Expression of hybrid <Emphasis Type="Italic">cry</Emphasis>3aM–<Emphasis Type="Italic">lic</Emphasis>BM2 genes in transgenic potatoes (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>)

The full-modified Bacillus thuringiensis cry3a (cry3aM) gene was designed and synthesized for effective expression in plants. A plant expression vector pC29RBCS-leader-cry3aM–licBM2 was constructed for potato transformation. In this vector, the cry3aM sequence was fused in reading frame with a new reporter gene (licBM2) and a leader sequence for the rbcs gene. The reporter gene encoded thermostable lichenase and the leader sequence encoded a signal peptide for transporting protein product to chloroplasts. The vector contained the light-inducible promoter for rbcs gene isolated from Arabidopsis thaliana. Transgenic plants were obtained by Agrobacterium mediated transformation using microtuber explants. Transgenic plantlets were selected by kanamycin resistance and confirmed as transgenic by PCR with specific primers, evaluation of lichenase activity, and bioassay of Colorado potato beetle neonate larvae. Promoter activity assays under light induction (kinetic analysis) using lichenase activity and bioassay both showed high and stable expression of hybrid genes in transgenic plantlets. Furthermore, the presence of lichenase as a reporter protein in the composition of hybrid protein was shown to facilitate selection and analysis of the expression level of hybrid genes in transgenic plants.     

复苏植物旋蒴苣苔早期光诱导蛋白的特征分析

随着全球气候变暖,旱胁迫越来越成为粮食生产的严重威胁。探索复苏植物耐受极端旱胁迫的机理,积累抗旱相关基因资源,对于植物适应逆境的基础研究,和农业可持续发展,均有重要的作用。旋蒴苣苔(B. hygrometrica)广泛分布于我国山区,是复苏植物的代表性物种,而且有高质量的全基因组序列可以分析利用。本研究对旋蒴苣苔(B. hygrometrica)基因组中的早期光诱导蛋白(ELIPs)基因家族进行挖掘和分析。旋蒴苣苔(B. hygrometrica)基因组中早期光诱导蛋白(ELIPs)基因的数目远高于其他植物(拟南芥,水稻,辣椒,番茄)。系统发育分析显示,早期光诱导蛋白(ELIPs)基因序列具有很高的植物特异性。这说明,早期光诱导蛋白(ELIPs)基因对不同植物进化和适应各自的生长环境具有重要的作用。对处于不同程度干旱状态的旋蒴苣苔(B. hygrometrica)叶片的基因表达分析,表明一部分早期光诱导蛋白(ELIPs)基因表现出很强的对旱处理的响应。不同的早期光诱导蛋白(ELIPs)基因表现出三种不同的响应模式。这些结果表明,旋蒴苣苔(B. hygrometrica)早期光诱导蛋白(ELIPs)基因参与植株应对旱胁迫,而且有精细的调控机制控制不同早期光诱导蛋白(ELIPs)基因的表达量。     

Expression of photosynthetic genes from the C4 plant, maize, in tobacco.

Summary Previous results from this laboratory have demonstrated the presence of genes for phosphoenolpyruvate carboxylase and pyruvate, orthophosphate dikinase in C₃ plants. The structure and light-enhanced expression of these genes is very similar to that of the genes found in the C₄ plant, maize. In order to investigate whether or not the regulation of these genes is similar in C₃ and C₄ plants, we have constructed chimeric genes using -glucuronidase as a reporter gene under the control of the maize promoters of the genes for phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, and the small subunit of ribulose bisphosphate carboxylase (RuBisCO). The chimeric genes were introduced into tobacco, a C₃ plant. These genes were expressed primarily in leaf and stem tissue and the expression was enhanced by light. Thus, as in C₄ plants, the genes are expressed in a tissue-specific and light-inducible manner in the C₃ plant. Since the expression of these genes is restricted to specific cells in leaf tissue of C₄ plants, we also investigated the spatial pattern of expression of the chimeric genes using histochemical analysis of -glucuronidase activity. High level expression of all of these genes was found in mesophyll cells. This included the small subunit of RuBisCO, which is not expressed in mesophyll cells but in bundle sheath cells in C₄ plants. This report describes similarities between C₃ and C₄ plants in regulating the expression of these genes.     

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 -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-pecific 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.     

The promoters of two carboxylases in a C4 plant (maize) direct cell-specific, light-regulated expression in a C3 plant (rice)

C₄ plants have two carboxylases which function in photosynthesis. One, phosphoenolpyruvate carboxylase (PEPC) is localized in mesophyll cells, and the other, ribulose bisphosphate carboxylase (RuBPC) is found in bundle sheath cells. In contrast, C₃ plants have only one photosynthetic carboxylase, RuBPC, which is localized in mesophyll cells. The expression of PEPC in C₃ mesophyll cells is quite low relative to PEPC expression in C₄ mesophyll cells. Two chimeric genes have been constructed consisting of the structural gene encoding β-glucuronidase (GUS) controlled by two promoters from C₄ (maize) photosynthetic genes: (i) the PEPC gene (pepc) and (ii) the small subunit of RuBPC (rbcS). These constructs were introduced into a C₃ cereal, rice. Both chimeric genes were expressed almost exclusively in mesophyll cells in the leaf blades and leaf sheaths at high levels, and no or very little activity was observed in other cells. The expression of both genes was also regulated by light. These observations indicate that the regulation systems which direct cell-specific and light-inducible expression of pepc and rbcS in C₄ plants are also present in C₃ plants. Nevertheless, expression of endogenous pepc in C₃ plants is very low in C₃ mesophyll cells, and the cell specificity of rbcS expression in C₃ plants differs from that in C₄ plants. Rice nuclear extracts were assayed for DNA-binding protein(s) which interact with a cis-regulatory element in the pepc promoter. Gel-retardation assays indicate that a nuclear protein with similar DNA-binding specificity to a maize nuclear protein is present in rice. The possibility that differences in pepc expression in a C₃ plant (rice) and C₄ plant (maize) may be the result of changes in cis-acting elements between pepc in rice and maize is discussed. It also appears that differences in the cellular localization of rbcS expression are probably due to changes in a trans-acting factor(s) required for rbcS expression.     

Light-induced expression of ipt from Agrobacterium tumefaciens results in cytokinin accumulation and osmotic stress symptoms in transgenic tobacco

Cytokinins are plant growth regulators that induce shoot formation, inhibit senescence and root growth. Experiments with hydroponically grown tobacco plants, however, indicated that exogenously applied cytokinin led to the accumulation of proline and osmotin. These responses were also associated with environmental stress reactions, such as salt stress, in many plant species. To test whether increased endogenous cytokinin accumulation led to NaCl stress symptoms, the gene ipt from Agrobacterium tumefaciens, encoding isopentenyl transferase, was transformed into Nicotiana tabacum cv. SR-1 under the control of the light-inducible rbcS-3A promoter from pea. In high light (300 mol PPFD m^-2 s^-1), ipt mRNA was detected and zeatin/zeatin glucoside levels were 10-fold higher than in control plants or when transformants were grown in low light (30 mol PPFD m^-2 s^-1). High light treatment was accompanied by increased levels of proline and osmotin when compared to low light grown transformed and untransformed control plants. Elevated in planta cytokinin levels induced responses also stimulated by salt stress, suggesting either common or overlapping signaling pathways are initiated independently by cytokinin and NaCl, setting in motion gene expression normally elicited by developmental processes such as flowering or environmental stress.Abbreviations IPT isopentenyl, transferase - rbcS-3A gene encoding a small subunit protein (SSU) of Rubisco from Pisum sativum - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

Agrobacterium-mediated transformation of Amaranthus hypochondriacus: light- and tissue-specific expression of a pea chlorophyll a/b-binding protein promoter

Mature embryos of Amaranthus hypochondriacus (amaranth) were used to develop an in vitro culture system for plant regeneration and genetic transformation. Plants were regenerated from embryo-derived callus cultivated on Murashige and Skoog medium supplemented with 10 μM 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-2-methoxybenzoic acid and 10% coconut liquid endosperm. Transgenic plants were obtained by inoculation of mature embryo explants with a disarmed Agrobacterium strain containing the plasmid pGV2260(pEsc4), which carried the genes encoding neomycin phosphotransferase type II and β-glucuronidase. The presence of transgenes in the genome of transformed amaranth plants and their progeny was demonstrated by Southern blot hybridization. Tissue specific and light-inducible expression directed by a pea chlorophyll a/b-binding protein promoter was observed in transgenic amaranth plants and their progeny. Received: 30 December 1996 / Revision received: 14 May 1997 / Accepted: 3 June 1997     

Towards Arabidopsis genome analysis: monitoring expression profiles of 1400 genes using cDNA microarrays

cDNA microarrays containing 1443 Arabidopsis thaliana genes were analyzed for expression profiles in major organs of Arabidopsis plants. Novel expression profiles were identified for many coding sequences with putative gene identifications. Expression patterns of novel sequences provided clues to their possible functions. The results demonstrate how microarrays containing a large number of Arabidopsis genes can provide a powerful tool for plant gene discovery, functional analysis and elucidation of genetic regulatory networks.     

Role of calcium ions in photosignaling processes in a plant cell

Ca²⁺ is an important structural and functional component of plant cells. During the last decade, Ca²⁺ attracted attention as a secondary messenger in signaling processes in plants to mediate the action of abiotic and biotic signals including light. The structural basis for Ca²⁺ signaling in plants, the generation of Ca²⁺ signatures and the nature of Ca²⁺ sensors are considered in relation to the functioning of plant photo-receptors phytochromes, cryptochromes, and phototropins. Special attention is focused upon genetic factors controlling the expression of light-inducible genes being closely related to above photoreceptors. The analysis of the achievements in the field of plant photoreceptor signal transduction and suggestions of some prospects for the future research were done.     

Regulation of per and cry Genes Reveals a Central Role for the D-Box Enhancer in Light-Dependent Gene Expression

Light serves as a key environmental signal for synchronizing the circadian clock with the day night cycle. The zebrafish represents an attractive model for exploring how light influences the vertebrate clock mechanism. Direct illumination of most fish tissues and cell lines induces expression of a broad range of genes including DNA repair, stress response and key clock genes. We have previously identified D- and E-box elements within the promoter of the zebrafish per2 gene that together direct light-induced gene expression. However, is the combined regulation by E- and D-boxes a general feature for all light-induced gene expression? We have tackled this question by examining the regulation of additional light-inducible genes. Our results demonstrate that with the exception of per2, all other genes tested are not induced by light upon blocking of de novo protein synthesis. We reveal that a single D-box serves as the principal light responsive element within the cry1a promoter. Furthermore, upon inhibition of protein synthesis D-box mediated gene expression is abolished while the E-box confers light driven activation as observed in the per2 gene. Given the existence of different photoreceptors in fish cells, our results implicate the D-box enhancer as a general convergence point for light driven signaling.     

Editing of an effector gene promoter sequence impacts plant‐Phytophthora interaction

Pathogen avirulence (Avr) effectors interplay with corresponding plant resistance (R) proteins and activate robust plant immune responses. Although the expression pattern of Avr genes has been tied to their functions for a long time, it is still not clear how Avr gene expression patterns impact plant‐microbe interactions. Here, we selected PsAvr3b, which shows a typical effector gene expression pattern from a soybean root pathogen Phytophthora sojae. To modulate gene expression, we engineered PsAvr3b promoter sequences by in situ substitution with promoter sequences from Actin (constitutive expression), PsXEG1 (early expression), and PsNLP1 (later expression) using the CRISPR/Cas9. PsAvr3b driven by different promoters resulted in distinct expression levels across all the tested infection time points. Importantly, those mutants with low PsAvr3b expression successfully colonized soybean plants carrying the cognate R gene Rps3b. To dissect the difference in plant responses to the PsAvr3b expression level, we conducted RNA‐sequencing of different infection samples at 24 h postinfection and found soybean immune genes, including a few previously unknown genes that are associated with resistance. Our study highlights that fine‐tuning in Avr gene expression impacts the compatibility of plant disease and provides clues to improve crop resistance in disease control management.     

Identification of genes regulated by dark adaptation and far‐red light illumination in roots of Arabidopsis thaliana*

Roots in the soil are illuminated by far‐red (FR) light passed through plant tissues in the daytime, and are in complete darkness at night. To evaluate whether gene expression of roots is affected by a dark‐FR light cycle, gene expression profiles were analysed for dark‐adapted versus light‐grown plants and for FR light‐illuminated versus dark‐adapted plants using the RIKEN Arabidopsis full‐length cDNA microarray (containing approximately 7000 independent, full‐length cDNA groups). Among candidate dark‐ and FR‐regulated genes, several were further analysed. Eleven dark‐inducible and five dark‐repressed genes were characterized. Almost all the dark‐inducible and –repressed genes were oppositely regulated by FR light illumination. The functions of dark‐ and FR‐responsive genes and the significance of FR light‐regulated gene expression in roots under ground are discussed.     

Identification of differentially expressed genes induced by <Emphasis Type="Italic">Bamboo mosaic virus</Emphasis> infection in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>by cDNA-amplified fragment length polymorphism

Background  

The genes of plants can be up- or down-regulated during viral infection to influence the replication of viruses. Identification of these differentially expressed genes could shed light on the defense systems employed by plants and the mechanisms involved in the adaption of viruses to plant cells. Differential gene expression in Nicotiana benthamiana plants in response to infection with Bamboo mosaic virus (BaMV) was revealed using cDNA-amplified fragment length polymorphism (AFLP).     

Heavy metal stress-inducible early light-inducible gene CaELIP from hot pepper (Capsicum annuum) shows broad expression patterns under various abiotic stresses and circadian rhythmicity

An early light-inducible protein gene (CaELIP) was isolated from a cDNA library of hot pepper (Capsicum annuum) that showed heavy metal stress-inducible expressions. This gene contains an open reading frame (ORF) encoding a protein of 160 amino acids, and the protein has significant homology with reported early light-inducible proteins from other plant species. Topology analysis for CaELIP suggested three transmembrane domains. Genomic DNA blot analysis showed that CaELIP is a single copy gene in hot pepper. The treatment of seedling roots of hot pepper with Cu induced ROS generation in the root, and the level of ROS generation was paralleled to the concentration of Cu that again was matched to the increase in the CaELIP expression level. Results suggested that expression of CaELIP can be induced by the ROS generated by the excessive Cu in the plant. Exogenous SA treatment significantly alleviated Cu-induced expression of CaELIP, while exogenous JA treatment aggravated expression of CaELIP under Cu stress. CaELIP showed a transient expression when exposing the plant to light for 1 h. CaELIP also showed an endogenous circadian rhythmicity with high expression level in the morning and decreased expression level thereafter. The expression of CaELIP was also induced by high or low temperature, high salinity, drought, and stress hormone ABA. Taken together, the results suggest that CaELIP would function in responding to environmental signals and possibly regulating the response to the abiotic stresses that can be related to the abiotic stress tolerance in plants.     

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.     

Genome-wide analysis of the family of light-harvesting chlorophyll a/b-binding proteins in arabidopsis and rice

Light-harvesting antenna system possesses an inherent property of photoprotection. The single-helix proteins found in cyanobacteria play role in photoprotection and/or pigment metabolism. The photoprotective functions are also manifested by the two- and four-helix proteins. The photoprotection mechanism evolved earlier to the mechanism of light-harvesting of the antenna complex. Here, the light-harvesting complex genes of photosystems I and II from Arabidopsis are enlisted, and almost similar set of genes are identified in rice. Also, the three-helix early light-inducible proteins (ELIPs), two-helix stress-enhanced proteins (SEPs) and one-helix high light-inducible proteins [one-helix proteins (OHPs)] are identified in rice. Interestingly, two independent genomic loci encoding PsbS protein are also identified with implications on additional mode of non-photochemical quenching (NPQ) mechanism in rice. A few additional LHC-related genes are also identified in rice (LOC_Os09g12540, LOC_Os02g03330). This is the first report of identification of light-harvesting complex genes and light-inducible genes in rice.Key words: Lhca and Lhcb proteins, Lhc proteins evolution, light-inducible proteins, protein alignment, PsbSThe light-harvesting proteins are present in different taxa. The proteins of light-harvesting systems from higher plants, cyano-bacteria, purple bacteria and green sulphur bacteria share no sequence similarity however little structural similarity can be seen.¹ Apparently, the light-harvesting systems in these different taxa might have evolved independently from each other.¹ To enable efficient transfer of excitation energy into the reaction centers, where charge separation takes place, different proteins are recruited in order to coordinate the photosynthetic pigment molecules. The light-harvesting and light dissipation are tightly coupled processes involving the higher plant light-harvesting antenna. Here, genome-wide analysis of the light-harvesting chlorophyll a/b-binding proteins and light-inducible proteins in Arabidopsis thaliana L. and Oryza sativa L. (rice) is conducted. This study wherein genes coding for antenna proteins are identified and named can be used as a nomenclature guide to the light-harvesting complex gene family members and their relatives in rice.