rbcS Gene Research Articles

Linking tocopherols with cellular signaling in plants

Linking tocopherols with cellular signaling in plants

Isolation and molecular characterization of rbcS in the unicellular green alga Nannochloris bacillaris (Chlorophyta, Trebouxiophyceae)

SUMMARY The small subunit of the chloroplast enzyme ribulose-1,5-bisphosphate carboxyase/oxygenase (Rubisco) is encoded by rbcS. We isolated and characterized three rbcS genes (NbrbcS1-1, NbrbcS1-2, and NbrbcS2) from the unicellular green alga Nannochloris bacillaris (Chlorophyta, Trebouxiophyceae). In the haploid N. bacillaris genome, each is a single-copy gene located on different chromosomes. Each mature peptide contains 140 amino acid residues. NbrbcS1-1 and NbrbcS1-2 are identical and share 80% identity with NbrbcS2, while the respective transit sequences share only approximately 48% identity with that of NbrbcS2. NbrbcS1-1 transcription was suppressed in the dark and recovered drastically after transfer to light. In contrast, NbrbcS1-2 and NbrbcS2 expression were not reduced after transfer from light to dark. In chlorophyllic tobacco cells containing green fluorescent protein fusion proteins of the transit sequences of each peptide, green fluorescent protein signals were localized on particles matching chloroplasts. The first introns of NbrbcS1-1 and NbrbcS1-2 are identical to the corresponding introns of 37 rbcS genes in eight embryophyte species. While the second intron is conserved in the green algae, the NbrbcS2 intron appears to involve sliding by one base pair. The NbrbcS1-1 and NbrbcS1-2 intron conserved in green algae and embryophytes might be an ancestral intron from nuclear-encoded rbcS.

Characterisation of the Dunaliella tertiolecta RbcS genes and their promoter activity in Chlamydomonas reinhardtii.

The availability of highly active homologous promoters and terminators is critical in the development of a transformation system for the unicellular microalga Dunaliella tertiolecta. To facilitate transformation of this species, we isolated and characterised two native ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit genes (RbcS) including flanking sequences. The two non-allelic cDNA sequences share approximately 80% identity and have approximately 60% identity to the RbcS genes of Chlamydomonas reinhardtii. The D. tertiolecta RbcS promoter and 3' untranslated regions were shown to drive expression of the bleomycin resistance gene (ble) in C. reinhardtii. This is the first demonstration of a heterologous algal promoter being used to drive transgene expression in C. reinhardtii. In addition, promoter deletions were shown to further increase transformation efficiency.

Thylakoid dismantling of damaged unfunctional chloroplasts modulates the Cab and RbcS gene expression in wheat leaves

Thylakoid membrane dismantling and Lhcb and RbcS nuclear gene expression have been analysed in leaves of wheat plants grown in high fluence rate light and deprived of photoprotective carotenoids by treatments with the two bleaching herbicides, either norflurazon or amitrole. The Lhcb transcript was not detectable in cells of norflurazon-supplied leaves, having chloroplasts totally devoid of both inner membranes and pigments. In contrast, a substantial amount of Lhcb mRNA could be found in cells of amitrole-treated leaves, whose severely damaged organelles still contained few strikingly altered and photosynthetically unfunctional thylakoids, as well as chlorophyll traces. A possible relationship between chlorophyll synthesis and Lhcb expression, with the transcript level depending on the rate of pigment production in photodamaged chloroplasts is discussed. Also the RbcS expression was linked to the chloroplast membrane photodamage. However, a detectable level of transcript was still produced in norflurazon-treated cells, despite complete thylakoid demolition. Thus, the wheat cell behaviour had to be placed between that of species, such as maize, in which the RbcS expression is broken off in these conditions, and that of species, such as pea, in which it is slightly lowered. Interestingly, the dramatically photodamaged chloroplasts still maintained the ability to synthesize proteins and this allowed SSU and LSU Rubisco subunits to be found in the organelles of both norflurazon- and amitrole-treated plants.

Liquid phase fluorescence in situ RT-PCR analysis for gene expression analysis in woody stems.

We explore a rapid in situ RT-PCR protocol for gene expression studies in woody stem tissues. In situ RT-PCR was performed using fluorescent dye-conjugated nucleic acid and the fluorescence signals derived from target RNAs were detected using confocal laser scanning microscopy. The signal to background ratio was greatly enhanced by performing two rounds of PCR reactions, first without the fluorescent dye and second with the dye. Using this protocol, we obtained strong gene-specific signals in secondary stem tissues. The signals were PCR-dependent, as shown by the lack of cytoplasmic signals in the tissue sections in which either DNA polymerase or primers were omitted from PCR reactions, and were RNA-dependent, as shown by great reduction of cytoplasmic signals when sections were treated with RNase before RT reactions. To verify our protocol, transcript localization of the rbcS gene was examined in secondary stems of hybrid aspen ( Populus tremula L. x tremuloides Michx.) and compared to the chlorophyll autofluorescence signal. The in situ RT-PCR signals form the rbcS gene and chlorophyll autofluorescence co-localized in the same cell types. The signal was also confirmed by Northern blot analysis of isolated RNA from the cambium and developing xylem, thus confirming the validity of the protocol. Some difficulties of in situ transcript localization and the interpretation of the signal distribution in the secondary tissues are discussed.

82 Taxonomic revision of sphaerotrichia divaricata (C. AG.) kylin (ectocarpales S.L., phaeophyceae), with a reappraisal of s. firma (GEPP) a. zinova from the northwestern pacific

Specimens of Sphaerotrichia in Japan can be classified into two morphotypes based on mode of branching and anatomy: 1) specimens having an obvious (traceable) main axis associated with more first‐order branches, and the distance between the branches is relatively constant; 2) specimens that branches more or less divaricately and the distance between branches tends to be shorter in the distal portion. Molecular analyses of North Atlantic and North Pacific Sphaerotrichia using ribo‐somal RNA (ITS1‐5.8S‐ITS2 and IGS regions between 26S and 5S rDNA) and Rubisco (almost complete rbcL gene and the spacer between rbcL and rbcS) gene sequences revealed that there are two major genetic groups (i.e. Group‐1 and Group‐2), whereas the sequence length variation in ITS1 suggested subdivisions of Group‐2 into two subgroups (Group‐2Atlantic and Group‐2Pacific). Specimens belonging to Group‐1 were generally epilithic, and morphologically corresponded to Sphaerotrichia firma (Gepp) A. Zinova. Specimens belonging to Group‐2 were epiphytic or epilithic and morphologically corresponded to S. divaricata (C. Ag.) Kylin.

Taxonomic revision of Sphaerotrichia divaricata (Ectocarpales, Phaeophyceae), with a reappraisal of S. firma from the north-west Pacific

Specimens of Sphaerotrichia in Japan can be classified into two morphotypes, based on their mode of branching and anatomy: (1) specimens that have an obvious (traceable) main axis associated with a relatively high number of first-order branches, and a relatively constant distance between branches; and (2) specimens that branch more or less divaricately, with shorter distances between branches in the distal portion. Molecular analyses of Sphaerotrichia from the North Atlantic and North Pacific using ribosomal RNA (ITS1–5.8S–ITS2 and intragenic spacer regions between 26S and 5S rDNA) and Rubisco (almost complete rbcL gene and the spacer between rbcL and rbcS) gene sequences revealed two major genetic groups, Group-1 and Group-2. The sequence length variation in ITS1 suggested subdivisions of Group-2 into two subgroups: Group-2Atlantic and Group-2Pacific. Specimens belonging to Group-1 were generally epilithic, and morphologically corresponded to S. firma. Specimens belonging to Group-2 were epiphytic or epilithic and morphologically corresponded to S. divaricata.

The promoter-terminator of chrysanthemum rbcS1 directs very high expression levels in plants.

Transgenic plants are increasingly used as production platforms for various proteins, yet protein expression levels in the range of the most abundant plant protein, ribulose-1,5-bisphosphate carboxylase have not yet been achieved by nuclear transformation. Suitable gene regulatory 5' and 3' elements are crucial to obtain adequate expression. In this study an abundantly transcribed member (rbcS1) of the ribulose-1,5-bisphosphate carboxylase small-subunit gene family of chrysanthemum (Chrysanthemum morifolium Ramat.) was cloned. The promoter of rbcS1 was found to be homologous to promoters of highly expressed rbcS gene members of the plant families Asteraceae, Fabaceae and Solanaceae. The regulatory 5' and 3' non-translated regions of rbcS1 were engineered to drive heterologous expression of various genes. In chrysanthemum, the homologous rbcS1 cassette resulted in a beta-glucuronidase (gusA) accumulation of, at maximum, 0.88% of total soluble protein (population mean 0.17%). In tobacco (Nicotiana tabacum L.), the gusA expression reached 10% of total soluble protein. The population mean of 2.7% was found to be 7- to 8-fold higher than for the commonly used cauliflower mosaic virus (CaMV) 35S promoter (population mean 0.34%). RbcS1-driven expression of sea anemone equistatin in potato (Solanum tuberosum L.), and potato cystatin in tomato (Lycopersicon esculentum Mill.) yielded maximum levels of 3-7% of total soluble protein. The results demonstrate, that the compact 2-kb rbcS1 expression cassette provides a novel nuclear transformation vector that generates plants with expression levels of up to 10% of total protein.

Multiple effects of antibiotics on chloroplast and nuclear gene expression.

Antibiotics are widely used to monitor signalling cascades within a plant cell, for example between the nucleus and chloroplasts, and to study the function of the photosynthetic machinery. In the present study, we attempted to test various antibiotics with respect to their expected modes of function and also to monitor their possible side effects on metabolic processes in mature leaves of pea (Pisum sativum L.). Streptomycin, despite its reported prokaryotic nature, prevented translation not only in the chloroplast, but also in the cytosol. Application of puromycin, an inhibitor of protein synthesis in both the pro- and eukaryotes, resulted in severe photoinhibition of photosystem II upon illumination, yet had no effect on plastid translation, thus implying a severe side effect on plastid metabolism. Prokaryotic-type translation inhibitors lincomycin, spectinomycin and erythromycin blocked translation in the chloroplast without any direct effects on cytoplasmic protein synthesis. More detailed studies with lincomycin, however, revealed a strong modulation of the expression of nuclear-encoded genes by slowing down the transcription rate of photosynthesis-related Lhcb and RbcS genes, and furthermore, lincomycin clearly decreased the phosphorylation level of the LHCII proteins.

Complementation of the nuclear antisense rbcS-induced photosynthesis deficiency by introducing an rbcS gene into the tobacco plastid genome.

The small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a nuclear gene-encoded protein that is imported into chloroplasts where it assembles with the large subunit (LS) after removal of the transit peptide to form Rubisco. We have explored the possibility that the severe deficiency in photosynthesis exhibited in nuclear transgenic tobacco (line alpha5) expressing antisense rbcS coding DNA that results in low SS and Rubisco protein content [Rodermel et al. (1988) Cell 55: 673] could be complemented by introducing a copy of the rbcS gene into its plastid genome through chloroplast transformation. Two independent lines of transplastomic plants were generated, in which the tobacco rbcS coding sequence, either with or without the transit sequence, was site-specifically integrated into the plastid genome. We found that compared with the antisense plants, expression of the plastid rbcS gene in the transplastomic plants resulted in very high mRNA abundance but no increased accumulation of the SS and Rubisco protein or improvement in plant growth and photosynthesis. Therefore, there is a limitation in efficient translation of the rbcS mRNA in the plastid or an incorrect processing and modification of the plastid-synthesized SS protein that might cause its rapid degradation.

Rubisco small subunit, chlorophyll a/b-binding protein and sucrose:fructan-6-fructosyl transferase gene expression and sugar status in single barley leaf cells in situ. Cell type specificity and induction by light.

We describe a highly efficient two-step single-cell reverse transcriptase-polymerase chain reaction technique for analyzing gene expression at the single-cell level. Good reproducibility and a linear dose response indicated that the technique has high specificity and sensitivity for detection and quantification of rare RNA. Actin could be used as an internal standard. The expression of message for Rubisco small subunit (RbcS), chlorophyll a/b-binding protein (Cab), sucrose (Suc):fructan-6-fructosyl transferase (6-SFT), and Actin were measured in individual photosynthetic cells of the barley (Hordeum vulgare) leaf. Only Actin was found in the non-photosynthetic epidermal cells. Cab, RbcS, and 6-SFT genes were expressed at a low level in mesophyll and parenchymatous bundle sheath (BS) cells when sampled from plants held in dark for 40 h. Expression increased considerably after illumination. The amount of 6-SFT, Cab, and RbcS transcript increased more in mesophyll cells than in the parenchymatous BS cells. The difference may be caused by different chloroplast structure and posttranscriptional control in mesophyll and BS cells. When similar single-cell samples were assayed for Suc, glucose, and fructan, there was high correlation between 6-SFT gene expression and Suc and glucose concentrations. This is consistent with Suc concentration being the trigger for transcription. Together with earlier demonstrations that the mesophyll cells have a higher sugar threshold for fructan polymerization, our data may indicate separate control of transcription and enzyme activity. Values for the sugar concentrations of the individual cell types are reported.

PHYLOGENETIC ANALYSIS OF 32 STRAINS OFVAUCHERIA(XANTHOPHYCEAE) USING THErbcL GENE AND ITS TWO FLANKING SPACER REGIONS1

The completerbcL gene was sequenced for 21 species and 32 strains ofVaucheriaand for five other Xanthophyceae (Asterosiphon dichotomus(Kützing) Rieth,Botrydium becharianumVischer,B. cystosumVischer,B. stoloniferumMitra,Tribonema intermixtumPascher). ThepsbA‐rbcL spacer, upstream of therbcL gene, and the RUBISCO spacer between therbcL andrbcS genes were also completely sequenced for theVaucheriastrains andAsterosiphon.ThepsbA‐rbcL spacer was the most variable region that was sequenced, and only the 3′ end of the spacer could be aligned. Phylogenetic analyses (maximum parsimony, neighbor joining, and maximum likelihood) were conducted using the DNA sequence and the amino acid sequence for therbcL gene, and a second analysis was conducted using a portion of thepsbA‐rbcL spacer +rbcL gene + RUBISCO spacer. All analyses showed thatVaucheriaspecies formed monophyletic clades that corresponded with morphologically based subgeneric sections, including the section Racemosae. Species producing a gametophore (= fruiting branch, bearing both an antheridium and oogonium) formed a monophyletic clade in all analyses. The nongametophore species sometimes formed a monophyletic clade but other times formed a basal grade. Pair‐wise comparisons of nucleotides and amino acids showed that for some species, numerous nucleotide changes resulted in relatively few amino acid changes. Consequently, phylogenetic analysis of the amino acids produced numerous trees, which in a strict consensus tree resulted in numerous polychotomies. An original strain ofV. terrestristhat was deposited in two culture collections over 25 years ago had identical sequences, suggesting no rapid change was occurring in the sequenced regions. Two strains ofV. prona, isolated from Europe and North America, had identical sequences. Other species, for which two or more strains were examined, had different sequences. These results suggest that cryptic species complexes exist withinVaucheriabecause therbcL gene is a conservative gene that is identical in other protists.

Transcritption regulation of soybean ribulose-1,5-bisphosphate carboxylase small subunit gene by external factors

Ribulose-1,5-bisphosphate carboxylase small subunit gene (rbcS) is present with multi-gene family in plant genome. In Glycine max, the rbcS polypeptide (EC4.1.1.39) is encoded by a gene family containing 4–8 members. Three full-length rbcS cDNA clones were isolated and characterized from soybean seedlings, and both of their nucleotide and amino acid sequences showed high similarity. Differential accumulation of the rbcS mRNA was observed among roots, hypocotyls, cotyledons, epicotyls and leaves. The rbcS genes were up-regulated by various external factors such as salicylic acid (SA), salt stress and drought stress. The expression level of rbcS genes after being treated by 2.0 mmol/L SA and 0.4% NaCl, respectively, is 2.5–3.0-fold as high as that of control sample. Moreover, soybean rbcS mRNA was accumulated with diurnal variation but easily influenced by light and low temperature.

Stable integration and functional expression of flounder growth hormone gene in transformed microalga, Chlorella ellipsoidea.

Chlorella is an attractive organism for complex recombinant protein production because of its eukaryotic characteristics and low cost for large-scale culture. Protoplasts of C. ellipsoidea were transformed with a vector containing the flounder growth hormone gene (fGH) under the control of the cauliflower mosaic virus 35S promoter, and the phleomycin resistance Sh ble gene under the control of the Chlamydomonas RBCS2 gene promoter. The presence of introduced DNA was first determined by PCR amplification of both the fGH and Sh ble genes from genomic DNA isolated from transformants and fGH protein expression was detected by immunoblot analysis. Over 400 microg of fGH protein expression per one liter culture containing 1 x 10(8) cells/ml was estimated by ELISA. Stable integration of introduced DNA was confirmed by Southern blot analysis of genomic DNA digested with restriction enzymes. The introduced DNA and fGH expression were detected after seven successive transfers in media devoid of phleomycin, but stably remained in the presence of the antibiotic. Flounder fry fed on the transformed Chlorella revealed a 25% growth increase after 30 days of feeding.

Chloroplast targeting signal of a rice rbcS gene enhances transgene expression

Previously, we reported that the presence of therbcS transit peptide (TP) sequence as a plastid-targetingsignal led to greatly enhanced accumulation of transgene products in transgenicrice plants. To study the mechanisms underlying the regulation of transgeneexpression by the TP sequence, we mutated the sequence(mTP) so that it did not localize fused heterologousproteins into plastids, while it retained a nearly identical nucleotidesequenceto its wild type. mTP was fused to a synthetic gene(sgfp) encoding a modified form of the green fluorescentprotein (sGFP) and linked to the rice rbcS promoter,generating an expression vector, rbcS-mTP-sgfp. To compareexpression levels in transgenic plants, two expression vectors wereconstructed:rbcS-sgfp for untargeted expression andrbcS-TP-sgfp for chloroplast-targeted expression. Severaltransgenic plants were produced for each construct by theAgrobacterium-mediated method. Confocal microscopicanalyses demonstrated that GFP fluorescence in therbcS-TP-sgfp-transformed lines was specifically localizedwithin chloroplasts, whereas fluorescence was detected within cytoplasm andnucleoplasm in the rbcS-mTP-sgfp- andrbcS-sgfp-transformed lines. Northern blot analysisindicated that mRNA levels were very different, depending on the type ofTP sequence employed. Transcript levels ofrbcS-TP-sgfp-transformed lines were 10 fold higher thanthose of either rbcS-mTP-sgfp- orrbcS-sgfp-transformed lines, whereas the levels of thelasttwo were comparable. Levels of sGFP accumulation were also correspondinglyhigher in rbcS-TP-sgfp-transformed lines than in the othertwo lines, as examined by immunoblot analyses. Thus, increased levels of sGFPaccumulation appeared to reflect increased mRNA levels; this was due to thepresence of the functional rbcS transit peptide. Theseresults suggest that a functional rbcS transit peptideenhances transgene expression in addition to targeting the gene products intochloroplasts.

Extracellular Calmodulin Stimulates Light-Independent RbcS-GUS Expression in Suspension-Cultured Cells of Transgenic Tobacco

The RbcS genes encode the small subunits of rubisco; the expression of these genes is controlled in a light-dependent and independent manner. It has been reported that intracellular calmodulin (CaM) is involved in light-dependent RbcS expression. In this report, the role of extracellular CaM in regulating expression of RbcS in darkness was examined. The time course of expression of RbcS-GUS and that of the secretion of CaM in the suspended transgenic tobacco cells in darkness were very similar. Both showed initial increase followed by decline with maximum CaM secretion preceding maximum GUS expression by 24 h. The concentration of CaM in the culture medium is regulated light independently. Purified CaM alone added to the media enhanced RbcS-GUS expression in darkness. The addition of membrane-impermeable CaM inhibitors, such as anti-CaM antiserum or W7-agarose, repressed the expression of RbcS-GUS in darkness, but this inhibitory effect was completely reversed by adding exogenous purified CaM. These results provide the first evidence that extracellular CaM is involved in the regulation of light-independent RbcS gene expression.

A Streptomyces rimosusaphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii

Although Chlamydomonas reinhardtii serves as the most popular algal model system, no efficient enzymatic selection marker for the nuclear transformation of wild-type cells is available. We sequenced an aminoglycoside 3′-phosphotransferase gene ( aph) from Streptomyces rimosus. Though the derived protein sequence is homologous to members of APH type V, it constitutes a new type, named APHVIII. Since the aphVIII gene has a codon bias similar to that of the nuclear genome of green algae, the aphVIII coding sequence was fused to the 5′- and 3′-untranslated regions of the C. reinhardtii rbcS2 gene. C. reinhardtii transformants were capable of inactivating the antibiotics paromomycin, kanamycin, and neomycin, to which wild-type cells are sensitive. After addition of the 5′-region of hsp70A as a second promoter and insertion of the rbcS2 intron I, the transformation rate increased to two transformants per 1×10 5 cells, which is close to the efficiency of transforming auxotrophic strains with the homologous marker arg7. Transformation with the promoter-less aphVIII led to random gene fusion at high frequency. In an aphVIII-based reporter gene assay we have found a so far unknown promoter activity of the 3′-untranslated region of rbcS2, that may promote antisense RNA synthesis from the rbcS2 gene in vivo. We conclude that the aphVIII gene is a useful marker for nuclear transformation and promoter tagging of C. reinhardtii wild-type and probably other green algae.

Regulation of Chloroplast Gene Expression is Affected in ali, a Novel Tobacco Albino Mutant

An albino mutant designated ali was isolated from an M 2population derived from a tobacco ( Nicotiana tabacum L. ‘BY-4’) plant which had been irradiated with a 14N-ion beam at an early stage of embryonic development. Leaf mesophyll cells of ali were devoid of developed chloroplasts, and no stacked thylakoid membranes could be detected in the plastids. The reduction of pigment and the arrest of chloroplast development in ali were associated with distinct alterations in gene expression. Expression of plastid-encoded genes for the photosynthesis system ( rbcL, psbA) was greatly reduced in ali, but at the transcript level plastid-encoded genes for 23S rDNA and 16S rDNA expression were only slightly reduced. Transcription of the nuclear-encoded Lhc and rbcS genes in the mutant was similar to that in the wild-type. There was no accumulation of Rubisco-L, Rubisco-S, and PSII-D1 polypeptides in the ali mutant. The number of chloroplasts per cell was similar in both ali and wild-type plants. These results suggest that ali is a novel albino mutant in which chloroplast gene expression is affected.

Inhibition of the light-independent synthesis of chlorophyll in pine cotyledons at low temperature.

Cotyledons of Japanese black pine (Pinus thunbergii) were yellow when they developed in darkness at 8 degrees C since the light-independent synthesis of chlorophyll was almost completely inhibited in these cotyledons. The level of chlorophyll in dark-grown cotyledons was less than one-twentieth of that in light-grown cotyledons at the same temperature. In the yellow cotyledons, levels of transcripts of cab, rbcS, rbcL and psbA genes were quite high. The large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase were also detected at relatively high levels in yellow cotyledons. However, the accumulation of the two apoproteins of the light-harvesting chlorophyll a/b-binding protein of PSII was limited because of the limited supply of chlorophyll.

Amitrole treatment of etiolated barley seedlings leads to deregulation of tetrapyrrole synthesis and to reduced expression of Lhc and RbcS genes.

The effect of amitrole, known as an inhibitor of carotenoid biosynthesis, upon tetrapyrrole biosynthesis and its regulation has been studied. Etiolated barley (Hordeum vulgare L.) seedlings, grown in 125 microM amitrole, accumulated high levels of 5-aminolevulinate, Mg-protoporphyrin, Mg-protoporphyrin monomethyl ester, and protochlorophyllide. The amitrole-treated seedlings did not form paracrystalline prolamellar bodies, and the induction of Lhc and RbcS gene expression was reduced by non-photooxidative, low-intensity light. None of these events was observed upon treatment of the seedlings with 100 microM norflurazon, another inhibitor of carotenoid biosynthesis. The effect of amitrole cannot be explained solely by interaction with a presumed feedback inhibition of 5-aminolevulinate synthesis since incubation with amitrole and 5-aminolevulinate indicated that deregulation also occurs at later steps of tetrapyrrole biosynthesis. A possible relationship between this deregulation and ultrastructural changes is discussed. In connection with previously published data, we discuss Mg-protoporphyrin and its monomethyl ester as possible candidates for a "plastid signal" that operates as a negative factor, reducing the expression of Lhc and RbcS genes in this higher plant.