Rubisco Synthesis Research Articles

Rubisco synthesis, turnover and degradation: some new thoughts on an old problem

Rubisco synthesis, turnover and degradation: some new thoughts on an old problem

A dynamic model of Rubisco turnover in cereal leaves

A simple, improved model of Rubisco synthesis and degradation in cereal leaves is developed using data obtained over the leaf lifespan to return maximum likelihood values for Rubisco proteolysis and biosynthesis. It assumes that the time course of leaf Rubisco content can be described using a log-normal curve, and degradation of the Rubisco pool occurs exponentially. Curve parameters give an insight into how Rubisco dynamics differ among treatments or genotypes; also, statistical analyses can be performed more easily, requiring fewer data and allowing more flexibility in sampling than previous studies. Predicted patterns of synthesis correlate well with independent rbc gene transcript data. Rubisco degradation is a first-order decay process, and biosynthesis correlates with leaf elongation rates. As Rubisco degradation takes place according to first-order kinetics, control of leaf Rubisco concentration must be exerted by adjustment of biosynthetic, rather than proteolytic, rates.

Brefeldin A Inhibits Circadian Remodeling of Chloroplast Structure in the Dinoflagellate Gonyaulax

Circadian increases in the rate of carbon fixation in the dinoflagellate Gonyaulax are correlated with extensive plastid remodeling. One marker for this remodeling is mobilization of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from the plastid periphery to plastid regions nearer the cell center called pyrenoids. Nuclear-encoded proteins such as Rubisco transit through the Golgi in dinoflagellates; hence, we blocked protein import into the plastids using Brefeldin A (BFA) to explore the mechanism for plastid remodeling. We find that pyrenoid formation normally occurs concurrently with increased Rubisco synthesis rates in vivo, and when BFA is given prior to the onset of Rubisco synthesis, pyrenoid formation is partially or completely inhibited by 0.1 or 0.3 microg/mL BFA, respectively. Rubisco synthesis itself is not affected, and BFA-treated cells accumulate Rubisco in novel structures we term BFA bodies. Interestingly, when given just after the onset of Rubisco synthesis, BFA delays but does not block Rubisco mobilization, suggesting that a timing signal for plastid remodeling is delivered to the organelles at the same time as newly synthesized Rubisco. BFA also inhibits the circadian increases in carbon fixation rates, supporting the hypothesis that the biochemical basis for this circadian rhythm may be Rubisco distribution within the plastid.

Carbon and Nitrogen Metabolism Related to Grain Formation in Two Different Senescent Types of Maize

Field experiments and plant analyses were carried out to investigate the changes of ribulose-1,5-bisphophate carboxyase/oxygenase, phosphoenolpyruvate carboxylase, and chlorophyll in two maize (Zea mays L.) cultivars and their relationship to grain formation as regulated by different nitrogen applications. Two maize hybrids included “Danyu 13,” an earlier senescent hybrid, and “Zhongdan 306,” a stay-green hybrid. There were four nitrogen (N) treatments: (1) N1: 0 kg N ha−1, (2) N2: 250 kg N ha−1 (1/5 N as basal application and 4/5 N as top-dressing at stalk elongation stage), (3) N3: 250 kg N ha−1 (1/5 N as basal, 2/5 N as top-dressing at stalk elongation stage and at tasselling stage, respectively), (4) N4: 400 kg N ha−1 (1/5 N as basal and 4/5 N as top-dressing at stalk elongation stage). Results showed that biological yield and grain yield for “Zhongdan 306” were higher than “Danyu 13” in each N treatment, and the maximum grain yield was obtained in N2 and N3 treatment for “Danyu 13” and “Zhongdan 306,” separately. The amount of N, chlorophyll (chl), and Rubisco-N in leaves for “Zhongdan 306” was clearly higher than “Danyu 13” in each N treatment during entire growth stage. Synthesis of Rubisco and chl, especially Rubisco, played a very important role in the nitrogen supply for grain formation for two hybrids. Excessive N application (N4 treatment) could not increase grain yield further in both hybrids. For “Zhongdan 306,” the carbon sources in grain were 87–96% of total carbon (C) from leaf photosynthesis and 5–14% of total C from mobilized C, while the nitrogen sources in grain were 69–90% of total nitrogen absorbed by roots from soil and 10–31% of total nitrogen from mobilized N. For “Danyu 13,” the C from photosynthesis and mobilized C accounted for 66–81% and 19–34% respectively. The mobilized N and N absorbed by roots from soil accounted for 48–68% and 32–53%, separately. That is, for stay-green variety, the C and N in grain was depended mainly on the larger retranslocation of photosynthetic C and N absorbed by roots during maturation, and smaller retranslocation of C and N existed in vegetative mass before silking stage, whereas that for earlier senescent hybrid, relied upon the larger retranslocation of C and N existed in vegetative mass before silking stage, and smaller retranslocation of C from photosynthesis and N absorbed by roots during maturation.

Cytokinin Effects on Ribulose‐1,5‐Bisphosphate Carboxylase/Oxygenase and Nitrogen Partitioning in Rice during Ripening

High yield rice (Oryza sativa L.) cultivar Akenohoshi can realize a higher rate of photosynthesis during ripening by maintaining a higher level of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) than cv. Nipponbare. To clarify the causal factors for the difference in the levels of Rubisco in leaves between rice cultivars and the mechanisms of action of cytokinins on the maintenance of high levels of Rubisco in leaves, the level of rbcL and rbcS transcripts and the nitrogen content of leaves were investigated. The Rubisco content remained high not only in plants given additional nitrogen fertilizer (NF) but also in plants sprayed with 6-benzylaminopurine (BA). There was a close correlation between the Rubisco content and the level of rbcL and rbcS mRNAs. The maintenance of high leaf levels of Rubisco and of rbcL and rbcS mRNAs was related to the high nitrogen content in leaves, irrespective of the treatments and cultivars. The maintenance of high leaf nitrogen content was caused by an increase in nitrogen partitioning to leaves and not by an increase in nitrogen absorption by plants treated with BA. These results indicate that there are two probable effects of BA on the leaf level of Rubisco. One is the direct effect of BA inducing the synthesis of Rubisco. The other is the indirect effect of BA inducing the effective partitioning of nitrogen to leaves. Cytokinins could account for the difference in the reduction in leaf level of Rubisco during senescence between the rice cultivars.

Amount of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) Protein and levels of mRNAs of rbcS and rbcL in the leaves at different positions in transgenic rice plants with decreased content of Rubisco

The differences in the amounts of various photosynthetic components, including ribulose- 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and chlorophyll (Chl), and the activities of chloroplastic fructose-1,6-bisphosphatase (Cp-FBPase), NADP-glyceraldehyde-3- phosphate dehydrogenase (NADP-G3PDH), ribulose-5-phosphate kinase (Ru5P kinase), and sucrose-phosphate synthase (SPS) enzymes were examined in leaf blades at different leaf positions on the main culms of transgenic rbcS antisense rice plants. The transcript levels of rbcS and rbcL mRNAs were also determined. All the leaves were collected when the flag leaves became fully expanded. Although the Rubisco content was much lower in the upper leaves in the rbcS antisense plants than in the wild-type plants, the difference was less pronounced in lower leaves. Whereas the levels of rbcS mRNA in all the leaves were depressed in the antisense plants, the levels of rbcL mRNA did not differ. Both rbcS and rbcL mRNA levels in lower leaves were very low in the wild-type and antisense plants. Chl content and the activities of Cp-FBPase, NADP-G3PDH, Ru5P kinase, and SPS were slightly higher in the antisense plants. However, the changes among the leaves at different positions were similar to those in the wild-type plants. These results suggest that a selectively slow decrease in the Rubisco content in lower leaves occurs in the rbcS antisense plants and that it is caused by the attenuation of degradation and not by the maintenance of Rubisco synthesis.

Changes in synthesis and degradation of Rubisco and LHCII with leaf age in rice (Oryza sativa L.) growing under supplementary UV‐B radiation

AbstractThe effects of supplementary ultraviolet‐B (UV‐B) radiation on the changes in synthesis and degradation of ribulose ‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) and light‐harvesting chlorophyll a/b binding protein of PSII (LHCII) were examined, as well as mRNA levels for small and large subunits of Rubisco (rbcS and rbcL, respectively) and LHCII (cab) with leaf age in UV‐sensitive rice (Norin 1) and UV‐resistant rice (Sasanishiki). Both Rubisco and LHCII were actively synthesized until the leaf had fully expanded, and then decreased with increasing leaf age. Synthesis of Rubisco, but not LHCII, was significantly suppressed by UV‐B in Norin 1. The degradation of Rubisco was enhanced by UV‐B around the time of leaf maturation in the two cultivars. The levels of rbcS and rbcL were reduced by UV‐B at the early stages after leaf emergence in both cultivars. Cab transcripts were first present at high levels in the two cultivars, but drastically decreased due to UV‐B treatment immediately after leaf emergence in Norin 1. It was shown that synthesis and degradation of Rubisco and LHCII greatly changed with leaf age: Rubisco synthesis was significantly suppressed by supplementary UV‐B radiation at the transcription step during the early leaf stages. It was also suggested that the difference in UV‐B sensitivity in Rubisco synthesis between the two rice cultivars might be due to specific suppression both transcriptionally and post‐transcriptionally.

Changes in the turnover of Rubisco and levels of mRNAs of rbcL and rbcS in rice leaves from emergence to senescence

AbstractChanges in the amount of ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco; EC 4·1·1·39) synthesized and degraded and the levels of rbcL and rbcS mRNAs were examined in the eighth leaf blades of rice from emergence to senescence. Synthesis of Rubisco was very active during leaf expansion, became quite low at the time of full expansion and then declined further during senescence. The changes in the levels of rbcL and rbcS mRNAs co‐ordinated approximately with those in the amount of Rubisco synthesized. Thus, it is suggested that the amount of Rubisco synthesized is determined primarily by the levels of rbcL and rbcS mRNAs during the life span of the leaves. Degradation of Rubisco started just before the time of full expansion and became far more active than its synthesis during senescence. Since the synthesis of Rubisco during senescence scarcely contributed to its amount, it can be concluded that the degradation of Rubisco is the major determinant for the amount of Rubisco in senescent leaves. The decline in the level of rbcL mRNA occurred much earlier in the developmental stage and proceeded at a much faster rate than that of rbcL DNA, indicating that the level of rbcL DNA is not a major determinant for the level of rbcL mRNA in senescent leaves of rice.

Insertional Mutants of Chlamydomonas reinhardtii That Require Elevated CO2 for Survival

Aquatic photosynthetic organisms live in quite variable conditions of CO(2) availability. To survive in limiting CO(2) conditions, Chlamydomonas reinhardtii and other microalgae show adaptive changes, such as induction of a CO(2)-concentrating mechanism, changes in cell organization, increased photorespiratory enzyme activity, induction of periplasmic carbonic anhydrase and specific polypeptides (mitochondrial carbonic anhydrases and putative chloroplast carrier proteins), and transient down-regulation in the synthesis of Rubisco. The signal for acclimation to limiting CO(2) in C. reinhardtii is unidentified, and it is not known how they sense a change of CO(2) level. The limiting CO(2) signals must be transduced into the changes in gene expression observed during acclimation, so mutational analyses should be helpful for investigating the signal transduction pathway for low CO(2) acclimation. Eight independently isolated mutants of C. reinhardtii that require high CO(2) for photoautotrophic growth were tested by complementation group analysis. These mutants are likely to be defective in some aspects of the acclimation to low CO(2) because they differ from wild type in their growth and in the expression patterns of five low CO(2)-inducible genes (Cah1, Mca1, Mca2, Ccp1, and Ccp2). Two of the new mutants formed a single complementation group along with the previously described mutant cia-5, which appears to be defective in the signal transduction pathway for low CO(2) acclimation. The other mutations represent six additional, independent complementation groups.

Insertional Mutants of Chlamydomonas reinhardtii That Require Elevated CO2 for Survival

Aquatic photosynthetic organisms live in quite variable conditions of CO2 availability. To survive in limiting CO2conditions, Chlamydomonas reinhardtii and other microalgae show adaptive changes, such as induction of a CO2-concentrating mechanism, changes in cell organization, increased photorespiratory enzyme activity, induction of periplasmic carbonic anhydrase and specific polypeptides (mitochondrial carbonic anhydrases and putative chloroplast carrier proteins), and transient down-regulation in the synthesis of Rubisco. The signal for acclimation to limiting CO2 in C. reinhardtii is unidentified, and it is not known how they sense a change of CO2 level. The limiting CO2 signals must be transduced into the changes in gene expression observed during acclimation, so mutational analyses should be helpful for investigating the signal transduction pathway for low CO2 acclimation. Eight independently isolated mutants of C. reinhardtiithat require high CO2 for photoautotrophic growth were tested by complementation group analysis. These mutants are likely to be defective in some aspects of the acclimation to low CO2because they differ from wild type in their growth and in the expression patterns of five low CO2-inducible genes (Cah1, Mca1, Mca2,Ccp1, and Ccp2). Two of the new mutants formed a single complementation group along with the previously described mutant cia-5, which appears to be defective in the signal transduction pathway for low CO2 acclimation. The other mutations represent six additional, independent complementation groups.

Effects of UVB radiation on protein turnover of Rubisco and LHCII in rice

We previously found that the amount of Rubisco dramatically decreased in leaves of UV-sensitive rice grown with supplemental UVB radiation, while the amount of LHCII was not so much reduced [Hidema et al. 1996, Plant Cell Physiology.]. This work was aimed to clarify the effects of UVB radiation on synthesis and degradation of Rubisco and LHCII in leaves of UV-resistant Sasanishiki and UV-sensitive Norin 1 rice. And we examined the changes in the amounts of Rubisco or LHCII synthesized and degraded using 15N tracer, and mRNA transcription levels of rbcL, rbcS, and cab genes by RT-PCT analysis, during leaf development. The amount of Rubisco synthesized in Norin 1 grown under supplemental UVB radiation remarkably decreased during leaf developing, while the amount of LHCII synthesized in leaves of both cultivars was scarcely affected by supplemental UVB. The degradation of Rubisco and LHCII could not be detected during leaf development until leaf fully expanded in both plants grown with or without supplemental UVB, and started immediately after full expansion of leave. The mRNA transcripts for rbcS in Norin 1 greatly reduced by supplemental UVB. These results indicate that the synthesis of Rubisco was remarkably inhibited by UVB radiation during leaf development, and that the degradation of Rubisco was accelerated by UVB after leaf fully expanded.

Production and characterization of a specific rubisco monoclonal antibody, and its use in rubisco quantification during Zantedeschia aethiopica spathe development.

Ribulose 1,5-bisphosphate carboxylase/oxygenase was purified from leaves of Zantedeschia aethiopica and used to immunize female Balb/c mice. Monoclonal antibodies (MAbs) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner. A random selected IgG2a subclass MAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose CL-4B, with a recovery of 84.3% and it was apparently homogeneous on native PAGE. The monoclonality of the purified MAb was determined by IEF. The MAb was highly specific for Rubisco from leaves of Z. aethiopica as determined by Western blotting and was used to determine the concentration of Rubisco protein by enzyme-linked immunoadsorbent assay (ELISA), at three distinct stages of Z. aethiopica spathe development and in the leaf. The results suggest de novo synthesis of Rubisco during the spathe regreening, which could explain, at least in part, the increase of photosynthetic activity observed during regreening.

Ozone-induced changes in biosynthesis of Rubisco and associated compensation to stress in foliage of hybrid poplar.

Experiments were conducted during the growing seasons of 1993-1995 to determine whether exposure to ozone (O(3)) affected the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in hybrid poplar, Populus maximowizii A. Henry x trichocarpa Torr. & A. Gray, Clone 245. As the canopy aged, the concentration of Rubisco decreased at a more rapid rate in lower leaves of hyrid poplar ramets subjected to chronic O(3) exposure in open-top chambers than in comparable foliage of plants grown in charcoal-filtered air. There was no difference in rate of synthesis of Rubisco between treatments, suggesting that loss of this protein in O(3)-treated leaves was caused by an accelerated rate of proteolysis. In foliage higher in the canopy, both concentration and rate of synthesis of Rubisco were stimulated by O(3) for a brief period when the leaves were young. Quantification of mRNA for the small (rbcS) and large (rbcL) transcripts of Rubisco did not reveal changes that were likely to reflect altered synthesis of Rubisco as a prime response to O(3). Analyses of Rubisco concentration and rate of Rubisco synthesis in foliage connected by vascular traces within the canopy indicated that loss of Rubisco in older leaves was associated with an increase in this protein in younger leaves higher in the canopy. These data support the notion that accelerated senescence may provide some compensatory benefit to the plant. In 1995, the rate of synthesis of Rubisco was almost always higher in O(3)-treated foliage than in nontreated foliage, even when the concentration of Rubisco was adversely affected by the O(3) treatment. Because accelerated foliar abscission in response to O(3) was minimal in 1995 compared to other years, we speculate that, when abscission is delayed, Rubisco synthesis and concentration become uncoupled.

Effect of salicylic acid on the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase in barley leaves

Hordeum vulgare L. (cv. Alfa) was grown for 7 days over a range of salicylic acid (SA) concentrations (100 μmol/L-1 mmol/L), and the effect on the level of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) was examined. The treated plants showed a decrease in the level of total soluble protein, in particular in the level of RubisCO. In the presence of 1 mmol/L SA the level of total soluble protein on a fresh weight basis was about 68% of the control, whereas the level of RubisCO was about 50% that of control plants. The percentage of inhibition on the small subunit was higher, as a result of which the small/large subunit ratio was lower for the experimental variants. When 7-day-old barley seedlings were supplied with SA through the transpiration stream for 24 h no changes in the levels of total soluble protein and RubisCO were observed. The influence of SA on the synthesis of RubisCO was followed by in vivo labelling with a 14C-amino acid mixure (14C-AAM). Results demonstrated that SA inhibited both the synthesis of total soluble protein and, more pronounced, the synthesis of RubisCO.

Sulfur Availability, Rubisco Content, and Photosynthetic Rate of Soybean

Understanding the role of S in soybean [Glycine max (L.) Merr.] growth is important because a deficiency of the S‐containing amino acids methionine and cysteine limits the nutritional value of soybean protein. As part of a broader study of S uptake and use, our objective was to quantify the effect of S availability on photosynthetic characteristics of soybean. Kenwood soybean was grown in a 3:1 mixture of Ida silt loam [fine‐silty, mixed (calcareous), mesic Typic Udorthent] and acid washed sand with varying amounts of S added as gypsum (CaSO4) in two greenhouse trials. Single‐leaf CO2‐exchange rate (CER), quantum efficiency, dark respiration, leaf S, leaf N, and Rubisco content were determined. There appeared to be a critical level of 35 mg S per kg mixture (about 85 mg S per plant) below which specific leaf S (mg m−2 leaf area) declined linearly with decreases in soil S availability. On average, the most S deficient plants had 70% lower CER, 42% less quantum efficiency, and 50% decline in dark respiration relative to plants receiving adequate S. Total leaf N and Rubisco content were linearly related to leaf S levels, and declined on average 62, and 95%, respectively, as S declined from approximately 120 to 40 mg S m−2 leaf area. There was a linear relation between CER and Rubisco content; however, the amount of nonstructural carbohydrates per unit leaf N increased two‐fold under S deficiency, suggesting that limitations on sink strength were greater than those on source capacity. Carbon dioxide exchange rate was linearly related to leaf S content at all sample dates, with a mean relation of 216 (μmol CO2 g−1 S s−1. The Rubisco fraction declined linearly from nearly 50 to <10% of soluble protein, as leaf soluble protein content declined under S deficiency. We postulate that the decline in the Rubisco fraction may be a function of increased relative importance of housekeeping enzymes for survival as protein levels decline, and/or down‐regulation of Rubisco synthesis in response to a build up of carbohydrates in the source leaves under S deficiency.

Control of the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit in cotyledons during dark growth of sugar beet seedlings

The expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit in cotyledons was investigated during germination and dark growth of sugar beet (Beta vulgaris L.) seedlings. The levels of large-subunit (LS) Rubisco were determined by immunodetection on western blots of SDS-PAGE-separated proteins, using highly specific monoclonal antibodies. Although rbcL transcripts of 1.6 kb were shown to be present at all stages of development, from imbibition to prolonged dark-growth, the 55 kDa LS Rubisco was not detectable in dry seeds and during early dark growth. At these early stages of development, the monoclonal antibodies detected significant levels of a 38 kDa protein. This protein was associated with the polysomal fraction and was found to disappear progressively during cotyledon development and proplastid-etioplast transition, while carotenoids, rbcL transcripts and the 55 kDa LS Rubisco accumulated. Abscisic acid and gibberellic acid, which affected the rate of germination and plastid development, correlatively modified the time-course of transition from the 38 kDa to the 55 kDa protein. Synthesis of LS Rubisco during proplastid-etioplast development is therefore shown to be regulated not only at the level of mRNA accumulation, but also at the translational level. Thus translational control is discussed in relation with ribosomal pausing, which has been described for a number of mRNAs in chloroplast systems.

Regulation of ribulose bisphosphate carboxylase gene expression in natural phytoplankton communities. I. Diel rhythms

Marine phytoplankton fix carbon dloxide primarily through the action of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the first enzyme in the Calvin Cycle. Although the regulation of this enzyme has been studied in algal cultures and higher plants, little is known regarding RubisCO regulation in natural phytoplankton populations. To determine if natural communities of phytoplankton utilize transcriptional regulation to control RubisCO expression, we investigated the diel relationship between ' carbon fixation and RublsCO large subunit (rbcL) transcript levels, rbcL DNA, chlorophyll a, autofluorescent cell counts and bacterial direct counts for natural communities of the southeastern Gulf of Mexico. Studies were performed with natural phytoplankton populations in Lagrangian studies or deck-top incubators as well as with a Prochlorococcus isolate in culture from the North Pacific Ocean. rbcL mRNA and DNA were detected by stringent hybridization using an antisense rbcL RNA gene probe originating from Synechococcus sp. PCC 6301. For natural communities, carbon fixation maxima (-0.1 to 0.7 pg C 1-' h-') occurred from early morning to mld afternoon, with minimal values at nlght. Peak levels of rbcL mRNA (3.6 to 22.2 ng 1-') almost always coincided with the time of maxlmum carbon fixation and were positively correlated. When natural phytoplankton popula t ion~ or the Prochlorococcus culture were maintained in continuous illuminat~on for 24 h, the same die1 pattern of RubisCO regulation was observed, implying that diel entra~ned rhythms in rbcL transcription occurred. The results thus indicate that natural phytoplankton communrties appear to regulate carbon fixatlon at least in part by transcriptional control of RubisCO synthesis and that such patterns appear to be rhythmlc in nature.

Rubisco Synthesis, Assembly, Mechanism, and Regulation

Rubisco Synthesis, Assembly, Mechanism, and Regulation

Rubisco Synthesis, Assembly, Mechanism, and Regulation.

abstractlon - H cls -enedlol o=o ribulose 1,5-P, / OPO', + 2P glycolate OPO', 3P Dglycerate OPO', 3-keto arabinitol 1 ,5-P2 Figure 4. The Reactions Catalyzed by Rubisco. The first intermediate of catalysis is the C2,C3 cis-enediol form of ribulose-bisphosphate (ribulose 1,5-P2) after abstraction of the C3 proton. The enediol can partition a number of ways, the majority into the products of carboxylation (upper reactions) or oxygenation (lwer reactions). However, a number of misprotonated isomers of ribulose-bisphosphate, for example, xylulose-bisphosphate, have been detected with the wild-type enzyme that are produced in quantity by mutations of specific amino acids involved in proton transfer. Phosphate elimination of the carbanion forms of intermediates are also produced by some mutants (see Morrell et al., 1994). R, -CHOH-CH20P03= ; 3P D-glycerate, 3-phosphoglycerate; 2P glyco- late, 2-phosphoglycolate. to bind. Coordination of the metal does not complete the ac- tive site but simply positions the carbamate and Mg2+ relative to those centers of the bisphosphate substrate, namely, the C2 carbonyl oxygen and C3 hydroxyl, that are involved in the catalytic events. With substrate bound in the correct orienta- tion (Lorimer et al., 1989), the C2 and C3 oxygen atoms complete the coordination sphere (Gutteridge and Lundqvist, 1994). The catalytic mechanism of the carboxylation of ribulose- bisphosphate can be depicted as five discrete partia1 reactions (Andrews and Lorimer, 1987). The first step has been estab- lished as the formation of an enediol intermediate of the bisphosphate substrate that is catalyzed by the enzyme in the absence of the second substrates,

O3 -induced degradation of Rubisco protein and loss of Rubisco mRNA in relation to leaf age in Solanum tuberosum L.

The effect of ozone (O3 ) on Rubisco degradation was investigated by dark incubation of potato plants (Solanum tuberosum L. cv. Norland) following exposure to charcoal-filtered air or 0.30μl l-1 O3 for 6 h. Rubisco small subunit (rbcS) mRNA levels declined dramatically in control foliage within 10.16 h of dark incubation; thus declines in Rubisco protein following a 48-h dark period were ascribed to proteolytic degradation. Foliage sampled from the 4th and 6th leaves from the apex, designated immature and mature, respectively, showed no effect of O3 on Rubisco protein content immediately following the exposure. However, the decline in Rubisco quantity during the dark incubation was significantly enhanced by prior treatment with O3 in the mature leaves. The immature leaves sustained a similar decline in Rubisco quantity in both O2 and control treatments. O3 had a significant effect on the relative quantity of rbcS mRNA in the immature leaves (sampled immediately following the O3 exposure). Levels of rbcS mRNA in mature leaves and Rubisco large subunit (rbcL) mRNA in both leaf ages were not significantly affected by O3 , There was no effect of O3 , on Rubisco quantity in immature or mature leave's of plants maintained in the greenhouse under a 16 h photoperiod far 48 h following the exposure. Thus the effect of short-term O3 exposure on processes affecting Rubisco synthesis and degradation may have been transient, and a more prolonged exposure would he necessary to effect a decline in Rubisco protein quantity in plants grown under a 16 h photoperiod. We concluded that O3 caused enhanced degradation of Rubisco in mature leaves of plants induced to senesce by dark incubation following O3 exposures. The potential for O3 -induced reduction in synthesis of Rubisco also exists.