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Tobacco genes induced by the bacterial effector protein AvrPto.

The type III effector protein AvrPto acts as a virulence factor in susceptible plants lacking a cognate resistance gene but triggers hypersensitive response and disease resistance in tomato plants carrying the Pto gene or in tobacco plants carrying an unknown resistance gene. To assist the characterization of cellular responses caused by AvrPto in the plant, a pathogen-free system was adopted to isolate genes up-regulated 12 h after induced expression of AvrPto. By using subtraction cloning and transgenic tobacco plants expressing avrPto as a transgene, we isolated 125 nonredundant cDNA clones that represent avrPto-response genes (ARG). In addition to genes that are known to be induced by Pto-avrPto recognition, a number of new genes were also isolated. Most of ARG showed a specific induction in tobacco plants challenged with incompatible or nonhost pathogens. The use of an avrPto mutant that selectively eliminated the avrPto recognition in tobacco demonstrated that the ARG were induced in a highly specific manner by the avirulence, instead of the virulence activity of avrPto.

Bacterial Proteins↗

A reporter gene system used to study developmental expression of alternative oxidase and isolate mitochondrial retrograde regulation mutants in Arabidopsis.

Perturbation of mitochondrial function causes altered nuclear gene expression in plants. To study this response, called mitochondrial retrograde regulation, and developmental gene expression, a transgenic Arabidopsis thaliana (Col-0) line containing a firefly luciferase gene controlled by a promoter region of the Arabidopsis alternative oxidase 1a gene (AtAOX1a) was created. The transgene and the endogenous gene were developmentally induced in young cotyledons to a level higher than in older cotyledons and leaves. Analysis of transgene expression suggests that this is true for emerging leaves as well. Antimycin A (AA), a mitochondrial electron transport chain inhibitor, and monofluroacetate (MFA), a TCA cycle inhibitor, induced expression of the transgene and the endogenous gene in parallel. The following comparative responses of the transgene to inhibitors were observed: (a) the response in cotyledons to AA treatment differed greatly in magnitude from the response in leaves; (b) the induction kinetics in cotyledons following MFA treatment differed greatly from the kinetics in leaves; and (c) the induction kinetics following MFA treatment differed from the kinetics of AA in both leaves and cotyledons. The transgenic line was used in a genetic screen to isolate mutants with greatly decreased transgene and AtAOX1a induction in response to AA. Some of these mutant lines showed greatly decreased induction by MFA, but one did not. Taken altogether, the data provide genetic evidence that suggests that induction of the AtAOX1a gene by distinct mitochondrial perturbations are via distinct, but overlapping signaling pathways that are tissue specific.

Antimycin A↗

Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression.

The Arabidopsis NPR1 gene is a positive regulator of inducible plant disease resistance. Expression of NPR1 is induced by pathogen infection or treatment with defense-inducing compounds such as salicylic acid (SA). Transgenic plants overexpressing NPR1 exhibit enhanced resistance to a broad spectrum of microbial pathogens, whereas plants underexpressing the gene are more susceptible to pathogen infection. These results suggest that regulation of NPR1 gene expression is important for the activation of plant defense responses. In the present study, we report the identification of W-box sequences in the promoter region of the NPR1 gene that are recognized specifically by SA-induced WRKY DNA binding proteins from Arabidopsis. Mutations in these W-box sequences abolished their recognition by WRKY DNA binding proteins, rendered the promoter unable to activate a downstream reporter gene, and compromised the ability of NPR1 to complement npr1 mutants for SA-induced defense gene expression and disease resistance. These results provide strong evidence that certain WRKY genes act upstream of NPR1 and positively regulate its expression during the activation of plant defense responses. Consistent with this model, we found that SA-induced expression of a number of WRKY genes was independent of NPR1.

Arabidopsis↗

Auxin regulated gene expression and gravitropism in plants.

Transcription of two gene families, SAURs and GH3, in soybean has been shown to be specifically induced by the plant hormone auxin. The SAUR mRNAs have been shown to accumulate on the lower half and disappear from the upper half of soybean hypocotyls during gravitropic curvature. The SAUR and GH3 promoters have been fused to the beta-glucuronidase (GUS) reporter gene and shown to be specifically induced by auxins in transgenic tobacco plants. Histochemical staining and quantitative GUS assays indicate that these auxin inducible promoters are activated on the lower half of transgenic tobacco stems undergoing gravitropic curvature. The auxin transport inhibitors, TIBA and NPA, block both gravitropic curvature and the activation of the auxin responsive promoters in transgenic tobacco stems. The auxin responsive elements (AuxREs) within the SAUR and GH3 promoters have been identified, and these AuxREs are likely to be the elements that are responsible for the increased expression of the SAUR and GH3 genes during gravitropism.

Base Sequence↗

Rice lipid transfer protein (LTP) genes belong to a complex multigene family and are differentially regulated.

Several cDNA clones encoding three different lipid transfer proteins (LTPs) have been isolated from rice (Oryza sativa L.) in order to analyse the complexity, the evolution and the expression of the LTP gene family. The mature proteins deduced from three clones exhibited a molecular mass of 9 kDa, in agreement with the molecular mass of other LTPs from plants. The clones were shown to be homologous in the coding region, while the 3' non-coding regions diverged strongly between the clones. The occurrence of at least three small multigene families encoding these proteins in rice was confirmed by Southern blot analysis. When compared with each other and with LTPs from other plants, the cluster including rice LTPs and other cereal LTPs indicated that these genes duplicated rather recently and independently in the different plant phyla. The expression pattern of each gene family was also investigated. Northern blot experiments demonstrated that they are differentially regulated in the different tissues analysed. Components such as salt, salicylic acid and abscisic acid were shown to modulate Ltp gene expression, depending on tissues and gene classes, suggesting a complex regulation of these genes.

Abscisic Acid↗

Mutations in LIKE HETEROCHROMATIN PROTEIN 1 affect flowering time and plant architecture in Arabidopsis.

In plants, recent studies have demonstrated links between the regulation of developmental processes and chromatin dynamics and organisation. Analysis of new mutations affecting overall plant architecture, leaf development and flowering time in Arabidopsis has allowed us to clone and characterise LHP1, the Drosophila heterochromatin protein 1 (HP1) homologue. LHP1 has the chromo and chromo shadow domains central to the function of animal proteins. Yeast two hybrid studies and in planta deletion experiments suggest similar modes of action in plants and animals via homodimer formation. In vivo localisation experiments revealed a specific subnuclear protein distribution in foci throughout the nucleus. Our data suggest that LHP1 may act as a main regulator of gene expression in plants, through formation of heterochromatin-like repressive complexes, to control developmental pathways involved in organ and cell size, and the vegetative to reproductive phase transition.

Amino Acid Sequence↗

Two PR-1 genes from tomato are differentially regulated and reveal a novel mode of expression for a pathogenesis-related gene during the hypersensitive response and development.

Pathogenesis-related (PR) proteins form a heterogeneous family of plant proteins that are likely to be involved in defense and are inducible by pathogen attacks. One group of PRs, represented by the subfamily PR-1, are low-molecular-weight proteins of unknown biochemical function. Here we describe the cloning and characterization of two closely related genes encoding a basic and an acidic PR-1 protein (PR1b1 and PR1a2) from tomato (Lycopersicon esculentum). We present a comparative study of the mode of transcriptional regulation of these two genes in transgenic tobacco plants using a series of promoter-GUS fusions. Unexpectedly, the chimeric PR1a2/GUS gene is not induced by pathogenic signals but instead shows constitutive expression with a reproducible developmental expression pattern. It is expressed in shoot meristems, trichomes, and cortical cells as well as in vascular and nearby tissues of the mature stem. This constitutive expression pattern may represent preemption of plant defenses against potential pathogens. Conversely, the chimeric PR1b1/GUS gene does not show any constitutive expression in the plant, but it is transcriptionally activated following pathogen attack. Upon infection by tobacco mosaic virus, the PR1b1 gene is strongly activated locally in tissues undergoing the hypersensitive response but not systemically in uninoculated tissues. Furthermore, its expression is induced by both salicylic acid and ethylene precursors, two signals that coexist and apparently mediate the activation of local defenses during the hypersensitive response. We speculate that the different mode of expression of the two genes presented here, together with that reported previously for the induction of other PR-1 genes in systemic, uninoculated tissues, may all be complementary and necessary for the plant to acquire an efficient refractory state to resist pathogen attacks.

Amino Acid Sequence↗

Characterization of the structure and expression of a highly conserved ribosomal protein gene, L9, from pea.

The eukaryotic ribosomal protein (RP) L9 is highly conserved in nature, and its gene is expressed to high levels in the actively growing tissues of pea. The transcriptional activity of the gene is highest in root, cambial and shoot meristems and immature tissues of the plant. Promoter deletion analysis using constructs employing the reporter gene gus were stably transferred into tobacco and revealed that the fully functional promoter is found in the first 316bp upstream from the start codon. Transgenic pea plants carrying one of these constructs show that translational efficiency mirrors gene transcription; gene expression appears to be developmentally regulated at the level of transcription. The coding region of the gene shares 80% amino acid homology with Arabidopsis and 76% homology with rice. Comparisons of the gene structure to that of the human, fruit fly, yeast, and Arabidopsis homologues reveal a close relationship in both promoter structure and intron insertion sites with the Arabidopsis gene. A nucleotide sequence alignment of the pea gene with other plant RP genes revealed that a sequence, -TTAGGGTTTT-, was commonly found in the forward and/or the inverted orientation at or near the TATA boxes of the promoters of these genes and may have a role in regulating the coordinate production of the RP genes in plants.

Amino Acid Sequence↗

Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis.

To investigate the regulation of gene expression during male gametophyte development, we analyzed the promoter activity of two different genes (LAT52 and LAT59) from tomato, isolated on the basis of their anther-specific expression. In transgenic tomato, tobacco and Arabidopsis plants containing the LAT52 promoter region fused to the beta-glucuronidase (GUS) gene, GUS activity was restricted to pollen. Transgenic tomato, tobacco and Arabidopsis plants containing the LAT59 promoter region fused to GUS also showed very high levels of GUS activity in pollen. However, low levels of expression of the LAT59 promoter construct were also detected in seeds and roots. With both constructs, the appearance of GUS activity in developing anthers was correlated with the onset of microspore mitosis and increased progressively until anthesis (pollen shed). Our results demonstrate co-ordinate regulation of the LAT52 and LAT59 promoters in developing microspores and suggest that the mechanisms that regulate pollen-specific gene expression are evolutionarily conserved.

Chimera↗

Anthocyanin regulatory mutations in pea: effects on gene expression and complementation by R-like genes of maize.

Anthocyanin production in higher plants is a function of the tissue considered and its developmental stage, and is modulated by environmental factors. In maize, the best characterized system, regulation of the pathway is achieved largely through the action of proteins with homology to the transcriptional factors encoded by myc and myb proto-oncogenes of animals; these homologues control the expression of structural genes and thus regulate the availability of anthocyanin biosynthetic enzymes. We have studied anthocyanin biosynthesis and its regulation in flowers of pea (Pisum sativum). Our results demonstrate a correlation between anthocyanin accumulation and steady-state mRNA levels for genes encoding chalcone synthase, flavanone 3 beta-hydroxylase, and dihydroflavonol 4-reductase in developing flowers. Patterns of expression for these biosynthetic genes in both a and a2 mutants confirm the regulatory roles of the two a loci. The reduced expression of all three biosynthetic genes in mutant lines suggests that genes acting both early and late in the anthocyanin biosynthetic pathway are controlled by a and a2. Particle bombardment of floral tissue demonstrates the ability of two maize R-like genes, Lc and R-S, but neither myb-like genes nor R-like genes from snapdragon or petunia, functionally to complement a and a2 mutations. We cannot distinguish whether a and a2 act coordinately or sequentially in anthocyanin regulation, but the epistatic action of maize R-like genes suggests that they mimic the action of a gene that normally functions downstream of both a and a2 in the regulatory cascade.

Acyltransferases↗

Dual regulation of a heat shock promoter during embryogenesis: stage-dependent role of heat shock elements.

Transgenic tobacco expression was analysed of chimeric genes with point mutations in the heat shock element (HSE) arrays of a small heat shock protein (sHSP) gene from sunflower: Ha hsp17.7 G4. The promoter was developmentally regulated during zygotic embryogenesis and responded to heat stress in vegetative tissues. Mutations in the HSE affected nucleotides crucial for human heat shock transcription factor 1 (HSF1) binding. They abolished the heat shock response of Ha hsp17.7 G4 and produced expression changes that demonstrated dual regulation of this promoter during embryogenesis. Thus, whereas activation of the chimeric genes during early maturation stages did not require intact HSE, expression at later desiccation stages was reduced by mutations in both the proximal (-57 to -89) and distal (-99 to -121) HSE. In contrast, two point mutations in the proximal HSE that did not severely affect gene expression during zygotic embryogenesis, eliminated the heat shock response of the same chimeric gene in vegetative organs. Therefore, by site-directed mutagenesis, it was possible to separate the heat shock response of Ha hsp17.7 G4 from its developmental regulation. The results indicate the co-existence, in a single promoter, of HSF-dependent and -independent regulation mechanisms that would control sHSP gene expression at different stages during plant embryogenesis.

Base Sequence↗

Transcriptome Analysis Reveals Key Drought-Stress-Responsive Genes in Two Bermudagrass Genotypes.

Drought inhibits grass development and survival. However, molecular-based studies on drought tolerance mechanisms in bermudagrass (Cynodon dactylon) remain scarce. Therefore, a drought-resistant bermudagrass (Tianshui) and a drought-sensitive (Zhengzhou) genotype were selected and subjected to 28 days of 50% drought stress. Leaves were sampled for RNA sequencing. Under drought stress, 2410 differentially expressed genes (DEGs) were discovered in which 1214 were upregulated (tolerant vs. sensitive) and 1196 downregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated that these specific DEGs are notably present in hormonal signal transduction pathways, flavonoids biogenesis, carbohydrate metabolic processes, abscisic acid-mediated pathways, MAPK signaling, and gluconeogenesis. Additionally, the plant hormone signal transduction pathway is predominantly linked to abscisic acid signal transduction, and many other plant hormones were also drought-responsive. The study specifically targeted genes associated with the antioxidant enzyme system, with a particular emphasis on responsive TFs such as MYB, bHLH, bZIP, GRAS, and WRKY. This study establishes the theoretical framework and identifies gene sources for the genetic enhancement and breeding of bermudagrass in the future.

Cynodon↗

Molecular cloning, structure and expression of an elicitor-inducible chitinase gene from pine trees.

We have cloned, sequenced, and examined the expression of genes from pine trees that appear to encode extracellular class II chitinase. Nucleotide sequence analysis indicates a coding sequence composed of three exons interrupted by two introns at locations identical to those found in other chitinase genes that possess introns. One of the genes, Pschi4, potentially encodes a protein that shares 62% amino acid sequence identity through the catalytic domain with class II chitinase from tobacco. In contrast, Pschi1 contains a stop codon in the first exon and may be a pseudogene. Pschi4 genes are conserved in several species of pine, and appear to comprise a small multigene family. Treatment of pine cell suspension cultures with the general elicitor chitosan induced Pschi4 expression. The regulatory sequences associated with the Pschi4 gene were sufficient to direct chitosan-inducible expression of Pschi4 in transgenic tobacco plants, which indicates that Pschi4 is an actively expressed member of the multigene family. The observation that the Pschi4 gene from pine (a gymnosperm) was appropriately regulated by chitosan in tobacco (an angiosperm) suggests that the signaling pathways that mediate chitosan-induced transcription are highly conserved in the plant kingdom.

Amino Acid Sequence↗

Tissue-specific and developmental pattern of expression of the rice sps1 gene.

Sucrose-phosphate synthase (SPS) is one of the key regulatory enzymes in carbon assimilation and partitioning in plants. SPS plays a central role in the production of sucrose in photosynthetic cells and in the conversion of starch or fatty acids into sucrose in germinating seeds. To explore the mechanisms that regulate the tissue-specific and developmental distribution of SPS, the expression pattern of rice (Oryza sativa) sps1 (GenBank accession no. U33175) was examined by in situ reverse transcriptase-polymerase chain reaction and the expression directed by the sps1 promoter using the beta-glucuronidase reporter gene. It was found that the expression of the rice sps1 gene is limited to mesophyll cells in leaves, the scutellum of germinating seedlings, and pollen of immature inflorescences. During leaf development, the sps1 promoter directs a basipetal pattern of expression that coincides with the distribution of SPS activity during the leaf sink-to-source transition. It was also found that during the vegetative part of the growth cycle, SPS expression and enzymatic activity are highest in the youngest fully expanded leaf. Additionally, it was observed that the expression of the sps1 promoter is regulated by light and dependent on plastid development in photosynthetic tissues, whereas expression in scutellum is independent of both light and plastid development.

Base Sequence↗

Cytokinin-induced abnormal shoot organogenesis is associated with elevated Knotted1-type homeobox gene expression in tobacco.

The molecular mechanisms that regulate the transcription of key developmental genes involved in shoot organogenesis have yet to be fully elucidated. However, it is clear that plant growth regulators, such as cytokinin, play a critical role in the differentiation of adventitious shoots. In Nicotiana tabacum zz100 leaf discs, high frequency shoot formation could be induced with 5 microM of the cytokinin N6-benzyladenine (BA). Increasing the exogenous BA concentration to greater than 20 microM resulted in stunted explants with abnormal shoot morphology and altered mineral composition. Explants with abnormal shoots did not appear to be hyperhydric. Abnormalities were, however, associated with an increase in the expression of a knotted1-type homeobox gene (TobH1) isolated from normal shoot-forming cultures. The results suggest that the development of cytokinin-induced abnormal shoot morphology possibly involves changes in TobH1 gene expression.

Adenine↗

Expression of a novel NAC domain-containing transcription factor (CaNAC1) is preferentially associated with incompatible interactions between chili pepper and pathogens.

We aim to isolate genes in chili pepper that are regulated during the hypersensitive response to infection by nonhost pathogens, with a view to elucidating the defense responses against pathogen attack. Among the 90 transcription factors initially characterized by reverse RNA gel blot analysis, a cDNA clone, CaNAC1 (Capsicum annuum NAC1) containing the plant-specific NAC domain motif was further characterized. Expression of the CaNAC1 gene was rapidly and specifically induced during incompatible interactions between pepper and bacterial or viral pathogens. Additionally, this gene was strongly induced by exogenously applied salicylic acid and ethephon, whereas methyl jasmonate only had a transient effect. A CaNAC1-smGFP (soluble modified green fluorescent protein) fusion protein localized to the nucleus following transfection into the epidermis of onion. Using the yeast system, we further disclose that the transcription activation domain of CaNAC1 is located in the C-terminal half of the protein. Our results collectively suggest that the plant-specific NAC domain protein, CaNAC1, may play a role in the regulation of defense responses in plants.

Amino Acid Sequence↗

Overexpression of PRE1 and its homologous genes activates Gibberellin-dependent responses in Arabidopsis thaliana.

Gibberellins control various aspects of growth and development. Here, we identified a gene, designated paclobutrazol resistance1 (PRE1), by screening Arabidopsis activation-tagged lines. PRE1 encodes a helix-loop-helix protein and belongs to a small gene family. Physiological and genetic analysis indicated that overexpression of PRE1 altered various aspects of gibberellin-dependent responses such as germination, elongation of hypocotyl/petiole, floral induction and fruit development, and suppressed gibberellin-deficient phenotypes of the ga2 mutant. Expression of some gibberellin-responsive genes was also affected by PRE1. Expression of PRE1 was shown to be early gibberellin inducible in the wild-type plants and under control of SPY and GAI, upstream negative regulators of gibberellin signaling. The shortened hypocotyl length phenotype of the gai-1 mutant was suppressed by PRE1 overexpression. Ectopic overexpression of each of the four PRE1-related genes conferred pleiotropic phenotypes similar to PRE1 overexpression, indicative of overlapping functions among the PRE gene family. Our results of gain-of-function studies suggest that PRE genes may have a regulatory role in gibberellin-dependent development in Arabidopsis thaliana.

Arabidopsis↗