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.

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.

ABSTRACTDELAY OF GERMINATION1(DOG1) represents a major quantitative locus for the genetic regulation of seed dormancy in Arabidopsis. Accumulation of DOG1 in seeds leads to deep dormancy and delayed germination. Here, we report that the conserved B3 DNA binding domains of the transcriptional repressors HIGH-LEVEL EXPRESSION OF SUGAR INDICIBLE GENE2/ VIVIPAROUS-1/ABSCISIC ACID INSENSITIVE 3-LIKE1 (HSI2/VAL1) and HSI2-LIKE1/ VIVIPAROUS-1/ABSCISIC ACID INSENSITIVE 3-LIKE2 (HSL1/VAL2), which play critical roles in the developmental transition from seed maturation to seedling growth, interact with RY elements in theDOG1proximal promoter leading to repression ofDOG1transcription during germination and seedling establishment.DOG1expression is partially de-repressed inhsi2/val1(hsi2-2) but not inhsl1/val2(hsl1-1) knockout mutants and is strongly upregulated in ahsi2/val1 hsl1/val2double mutant, indicating that HSI2/VAL1 and HSL1/VAL2 act redundantly to repressDOG1expression. HSI2/VAL1 and HSL1/VAL2 form homo- and hetero-dimersin vivo, and dimerization is dependent on the HSI2/VAL1 PHD-like domain. Complementation ofhsi2-2with HSI2/VAL1 harboring a disrupted plant homeodomain (PHD)-like domain results in stronger de-repression ofDOG1expression than thehsi2-2knockout, indicating that the PHD-like domain plays a critical role in mediating functional interactions between HSI2/VAL1 and HSL1/VAL2. Both HSI2/VAL1 and HSL1/VAL2 interact with components of polycomb repressive complex 2 (PRC2), including CURLY LEAF and MULTICOPY SUPPRESSOR OF IRA1 (MSI1), along with LIKE HETERCHROMATIN PROTEIN 1 (LHP1), which are involved in the deposition and expansion of histone H3 lysine 27 trimethylation (H3K27me3) marks in repressive chromatin. Thus, HSI2/VAL1 HSL1/VAL2-dependent recruitment of PRC2 leads to silencing ofDOG1through the deposition of H3K27me3.

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.

Plants have evolved complex molecular, cellular and physiological mechanisms to respond to environmental stressors. Because of the inherent complexity of this response, genetic manipulation to substantially improve water deficit tolerance, particularly in agricultural crops, has been largely unsuccessful, as the improvements are frequently accompanied by slower growth and delayed reproduction. Here, we ectopically express two abiotic stress-responsive bZIP AREB/ABF transcription factor orthologs, Arabidopsis ABF3 and Gossypium hirsutum ABF2D, in G. hirsutum, to compare the effects of exogenous and endogenous AREB/ABF transgene overexpression on dehydration resilience. Our results show that ectopic expression of each of these orthologs increases dehydration resilience, although these increases are accompanied by slower growth. These phenotypic effects are proportional to the ectopic expression level in the GhABF2D transgenic plants, while the phenotypes of all of the AtABF3 transgenic plants are similar, largely independent of ectopic expression level, possibly indicating differential post-transcriptional regulation of these transgenes. Our results indicate that overexpression of exogenous and endogenous ABF homologs in G. hirsutum substantially increases drought resilience, primarily through stomatal regulation, negatively impacting transpiration and photosynthetic productivity.

The AREB/ABF bZIP transcription factors play a pivotal role in abscisic acid-dependent abiotic stress-responsive gene expression. Despite the perennial damage and reduced productivity that result from water-deficit and unpredictable early season temperature fluctuations, these critical genes have not been previously examined in upland cotton (Gossypium hirsutum). Here, we report the isolation of the G. hirsutum ABF homologs, characterization of their expression patterns in response to abiotic stress treatments, and examination of their functions through heterologous ectopic expression in Arabidopsis. As expected for an allotetraploid, G. hirsutum ABF homologs are present in the genome as homeologous pairs. These genes are differentially expressed, both among the homologs and within the homeologous pairs, in response to exogenous abscisic acid (ABA) application, dehydration, and chilling temperatures. Furthermore, heterologous ectopic expression of many of the G. hirsutum ABF genes in Arabidopsis conferred increased tolerance to water deficit and osmotic stress, as well as cold tolerance, in a gene specific manner. These results indicate the G. hirsutum ABF homologs are functional in Arabidopsis and, as in other species, are likely to play an essential role in the abiotic stress response. Highlight The Gossypium hirsutum ABF homeologs are differentially expressed in response to abiotic stress, and their ectopic expression in Arabidopsis can confer increased water deficit tolerance.

Arabidopsis thaliana Stress Associated Protein 9 (AtSAP9) is a member of the A20/AN1 zinc finger protein family known to play important roles in plant stress responses and in the mammalian immune response. Although SAPs of several plant species were shown to be involved in abiotic stress responses, the underlying molecular mechanisms are largely unknown, and little is known about the involvement of SAPs in plant disease responses. Expression of SAP9 in Arabidopsis is up-regulated in response to dehydration, cold, salinity and abscisic acid (ABA), as well as pathogen infection. Constitutive expression of AtSAP9 in Arabidopsis leads to increased sensitivity to ABA and osmotic stress during germination and post-germinative development. Plants that overexpress AtSAP9 also showed increased susceptibility to infection by non-host pathogen Pseudomonas syringae pv. phaseolicola, indicating a potential role of AtSAP9 in disease resistance. AtSAP9 was found to interact with RADIATION SENSITIVE23d (Rad23d), a shuttle factor for the transport of ubiquitinated substrates to the proteasome, and it is co-localized with Rad23d in the nucleus. Thus, AtSAP9 may promote the protein degradation process by mediating the interaction of ubiquitinated targets with Rad23d. Taken together, these results indicate that AtSAP9 regulates abiotic and biotic stress responses, possibly via the ubiquitination/proteasome pathway.

Transgenic cotton lines that ectopically express a cotton germin-like protein (GLP) were screened for resistance/tolerance factors to the beet armyworm (BAW) Spodoptera exigua (Hubner) via feeding assays. The number of BAW eggs that successfully hatched was not statistically different at 72 h after infestation for wild-type cotton plants (Gossypium hirsutum L. c.v. 'Coker 312') or plants of 4 independent transgenic lines (ABP-A, ABP-B and ABP-C and ABP-D). However, the damage caused by these same larvae at 72 h was higher for 'Coker 312' and line ABP-D when compared to ABP-A, ABP-B and ABP-C transgenic plants. Larval live weights were also significantly higher for Coker 312 and ABP-D at 5, 7, and 14 d when compared to ABP-A, ABP-B and ABP-C. The percentage of larvae that successfully completed pupation was significantly higher for BAW larvae fed 'Coker 312' and ABP-D tissue compared to the other 3 lines. These feeding bioassays show the potential for using cotton germin like protein to improve resistance or tolerance for BAW attacking cotton.

The LOS2 gene in Arabidopsis encodes an enolase with 72% amino acid sequence identity with human ENO1. In mammalian cells, the α-enolase (ENO1) gene encodes both a 48 kDa glycolytic enzyme and a 37 kDa transcriptional suppressor protein that are targeted to different cellular compartments. The tumor suppressor c-myc binding protein (MBP-1), which is alternatively translated from the second start codon of ENO1 transcripts, is preferentially localized in nuclei while α-enolase is found in the cytoplasm. We report here that an Arabidopsis MBP-1-like protein (AtMBP-1) is alternatively translated from full-length LOS2 transcripts using a second start codon. Like mammalian MBP-1, this truncated form of LOS2 has little, if any, enolase activity, indicating that an intact N-terminal region of LOS2 is critical for catalysis. AtMBP-1 has a short half-life in vivo and is stabilized by the proteasome inhibitor MG132, indicating that it is degraded via the ubiquitin-dependent proteasome pathway. Arabidopsis plants that over-express AtMBP-1 are hypersensitive to abscisic acid (ABA) during seed germination and show defects in vegetative growth and lateral stem development. AtMBP-1 interacts directly with the E3 ubiquitin ligase AtSAP5 and co-expression of these proteins resulted in destabilization of AtMBP-1 in vivo and abolished the developmental defects associated with AtMBP-1 over-expression. Thus, AtMBP-1 is as a bona fide alternative translation product of LOS2. Accumulation of this factor is limited by ubiquitin-dependent destabilization, apparently mediated by AtSAP5.

The regulation of gene expression is a key factor in plant acclimation to stress, and it is thought that manipulation of the expression of critical stress-responsive genes should ultimately provide increased protection against abiotic stress. The aim of this study was to test the hypothesis that the ectopic expression of the AtSAP5 (AT3G12630) gene in transgenic cotton (Gossypium hirsutum, cv. Coker 312) will improve tolerance to drought and heat stress by up-regulating the expression of endogenous stress-responsive genes. The SAP5 gene is a member of the stress-associated family of genes that encode proteins containing A20/AN1 zinc finger domains. Under non-stressful conditions, cotton plants that expressed the AtSAP5 gene showed elevated expression of at least four genes normally induced during water deficit or heat stress. The rate of net CO2 assimilation A for three of four transgenic lines tested was less sensitive to rapidly developing water deficit over 4d than untransformed wild-type plants, but the recovery of A following drought was not significantly affected. The enhanced protection of photosynthesis during drought was determined to be primarily at the biochemical level, since the extent of stomatal closure was not significantly different for all genotypes. Expression of AtSAP5 resulted in the complete protection of photosystem (PS) II complexes from photodamage at mid-day after 4d of drought, whereas wild-type plants experienced a 20% decline in active photosystem II (PSII) complexes. In addition, enhanced protection of seedling growth and leaf viability was associated with the expression of AtSAP5. Since A for the transgenic plants was significantly more heat tolerant than A for wild-type plants, we conclude that ectopic expression of SAP genes is a potentially viable approach to improving carbon gain and productivity for cotton grown in semi-arid regions with severe drought and heat stress.

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.

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.