Green Tissue-specific Expression Research Articles

Isolation and Functional Characterization of a Green-Tissue Promoter in Japonica Rice (Oryza sativa subsp. Japonica).

Simple SummaryTransgenic applications have largely focused on constitutive promoters in plants. However, strong and continuous over-expression of certain genes may be redundant and even harmful to plant growth. Thus, tissue-specific promoters are the most suitable for regulating target gene expression. Although several tissue-specific promoters have been identified, the regulatory mechanism of tissue-specific gene expression remains unclear. By a series of GUS staining of 5′ and 3′ deletions, we uncover tissue-specific cis-acting elements in GSX7R, including ten light-responsive elements. The results reveal that GSX7R is a reverse green tissue-specific promoter, except in endosperm. In contrast, strong tissue-specific promoters that can be used for rice improvements are limited. In this study, we successfully showed that the GSX7R promoter can drive the Cry1Ab gene to resistant rice yellow stem borer. In addition, our study demonstrates an effective promoter to drive foreign genes for crop improvement.Plant promoters play a vital role in the initiation and regulation of gene transcription. In this study, a rice protein/gene of unknown expression, named Os8GSX7, was gained from a rice T-DNA capture line. The semi-quantitative RT-PCR analysis showed that the gene was only expressed in root, glume, and flower, but not in stem, leaf, embryo, and endosperm of japonica rice. The GUS activity analysis of the GSX7R promoter showed that it was a reverse green tissue expression promoter, except in endosperm. The forward promoter of GSX7 cannot normally drive the expression of the foreign GUS gene, while the reverse promoter of GSX7 is a green tissue-specific expression promoter, which can drive the expression of the foreign GUS gene. The region from −2097 to −1543 bp was the key region for controlling the green tissue-specific expression. The regulatory sequences with different lengths from the 2097 bp reverse sequence from the upstream region of the Os8GSX7 were fused with the GUS reporter gene and stably expressed in rice. Furthermore, transgenic rice plants carrying Cry1Ab encoding Bacillus thuringiensis endotoxin, regulated by GSX7R, were resistant to yellow stem borer. The analysis suggested that 10 light responsive elements of tissue-specific expression were found, including ACE, Box4, CAT-box, G-Box, G-box, GATA motif, GC motif, I-box, Sp1, and chs-unit1 M1. In addition, the results of 5′ and 3′ deletions further speculated that ACE and I-box may be the key elements for determining the green tissue-specific expression of GSX7R promoter.

DEMETER plays a role in DNA demethylation and disease response in somatic tissues of Arabidopsis

ABSTRACTDNA demethylases function in conjunction with DNA methyltransferases to modulate genomic DNA methylation levels in plants. The Arabidopsis genome contains four DNA demethylase genes, DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1) also known as DEMETER-LIKE 1 (DML1), DML2, and DML3. While ROS1, DML2, and DML3 were shown to function in disease response in somatic tissues, DME has been thought to function only in reproductive tissues to maintain the maternal-specific expression pattern of a subset of imprinted genes. Here we used promoter:β-glucuronidase (GUS) fusion constructs to show that DME is constitutively expressed throughout the plant, and that ROS1, DML2, and DML3 have tissue-specific expression patterns. Loss-of-function mutations in DME cause seed abortion and therefore viable DME mutants are not available for gene function analysis. We knocked down DME expression in a triple ros1 dml2 dml3 (rdd) mutant background using green tissue-specific expression of a hairpin RNA transgene (RNAi), generating a viable ‘quadruple’ demethylase mutant line. We show that this rdd DME RNAi line has enhanced disease susceptibility to Fusarium oxysporum infection compared to the rdd triple mutant. Furthermore, several defence-related genes, previously shown to be repressed in rdd, were further repressed in the rdd DME RNAi plants. DNA methylation analysis of two of these genes revealed increased differential promoter DNA methylation in rdd DME RNAi plants compared to WT, beyond the difference observed in the parental rdd plants. These results indicate that DME contributes to DNA demethylase activity and disease response in somatic tissues.

Switchgrass (Panicum virgatum L.) promoters for green tissue-specific expression of the MYB4 transcription factor for reduced-recalcitrance transgenic switchgrass

BackgroundGenetic engineering of switchgrass (Panicum virgatum L.) for reduced cell wall recalcitrance and improved biofuel production has been a long pursued goal. Up to now, constitutive promoters have been used to direct the expression of cell wall biosynthesis genes toward attaining that goal. While generally sufficient to gauge a transgene’s effects in the heterologous host, constitutive overexpression often leads to undesirable plant phenotypic effects. Green tissue-specific promoters from switchgrass are potentially valuable to directly alter cell wall traits exclusively in harvestable aboveground biomass while not changing root phenotypes.ResultsWe identified and functionally characterized three switchgrass green tissue-specific promoters and assessed marker gene expression patterns and intensity in stably transformed rice (Oryza sativa L.), and then used them to direct the expression of the switchgrass MYB4 (PvMYB4) transcription factor gene in transgenic switchgrass to endow reduced recalcitrance in aboveground biomass. These promoters correspond to photosynthesis-related light-harvesting complex II chlorophyll-a/b binding gene (PvLhcb), phosphoenolpyruvate carboxylase (PvPEPC), and the photosystem II 10 kDa R subunit (PvPsbR). Real-time RT-PCR analysis detected their strong expression in the aboveground tissues including leaf blades, leaf sheaths, internodes, inflorescences, and nodes of switchgrass, which was tightly up-regulated by light. Stable transgenic rice expressing the GUS reporter under the control of each promoter (756–2005 bp in length) further confirmed their strong expression patterns in leaves and stems. With the exception of the serial promoter deletions of PvLhcb, all GUS marker patterns under the control of each 5′-end serial promoter deletion were not different from that conveyed by their respective promoters. All of the shortest promoter fragments (199–275 bp in length) conveyed strong green tissue-specific GUS expression in transgenic rice. PvMYB4 is a master repressor of lignin biosynthesis. The green tissue-specific expression of PvMYB4 via each promoter in transgenic switchgrass led to significant gains in saccharification efficiency, decreased lignin, and decreased S/G lignin ratios. In contrast to constitutive overexpression of PvMYB4, which negatively impacts switchgrass root growth, plant growth was not compromised in green tissue-expressed PvMYB4 switchgrass plants in the current study.ConclusionsEach of the newly described green tissue-specific promoters from switchgrass has utility to change cell wall biosynthesis exclusively in aboveground harvestable biomass without altering root systems. The truncated green tissue promoters are very short and should be useful for targeted expression in a number of monocots to improve shoot traits while restricting gene expression from roots. Green tissue-specific expression of PvMYB4 is an effective strategy for improvement of transgenic feedstocks.

Cloning and Functional Analysis of Green Tissue-specific Expression Promoter of Common Wild Rice(Oryza rufipogon Griff.)

Cloning and Functional Analysis of Green Tissue-specific Expression Promoter of Common Wild Rice(Oryza rufipogon Griff.)

Characterization of a strong green tissue-specific motif in rice photosystem I gene promoter Ppsak

Characterization of green tissue-specific promoters helps facilitate genetic improvement in crops. Here, we isolated a novel green tissue-specific expression gene Psak encoding photosystem I protein in rice through RT-PCR analysis. The 5′ flanking region from −635 to +60 (transcription start site of Psak as +1) covering parts of Psak gene and its promoter Ppsak was identified as be critical for green tissue-specific expression (including leaves and stems) in rice. Further promoter deletion analyses demonstrated that the promoter region from −559 to −232 was necessary and sufficient for green tissue-specific expression of Psak gene. Histochemical assays showed that the GUS expression of this 267-bp region was highly presented in leaves and stems, but reduced or absent in other tissues examined. The GUS expression level of this core promoter region could be 14.6- to 21.6-fold higher than that of the full-length region of Ppsak promoter in leaves and stems. These results combined with cis-acting elements prediction in Ppsak promoter suggested that the TCT motif located in this promoter played a key role in strong green tissue-specific expression.

Tissue-specific expression of Arabidopsis NPR1 gene in rice for sheath blight resistance without compromising phenotypic cost

Rice sheath blight disease, caused by the fungus Rhizoctonia solani, is considered the second most important disease of rice after blast. NPR1 (non expressor of PR1) is the central regulator of systemic acquired resistance (SAR) conferring broad spectrum resistance to various pathogens. Previous reports have indicated that constitutive expression of the Arabidopsis thaliana NPR1 (AtNPR1) gene results in disease resistance in rice but has a negative impact on growth and agronomic traits. Here, we report that green tissue-specific expression of AtNPR1 in rice confers resistance to the sheath blight pathogen, with no concomitant abnormalities in plant growth and yield parameters. Elevated levels of NPR1 activated the defence pathway in the transgenic plants by inducing expression of endogenous genes such as PR1b, RC24, and PR10A. Enhanced sheath blight resistance of the transgenic plants was evaluated using three different bioassay systems. A partially isolated toxin from R. solani was used in the bioassays to measure the resistance level. Studies of the phenotype and yield showed that the transgenic plants did not exhibit any kind of phenotypic imbalances. Our results demonstrate that green tissue-specific expression of AtNPR1 is an effective strategy for controlling the sheath blight pathogen. The present work in rice can be extended to other crop plants severely damaged by the pathogen.

Novel green tissue-specific synthetic promoters and cis-regulatory elements in rice.

As an important part of synthetic biology, synthetic promoter has gradually become a hotspot in current biology. The purposes of the present study were to synthesize green tissue-specific promoters and to discover green tissue-specific cis-elements. We first assembled several regulatory sequences related to tissue-specific expression in different combinations, aiming to obtain novel green tissue-specific synthetic promoters. GUS assays of the transgenic plants indicated 5 synthetic promoters showed green tissue-specific expression patterns and different expression efficiencies in various tissues. Subsequently, we scanned and counted the cis-elements in different tissue-specific promoters based on the plant cis-elements database PLACE and the rice cDNA microarray database CREP for green tissue-specific cis-element discovery, resulting in 10 potential cis-elements. The flanking sequence of one potential core element (GEAT) was predicted by bioinformatics. Then, the combination of GEAT and its flanking sequence was functionally identified with synthetic promoter. GUS assays of the transgenic plants proved its green tissue-specificity. Furthermore, the function of GEAT flanking sequence was analyzed in detail with site-directed mutagenesis. Our study provides an example for the synthesis of rice tissue-specific promoters and develops a feasible method for screening and functional identification of tissue-specific cis-elements with their flanking sequences at the genome-wide level in rice.

Characterization of the rice RbcS3 promoter and its transit peptide for use in chloroplast-targeted expression

Here, we report characterization of an RbcS promoter and its transit peptide for use in chloroplast-targeted expression. RbcS3 that encodes a small subunit of ribulose bisphosphate carboxylase/oxygenase is of great interest due to its green tissue-specific expression such as leaf and leaf sheaths. We isolated the RbcS3 promoter and its transit peptide sequence and linked to GFP for characterization in transgenic plants. Our qRT-PCR analysis on leaves and roots of transgenic plants revealed that GFP transcript levels were higher by fivefold in leaves of RbcS3:TP3:GFP transgenic lines than in those of the RbcS3:GFP transgenic lines, suggesting that the transcript levels were increased by chloroplast-targeted expression, as shown in previously characterized RbcS1:TP1 system. The transit peptide sequence TP3 was sufficient to target GFP to chloroplasts as observed in protoplasts of RbcS3:TP3:GFP transgenic lines. GFP fluorescence in RbcS3:GFP plants was detected only in the cytosol. The GFP protein levels in leaves of RbcS3:TP3:GFP plants were 0.12–0.23 % of the total soluble leaf proteins. These levels were higher or comparable to those of PGD1:GFP plants (GFP driven by a constitutive promoter PGD1). Taken together, these results indicate that the fusion of the RbcS3 promoter to its transit peptide, RbcS3:TP3, provides a chloroplast-targeting expression system.

The sweet potato RbcS gene (IbRbcS1) promoter confers high-level and green tissue-specific expression of the GUS reporter gene in transgenic Arabidopsis

Sweet potato is an important crop because of its high yield and biomass production. We herein investigated the potential of the promoter activity of a small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcS) from sweet potato (Ipomoea batatas) in order to develop the high expression system of exogenous DNA in Arabidopsis. We isolated two different cDNAs (IbRbcS1 and IbRbcS2) encoding RbcS from sweet potato. Their predicted amino acid sequences were well conserved with the mature RbcS protein of other plants. The tissue-specific expression patterns of these two genes revealed that expression of IbRbcS1 was specific to green tissue, whereas that of IbRbcS2 was non-photosynthetic tissues such as roots and tubers. These results suggested that IbRbcS1 was predominantly expressed in the green tissue-specific of sweet potato over IbRbcS2. Therefore, the IbRbcS1 promoter was transformed into Arabidopsis along with β-glucuronidase (GUS) as a reporter gene. GUS staining and semi-quantitative RT-PCR showed that the IbRbcS1 promoter conferred the expression of the GUS reporter gene in green tissue-specific and light-inducible manners. Furthermore, qPCR showed that the expression levels of GUS reporter gene in IbRbcS1 pro:GUS were same as those in CaMV 35S pro:GUS plants. These results suggest that the IbRbcS1 promoter is a potentially strong foreign gene expression system for genetic transformation in plants.

Green tissue-specific analysis of a cloned rbcS promoter from Lemna gibba

Many plant genetic engineering taskss require the spatial expression of genes which in turn depends upon the availability of specific promoters. The present paper analyses the green-tissue characteristics of a new L. gibba rbcS promoter driving the expression of the gus gene in transgenic tobacco. A 1491 bp rbcS (small subunit of ribulose bisphosphate carboxylase) promoter was isolated from Lemna gibba. The sequence analysis revealed that this promoter is different from the previously reported rbcS promoter and is named SSU5C. A 1438 bp fragment of the SSU5C promoter was fused with the gus gene and transgenic tobacco plants were generated. The analysis of T<sub>1</sub> tobacco p1438-gus revealed that GUS expression driven by the SSU5C promoter was detected in the green part of vegetative organs. The promoter deletion analysis confirmed a region from position –152 to –49 relative to the start of transcription containing boxes X, Y and Z, while a positive regulatory region conferred green tissue-specific expression. Further functional analysis of constructs of box-X, Y, Z, which was fused with the basal SSU5C promoter, confirmed that the boxes X, Y and Z represent the new minimized functional promoter, respectively, and are able to direct green tissue-specific expression. This promoter may be used for gene expression in a tissue-specific manner in plant molecular breeding.

Two novel positive cis-regulatory elements involved in green tissue-specific promoter activity in rice (Oryza sativa L ssp.)

In plant genetic engineering, using tissue-specific promoters to control the expression of target gene is an effective way to avoid potential negative effects of using constitutive promoter, such as metabolic burden and so on. However, until now, there are few tissue-specific promoters with strong and reliable expression that could be used in crop biotechnology application. In this study, based on microarray and RT-PCR data, we identified a rice green tissue-specific expression gene DX1 (LOC_Os12g33120). The expression pattern of DX1 gene promoter was examined by using the β-glucuronidase (GUS) reporter gene and analyzed in transgenic rice plants in different tissues. Histochemical assays and quantitative analyses of GUS activity confirmed that P (DX1):GUS was highly expressed in green tissues. To identify the regulatory elements controlling the expression of the DX1 gene, a series of 5' and 3' deletions of DX1 promoter were fused to GUS gene and stably introduced into rice plants. In addition, gel mobility shift assays and site-directed mutagenesis studies were used, allowing for the identification of two novel tissue-specific cis-acting elements (GSE1 and GSE2) within P(DX1). GSE1 acted as a positive regulator in all green tissues (leaf, sheath, stem and panicle). Compared with GSE1, GSE2 acted as a positive regulator only in sheath and stem tissue, and had a weaker effect on gene expression. In addition, P(DX1):GUS was not expressed in anther and seed, this characteristic reduced the potential ecological risk and potential food safety issues. Taken together, our results strongly suggest that the identified promoter, P(DX1), and its cis regulatory elements, GSE1 and GSE2, are potentially useful in the field of rice transgenic breeding. We have isolated and characterized the rice green tissue-specific promoter P(DX1), and identified two novel positive cis-acting elements in P(DX1).

TheLP2leucine‐rich repeat receptor kinase gene promoter directs organ‐specific, light‐responsive expression in transgenic rice

Biotechnologists seeking to limit gene expression to nonseed tissues of genetically engineered cereal crops have only a few choices of well characterized organ-specific promoters. We have isolated and characterized the promoter of the rice Leaf Panicle 2 gene (LP2, Os02g40240). The LP2 gene encodes a leucine-rich repeat-receptor kinase-like protein that is strongly expressed in leaves and other photosynthetic tissues. Transgenic rice plants containing an LP2 promoter-GUS::GFP bifunctional reporter gene displayed an organ-specific pattern of expression. This expression corresponded to transcript levels observed on RNA blots of various rice organs and microarray gene expression data. The strongest beta-glucuronidase activity was observed in histochemically stained mesophyll cells, but other green tissues and leaf cell types including epidermal cells also exhibited expression. Low or undetectable levels of LP2 transcript and LP2-mediated reporter gene expression were observed in roots, mature seeds, and reproductive tissues. The LP2 promoter is highly responsive to light and only weak expression was detected in etiolated rice seedlings. The specificity and strength of the LP2 promoter suggests that this promoter will be a useful control element for green tissue-specific expression in rice and potentially other plants. Organ-specific promoters like LP2 will enable precise, localized expression of transgenes in biotechnology-derived crops and limit the potential of unintended impacts on plant physiology and the environment.

Identification of potential protein regulators bound to the tissue‐specific positive and negative cis‐acting elements of a green tissue‐specific promoter in rice

Although some tissue-specific cis-acting elements have been identified, the molecular mechanisms of tissue-specific gene expression remain elusive. Here, we report the identification by a yeast one-hybrid screen of five proteins, Os10g31330/glycine-rich, Os01g10400/metallothionein-like, Os05g51180/nucleic acid-binding, Os05g37930/unknown and Os01g01689/phosphatidylinositol kinase that bound to either the positive or negative tissue-specific cis elements of a rice promoter from the green tissue-specific D54O gene. These proteins are localised in the nucleus and the genes encoding them are differentially expressed in different tissues, further suggesting their putative roles in regulating gene expression. These results suggest that the green tissue-specific expression of the D54O gene may be regulated by the interaction of multiple proteins with cis elements in the promoter region.