Phosphoproteomic analysis of SnRK2 QKO of physcomitrella patens
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ABSTRACT:
ORGANISM(S): Cellular Organisms
SUBMITTER: Shinnosuke Ishikawa
PROVIDER: PXD010928 | JPOST Repository | Wed Aug 28 00:00:00 BST 2019
REPOSITORIES: jPOST
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Project description:Abscisic acid (ABA) is a key hormone for plant growth, development and stress adaptation. Perception of ABA through four types of receptors has been reported. We show here that impairment of ABA perception through the PYR/PYL/RCAR branch reduces vegetative growth and seed production, and leads to a severely open stomata and dramatic ABA insensitive phenotype, even though other branches for ABA perception remain functional. Arabidopsis sextuple mutant impaired in 6 PYR/PYL receptors, namely PYR1, PYL1, PYL2, PYL4, PYL5 and PYL8, was able to germinate and grow even on 100 mM ABA. Whole-rosette stomatal conductance (Gst) measurements revealed that leaf transpiration in the sextuple pyr/pyl mutant was higher than in the ABA-deficient aba3-1 or ABA-insensitive snrk2.6 mutants. The gradually increasing Gst values of plants lacking three, four, five and six PYR/PYLs indicate quantitative regulation of stomatal aperture by this family of receptors. The sextuple mutant lacked ABA-mediated activation of SnRK2s and ABA-responsive gene expression was dramatically impaired as was reported in snrk2.2/2.3/2.6. In summary, these results show that ABA perception by PYR/PYLs plays a major role to regulate seed germination and establishment, basal ABA signaling required for vegetative and reproductive growth, stomatal aperture and transcriptional response to the hormone. Four biological replicates were generated for the 3 genotypes, pyr1pyl1pyl2pyl4pyl5pyl8 sextuple mutant, snrk2.2/2.3/2.6 triple mutant, and Col-0. All the samples were treated with ABA for 3 hours before harvesting. The four Col-0 samples were mix to create an unique reference Col-0 sample. Two comparasion were made, fisrt one, PYR/PYL sextuple mutant versus Col-0 reference sample and second one, SnRK2 triple mutant versus Col-0 reference sample. In each comparasion four biological replicates were made. Replicas number 1 and 2 were labeled with Cy5 for the mutant sample and Cy3 for the Col-0 reference sample, while the other two replicas,#3 and #4, were reversed-labeled.
2012-11-30 | E-GEOD-36692 | biostudies-arrayexpress
Project description:Resistance to drought stress is fundamental to plant survival and development. Abscisic acid (ABA) is one of the major hormones involved in different types of abiotic and biotic stress responses. ABA intracellular signaling has been extensively explored in Arabidopsis thaliana and occurs via a phosphorylation cascade mediated by three related protein kinases, denominated SnRK2s (SNF1-related protein kinases). However, the role of ABA signaling and the biochemistry of SnRK2 in crop plants remains underexplored. Considering the importance of the ABA hormone in abiotic stress tolerance, here we investigated the regulatory mechanism of sugarcane SnRK2s - known as SAPKs (Stress/ABA-activated Protein Kinases). The crystal structure of ScSAPK10 revealed the characteristic SnRK2 family architecture, in which the regulatory SnRK2-box interacts with the kinase domain αC helix. To study sugarcane SnRK2 regulation, we produced a series of mutants for the protein regulatory domains SnRK2-box and ABA-box. Surprisingly, mutations in the SnRK2-box did not drastically affect sugarcane SnRK2 activity, in contrast to previous observations for the homologous proteins in Arabidopsis. Also, we found that the ABA-box might have a role in SnRK2 activation in the absence of PP2C phosphatase. Taken together, our results demonstrate that both C-terminal domains of sugarcane SnRK2 proteins play a role in protein activation and activity.
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Project description:The endosperm is a nutritive tissue supporting embryo growth in flowering plants. Most commonly, the endosperm initially develops as a coenocyte (multinucleate cell) and then cellularizes. This process of cellularization is frequently disrupted in hybrid seeds generated by crosses between different flowering plant species or plants that differ in ploidy, resulting in embryo arrest and seed lethality. The reason for embryo arrest upon cellularization failure remains unclear. In this study, we show that triploid Arabidopsis thaliana embryos surrounded by uncellularized endosperm mount an osmotic stress response that is connected to increased levels of abscisic acid (ABA) and enhanced ABA responses. Impairing ABA biosynthesis and signalling aggravated triploid seed abortion, while increasing endogenous ABA levels as well as the exogenous application of ABA induced endosperm cellularization and suppressed embryo growth arrest. Taking these results together, we propose that endosperm cellularization is required to establish dehydration tolerance in the developing embryo, ensuring its survival during seed maturation.
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Project description:To understand the Abscisic Acid (ABA) signaling in response to dehydration stress, we performed analysis of gene expression using Arabidopsis wild-type plants and the nced3-2 mutant under dehydration stress. The nced3-2 mutant is an Arabidopsis T-DNA tagged knock-out mutant of the NCED3 gene, which has an essential role in dehydration-inducible ABA biosynthesis. Arabidopsis plants were grown in in soil (verdenite 40 mmΦ, Verde Co., Ltd., Kanagawa, Japan) in a cell strainer (Falcon, 40 μm; Corning Inc., NY, USA). Plants were grown at 22°C for 3 weeks under illumination (40â60 μmol m-2s-1; 16 h light/8 h darkness). Three-week-old plants were exposed to dehydration stress by being denied water for 6, 24, 48, or 72 h.
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Project description:Multiple transcription factors (TFs) play essential roles in plants under abiotic stress, one of the most challenging conditions of plant survival. However, how these multiple TFs cooperate in abiotic stress responses still remains largely unknown. In this study, we provide evidence that a novel NAC (NAM, ATAF1/2, and CUC2) transcription factor (ANAC096) cooperates with bZIP-type TFs [ABRE-binding factor/ABRE-binding protein (ABF/AREB)] in ensuring survival under dehydration and osmotic stress conditions. Intriguingly, ANAC096 directly interacted with ABF2 and ABF4, but not with ABF3, both in vitro and in vivo. ANAC096 and ABF2 synergistically activated RD29A transcription. The genome-wide gene expression analysis revealed that a major proportion of ABA-responsive genes are under the transcriptional regulation of ANAC096.An Arabidopsis mutant, anac096, was hyposensitive to exogenous abscisic acid (ABA), and showed impaired ABA-induced stomatal closure and increased water loss under dehydration stress conditions. Furthermore, the anac096 abf2 abf4 triple mutant was much more sensitive to dehydration and osmotic stresses than the anac096 single mutant or the abf2 abf4 double-mutant. Based on these results, we propose that ANAC096 is involved in a synergistic relationship with a subset of ABFs for the transcriptional activation of ABA-inducible genes in response to dehydration and osmotic stresses.
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Project description:The signaling pathway of the phytohormone abscisic acid (ABA) regulates responses towards abiotic stress such as drought and high osmotic conditions. The multitude of functionally redundant components involved in ABA signaling, poses a major challenge for elucidating the largely unresolved response selectivity. We decided rebuilding single linear ABA signaling pathways in yeast for combinatoric permutation of ABA receptors and coreceptors, as well as the response-mediating SnRK2 protein kinases and their targeted transcription factors to drive luciferase expression in a heterologous host. We show that SnRK2s differ in the regulation by ABA receptor complexes, affect ABA responsiveness of the pathway, and differ in their transactivation activity but have similar preferences for ABA-responsive transcription factors. SnRK2s thought to act ABA-independently and known to be activated under osmotic stress in plants were regulated by ABA receptor complexes in yeast and competed with ABA receptor components in an ABA-dependent manner in plant tissue. The study reveals the suitability of the yeast system for analysis of ABA signaling factors and allowing the future dissection of ligand-receptor specificities in a functional response pathway. The analysis provides new insights into SnRK2 regulation indicating that four SnRK2 members of the osmotic stress response are tightly embedded into the ABA signaling pathway.
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Project description:Large gene families and the frequent overlapping functions of homologous genes remain a major challenge for functional genetics in plants. In recent years, the development of homology-based gene silencing using computationally generated artificial microRNAs (amiRNAs) has been demonstrated to be a promising tool for functional genomics in plants. In this study, through a forward genetics screen, we isolated an abscisic acid-insensitive amiRNA line targeting five previously uncharacterized F-box Insensitive to ABA (FIA) genes. Notably, a triple mutant in the identified FIA genes FIA1, FIA3 and FIA4 (f1/f3/f4), that are expressed in germinating seeds, exhibited ABA insensitivity in the inhibition of seed germination. In contrast, this ABA insensitivity was not observed in a double mutant of two FIA genes FIA1 and FIA4 (f1/f4). Further investigation of the FIA1 interactome using F box decoy lines revealed the gibberellin (GA)-responsive GASA1 protein that has been reported to encode a small stress signaling peptide as an interacting partner. We found that abscisic acid promoted the ubiquitination of GASA1 in Arabidopsis, leading to its degradation via the 26S proteasome pathway. Together, our study reveals that abscisic acid represses seed germination through FIA proteins and regulates the FIA interactor, GA-responsive GASA1.
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