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Plant defense responses at stomata and apoplast are the most important early events during plant-bacteria interactions. The key components for the signaling of stomatal defense and nonhost resistance have not been fully characterized. Here we report the newly identified small GTPase, Nucleolar GTP-binding protein 1 (NOG1), functions for plant immunity against bacterial pathogens. Virus-induced gene silencing of NOG1 compromised nonhost resistance in N. benthamiana and tomato. Comparative genomic analysis showed that two NOG1 copies are present in all known plant species: NOG1-1 and NOG1-2. Gene downregulation and overexpression studies of NOG1-1 and NOG1-2 in Arabidopsis revealed the novel function of these genes in nonhost resistance and stomatal defense against bacterial pathogens, respectively. Specially, NOG1-2 regulates guard cell signaling in response to biotic and abiotic stimuli through jasmonic acid (JA)- and abscisic acid (ABA)-mediated pathways. The results here provide valuable information on the new functional role of small GTPase, NOG1, in guard cell signaling and early plant defense in response to bacterial pathogens.

Background SEVEN IN ABSENTIA (SINA) is a RING domain-containing ubiquitin ligase involved in Drosophila eye formation. SINA-like proteins in plants are involved in several signaling pathways. Of the 18 SINA-like proteins identified in Arabidopsis, SEVEN IN ABSENTIA 2 (SINA2) lacks a canonical RING domain and is thought to lack ubiquitin ligase activity. Results Our results show that SINA2 has E3 ligase activity in vitro, raising the possibility that a modified B-box domain may compensate for its lack of a RING domain. SINA2 physically interacts with the nuclear protein CYCLIN-DEPENDENT KINASE G1 (CDKG1), which acts as a positive regulator of plant responses to abiotic stress. CDKG1 is expressed in multiple tissues and its expression increased in response to abscisic acid (ABA) and osmotic stress. Transgenic Arabidopsis plants that ectopically express CDKG1 exhibit increased tolerance to ABA and osmotic stress treatments during seed germination and cotyledon development, while the loss-of-function cdkg1 mutant plants show reduced tolerance to ABA and osmotic stress treatments. Moreover, CDKG1-dependent phosphorylation of SINA2 positively affects its E3 ubiquitin ligase activity. Conclusions Based on these results, we propose that CDKG1 modulates SINA2 ubiquitin ligase activity to regulate its effect on plant responses to ABA and osmotic stress.

• DELAY OF GERMINATION1 (DOG1) is a primary regulator of seed dormancy. Accumulation of DOG1 in seeds leads to deep dormancy and delayed germination in Arabidopsis. B3 domain-containing transcriptional repressors HSI2/VAL1 and HSL1/VAL2 silence seed dormancy and enable the subsequent germination and seedling growth. However, the roles of HSI2 and HSL1 in regulation of DOG1 expression and seed dormancy remain elusive. • Seed dormancy was analysed by measurement of maximum germination percentage of freshly harvested Arabidopsis seeds. In vivo protein–protein interaction analysis, ChIP-qPCR and EMSA were performed and suggested that HSI2 and HSL1 can form dimers to directly regulate DOG1. • HSI2 and HSL1 dimers interact with RY elements at DOG1 promoter. Both B3 and PHD-like domains are required for enrichment of HSI2 and HSL1 at the DOG1 promoter. HSI2 and HSL1 recruit components of polycomb-group proteins, including CURLY LEAF (CLF) and LIKE HETERCHROMATIN PROTEIN 1 (LHP1), for consequent deposition of H3K27me3 marks, leading to repression of DOG1 expression. • Our findings suggest that HSI2- and HSL1-dependent histone methylation plays critical roles in regulation of seed dormancy during seed germination and early seedling growth.

Gene expression during seed development in Arabidopsis thaliana is controlled by transcription factors including LEAFY COTYLEDON1 (LEC1) and LEC2, ABA INSENSITIVE3 (ABI3), FUSCA3 (FUS3), known as LAFL proteins, and AGAMOUS-LIKE15 (AGL15). The transition from seed maturation to germination and seedling growth requires the transcriptional silencing of these seed maturation-specific factors leading to downregulation of structural genes including those that encode seed storage proteins, oleosins, and dehydrins. During seed germination and vegetative growth, B3-domain protein HSI2/VAL1 is required for the transcriptional silencing of LAFL genes. Here, we report chromatin immunoprecipitation analysis indicating that HSI2/VAL1 binds to the upstream sequences of the AGL15 gene but not at LEC1, ABI3, FUS3, or LEC2 loci. Functional analysis indicates that the HSI2/VAL1 B3 domain interacts with two RY elements upstream of the AGL15 coding region and at least one of them is required for HSI2/VAL1-dependent AGL15 repression. Expression analysis of the major seed maturation regulatory genes LEC1, ABI3, FUS3, and LEC2 in different genetic backgrounds demonstrates that HSI2/VAL1 is epistatic to AGL15 and represses the seed maturation regulatory program through downregulation of AGL15 by deposition of H3K27me3 at this locus. This hypothesis is further supported by results that show that HSI2/VAL1 physically interacts with the Polycomb Repressive Complex 2 component protein MSI1, which is also enriched at the AGL15 locus.

The aging pathway in flowering regulation is controlled mainly by microRNA156 (miR156). Studies in Arabidopsis thaliana reveal that nine miR156-targeted SQUAMOSA PROMOTER BINDING-LIKE (SPL) genes are involved in the control of flowering. However, the roles of SPLs in flowering remain elusive in grasses. Inflorescence development in switchgrass was characterized using scanning electron microscopy (SEM). Microarray, quantitative reverse transcription polymerase chain reaction (qRT-PCR), chromatin immunoprecipitation (ChIP)-PCR and EMSA were used to identify regulators of phase transition and flowering. Gene function was characterized by downregulation and overexpression of the target genes. Overexpression of SPL7 and SPL8 promotes flowering, whereas downregulation of individual genes moderately delays flowering. Simultaneous downregulation of SPL7/SPL8 results in extremely delayed or nonflowering plants. Furthermore, downregulation of both genes leads to a vegetative-to-reproductive reversion in the inflorescence, a phenomenon that has not been reported in any other grasses. Detailed analyses demonstrate that SPL7 and SPL8 induce phase transition and flowering in grasses by directly upregulating SEPALLATA3 (SEP3) and MADS32. Thus, the SPL7/8 pathway represents a novel regulatory mechanism in grasses that is largely different from that in Arabidopsis. Additionally, genetic modification of SPL7 and SPL8 results in much taller plants with significantly increased biomass yield and sugar release.

Gibberellic acid (GA) is both necessary and sufficient to promote fiber elongation in cultured fertilized ovules of the upland cotton variety Coker 312. This is likely due to the temporal and spatial regulation of GA biosynthesis, perception, and subsequent signal transduction that leads to alterations in gene expression and morphology. Our results indicate that the initiation of fiber elongation by the application of GA to cultured ovules corresponds with increased expression of genes that encode xyloglucan endotransglycosylase/hydrolase (XTH) and expansin (EXP) that are involved in promoting cell elongation. To gain a better understanding of the GA signaling components in cotton, that lead to such changes in gene expression, two GA receptor genes (GhGID1a and GhGID1b) and two DELLA protein genes (GhSLR1a and GhSLR1b) that are orthologous to the rice GA receptor (GID1) and the rice DELLA gene (SLR1), respectively, were characterized. Similar to the GA biosynthetic genes, expression of GhGID1a and GhGID1b is under the negative regulation by GA while GA positively regulates GhSLR1a. Recombinant GST-GhGID1s showed GA-binding activity in vitro that was augmented in the presence of GhSLR1a, GhSLR1b, or rice SLR1, indicating complex formation between the receptors and repressor proteins. This was further supported by the GA-dependent interaction of these proteins in yeast cells. Ectopic expression of the GhGID1a in the rice gid1-3 mutant plants rescued the GA-insensitive dwarf phenotype, which demonstrates that it is a functional GA receptor. Furthermore, ectopic expression of GhSLR1b in wild type Arabidopsis led to reduced growth and upregulated expression of DELLA-responsive genes.

AtSAP5, one of approximately 14 members of the Stress Associated Protein gene family in Arabidopsis, was identified by its expression in response to salinity, osmotic, drought and cold stress. AtSAP5 shows strong homology to OSISAP1, an A20/AN1-type zinc finger protein implicated in stress tolerance in rice. To evaluate the function of AtSAP5 in the regulation of abiotic stress responses, transgenic Arabidopsis plants that over-express AtSAP5 ( 35S::AtSAP5 ) were characterized, along with wild-type and T-DNA knock-down plants. Plants that over-express AtSAP5 showed increased tolerance to environmental challenges including salt stress, osmotic stress and water deficit. Comparison of gene expression patterns between 35S::AtSAP5 transgenic plants and wild-type plants under normal conditions and water deficit stress indicated that over-expression of AtSAP5 correlates with up-regulation of drought stress responsive gene expression. Analysis of transgenic plants that express GFP-AtSAP5 showed that it is localized primarily in nuclei of root cells and recombinant AtSAP5 has E3 ubiquitin ligase activity in vitro. These results indicate that AtSAP5 has E3 ligase activity and acts as a positive regulator of stress responses in Arabidopsis.

• WOX family transcription factors regulate multiple developmental programs. The intermediate clade transcriptional activator WOX9 functions together with the modern clade transcriptional repressor WOX genes in embryogenesis and meristems maintenance, but the mechanism of this interaction is unclear. • STF and LAM1 are WOX1 orthologs required for leaf blade outgrowth in Medicago truncatula and Nicotiana sylvestris, respectively. Using biochemical methods and genome editing technology, here we show that WOX9 is an abaxial factor and functions antagonistically to STF and LAM1 to regulate leaf blade development. • While NsWOX9 ectopic expression enhances the lam1 mutant phenotype, and antisense expression partially rescues the lam1 mutant, both overexpression and knockout of NsWOX9 in N. sylvestris resulted in a range of severe leaf blade distortions, indicating important role in blade development. Our results indicate that direct repression of WOX9 by WUS clade repressor STF/LAM1 is required for correct blade architecture and patterning in M. truncatula and N. sylvestris. • These findings suggest that controlling transcriptional activation and repression mechanisms by direct interaction of activator and repressor WOX genes may be required for cell proliferation and differentiation homeostasis, and could be an evolutionarily conserved mechanism for the development of complex and diverse morphology in flowering plants.

Plant cell wall extensibility is mediated, in part, by xyloglucan endotransglycosylases/hydrolases (XTH) that are able to cleave and reattach xyloglucan polymers that make up the hemicelluloses matrix of type I cell walls. In Arabidopsis and other plants, XTHs are encoded by relatively large gene families that are regulated in specific spatial and temporal patterns. In silico screening of a cotton expressed sequence tag (EST) database identified 23 sequences with close sequence similarity to Arabidopsis XTH coding sequences. Analysis of full-length cotton cDNAs derived from these ESTs allow for the identification of three distinct GhXTH cDNAs (denoted GhXTH1, GhXTH2 and GhXTH3) based primarily on their 3′ untranslated sequences. The three GhXTH genes were expressed differently with GhXTH1 predominantly expressed in elongating cotton fibers. The function of GhXTH1 in mediating cotton fiber elongation was analyzed in transgenic cotton plants that express a transgene consisting of the GhXTH1 coding sequence under transcriptional control of the CaMV 35S promoter. Plants that over-expressed GhXTH1 had increased XTH activity and produced mature cotton fibers that were between 15 and 20% longer than wild-type cotton plants under both greenhouse and field growth conditions. Segregation analysis showed that the 35S::GhXTH1 transgene acts as a dominant fiber length allele in transgenic cotton. These results confirm that GhXTH1 is the predominant XTH in elongating fibers and its expression limits cotton fiber elongation.

Suppression of brassinosteroid (BR) biosynthesis in cotton ovules by treatment with brassinazole inhibits fiber formation, indicating that BR plays an important role in cotton fiber development. Plant responses to brassinosteroids (BR) are mediated through a plasma membrane-bound leucine-rich repeat (LRR) receptor-like protein kinase known as BRI1. Mutations in the BRI1 genes of several species result in dwarfed plants with reduced sensitivity to BR. A single expressed sequence tag (EST) from cotton with strong sequence similarity to Arabidopsis BRI1 ( AtBRI1 ) was identified in a search of publicly available databases. With this EST as a starting point, full-length cDNAs and genomic coding sequences from upland cotton ( Gossypium hirsutum ) BRI1 ( GhBRI1 ) were obtained and characterized. Ectopic expression of this coding sequence in BR-insensitive Arabidopsis plants resulted in recovery of normal growth indicating that GhBRI1 is a functional homologue of AtBRI1. G. hirsutum is an allotetraploid (AADD) derived from diploid ancestors. Analysis of several GhBRI1 cDNAs showed two distinct sequences indicating the presence of two GhBRI1 genes, denoted GhBRI1-1 and GhBRI1-2. Sequence comparisons between these GhBRI1 coding sequences and those from related A and D genome diploid Gossypium species ( G. arboreum and G. thurberi ) indicated that GhBRI1-1 is likely to the A sub-genome orthologue while GhBRI1-2 is from the D sub-genome.