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Showing papers in "The Plant Cell in 2003"


Journal ArticleDOI
TL;DR: Results indicate that both AtMYC2 and AtMYB2 proteins function as transcriptional activators in ABA-inducible gene expression under drought stress in plants.
Abstract: In Arabidopsis, the induction of a dehydration-responsive gene, rd22, is mediated by abscisic acid (ABA). We reported previously that MYC and MYB recognition sites in the rd22 promoter region function as cis-acting elements in the drought- and ABA-induced gene expression of rd22. bHLH- and MYB-related transcription factors, rd22BP1 (renamed AtMYC2) and AtMYB2, interact specifically with the MYC and MYB recognition sites, respectively, in vitro and activate the transcription of the β-glucuronidase reporter gene driven by the MYC and MYB recognition sites in Arabidopsis leaf protoplasts. Here, we show that transgenic plants overexpressing AtMYC2 and/or AtMYB2 cDNAs have higher sensitivity to ABA. The ABA-induced gene expression of rd22 and AtADH1 was enhanced in these transgenic plants. Microarray analysis of the transgenic plants overexpressing both AtMYC2 and AtMYB2 cDNAs revealed that several ABA-inducible genes also are upregulated in the transgenic plants. By contrast, a Ds insertion mutant of the AtMYC2 gene was less sensitive to ABA and showed significantly decreased ABA-induced gene expression of rd22 and AtADH1. These results indicate that both AtMYC2 and AtMYB2 proteins function as transcriptional activators in ABA-inducible gene expression under drought stress in plants.

1,931 citations


Journal ArticleDOI
Milo Aukerman1, Hajime Sakai1
TL;DR: It is demonstrated that miRNA 172 (miR172) causes early flowering and disrupts the specification of floral organ identity when overexpressed in Arabidopsis through an activation-tagging approach, supporting the notion that miR172 regulates flowering time by downregulating AP2-like target genes.
Abstract: MicroRNAs (miRNAs) are ∼21-nucleotide noncoding RNAs that have been identified in both animals and plants. Although in animals there is direct evidence implicating particular miRNAs in the control of developmental timing, to date it is not known whether plant miRNAs also play a role in regulating temporal transitions. Through an activation-tagging approach, we demonstrate that miRNA 172 (miR172) causes early flowering and disrupts the specification of floral organ identity when overexpressed in Arabidopsis. miR172 normally is expressed in a temporal manner, consistent with its proposed role in flowering time control. The regulatory target of miR172 is a subfamily of APETALA2 (AP2) transcription factor genes. We present evidence that miR172 downregulates these target genes by a translational mechanism rather than by RNA cleavage. Gain-of-function and loss-of-function analyses indicate that two of the AP2-like target genes normally act as floral repressors, supporting the notion that miR172 regulates flowering time by downregulating AP2-like target genes.

1,848 citations


Journal ArticleDOI
TL;DR: The observed diversity of these NBS-LRR proteins indicates the variety of recognition molecules available in an individual genotype to detect diverse biotic challenges.
Abstract: The Arabidopsis genome contains ∼200 genes that encode proteins with similarity to the nucleotide binding site and other domains characteristic of plant resistance proteins. Through a reiterative process of sequence analysis and reannotation, we identified 149 NBS-LRR–encoding genes in the Arabidopsis (ecotype Columbia) genomic sequence. Fifty-six of these genes were corrected from earlier annotations. At least 12 are predicted to be pseudogenes. As described previously, two distinct groups of sequences were identified: those that encoded an N-terminal domain with Toll/Interleukin-1 Receptor homology (TIR-NBS-LRR, or TNL), and those that encoded an N-terminal coiled-coil motif (CC-NBS-LRR, or CNL). The encoded proteins are distinct from the 58 predicted adapter proteins in the previously described TIR-X, TIR-NBS, and CC-NBS groups. Classification based on protein domains, intron positions, sequence conservation, and genome distribution defined four subgroups of CNL proteins, eight subgroups of TNL proteins, and a pair of divergent NL proteins that lack a defined N-terminal motif. CNL proteins generally were encoded in single exons, although two subclasses were identified that contained introns in unique positions. TNL proteins were encoded in modular exons, with conserved intron positions separating distinct protein domains. Conserved motifs were identified in the LRRs of both CNL and TNL proteins. In contrast to CNL proteins, TNL proteins contained large and variable C-terminal domains. The extant distribution and diversity of the NBS-LRR sequences has been generated by extensive duplication and ectopic rearrangements that involved segmental duplications as well as microscale events. The observed diversity of these NBS-LRR proteins indicates the variety of recognition molecules available in an individual genotype to detect diverse biotic challenges.

1,503 citations


Journal ArticleDOI
TL;DR: The results are consistent with the hypothesis that cytokinins have central, but opposite, regulatory functions in root and shoot meristems and indicate that a fine-tuned control of catabolism plays an important role in ensuring the proper regulation of cytokinin functions.
Abstract: Cytokinins are hormones that regulate cell division and development. As a result of a lack of specific mutants and biochemical tools, it has not been possible to study the consequences of cytokinin deficiency. Cytokinin-deficient plants are expected to yield information about processes in which cytokinins are limiting and that, therefore, they might regulate. We have engineered transgenic Arabidopsis plants that overexpress individually six different members of the cytokinin oxidase/dehydrogenase (AtCKX) gene family and have undertaken a detailed phenotypic analysis. Transgenic plants had increased cytokinin breakdown (30 to 45% of wild-type cytokinin content) and reduced expression of the cytokinin reporter gene ARR5:GUS (beta-glucuronidase). Cytokinin deficiency resulted in diminished activity of the vegetative and floral shoot apical meristems and leaf primordia, indicating an absolute requirement for the hormone. By contrast, cytokinins are negative regulators of root growth and lateral root formation. We show that the increased growth of the primary root is linked to an enhanced meristematic cell number, suggesting that cytokinins control the exit of cells from the root meristem. Different AtCKX-green fluorescent protein fusion proteins were localized to the vacuoles or the endoplasmic reticulum and possibly to the extracellular space, indicating that subcellular compartmentation plays an important role in cytokinin biology. Analyses of promoter:GUS fusion genes showed differential expression of AtCKX genes during plant development, the activity being confined predominantly to zones of active growth. Our results are consistent with the hypothesis that cytokinins have central, but opposite, regulatory functions in root and shoot meristems and indicate that a fine-tuned control of catabolism plays an important role in ensuring the proper regulation of cytokinin functions.

1,365 citations


Journal ArticleDOI
TL;DR: The results suggest that ERF1 acts downstream of the intersection between ethylene and jasmonate pathways and suggest that this transcription factor is a key element in the integration of both signals for the regulation of defense response genes.
Abstract: Cross-talk between ethylene and jasmonate signaling pathways determines the activation of a set of defense responses against pathogens and herbivores. However, the molecular mechanisms that underlie this cross-talk are poorly understood. Here, we show that ethylene and jasmonate pathways converge in the transcriptional activation of ETHYLENE RESPONSE FACTOR1 (ERF1), which encodes a transcription factor that regulates the expression of pathogen response genes that prevent disease progression. The expression of ERF1 can be activated rapidly by ethylene or jasmonate and can be activated synergistically by both hormones. In addition, both signaling pathways are required simultaneously to activate ERF1, because mutations that block any of them prevent ERF1 induction by any of these hormones either alone or in combination. Furthermore, 35S:ERF1 expression can rescue the defense response defects of coi1 (coronative insensitive1) and ein2 (ethylene insensitive2); therefore, it is a likely downstream component of both ethylene and jasmonate signaling pathways. Transcriptome analysis in Col;35S:ERF1 transgenic plants and ethylene/jasmonate-treated wild-type plants further supports the notion that ERF1 regulates in vivo the expression of a large number of genes responsive to both ethylene and jasmonate. These results suggest that ERF1 acts downstream of the intersection between ethylene and jasmonate pathways and suggest that this transcription factor is a key element in the integration of both signals for the regulation of defense response genes.

1,240 citations


Journal ArticleDOI
TL;DR: Observations suggest that the Arabidopsis bHLH proteins have the potential to participate in an extensive set of combinatorial interactions, endowing them with the capacity to be involved in the regulation of a multiplicity of transcriptional programs.
Abstract: The basic/helix-loop-helix (bHLH) proteins are a superfamily of transcription factors that bind as dimers to specific DNA target sites and that have been well characterized in nonplant eukaryotes as important regulatory components in diverse biological processes. Based on evidence that the bHLH protein PIF3 is a direct phytochrome reaction partner in the photoreceptor’s signaling network, we have undertaken a comprehensive computational analysis of the Arabidopsis genome sequence databases to define the scope and features of the bHLH family. Using a set of criteria derived from a previously defined consensus motif, we identified 147 bHLH protein‐encoding genes, making this one of the largest transcription factor families in Arabidopsis. Phylogenetic analysis of the bHLH domain sequences permits classification of these genes into 21 subfamilies. The evolutionary and potential functional relationships implied by this analysis are supported by other criteria, including the chromosomal distribution of these genes relative to duplicated genome segments, the conservation of variant exon/intron structural patterns, and the predicted DNA binding activities within subfamilies. Considerable diversity in DNA binding site specificity among family members is predicted, and marked divergence in protein sequence outside of the conserved bHLH domain is observed. Together with the established propensity of bHLH factors to engage in varying degrees of homodimerization and heterodimerization, these observations suggest that the Arabidopsis bHLH proteins have the potential to participate in an extensive set of combinatorial interactions, endowing them with the capacity to be involved in the regulation of a multiplicity of transcriptional programs. We provide evidence from yeast two-hybrid and in vitro binding assays that two related phytochrome-interacting members in the Arabidopsis family, PIF3 and PIF4, can form both homodimers and heterodimers and that all three dimeric configurations can bind specifically to the G-box DNA sequence motif CACGTG. These data are consistent, in principle, with the operation of this combinatorial mechanism in Arabidopsis.

1,094 citations


Journal ArticleDOI
TL;DR: Analysis of the Arabidopsis mutant npr1 revealed that the antagonistic effect of SA on JA signaling requires the regulatory protein NPR1, indicating that cross-talk between SA and JA is modulated through a novel function of NPR1 in the cytosol.
Abstract: Plant defenses against pathogens and insects are regulated differentially by cross-communicating signal transduction pathways in which salicylic acid (SA) and jasmonic acid (JA) play key roles. In this study, we investigated the molecular mechanism of the antagonistic effect of SA on JA signaling. Arabidopsis plants unable to accumulate SA produced 25-fold higher levels of JA and showed enhanced expression of the JA-responsive genes LOX2, PDF1.2, and VSP in response to infection by Pseudomonas syringae pv tomato DC3000, indicating that in wild-type plants, pathogen-induced SA accumulation is associated with the suppression of JA signaling. Analysis of the Arabidopsis mutant npr1, which is impaired in SA signal transduction, revealed that the antagonistic effect of SA on JA signaling requires the regulatory protein NPR1. Nuclear localization of NPR1, which is essential for SA-mediated defense gene expression, is not required for the suppression of JA signaling, indicating that cross-talk between SA and JA is modulated through a novel function of NPR1 in the cytosol.

1,088 citations


Journal ArticleDOI
TL;DR: In situ hybridization analysis demonstrated that the expression of GA-responsive genes is not restricted to the predicted site of GA biosynthesis, suggesting that GA itself, or GA signals, is transmitted across different cell types during Arabidopsis seed germination.
Abstract: The hormone-mediated control of plant growth and development involves both synthesis and response. Previous studies have shown that gibberellin (GA) plays an essential role in Arabidopsis seed germination. To learn how GA stimulates seed germination, we performed comprehensive analyses of GA biosynthesis and response using gas chromatography–mass spectrometry and oligonucleotide-based DNA microarray analysis. In addition, spatial correlations between GA biosynthesis and response were assessed by in situ hybridization. We identified a number of transcripts, the abundance of which is modulated upon exposure to exogenous GA. A subset of these GA-regulated genes was expressed in accordance with an increase in endogenous active GA levels, which occurs just before radicle emergence. The GA-responsive genes identified include those responsible for synthesis, transport, and signaling of other hormones, suggesting the presence of uncharacterized crosstalk between GA and other hormones. In situ hybridization analysis demonstrated that the expression of GA-responsive genes is not restricted to the predicted site of GA biosynthesis, suggesting that GA itself, or GA signals, is transmitted across different cell types during Arabidopsis seed germination.

869 citations


Journal ArticleDOI
TL;DR: Results with transfected protoplasts reported here show that ARFs with Q-rich MRs function as activators, whereas most, if not all other ARFs, function as repressors.
Abstract: Auxin response factors (ARFs) are transcription factors that bind to TGTCTC auxin response elements in promoters of early auxin response genes. ARFs have a conserved N-terminal DNA binding domain (DBD) and in most cases a conserved C-terminal dimerization domain (CTD). The ARF CTD is related in amino acid sequence to motifs III and IV found in Aux/IAA proteins. Just C terminal to the DBD, ARFs contain a nonconserved region referred to as the middle region (MR), which has been proposed to function as a transcriptional repression or activation domain. Results with transfected protoplasts reported here show that ARFs with Q-rich MRs function as activators, whereas most, if not all other ARFs, function as repressors. ARF DBDs alone are sufficient to recruit ARFs to their DNA target sites, and auxin does not influence this recruitment. ARF MRs alone function as activation or repression domains when targeted to reporter genes via a yeast Gal4 DBD, and auxin does not influence the potency of activation or repression. ARF CTDs, along with a Q-rich MR, are required for an auxin response whether the MRs plus CTDs are recruited to a promoter by an ARF DBD or by a Gal4 DBD. The auxin response is mediated by the recruitment of Aux/IAA proteins to promoters that contain a DNA binding protein with a Q-rich MR and an attached CTD.

812 citations


Journal ArticleDOI
TL;DR: Results strongly suggest that OsMAPK5 can positively regulate drought, salt, and cold tolerance and negatively modulate PR gene expression and broad-spectrum disease resistance.
Abstract: Mitogen-activated protein kinase (MAPK) cascades play an important role in mediating stress responses in eukaryotic organisms. However, little is known about the role of MAPKs in modulating the interaction of defense pathways activated by biotic and abiotic factors. In this study, we have isolated and functionally characterized a stress-responsive MAPK gene (OsMAPK5) from rice. OsMAPK5 is a single-copy gene but can generate at least two differentially spliced transcripts. The OsMAPK5 gene, its protein, and kinase activity were inducible by abscisic acid as well as various biotic (pathogen infection) and abiotic (wounding, drought, salt, and cold) stresses. To determine its biological function, we generated and analyzed transgenic rice plants with overexpression (using the 35S promoter of Cauliflower mosaic virus) or suppression (using double-stranded RNA interference [dsRNAi]) of OsMAPK5. Interestingly, suppression of OsMAPK5 expression and its kinase activity resulted in the constitutive expression of pathogenesis-related (PR) genes such as PR1 and PR10 in the dsRNAi transgenic plants and significantly enhanced resistance to fungal (Magnaporthe grisea) and bacterial (Burkholderia glumae) pathogens. However, these same dsRNAi lines had significant reductions in drought, salt, and cold tolerance. By contrast, overexpression lines exhibited increased OsMAPK5 kinase activity and increased tolerance to drought, salt, and cold stresses. These results strongly suggest that OsMAPK5 can positively regulate drought, salt, and cold tolerance and negatively modulate PR gene expression and broad-spectrum disease resistance.

780 citations


Journal ArticleDOI
TL;DR: This study provides a solid base for functional genomics studies into this important family of plant regulatory genes, including the poorly characterized group of M-type MADS-box proteins.
Abstract: MADS-box transcription factors are key regulators of several plant development processes Analysis of the complete Arabidopsis genome sequence revealed 107 genes encoding MADS-box proteins, of which 84% are of unknown function Here, we provide a complete overview of this family, describing the gene structure, gene expression, genome localization, protein motif organization, and phylogenetic relationship of each member We have divided this transcription factor family into five groups (named MIKC, Mα, Mβ, Mγ, and Mδ) based on the phylogenetic relationships of the conserved MADS-box domain This study provides a solid base for functional genomics studies into this important family of plant regulatory genes, including the poorly characterized group of M-type MADS-box proteins MADS-box genes also constitute an excellent system with which to study the evolution of complex gene families in higher plants

Journal ArticleDOI
TL;DR: Despite high similarity between responses mediated by the R genes R PS2 and RPM1 in wild-type plants, RPS2-mediated responses were strongly suppressed by the ndr1 mutation and the NahG transgene, whereas RPM1- mediated responses were not, which is consistent with the resistance phenotypes of these plants.
Abstract: We performed large-scale mRNA expression profiling using an Affymetrix GeneChip to study Arabidopsis responses to the bacterial pathogen Pseudomonas syringae. The interactions were compatible (virulent bacteria) or incompatible (avirulent bacteria), including a nonhost interaction and interactions mediated by two different avirulence gene–resistance (R) gene combinations. Approximately 2000 of the ∼8000 genes monitored showed reproducible significant expression level changes in at least one of the interactions. Analysis of biological variation suggested that the system behavior of the plant response in an incompatible interaction was robust but that of a compatible interaction was not. A large part of the difference between incompatible and compatible interactions can be explained quantitatively. Despite high similarity between responses mediated by the R genes RPS2 and RPM1 in wild-type plants, RPS2-mediated responses were strongly suppressed by the ndr1 mutation and the NahG transgene, whereas RPM1-mediated responses were not. This finding is consistent with the resistance phenotypes of these plants. We propose a simple quantitative model with a saturating response curve that approximates the overall behavior of this plant-pathogen system.

Journal ArticleDOI
TL;DR: Results demonstrate that singlet oxygen does not act primarily as a toxin but rather as a signal that activates several stress-response pathways and its biological activity in Arabidopsis exhibits a high degree of specificity that seems to be derived from the chemical identity of this reactive oxygen species and/or the intracellular location at which it is generated.
Abstract: The conditional fluorescent (flu) mutant of Arabidopsis accumulates the photosensitizer protochlorophyllide in the dark. After a dark-to-light shift, the generation of singlet oxygen, a nonradical reactive oxygen species, starts within the first minute of illumination and was shown to be confined to plastids. Immediately after the shift, plants stopped growing and developed necrotic lesions. These early stress responses of the flu mutant do not seem to result merely from physicochemical damage. Peroxidation of chloroplast membrane lipids in these plants started rapidly and led to the transient and selective accumulation of a stereospecific and regiospecific isomer of hydroxyoctadecatrieonic acid, free (13S)-HOTE, that could be attributed almost exclusively to the enzymatic oxidation of linolenic acid. Within the first 15 min of reillumination, distinct sets of genes were activated that were different from those induced by superoxide/hydrogen peroxide. Collectively, these results demonstrate that singlet oxygen does not act primarily as a toxin but rather as a signal that activates several stress-response pathways. Its biological activity in Arabidopsis exhibits a high degree of specificity that seems to be derived from the chemical identity of this reactive oxygen species and/or the intracellular location at which it is generated.

Journal ArticleDOI
TL;DR: In this paper, two Respiratory burst oxidase homologs (rboh genes) have been implicated in active oxygen species (AOS) generation and in hypersensitive response (HR) using virus-induced gene silencing (VIGS).
Abstract: Active oxygen species (AOS) are responsible for triggering defense responses in plants. Respiratory burst oxidase homologs (rboh genes) have been implicated in AOS generation. We have isolated two rboh cDNAs, NbrbohA and NbrbohB, from Nicotiana benthamiana leaves. NbrbohA was expressed constitutively at a low level and the transcripts were increased after mechanical stress of control leaf infiltration, whereas NbrbohB was induced specifically by the protein elicitor INF1 from the potato pathogen Phytophthora infestans. We examined the function of the Nbrboh genes in AOS generation and in the hypersensitive response (HR) using virus-induced gene silencing (VIGS). VIGS indicated that both genes are required for H2O2 accumulation and for resistance to Phytophthora. VIGS of Nbrboh genes also led to a reduction and delay of HR cell death caused by INF1. We further demonstrate that the induction of HR-like cell death by overexpression of a constitutively active mutant of a mitogen-activated protein kinase kinase, MEKDD, is compromised by VIGS of NbrbohB. We found that MEKDD induced NbrbohB but not NbrbohA. This work provides genetic evidence for the involvement of a mitogen-activated protein kinase cascade in the regulation of rboh genes.

Journal ArticleDOI
TL;DR: The predicted SLY1 amino acid sequence is highly conserved among plants, indicating a key role in GA response, and suggests SCFSLY1-targeted degradation of RGA through the 26S proteasome pathway.
Abstract: The Arabidopsis SLY1 (SLEEPY1) gene positively regulates gibberellin (GA) signaling. Positional cloning of SLY1 revealed that it encodes a putative F-box protein. This result suggests that SLY1 is the F-box subunit of an SCF E3 ubiquitin ligase that regulates GA responses. The DELLA domain protein RGA (repressor of ga1-3) is a repressor of GA response that appears to undergo GA-stimulated protein degradation. RGA is a potential substrate of SLY1, because sly1 mutations cause a significant increase in RGA protein accumulation even after GA treatment. This result suggests SCFSLY1-targeted degradation of RGA through the 26S proteasome pathway. Further support for this model is provided by the observation that an rga null allele partially suppresses the sly1-10 mutant phenotype. The predicted SLY1 amino acid sequence is highly conserved among plants, indicating a key role in GA response.

Journal ArticleDOI
TL;DR: It is reported that although TGA1 does not interact with NPR1 in yeast two-hybrid assays, treatment with salicylic acid induces the interaction between these proteins in Arabidopsis leaves, and it is indicated that T GA1 relies on the oxidation state of Cys residues to mediate the interaction withNPR1.
Abstract: The Arabidopsis NPR1 protein is essential for regulating salicylic acid–dependent gene expression during systemic acquired resistance. NPR1 interacts differentially with members of the TGA class of basic domain/Leu zipper transcription factors and regulates their DNA binding activity. Here, we report that although TGA1 does not interact with NPR1 in yeast two-hybrid assays, treatment with salicylic acid induces the interaction between these proteins in Arabidopsis leaves. This phenomenon is correlated with a reduction of TGA1 Cys residues. Furthermore, site-directed mutagenesis of TGA1 Cys-260 and Cys-266 enables the interaction with NPR1 in yeast and Arabidopsis. Together, these results indicate that TGA1 relies on the oxidation state of Cys residues to mediate the interaction with NPR1. An intramolecular disulfide bridge in TGA1 precludes interaction with NPR1, and NPR1 can only stimulate the DNA binding activity of the reduced form of TGA1. Unlike its animal and yeast counterparts, the DNA binding activity of TGA1 is not redox regulated; however, this property is conferred by interaction with the NPR1 cofactor.

Journal ArticleDOI
TL;DR: In this article, the authors used the vtc1 mutant to provide a molecular signature for vitamin C deficiency in plants and showed that 171 genes are expressed differentially in the mutant compared with the wild type and transcript changes indicate that growth and development are constrained by the modulation of abscisic acid signaling.
Abstract: Vitamin C deficiency in the Arabidopsis mutant vtc1 causes slow growth and late flowering. This is not attributable to changes in photosynthesis or increased oxidative stress. We have used the vtc1 mutant to provide a molecular signature for vitamin C deficiency in plants. Using statistical analysis, we show that 171 genes are expressed differentially in vtc1 compared with the wild type. Many defense genes are activated, particularly those that encode pathogenesis-related proteins. Furthermore, transcript changes indicate that growth and development are constrained in vtc1 by the modulation of abscisic acid signaling. Abscisic acid contents are significantly higher in vtc1 than in the wild type. Key features of the molecular signature of ascorbate deficiency can be reversed by incubating vtc1 leaf discs in ascorbate. This finding provides evidence that many of the observed effects on transcript abundance in vtc1 result from ascorbate deficiency. Hence, through modifying gene expression, vitamin C contents not only act to regulate defense and survival but also act via phytohormones to modulate plant growth under optimal conditions.

Journal ArticleDOI
TL;DR: The value of Arabidopsis for studies of the biosynthesis and ecological role of terpenoids is demonstrated and new insights into their metabolism in wild-type and transgenic plants are provided.
Abstract: Volatile components, such as terpenoids, are emitted from aerial parts of plants and play a major role in the interaction between plants and their environment. Analysis of the composition and emission pattern of volatiles in the model plant Arabidopsis showed that a range of volatile components are released, primarily from flowers. Most of the volatiles detected were monoterpenes and sesquiterpenes, which in contrast to other volatiles showed a diurnal emission pattern. The active terpenoid metabolism in wild-type Arabidopsis provoked us to conduct an additional set of experiments in which transgenic Arabidopsis overexpressing two different terpene synthases were generated. Leaves of transgenic plants constitutively expressing a dual linalool/nerolidol synthase in the plastids (FaNES1) produced linalool and its glycosylated and hydroxylated derivatives. The sum of glycosylated components was in some of the transgenic lines up to 40- to 60-fold higher than the sum of the corresponding free alcohols. Surprisingly, we also detected the production and emission of nerolidol, albeit at a low level, suggesting that a small pool of its precursor farnesyl diphosphate is present in the plastids. Transgenic lines with strong transgene expression showed growth retardation, possibly as a result of the depletion of isoprenoid precursors in the plastids. In dual-choice assays with Myzus persicae, the FaNES1-expressing lines significantly repelled the aphids. Overexpression of a typical cytosolic sesquiterpene synthase resulted in the production of only trace amounts of the expected sesquiterpene, suggesting tight control of the cytosolic pool of farnesyl diphosphate, the precursor for sesquiterpenoid biosynthesis. This study further demonstrates the value of Arabidopsis for studies of the biosynthesis and ecological role of terpenoids and provides new insights into their metabolism in wild-type and transgenic plants.

Journal ArticleDOI
TL;DR: Suppression subtractive hybridization and RNA hybridization analysis of the purple tomato plants indicated that the overexpression of ANT1 caused the upregulation of genes that encode proteins in both the early and later steps of anthocyanidin biosynthesis as well as genes involved in the glycosylation and transport of Anthocyanins into the vacuole.
Abstract: We have developed a high-throughput T-DNA insertional mutagenesis program in tomato using activation tagging to identify genes that regulate metabolic pathways. One of the activation-tagged insertion lines (ant1) showed intense purple pigmentation from the very early stage of shoot formation in culture, reflecting activation of the biosynthetic pathway leading to anthocyanin accumulation. The purple coloration resulted from the overexpression of a gene that encodes a MYB transcription factor. Vegetative tissues of ant1 plants displayed intense purple color, and the fruit showed purple spotting on the epidermis and pericarp. The gene-to-trait relationship of ant1 was confirmed by the overexpression of ANT1 in transgenic tomato and in tobacco under the control of a constitutive promoter. Suppression subtractive hybridization and RNA hybridization analysis of the purple tomato plants indicated that the overexpression of ANT1 caused the upregulation of genes that encode proteins in both the early and later steps of anthocyanidin biosynthesis as well as genes involved in the glycosylation and transport of anthocyanins into the vacuole.

Journal ArticleDOI
TL;DR: The results suggest that BOS1 mediates responses to signals, possibly mediated by reactive oxygen intermediates from both biotic and abiotic stress agents, and suggests a common host response strategy against these pathogens.
Abstract: The molecular and cellular mechanisms involved in plant resistance to the necrotrophic fungal pathogen Botrytis cinerea and their genetic control are poorly understood. Botrytis causes severe disease in a wide range of plant species, both in the field and in postharvest situations, resulting in significant economic losses. We have isolated the BOS1 (BOTRYTIS-SUSCEPTIBLE1) gene of Arabidopsis based on a T-DNA insertion allele that resulted in increased susceptibility to Botrytis infection. The BOS1 gene is required to restrict the spread of another necrotrophic pathogen, Alternaria brassicicola, suggesting a common host response strategy against these pathogens. In the case of the biotrophic pathogens Pseudomonas syringae pv tomato and the oomycete parasite Peronospora parasitica, bos1 exhibits enhanced disease symptoms, but pathogen growth is similar in bos1 and wild-type plants. Strikingly, bos1 plants have impaired tolerance to water deficit, increased salinity, and oxidative stress. Botrytis infection induces the expression of the BOS1 gene. This increased expression is severely impaired in the coi1 mutant, suggesting an interaction of BOS1 with the jasmonate signaling pathway. BOS1 encodes an R2R3MYB transcription factor protein, and our results suggest that it mediates responses to signals, possibly mediated by reactive oxygen intermediates from both biotic and abiotic stress agents.

Journal ArticleDOI
TL;DR: The data indicate that SUSIBA2 binds to the SURE elements in the barley iso1 promoter as an activator and demonstrate the involvement of a WRKY protein in carbohydrate anabolism.
Abstract: SURE (sugar responsive) is a cis element in plant sugar signaling. The SURE element was reported first for potato, in which it confers sugar responsiveness to the patatin promoter. A SURE binding transcription factor has not been isolated. We have isolated a transcription factor cDNA from barley and purified the corresponding protein. The transcription factor, SUSIBA2 (sugar signaling in barley), belongs to the WRKY proteins and was shown to bind to SURE and W-box elements but not to the SP8a element in the iso1 promoter. Nuclear localization of SUSIBA2 was demonstrated in a transient assay system with a SUSIBA2:green fluorescent protein fusion protein. Exploiting the novel transcription factor oligodeoxynucleotide decoy strategy with transformed barley endosperm provided experimental evidence for the importance of the SURE elements in iso1 transcription. Antibodies against SUSIBA2 were produced, and the expression pattern for susiba2 was determined at the RNA and protein levels. It was found that susiba2 is expressed in endosperm but not in leaves. Transcription of susiba2 is sugar inducible, and ectopic susiba2 expression was obtained in sugar-treated leaves. Likewise, binding to SURE elements was observed for nuclear extracts from sugar-treated but not from control barley leaves. The temporal expression of susiba2 in barley endosperm followed that of iso1 and endogenous sucrose levels, with a peak at ∼12 days after pollination. Our data indicate that SUSIBA2 binds to the SURE elements in the barley iso1 promoter as an activator. Furthermore, they show that SUSIBA2 is a regulatory transcription factor in starch synthesis and demonstrate the involvement of a WRKY protein in carbohydrate anabolism. Orthologs to SUSIBA2 were isolated from rice and wheat endosperm.

Journal ArticleDOI
TL;DR: The target site specificity of the rice retrotransposon Tos17, which can be activated by tissue culture, is examined, and it is shown that insertion events are three times more frequent in genic regions than in intergenic regions.
Abstract: Because retrotransposons are the major component of plant genomes, analysis of the target site selection of retrotransposons is important for understanding the structure and evolution of plant genomes. Here, we examined the target site specificity of the rice retrotransposon Tos17, which can be activated by tissue culture. We have produced 47,196 Tos17-induced insertion mutants of rice. This mutant population carries ∼500,000 insertions. We analyzed >42,000 flanking sequences of newly transposed Tos17 copies from 4316 mutant lines. More than 20,000 unique loci were assigned on the rice genomic sequence. Analysis of these sequences showed that insertion events are three times more frequent in genic regions than in intergenic regions. Consistent with this result, Tos17 was shown to prefer gene-dense regions over centromeric heterochromatin regions. Analysis of insertion target sequences revealed a palindromic consensus sequence, ANGTT-TSD-AACNT, flanking the 5-bp target site duplication. Although insertion targets are distributed throughout the chromosomes, they tend to cluster, and 76% of the clusters are located in genic regions. The mechanisms of target site selection by Tos17, the utility of the mutant lines, and the knockout gene database are discussed.

Journal ArticleDOI
TL;DR: Loss of complex I function reveals effective antioxidant crosstalk and acclimation between the mitochondria and other organelles to maintain whole cell redox balance and is associated with higher tolerance to ozone and Tobacco mosaic virus.
Abstract: To explore the role of plant mitochondria in the regulation of cellular redox homeostasis and stress resistance, we exploited a Nicotiana sylvestris mitochondrial mutant. The cytoplasmic male-sterile mutant (CMSII) is impaired in complex I function and displays enhanced nonphosphorylating rotenone-insensitive [NAD(P)H dehydrogenases] and cyanide-insensitive (alternative oxidase) respiration. Loss of complex I function is not associated with increased oxidative stress, as shown by decreased leaf H(2)O(2) and the maintenance of glutathione and ascorbate content and redox state. However, the expression and activity of several antioxidant enzymes are modified in CMSII. In particular, diurnal patterns of alternative oxidase expression are lost, the relative importance of the different catalase isoforms is modified, and the transcripts, protein, and activity of cytosolic ascorbate peroxidase are enhanced markedly. Thus, loss of complex I function reveals effective antioxidant crosstalk and acclimation between the mitochondria and other organelles to maintain whole cell redox balance. This reorchestration of the cellular antioxidative system is associated with higher tolerance to ozone and Tobacco mosaic virus.

Journal ArticleDOI
TL;DR: Depletion of callose from papillae in gsl5 plants marginally enhanced the penetration of the grass powdery mildew fungus Blumeria graminis on the nonhost Arabidopsis, providing strong genetic evidence that the GSL genes of higher plants encode proteins that are essential for callose formation.
Abstract: Arabidopsis was transformed with double-stranded RNA interference (dsRNAi) constructs designed to silence three putative callose synthase genes: GLUCAN SYNTHASE-LIKE5 (GSL5), GSL6, and GSL11. Both wound callose and papillary callose were absent in lines transformed with GSL5 dsRNAi and in a corresponding sequence-indexed GSL5 T-DNA insertion line but were unaffected in GSL6 and GSL11 dsRNAi lines. These data provide strong genetic evidence that the GSL genes of higher plants encode proteins that are essential for callose formation. Deposition of callosic plugs, or papillae, at sites of fungal penetration is a widely recognized early response of host plants to microbial attack and has been implicated in impeding entry of the fungus. Depletion of callose from papillae in gsl5 plants marginally enhanced the penetration of the grass powdery mildew fungus Blumeria graminis on the nonhost Arabidopsis. Paradoxically, the absence of callose in papillae or haustorial complexes correlated with the effective growth cessation of several normally virulent powdery mildew species and of Peronospora parasitica.

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TL;DR: It is concluded that D2/CYP90D2 catalyzes the steps from 6-deoxoteasterone to 3-dehydro-6- deoxoteastersone and from teasteroneto 3- dehydroteasterone in the late BR biosynthesis pathway.
Abstract: We characterized a rice dwarf mutant, ebisu dwarf (d2). It showed the pleiotropic abnormal phenotype similar to that of the rice brassinosteroid (BR)-insensitive mutant, d61. The dwarf phenotype of d2 was rescued by exogenous brassinolide treatment. The accumulation profile of BR intermediates in the d2 mutants confirmed that these plants are deficient in late BR biosynthesis. We cloned the D2 gene by map-based cloning. The D2 gene encoded a novel cytochrome P450 classified in CYP90D that is highly similar to the reported BR synthesis enzymes. Introduction of the wild D2 gene into d2-1 rescued the abnormal phenotype of the mutants. In feeding experiments, 3-dehydro-6-deoxoteasterone, 3-dehydroteasterone, and brassinolide effectively caused the lamina joints of the d2 plants to bend, whereas more upstream compounds did not cause bending. Based on these results, we conclude that D2/CYP90D2 catalyzes the steps from 6-deoxoteasterone to 3-dehydro-6-deoxoteasterone and from teasterone to 3-dehydroteasterone in the late BR biosynthesis pathway.

Journal ArticleDOI
TL;DR: Heterosis, or hybrid vigor, refers to the phenomenon that progeny of diverse inbred varieties exhibit greater biomass, speed of development, and fertility than the better of the two parents.
Abstract: Heterosis, or hybrid vigor, refers to the phenomenon that progeny of diverse inbred varieties exhibit greater biomass, speed of development, and fertility than the better of the two parents ([Figure 1][1]). This phenomenon has been exploited extensively in crop production and has been a powerful

Journal ArticleDOI
TL;DR: It is shown that ectopic expression of either the STK or SHP gene is sufficient to induce the transformation of sepals into carpeloid organs bearing ovules and that the SEP proteins, known to form multimeric complexes in the control of flower organ identity, also form complexes to control normal ovule development.
Abstract: The AGAMOUS (AG) gene is necessary for stamen and carpel development and is part of a monophyletic clade of MADS-box genes that also includes SHATTERPROOF1 (SHP1), SHP2, and SEEDSTICK (STK). Here, we show that ectopic expression of either the STK or SHP gene is sufficient to induce the transformation of sepals into carpeloid organs bearing ovules. Moreover, the fact that these organ transformations occur when the STK gene is expressed ectopically in ag mutants shows that STK can promote carpel development in the absence of AG activity. We also show that STK, AG, SHP1, and SHP2 can form multimeric complexes and that these interactions require the SEPALLATA (SEP) MADS-box proteins. We provide genetic evidence for this role of the SEP proteins by showing that a reduction in SEP activity leads to the loss of normal ovule development, similar to what occurs in stk shp1 shp2 triple mutants. Together, these results indicate that the SEP proteins, which are known to form multimeric complexes in the control of flower organ identity, also form complexes to control normal ovule development.

Journal ArticleDOI
TL;DR: Although the resistance signaling in snc1 was fully dependent on PAD4, it was only partially affected by blocking salicylic acid synthesis, suggesting thatsnc1 activates both SA-dependent and SA-independent resistance pathways.
Abstract: Plants have evolved sophisticated defense mechanisms against pathogen infections, during which resistance (R) genes play central roles in recognizing pathogens and initiating defense cascades. Most of the cloned R genes share two common domains: the central domain, which encodes a nucleotide binding adaptor shared by APAF-1, certain R proteins, and CED-4 (NB-ARC), plus a C-terminal region that encodes Leu-rich repeats (LRR). In Arabidopsis, a dominant mutant, suppressor of npr1-1, constitutive 1 (snc1), was identified previously that constitutively expresses pathogenesis-related (PR) genes and resistance against both Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2. The snc1 mutation was mapped to the RPP4 cluster. In snc1, one of the TIR-NB-LRR–type R genes contains a point mutation that results in a single amino acid change from Glu to Lys in the region between NB-ARC and LRR. Deletions of this R gene in snc1 reverted the plants to wild-type morphology and completely abolished constitutive PR gene expression and disease resistance. The constitutive activation of the defense responses was not the result of the overexpression of the R gene, because its expression level was not altered in snc1. Our data suggest that the point mutation in snc1 renders the R gene constitutively active without interaction with pathogens. To analyze signal transduction pathways downstream of snc1, epistasis analyses between snc1 and pad4-1 or eds5-3 were performed. Although the resistance signaling in snc1 was fully dependent on PAD4, it was only partially affected by blocking salicylic acid (SA) synthesis, suggesting that snc1 activates both SA-dependent and SA-independent resistance pathways.

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TL;DR: It is demonstrated that Sus plays a rate-limiting role in the initiation and elongation of the single-celled fibers and that suppression of Sus only in the maternal seed tissue represses fiber development without affecting embryo development and seed size.
Abstract: Cotton is the most important textile crop as a result of its long cellulose-enriched mature fibers. These single-celled hairs initiate at anthesis from the ovule epidermis. To date, genes proven to be critical for fiber development have not been identified. Here, we examined the role of the sucrose synthase gene (Sus) in cotton fiber and seed by transforming cotton with Sus suppression constructs. We focused our analysis on 0 to 3 days after anthesis (DAA) for early fiber development and 25 DAA, when the fiber and seed are maximal in size. Suppression of Sus activity by 70% or more in the ovule epidermis led to a fiberless phenotype. The fiber initials in those ovules were fewer and shrunken or collapsed. The level of Sus suppression correlated strongly with the degree of inhibition of fiber initiation and elongation, probably as a result of the reduction of hexoses. By 25 DAA, a portion of the seeds in the fruit showed Sus suppression only in the seed coat fibers and transfer cells but not in the endosperm and embryo. These transgenic seeds were identical to wild-type seeds except for much reduced fiber growth. However, the remaining seeds in the fruit showed Sus suppression both in the seed coat and in the endosperm and embryo. These seeds were shrunken with loss of the transfer cells and were <5% of wild-type seed weight. These results demonstrate that Sus plays a rate-limiting role in the initiation and elongation of the single-celled fibers. These analyses also show that suppression of Sus only in the maternal seed tissue represses fiber development without affecting embryo development and seed size. Additional suppression in the endosperm and embryo inhibits their own development, which blocks the formation of adjacent seed coat transfer cells and arrests seed development entirely.

Journal ArticleDOI
TL;DR: Analysis of a CUC3 hypomorph and a putative cuc3 null mutant indicates that C UC3 function is partially redundant with that of CUC1 and CUC2 in the establishment of the cotyledon boundary and the shoot meristem, thus revealing an even higher degree of redundancy in this class of genes than was thought previously.
Abstract: From an enhancer trap screen for genes expressed in Arabidopsis embryos, we identified a gene expressed from the octant stage onward in the boundary between the two presumptive cotyledons and in a variety of postembryonic organ and meristem boundaries. This gene, CUP-SHAPED COTYLEDON3 (CUC3), encodes a putative NAC-domain transcription factor that is homologous with CUC1 and CUC2. Analysis of a CUC3 hypomorph and a putative cuc3 null mutant indicates that CUC3 function is partially redundant with that of CUC1 and CUC2 in the establishment of the cotyledon boundary and the shoot meristem, thus revealing an even higher degree of redundancy in this class of genes than was thought previously. The CUC3 expression pattern, the cuc3 phenotypes, and CUC3 expression in a series of shoot meristem mutants and transgenes suggest a primary role for CUC3 in the establishment of boundaries that contain cells with low proliferation and/or differentiation rates. The CUC-mediated establishment of such boundaries may be essential for the initiation of shoot meristems.