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

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

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.     

Purification and characterization of the phosphoenolpyruvate carboxylase from the facultative chemolithotrophThiobacillus novellus (ATCC 8093)

Phosphoenolpyruvate carboxylase (EC 4.1.1.31), which catalyzes the carboxylation of phosphoenolpyruvate to produce oxaloacetate was purified 465-fold from extracts of organotrophically grownThiobacillus novellus. Nondenaturing polyacrylamide gel electrophoresis (PAGE) of the purified enzyme revealed the presence of two bands after staining with Buffalo Black. Gels stained with Fast Violet B after incubation with PEP, HCO₃ ^-, Mg²⁺ and acetyl CoA also showed two bands of activity with the faster moving the more active of the two. Sodium dodecylsulfate (SDS)-PAGE of the enzyme heated at 100°C for 5 min revealed the presence of three intensely stained bands of M_r 95 K, 51 K, and 28 K. However, electrophoresis of the enzyme heated for 2 min showed a single band of about 100 K, indicating that the preparation was likely homogeneous. The 51 K and 28 K subunits are thus products of the 95 K subunit. Gel filtration studies of the native enzyme yielded a M_r of 360 K. Therefore, the enzyme is a tetramer. The optimum pH in Tris buffer was 8.0, with K_m for PEP 0.64 mM, HCO₃ ^- 0.11 mM, and acetyl CoA a potent activator, 1.3 M. A divalent cation best served by Mg²⁺ gave sigmoidal initial velocity plots. Hill plots of the data gave coefficients (n_H) of 2.6. None of the metabolites tested, nucleotide triophosphates excepted, significantly affected enzyme activity. Binding studies with¹⁴C-labelled PEP revealed the binding of about 20 moles PEP per mole (360,000 g) of PEPC. Initial velocity studies suggest that the reaction is catalyzed by a random Bi Bi mechanism. Despite the lack of inhibition by certain metabolites, the enzyme's function is probably anaplerotic.Supported by an operating grant from NSERC to AMC.     

C(4) Acid Metabolism and Dark CO(2) Fixation in a Submersed Aquatic Macrophyte (Hydrilla verticillata)

The CO₂ compensation point of the submersed aquatic macrophyte Hydrilla verticillata varied from high (above 50 microliters per liter) to low (10 to 25 microliters per liter) values, depending on the growth conditions. Plants from the lake in winter or after incubation in an 11 C/9-hour photoperiod had high values, whereas summer plants or those incubated in a 27 C/14-hour photoperiod had low values. The plants with low CO₂ compensation points exhibited dark ¹⁴CO₂ fixation rates that were up to 30% of the light fixation rates. This fixation reduced respiratory CO₂ loss, but did not result in a net uptake of CO₂ at night. The low compensation point plants also showed diurnal fluctuations in titratable acid, such as occur in Crassulacean acid metabolism plants. However, dark fixation and diurnal acid fluctuations were negligible in Hydrilla plants with high CO₂ compensation points.     

Influence of photoperiod on tuber production in various races of Hydrilla (Hydrilla verticillata)

The potential for asexual reproduction throughproduction of tubers was evaluated in various races ofan International Hydrilla [Hydrilla verticillata (L.f.)Royle] germplasm collection. Interracecomparisons were made under both shortday and longdayphotoperiods in environmental growth chambers. Allthe races evaluated produced rates varying from91-7182 tubers per sq m of production under shortdayconditions. Tuber production occurred under longdayphotoperiods in several races. Under propertemperature conditions, all the monoecious racescurrently established in the U.S. appear capable oftuber production throughout the year.     

Biosorption and Desorption of Lead(II) by Hydrilla verticillata

The potential of nonliving biomass of Hydrilla verticillata to adsorb Pb(II) from an aqueous solution containing very low concentrations of Pb(II) was determined in this study. Effects of shaking time, contact time, biosorbent dosage, pH of the medium, and initial Pb(II) concentration on metal-biosorbent interactions were studied through batch adsorption experiments. Maximum Pb(II) removal was obtained after 2 h of shaking. Adsorption capacity at the equilibrium increased with increasing initial Pb(II) concentration, whereas it decreased with increasing biosorbent dosage. The optimum pH of the biosorption was 4.0. Surface titrations showed that the surface of the biosorbent was positively charged at low pH and negatively charged at pH higher than 3.6. Fourier transform infrared (FT-IR) spectra of the biosorbent confirmed the involvement of hydroxyl and C?O of acylamide functional groups on the biosorbent surface in the Pb(II) binding process. Kinetic and equilibrium data showed that the adsorption process followed the pseudo-second-order kinetic model and both Langmuir and Freundlich isothermal models. The mean adsorption energy showed that the adsorption of Pb(II) was physical in nature. The monolayer adsorption capacity of Pb(II) was 125 mg g^?1. The desorption of Pb(II) from the biosorbent by selected desorbing solutions were HNO₃ > Na₂CO₃ > NaOH > NaNO₃.     

Regulation and Localization of Key Enzymes during the Induction of Kranz-Less,C4-Type Photosynthesis in Hydrilla verticillata

Kranz-less, C4-type photosynthesis was induced in the submersed monocot Hydrilla verticillata (L.f.) Royle. During a 12-d induction period the CO2 compensation point and O2 inhibition of photosynthesis declined linearly. Phosphoenolpyruvate carboxylase (PEPC) activity increased 16-fold, with the major increase occurring within 3 d. Asparagine and alanine aminotransferases were also induced rapidly. Pyruvate orthophosphate dikinase (PPDK) and NADP-malic enzyme (ME) activities increased 10-fold but slowly over 15 d. Total ribulose-1,5-bisphosphate carboxylase/oxygenase activity did not increase, and its activation declined from 82 to 50%. Western blots for PEPC, PPDK, and NADP-ME indicated that increased protein levels were involved in their induction. The H. verticillata NADP-ME polypeptide was larger (90 kD) than the maize C4 enzyme (62 kD). PEPC and PPDK exhibited up-regulation in the light. Subcellular fractionation of C4-type leaves showed that PEPC was cytosolic, whereas PPDK and NADP-ME were located in the chloroplasts. The O2 inhibition of photosynthesis was doubled when C4-type but not C3-type leaves were exposed to diethyl oxalacetate, a PEPC inhibitor. The data are consistent with a C4-cycle concentrating CO2 in H. verticillata chloroplasts and indicate that Kranz anatomy is not obligatory for C4-type photosynthesis. H. verticillata predates modern terrestrial C4 monocots; therefore, this inducible CO2-concentrating mechanism may represent an ancient form of C4 photosynthesis.     

Photosynthetic Enzyme Activities and Localization in Mollugo verticillata Populations Differing in the Levels of C(3) and C(4) Cycle Operation

Ecotypic differences in the photosynthetic carbon metabolism of Mollugo verticillata were studied. Variations in C₃ and C₄ cycle activity are apparently due to differences in the activities of enzymes associated with each pathway. Compared to C₄ plants, the activities of C₄ pathway enzymes were generally lower in M. verticillata, with the exception of the decarboxylase enzyme, NAD malic enzyme. The combined total carboxylase enzyme activity of M. verticillata was greater than that of C₃ plants, possibly accounting for the high photosynthetic rates of this species. Unlike either C₃ or C₄ plants, ribulose bisphosphate carboxylase was present in both mesophyll and bundle sheath cell chloroplasts in M. verticillata. The localization of this enzyme in both cells in this plant, in conjunction with an efficient C₄ acid decarboxylation mechanism most likely localized in bundle sheath cell mitochondria, may account for intermediate photorespiration levels previously observed in this species.     

Tree stem phosphoenolpyruvate carboxylase (PEPC): lack of biochemical and localization evidence for a C4-like photosynthesis system

Here, the kinetic properties and immunolocalization of phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in young stems of Fagus sylvatica were investigated. The aim of the study was to test the hypothesis that there is a C4-like photosynthesis system in the stems of this C3 tree species. The activity, optimal pH and L-malate sensitivity of PEPC, and the Michaelis-Menten constant (Km) for phosphoenolpyruvate (PEP), were measured in protein extracts from current-year stems and leaves. A gel blot experiment and immunolocalization studies were performed to examine the isozyme complexity of PEPC and the tissue distribution of PEPC and Rubisco in stems. Leaf and stem PEPCs exhibited similar, classical values characteristic of C3 PEPCs, with an optimal pH of c. 7.8, a Km for PEP of c. 0.3 mM and a IC50 for L-malate (the L-malate concentration that inhibits 50% of PEPC activity at the Km for PEP) of c. 0.1 mM. Western blot analysis showed the presence of two PEPC subunits (molecular mass c. 110 kDa) both in leaves and in stems. Immunogold labelling did not reveal any differential localization of PEPC and Rubisco, neither between nor inside cells. This study suggests that C4-type photosynthesis does not occur in stems of F. sylvatica and underlines the importance of PEPC in nonphotosynthetic carbon fixation by most stem tissues (fixation of respired CO2 and fixation via the anaplerotic pathway).     

Light/dark modulation of phosphoenolpyruvate carboxylase in C3 and C4 species

In this report, the effects of light on the activity and allosteric properties of phosphoenolpyruvate (PEP) carboxylase were examined in newly matured leaves of several C₃ and C₄ species. Illumination of previously darkened leaves increased the enzyme activity 1.1 to 1.3 fold in C₃ species and 1.4 to 2.3 fold in C₄ species, when assayed under suboptimal conditions (pH 7) without allosteric effectors. The sensitivities of PEP carboxylase to the allosteric effectors malate and glucose-6-phosphate were markedly different between C₃ and C₄ species. In the presence of 5 mM malate, the activity of the enzyme extracted from illuminated leaves was 3 to 10 fold higher than that from darkened leaves in C₄ species due to reduced malate inhibition of the enzyme from illuminated leaves, whereas it increased only slightly in C₃ species. The Ki(malate) for the enzyme increased about 3 fold by illumination in C₄ species, but increased only slightly in C₃ species. Also, the addition of the positive effector glucose-6-phosphate provided much greater protection against malate inhibition of the enzyme from C₄ species than C₃ species. Feeding nitrate to excised leaves of nitrogen deficient plants enhanced the degree of light activation of PEP carboxylase in the C₄ species maize, but had little or no effect in the C₃ species wheat. These results suggest that post-translational modification by light affects the activity and allosteric properties of PEP carboxylase to a much greater extend in C₄ than in C₃ species.     

低浓度CO2对水车前和黑藻光合特性的影响

本实验通过对沉水植物水车前（offelia alismoides）和黑藻（Hydrilla verticillata）进行低浓度C02诱导,研究其光合特性的变化。研究显示,诱导后,水车前的PEPC／Rubisco活性比值由0．45上升到4．17,黑藻由0．47上升到4．17,两种植物的C4途径光合酶PEPC和NAD—ME的活性升高了10倍左右,其他C4光合酶如NAD—MDH和PPDK的活性也大幅升高;诱导后,水车前和黑藻的其他光合特性也发生了显著变化：净光合速率比对照分别提高了50．8％和74．1％,O2对处理组光合速率的抑制分别为对照组的35％和60％,叶绿素荧光基本参数也有显著性变化。研究结果表明,经低浓度C02诱导,水车前和黑藻光合碳同化途径可能由C3转变成为C4。     

Molecular biology of C4 phosphoenolpyruvate carboxylase: Structure,regulation and genetic engineering

Three to four families of nuclear genes encode different isoforms of phosphoenolpyruvate (PEP) carboxylase (PEPC): C₄-specific, C₃ or etiolated, CAM and root forms. C₄ leaf PEPC is encoded by a single gene (ppc) in sorghum and maize, but multiple genes in the C₄-dicot Flaveria trinervia. Selective expression of ppc in only C₄-mesophyll cells is proposed to be due to nuclear factors, DNA methylation and a distinct gene promoter. Deduced amino acid sequences of C₄-PEPC pinpoint the phosphorylatable serine near the N-terminus, C₄-specific valine and serine residues near the C-terminus, conserved cysteine, lysine and histidine residues and PEP binding/catalytic sites. During the PEPC reaction, PEP and bicarbonate are first converted into carboxyphosphate and the enolate of pyruvate. Carboxyphosphate decomposes within the active site into Pi and CO₂, the latter combining with the enolate to form oxalacetate. Besides carboxylation, PEPC catalyzes a HCO₃ ^--dependent hydrolysis of PEP to yield pyruvate and Pi. Post-translational regulation of PEPC occurs by a phosphorylation/dephosphorylation cascade in vivo and by reversible enzyme oligomerization in vitro. The interrelation between phosphorylation and oligomerization of the enzyme is not clear. PEPC-protein kinase (PEPC-PK), the enzyme responsible for phosphorylation of PEPC, has been studied extensively while only limited information is available on the protein phosphatase 2A capable of dephosphorylating PEPC. The C₄ ppc was cloned and expressed in Escherichia coli as well as tobacco. The transformed E. coli produced a functional/phosphorylatable C₄ PEPC and the transgenic tobacco plants expressed both C₃ and C₄ isoforms. Site-directed mutagenesis of ppc indicates the importance of His¹³⁸, His⁵⁷⁹ and Arg⁵⁸⁷ in catalysis and/or substrate-binding by the E. coli enzyme, Ser⁸ in the regulation of sorghum PEPC. Important areas for further research on C₄ PEPC are: mechanism of transduction of light signal during photoactivation of PEPC-PK and PEPC in leaves, extensive use of site-directed mutagenesis to precisely identify other key amino acid residues, changes in quarternary structure of PEPC in vivo, a high-resolution crystal structure, and hormonal regulation of PEPC expression.Abbreviations OAA oxalacetate - PEP phosphoenolpyruvate - PEPC PEP carboxylase - PEPC-PK PEPC-protein kinase - PPDK pyruvate, orthophosphate dikinase - Rubisco ribulose 1,5-bis-phosphate carboxylase/oxygenase - CAM Crassulacean acid metabolism     

Two independent C4 origins in Aristidoideae (Poaceae) revealed by the recruitment of distinct phosphoenolpyruvate carboxylase genes

Determining the number of evolutions of an adaptive novelty is primordial to understand its evolutionary significance. C(4) photosynthesis, an adaptation to low CO(2) atmospheric concentration and high temperature, evolved multiple times, but the number of convergent evolutions is still debated. In Poaceae phylogeny, numerous C(4) groups are separated by C(3) taxa, but whether these correspond to independent C(4) origins or a few C(4) evolutions followed by reversals is controversial. The Aristidoideae subfamily is formed by two C(4) genera, Aristida and Stipagrostis, separated by the C(3) genus Sartidia. In the current study, we investigated the evolutionary history of genes encoding phosphoenolpyruvate carboxylases (PEPC) to shed light on the photosynthetic transitions that occurred in Aristidoideae. We identified six distinct PEPC gene lineages that appeared through several rounds of gene duplications before or early during grass diversification. The gene lineage encoding the C(4) PEPC of Stipagrostis differs from those of the other C(4) grasses, including Aristida. These distinct origins of C(4) PEPC genes from these two Aristidoideae genera unequivocally indicate that they integrated the C(4) pathway independently. This highlights the importance of candidate-gene studies when inferring the evolutionary history of a character such as C(4) photosynthesis, one of the greatest evolutionary successes in plant history.     

Evolution of C(4) phosphoenolpyruvate carboxylase in Flaveria: determinants for high tolerance towards the inhibitor L-malate

During the evolution of angiosperms, C4 phosphoenolpyruvate carboxylases have evolved several times independently from ancestral non-photosynthetic isoforms. They show distinct kinetic and regulatory properties when compared with the C3 isozymes. To identify the evolutionary alterations which are responsible for C4-specific properties, particularly the increased tolerance towards the allosteric inhibitor L-malate, the photosynthetic phosphoenolpyruvate carboxylase of Flaveria trinervia Mohr C4 and its ortholog from the closely related C3 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 C4 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 C3 and the C4 enzyme towards this allosteric activator. In addition, it was possible to demonstrate that the only C4-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 C4 enzyme.     

Ingenious floral structure drives explosive pollination in Hydrilla verticillata (Hydrocharitaceae)

• In explosive pollination, many structures and mechanisms have evolved to achieve high‐speed stamen movement. The male flower of the submerged plant Hydrilla verticillata is reported to be able to release pollen explosively some time after leaving the mother plant time, but the mechanism of stamen movement and the related functional structure in this species are unclear.
• In this study, we observed the male flower structure and pollen dispersal process of H. verticillata. We analysed the stamen movements during the pollen dispersal process and conducted several controlled experiments to study the process of storage and release of elastic potential energy in explosive pollination.
• When the male flower of H. verticillata is bound to the united bracts, the sepals accumulate elastic potential energy through the expansion of basal extensor cells. After the male flower is liberated from the mother plant, the stamens unfold rapidly with the sepals under adhesion and transfer the elastic potential energy to the filament in seconds. Once stamens unfold to a critical angle, at which the elasticity of the filament just exceeds the adhesion between sepals and anthers, the stamens automatically rebound and release pollen in milliseconds.
• These results reveal that Catapult‐like stamens, spoon‐shaped sepals and enclosed united bracts in the spathe together constitute the functional structure in rapid stamen movement of H. verticillata. They ensure that the pollen can be released on the water surface, and thus adapt successfully to the pollen‐epihydrophilous pollination.

     

Metabolite activation of crassulacean Acid metabolism and c(4) phosphoenolpyruvate carboxylase

The effects of glycine, alanine, serine, and various phosphorylated metabolites on the activity of phosphoenolpyruvate (PEP) carboxylase from Zea mays and Crassula argentea were studied. The maize enzyme was found to be activated by amino acids at a site that is separate from the glucose 6-phosphate binding site. The combination of glycine and glucose 6-phosphate synergistically reduced the apparent K_m of the enzyme for PEP and increased the apparent V_max. Of the amino acids tested, glycine showed the lowest apparent K_a and caused the greatest activation. d-Isomers of alanine and serine were more effective activators than the l-isomers. Unlike the maize enzyme, the Crassula enzyme was not activated by amino acids. Activation of either the Crassula or maize enzyme by glucose 6-phosphate occurred without dephosphorylation of the activator molecule. Furthermore, the Crassula enzyme was activated by two compounds containing phosphonate groups whose carbon-phosphorus bonds were not cleaved by the enzyme. A study of analogs of glucose 6-phosphate with Crassula PEP carboxylase revealed that the identity of the ring heteroatom was a significant structural feature affecting activation. Activation was not highly sensitive to the orientation of the hydroxyl group at the second or fourth carbon positions or to the presence of a hydroxyl group at the second position. However, the position of the phosphate group was found to be a significant factor.