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WRKY transcription factors play a central role in controlling plant organ senescence; however, it is unclear whether and how they regulate petal senescence in the widely grown ornamental plant tulip (Tulipa gesneriana). In this study, we report that TgWRKY75 promotes petal senescence by enhancing the synthesis of both abscisic acid (ABA) and salicylic acid (SA) in tulip and in transgenic Arabidopsis. The expression level of TgWRKY75 was up-regulated in senescent petals, and exogenous ABA or SA treatment induced its expression. The endogenous contents of ABA and SA significantly increased during petal senescence and in response to TgWRKY75 overexpression. Two SA synthesis-related genes, TgICS1 and TgPAL1, were identified as direct targets of TgWRKY75, which binds to their promoters. In parallel, TgWRKY75 activated the expression of the ABA biosynthesis-related gene TgNCED3 via directly binding to its promoter region. Site mutation of the W-box core motif located in the promoters of TgICS1, TgPAL1, and TgNCED3 eliminated their interactions with TgWRKY75. In summary, our study demonstrates a dual regulation of ABA and SA biosynthesis by TgWRKY75, revealing a synergistic process of tulip petal senescence through feedback regulation between TgWRKY75 and the accumulation of ABA and SA., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

To bear harsh environmental threats, plants have developed complex protection mechanisms involving phytohormones, counting abscisic acid (ABA). The function of the F-Box family containing the Domain of Unknown Function 295 (DUF295) has not yet been comprehensively characterized in Arabidopsis (Arabidopsis thaliana). In this study, we evaluated the function of a putative member of the F-Box/DUF295 family in Arabidopsis, F-box/DUF295 Brassiceae specific 2 (FDB2). We found that FDB2 expression was suppressed by ABA and abiotic stresses. FDB2 overexpression (OE) reduced ABA sensitivity during seed germination and seedling growth, but enhanced ABA-sensitivity of seed germination and seedling growth in fdb2 mutants was scored. When treated with ABA, expressions of ABI3, ABI4 and ABI5 showed decreased in OE lines but increased in fdb2 mutants. In addition, ABA-induced FDB2 degradation exhibited sensitive to MG132, suggesting that FDB2 degradation by ABA might be mediated by the ubiquitin-26S proteasome system. Moreover, ABA-induced significant over-accumulation of reactive oxygen species (ROS) at the root tips of fdb2 mutants was observed, this phenomenon was correlated to reduced activities of a set of ROS scavengers in fdb2 mutants relative to Col-0. In summary, our results suggest that Arabidopsis FDB2 is involved in ABA-mediated inhibition of seed germination, seedling growth including modulation of ROS homeostasis in roots., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Abscisic acid (ABA) hydroxylation is an important pathway for ABA inactivation and homeostasis maintenance. Here, we discover a new downstream catabolite of neophaseic acid (neoPA) in the ABA 9'-hydroxyl pathway and identify it as epi-neodihydrophaseic acid (epi-neoDPA) by comparing its accurate mass, retention time, and MS n spectra with those of our chemically synthesized epi-neoDPA. Analyses of Arabidopsis seed germination and ABA-related gene expression reveal that neoPA rather than epi-neoDPA possesses ABA-like hormonal activity. In vitro enzyme activity tests of prokaryotic recombinant protein reveal that NeoPAR1 (neoPA reductase 1) identified from Arabidopsis converts neoPA into epi-neoDPA. Site-directed mutation at Tyr163 in the conserved motif of NeoPAR1 abolishes the catalytic activity of NeoPAR1. Accelerated seed germination was observed in NeoPAR1 knockdown and knockout mutants, whereas retarded seed germination and the accumulation of epi-neoDPA and ABA were observed in NeoPAR1 overexpression lines, suggesting that NeoPAR1 is involved in seed germination and maintenance of ABA homeostasis., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Melatonin is known to involve multiple physiological actions in plants. Herein, we found that exogenous melatonin inhibited the Arabidopsis seedling growth through the elevated abscisic acid (ABA) levels, and the elevated ABA was ascribed to the upregulation of 9-cis-epoxycarotenoid dioxygenase genes (NCEDs) in the ABA biosynthesis pathway. We also found that the overexpression lines of the melatonin receptor gene PMTR1 (also known as Cand2) yielded smaller seeds and germinated slower than the wild type, whereas PMTR1-knockout mutants produced larger seeds and germinated faster than the wild type. During the seed development, the accumulation peak of ABA was higher in the PMTR1-knockout mutant, while it was lower in the PMTR1-overexpression line than that in the wild type. In the dry seeds and imbibed seeds, the PMTR1-overexpression line accumulated higher ABA levels, while the PMTR1-knockout contained less ABA than the wild type. In summary, our findings suggest that PMTR1 is involved in ABA-mediated seed development and germination in Arabidopsis., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Phaseic acid (PA), a main catabolite of abscisic acid (ABA), is structurally related to ABA and possesses ABA-like hormonal activity. However, the comprehensive metabolism pathway and roles of PA are not well understood. Here, using homologous alignment and expression pattern analysis, we identified in Arabidopsis the previously named CRL1 (Cinnamoyl coA: NADP oxidoreductase-like 1) as a PA reductase that catalyses PA to dihydrophaseic acid. The function of CRL1 and the potential role of PA were studied in transgenic CRL1 plants. Overexpression of CRL1 resulted in decreased ABA sensitivity in seed germination and attenuated drought tolerance. In contrast, increased ABA sensitivity and elevated drought tolerance was observed in down-regulated and loss-of-function crl1 mutants. Tyr162 in the conserved motif is the key residue in CRL1 to catalyse PA. Accelerated seed germination and earlier flowering phenotype were also observed in overexpressing lines, while retarded seed germination and delayed flowering occurred in crl1 mutants which accumulated more PA, but less dihydrophaseic acid than the wild type. This study demonstrates that PA plays diverse functions in drought tolerance, seed germination and flowering in an ABA-like manner, which may increase the adaptive plasticity of plants., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Seed aging is the gradual decline in seed vigor, during which programmed cell death (PCD) occurs. The functions of nitric oxide (NO) are exerted through protein S-nitrosylation, a reversible post-translational modification. During seed aging, more than 80 proteins are S-nitrosylated, but the particular role of individual proteins is unknown. Here, we showed that the S-nitrosylation level of glyceraldehyde 3-phosphate dehydrogenase (UpGAPDH) in elm (Ulmus pumila L.) seeds increased after controlled deterioration treatment. UpGAPDH was S-nitrosylated at Cys154 during S-nitrosoglutathione (GSNO) treatment, and its oligomerization was triggered both in vitro and in elm seeds. Interestingly, UpGAPDH interacted with the mitochondrial voltage-dependent anion channel in an S-nitrosylation-dependent way. Some UpGAPDH-green fluorescent protein in Arabidopsis protoplasts co-localized with mitochondria during the GSNO treatment, while the S-nitrosylation-defective UpGAPDH C154S-GFP protein did not. Seeds of oxUpGAPDH lines showed cell death and lost seed vigor rapidly during controlled deterioration treatment-triggered seed aging, while those overexpressing S-nitrosylation-defective UpGAPDH-Cys154 did not. Our results suggest that S-nitrosylation of UpGAPDH may accelerate cell death and seed deterioration during controlled deterioration treatment. These results provide new insights into the effects of UpGAPDH S-nitrosylation on protein interactions and seed aging., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Brassinosteroids (BRs) are endogenous plant growth-promoting hormones affecting growth and development during the entire life cycle of plants. Naturally occurring BRs can be classified into C 27 -, C 28 -, or C 29 -BRs based on the nature of the alkyl groups occupying the C-24 position in the side chain of the 5a-cholestane carbon skeleton. However, while C 27 -BRs exhibit similar bioactivities to C 28 - and C 29 -BRs, the biosynthetic pathways of C 27 -BRs in plants have not yet been clearly characterized. In addition to a lack of biochemical and enzymatic evidence regarding the biosynthetic pathways of C 27 -BRs, even most of the intermediate compounds on their pathways have not been explored and identified due to the lower endogenous levels of C 27 -BRs. Therefore, the development of highly sensitive analytical methods is essential for studying the biosynthetic pathways and physiological functions of C 27 -BRs. Accordingly, this study establishes qualitative and quantitative methods for identifying and detecting C 27 -, C 28 -, and C 29 -BRs using a newly synthesized boronic acid reagent denoted as 2-methyl-4-phenylaminomethylphenylboronic acid (2-methyl-4-PAMBA) in conjunction with liquid chromatography-mass spectrometry (LC-MS). Labeling with 2-methyl-4-PAMBA provides derivatives with excellent stability, and the detection sensitivities of BRs, particularly for C 27 -BRs, are dramatically improved. The limits of detection (with a signal-to-noise ratio of 3) for six BRs, including 2 C 27 -BRs (28-norCS and 28-norBL), 3 C 28 -BRs (CS, BL, and TY), and a single C 29 -BR (28-homoBL), are found to be 0.10-1.68 pg/mL after labeling with 2-methyl-4-PAMBA. Finally, the proposed analytical method is successfully applied for the detection of endogenous BRs in small mass samples of Oryza sativa seedlings, Rape flowers, Arabidopsis shoots, and Arabidopsis flowers. In addition, a method for profiling potential BRs in plants is also developed using LC-MS in multiple reaction monitoring scan mode assisted by 2-methyl-4-PAMBA and 2-methyl-4-PAMBA-d 5 labeling. The developed method is able to identify 10 potential BRs in a Rape flower extract. The proposed quantitative and qualitative methods established by 2-methyl-4-PAMBA labeling are helpful for facilitating an understanding of the physiological functions and biosynthetic pathways of BRs, particularly for C 27 -BRs. Graphical abstract.

Seed storage proteins (SSPs) provide free amino acids and energy for the process of seed germination. Although degradation of SSPs by the aspartic proteases isolated from seeds has been documented in vitro, there is still no genetic evidence for involvement of aspartic proteases in seed germination. Here we report that the aspartic protease ASPG1 (ASPARTIC PROTEASE IN GUARD CELL 1) plays an important role in the process of dormancy, viability and germination of Arabidopsis seeds. We show that aspg1-1 mutants have enhanced seed dormancy and reduced seed viability. A significant increase in expression of DELLA genes which act as repressors in the gibberellic acid signal transduction pathway were detected in aspg1-1 during seed germination. Seed germination of aspg1-1 mutants was more sensitive to treatment with paclobutrazol (PAC; a gibberellic acid biosynthesis inhibitor). In contrast, seed germination of ASPG1 overexpression (OE) transgenic lines showed resistant to PAC. The degradation of SSPs in germinating seeds was severely impaired in aspg1-1 mutants. Moreover, the development of aspg1-1 young seedlings was arrested when grown on the nutrient-free medium. Thus ASPG1 is important for seed dormancy, seed longevity and seed germination, and its function is associated with degradation of SSPs and regulation of gibberellic acid signaling in Arabidopsis.

The phytohormones gibberellic acid (GA) and abscisic acid (ABA) antagonistically control seed germination. High levels of GA favor seed germination, whereas high levels of ABA hinder this process. The direct relationship between GA biosynthesis and seed germination ability need further investigation. Here, we identified the ABA-insensitive gain-of-function mutant germination insensitive to ABA mutant 2 (gim2) by screening a population of XVE T-DNA-tagged mutant lines. Based on two loss-of-function gim2-ko mutant lines, the disruption of GIM2 function caused a delay in seed germination. By contrast, upregulation of GIM2 accelerated seed germination, as observed in transgenic lines overexpressing GIM2 (OE). We detected a reduction in endogenous bioactive GA levels and an increase in endogenous ABA levels in the gim2-ko mutants compared to wild type. Conversely, the OE lines had increased endogenous bioactive GA levels and decreased endogenous ABA levels. The expression levels of a set of GA- and/or ABA-related genes were altered in both the gim2-ko mutants and the OE lines. We confirmed that GIM2 has dioxygenase activity using an in vitro enzyme assay, observing that GIM2 can oxidize GA 12 . Hence, our characterization of GIM2 demonstrates that it plays a role in seed germination by affecting the GA metabolic pathway in Arabidopsis., (© 2017 Institute of Botany, Chinese Academy of Sciences.)

Plants have developed complex mechanisms to respond to salt stress, depending on secondary messenger-mediated stress perception and signal transduction. Nitric oxide (NO) is widely known as a 'jack-of-all-trades' in stress responses. However, NO-mediated crosstalk between plant hormones remains unclear. In this study, we found that salt stabilized both AUXIN/INDOLE-3-ACETIC ACID 17 (Aux/IAA17) and RGA-LIKE3 (RGL3) proteins due to salt-induced NO production. Salt-induced NO overaccumulation and IAA17 overexpression decreased the transcripts of GA3ox genes, resulting in lower bioactive GA4. Further investigation showed that IAA17 directly interacted with RGL3 and increased its protein stability. Consistently, RGL3 stabilized IAA17 protein through inhibiting the interaction of TIR1 and IAA17 by competitively binding to IAA17. Moreover, both IAA17 and RGL3 conferred salt stress resistance. Overexpression of IAA17 and RGL3 partially alleviated the inhibitory effect of NO deficiency on salt resistance, whereas the iaa17 and rgl3 mutants displayed reduced responsiveness to NO-promoted salt resistance. Thus, the associations between IAA17 and gibberellin (GA) synthesis and signal transduction, and between the IAA17-interacting complex and the NO-mediated salt stress response were revealed based on physiological and genetic approaches. We conclude that integration of IAA17 and RGL3 is an essential component of NO-mediated salt stress response., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Drought stress is one of the disadvantageous environmental conditions for plant growth and reproduction. Given the importance of abscisic acid (ABA) to plant growth and abiotic stress responses, identification of novel components involved in ABA signalling transduction is critical. In this study, we screened numerous Arabidopsis thaliana mutants by seed germination assay and identified a mutant mlp43 (major latex protein-like 43) with decreased ABA sensitivity in seed germination. The mlp43 mutant was sensitive to drought stress while the MLP43-overexpressed transgenic plants were drought tolerant. The tissue-specific expression pattern analysis showed that MLP43 was predominantly expressed in cotyledons, primary roots and apical meristems, and a subcellular localization study indicated that MLP43 was localized in the nucleus and cytoplasm. Physiological and biochemical analyses indicated that MLP43 functioned as a positive regulator in ABA- and drought-stress responses in Arabidopsis through regulating water loss efficiency, electrolyte leakage, ROS levels, and as well as ABA-responsive gene expression. Moreover, metabolite profiling analysis indicated that MLP43 could modulate the production of primary metabolites under drought stress conditions. Reconstitution of ABA signalling components in Arabidopsis protoplasts indicated that MLP43 was involved in ABA signalling transduction and acted upstream of SnRK2s by directly interacting with SnRK2.6 and ABF1 in a yeast two-hybrid assay. Moreover, ABA and drought stress down-regulated MLP43 expression as a negative feedback loop regulation to the performance of MLP43 in ABA and drought stress responses. Therefore, this study provided new insights for interpretation of physiological and molecular mechanisms of Arabidopsis MLP43 mediating ABA signalling transduction and drought stress responses., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Phospholipase C (PLC) is an enzyme that plays crucial roles in various signal transduction pathways in mammalian cells. However, the role of PLC in plant development is poorly understood. Here we report involvement of PLC2 in auxin-mediated reproductive development in Arabidopsis. Disruption of PLC2 led to sterility, indicating a significant role for PLC2 in reproductive development. Development of both male and female gametophytes was severely perturbed in plc2 mutants. Moreover, elevated auxin levels were observed in plc2 floral tissues, suggesting that the infertility of plc2 plants may be associated with increased auxin concentrations in the reproductive organs. We show that expression levels of the auxin reporters DR5:GUS and DR5:GFP were elevated in plc2 anthers and ovules. In addition, we found that expression of the auxin biosynthetic YUCCA genes was increased in plc2 plants. We conclude that PLC2 is involved in auxin biosynthesis and signaling, thus modulating development of both male and female gametophytes in Arabidopsis., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

Abscisic acid (ABA) is an important phytohormone that functions in seed germination, plant development, and multiple stress responses. Arabidopsis Peroxisome defective 2 (AtPED2) (also known as AtPEXOXIN14, AtPEX14), is involved in the intracellular transport of thiolase from the cytosol to glyoxysomes, and perosisomal matrix protein import in plants. In this study, we assigned a new role for AtPED2 in drought stress resistance. The transcript level of AtPED2 was downregulated by ABA and abiotic stress treatments. AtPED2 knockout mutants were insensitive to ABA-mediated seed germination, primary root elongation, and stomatal response, while AtPED2 over-expressing plants were sensitive to ABA in comparison to wide type (WT). AtPED2 also positively regulated drought stress resistance, as evidenced by the changes of water loss rate, electrolyte leakage, and survival rate. Notably, AtPED2 positively modulated expression of several stress-responsive genes (RAB18, RD22, RD29A, and RD29B), positively affected underlying antioxidant enzyme activities and negatively regulated reactive oxygen species (ROS) level under drought stress conditions. Moreover, multiple carbon metabolites including amino acids, organic acids, sugars, sugar alcohols, and aromatic amines were also positively regulated by AtPED2. Taken together, these results indicated a positive role for AtPED2 in drought resistance, through modulation of stress-responsive genes expression, ROS metabolism, and metabolic homeostasis, at least partially., (© 2015 Institute of Botany, Chinese Academy of Sciences.)

Hydrogen sulfide (H2S) is an important gaseous molecule in various plant developmental processes and plant stress responses. In this study, the transgenic Arabidopsis thaliana plants with modulated expressions of two cysteine desulfhydrases, and exogenous H2S donor (sodium hydrosulfide, NaHS) and H2S scavenger (hypotaurine, HT) pre-treated plants were used to dissect the involvement of H2S in plant stress responses. The cysteine desulfhydrases overexpressing plants and NaHS pre-treated plants exhibited higher endogenous H2S level and improved abiotic stress tolerance and biotic stress resistance, while cysteine desulfhydrases knockdown plants and HT pre-treated plants displayed lower endogenous H2S level and decreased stress resistance. Moreover, H2S upregulated the transcripts of multiple abiotic and biotic stress-related genes, and inhibited reactive oxygen species (ROS) accumulation. Interestingly, MIR393-mediated auxin signaling including MIR393a/b and their target genes (TIR1, AFB1, AFB2, and AFB3) was transcriptionally regulated by H2S, and was related with H2S-induced antibacterial resistance. Moreover, H2S regulated 50 carbon metabolites including amino acids, organic acids, sugars, sugar alcohols, and aromatic amines. Taken together, these results indicated that cysteine desulfhydrase and H2S conferred abiotic stress tolerance and biotic stress resistance, via affecting the stress-related gene expressions, ROS metabolism, metabolic homeostasis, and MIR393-targeted auxin receptors., (© 2014 Institute of Botany, Chinese Academy of Sciences.)

Melatonin (N-acetyl-5-methoxytryptamine) serves as an important signal molecule during plant developmental processes and multiple abiotic stress responses. However, the involvement of melatonin in thermotolerance and the underlying molecular mechanism in Arabidopsis were largely unknown. In this study, we found that the endogenous melatonin level in Arabidopsis leaves was significantly induced by heat stress treatment, and exogenous melatonin treatment conferred improved thermotolerance in Arabidopsis. The transcript levels of class A1 heat-shock factors (HSFA1s), which serve as the master regulators of heat stress responses, were significantly upregulated by heat stress and exogenous melatonin treatment in Arabidopsis. Notably, exogenous melatonin-enhanced thermotolerance was largely alleviated in HSFA1s quadruple knockout (QK) mutants, and HSFA1s-activated transcripts of heat-responsive genes (HSFA2, heat stress-associated 32 (HSA32), heat-shock protein 90 (HSP90), and 101 (HSP101)) might be contributed to melatonin-mediated thermotolerance. Taken together, this study provided direct link between melatonin and thermotolerance and indicated the involvement of HSFA1s-activated heat-responsive genes in melatonin-mediated thermotolerance in Arabidopsis., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Auxin is a well-known plant phytohormone that is involved in multiple plant growth processes and stress responses. In this study, auxin response was significantly modulated under drought stress condition. The iaaM-OX transgenic lines with higher endogenous indole-3-acetic acid (IAA) level and IAA pre-treated wild type (WT) plants exhibited enhanced drought stress resistance, while the yuc1yuc2yuc6 triple mutants with lower endogenous IAA level showed decreased stress resistance in comparison to non-treated WT plants. Additionally, endogenous and exogenous auxin positively modulated the expression levels of multiple abiotic stress-related genes (RAB18, RD22, RD29A, RD29B, DREB2A, and DREB2B), and positively affected reactive oxygen species (ROS) metabolism and underlying antioxidant enzyme activities. Moreover, auxin significantly modulated some carbon metabolites including amino acids, organic acids, sugars, sugar alcohols and aromatic amines. Notably, endogenous and exogenous auxin positively modulated root architecture especially the lateral root number. Taken together, this study demonstrated that auxin might participate in the positive regulation of drought stress resistance, through regulation of root architecture, ABA-responsive genes expression, ROS metabolism, and metabolic homeostasis, at least partially., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Nitric oxide (NO) is involved in plant responses to many environmental stresses. Transgenic Arabidopsis lines that constitutively express rat neuronal NO synthase (nNOS) were described recently. In this study, it is reported that the nNOS transgenic Arabidopsis plants displayed high levels of osmolytes and increased antioxidant enzyme activities. Transcriptomic analysis identified 601 or 510 genes that were differentially expressed as a consequence of drought stress or nNOS transformation, respectively. Pathway and gene ontology (GO) term enrichment analyses revealed that genes involved in photosynthesis, redox, stress, and phytohormone and secondary metabolism were greatly affected by the nNOS transgene. Several CBF genes and members of zinc finger gene families, which are known to regulate transcription in the stress response, were changed by the nNOS transgene. Genes regulated by both the nNOS transgene and abscisic acid (ABA) treatments were compared and identified, including those for two ABA receptors (AtPYL4 and AtPYL5). Moreover, overexpression of AtPYL4 and AtPYL5 enhanced drought resistance, antioxidant enzyme activity, and osmolyte levels. These observations increase our understanding of the role of NO in drought stress response in Arabidopsis., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Several eukaryotic Heme-associated proteins (HAPs) have been reported to bind specifically to DNA fragments containing CCAAT-box; however, the physiological functions and direct targets of these HAP proteins in plants remain unclear. In this study, we showed that AtHAP5A as a transcription factor interacted with CCAAT motif in vivo, and AtXTH21, one direct target of AtHAP5A, was involved in freezing stress resistance. The AtHAP5A overexpressing plants were more tolerant, whereas the loss-of-function mutant of AtHAP5A was more sensitive to freezing stress than wild-type plants. Chromatin immunoprecipitation (ChIP) assay demonstrated that AtHAP5A could bind to five fragments that contained CCAAT motifs in the AtXTH21 promoter. Similarly, the AtXTH21 overexpressing plants exhibited improved freezing resistance, while xth21 knockdown mutants displayed decreased freezing resistance. Notably, the modulated freezing resistance of AtHAP5A overexpressing plants and knockout mutant could be reversed by the xth21 mutant and AtXTH21 overexpressing plants, respectively, indicating that AtHAP5A might act upstream of AtXTH21 in freezing stress. Additionally, modulation of AtHAP5A and AtXTH21 expression had the same effects on abscisic acid (ABA) sensitivity and reactive oxygen species (ROS) metabolism. Taken together, these results demonstrated that AtHAP5A modulates freezing stress resistance in Arabidopsis through binding to the CCAAT motif of AtXTH21., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)

Abscisic acid (ABA) is an important signaling molecule with multiple biological functions in seed germination, plant development and stress responses. ALTERED MERISTEM PROGRAM 1 (AMP1), encoding a putative glutamate carboxypeptidase, is involved in plant growth, morphogenesis and seed dormancy. In this study, we assigned new role for AtAMP1 in ABA signaling and dehydration stress. AtAMP1 was transcriptionally down-regulated by ABA. Loss-of-function mutant of AtAMP1 (amp1-1, encoding a premature stop codon in AtAMP1) resulted in hypersensitive phenotypes toward ABA-mediated seed germination and primary root elongation. The amp1-1 mutant also exhibited enhanced dehydration resistance, as evidenced by the changes of electrolyte leakage (EL), water loss rate and survival rate. Notably, the amp1-1 lines exhibited higher expression levels of ABA-responsive genes (RAB18, RD29A and RD29B), higher concentration of proline and lower reactive oxygen species (ROS) levels (H2O2 and O2(-)) after ABA and dehydration treatments than those of wild type. Taken together, these observations indicated a negative role for AtAMP1 in ABA-mediated seed germination, seedling development and dehydration stress response., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Arginine is an important medium for the transport and storage of nitrogen, and arginase (also known as arginine amidohydrolase, ARGAH) is responsible for catalyse of arginine into ornithine and urea in plants. In this study, the impact of AtARGAHs on abiotic stress response was investigated by manipulating AtARGAHs expression. In the knockout mutants of AtARGAHs, enhanced tolerances were observed to multiple abiotic stresses including water deficit, salt, and freezing stresses, while AtARGAH1- and AtARGAH2-overexpressing lines exhibited reduced abiotic stress tolerances compared to the wild type. Consistently, the enhanced tolerances were confirmed by the changes of physiological parameters including electrolyte leakage, water loss rate, stomatal aperture, and survival rate. Interestingly, the direct downstream products of arginine catabolism including polyamines and nitric oxide (NO) concentrations significantly increased in the AtARGAHs-knockout lines, but decreased in overexpressing lines under control conditions. Additionally, the AtARGAHs-overexpressing and -knockout lines displayed significantly reduced relative arginine (% of total free amino acids) relative to the wild type. Similarly, reactive oxygen species accumulation was remarkably regulated by AtARGAHs under abiotic stress conditions, as shown from hydrogen peroxide (H2O2), superoxide radical ( ) concentrations, and antioxidant enzyme activities. Taken together, this is the first report, as far as is known, to provide evidence that AtARGAHs negatively regulate many abiotic stress tolerances, at least partially, attribute to their roles in modulating arginine metabolism and reactive oxygen species accumulation. Biotechnological strategy based on manipulation of AtARGAHs expression will be valuable for future crop breeding.

Seed germination and flowering initiation are both transitions responding to similar seasonal cues. This study shows that ABSCISIC ACID-INSENSITIVE MUTANT 5 (ABI5), a bZIP transcription factor, which plays an important role in the abscisic acid (ABA)-arrested seed germination, is robustly associated with the floral transition in Arabidopsis. Under long-day conditions, overexpression of ABI5 could delay floral transition through upregulating FLOWERING LOCUS C (FLC) expression. In contrast, ectopically overexpressing FLC in an abi5 mutant reversed the earlier flowering phenotype. Further analysis indicated that transactivation of FLC could be promoted by ABI5 and/or other abscisic acid-responsive element (ABRE)-binding factors (ABFs). The expression of FLC that was promoted by ABI5 and/or other ABFs could be blocked in a triple SNF1-related protein kinase (SnRK) mutant, snrk2.2/2.3/2.6, despite the presence of ABA. In sharp contrast, when SnRK2.6 was coexpressed, the reduction of transactivity of FLC was reverted in mesophyll protoplasts of snrk2.2/2.3/2.6. Additional results from analysing transgenic plants carrying mutations of phosphoamino acids (ABI5 ( S42AS145AT201A )), which are conserved in ABI5, suggested that SnRK2-mediated ABI5 and/or ABF phosphorylation may be crucial for promoting FLC expression. The transgenic plants ABI5 ( S42AS145AT201A ) were insensitive to ABA in seed germination, in addition to having an earlier flowering phenotype. Direct binding of ABI5 to the ABRE/G-box promoter elements existing in FLC was demonstrated by chromatin immunoprecipitation. Mutations at the ABRE/G-box regions in FLC promoter sequences abolished the ABI5-promoted transactivation of FLC. In summary, these results may decipher the inhibitory effect of ABA on floral transition in Arabidopsis.

Drought is one of the most severe environmental stresses affecting plant growth and limiting crop production. Although many genes involved in adaptation to drought stress have been disclosed, the relevant molecular mechanisms are far from understood. This study describes an Arabidopsis gene, ASPG1 (ASPARTIC PROTEASE IN GUARD CELL 1), that may function in drought avoidance through abscisic acid (ABA) signalling in guard cells. Overexpression of the ASPG1 gene enhanced ABA sensitivity in guard cells and reduced water loss in ectopically overexpressing ASPG1 (ASPG1-OE) transgenic plants. In ASPG1-OE plants, some downstream targets in ABA and/or drought-signalling pathways were altered at various levels, suggesting the involvement of ASPG1 in ABA-dependent drought avoidance in Arabidopsis. By analysing the activities of several antioxidases including superoxide dismutase and catalase in ASPG1-OE plants, the existence was demonstrated of an effective detoxification system for drought avoidance in these plants. Analysis of ProASPG1-GUS lines showed a predominant guard cell expression pattern in various aerial tissues. Moreover, the protease activity of ASPG1 was characterized in vitro, and two aspartic acid sites, D180 and D379, were found to be key residues for ASPG1 aspartic protease activity in response to ABA. In summary, these findings suggest that functional ASPG1 may be involved in ABA-dependent responsiveness and that overexpression of the ASPG1 gene can confer drought avoidance in Arabidopsis.

Abscisic acid (ABA) and cytokinin are key hormones controlling plant development. How ABA and cytokinin interplay to control the transition from a dry seed into a young seedling remains elusive. Here we undertook a gain-of-function genetic screen to identify ABA-insensitive mutants during seed germination in Arabidopsis using an estradiol-inducible approach. In the presence of estradiol, one of these mutants gim1 (germination insensitive to ABA mutant 1) exhibited an elevated level of cytokinin that was attributed to the estradiol-induced expression of AtIPT8 that encodes an isopentenyltransferase for the biosynthesis of cytokinins. Our data on OE-2 and Com-1 transgenic plants carrying the ectopically expressing AtIPT8 gene indicated that the elevation of cytokinin level was responsible for the ABA-insensitivity of gim1 seed germination. Further analyses on alterations of gene transcriptomes in the gim1 mutant demonstrated that the expression of some ABA-inducible genes, including ABI5, was reduced, and could not be restored by exogenous ABA treatment. Moreover, we also failed to observe the ABA-mediated repression of a family of cytokinin signal transducers and transcription repressors called type-A ARR4, ARR5 and ARR6 in the gim1 seedlings. Further analysis demonstrated that type-A ARR4, ARR5 and ARR6 could negatively regulate ABI5 expression, and the physical interaction of ABI5 and type-A ARR4, ARR5 and ARR6 proteins was detected. In summary, our study suggests that the interaction of ABA and cytokinin during seed germination and seedling growth can be mediated by the interplay of transcriptional regulators in Arabidopsis., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Abstract: The phytohormones gibberellic acid (GA) and abscisic acid (ABA) antagonistically control seed germination. High levels of GA favor seed germination, whereas high levels of ABA hinder this process. The direct relationship between GA biosynthesis and seed germination ability need further investigation. Here, we identified the ABA‐insensitive gain‐of‐function mutant germination insensitive to ABA mutant 2 (gim2) by screening a population of XVE T‐DNA‐tagged mutant lines. Based on two loss‐of‐function gim2‐ko mutant lines, the disruption of GIM2 function caused a delay in seed germination. By contrast, upregulation of GIM2 accelerated seed germination, as observed in transgenic lines overexpressing GIM2 (OE). We detected a reduction in endogenous bioactive GA levels and an increase in endogenous ABA levels in the gim2‐ko mutants compared to wild type. Conversely, the OE lines had increased endogenous bioactive GA levels and decreased endogenous ABA levels. The expression levels of a set of GA‐ and/or ABA‐related genes were altered in both the gim2‐ko mutants and the OE lines. We confirmed that GIM2 has dioxygenase activity using an in vitro enzyme assay, observing that GIM2 can oxidize GA12. Hence, our characterization of GIM2 demonstrates that it plays a role in seed germination by affecting the GA metabolic pathway in Arabidopsis. [ABSTRACT FROM AUTHOR]

Several eukaryotic Heme-associated proteins (HAPs) have been reported to bind specifically to DNA fragments containing CCAAT-box; however, the physiological functions and direct targets of these HAP proteins in plants remain unclear., In this study, we showed that At HAP5A as a transcription factor interacted with CCAAT motif in vivo, and AtXTH21, one direct target of At HAP5A, was involved in freezing stress resistance. The AtHAP5A overexpressing plants were more tolerant, whereas the loss-of-function mutant of AtHAP5A was more sensitive to freezing stress than wild-type plants. Chromatin immunoprecipitation (Ch IP) assay demonstrated that At HAP5A could bind to five fragments that contained CCAAT motifs in the AtXTH21 promoter., Similarly, the AtXTH21 overexpressing plants exhibited improved freezing resistance, while xth21 knockdown mutants displayed decreased freezing resistance. Notably, the modulated freezing resistance of AtHAP5A overexpressing plants and knockout mutant could be reversed by the xth21 mutant and AtXTH21 overexpressing plants, respectively, indicating that AtHAP5A might act upstream of AtXTH21 in freezing stress. Additionally, modulation of AtHAP5A and AtXTH21 expression had the same effects on abscisic acid ( ABA) sensitivity and reactive oxygen species ( ROS) metabolism., Taken together, these results demonstrated that At HAP5A modulates freezing stress resistance in Arabidopsis through binding to the CCAAT motif of AtXTH21. [ABSTRACT FROM AUTHOR]