be the experimental system of choice. Flaveria contains closely related C3, C3-C4, and C4 species that can be ordered by their gradual increase in C4 photosynthetic traits. The C4 PEPC of F. trinervia, which is encoded by the ppcA gene class, possesses typical kinetic and regulatory features of a C4-type PEPC. Its nearest neighbor is the orthologous ppcA gene of the C3 species F. pringlei. This latter gene encodes a typical nonphotosynthetic C3-type PEPC which is believed to be similar to the C3 ancestral PEPC. This pair of orthologous PEPCs has been used to map C4-specific molecular determinants for the kinetic and regulatory characteristics of C4 PEPCs. The most notable finding from these investigations was the identification of a C4 PEPC invariant site-specific mutation from alanine (C3) to serine (C4) at position 774 that was a necessary and late step in the evolution of C3 to C4 PEPC. The C3-C4 intermediate ppcA PEPCs are used to identify the sequence of events leading from a C3- to a C4-type PEPC.     

The future of C4 research--maize, Flaveria or Cleome?

C4 photosynthesis has evolved multiple times among the angiosperms: the spatial rearrangement of the photosynthetic apparatus, combined with alterations to the leaf structure, allows CO2 to be concentrated around Rubisco. Higher CO2 concentrations at Rubisco decrease the rate of oxygenation and therefore reduce the amount of energy lost through photorespiration. C4 plants are particularly prevalent in tropical and subtropical regions because they can sustain higher rates of net photosynthesis; they also represent some of our most productive crops. To date, most progress in identifying genes crucial for C4 photosynthesis has been made using maize and Flaveria. We propose that Cleome, the most closely related genus containing C4 species to the C3 model Arabidopsis, be used together with Arabidopsis resources to accelerate our progress in elucidating the genetic basis of C4 photosynthesis.     

Molecular evolution of C4 phospho<Emphasis Type="Italic">enol</Emphasis>pyruvate carboxylase in the genus<Emphasis Type="Italic"> Flaveria</Emphasis>—a gradual increase from C3 to C4 characteristics

In order to elucidate the discrete steps in phospho enolpyruvate carboxylase (PEPC) evolution concerning K(m)-PEP and malate tolerance a comparison was made between C3, C3-C4 and C4 species of the dicot genus Flaveria. The PEPCs of this genus are encoded by a gene family comprising three classes: ppcA, ppcB and ppcC [J. Hermans and P. Westhoff (1990) Mol Gen Genet 224:459-468, (1992) Mol Gen Genet 234:275-284]. The ppcA of F trinervia (C4) codes for the C4 PEPC isoform but other plants of the genus contain ppcA orthologues too. The C3 plant F. pringlei showed the lowest levels of ppcA PEPC mRNA followed by F. pubescens (C3-C4) while the C4-like plant F. brownii displayed RNA amounts close to the C4 species F. trinervia. In contrast to the similar expression profiles of F. brownii (C4-like) and F. trinervia (C4) the PEPC amino acid sequence of F. brownii was more similar to the C3 and C3-C4 ppcA PEPCs than to the C4 PEPC. Similarly, the C3, C3-C4 and C4-like ppcA PEPCs showed almost identical PEP saturation kinetics when activated by glucose-6-phosphate ( K(m)-PEP: 17-20 microM) while the K(m)-PEP for the C4 PEPC was determined to be 53 microM. However, without activation the ppcA PEPCs of F. pubescens and F. brownii displayed C3-C4 intermediate values. A similar picture was obtained when the malate sensitivities were compared. In the non-activated state the F. trinervia (C4) enzyme was 10 times more tolerant to malate than the F. pringlei counterpart. The ppcA enzymes of F. pubescens (C3-C4) and F. brownii (C4-like) displayed intermediate values. In contrast, the inclusion of 5 mM glucose-6-phosphate in the reaction mixture changed the order totally. Interestingly, the activation rendered the C4 enzyme about 50% less tolerant to malate than the C3 PEPC. The activation had a positive effect on malate tolerance of the F. pubescens (C3-C4) PEPC while the ppcA PEPC of F. brownii (C4-like) was almost unaffected.