ABI5 Gene Research Articles

Genome-wide identification and expression profiling of the ABI5 gene family in foxtail millet (Setaria italica)

BackgroundABA Insensitive 5 (ABI5) is a basic leucine zipper transcription factor that crucially influences plant growth, development, and stress response. However, there is minimal research on the ABI5 family in foxtail millet.ResultsIn this study, 16 ABI5 genes were identified in foxtail millet, and their sequence composition, gene structures, cis-acting elements, chromosome positions, and gene replication events were analyzed. To more thoroughly evaluate the developmental mechanisms of the SiABI5 family during evolution, we selected three dicotyledons (S. lycopersicum, A. thaliana, F. tataricum) and three (Z. mays, O. sativa, S. bicolor) specific representative monocotyledons associated with foxtail millet for comparative homology mapping. The results showed that foxtail millet ABI5 genes had the best homology with maize. A promoter sequence analysis showed that the SiABI5s contain numerous cis-acting elements related to hormone and stress responses, indicating that the regulation of SiABI5 expression was complex. The expression responses of 16 genes in different tissues, seed germination, and ear development were analyzed. A total of six representative genes were targeted from five subfamilies to characterize their gene expression responses to four different abiotic stresses. Overexpression of SiABI5.12 confers tolerance to osmotic stress in transgenic Arabidopsis thaliana, which demonstrated the function of SiABI5 responded to abiotic stress.ConclusionsIn summary, our research results comprehensively characterized the SiABI5 family and can provide a valuable reference for demonstrating the role of SiABI5s in regulating abiotic stress responses in foxtail millet.

Characterization and expression analysis of SnRK2, PYL, and ABF/ AREB/ ABI5 gene families in sweet potato.

Abscisic acid (ABA) signaling in plants is essential to several aspects of plant development, such as tolerance to environmental stresses and growth. ABA signaling is also important for storage organ formation in crops, such as sweet potato. However, the repertoire of I. batatas ABA signaling gene families has not yet been fully characterized, so that it is unclear which members of these families are necessary for tuberization. Therefore, genome-wide identification of the sweet potato ABF/ AREB/ ABI5, SnRK2, and PYL gene families was performed, along with phylogenetic, motif, cis-regulatory element (CRE), and expression analyses. Nine ABF, eight SnRK2, and eleven PYL gene family members were identified, and there was high sequence conservation among these proteins that were revealed by phylogenetic and motif analyses. The promoter sequences of these genes had multiple CREs that were involved in hormone responses and stress responses. In silico and qRT-PCR expression analyses revealed that these genes were expressed in various tissues and that IbABF3, IbABF4, IbDPBF3, IbDPBF4, IbPYL4, IbSnRK2.1, and IbSnRK2.2 were significantly expressed during storage root development. These results are an important reference that can be used for functional validation studies to better understand how ABA signaling elicits storage root formation at the molecular level.

SLs signal transduction gene CsMAX2 of cucumber positively regulated to salt, drought and ABA stress in Arabidopsis thaliana L.

Recent studies have demonstrated that strigolactones (SLs) participate in the regulation of stress adaptation, however, the mechanisms remain elusive. MAX2 (MORE AXILLARY GROWTH2) is the key gene in the signal transduction pathway of SLs. This study aimed to clone and functionally characterize the CsMAX2 gene of cucumber in Arabidopsis. The results showed that the expression levels of the CsMAX2 gene changed significantly after salt, drought, and ABA stresses in cucumber. Moreover, the overexpression of CsMAX2 promoted stress tolerance and increased the germination rate and root length of Arabidopsis thaliana. Meanwhile, the content of chlorophyll increased and malondialdehyde decreased in CsMAX2 OE lines under salt and drought stresses. Additionally, the expression levels of stress-related marker genes, especially AREB1 and COR15A, were significantly upregulated under salt stress, while the expression levels of all genes were upregulated under drought stress, except ABI4 and ABI5 genes. The level of NCED3 continued to rise under both salt and drought stresses. In addition, D10 and D27 gene expression level also showed a continuous increase under ABA stress. The result suggested the interaction between SL and ABA in the process of adapting to stress. Overall, CsMAX2 could positively regulate salt, drought, and ABA stress resistance, and this process correlated with ABA transduction.

Overexpression of the Salix matsudana SmAP2-17 gene improves Arabidopsis salinity tolerance by enhancing the expression of SOS3 and ABI5

BackgroundSalix matsudana (Koidz.) is a widely planted ornamental allotetraploid tree species. Genetic engineering can be used to enhance the tolerance of this species to soil salinization, endowing varieties with the ability to grow along coastlines, thereby mitigating afforestation and protecting the environment. The AP2/ERF family of transcription factors (TFs) plays multidimensional roles in plant biotic/abiotic stress tolerance and plant development. In this study, we cloned the SmAP2-17 gene and performed functional analysis of its role in salt tolerance. This study aims to identify key genes for future breeding of stress-resistant varieties of Salix matsudana.ResultsSmAP2-17 was predicted to be a homolog of AP2-like ethylene-responsive transcription factor ANT isoform X2 from Arabidopsis, with a predicted ORF of 2058 bp encoding an estimated protein of 685 amino acids containing two conserved AP2 domains (PF00847.20). SmAP2-17 had a constitutive expression pattern and was localized to the nucleus. The overexpression of the native SmAP2-17 CDS sequence in Arabidopsis did not increase salt tolerance because of the reduced expression level of ectopic SmAP2-17, potentially caused by salt-induced RNAi. Transgenic lines with high expression of optimized SmAP2-17 CDS under salt stress showed enhanced tolerance to salt. Moreover, the expression of general stress marker genes and important salt stress signaling genes, including RD29A, ABI5, SOS3, AtHKT1, and RBohF, were upregulated in SmAP2-17-overexpressed lines, with expression levels consistent with that of SmAP2-17 or optimized SmAP2-17. Promoter activity analysis using dual luciferase analysis showed that SmAP2-17 could bind the promoters of SOS3 and ABI5 to activate their expression, which plays a key role in regulating salt tolerance.ConclusionsThe SmAP2-17 gene isolated from Salix matsudana (Koidz.) is a positive regulator that improves the resistance of transgenic plants to salt stress by upregulating SOS3 and ABI5 genes. This study provides a potential functional gene resource for future generation of salt-resistant Salix lines by genetic engineering.

The miR159‐MYB33‐ABI5 module regulates seed germination in Arabidopsis

AbstractDrought stress restricts crop productivity and exacerbates food shortages. The plant hormone, abscisic acid (ABA), has been shown to be a pivotal player in the regulation of drought tolerance and seed germination in plants. ABA accumulates under abiotic stresses to promote miR159 expression. miR159 is an ancient and conserved plant miRNA that plays diverse roles in plant development, seed germination, and drought response in Arabidopsis. Our previous studies demonstrated that miR159 regulates the vegetative phase change by repressing the ABI5 activation and thereafter preventing hyperactivation of miR156. However, whether the miR159‐MYB33‐ABI5 module plays a role in seed germination and drought response, and if so, how they interact genetically, remain largely unexplored. Here, we show that loss‐of‐function of miR159 (mir159ab) confers enhanced drought tolerance and hypersensitivity of seed germination to ABA. Genetic analyses demonstrated that loss‐of‐function mutation in the ABI5 gene suppresses the hypersensitivity of mir159ab to ABA, and the insensitivity of myb33 seeds to ABA treatment is ABI5 dependent. ABI5 functions downstream of MYB33 and miR159 in response to ABA. Therefore, our results uncover a new role for the miR159‐MYB33‐ABI5 module in the regulation of drought response and seed germination in plants.

ENAP1 retrains seed germination via H3K9 acetylation mediated positive feedback regulation of ABI5.

Histone acetylation is involved in the regulation of seed germination. The transcription factor ABI5 plays an essential role in ABA- inhibited seed germination. However, the molecular mechanism of how ABI5 and histone acetylation coordinate to regulate gene expression during seed germination is still ambiguous. Here, we show that ENAP1 interacts with ABI5 and they co-bind to ABA responsive genes including ABI5 itself. The hypersensitivity to ABA of ENAP1ox seeds germination is recovered by the abi5 null mutation. ABA enhances H3K9Ac enrichment in the promoter regions as well as the transcription of target genes co-bound by ENAP1 and ABI5, which requires both ENAP1 and ABI5. ABI5 gene is directly regulated by ENAP1 and ABI5. In the enap1 deficient mutant, H3K9Ac enrichment and the binding activity of ABI5 in its own promoter region, along with ABI5 transcription and protein levels are all reduced; while in the abi5-1 mutant, the H3K9Ac enrichment and ENAP1 binding activity in ABI5 promoter are decreased, suggesting that ENAP1 and ABI5 function together to regulate ABI5- mediated positive feedback regulation. Overall, our research reveals a new molecular mechanism by which ENAP1 regulates H3K9 acetylation and mediates the positive feedback regulation of ABI5 to inhibit seed germination.

Bacterial Quorum-Sensing Signaling-Related drr1 Mutant Influences Abscisic Acid Responsiveness in Arabidopsis thaliana L.

N-acyl-L-homoserine lactones (AHLs) are involved in cell-to-cell communication in Gram-negative bacteria through a process termed quorum-sensing (QS). In this report, we evaluated the response of Arabidopsis thaliana primary roots to abscisic acid (ABA) in wild-type (WT) and decanamide resistant root 1 (drr1) mutant, previously reported to be resistant to N-decanoyl-L-homoserine lactone (C10-HL). When compared to WT seedlings, drr1 mutants were hypersensitive to ABA and had primary roots shorter, which correlated with lower cell division in meristems, a higher concentration of endogenous ABA, and a greater expression of ABI5 gene; and this shortened primary root phenotype was reversed in drr1abi5 double mutants. An analysis of expression of ABSCISIC ACID INSENSITIVE 4 (ABI4) showed an ABA-inducible pattern in primary root tips, which was further increased in drr1 mutant seedlings. Comparison of seed germination in WT, drr1, abi5, and drr1abi5 lines showed higher germination percentages in the following order under control condition: abi5 > drr1abi5 > WT > drr1, while abi5 and drr1abi5 germinated faster and drr1 germinated slower with respect to WT under ABA condition. Taken together, our results suggest that DRR1 is a negative regulator of ABA signaling probably acting upstream of the transcription factors ABI4 and ABI5, which influence ABA responsiveness in primary roots and seed germination.

Wheat Germination Is Dependent on Plant Target of Rapamycin Signaling.

Germination is a process of seed sprouting that facilitates embryo growth. The breakdown of reserved starch in the endosperm into simple sugars is essential for seed germination and subsequent seedling growth. At the early stage of germination, gibberellic acid (GA) activates transcription factor GAMYB to promote de novo synthesis of isoforms of α-amylase in the aleurone layer and scutellar epithelium of the embryo. Here, we demonstrate that wheat germination is regulated by plant target of rapamycin (TOR) signaling. TOR is a central component of the essential-nutrient–dependent pathway controlling cell growth in all eukaryotes. It is known that rapamycin, a highly specific allosteric inhibitor of TOR, is effective in yeast and animal cells but ineffective in most of higher plants likely owing to structural differences in ubiquitous rapamycin receptor FKBP12. The action of rapamycin on wheat growth has not been studied. Our data show that rapamycin inhibits germination of wheat seeds and of their isolated embryos in a dose-dependent manner. The involvement of Triticum aestivum TOR (TaTOR) in wheat germination was consistent with the suppression of wheat embryo growth by specific inhibitors of the TOR kinase: pp242 or torin1. Rapamycin or torin1 interfered with GA function in germination because of a potent inhibitory effect on α-amylase and GAMYB gene expression. The TOR inhibitors selectively targeted the GA-dependent gene expression, whereas expression of the abscisic acid-dependent ABI5 gene was not affected by either rapamycin or torin1. To determine whether the TaTOR kinase activation takes place during wheat germination, we examined phosphorylation of a ribosomal protein, T. aestivum S6 kinase 1 (TaS6K1; a substrate of TOR). The phosphorylation of serine 467 (S467) in a hydrophobic motif on TaS6K1 was induced in a process of germination triggered by GA. Moreover, the germination-induced phosphorylation of TaS6K1 on S467 was dependent on TaTOR and was inhibited by rapamycin or torin1. Besides, a gibberellin biosynthesis inhibitor (paclobutrazol; PBZ) blocked not only α-amylase gene expression but also TaS6K1 phosphorylation in wheat embryos. Thus, a hormonal action of GA turns on the synthesis of α-amylase in wheat germination via activation of the TaTOR–S6K1 signaling pathway.

Ectopic expression of UGT75D1, a glycosyltransferase preferring indole-3-butyric acid, modulates cotyledon development and stress tolerance in seed germination of Arabidopsis thaliana.

The formation of auxin glucose conjugate is proposed to be one of the molecular modifications controlling auxin homeostasis. However, the involved mechanisms and relevant physiological significances are largely unknown or poorly understood. In this study, Arabidopsis UGT75D1 was at the first time identified to be an indole-3-butyric acid (IBA) preferring glycosyltransferase. Assessment of enzyme activity and IBA conjugates in transgenic plants ectopically expressing UGT75D1 indicated that the UGT75D1 catalytic specificity was maintained in planta. It was found that the expression pattern of UGT75D1 was specific in germinating seeds. Consistently, we found that transgenic seedlings with over-produced UGT75D1 exhibited smaller cotyledons and cotyledon epidermal cells than the wild type. In addition, UGT75D1 was found to be up-regulated under mannitol, salt and ABA treatments and the over-expression lines were tolerant to osmotic and salt stresses during germination, resulting in an increased germination rate. Quantitative RT-PCR analysis revealed that the mRNA levels of ABA INSENSITIVE 3 (ABI3) and ABI5 gene in ABA signaling were substantially down-regulated in the transgenic lines under stress treatments. Interestingly, AUXIN RESPONSE FACTOR 16 (ARF16) gene of transgenic lines was also dramatically down-regulated under the same stress conditions. Since ARF16 functions as an activator of ABI3 transcription, we supposed that UGT75D1 might play a role in stress tolerance during germination through modulating ARF16-ABI3 signaling. Taken together, our work indicated that, serving as the IBA preferring glycosyltransferase but distinct from other auxin glycosyltransferases identified so far, UGT75D1 might be a very important player mediating a crosstalk between cotyledon development and stress tolerance of germination at the early stage of plant growth.

AtGRDP1 Gene Encoding a Glycine-Rich Domain Protein Is Involved in Germination and Responds to ABA Signalling

Seed maturation and germination involve changes in gene expression, as well as physiological and metabolic events; however, much remains to be learned. In plants, abiotic stress affects germination, growth and development, significantly reducing productivity, and in some cases, causing plant death. AtGRDP1 is a novel protein that contains a short glycine-rich domain, a DUF1399 domain, and a putative RNP motif. Expression analysis showed that AtGRDP1 gene is modulated in response to NaCl, LiCl, mannitol, sorbitol, glucose, and exogenous ABA. In order to characterise the AtGRDP1 gene, null mutant and overerexpressing lines were obtained. The Atgrdp1-null mutant line showed an increased sensitivity to salt and osmotic stress in germination and cotyledon development, whereas 35S::AtGRDP1 overerexpressing lines resulted in increased tolerance to abiotic stress. Interestingly, 35S::AtGRDP1 overerexpressing lines showed resistance to ABA, resembling a well-known ABI phenotype, whereas the disruption of AtGRDP1 gene resulted in ABA hypersensitivity, mimicking the ABI3-overexpression phenotype. Furthermore, we analysed the ABI3 and ABI5 genes, which are central regulators in ABA signalling, in Atgrdp1-null mutant and 35S::AtGRDP1 overerexpressing lines. Under ABA treatments, Atgrdp1-null mutant seedlings showed higher ABI3 and ABI5 transcript levels, whereas in 35S::AtGRDP1 overexpressing line, the ABI3 and ABI5 transcripts were repressed. These results suggest that AtGRDP1 gene plays a regulatory role in ABA signalling and tolerance to abiotic stress.

Cooperation of three WRKY-domain transcription factors WRKY18, WRKY40, and WRKY60 in repressing two ABA-responsive genes ABI4 and ABI5 in Arabidopsis.

Three evolutionarily closely related WRKY-domain transcription factors WRKY18, WRKY40, and WRKY60 in Arabidopsis were previously identified as negative abscisic acid (ABA) signalling regulators, of which WRKY40 regulates ABI4 and ABI5 expression, but it remains unclear whether and how the three transcription factors cooperate to regulate expression of ABI4 and ABI5. In the present experiments, it was shown that WRKY18 and WRKY60, like WRKY40, interact with the W-box in the promoters of ABI4 and ABI5 genes, though the three WRKYs have their own preferential binding domains in the two promoters. WRKY18 and WRKY60, together with WRKY40, inhibit expression of the ABI5 and/or ABI4 genes, which is consistent with their negative roles in ABA signalling. Further, genetic evidence is provided that mutations of ABI4 and ABI5 genes suppress ABA-hypersensitive phenotypes of the null mutant alleles of WRKY18 and WRKY60 genes, demonstrating that ABI4 and ABI5 function downstream of these two WRKY transcription factors in ABA signalling. A working model of cooperation of the three WRKYs in repressing ABI4 and ABI5 expression is proposed, in which the three WRKYs antagonize or aid each other in a highly complex manner. These findings help to understand the complex mechanisms of WRKY-mediated ABA signal transduction.

The Mg-Chelatase H Subunit ofArabidopsisAntagonizes a Group of WRKY Transcription Repressors to Relieve ABA-Responsive Genes of Inhibition

The phytohormone abscisic acid (ABA) plays a vital role in plant development and response to environmental challenges, but the complex networks of ABA signaling pathways are poorly understood. We previously reported that a chloroplast protein, the magnesium-protoporphyrin IX chelatase H subunit (CHLH/ABAR), functions as a receptor for ABA in Arabidopsis thaliana. Here, we report that ABAR spans the chloroplast envelope and that the cytosolic C terminus of ABAR interacts with a group of WRKY transcription factors (WRKY40, WRKY18, and WRKY60) that function as negative regulators of ABA signaling in seed germination and postgermination growth. WRKY40, a central negative regulator, inhibits expression of ABA-responsive genes, such as ABI5. In response to a high level of ABA signal that recruits WRKY40 from the nucleus to the cytosol and promotes ABAR-WRKY40 interaction, ABAR relieves the ABI5 gene of inhibition by repressing WRKY40 expression. These findings describe a unique ABA signaling pathway from the early signaling events to downstream gene expression.

Role of HY5 in abscisic acid response in seeds and seedlings

Seed dormancy offers plants an adaptive advantage that is crucial to their successful colonization of land. The decision to make the transition from a dormant seed to a photoautotrophic seedling is a result of a complex interaction of internal hormonal signals and external stimuli such as water, temperature, and light. We recently showed that HY5, a well-characterized component in the light signaling pathway, also mediates abscisic acid (ABA) response during seed germination, early seedling growth, and root development in Arabidopsis. We proposed that HY5 regulates these ABA responses partly by directly activating the transcription factor gene ABI5. By analyzing the premature germination of hy5 and abi5 single and double mutants, here we demonstrated that HY5 also positively controls seed maturation and dormancy, likely through direct activation of the ABI5 gene. The contrasting role of light regulation of seed development and germination may be important for the adaptation of plants to the environment. Addendum to: Chen H, Zhang J, Neff MM, Hong S-W, Zhang H, Deng X-W, Xiong L. Integration of light and abscisic acid signaling during seed germination and early seedling development. Proc Natl Acad Sci USA 2008; 105:4495-500.

ABI5 Interacts with Abscisic Acid Signaling Effectors in Rice Protoplasts

Abscisic acid (ABA) regulates seed maturation, germination, and adaptation of vegetative tissues to environmental stresses. The mechanisms of ABA action and the specificity conferred by signaling components in overlapping pathways are not completely understood. The ABI5 gene (ABA insensitive 5) of Arabidopsis encodes a basic leucine zipper factor required for ABA response in the seed and vegetative tissues. Using transient gene expression in rice protoplasts, we provide evidence for the functional interactions of ABI5 with ABA signaling effectors VP1 (viviparous 1) and ABI1 (ABA insensitive 1). Co-transformation experiments with ABI5 cDNA constructs resulted in specific transactivation of the ABA-inducible wheat Em, Arabidopsis AtEm6, bean beta-Phaseolin, and barley HVA1 and HVA22 promoters. Furthermore, ABI5 interacted synergistically with ABA and co-expressed VP1, indicating that ABI5 is involved in ABA-regulated transcription mediated by VP1. ABI5-mediated transactivation was inhibited by overexpression of abi1-1, the dominant-negative allele of the protein phosphatase ABI1, and by 1-butanol, a competitive inhibitor of phospholipase D involved in ABA signaling. Lanthanum, a trivalent ion that acts as an agonist of ABA signaling, potentiated ABI5 transactivation. These results demonstrate that ABI5 is a key target of a conserved ABA signaling pathway in plants.

The Arabidopsis sugar-insensitive mutants sis4 and sis5 are defective in abscisic acid synthesis and response.

Although soluble sugar levels affect many aspects of plant development and physiology, little is known about the mechanisms by which plants respond to sugar. Here we report the isolation of 13 sugar-insensitive (sis) mutants of Arabidopsis that, unlike wild-type plants, are able to form expanded cotyledons and true leaves when germinated on media containing high concentrations of glucose or sucrose. The sis4 and sis5 mutants are allelic to the ABA-biosynthesis mutant aba2 and the ABA-insensitive mutant abi4, respectively. In addition to being insensitive to glucose and sucrose, the sis4/aba2 and sis5/abi4 mutants also display decreased sensitivity to the inhibitory effects of mannose on early seedling development. Mutations in the ABI5 gene, but not mutations in the ABI1, ABI2 or ABI3 genes, also lead to weak glucose- and mannose-insensitive phenotypes. Wild-type and mutant plants show similar responses to the effects of exogenous sugar on chlorophyll and anthocyanin accumulation, indicating that the mutants are not defective in all sugar responses. These results indicate that defects in ABA metabolism and some, but not all, defects in ABA response can also alter response to exogenous sugar.

The Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine zipper transcription factor.

The Arabidopsis abscisic acid (ABA)-insensitive abi5 mutants have pleiotropic defects in ABA response, including decreased sensitivity to ABA inhibition of germination and altered expression of some ABA-regulated genes. We isolated the ABI5 gene by using a positional cloning approach and found that it encodes a member of the basic leucine zipper transcription factor family. The previously characterized abi5-1 allele encodes a protein that lacks the DNA binding and dimerization domains required for ABI5 function. Analyses of ABI5 expression provide evidence for ABA regulation, cross-regulation by other ABI genes, and possibly autoregulation. Comparison of seed and ABA-inducible vegetative gene expression in wild-type and abi5-1 plants indicates that ABI5 regulates a subset of late embryogenesis-abundant genes during both developmental stages.