Can the cold tolerance of C4 photosynthesis in Miscanthus x giganteus relative to Zea mays be explained by differences in activities and thermal properties of Rubisco?

The previous investigations show that the amount and activity of Rubisco appears the major limitation to effective C(4) photosynthesis at low temperatures. The chilling-tolerant and bioenergy feedstock species Miscanthus x giganteus (M. x giganteus) is exceptionally productive among C(4) grasses in cold climates. It is able to develop photosynthetically active leaves at temperatures 6 degrees C below the minimum for maize, and achieves a productivity even at 52 degrees N that exceeds that of the most productive C(3) crops at this latitude. This study investigates whether this unusual low temperature tolerance can be attributed to differences in the amount or kinetic properties of Rubisco relative to maize. An efficient protocol was developed to purify large amounts of functional Rubisco from C(4) leaves. The maximum carboxylation activities (V(max)), activation states, catalytic rates per active site (K(cat)) and activation energies (E(a)) of purified Rubisco and Rubisco in crude leaf extracts were determined for M. x giganteus grown at 14 degrees C and 25 degrees C, and maize grown at 25 degrees C. The sequences of M. x giganteus Rubisco small subunit mRNA are highly conserved, and 91% identical to those of maize. Although there were a few differences between the species in the translated protein sequences, there were no significant differences in the catalytic properties (V(max), K(cat), and E(a)) for purified Rubisco, nor was there any effect of growth temperature in M. x giganteus on these kinetic properties. Extracted activities were close to the observed rates of CO(2) assimilation by the leaves in vivo. On a leaf area basis the extracted activities and activation state of Rubisco did not differ significantly, either between the two species or between growth temperatures. The activation state of Rubisco in leaf extracts showed no significant difference between warm and cold-grown M. x giganteus. In total, these results suggest that the ability of M. x giganteus to be productive and maintain photosynthetically competent leaves at low temperature does not result from low temperature acclimation or adaptation of the catalytic properties of Rubisco.     

Sensitivity of photosynthesis in a C4 plant,maize, to heat stress

Our objective was to determine the sensitivity of components of the photosynthetic apparatus of maize (Zea mays), a C4 plant, to high temperature stress. Net photosynthesis (Pn) was inhibited at leaf temperatures above 38 degrees C, and the inhibition was much more severe when the temperature was increased rapidly rather than gradually. Transpiration rate increased progressively with leaf temperature, indicating that inhibition was not associated with stomatal closure. Nonphotochemical fluorescence quenching (qN) increased at leaf temperatures above 30 degrees C, indicating increased thylakoid energization even at temperatures that did not inhibit Pn. Compared with CO(2) assimilation, the maximum quantum yield of photosystem II (F(v)/F(m)) was relatively insensitive to leaf temperatures up to 45 degrees C. The activation state of phosphoenolpyruvate carboxylase decreased marginally at leaf temperatures above 40 degrees C, and the activity of pyruvate phosphate dikinase was insensitive to temperature up to 45 degrees C. The activation state of Rubisco decreased at temperatures exceeding 32.5 degrees C, with nearly complete inactivation at 45 degrees C. Levels of 3-phosphoglyceric acid and ribulose-1,5-bisphosphate decreased and increased, respectively, as leaf temperature increased, consistent with the decrease in Rubisco activation. When leaf temperature was increased gradually, Rubisco activation acclimated in a similar manner as Pn, and acclimation was associated with the expression of a new activase polypeptide. Rates of Pn calculated solely from the kinetics of Rubisco were remarkably similar to measured rates if the calculation included adjustment for temperature effects on Rubisco activation. We conclude that inactivation of Rubisco was the primary constraint on the rate of Pn of maize leaves as leaf temperature increased above 30 degrees C.     

C4 photosynthesis at low temperature. A study using transgenic plants with reduced amounts of Rubisco

C(4) plants are rare in the cool climates characteristic of high latitudes and elevations, but the reasons for this are unclear. We tested the hypothesis that CO(2) fixation by Rubisco is the rate-limiting step during C(4) photosynthesis at cool temperatures. We measured photosynthesis and chlorophyll fluorescence from 6 degrees C to 40 degrees C, and in vitro Rubisco and phosphoenolpyruvate carboxylase activity from 0 degrees C to 42 degrees C, in Flaveria bidentis modified by an antisense construct (targeted to the nuclear-encoded small subunit of Rubisco, anti-RbcS) to have 49% and 32% of the wild-type Rubisco content. Photosynthesis was reduced at all temperatures in the anti-Rbcs plants, but the thermal optimum for photosynthesis (35 degrees C) did not differ. The in vitro turnover rate (kcat) of fully carbamylated Rubisco was 3.8 mol mol(-)(1) s(-)(1) at 24 degrees C, regardless of genotype. The in vitro kcat (Rubisco Vcmax per catalytic site) and in vivo kcat (gross photosynthesis per Rubisco catalytic site) were the same below 20 degrees C, but at warmer temperatures, the in vitro capacity of the enzyme exceeded the realized rate of photosynthesis. The quantum requirement of CO(2) assimilation increased below 25 degrees C in all genotypes, suggesting greater leakage of CO(2) from the bundle sheath. The Rubisco flux control coefficient was 0.68 at the thermal optimum and increased to 0.99 at 6 degrees C. Our results thus demonstrate that Rubisco capacity is a principle control over the rate of C(4) photosynthesis at low temperatures. On the basis of these results, we propose that the lack of C(4) success in cool climates reflects a constraint imposed by having less Rubisco than their C(3) competitors.     

A rapid screening method to detect specific inhibitors of pyruvate orthophosphate dikinase as leads for C4 plant-selective herbicides

Plants using the C(4) photosynthetic pathway are highly represented among the world's worst weeds, with only 4 C(4) species being agriculturally productive (maize, sorghum, millet, and sugar cane). With the C(4) acid cycle operating as a biochemical appendage of C(3) photosynthesis, the additional enzymes involved in C(4) photosynthesis represent an attractive target for the development of weed-specific herbicides. The rate-limiting enzyme of this metabolic pathway is pyruvate orthophosphate dikinase (PPDK). PPDK, coupled with phosphoenolpyruvate carboxylase and nicotinamide adenine dinucleotide-malate dehydrogenase, was used to develop a microplate-based assay to detect inhibitors of enzymes of the C(4) acid cycle. The resulting assay had a Z' factor of 0.61, making it a high-quality assay able to reliably identify active test samples. Organic extracts of 6679 marine macroscopic organisms were tested within the assay, and 343 were identified that inhibited the 3 enzyme-coupled reaction. A high confirmation rate was achieved, with 95% of these hit extracts proving active again upon retesting. Sequential addition of phosphoenolpyruvate and oxaloacetate to the assay facilitated identification of 83 extracts that specifically inhibited PPDK.     

Development of structural and biochemical characteristics of C(4) photosynthesis in two types of Kranz anatomy in genus Suaeda (family Chenopodiaceae)

Genus Suaeda (family Chenopodiaceae, subfamily Suaedoideae) has two structural types of Kranz anatomy consisting of a single compound Kranz unit enclosing vascular tissue. One, represented by Suaeda taxifolia, has mesophyll (M) and bundle sheath (BS) cells distributed around the leaf periphery. The second, represented by Suaeda eltonica, has M and BS surrounding vascular bundles in the central plane. In both, structural and biochemical development of C(4) occurs basipetally, as observed by analysis of the maturation gradient on longitudinal leaf sections. This progression in development was also observed in mid-sections of young, intermediate, and mature leaves in both species, with three clear stages: (i) monomorphic chloroplasts in the two cell types in younger tissue with immunolocalization and in situ hybridization showing ribulose bisphosphate carboxylase oxygenase (Rubisco) preferentially localized in BS chloroplasts, and increasing in parallel with the establishment of Kranz anatomy; (ii) vacuolization and selective organelle positioning in BS cells, with occurrence of phosphoenolpyruvate carboxylase (PEPC) and immunolocalization showing that it is preferentially in M cells; (iii) establishment of chloroplast dimorphism and mitochondrial differentiation in mature tissue and full expression of C(4) biochemistry including pyruvate, Pi dikinase (PPDK) and NAD-malic enzyme (NAD-ME). Accumulation of rbcL mRNA preceded its peptide expression, occurring prior to organelle positioning and differentiation. During development there was sequential expression and increase in levels of Rubisco and PEPC followed by NAD-ME and PPDK, and an increase in the (13)C/(12)C isotope composition of leaves to values characteristic of C(4) photosynthesis. The findings indicate that these two forms of NAD-ME type C(4) photosynthesis evolved in parallel within the subfamily with similar ontogenetic programmes.     

Adaptation responses in C4 photosynthesis of maize under salinity

The effect of salinity on C(4) photosynthesis was examined in leaves of maize, a NADP-malic enzyme (NADP-ME) type C(4) species. Potted plants with the fourth leaf blade fully developed were treated with 3% NaCl solution for 5d. Under salt treatment, the activities of pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPCase), NADP-dependent malate dehydrogenase (NADP-MDH) and NAD-dependent malate dehydrogenase (NAD-MDH), which are derived mainly from mesophyll cells, increased, whereas those of NADP-ME and ribulose-1,5-bisphosphate carboxylase, which are derived mainly from bundle sheath cells (BSCs), decreased. Immunocytochemical studies by electron microscopy revealed that PPDK protein increased, while the content of ribulose-1,5-bisphosphate carboxylase/oxygenase protein decreased under salinity. In salt-treated plants, the photosynthetic metabolites malate, pyruvate and starch decreased by 40, 89 and 81%, respectively. Gas-exchange analysis revealed that the net photosynthetic rate, the transpiration rate, stomatal conductance (g(s)) and the intercellular CO(2) concentration decreased strongly in salt-treated plants. The carbon isotope ratio (δ(13)C) in these plants was significantly lower than that in control. These findings suggest that the decrease in photosynthetic metabolites under salinity was induced by a reduction in gas-exchange. Moreover, in addition to the decrease in g(s), the decrease in enzyme activities in BSCs was responsible for the decline of C(4) photosynthesis. The increase of PPDK, PEPCase, NADP-MDH, and NAD-MDH activities and the decrease of NADP-ME activity are interpreted as adaptation responses to salinity.     

Low growth temperatures modify the efficiency of light use by photosystem II for CO2 assimilation in leaves of two chilling-tolerant C4 species, Cyperus longus L. and Miscanthus x giganteus

Two C4 plants, Miscanthus x giganteus and Cyperus longus L., were grown at suboptimal growth temperatures and the relationships between the quantum efficiencies of photosynthetic electron transport through photosystem II (PSII) (PSII operating efficiency; Fq'/Fm') and CO2 assimilation (phiCO2) in leaves were examined. When M. x giganteus was grown at 10 degrees C, the ratio of the PSII operating efficiency to phiCO2 increased relative to that found in leaves grown at 14 and 25 degrees C. Similar increases in the Fq'/Fm': phiCO2 occurred in the leaves of two C. longus ecotypes when the plants were grown at 17 degrees C, compared to 25 degrees C. These elevations of Fq'/Fm': phiCO2 at low growth temperatures were not attributable to the development of anthocyanins, as has been suggested for maize, and were indicative of the operation of an alternative sink to CO2 assimilation for photosynthetic reducing equivalents, possibly oxygen reduction via a Mehler reaction, which would act as a mechanism for protection of PSII from photoinactivation and damage. Furthermore, in M. x giganteus grown at 10 degrees C, further protection of PSII was effected by a 20-fold increase in zeaxanthin content in dark-adapted leaves, which was associated with much higher levels of non-photochemical quenching of excitation energy, compared to that observed in leaves grown at 14 and 25 degrees C. These differences may explain the long growing season and remarkable productivity of this C4 plant in cool climates, even in comparison to other C4 species such as C. longus, which occur naturally in such climates.     

The temperature response of C(3) and C(4) photosynthesis

We review the current understanding of the temperature responses of C(3) and C(4) photosynthesis across thermal ranges that do not harm the photosynthetic apparatus. In C(3) species, photosynthesis is classically considered to be limited by the capacities of ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco), ribulose bisphosphate (RuBP) regeneration or P(i) regeneration. Using both theoretical and empirical evidence, we describe the temperature response of instantaneous net CO(2) assimilation rate (A) in terms of these limitations, and evaluate possible limitations on A at elevated temperatures arising from heat-induced lability of Rubisco activase. In C(3) plants, Rubisco capacity is the predominant limitation on A across a wide range of temperatures at low CO(2) (<300 microbar), while at elevated CO(2), the limitation shifts to P(i) regeneration capacity at suboptimal temperatures, and either electron transport capacity or Rubisco activase capacity at supraoptimal temperatures. In C(4) plants, Rubisco capacity limits A below 20 degrees C in chilling-tolerant species, but the control over A at elevated temperature remains uncertain. Acclimation of C(3) photosynthesis to suboptimal growth temperature is commonly associated with a disproportional enhancement of the P(i) regeneration capacity. Above the thermal optimum, acclimation of A to increasing growth temperature is associated with increased electron transport capacity and/or greater heat stability of Rubisco activase. In many C(4) species from warm habitats, acclimation to cooler growth conditions increases levels of Rubisco and C(4) cycle enzymes which then enhance A below the thermal optimum. By contrast, few C(4) species adapted to cooler habitats increase Rubisco content during acclimation to reduced growth temperature; as a result, A changes little at suboptimal temperatures. Global change is likely to cause a widespread shift in patterns of photosynthetic limitation in higher plants. Limitations in electron transport and Rubisco activase capacity should be more common in the warmer, high CO(2) conditions expected by the end of the century.     

Functional analysis of corn husk photosynthesis

The husk surrounding the ear of corn/maize (Zea mays) has widely spaced veins with a number of interveinal mesophyll (M) cells and has been described as operating a partial C(3) photosynthetic pathway, in contrast to its leaves, which use the C(4) photosynthetic pathway. Here, we characterized photosynthesis in maize husk and leaf by measuring combined gas exchange and carbon isotope discrimination, the oxygen dependence of the CO(2) compensation point, and photosynthetic enzyme activity and localization together with anatomy. The CO(2) assimilation rate in the husk was less than that in the leaves and did not saturate at high CO(2), indicating CO(2) diffusion limitations. However, maximal photosynthetic rates were similar between the leaf and husk when expressed on a chlorophyll basis. The CO(2) compensation points of the husk were high compared with the leaf but did not vary with oxygen concentration. This and the low carbon isotope discrimination measured concurrently with gas exchange in the husk and leaf suggested C(4)-like photosynthesis in the husk. However, both Rubisco activity and the ratio of phosphoenolpyruvate carboxylase to Rubisco activity were reduced in the husk. Immunolocalization studies showed that phosphoenolpyruvate carboxylase is specifically localized in the layer of M cells surrounding the bundle sheath cells, while Rubisco and glycine decarboxylase were enriched in bundle sheath cells but also present in M cells. We conclude that maize husk operates C(4) photosynthesis dispersed around the widely spaced veins (analogous to leaves) in a diffusion-limited manner due to low M surface area exposed to intercellular air space, with the functional role of Rubisco and glycine decarboxylase in distant M yet to be explained.     

热玫瑰小双孢菌来源的丙酮酸磷酸双激酶的表达及应用

以热玫瑰小双孢菌基因组DNA为模板, 通过PCR扩增得到了编码PPDK的基因, 将此基因片段插入到表达载体pET28a(+)中构建得到了重组表达质粒pET28a(+)-PPDK, 将重组表达质粒pET28a(+)-PPDK转化到大肠杆菌BL21(DE3)中, 经过IPTG诱导, 重组菌成功表达了N端带有6-His Tag的重组PPDK。经SDS-PAGE分析, 重组PPDK单体分子量为101 kD。经过镍亲和层析和超滤后, 重组PPDK蛋白基本达到电泳纯, 并被成功应用于焦测序中。     

热玫瑰小双孢菌来源的丙酮酸磷酸双激酶的表达及应用

以热玫瑰小双孢菌基因组DNA为模板, 通过PCR扩增得到了编码PPDK的基因, 将此基因片段插入到表达载体pET28a(+)中构建得到了重组表达质粒pET28a(+)-PPDK, 将重组表达质粒pET28a(+)-PPDK转化到大肠杆菌BL21(DE3)中, 经过IPTG诱导, 重组菌成功表达了N端带有6-His Tag的重组PPDK。经SDS-PAGE分析, 重组PPDK单体分子量为101 kD。经过镍亲和层析和超滤后, 重组PPDK蛋白基本达到电泳纯, 并被成功应用于焦测序中。     

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.     

Immunogold localization of photosynthetic enzymes in leaves of various C4 plants, with particular reference to pyruvate orthophosphate dikinase

Cellular localization of photosynthetic enzymes was investigated by immunogold electron microscopy for leaves of nine C4 grasses (three NADP-malic enzyme (NADP-ME)subtype species, three NAD-malic enzyme (NAD-ME) subtype species, and three phosphoenolpyruvate carboxykinase (PCK) subtype species), two C4 sedges (NADP-ME subtype species) and two C4 dicots (an NADP-ME and an NADP/NAD-ME subtype species). In leaves of all species, immunogold labelling was present for phosphoenolpyruvate carboxylase in the cytosol of the mesophyll cells (MC) and for ribulose-1,5-bisphosphate carboxylase/oxygenase in the chloroplasts of the bundle sheath cells (BSC). However, considerable specific variation was found in the intercellular patterns of labelling for pyruvate orthophosphate dikinase (PPDK). In the NADP-ME grasses, two NAD-ME grasses, and the dicots, significant labelling for PPDK was present in the both the BSC and the MC chloroplasts. In the other NAD-ME grass, the PCK grasses, and the sedges, labelling for PPDK was present almost exclusively in the chloroplasts of the MC. These patterns were observed in the leaves of both young seedlings and mature plants. These results indicate that the accumulation of PPDK in leaves of C4 plants is not necessarily restricted to the MC, although the chloroplasts of the MC accumulate more than those of the BSC.Key words: C4 plants, immunolocalization, phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, ribulose-1,5-bisphosphate carboxylase/oxygenase.      

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.     

Variation in the k(cat) of Rubisco in C(3) and C(4) plants and some implications for photosynthetic performance at high and low temperature

The capacity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to consume RuBP is a major limitation on the rate of net CO(2) assimilation (A) in C(3) and C(4) plants. The pattern of Rubisco limitation differs between the two photosynthetic types, as shown by comparisons of temperature and CO(2) responses of A and Rubisco activity from C(3) and C(4) species. In C(3) species, Rubisco capacity is the primary limitation on A at light saturation and CO(2) concentrations below the current atmospheric value of 37 Pa, particularly near the temperature optimum. Below 20 degrees C, C(3) photosynthesis at 37 and 68 Pa is often limited by the capacity to regenerate phosphate for photophosphorylation. In C(4) plants, the Rubisco capacity is equivalent to A below 18 degrees C, but exceeds the photosynthetic capacity above 25 degrees C, indicating that Rubisco is an important limitation at cool but not warm temperatures. A comparison of the catalytic efficiency of Rubisco (k(cat) in mol CO(2) mol(-1) Rubisco active sites s(-1)) from 17 C(3) and C(4) plants showed that Rubisco from C(4) species, and C(3) species originating in cool environments, had higher k(cat) than Rubisco from C(3) species originating in warm environments. This indicates that Rubisco evolved to improve performance in the environment that plants normally experience. In C(4) plants, and C(3) species from cool environments, Rubisco often operates near CO(2) saturation, so that increases in k(cat) would enhance A. In warm-habitat C(4) species, Rubisco often operates at CO(2) concentrations below the K(m) for CO(2). Because k(cat) and K(m) vary proportionally, the low k(cat) indicates that Rubisco has been modified in a manner that reduces K(m) and thus increases the affinity for CO(2) in C(3) species from warm climates.