rbcS Promoter Research Articles

Efficient and stable transgene suppression via RNAi in field-grown poplars

The efficiency and stability of RNA interference (RNAi) in perennial species, particularly in natural environments, is poorly understood. We studied 56 independent poplar RNAi transgenic events in the field over 2 years. A resident BAR transgene was targeted with two different types of RNAi constructs: a 475-bp IR of the promoter sequence and a 275-bp IR of the coding sequence, each with and without the presence of flanking matrix attachment regions (MARs). RNAi directed at the coding sequence was a strong inducer of gene silencing; 80% of the transgenic events showed more than 90% suppression. In contrast, RNAi targeting the promoter resulted in only 6% of transgenic events showing more than 90% suppression. The degree of suppression varied widely but was highly stable in each event over 2 years in the field, and had no association with insert copy number or the presence of MARs. RNAi remained stable during a winter to summer seasonal cycle, a time when expression of the targeted transgene driven by an rbcS promoter varied widely. When strong gene suppression was induced by an IR directed at the promoter sequence, it was accompanied by methylation of the homologous promoter region. DNA methylation was also observed in the coding region of highly suppressed events containing an IR directed at the coding sequence; however, the methylation degree and pattern varied widely among those suppressed events. Our results suggest that RNAi can be highly effective for functional genomics and biotechnology of perennial plants.

Phylogenetic and molecular analysis of the ribulose-1,5-bisphosphate carboxylase small subunit gene family in banana

Despite being the number one fruit crop in the world, very little is known about the phylogeny and molecular biology of banana (Musa spp.). Six banana rbcS gene families encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from six different Musa spp. are presented. For a comprehensive phylogenetic study using Musa rbcS genes, a total of 57 distinct rbcS sequences was isolated from six accessions that contained different combinations of the A and B ancestral/parental genomes. As a result, five of the six members of the rbcS gene family could be affiliated with the A and/or B Musa genomes and at least three of the six gene families most likely existed before Musa A and B genomes separated. By combining sequence data with quantitative real-time PCR it was determined that the different Musa rbcS gene family members are also often multiply represented in each genome, with the highest copy numbers in the B genome. Expression of some of the rbcS genes varied in intensity and in different tissues indicating differences in regulation. To analyse and compare regulatory sequences of Musa rbcS genes, promoter and terminator regions were cloned for three Musa rbcS genes. Transient transformation assays using promoter-reporter-terminator constructs in maize, wheat, and sugarcane demonstrated that the rbcS-Ma1, rbcS-Ma3, and rbcS-Ma5 promoters could be useful for transgene expression in heterologous expression systems. Furthermore, the rbcS-Ma1 terminator resulted in a 2-fold increase of transgene expression when directly compared with the widely used Nos terminator.

Increased Rubisco Content in Transgenic Rice Transformed with the ‘Sense’ rbcS Gene

Rice (Oryza sativa L.) plants with substantially increased Rubisco content were obtained by Agrobacterium-mediated transformation with the rice rbcS sense gene under the control of the rice rbcS promoter. The primary transformants were screened for the ratio of Rubisco to leaf-N content, and the transformants with >120% wild-type levels of Rubisco were selected. In the progeny of the selected lines of the transformants, the mRNA levels of one member of the rbcS gene family were increased from 3.9- to 6.2-fold, whereas those of other members of the rbcS gene family were unchanged. The total levels of rbcS mRNA were increased from 2.1- to 2.8-fold. The levels of rbcL mRNA were increased from 1.2- to 1.9-fold. Rubisco protein content was significantly increased by 30% on a leaf area basis. The ratio of Rubisco-N to leaf-N was also increased by 10-20%, irrespective of N treatment. The specific activity of Rubisco per unit of enzyme protein was not different. However, light-saturated photosynthesis was not enhanced even when the rate was measured at low [CO2] where Rubisco becomes limiting for photosynthesis. Some lines showed lower photosynthesis at high [CO2] (>60 Pa). We conclude that introduction of additional sense rbcS leads to overexpression of rbcS and that this overexpression slightly up-regulates the gene expression of rbcL at the transcript level and enhances the amount of Rubisco holoenzyme. However, overproduction of Rubisco protein does not improve photosynthesis.

Cloning of new rubisco promoters from Brassica rapa and determination of their activity in stably transformed Brassica napus and Nicotiana tabacum plants

The aim of our study was to identify the highest expressing rubisco small subunit (RbcS) promoters (pRbcS) from the cotyledons of germinating seedlings of Brassica rapa var. oleifera to drive high-level and preferably stage-specific transgenic protein expression in Brassicaceae plants. We cloned four new pRbcS promoters using several approaches, including the construction of a cDNA library and use of genome walking technique. Real-time PCR analysis of RbcS mRNA expression clearly showed that two of these promoters exhibited the highest activity on the germination stage of plant development. We used gusA expression as a reporter of promoter activity in Brassica napus and Nicotiana tabacum plants that were transformed with the constructs using an Agrobacterium-mediated transformation strategy. The mRNA level of RbcS and of gusA was quantified in transformed plants. The data obtained demonstrate that the promoter most active in seedlings under native conditions was also most active in transgenic constructs at the same stage of plant development. The fine structure of the promoters is discussed herein.

PHYTOREMEDIATION WITH TRANSGENIC PLANTS

Genetic manipulation of environmentally important plants can produce elite plant lines with enhanced remediation abilities. Introduction of key genes to increase the remediation ability is an obvious approach and significant progress has been made in recent years in producing genetically modified plants from several species. For improvement of heavy metal accumulation we assembled plant expression cassettes from the fission yeast HMT1 gene but they all failed to express in different plant hosts. Another phytoremediation expression cassette was constructed from the salt marsh worm (Amphitrite ornata) dehaloperoxidase gene and introduced into Arabidopsis and tobacco model systems with more success. Both steady state mRNA and activity have been demonstrated in the transgenic plants. We also have developed a green tissue(leaf-) specific mercuric reductase (merA) expression cassette using the wheat rbcS promoter to enhance merA expression in fully expanded leaves, decreasing mercury content and enhancing mercury volatilization. We have developed tissue culture methods in our lab for a number of wetland plants of various ecological requirements including Spartina alterniflora. An in vitro mass selection system for elite lines has been developed for several species using the Liquid Lab Rocker system of Southern Sun Biosystems (Hodges, SC, USA). Using the 35S and Ubi constitutive promoters controlling the organomercurial lyase (merB) and (merA) genes transgenic Spartina lines were obtained carrying and expressing both genes as indicated by the presence of mRNAs and the resistance to ionic and organic mercury. Gene transfer experiments are in progress with other wetland plants such as Arundo, Phragmites, Typha, and Schoenoplectus species.

Expression of the large isoform of ribulose-1,5-bisphosphate carboxylase/oxygenase activase gene driven by rbcS promoter in Oryza sativa enhances the photosynthetic capacity

In order to investigate the effect of large isoform of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) activase (RuBPCO-A) on photosynthesis, cDNA of the enzyme (rca) was transferred to rice cultivars (Oryza sativa f. japonica cv. Nipponbare) under the control of RuBPCO small subunit gene promoter (rbcS) via Agrobacterium tumefaciens-mediated transformation. Transgenic rice plants were identified by polymerase chain reaction (PCR) and Southern and Western blot analyses. Net photosynthetic rate (P N) values of the T1 transgenic lines 34 (T34) and 40 (T40) were 45.26 and 46.32 % higher than that of the control plants, respectively. At the same time, their carboxylation efficiency and RuBPCO initial activity, quantum yield of electron transport in photosystem 2 (ΦPS2), and steady state photochemical fluorescence quenching (qP) increased. In addition, heading time of the transgenic rice was advanced. Thus increasing the amount of large isoform of RuBPCO-A in the transgenic rice might have a stimulatory effect on both photosynthesis and plant growth.

Differential Expression of rolC Results in Unique Plant Phenotypes

Expression of the rolC gene in plants has been shown to cause pleiotropic effects, including decreased height. The effects of differential rolC gene expression on plant height, leaf color, root growth, leaf size, corolla length, and stem diameter were determined. Differential expression of rolC in Nicotiana tabacum L. `Samsun' plants was achieved using the 35S promoter, the light-inducible rbcS promoter, or the native rolC promoter. Sixteen plants from the T1 generation—six with the 35S promoter, six with the rbcS promoter, and four with the native rolC promoter—and non-transformed controls were measured for height, internode length, branch number, bud size, corolla length and diameter, root growth, and the number of days to flowering. Steady state mRNA levels of rolC were measured in roots, stems, and leaves to assess relationships between rolC expression level in specific tissues and phenotypes. Plants expressing rolC showed a wide range of phenotypes, with the largest changes in plants expressing rolC using the 35S promoter, which also had the highest rolC mRNA levels. Plants expressing rolC with the rolC or rbcS promoter had significant changes for many measured traits, despite rolC mRNA levels that were not significantly different from non-transformed controls. In general, as rolC mRNA levels increased, so did the severity of the rolC phenotype observed. Three plants, A4, A7, and B9, had unique combinations of traits that did not follow this general trend. Transformation with rolC can be useful in ornamental crops where smaller cultivars are desired.

Genomic approaches to the discovery of promoters for sustained expression in cotton (Gossypium hirsutum L.) under field conditions: expression analysis in transgenic cotton and Arabidopsis of a Rubisco small subunit promoter identified using EST sequence analysis and cDNA microarrays

Biotechnology requires robust and predictable expression of transgenes. Most commercial Genetically Modified (GM) crops contain the viral 35S promoter to drive insecticide and herbicide resistance genes. In cotton there have been reductions in efficacy of Bacillus thuringiensis toxin (Bt) expressing plants late in the season that have been attributed to reductions in promoter activity. We have used genomic approaches to identify cotton genes whose expression remains high during the season to find promoters that might better maintain expression of transgenes in the field. A cDNA library from young late season leaves was used to generate about 2000 ESTs. Clustering of ESTs was used to determine relative transcript abundance and identify the most highly-expressed genes. These were primarily photosynthetic and housekeeping genes and some metabolic genes. The ESTs were printed to a small cDNA microarray and probed with both early- and late- season leaf mRNAs. Absolute fluorescence levels were used to rank genes and confirm the EST abundance data. Candidate genes, including the small subunit of Rubisco (RbcS) were selected. An RbcS promoter (Genbank Accession DQ648074) was isolated and analysed in both Arabidopsis and cotton linked to a GUS reporter gene. Expression of the reporter gene was consistently high in green tissues throughout the life cycle of cotton in the glasshouse, and in the field. A number of other candidate promoters have been identified that may be useful in a variety of biotechnology applications.

Light-regulated overexpression of an Arabidopsis phytochrome A gene in rice alters plant architecture and increases grain yield

The phytochromes are a family of red/far-red light absorbing photoreceptors that control plant developmental and metabolic processes in response to changes in the light environment. We report here the overexpression of Arabidopsis thaliana PHYTOCHROME A (PHYA) gene in a commercially important indica rice variety (Oryza sativa L. Pusa Basmati-1). The expression of the transgene was driven by the light-regulated and tissue-specific rice rbcS promoter. Several independent homozygous sixth generation (T(5)) transgenic lines were characterized and shown to accumulate relatively high levels of PHYA protein in the light. Under both far-red and red light, PHYA-overexpressing lines showed inhibition of the coleoptile extension in comparison to non-transgenic seedlings. Furthermore, compared with non-transgenic rice plants, mature transgenic plants showed significant reduction in plant height, internode length and internode diameter (including differences in cell size and number), and produced an increased number of panicles per plant. Under greenhouse conditions, rice grain yield was 6-21% higher in three PHYA-overexpressing lines than in non-transgenic plants. These results demonstrate the potential of manipulating light signal-transduction pathways to minimize the problems of lodging in basmati/aromatic rice and to enhance grain productivity.

Sugar and ABA responsiveness of a minimal RBCS light‐responsive unit is mediated by direct binding of ABI4

Photosynthesis-associated nuclear genes (PhANGs) are able to respond to multiple environmental and developmental signals, including light, sugars and abscisic acid (ABA). PhANGs have been extensively studied at the level of transcriptional regulation and several cis-acting elements important for light responsiveness have been identified in their promoter sequences. However, the regulatory elements involved in sugar and ABA regulation of PhANGs have not been completely characterized. Using conserved modular arrangement 5 (CMA5), a previously characterized minimal light-responsive unit, we show that in Arabidopsis thaliana this unit responds not only positively to light signals, but also negatively to sugars and ABA. The latter responses were found to be impaired in the abi4 mutant, indicating that ABSCISIC ACID INSENSITIVE-4 (ABI4) is a regulator involved in sugar and ABA repression of this minimal regulatory unit. Furthermore, we report a new sequence element conserved in several rbcS promoters, herewith named S-box, which is important for the sugar and ABA responsiveness of CMA5. This sequence corresponds to a putative ABI4-binding site, which is in fact bound by the Arabidopsis ABI4 protein in vitro. The S-box is closely associated with the G-box present in CMA5, and this association is conserved in the promoters of several RBCS genes. This phylogenetically conserved promoter feature probably reflects a common regulatory mechanism and identifies a point of convergence between light- and sugar-signaling pathways.

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.

A Comparison of Constitutive Promoters for Expression of Transgenes in Alfalfa (Medicago Sativa)

The activity of constitutive promoters was compared in transgenic alfalfa plants using two marker genes. Three promoters, the 35S promoter from cauliflower mosaic virus (CaMV), the cassava vein mosaic virus (CsVMV) promoter, and the sugarcane bacilliform badnavirus (ScBV) promoter were each fused to the beta-glucuronidase (gusA) gene. The highest GUS enzyme activity was obtained using the CsVMV promoter and all alfalfa cells assayed by in situ staining had high levels of enzyme activity. The 35S promoter was expressed in leaves, roots, and stems at moderate levels, but the promoter was not active in stem pith cells, root cortical cells, or in the symbiotic zones of nodules. The ScBV promoter was active primarily in vascular tissues throughout the plant. In leaves, GUS activity driven by the CsVMV promoter was approximately 24-fold greater than the activity from the 35S promoter and 38-fold greater than the activity from the ScBV promoter. Five promoters, the double 35S promoter, figwort mosaic virus (FMV) promoter, CsVMV promoter, ScBV promoter, and alfalfa small subunit Rubisco (RbcS) promoter were used to control expression of a cDNA from Trichoderma atroviride encoding an endochitinase (ech42). Highest chitinase activity in leaves, roots, and root nodules was obtained in plants containing the CsVMV:ech42 transgene. Plants expressing the endochitinase were challenged with Phoma medicaginis var. medicaginis, the causal agent of spring black stem and leaf spot of alfalfa. Although endochitinase activity in leaves of transgenic plants was 50- to 2650-fold greater than activity in control plants, none of the transgenic plants showed a consistent increase in disease resistance compared to controls. The high constitutive levels of both GUS and endochitinase activity obtained demonstrate that the CsVMV promoter is useful for high-level transgene expression in alfalfa.

Characterization of sunlight-grown transgenic rice plants expressing Arabidopsis phytochrome A

Transgenic rice (Oryza sativa) overexpressing Arabidopsis phytochrome A (phyA) was cultivated up to the T3 generation in paddy to elucidate the role of phyA in determining the plant architecture and the productivity of sunlight-grown rice plants. PhyA is light-labile and controls plant growth in response to the far-red light-dependent high-irradiance response as well as the very low fluence response. The Arabidopsis phyA gene linked to the rice rbcS promoter was transformed into embryogenic rice calli, and the calli were regenerated to whole plants. Compared to wild-type seedlings, the rbcS::PHYA transgenic seedlings contained more phyA when grown in the dark, and at least 10-fold more phyA when exposed to white light. When grown in paddy, the phyA transgenic plants in general exhibited reduced plant height (dwarfing), larger grain size, higher chlorophyll content, smaller tiller number, and low grain fertility compared to wild-type plants. The heading stage was not significantly changed. However, it is likely that a certain level of phyA is a prerequisite for induction of such changes. It is suggested that phyA overproduction in rice could be a useful tool to improve rice grain productivity by the larger grain size that increases grain yield and the dwarfing that tolerates lodging-associated damage.

Differential Expression of rolC Results in Unique Plant Phenotypes

As residential lot sizes decrease, there is an increased demand for new, small-statured landscape plants to fit into the smaller lots. One promising method to create smaller plants is by introducing a dwarfing gene into a plant of interest. A dwarfing gene that has been identified is the rolC gene from Agrobacterium rhizogenes. Expression of rolC in plants has been shown to cause decreased height and internode length, increased branching, and modified leaf size in a several species. Although the effects of the rolC gene have been well characterized for many plant species, most research has concerned the native promoter or the CaMV 35S promoter. Less research has been done with additional promoters or comparing the results from different promoters. In this study we examined the effects of three separate gene constructs, all containing rolC driven under either the 35S promoter, the light inducible rbcs promoter, or the native rolC promoter in tobacco. Plants transformed with these constructs ranged widely for height and other phenotypic traits. Representative plants were crossed back to wild-type tobacco. Plants from this next generation, six with the 35S promoter, six with the rbcs promoter and four with the native rolC promoter, were measured for traits such as height, days to flower, number of branches and internode length. RolC RNA expression levels were also measured in roots, stems, and leaves to determine correlations between rolC expression level in specific tissues and the observed phenotype. Information about these relationships can be used to provide insight into the use of rolC in ornamental plants and the potential to modify its phenotypic effects by controlling expression level.

Co-expression of a modified maize ribosome-inactivating protein and a rice basic chitinase gene in transgenic rice plants confers enhanced resistance to sheath blight.

Chitinases, beta-1,3-glucanases, and ribosome-inactivating proteins are reported to have antifungal activity in plants. With the aim of producing fungus-resistant transgenic plants, we co-expressed a modified maize ribosome-inactivating protein gene, MOD1, and a rice basic chitinase gene, RCH10, in transgenic rice plants. A construct containing MOD1 and RCH10 under the control of the rice rbcS and Act1 promoters, respectively, was co-transformed with a plasmid containing the herbicide-resistance gene bar as a selection marker into rice by particle bombardment. Several transformants analyzed by genomic Southern-blot hybridization demonstrated integration of multiple copies of the foreign gene into rice chromosomes. Immunoblot experiments showed that MOD1 formed approximately 0.5% of the total soluble protein in transgenic leaves. RCH10 expression was examined using the native polyacrylamide-overlay gel method, and high RCH10 activity was observed in leaf tissues where endogenous RCH10 is not expressed. R1 plants were analyzed in a similar way, and the Southern-blot patterns and levels of transgene expression remained the same as in the parental line. Analysis of the response of R2 plants to three fungal pathogens of rice, Rhizoctonia solani, Bipolaris oryzae, and Magnaporthe grisea, indicated statistically significant symptom reduction only in the case of R. solani (sheath blight). The increased resistance co-segregated with herbicide tolerance, reflecting a correlation between the resistance phenotype and transgene expression.

Mycorrhizal colonization of transgenic aspen in a field trial.

Mycorrhizal colonization of genetically modified hybrid aspen (Populus tremula x P. tremuloides Michx.) was investigated over 15 months in a field experiment. The aspen carried the rolC gene from Agrobacterium rhizogenes under control of either the constitutive cauliflower mosaic virus 35S promoter or the light-inducible rbcS promoter. Arbuscular mycorrhizas (AMs) were rare in all root samples, while fully developed ectomycorrhizas (EMs) were found in all samples. No significant differences in the degree of mycorrhizal colonization between aspen lines were seen with either AMs or EMs. The EM community on the release area was dominated by four fungal species that formed more than 90% of all mycorrhizas, while eleven EM types were found occasionally. Mycorrhizal diversity did not differ between transgenic and non-transgenic trees. The structure of mycorrhizal communities was similar for most aspen lines. The sole significant difference was found in the abundance and development of one of the four common EM morphotypes, which was rare and poorly developed on roots from the transgenic aspen line Esch5:35S-rolC-#5 compared with non-transgenic controls. This effect is clone specific as the formation of this EM type was not affected by the transgene expression in the other transgenic line, Esch5:35S-rolC-#1. This is the first demonstration of a clonal effect influencing the ability of a transgenic plant to form a mycorrhizal symbiosis with a potential fungal partner.

Asexual production of selectable marker-free transgenic woody plants, vegetatively propagated species.

We describe here a practical system for generating selectable marker-free transgenic woody plants independent of sexual crossing. We previously reported that the GST-MAT vector system could produce marker-free transgenic tobacco plants containing a single-copy transgene at high frequency. The GST-MAT vector system consists of a DNA excision cassette of the R/RS site-specific recombination system from Zygosaccharomyces rouxii into which the isopentenyltransferase gene from Agrobacterium tumefaciens has been inserted. In this study, we applied this new GST-MAT vector to hybrid aspen (Populus Sieboldii X Populus grandidentata), a model of vegetatively propagated plant species, to produce selectable marker-free transgenic woody plants. In the new GST-MAT vector, the chimeric ipt gene fused with a light-inducible rbcS promoter efficiently produced transgenic ipt-shooty with GUS activity from 38.0% of infected stems. Upon excision of the R and ipt genes between RS sites, regulated by the inducible promoter of the maize glutathione-S-transferase (GST-II-27) gene, 3 (21.4%) transgenic hybrid aspen plants with marker-free and normal phenotype were generated from 14 ipt-shooty lines within 2 months after cutting induction. These results suggest that the MAT-vector system might be useful for removing a selectable marker gene independent of sexual crossing in vegetatively propagated species.

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.

Promoter strength and tissue specificity effects on growth of tomato plants transformed with maize sucrose-phosphate synthase

When sucrose-phosphate synthase (SPS; EC 2.4.1.14) is expressed in tomato (Lycopersicon esculentum Mill.) from a ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) small subunit (rbcS) promoter, yields are often unchanged but when SPS is expressed from a Cauliflower Mosaic Virus 35S promoter, yield is enhanced up to 80%. Two explanations for this phenomenon are (i) that expression of SPS in tissues other than leaves accounts for the increased yield or (ii) that the lower level of expression directed by the 35S promoter is more beneficial than the high level of expression directed by the rbcS promoter. To test the first hypothesis, we conducted a reciprocal graft experiment, which showed that root SPS activity did not substantially affect growth. To test the second hypothesis, we conducted a field trial using a backcrossed, segregating, population of SPS-transformed plants derived from 35S and rbcS lines. The optimal dose of SPS activity for growth was approximately twice that of the wild type regardless of which promoter was used. The effect of SPS on growth was the result of a shift in partitioning of carbon among starch, sucrose, and ionic compounds (primarily amino acids), rather than of an increase in net photosynthesis. Excessive SPS activity resulted in a decreased rate of amino acid synthesis, which could explain the non-linear response of plant growth to the level of SPS expression.

The promoter of rbcS in a C3 plant (rice) directs organ-specific, light-dependent expression in a C4 plant (maize), but does not confer bundle sheath cell-specific expression.

The small subunit of ribulose-bisphosphate carboxylase (Rubisco), encoded by rbcS, is essential for photosynthesis in both C3 and C4 plants, even though the cell specificity of rbcS expression is different between C3 and C4 plants. The C3 rbcS is specifically expressed in mesophyll cells, while the C4 rbcS is expressed in bundle sheath cells, and not mesophyll cells. Two chimeric genes were constructed consisting of the structural gene encoding beta-glucuronidase (GUS) controlled by the two promoters from maize (C4) and rice (C3) rbcS genes. These constructs were introduced into a C4 plant, maize. Both chimeric genes were specifically expressed in photosynthetic organs, such as leaf blade, but not in non-photosynthetic organs. The expressions of the genes were also regulated by light. However, the rice promoter drove the GUS activity mainly in mesophyll cells and relatively low in bundle sheath cells, while the maize rbcS promoter induced the activity specifically in bundle sheath cells. These results suggest that the rice promoter contains some cis-acting elements responding in an organ-specific and light-inducible regulation manner in maize but does not contain element(s) for bundle sheath cell-specific expression, while the maize promoter does contain such element(s). Based on this result, we discuss the similarities and differences between the rice (C3) and maize (C4) rbcS promoter in terms of the evolution of the C4 photosynthetic gene.