Cytokinin treatment of embryos inhibits the synthesis of chloroplast proteins in Norway spruce

A pulse treatment of Norway spruce (Picea abies (L.) Karst) embryos with the cytokinin N⁶-benzyladenine induces the formation of adventitious buds from subepidermal cells in the hypocotyl and cotyledons. In addition the treatment also inhibits elongation growth, a key process during germination. In this report we demonstrate that these effects on development of the plant are associated with a suppression of the accumulation of several major chloroplast proteins during germination. These proteins include the large subunit of ribulose bisphosphate/carboxylase oxygenase, two subunits of the chloroplast ATPase, protochlorophyllide reductase and a 23000-M_r component of photosystem II. For two nuclear-encoded proteins, the small subunit of ribulose bisphosphate carboxylase/oxygenase and the light-harvesting chlorophyll a/b-binding protein, a corresponding suppression of the increase in the steady-state amounts of mRNA is recorded. The suppression of chloroplast protein synthesis is consistant with the previously documented delay in greening that results from cytokinin treatment, but the effect is opposite to that found in other plants, where cytokinins promote the synthesis of chloroplast proteins, and stimulate chloroplast biogenesis. We believe that this difference is explained by the cytokinin primarily suppressing organ development, and a strict dependance of chloroplast biogenesis on the developmental state of the organs.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - CF1 coupling-factor 1 of chloroplast ATPase - LHCP light-harvesting chlorophyll a/b-binding protein - LSU large subunit of Rubisco - NADPH-protochlorophyllide oxidoreductase Pchlide reductase - SDS sodium dodecyl sulfate - SSU small subunit of Rubisco We thank K. Hutchison (Dept. of Biochemistry, University of Maine, Orono, Maine, USA) and P. Gustafsson (Dept. of Plant Physiology, University of Umeå, Sweden) for providing the Larix and Pinus clones, and M. Ryberg (Dept. of Plant Physiology, University of Göteborg, Sweden), R. Ölmüller (Botanisches Institut, Universität München, FRG) and W. Lockau (Institut für Botanik, Universität Regensburg, FRG), for the gift of antisera towards Pchlide reductase, RuBPCase and LHCP, and ATPase, respectively. Supported by the Swedish Council for Forestry and Agricultural Research and the Swedish Natural Sciences Research Council.     

Nuclear DNA regulates the level of ribulose 1,5-bisphosphate carboxylase oxygenase in Medicago sativa L.

Summary The response to selection for leaf proteins was studied during three selection cycles. Selection for high total nitrogen content showed 75% heritability, and the levels of both ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco) and cytoplasmic protein were strongly under nuclear DNA control. High and low protein content were correlated with chloroplast area. Although the amounts of nuclear DNA were similar, the ratio of Rubisco/DNA and chlorophyll/DNA changed during the selection process. It can be concluded that the levels of Rubisco achieved in mature plants of M. sativa are under nuclear DNA control. The possible involvement of small subunit (SSU) genes in controlling these levels is discussed.     

The two genes for the small subunit of RuBP Carboxylase/oxygenase are closely linked in Chlamydomonas reinhardtii

Summary Ribulose bisphosphate carboxylase-oxygenase (Rubisco) is a key enzyme in the photosynthetic fixation of CO₂ by the chloroplast. The synthesis of the enzyme is an example of the cooperation between the chloroplast and the nucleocytoplasmic compartments, as it is assembled from subunits encoded in the two respective genomes. I have used a synthetic oligonucleotide probe to isolate the nuclear Rubisco small subunit genes (rbcS) directly from a genomic library of Chlamydomonas reinhardtii DNA. They constitute only a small family: there are two rbcS genes, and an additional related sequence, in the C. reinhardtii genome. All three are clustered within 11kb at a single locus, and should thus be particularly well suited for genetic manipulation. The pattern of expression of rbcS RNA is dependent on the growth conditions.     

Questions about the complexity of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) has played a central role in our understanding of chloroplast biogenesis and photosynthesis. In particular, its catalysis of the rate-limiting step of CO₂ fixation, and the mutual competition of CO₂ and O₂ at the active site, makes Rubisco a prime focus for genetically engineering an increase in photosynthetic productivity. Although it remains difficult to manipulate the chloroplast-encoded large subunit and nuclear-encoded small subunit of crop plants, much has been learned about the structure/function relationships of Rubisco by expressing prokaryotic genes in Escherichia coli or by exploiting classical genetics and chloroplast transformation of the green alga Chlamydomonas reinhardtii. However, the complexity of chloroplast Rubisco in land plants cannot be completely addressed with the existing model organisms. Two subunits encoded in different genetic compartments have coevolved in the formation of the Rubisco holoenzyme, but the function of the small subunit remains largely unknown. The subunits are posttranslationally modified, assembled via a complex process, and degraded in regulated ways. There is also a second chloroplast protein, Rubisco activase, that is responsible for removing inhibitory molecules from the large-subunit active site. Many of these complex interactions and processes display species specificity. This means that attempts to engineer or discover a better Rubisco may be futile if one cannot transfer the better enzyme to a compatible host. We must frame the questions that address this problem of chloroplast-Rubisco complexity. We must work harder to find the answers.     

Assembly of in Vitro-Synthesized Large Subunits into Ribulose Bisphosphate Carboxylase/Oxygenase Is Sensitive to CI-, Requires ATP,and Does Not Proceed When Large Subunits Are Synthesized at Temperatures [greater than or equal to]32[deg]C

In higher plants, ribulose bisphosphate carboxylase/oxygenase (Rubisco) consists of eight large "L" subunits, synthesized in chloroplasts, and eight small "S" subunits, synthesized as precursors in the cytosol. Assembly of these into holoenzyme occurs in the chloroplast stroma after import and processing of the S subunits. A chloroplast chaperonin interacts with the L subunits, which dissociate from the chaperonin before they assemble into holoenzyme. Our laboratory has reported L subunit assembly into Rubisco in chloroplast extracts after protein synthesis in leaves, intact chloroplasts, and most recently in membrane-free chloroplast extracts. We report here that the incorporation of in vitro-synthesized L subunits into holoenzyme depends on the conditions of L subunit synthesis. Rubisco assembly did not occur after L subunit synthesis at 160 mM KCI. When L subunit synthesis occurred at approximately 70 mM KCI, assembly depended on the temperature at which L subunit synthesis took place. These phenomena were the result of postsynthetic events taking place during incubation for protein synthesis. We separated these events from protein synthesis by lowering the temperature during protein synthesis. Lower temperatures supported the synthesis of full-length Rubisco L subunits. The assembly of these completed L subunits into Rubisco required intervening incubation with ATP, before addition of S subunits. ATP treatment mobilized L subunits from a complex with the chloroplast chaperonin 60 oligomer. Addition of 130 mM KCI at the beginning of the intervening incubation with ATP blocked the incorporation of L subunits into Rubisco. The inhibitory effect of high KCI was due to CI- and came after association of newly synthesized L subunits with chaperonin 60, but before S subunit addition. It is interesting that L subunits synthesized at [greater than or equal to]32[deg]C failed to assemble into Rubisco under any conditions. These results agree with previous results obtained in this laboratory using newly synthesized L subunits made in intact chloroplasts. They also show that assembly of in vitro-synthesized L subunits into Rubisco requires ATP, that CI- inhibits Rubisco assembly, and that synthesis temperature affects subsequent assembly competence of L subunits.     

A protein with an inactive pterin‐4a‐carbinolamine dehydratase domain is required for Rubisco biogenesis in plants

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation in the Calvin–Benson pathway. Incomplete knowledge of the assembly pathway of chloroplast Rubisco has hampered efforts to fully delineate the enzyme's properties, or seek improved catalytic characteristics via directed evolution. Here we report that a Mu transposon insertion in the Zea mays (maize) gene encoding a chloroplast dimerization co‐factor of hepatocyte nuclear factor 1 (DCoH)/pterin‐4α‐carbinolamine dehydratases (PCD)‐like protein is the causative mutation in a seedling‐lethal, Rubisco‐deficient mutant named Rubisco accumulation factor 2 (raf2‐1). In raf2 mutants newly synthesized Rubisco large subunit accumulates in a high‐molecular weight complex, the formation of which requires a specific chaperonin 60‐kDa isoform. Analogous observations had been made previously with maize mutants lacking the Rubisco biogenesis proteins RAF1 and BSD2. Chemical cross‐linking of maize leaves followed by immunoprecipitation with antibodies to RAF2, RAF1 or BSD2 demonstrated co‐immunoprecipitation of each with Rubisco small subunit, and to a lesser extent, co‐immunoprecipitation with Rubisco large subunit. We propose that RAF2, RAF1 and BSD2 form transient complexes with the Rubisco small subunit, which in turn assembles with the large subunit as it is released from chaperonins.     

Cytokinin-controlled ribulose 1,5-bisphosphate carboxylase gene expression in pumpkin cotyledons

Treatment of etiolated and excisedCucurbita cotyledons with exogenous cytokinin (benzyladenine) in darkness or light results in a marked stimulation of Rubisco activity, content of enzyme protein, and incorporation of labelled precursors into it, indicating cytokinin-stimulatedde novo synthesis of the enzyme. Cell-free translations of RNA in the wheat germ andE. coli systems show an increase in both large and small subunit mRNA amounts relative to the increase of total RNA under the influence of the phytohormone and light. This increase in the level of translatable RNA is confirmed by RNA hybridization with the Rubisco large subunit gene of spinach. In addition, our results demonstrating additive effects of benzyladenine and light in cotyledon and chloroplast development suggest that the two factors co-act independently in the causal sequence of Rubisco gene expression. The data are discussed in a general view of cytokinin action in gene expression steps. Parts of the results have been obtained by cooperation with Drs. N. L. Klyachko, E. Romanko, and O. N. Kulaeva, Institute Plant Physiology, Acad. Sci. USSR, Moscow (cf. Lerbset al. 1984).     

Regulation of chlorophyll and Rubisco levels in embryonic cotyledons of Phaseolus vulgaris

While deep within the maternal tissues (pods and testa), cotyledons of the bean (Phaseolus vulgaris L.) green and the plastids differentiate as chloroplasts. At the time of seed maturation the chloroplasts dedifferentiate and the green color is lost. We have used Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and chlorophyll to study chloroembryo development. Chlorophyll levels and Rubisco activity increase early in embryonic development then decline as the cotyledons enter the maturation phase. Rubisco accumulation follows a strong temporal pattern over the course of embryo development, and furthermore, occurs in total darkness. Therefore, accumulation of Rubisco during embryogenesis may occur in response to developmental signals. In embryos developed in total darkness, Rubisco accumulation was uncoupled from chlorophyll accumulation. Exposure of isolated cotyledons to abscisic acid (ABA) resulted in loss of chlorophyll and decline in Rubisco levels comparable to those seen in normal embryogenesis. This indicates that the decline in Rubisco in chloroembryos in vivo results from factors such as ABA that signal the onset of maturation. The results show that ABA not only enhances the accumulation of some proteins (e.g. storage proteins), but also depresses the accumulation of others during embryogeny.Abbreviations Rubisco ribulose-1,5-bisphosphate-carboxylase/oxygenase (EC 4.1.1.39) - LSU large subunit of Rubisco - SSU small subunit of Rubisco - ABA abscisic acid - FW fresh weight     

Gene activation in suspension-cultured cells of Arabidopsis thaliana during blue-light-dependent plantlet regeneration

In cell-suspension cultures of Arabidopsis thaliana (L.) Heynh., transfer to auxin-free medium initiates regeneration leading to the formation of numerous rootlets around day 5. This process is promoted by continuous irradiation of the cell cultures with blue light (400–500 nm) while red light (600–700 nm) is ineffective in this respect. During the course of this process, two mRNA species, encoding, respectively, chalcone synthase and a plasmalemma channel protein, transiently accumulate. A second temporary increase in the steady-state level of these mRNAs is correlated with the onset of chloroplast development after 13–17 d of blue-light exposure of the cell cultures. During this cellular differentiation process a number of mRNAs start to accumulate which specify prominent plastid proteins: the small and the large subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (SSU and LSU), respectively the light-harvesting chlorophyll-a/b protein II (LHCPII). These findings are in accordance with those obtained with carrot suspension cultures where a clear sequence of development, i.e. the formation of somatic embryos followed by bluelight-dependent chloroplast differentiation, has also been observed.Abbreviations AthH2 intrinsic membrane protein of Arabidopsis thaliana (gene) - CHS chalcone-synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - EFR energy fluence rate - LHCPII cab light harvesting chlorophyll-a/b protein of photosystem II (gene) - LSU rbcL large subunit of Rubisco - SSU rbcS small subunit of Rubisco - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase Dedicated to Prof. Wolfhart Rüdiger on the occasion of this 60th birthdayThe research was supported by the Deutsche Forschungsgemeinschaft. We thank Mrs. I. Liebscher for her competent assistance. For the generous gift of cloned gene sequences we thank Prof. Dr. G. Link (Pflanzliche Zellphysiologie, Bochum, Germany), Dr. A. Batschauer (Biologisches Institut II/Botanik, Freiburg, Germany) and Dr. B. Weißhaar (MPI für Züchtungsforschung, Köln, Germany).     

Changes in ribulosebisphosphate carboxylase/oxygenase and ribulose 5-phosphate kinase abundances and photosynthetic capacity during leaf senescence

The abundances of ribulose-1,5-bisphosphate carboxylate/oxygenase (Rubisco) and ribulose-5-phosphate (Ru5P) kinase in field-grown soybean (Glycine max L. Merr.) leaves were quantified by a Western blot technique and related to changes in chlorophyll and photosynthetic capacity during senescence. Even though the leaf content of Rubisco was approximately 80-fold greater than that of Ru5P kinase, the decline in the levels of these two Calvin cycle enzymes occurred in parallel during the senescence of the leaves. Moreover, the decrease in the content of Rubisco was accompanied by parallel decreases of both the large and small subunits of this enzyme but not by an accumulation of altered large or small subunit isoforms. With increasing senescence, decreases in abundances of Rubisco, Ru5P kinase and chlorophyll were closely correlated with the decline in photosynthetic capacity; thus, the specific photosynthetic capacity when expressed per abundance of any of these parameters was rather constant despite an 8-fold decrease in photosynthetic capacity. These results suggest that during senescence of soybean leaves the chloroplast is subject to autolysis by mechanisms causing an approximately 80-fold greater rate of loss of Rubisco than Ru5P kinase.Jointly supported by the United States Department of Agricultural Research Service and the Kentucky Agricultural Experiment Station, Lexington (paper No. 88 3 286).Mention of a commercial product does not constitute endorsement by the United States Department of Agriculture.     

Effects of in vitro tissue culture conditions and acclimatization on the contents of Rubisco, leaf soluble proteins, photosynthetic pigments, and C/N ratio

The levels of two subunits of chloroplast ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), total soluble proteins, carbon and leaf nitrogen content, and photosynthetic pigments in various plants (avocado, oak, olive, and strawberry) grown in vitro and ex vitro were analysed. Compared to ex vitro grown plants, micropropagated avocado, oak, and strawberry showed a markable decrease in large subunit Rubisco. However, the small subunit only decreased in strawberry and oak. Contrary to this, olive did not reveal any difference in the level of either subunit. The C/N ratio increased significantly in in vitro grown plants, except in the case of olive, where an opposite behaviour was found. Leaf chlorophyll concentration on unit mass basis was higher in all the in vitro plants than in those of greenhouse- grown plants. Only avocado plantlets showed a statistically significant decrease in total soluble proteins. Further, overall data suggest that in vitro cultural conditions have a species-specific influence on large and small subunits of Rubisco, independent of the protein, chlorophyll, or nitrogen level.     

Abundance of the Major Chloroplast Polypeptides during Development and Ripening of Tomato Fruits: An Immunological Study

During maturation and ripening of tomato (Lycopersicon esculentum, cv Tamar) fruits, there are differential changes in the steady state levels of chloroplast proteins. Western blot analysis indicated that with the exception of the core polypeptide of photosystem I (PSI) (subunit I) the whole complex disappears during the transition of chloroplast to chromoplast. The amounts of the core polypeptide of photosystem II (PSII) (43 kilodaltons) and the light harvesting chlorophyll protein complex increase during maturation and decrease thereafter. In contrast, the 33 kilodalton subunit of PSII is found at the highest levels from the early recorded stages and decreases gradually until late stages of ripening. The level of cytochrome f decreases slowly during the maturation and ripening process, whereas the Rieske protein of the same complex disappears at a faster rate. There are also differential changes in the subunits of the chloroplast coupling factor·ATPase complex; α and β subunits increase during maturation, whereas the level of the γ subunit is already maximal at the earliest recorded stage of development and depleted thereafter. The two subunits of the ribulose-1,5 bisphosphate carboxylase increase in abundance during chloroplast maturation and gradually disappear after the transition from chloroplast to chromoplast. However, there are substantial differences in the rates of increase and disappearance of the large and small subunits of this enzyme. This imbalance is attributed to different regulation of nuclear and chloroplast gene expression. In addition, the steady state levels of chloroplastic superoxide dismutase and phosphoenolpyruvate carboxylase have been followed. Both enzymes reach their maxima at the final stages of ripening. This increase coincides with the climacteric rise of CO₂ release.     

Expression of Acidothermus cellulolyticus endoglucanase E1 in transgenic tobacco: biochemical characteristics and physiological effects

The expression of the Acidothermus cellulolyticus endoglucanase E1 gene in transgenic tobacco (Nicotiana tabacum) was examined in this study, where E1 coding sequence was transcribed under the control of a leaf specific Rubisco small subunit promoter (tomato RbcS-3C). Targeting the E1 protein to the chloroplast was established using a chloroplast transit peptide of Rubisco small subunit protein (tomato RbcS-2A) and confirmed by immunocytochemistry. The E1 produced in transgenic tobacco plants was found to be biologically active, and to accumulate in leaves at levels of up to 1.35% of total soluble protein. Optimum temperature and pH for E1 enzyme activity in leaf extracts were 81°C and 5.25, respectively. E1 activity remained constant on a gram fresh leaf weight basis, but dramatically increased on a total leaf soluble protein basis as leaves aged, or when leaf discs were dehydrated. E1 protein in old leaves, or after 5h dehydration, was partially degraded although E1 activity remained constant. Transgenic plants exhibited normal growth and developmental characteristics with photosynthetic rates similar to those of untransformed SR1 tobacco plants. Results from these biochemical and physiological analyses suggest that the chloroplast is a suitable cellular compartment for accumulation of the hydrolytic E1 enzyme.     

Interaction, functional relations and evolution of large and small subunits in Rubisco from prokaryota and eukaryota

In early biological evolution anoxygenic photosynthetic bacteria may have been established through the acquisition of ribulose bisphosphate carboxylase-oxygenase (Rubisco). The establishment of cyanobacteria may have followed and led to the production of atmospheric oxygen. It has been postulated that a unicellular cyanobacterium evolved to cyanelles which were evolutionary precursors of chloroplasts of both green and non-green algae. The latter probably diverged from ancestors of green algae as evidenced by the occurrence of large (L) and small (S) subunit genes for Rubisco in the chloroplast genome of the chromophytic algae Olisthodiscus luteus. In contrast, the gene for the S subunit was integrated into the nucleus in the evolution of green algae and higher plants. The evolutionary advantages of this integration are uncertain because the function of S subunits is unknown. Recently, two forms of Rubisco (L8 and L8S8) of almost equivalent carboxylase and oxygenase activity have been isolated from the photosynthetic bacterium Chromatium vinosum. This observation perpetuates the enigma of S subunit function. Current breakthroughs are imminent, however, in our understanding of the function of catalytic L subunits because of the application of deoxyoligonucleotide-directed mutagenesis. Especially interesting mutated Rubisco molecules may have either enhanced carboxylase activity or higher carboxylase:oxygenase ratios. Tests of expression, however, must await the insertion of modified genes into the nucleus and chloroplasts. Methodology to accomplish chloroplast transformation is as yet unavailable. Recently, we have obtained the first transformation of cyanobacteria by a colE1 plasmid. We regard this transformation as an appropriate model for chloroplast transformation.     

Increased zinc content in transplastomic tobacco plants expressing a polyhistidine-tagged Rubisco large subunit

Rubisco is a hexadecameric enzyme composed of two subunits: a small subunit (SSU) encoded by a nuclear gene (rbcS), and a large subunit (LSU) encoded by a plastid gene (rbcL). Due to its high abundance, Rubisco represents an interesting target to express peptides or small proteins as fusion products at high levels. In an attempt to modify the plant metal content, a polyhistidine sequence was fused to Rubisco, the most abundant protein of plants. Plastid transformation was used to express a polyhistidine (6x) fused to the C-terminal extremity of the tobacco LSU. Transplastomic tobacco plants were generated by cotransformation of polyethylene glycol-treated protoplasts using two vectors: one containing the 16SrDNA marker gene, conferring spectinomycin resistance, and the other the polyhistidine-tagged rbcL gene. Homoplasmic plants containing L8-(His)6S8 as a single enzyme species were obtained. These plants contained normal Rubisco amounts and activity and displayed normal photosynthetic properties and growth. Interestingly, transplastomic plants accumulated higher zinc amounts than the wild-type when grown on zinc-enriched media. The highest zinc increase observed exceeded the estimated chelating ability of the polyhistidine sequence, indicating a perturbation in intracellular zinc homeostasis. We discuss the possibility of using Rubisco to express foreign peptides as fusion products and to confer new properties to higher plants.     

小球藻Rubisco在叶绿体中的免疫金标定位

应用免疫技术对Rubisco在中国小球藻(Chlorellaspp.640909)叶绿体中进行了分子定位及Native-PAGE电泳、SDS-PAGE电泳及其Westen印迹分析,并对小球藻淀粉核(Pyrenoid)超微结构进行了观察.结果显示Native-PAGE电泳图谱主要为一条主带,Westen印迹反应证明该条带即为Rubisco酶,SDS-PAGE电泳及其Western印迹图谱显示Rubisco大亚基分子量大约为55kD.中国小球藻淀粉核为椭圆形,被淀粉鞘所包围,中央有一条由2个类囊体组成的纵向通道,并在蛋白核内段处稍膨胀.淀粉核与叶绿体基质存在多处联系.免疫分子定位显示Rubisco大亚基和全酶分子主要分布于叶绿体的淀粉核上,且Rubisco在淀粉鞘部位也有少量分布,极少部分分布在叶绿体基质中,表明叶绿体淀粉核与光合作用关系密切.Rubisco聚集于淀粉核可能有利于藻类对CO2固定.     

A mutation in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase that reduces the rate of its incorporation into holoenzyme

A mutant of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), in which Arg53 is replaced by Glu, was synthesized and imported into isolated chloroplasts. The mutant protein was efficiently imported into the chloroplast and correctly processed to the mature size. Like the wild type protein, it was stable over a period of at least 2 h. Unlike the wilk-type protein however, most of the mutant protein was not assembled with holo-Rubisco at the end of a 10-min import reaction. It migrated instead as a diffused band on a non-denaturing gel, slower than the precursor protein, but faster than the holoenzyme. The level of the unassembled mutant protein in the stroma decreased with time, while its level in the assembled fraction has increased, indicating that this protein is a slowly-assembled, rather than a non-assembled, mutant of the small suubunit of Rubisco. Accumulation of the mutant protein in the holoenzyme fraction was dependent on ATP and light. The transient species, migrating faster than the holoenzyme but slower than the precursor protein, may represent an intermediate in the assembly process of the small subunit of RubiscoAbbreviations LSU large subunit of Rubisco - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SSU small subunit of Rubisco