Chemical modification of arginine alleviates the decline in activity during catalysis of spinach Rubisco

Arginine residues of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were chemically modified with phenylglyoxal (PhG). PhG inactivated Rubisco with a half-time of 20-25 min. An inclusion of a catalytic product, 3-phosphoglycerate (PGA), protected Rubisco from inactivation and delayed the half-time to 60-90 min. Peptide mapping and sequencing of Rubisco modified for 60 min with radiolabeled PhG in the presence of 10mM PGA revealed that Arg187, Arg258, and Arg431 of the large subunit were modified. The extent and rate of the decline in activity during catalysis (fallover phenomenon) were reduced by the modification. This is the first report identifying PhG-modified arginine residues and to demonstrate the effect of the modification of arginine residues on the kinetics of fallover.     

Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis Is Not Due to Decarbamylation of the Catalytic Site

An investigation was made of the proposal that the slow inactivation of ribulosebisphosphate carboxylase (Rubisco) activity, which occurs during in vitro assays, is due to decarbamylation of the enzyme. The level of carbamylation was compared with catalytic activity during assay conditions in which activity was both increasing and decreasing. Carbamylation level was measured using the reaction-intermediate analogue 2' -carboxy-D-arabinitol-1, 5-bisphosphate (carboxyarabinitol-P(2)). A dual isotope procedure was used in which [(3)H]carboxyarabinitol-P(2) measured total active sites and (14)CO(2) reported the level of carbamylation. The efficacy of the procedure was verified both in the presence and in the absence of the substrate d-ribulose-1, 5-bisphosphate (ribulose-P(2)). These measurements showed that changes in activity during assays were not correlated with carbamylation status. Inactivation during assays initiated with both fully and partially carbamylated enzyme was not associated with any change in carbamylation level. This implies that the loss of activity during assays is not due to ribulose-P(2) binding and sequestering the E form of the enzyme. Ribulose-P(2) did not appear to alter the equilibrium between carbamylated and uncarbamylated enzyme, but it did slow the rate at which enzyme was both decarbamylated and carbamylated. The most likely explanation for the loss of activity during assays appears to be the sequestration of carbamylated, Mg(2+)-bound active sites by an inhibitor.     

Subsaturating Ribulose-1,5-Bisphosphate Concentration Promotes Inactivation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) (Studies Using Continuous Substrate Addition in the Presence and Absence of Rubisco Activase)

We developed a continuous-addition method for maintaining subsaturating concentrations of ribulose-1,5-bisphosphate (RuBP) for several minutes, while simultaneously monitoring its consumption by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This method enabled us to observe the effects of subsaturating RuBP and CO2 concentrations on the activity of Rubisco during much longer periods than previously studied. At saturating CO2, the activity of the enzyme declined faster when RuBP was maintained at concentrations near its Km value than when RuBP was saturating. At saturating RuBP, activity declined faster at limiting than at saturating CO2, in accordance with previous observations. The most rapid decline in activity occurred when both CO2 and RuBP concentrations were subsaturating. The activity loss was accompanied by decarbamylation of the enzyme, even though the enzyme was maintained at the same CO2 concentration before and after exposure to RuBP. Rubisco activase ameliorated the decline in activity at subsaturating CO2 and RuBP concentrations. The results are consistent with a proposed mechanism for regulating the carbamylation of Rubisco, which postulates that Rubisco activase counteracts Rubisco's unfavorable carbamylation equilibrium in the presence of RuBP by accelerating, in an ATP-dependent manner, the release of RuBP from its complex with uncarbamylated sites.     

Mutagenesis at two distinct phosphate-binding sites unravels their differential roles in regulation of Rubisco activation and catalysis

Orthophosphate (P(i)) has two antagonistic effects on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), stimulation of activation and inhibition of catalysis by competition with the substrate RuBP. The enzyme binds P(i) at three distinct sites, two within the catalytic site (where 1P and 5P of ribulose 1,5-bisphosphate [RuBP] bind), and the third at the latch site (a positively charged pocket involved in active-site closure during catalysis). We examined the role of the latch and 5P sites in regulation of Rubisco activation and catalysis by introducing specific mutations in the enzyme of the cyanobacterium Synechocystis sp. strain PCC 6803. Whereas mutations at both sites abolished the P(i)-stimulated Rubisco activation, substitution of residues at the 5P site, but not at the latch site, affected the P(i) inhibition of Rubisco catalysis. Although some of these mutations substantially reduced the catalytic turnover of Rubisco and increased the K(m)(RuBP), they had little to moderate effect on the rate of photosynthesis and no effect on photoautotrophic growth. These findings suggest that in cyanobacteria, Rubisco does not limit photosynthesis to the extent previously estimated. These results indicate that both the latch and 5P sites participate in regulation of Rubisco activation, whereas P(i) binding only at the 5P site inhibits catalysis in a competitive manner.     

2-Carboxy-D-arabinitol 1-phosphate (CA1P) phosphatase: evidence for a wider role in plant Rubisco regulation

The genes for CA1Pase (2-carboxy-D-arabinitol-1-bisphosphate phosphatase) from French bean, wheat, Arabidopsis and tobacco were identified and cloned. The deduced protein sequence included an N-terminal motif identical with the PGM (phosphoglycerate mutase) active site sequence [LIVM]-x-R-H-G-[EQ]-x-x-[WN]. The corresponding gene from wheat coded for an enzyme with the properties published for CA1Pase. The expressed protein lacked PGM activity but rapidly dephosphorylated 2,3-DPG (2,3-diphosphoglycerate) to 2-phosphoglycerate. DTT (dithiothreitol) activation and GSSG inactivation of this enzyme was pH-sensitive, the greatest difference being apparent at pH 8. The presence of the expressed protein during in vitro measurement of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activity prevented a progressive decline in Rubisco turnover. This was due to the removal of an inhibitory bisphosphate that was present in the RuBP (ribulose-1,5-bisphosphate) preparation, and was found to be PDBP (D-glycero-2,3-pentodiulose-1,5-bisphosphate). The substrate specificity of the expressed protein indicates a role for CA1Pase in the removal of 'misfire' products of Rubisco.     

Oxygen-dependent H2O2 production by Rubisco

Oxygen and ribulose-1,5-bisphosphate dependent, H(2)O(2) production was observed with several wild type Rubisco enzymes using a sensitive assay. H(2)O(2) and d-glycero-2,3-pentodiulose-1,5-bisphosphate, a known and potent inhibitor of Rubisco activity, are predicted products arising from elimination of H(2)O(2) from a peroxyketone intermediate, specific to oxygenase activity. Parallel assays using varying CO(2) and O(2) concentrations revealed that the partitioning to H(2)O(2) during O(2) consumption by spinach Rubisco was constant at 1/260-1/270. High temperature (38 degrees C), which reduces Rubisco specificity for CO(2) versus O(2), increased the rates of H(2)O(2) production and O(2) consumption, resulting in a small increase in partitioning to H(2)O(2) (1/210). Two Rubiscos with lower specificity than spinach exhibited greater partitioning to H(2)O(2) during catalysis: Chlamydomonas reinhardtii (1/200); and Rhodospirillum rubrum (1/150).     

Evolving improved Synechococcus Rubisco functional expression in Escherichia coli

The photosynthetic CO2-fixing enzyme Rubisco [ribulose-P(2) (D-ribulose-1,5-bisphosphate) carboxylase/oxygenase] has long been a target for engineering kinetic improvements. Towards this goal we used an RDE (Rubisco-dependent Escherichia coli) selection system to evolve Synechococcus PCC6301 Form I Rubisco under different selection pressures. In the fastest growing colonies, the Rubisco L (large) subunit substitutions I174V, Q212L, M262T, F345L or F345I were repeatedly selected and shown to increase functional Rubisco expression 4- to 7-fold in the RDE and 5- to 17-fold when expressed in XL1-Blue E. coli. Introducing the F345I L-subunit substitution into Synechococcus PCC7002 Rubisco improved its functional expression 11-fold in XL1-Blue cells but could not elicit functional Arabidopsis Rubisco expression in the bacterium. The L subunit substitutions L161M and M169L were complementary in improving Rubisco yield 11-fold, whereas individually they improved yield approximately 5-fold. In XL1-Blue cells, additional GroE chaperonin enhanced expression of the I174V, Q212L and M262T mutant Rubiscos but engendered little change in the yield of the more assembly-competent F345I or F345L mutants. In contrast, the Rubisco chaperone RbcX stimulated functional assembly of wild-type and mutant Rubiscos. The kinetic properties of the mutated Rubiscos varied with noticeable reductions in carboxylation and oxygenation efficiency accompanying the Q212L mutation and a 2-fold increase in K(ribulose-P2) (K(M) for the substrate ribulose-P2) for the F345L mutant, which was contrary to the approximately 30% reductions in K(ribulose-P2) for the other mutants. These results confirm the RDE systems versatility for identifying mutations that improve functional Rubisco expression in E. coli and provide an impetus for developing the system to screen for kinetic improvements.     

2-(4-Bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, a new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. Determination of reaction parameters and characterization of an active site peptide

A new affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum, 2-(4-bromoacetamido)anilino-2-deoxypentitol 1,5-bisphosphate, has been prepared, Reductive amination of ribulose-P2 with p-phenylenediamine in the presence of sodium cyanoborohydride yielded an epimeric mixture which was resolved by chromatography on quaternary aminoethyl-Sephadex. Subsequent bromoacetylation of the isolated amino bisphosphates gave reagents A and B (ribo and arabino epimers of 2-(4-bromoacetamido) anilino-2-deoxypentitol 1,5-bisphosphate) which were competitive inhibitors of the carboxylase with Ki values of 705 and 104 microM, respectively. Reagent A exhibited no time-dependent effects on the carboxylase in either the deactivated or activated state. Incubation of the enzyme with reagent B in the presence of the essential activators CO2 and Mg2+, however, resulted in an irreversible, time-dependent loss of activity, with a Kinact of 125 microM and a minimal half-time of 7.3 min. Covalent incorporation of [14C]reagent B was directly proportional to the loss of activity, with total inactivation correlating with an incorporation of 1.1 mol of reagent/mol of subunit. Inclusion of the competitive inhibitor 2-carboxyribitol 1,5-bisphosphate protected against inactivation with a concomitant reduction in incorporation. Neither reagent affected the activity of spinach carboxylase. Fractionation of [14C]reagent B-modified enzyme on DEAE-cellulose, subsequent to carboxymethylation and tryptic digestion, revealed two major radioactive peaks of approximately equal area. Digestion of each peak with alkaline phosphatase and rechromatography on DEAE-cellulose resulted in pure peptides I and II. The peptides were identical except in the site of labeling: peptide I contained a modified cysteinyl residue while peptide II contained a modified histidyl residue. Automated Edman degradation established the sequence as (sequence in text) which is located near the NH2 terminus of the enzyme. The lack of reactivity with the spinach enzyme is explained by the deletion of the histidyl residue and the replacement of cysteine by tryptophan in the eukaryotic species. Although the nonconservation of the modified residues argues against a functional role other than maintenance of structural integrity, the extensive homology in this region among seven different species of carboxylase is compatible with the region comprising a portion of the active site.     

两个品种烟草叶片发育过程中几种光合参数变化的比较

比较烟草2个品种‘NC89’和‘JYH’叶片发育过程中几个光合参数变化的结果表明,烟草叶片发育过程中光合速率变化表现为上升期、高值持续期（APD）和速降期,叶绿素含量变化经历上升期、相对稳定期（RSP）和速降期。光合功能衰退过程中,核酮糖．1,5-二磷酸羧化酶（RuBPCase）活性比电子传递活性下降快。可逆衰退阶段的2个品种类囊体膜多肽组分和‘NC89’的核酮糖-1,5-二磷酸羧化酶加氧酶（Rubisco）大亚基基本上无变化;不可逆衰退阶段的2个品种类囊体膜多肽组分、Rubisco大小亚基均快速降解,尤其是光系统Ⅱ（PSⅡ）复合体和Rubisco小亚基。‘JYH’的叶龄为10-40d的叶中各光合参数与‘NC89’的差别不大,但‘JYH’的光合功能期短,光合功能衰退过程中光合电子传递与碳同化失衡较严重,光合功能衰退比‘NC89’早而迅速。     

Rubisco activity of scenedesmus ecornis: What is wrong with initial activity determination?

A procedure was devised to measure the initial and total Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activities for the green microalga, Scenedesmus ecornis. Total Rubisco activities corresponded well with photosynthetic carbon assimilation rates. Initial activities ranged from 10 to 40% of the total activities and did not correlate with photosynthetic rates. Investigations into potential causes of the reduced initial activities yielded modest increases in percentage of the total activity. Values of K_m for ribulose-1,5-bisphosphate (RuBP) were similar for both initial and CO₂-Mg²⁺ activated enzyme. Total activities increased with increasing concentrations of RuBP to 400 μm, the assay concentration. However, concentrations above the K_m, 25 μm RuBP, were inhibitory for the initial Rubisco form. Inhibition increased with increasing RuBP concentration. The addition of Mg²⁺ in the extraction solution did not prevent RuBP inhibition. The results suggest that the low initial Rubisco activities are principally due to decarbamylation of the active sites of the enzyme during extraction.     

Rubisco activity in Mediterranean species is regulated by the chloroplastic CO2 concentration under water stress

Water stress decreases the availability of the gaseous substrate for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) by decreasing leaf conductance to CO(2). In spite of limiting photosynthetic carbon assimilation, especially in those environments where drought is the predominant factor affecting plant growth and yield, the effects of water deprivation on the mechanisms that control Rubisco activity are unclear. In the present study, 11 Mediterranean species, representing different growth forms, were subject to increasing levels of drought stress, the most severe one followed by rewatering. The results confirmed species-specific patterns in the decrease in the initial activity and activation state of Rubisco as drought stress and leaf dehydration intensified. Nevertheless, all species followed roughly the same trend when Rubisco activity was related to stomatal conductance (g(s)) and chloroplastic CO(2) concentration (C(c)), suggesting that deactivation of Rubisco sites could be induced by low C(c), as a result of water stress. The threshold level of C(c) that triggered Rubisco deactivation was dependent on leaf characteristics and was related to the maximum attained for each species under non-stressing conditions. Those species adapted to low C(c) were more capable of maintaining active Rubisco as drought stress intensified.     

Rapid heating of intact leaves reveals initial effects of stromal oxidation on photosynthesis

Heat stress in leaves under natural conditions is characterized by rapid fluctuations in temperature. These fluctuations can be on the order of 10 degrees C in 7 s. By using a specially modified gas-exchange chamber, these conditions were mimicked in the laboratory to analyse the biochemical response to heat spikes. The decline in ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco) activity during prolonged heat stress is generally associated with an increase in ribulose 1,5-bisphosphate (RuBP) levels. However, rapid heating caused an initial decline in RuBP which was subsequently followed by a small decline in Rubisco carbamylation. The ratio of RuBP to Rubisco sites declined from a saturating concentration to a sub-saturating concentration, providing a possible mechanism for the decarbamylation of Rubisco. If RuBP is saturating (>1.8 RuBP Rubisco site(-1)), it acts as a cap on the catalytic site and keeps Rubisco activated. Measurements of triose-phosphate levels and NADP-malate dehydrogenase activation (a stromal redox proxy) indicated that the regeneration of RuBP by the Calvin cycle was limited by the availability of redox power.     

Rubisco regulation: a role for inhibitors

In photosynthesis Rubisco catalyses the assimilation of CO(2) by the carboxylation of ribulose-1,5-bisphosphate. However, the catalytic properties of Rubisco are not optimal for current or projected environments and limit the efficiency of photosynthesis. Rubisco activity is highly regulated in response to short-term fluctuations in the environment, although such regulation may not be optimally poised for crop productivity. The regulation of Rubisco activity in higher plants is reviewed here, including the role of Rubisco activase, tight binding inhibitors, and the impact of abiotic stress upon them.     

Covalent modification of a highly reactive and essential lysine residue of ribulose-1,5-bisphosphate carboxylase/oxygenase activase.

Chemical modification of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase with water-soluble N-hydroxysuccinimide esters was used to identify a reactive lysyl residue that is essential for activity. Incubation of Rubisco activase with sulfosuccinimidyl-7-amino-4-methylcoumarin-3-acetate (AMCA-sulfo-NHS) or sulfosuccinimidyl-acetate (sulfo-NHS-acetate) caused progressive inactivation of ATPase activity and concomitant loss of the ability to activate Rubisco. AMCA-sulfo-NHS was the more potent inactivator of Rubisco activase, exhibiting a second-order rate constant for inactivation of 239 M-1 s-1 compared to 21 M-1 s-1 for sulfo-NHS-acetate. Inactivation of enzyme activity by AMCA-sulfo-NHS correlated with the incorporation of 1.9 mol of AMCA per mol of 42-kD Rubisco activase monomer. ADP, a competitive inhibitor of Rubisco activase, afforded considerable protection against inactivation of Rubisco activase and decreased the amount of AMCA incorporated into the Rubisco activase monomer. Sequence analysis of the major labeled peptide from AMCA-sulfo-NHS-modified enzyme showed that the primary site of modification was lysine-247 (K247) in the tetrapeptide methionine-glutamic acid-lysine-phenylalanine. Upon complete inactivation of ATPase activity, modification of K247 accounted for 1 mol of AMCA incorporated per mol of Rubisco activase monomer. Photoaffinity labeling of AMCA-sulfo-NHS- and sulfo-NHS-acetate-modified Rubisco activase with ATP analogs derivatized on either the adenine base or on the gamma-phosphate showed that K247 is not essential for the binding of adenine nucleotides per se. Instead, the data indicated that the essentiality of K247 is probably due to an involvement of this highly reactive, species-invariant residue in an obligatory interaction that occurs between the protein and the nucleotide phosphate during catalysis.     

The Loss of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Caused by 24-Hour Rain Treatment Fully Explains the Decrease in the Photosynthetic Rate in Bean Leaves

Recently, we have found that simulated rainfall causes a chronic inhibition of leaf photosynthesis in Phaseolus vulgaris (M. Ishibashi and I. Terashima [1995] Plant Cell Environ 18: 431-438). Mechanisms of this inhibition were examined in the present study. After the plants were treated with continuous mist for 24 h and then dried to unwet conditions, light-saturated photosynthetic rates of the leaves measured at 35 Pa ambient CO2 decreased to one-half of the control level. The extractable activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) also decreased to the same extent. Unexpectedly, this decline was due not to the lowered activation state but to the decrease in the amount of Rubisco. Before or after the "rain" treatment, the relationship between the net photosynthetic rate and the amount of Rubisco was expressed as a unique linear function with a small intercept (r2 = 0.84). From these it was inferred that the main cause of the rain-induced decline in photo-synthetic rate was the loss in amount of Rubisco.     

Use of Transgenic Plants with Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Antisense DNA to Evaluate the Rate Limitation of Photosynthesis under Water Stress

The biochemical lesion that causes impaired chloroplast metabolism (and, hence, photosynthetic capacity) in plants exposed to water deficits is still a subject of controversy. In this study we used tobacco (Nicotiana tabacum L.) transformed with "antisense" ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) DNA sequences to evaluate whether Rubisco or some other enzymic step in the photosynthetic carbon reduction cycle pathway rate limits photosynthesis at low leaf water potential ([psi]w). These transformants, along with the wild-type material, provided a novel model system allowing for an evaluation of photosynthetic response to water stress in near-isogenic plants with widely varying levels of functional Rubisco. It was determined that impaired chloroplast metabolism (rather than decreased leaf conductance to CO2) was the major cause of photosynthetic inhibition as leaf [psi]w declined. Significantly, the extent of photosynthetic inhibition at low [psi]w was identical in wild-type and transformed plants. Decreasing Rubisco activity by 68% did not sensitize photosynthetic capacity to water stress. It was hypothesized that, if water stress effects on Rubisco caused photosynthetic inhibition under stress, an increase in the steady-state level of the substrate for this enzyme, ribulose 1,5-bisphosphate (RuBP), would be associated with stress-induced photosynthetic inhibition. Steady-state levels of RuBP were reduced as leaf [psi]w declined, even in transformed plants with low levels of Rubisco. Based on the similarity in photosynthetic response to water stress in wild-type and transformed plants, the reduction in RuBP as stress developed, and studies that demonstrated that ATP supply did not rate limit photosynthesis under stress, we concluded that stress effects on an enzymic step involved in RuBP regeneration caused impaired chloroplast metabolism and photosynthetic inhibition in plants exposed to water deficits.     

Inhibition and stimulation of ribulose-1,5-bisphosphate carboxylase/oxygenase by glyoxylate

Glyoxylate is a slowly reversible inhibitor of the CO2/Mg2+-activated form of ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach leaves. Inactivation occurred with an apparent dissociation constant of 3.3 mM and a maximum pseudo-first-order rate constant of 7 X 10(-3) s-1. The rate constant for reactivation was 1.2 X 10(-2) s-1. Glyoxylate did not cause differential inhibition of ribulosebisphosphate carboxylase or oxygenase activities. 6-Phosphogluconate protected the enzyme from inactivation by glyoxylate. Glyoxylate was incorporated irreversibly into the large subunit of ribulosebisphosphate carboxylase after reduction with sodium borohydride. Activated enzyme incorporated 1.3 mol of glyoxylate per mole protomer, while enzyme treated with carboxyarabinitol 1,5-bisphosphate (CABP) to protect the active sites incorporated only 0.3 mol glyoxylate per mole protomer. The data suggest that glyoxylate forms a Schiff base with a lysyl residue in the region of the catalytic site. Glyoxylate stimulated the activity of the unactivated enzyme by about twofold. Pseudo-first-order inactivation also occurred with the unactivated enzyme after the initial stimulation by glyoxylate, although at a much slower rate than with the activated enzyme. Glyoxylate treatment of partially activated enzyme did not stimulate formation of the quaternary complex of enzyme X CO2 X Mg2+ X CABP.     

Role of the small subunit in ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis, but O2 competes with CO2 for substrate ribulose 1,5-bisphosphate, leading to the loss of fixed carbon. Interest in genetically engineering improvements in carboxylation catalytic efficiency and CO2/O2 specificity has focused on the chloroplast-encoded large subunit because it contains the active site. However, there is another type of subunit in the holoenzyme of plants, which, like the large subunit, is present in eight copies. The role of these nuclear-encoded small subunits in Rubisco structure and function is poorly understood. Small subunits may have originated during evolution to concentrate large-subunit active sites, but the extensive divergence of structures among prokaryotes, algae, and land plants seems to indicate that small subunits have more-specialized functions. Furthermore, plants and green algae contain families of differentially expressed small subunits, raising the possibility that these subunits may regulate the structure or function of Rubisco. Studies of interspecific hybrid enzymes have indicated that small subunits are required for maximal catalysis and, in several cases, contribute to CO2/O2 specificity. Although small-subunit genetic engineering remains difficult in land plants, directed mutagenesis of cyanobacterial and green-algal genes has identified specific structural regions that influence catalytic efficiency and CO2/O2 specificity. It is thus apparent that small subunits will need to be taken into account as strategies are developed for creating better Rubisco enzymes.     

Effects of Rubisco kinetics and Rubisco activation state on the temperature dependence of the photosynthetic rate in spinach leaves from contrasting growth temperatures

Recently, several studies reported that the optimum temperature for the initial slope [IS(Ci)] of the light-saturated photosynthetic rate (A) versus intercellular CO2 concentration (Ci) curve changed, depending on the growth temperature. However, few studies compare IS(Ci) with ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) properties. Here, we assessed Rubisco activation state and in vitro Rubisco kinetics, the main determinants of IS(Ci), in spinach leaves grown at 30/25 [high temperature (HT)] and 15/10 degrees C [low temperature (LT)]. We measured Rubisco activation state and A at a CO2 concentration of 360 microL L(-1) (A360) at various temperatures. In both HT and LT leaves, the Rubisco activation state decreased with increasing temperatures above the optimum temperatures for A360, while the activation state remained high at lower temperatures. To compare Rubisco characteristics, temperature dependences of the maximum rate of ribulose 1,5-bisphosphate (RuBP) carboxylation (Vcmax), specificity factor (Sc/o) and thermal stability were examined. We also examined Vcmax, and thermal stability in the leaves that were transferred from HT to LT conditions and were subsequently kept under LT conditions for 2 weeks (HL). Rubisco purified from HT, LT and HL leaves are called HT, LT and HL Rubisco, respectively. Thermal stabilities of LT and HL Rubisco were similar and lower than that of HT Rubisco. Both Vcmax and Sc/o in LT Rubisco were higher than those of HT Rubisco at low temperatures, while these were lower at high temperatures. Vcmax in HL Rubisco were similar to those of LT Rubisco at low temperatures, and to those of HT Rubisco at high temperatures. The predicted photosynthetic rates, taking account of the Rubisco kinetics and the Rubisco activation state, agreed well with A360 in both HT and LT leaves. This study suggests that photosynthetic performance is largely determined by the Rubisco kinetics at low temperature and by Rubisco Kinetics and the Rubisco activation state at high temperature.     

Metabolism of 2-carboxyarabinitol 1-phosphate and regulation of ribulose-1,5-bisphosphate carboxylase activity

Metabolism of 2-carboxy-D-arabinitol 1-phosphate (CA1P) is an important component in the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase (Rubisco) activity and whole leaf photosynthetic CO₂ assimilation in many species, and functions as one mechanism for regulating Rubisco activity when photosynthesis is light-limited. Species differ in their capacity to accumulate CA1P, ranging from those which can synthesize levels of this compound approaching or in excess of the Rubisco catalytic site concentration, to those which apparently lack the capacity for CA1P synthesis. CA1P is structurally related to the six carbon transition state intermediate of the carboxylation reaction and binds tightly to the carbamylated catalytic site of Rubisco, making that site unavailable for catalysis. Under steady-state, the concentration of CA1P in the leaf is highest at low photon flux density (PFD) or in the dark. Degradation of CA1P and recovery of Rubisco activity requires light and is stimulated by increasing PFD. The initial degradation reaction is catalyzed by an enzyme located in the chloroplast stroma, CA1P phosphatase, which yields carboxyarabinitol (CA) and inorganic phosphate as its products. The pathway of CA metabolism in the plant remains to be determined. Synthesis of CA1P occurs in the dark, and in Phaseolus vulgaris this process has been shown to be stimulated by low PFD. The pathway of CA1P synthesis and its relationship to the degradative pathway remains unknown at the present time. The discovery of the existence of this previously unknown carbon pathway in photosynthesis indicates that we still have much to learn concerning the regulation of Rubisco activity and photosynthesis.Abbreviations CA 2-carboxy-D-arabinitol - CA1P 2-carboxy-D-arabinitol 1-phosphate - CABP 2-carboxy-D-arabinitol-1,5-bisphosphate (transition state analog) - PFD photon flux density - P₁ inorganic phosphate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) - RuBP ribulose-1,5-bisphosphate