Plant and Cell Physiology

We have developed a high-frequency method for Agrobacterium-mediated gene targeting by combining an efficient transformation system using rice suspension-cultured calli and a positive/negative selection system. Compared with the conventional transformation system using calli on solid medium, transformation using suspension-cultured calli resulted in a 5- to 10-fold increase in the number of resistant calli per weight of starting material after positive/negative selection. Homologous recombination occurred in about 1.5% of the positive/negative selected calli. To evaluate the efficacy of our method, we show in this report that knockout rice plants containing either a disrupted Waxy (granule-bound starch synthase) or a disrupted Xyl (β1,2-xylosyltransferase) gene can be easily obtained by homologous recombination. Study of gene function using homologous recombination in higher plants can now be considered routine work as a direct result of this technical advance.

A 1,3-rβ-glucanase purified from rice grain is a 33 kDa monomer with a pI of ≥10.4. The enzyme was determined to be an endo-1,3-rβ-glucanase (EC 3.2.1.39) by end product analysis using Laminaria digitata laminarin as substrate. Its amino-terminal amino acid sequence revealed strong homology to an endo-1,3-rβ-glucanase from barley.

One of the most rapid responses to aluminum (Al) stress in plants is enhanced synthesis and deposition of 1,3-beta-D-glucans (callose) in root tips. Ironically, Al-induced synthesis and deposition of callose occurs in vivo, despite evidence from in vitro systems that suggests that Al is a powerful inhibitor of 1,3-beta-D-glucan synthase. We set out to test the hypothesis that an Al-induced increase in the activity of free calcium in the cytoplasm ([Ca(2+)](cyt)) is the trigger for enhanced synthesis of callose in in vivo systems, an effect that would not be observed in in vitro systems. Root tips of an Al-sensitive cultivar of Triticum aestivum were treated with Al (0-100 microM) or the Ca ionophore A23187 (0-3 micro M) for 3-24 h, and the effects on [Ca(2+)](cyt) and synthesis of callose were measured using confocal laser scanning microscopy. Treatment with Al induced a rapid increase in both [Ca(2+)](cyt) (4.7-fold) and synthesis of callose (30-fold). Treatment with the Ca ionophore, A23187, also elicited an increase in [Ca(2+)](cyt) (6.6-fold). Despite a greater increase in [Ca(2+)](cyt) in the presence of A23187, this increase was accompanied by a smaller increase in callose deposition (11-fold) than was observed in the presence of Al. These data suggest that an increase in [Ca(2+)](cyt) is not the only factor modulating increases in callose synthesis and deposition in the presence of Al.

A cDNA for a pathogenesis-related endo-beta-1,3-glucanase isolated from soybean, was fused to an anther tapetum-specific promoter (Osg6B promoter) isolated from rice and the resulting chimeric gene was introduced into tobacco. The Osg6B promoter became active in the anther tapetum during formation of tetrads and the tapetal glucanase activity in the transgenic plants caused in a significant reduction in the number of fertile pollen grains. Most of the pollen grains were aberrant in shape, lacked germinal apertures and aggregate of the pollen grains. Granules of beta-1,3-glucan, which have not previously been reported, were often observed to adhere to the surface of the pollen grains. Further observations revealed that the callose wall was almost absent in the pollen tetrads of transgenic plants. In wild-type plants, by contrast, the tetrads were surrounded by callose that was degraded soon after the tetrad stage to release free microspores. Thus, the introduced gene for endo-beta-1,3-endoglucanase under the control of the Osg6B promoter caused digestion of the callose wall at the beginning of the tetrad stage, a time that was just a little earlier than the time at which endogenous glucanase activity normal appears. These results demonstrate that premature dissolution of the callose wall in pollen tetrads causes male sterility and suggest that the time at which tapetally produced glucanase is activate is critical for the normal development of microspores.

We isolated cDNA clones (pSgPG1 through pSgPG4, pSgPME1 and pSgGN1) for the polygalacturonases (PGs), pectin methylesterase (PME) and β-1,3-glucanase (GN) that are expressed specifically in male flowers of the dioecious willow (Salix gilgiana Seemen). The structural characteristics of the deduced proteins, designated SgPGs, SgPME1 and SgGN1, respectively, suggest that these enzymes function in pollens or anthers. The four SgPGs were more than 91.9% homologous to one another at the amino acid level, indicating that their genes are members of a single family. Although the expression of the SgPGs, SgPME1 and SgGN1 was specific to male catkins (inflorescences), these genes were found in the genomes of both male and female plants. The expression of the transcripts of SgPGs, SgPME1 and SgGN1 was regulated developmentally in male reproductive organs. Maximal expression of SgPGs and SgPME1 was detected when male flowers were fully open and mature, while maximal expression of SgGN1 occurred at an earlier time. In situ hybridization revealed that the expression of SgPGs and SgPME1 was restricted to mature pollen grains after microspore mitosis. These results suggest that the pollen-specific or antherspecific expression of genes for PGs, PME and GN occurs in a dioecious plant, willow, just as it does in monoecious plants, and that the expression of these genes is related to the developmental stage of pollen grains during male gametogenesis.

A cDNA (TAC1) and genomic clone (cel5) encoding an endo-beta-1,4-glucanase (EGase) were identified from tomato (Lycopersicon esculentum Mill., cv. Rutgers). The cel5 gene is expressed in pistils, flower pedicel and leaf abscission zones, and ripening fruit. The genomic sequence includes a 22 bp 5' upstream sequence that is conserved in a closely related peach EGase gene, ppEG1.

A cDNA for poplar endo-1,4-beta-glucanase was cloned by use of a synthetic oligonucleotide as probe. The probe was designed on the basis of the N-terminal amino acid sequence of the beta-glucanase from suspension-cultured poplar cells (Populus alba L.), and by complete nucleotide sequence of the cDNA was determined. The 1,614-bp cDNA contained an open reading frame of 1,482 base pairs, encoding 494 amino acids. Removal of a putative signal sequence from the deduced amino acid sequence of the polypeptide yielded a mature protein of 467 amino acids. Comparison of deduced amino acid sequences revealed that the poplar endo-1,4-beta-glucanase was 80% and 70% identity to avocado fruit and bean abscission endo-1,4-beta-glucanases, respectively. Hydropathy plot analysis of the deduced amino acid sequence suggests that poplar endo-1,4-beta-glucanase belongs to family E in terms of the cellulase catalytic domain, and avocado fruit and abscission bean endo-1,4-beta-glucanases also belong to this family. 2,4-D markedly increased the level of the endo-1,4-beta-glucanase mRNA in cultured cells, while GA3, benzyladenine and abscisic acid each repressed transcription of this mRNA. The transcript was also detected in the roots and stems of intact plants, although the level of mRNA was much lower in intact tissues than in cultured cells. Genomic Southern analysis indicated that a small family of gene for endo-1,4-beta-glucanase exists in poplar.

We report the cloning of a glycoside hydrolase family (GHF) 9 gene of rice (Oryza sativa L. cv. Sasanishiki), OsCel9A, corresponding to the auxin-induced 51 kDa endo-1,4-beta-glucanase (EGase). This enzyme reveals a broad substrate specificity with respect to sugar backbones (glucose and xylose) in beta-1,4-glycans of type II cell wall. OsCel9A encodes a 640 amino acid polypeptide and is an ortholog of TomCel8, a tomato EGase containing a carbohydrate-binding module (CBM) 2 sequence at its C-terminus. The expression of four rice EGase genes including OsCel9A showed different patterns of organ specificity and responses to auxin. OsCel9A was preferentially expressed during the initiation of lateral roots or subcultured root calli, but was hardly expressed during auxin-induced coleoptile elongation or in seed calli, in contrast to OsCel9D, a KORRIGAN (KOR) homolog. In situ localization of OsCel9A transcripts demonstrated that its expression was specifically up-regulated in lateral root primordia (LRP). Northern blotting analysis showed the presence of a single product of OsCel9A. In contrast, both mass spectrometric analyses of peptide fragments from purified 51 kDa EGase proteins and immunogel blot analysis of EGase proteins in root extracts using two antibodies against internal peptide sequences of OsCel9A revealed that the entire CBM2 region was post-translationally truncated from the 67 kDa nascent protein to generate 51 kDa EGase isoforms. Analyses of auxin concentration and time course dependence of accumulation of two EGase isoforms suggested that the translation and post-translational CBM2 truncation of the OsCel9A gene may participate in lateral root development.

Endo-1,4-beta-glucanase induced by treatment of pea seedlings with 2,4-D was extracted from a preparation of the walls of epicotyl cells. The beta-glucanase was purified by chromatography on DEAE-cellulose, affinity chromatography on Con A-Sepharose and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The activity of beta-glucanase was retained after removal of SDS and extraction from polyacrylamide gels. The band of a protein (46 kDa), that corresponded to the activity of endo-1,4-beta-glucanase, was injected directly into mice for preparation of antiserum and the protein was also subjected to amino acid sequencing after blotting onto a membrane. Western blot analysis showed that the antiserum obtained bound to a 46-kDa polypeptide and recognized endo-1,4-beta-glucanase. The N-terminal sequence of the 46-kDa polypeptide revealed some homology to abscission endo-1,4-beta-glucanases of bean and avocado fruit.

Recent studies have highlighted the involvement of membrane-anchored endo-�-1,4-glucanases in cellulose biosynthesis in plants, suggesting that there are parallels with Agrobacterium tumefaciens and other bacteria which also require endo-�-1,4-glucanases for cellulose synthesis. This review summarises recent literature on endo-�-1,4-glucanases and their role in plant development and addresses the possible functions of membrane-anchored isoforms in the synthesis of cellulose. Keywords: Cellulose — KORRIGAN — Membrane-anchored endo-�-1,4-glucanase.

Unraveling the role of genes annotated as protein of unknown function is of importance in progression of plant science. l-Galactono-1,4-lactone (l-GalL) is the terminal precursor for ascorbic acid (AsA) biosynthesis in Arabidopsis thaliana, and a previous study showed two DUF (domains of unknown function) 642 family genes (At1g80240 and At5g25460, designated as DGR1 and DGR2, respectively) to be sensitive to it. In this work, leaves from wild-type Arabidopsis were fed with d-glucose, l-galactose, l-GalL and AsA, and the expression level of the At1g80240 and At5g25460 genes showed a specific response to l-GalL, but not to the other supplements despite the increases of the tissue AsA contents. Analysis of promoter–β-glucuronidase (GUS) transgenic plants showed the two genes to be complementarily expressed at the root apex and in the rest of the root excluding the apex, respectively, in both young and old seedlings, and to be expressed at the leaf primordia. The GUS activity under the control of the At5g25460 promoter was high in the cotyledon and leaf veins of young seedlings. These findings were consistent with the results of quantitative real-time PCR. Interestingly, the T-DNA insertion mutant of At5g25460 (SALK_125079) displayed shorter roots and smaller rosettes than Col-0; however, no phenotypic difference was observed between the T-DNA insertion mutant of At1g80240 and the wild type. This is the first report on the expression and functional analysis of these two DUF642 family genes, with the results revealing the contribution of DGR genes to the development of Arabidopsis.

A temperature-sensitive, elongation-deficient mutant of Arabidopsis thaliana was isolated. At the non-permissive temperature of 31 degrees C, the mutation impaired tissue elongation; otherwise, tissue development was normal. Hypocotyl cells that had established cell walls at 21 degrees C under light-dark cycles ceased elongation and swelled when the mutant was shifted to 31 degrees C and darkness, indicating that the affected gene is essential for cell elongation. Analysis of the cell walls of mutant plants grown at 31 degrees C revealed that the cellulose content was reduced to 40% and the pectin content was increased to 162% of the corresponding values for the wild type grown at the same temperature. The increased amounts of pectin in the mutant were bound tightly to cellulose microfibrils. No change in the content of hemicellulose was apparent in the 31 degrees C-adapted mutant. Field emission-scanning electron microscopy suggested that the structure of cellulose bundles was affected by the mutation; X-ray diffraction, however, revealed no change in the crystallite size of cellulose microfibrils. The regeneration of cellulose microfibrils from naked mutant protoplasts was substantially delayed at 31 degrees C. The recessive mutation was mapped to chromosome V, and map-based cloning identified it as a single G-->A transition (resulting in a Gly(429)-->Arg substitution) in KORRIGAN, which encodes a putative membrane-bound endo-1,4-beta-glucanase. These results demonstrate that the product of this gene is required for cellulose synthesis.

Phospholipid metabolism is involved in hyperosmotic-stress responses in plants. To investigate the role of phosphoinositide-specific phospholipase C (PI-PLC)-a key enzyme in phosphoinositide turnover-in hyperosmotic-stress signaling, we analyzed changes in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) content in response to hyperosmotic shock or salinity in Arabidopsis thaliana T87 cultured cells. Within a few s, a hyperosmotic shock, caused by mannitol, NaCl, or dehydration, induced a rapid and transient increase in Ins(1,4,5)P3. However, no transient increase was detected in cells treated with ABA. Neomycin and U73122, inhibitors of PI-PLC, inhibited the increase in Ins(1,4,5)P3 caused by the hyperosmotic shock. A rapid increase in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in response to the hyperosmotic shock also occurred, but the rate of increase was much slower than that of Ins(1,4,5)P3. These findings indicate that the transient Ins(1,4,5)P3 production was due to the activation of PI-PLC in response to hyperosmotic stress. PI-PLC inhibitors also inhibited hyperosmotic stress-responsive expression of some dehydration-inducible genes, such as rd29A (lti78/cor78) and rd17 (cor47), that are controlled by the DRE/CRT cis-acting element but did not inhibit hyperosmotic stress-responsive expression of ABA-inducible genes, such as rd20. Taken together, these results suggest the involvement of PI-PLC and Ins(1,4,5)P3 in an ABA-independent hyperosmotic-stress signal transduction pathway in higher plants.

The biphasic reaction course, fallover, of carboxyla-tion catalysed by ribulose 1,5-bisphosphate carboxylase/ox-ygenase (RuBisCO) has been known as a characteristic of the enzyme from higher land plants. Fallover consists of hysteresis in the reaction seen during the initial several minutes and a very slow suicide inhibition by inhibitors formed from the substrate ribulose-l,5-bisphosphate (RuBP). This study examined the relationship between occurrence of fallover and non-catalytic RuBP-binding sites, and the putative hysteresis-inducible sites (Lys-21 and Lys-30S of the large subunit in spinach RuBisCO) amongst RuBisCOs of a wide variety of photosynthetic organisms. Fallover could be detected by following the course of the carboxylase reaction at 1 mM RuBP and the non-catalytic binding sites by alleviation of fallover at 5 mM RuBP. RuBisCO from Euglena gracilis showed the same linear reaction course at both RuBP concentrations, indicating an association between an absence of fallover and an absence of the non-catalytic binding sites. This was supported by the results of an equilibrium binding assay for this enzyme with a transition state analogue. Green macroalgae and non-green algae contained the plant-type, fallover enzyme. RuBisCOs from Conjugatae, Closterium ehrenbergii, Gona-tozygon monotaenium and Netrium digitus, showed a much smaller decrease in activity at 1 mM RuBP than the spinach enzyme and the reaction courses of these enzymes at 5 mM RuBP were almost linear. RuBisCO of a primitive type Conjugatae, Mesotaenium caldariorum, showed the same linear course at both RuBP concentrations. Sequencing of rbcL of these organisms indicated that Lys-305 was changed into arginine with Lys-21 conserved.

Glycation is a process whereby sugar molecules form a covalent adduct with protein amino groups. In this study, we used ascorbic acid (AsA) as a glycating agent and purified cucumber (Cucumis sativus L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a model protein in chloroplast tissues, and examined effects of glycation on the activity and susceptibility of Rubisco to proteases. Glycation proceeded via two phases during incubation with AsA and Rubisco in vitro at physiological conditions (10 mM AsA, pH 7.5, 25 degrees C in the presence of atmospheric oxygen). At the early stage of glycation (phase 1), the amount of AsA attaching to Rubisco increased at an almost linear rate (0.5-0.7 mol AsA incorporated (mol Rubisco)(-1) d(-1)). By Western blotting using monoclonal antibodies recognizing glycation adducts, a major glycation adduct, N( epsilon )-(carboxymethyl)lysine was detected. At the late stage of glycation (phase 2), incorporation of AsA reached saturation, and a glycation adduct, pentosidine mediating intramolecular cross-linking, was detected corresponding to formation of high molecular weight aggregates cross-linked between subunits. Glycation led to a decrease in Rubisco activity (half-life about 7-8 d). Furthermore, glycated Rubisco of phase 2 drastically increased protease susceptibility in contrast to unchanged susceptibility of glycated Rubisco of phase 1 compared to that of native Rubisco. Results obtained here suggest that AsA is possibly an important factor in the loss of activity and turnover of Rubisco.

Previous studies have demonstrated that the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase (Rubisco) is site-specifically cleaved by a hydroxyl radical (·OH) generated in the illuminated chloroplast lysates or by an artificial ·OH-generating system. However, it is not known whether such cleavage of the LSU by reactive oxygen species (ROS) actually occurs in an intact leaf. When leaf discs of chilling-sensitive cucumber (Cucumis sativus L.) were illuminated at 4°C, five major fragments of the LSU were observed. This fragmentation was completely inhibited by ROS scavengers, such as n-propyl gallate (for ·OH) and 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron) (for superoxide). FeSO4 stimulated this fragmentation, whereas an iron-specific chelator, deferoxamine, suppressed it. Furthermore, such fragments were identical to those generated from the purified Rubisco by an ·OH-generating system in vitro on two-dimensional PAGE. These results indicate that the direct fragmentation of the LSU by reacive oxygen species also occurs in an intact leaf.

The operon encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the cyanobacterium Synechococcus sp. PCC7002 contains three rbc genes, rbcL, rbcX and rbcS, in this order. Introduction of translational frameshift into the rbcX gene resulted in a significant decrease in the production of large (RbcL) and small (RbcS) subunits of the Rubisco protein in Synechococcus sp. PCC7002 and in Escherichia coli. To investigate the function of the rbcX gene product (RbcX), we constructed the expression plasmid for the rbcX gene and examined the effects of RbcX on the recombinant Rubisco production in Escherichia coli. The coexpression experiments revealed that RbcX had marked effects on the production of large and small subunits of Rubisco without any significant influence on the mRNA level of rbc genes and/or the post-translational assembly of the Rubisco protein. The present rbcX coexpression system provides a novel and useful method for investigating the Rubisco maturation pathway.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is composed of small subunits (SSs) encoded by rbcS on the nuclear genome and large subunits (LSs) encoded by rbcL on the chloroplast genome, and it is localized in the chloroplast stroma. Constitutive knockdown of the rbcS gene reportedly causes a reduction in LS quantity and the level of translation in tobacco and the unicellular green alga Chlamydomonas. Constitutively knockdown of the rbcS gene also causes a reduction in photosynthesis, which influences the expression of photosynthetic genes, including the rbcL gene. Here, to investigate the influence of the knockdown of the rbcS gene on the expression of the rbcL gene under normal photosynthetic conditions, we generated transgenic tobacco plants in which the amount of endogenous rbcS mRNA can be reduced by inducible expression of antisense rbcS mRNA with dexamethasone (DEX) treatment at later stages of growth. In already expanded leaves, after DEX treatment, the level of photosynthesis, RuBisCO quantity and the chloroplast ultrastructure were normal, but the amount of rbcS mRNA was reduced. An in vivo pulse labeling experiment and polysome analysis showed that LSs were translated at the same rate as in wild-type leaves. On the other hand, in newly emerging leaves, the rbcS mRNA quantity, the level of photosynthesis and the quantity of RuBisCO were reduced, and chloroplasts failed to develop. In these leaves, the level of LS translation was inhibited, as previously described. These results suggest that LS translation is regulated in an SS-independent manner in expanded leaves under normal photosynthetic conditions.

Net photosynthetic rates (Pns) in leaves were compared between rice plants grown in ambient air control and free-air CO2 enrichment (FACE, about 200 micromol mol(-1) above ambient) treatment rings. When measured at the same CO2 concentration, the Pn of FACE leaves decreased significantly, indicating that photosynthetic acclimation to high CO2 occurs. Although stomatal conductance (Gs) in FACE leaves was markedly decreased, intercellular CO2 concentrations (Ci) were almost the same in FACE and ambient leaves, indicating that the photosynthetic acclimation is not caused by the decreased Gs. Furthermore, carboxylation efficiency and maximal Pn, both light and CO2-saturated Pn, were decreased in FACE leaves, as shown by the Pn-Ci curves. In addition, the soluble protein, Rubisco (ribulose-1,5-bisphosphate caboxylase/oxygenase), and its activase contents as well as the sucrose-phosphate synthase activity decreased significantly, while some soluble sugar, inorganic phosphate, chlorophyll and light-harvesting complex II (LHC II) contents increased in FACE leaves. It appears that the photosynthetic acclimation in rice leaves is related to both ribulose-1,5-bisphosphate (RuBP) carboxylation limitation and RuBP regeneration limitation.

Immunocytochemical electron-microscopic observation indicated that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) and/or its degradation products are localized in small spherical bodies having a diameter of 0.4-1.2 micro m in naturally senescing leaves of wheat (Triticum aestivum L.). These Rubisco-containing bodies (RCBs) were found in the cytoplasm and in the vacuole. RCBs contained another stromal protein, chloroplastic glutamine synthetase, but not thylakoid proteins. Ultrastructural analysis suggested that RCBs had double membranes, which seemed to be derived from the chloroplast envelope, and that RCBs were further surrounded by the other membrane structures in the cytoplasm. The appearance of RCBs was the most remarkable when the amount of Rubisco started to decrease at the early phase of leaf senescence. These results suggest that RCBs might be involved in the degradation process of Rubisco outside of chloroplasts during leaf senescence.

We have studied source-sink relationships with a model consisting of single-rooted leaves without petioles. We previously reported that the rate of photosynthesis decreased when C4 model plants prepared from Amaranthus cruentus leaves were subjected to sink-limited conditions by exposure to continuous light for a few days. It was suggested that the inhibition is due to a coordinated decrease in the activity of ribulose-1,5-bisphosphate carboxylase (RuBPcase) and phosphoenol-pyruvate carboxylase (PEPcase), both essential enzymes for photosynthesis in C4 plants. We further investigated the mechanisms behind the decreased activity of RuBPcase, PEPcase, NAD-malic enzyme and NAD-malate dehydrogenase. The results suggested that (1) the initial activity of RuBPcase is suppressed by a lowering of the P(i) level in chloroplasts, (2) the inhibition of PEPcase is due to dephosphorylation of the enzyme via the inhibition of PEPcase kinase and PEPcase phosphatase, (3) the inhibition of NAD-malic enzyme and NAD-malate dehydrogenase is derived from the oxidation of these enzymes, and (4) some proteinous factor(s) may be involved in the inhibition of the activity of these latter three enzymes. The significance of a coordinated decrease in these enzymes in response to a change in the source-sink balance is discussed.

We analyzed the promoter of the genes encoding the ribulose-1,5-bisphosphate carboxylase/oxygenase (rbc) in the cyanobacterium Synechococcus sp. PCC7002 and localized the CO(2)-regulatory element. Cyanobacterial transformants were constructed with several DNA segments of the rbc promoter fused to the chloramphenicol acetyltransferase (CAT) gene, and their acetyltransferase activities were analyzed under 0.03% and 1% CO(2) conditions. We found that the AT-rich element localized from -262 to -291 relative to the rbc translation-starting site was required for CO(2)-dependent repression. Fluorescent-labeled oligonucleotide probes of identical sequence to the AT-rich element were reacted with protein extracts from cells cultured under conditions of low and high CO(2) atmospheric content. We detected a gel retardation complex of a strong signal intensity in extracts from cells cultured under 15% CO(2), but only a weak signal from cells cultured under 1% CO(2). Moreover, a DNA affinity precipitation assay identified a 16-kDa protein that bound to nucleotide sequences within the AT-rich element. The partial amino acid sequence of the protein was similar to the deduced protein sequences of ORF129 and ORF155 from Synechocystis 6803. Our findings suggest that the AT-rich element plays a role as a negative CO(2)-regulatory element and its trans-acting factor possibly regulates the rbc transcription in response to CO(2) levels.

Lysates of chloroplasts isolated from wheat (Triticum aestivum L. cv. Aoba) leaves were incubated on ice (pH 5.7) for 0 to 60 min in light (15 mumol quanta m-2 s-1), and degradation of the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco: EC 4.1.1.39) was analyzed by applying immunoblotting with site-specific antibodies against the N-terminal, internal, and C-terminal amino acid sequences of the LSU of wheat Rubisco. The most dominant product of the breakdown of the LSU and that which was first to appear was an apparent molecular mass of 37-kDa fragment containing the N-terminal region of the LSU. A 16-kDa fragment containing the C-terminal region of the LSU was concomitantly seen. This fragmentation of the LSU was inhibited in the presence of EDTA or 1,10-phenanthroline. The addition of active oxygen scavengers, catalase (for H2O2) and n-propyl gallate (for hydroxyl radical) to the lysates also inhibited the fragmentation. When the purified Rubisco from wheat leaves was exposed to a hydroxyl radical-generating system comprising H2O2, FeSO4 and ascorbic acid, the LSU was degraded in the same manner as observed in the chloroplast lysates. The results suggest that the large subunit of Rubisco was directly degraded to the 37-kDa fragment containing the N-terminal region and the 16-kDa fragment containing the C-terminal region of the LSU by active oxygen, probably the hydroxyl radical, generated in the lysates of chloroplasts.

Translational regulation plays a key role in light-induced expression of photosynthesis-related genes at various levels in chloroplasts. We here present the results suggesting a mechanism for light-induced translation of the rbcL mRNA encoding the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase (Rubisco). When 8-day-old dark-grown barley seedlings that have low plastid translation activity were illuminated for 16 h, a dramatic increase in synthesis of large subunit of Rubisco and global activation of plastid protein synthesis occurred. While an increase in polysome-associated rbcL mRNA was observed upon illumination for 16 h, the abundance of translation initiation complexes bound to rbcL mRNA remained constant, indicating that translation elongation might be controlled during this dark-to-light transition. Toeprinting of soluble rbcL polysomes after in organello plastid translation showed that ribosomes of rbcL translation initiation complexes could read-out into elongating ribosomes in illuminated plastids whereas in dark-grown plastids, read-out of ribosomes of translation initiation complexes was inhibited. Moreover, new rounds of translation initiation could also occur in illuminated plastids, but not in dark-grown plastids. These results suggest that translation initiation complexes for rbcL are normally formed in the dark, but the transition step of translation initiation complexes entering the elongation phase of protein synthesis and/or the elongation step might be inhibited, and this inhibition seems to be released upon illumination. The release of such a translational block upon illumination may contribute to light-activated translation of the rbcL mRNA.