|
|
| Line 4: |
Line 4: |
| | ==Annotated Information== | | ==Annotated Information== |
| | ===Function=== | | ===Function=== |
| − | Firstly, overexpression of ''OsAREB1'' alters seedling sensitivity to ABA and glucose and OsAREB1 might have a crucial role in these two
| + | * '''''OsbZIP46''''' is one member of the third subfamily of bZIP transcription factors in rice (Oryza sativa) |
| − | signaling pathways. Roots of transgenic plants were hypersensitive to ABA. Also,transgenic seeds were hypersensitive to glucose in germination period.
| + | * '''''OsbZIP46''''' is a positive regulator of ABA signaling and drought stress tolerance of rice depending on its activation. |
| | + | * The stress-related genes activated by '''''OsbZIP46CA1''''' are largely different from those activated by the other rice ABF/AREB homologs (such as OsbZIP23), further implying the value of '''''OsbZIP46CA1''''' in genetic engineering of drought tolerance. |
| | | | |
| − | Secondly, 35S-OsAREB1 plants enhanced the resistance to drought and heat.Transgenic seeds can hold more water to stand against drought condition and up-regulate stress-related genes, such as ''RD29A'', ''RD29B''.
| + | ===Phenotypic analysis=== |
| − | | + | * Overexpression of the native '''''OsbZIP46''''' gene increased ABA sensitivity but had no positive effect on drought resistance. |
| − | Thirdly, OsAREB1 delay the flowering time.by down-regulating the expression of flowering-related genes, such
| + | * The activation domain of '''''OsbZIP46''''' was defined by a series of deletions, and a region (domain D) was identified as having a negative effect on the activation. We produced a constitutive active form of '''''OsbZIP46''''' (OsbZIP46CA1) with a deletion of domain D. Overexpression of '''''OsbZIP46CA1''''' in rice significantly increased tolerance to drought and osmotic stresses. |
| − | as ''FT'', SOC1, ''LFY''and ''AP1''.
| + | * Gene chip analysis of the two overexpressors (native '''''OsbZIP46''''' and the constitutive active form '''''OsbZIP46CA'''''1) revealed that a large number of stress-related genes, many of them predicted to be downstream genes of ABF/ AREBs, were activated in the '''''OsbZIP46CA1 '''''overexpressor but not (even down-regulated) in the '''''OsbZIP46''''' overexpressor. |
| − | | + | * '''''OsbZIP46''''' can interact with homologs of SnRK2 protein kinases that phosphorylate ABFs in Arabidopsis. |
| − | In other work ,A number of transcription factors
| |
| − | (TFs) regulate stress-responsive gene expression. OsDREB1s and OsDREB2s were identified as abiotic-stress | |
| − | responsive TFs that belong to the AP2/ERF family. Similar to Arabidopsis, these DREB regulons were most likely not
| |
| − | involved in the abscisic acid (ABA) pathway. OsAREBs such as OsAREB1 were identified as key components in ABAdependent transcriptional networks in rice.
| |
| − | <ref name="refC" />
| |
| − | The abscisic acid (ABA) responsive element (ABRE)
| |
| − | binding protein (AREB)/ABRE binding factor (ABF) regulon functions in ABA-dependent gene expression
| |
| − | under osmotic stress conditions
| |
| − | | |
| − | Also,in ''Arabidopsis'', bZIP-type transcription factors AREBs/ABFs bind an abscisic acid
| |
| − | (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression inArabidopsis. Because AREB1 | |
| − | overexpression could not induce downstream gene expression,
| |
| − | activation of AREB1 requires ABA-dependent posttranscriptional
| |
| − | modification. We confirmed that ABA activated 42-kDa kinase
| |
| − | activity, which, in turn, phosphorylated Ser/Thr residues of R-XX-S/T sites in the conserved regions of AREB1. Amino acid substitutions of R-X-X-S/T sites to Ala suppressed transactivation activity, and multiple substitution of these sites resulted in almost
| |
| − | complete suppression of transactivation activity in transient assays. In contrast, substitution of the Ser/Thr residues to Asp
| |
| − | resulted in high transactivation activity without exogenous ABA
| |
| − | application. A phosphorylated, transcriptionally active form was
| |
| − | achieved by substitution of Ser/Thr in all conserved R-X-X-S/T sites
| |
| − | to Asp. Transgenic plants overexpressing the phosphorylated active form of AREB1 expressed many ABA-inducible genes, such as
| |
| − | RD29B, without ABA treatment. These results indicate that the
| |
| − | ABA-dependent multisite phosphorylation of AREB1 regulates its
| |
| − | own activation in plants.<ref name="refD" />
| |
| − | | |
| − | The phytohormone abscisic acid (ABA) plays important roles in
| |
| − | seed maturation and dormancy and is also involved in the
| |
| − | adaptation of vegetative tissues to abiotic environmental stresses,
| |
| − | such as drought and high salinity. ABA promotes stomatal closure
| |
| − | in guard cells and regulates the expression of many genes, the | |
| − | products of which may function in dehydration tolerance in both
| |
| − | vegetative tissues and seeds. Many ABA-inducible genes contain a
| |
| − | conserved element named ABA-responsive element (ABRE) (PyACGTGG/TC) in their promoter regions. The ABRE functions as
| |
| − | a cis-acting element and is involved in ABA-responsive gene expression.Each AREB protein
| |
| − | contained a single bZIP-type DNA-binding domain, and expression
| |
| − | ofAREB1andAREB2was up-regulated by ABA, drought, and
| |
| − | high-salinity stresses, shown to function as trans-acting activators by
| |
| − | using transient expression in protoplasts
| |
| | | | |
| | ===Expression=== | | ===Expression=== |
| − | Expression patterns of the ''OsAREB1'' gene under various environmental stresses and hormones were analyzed by RT-PCR. | + | * Expression of '''''OsbZIP46''''' was strongly induced by drought, heat, hydrogen peroxide, and abscisic acid (ABA) treatment; however, it was not induced by salt and cold stresses. |
| − | OsAREB1 gene was induced within 1 or 2 h under 100 μM ABA and 15% PEG 6,000 treatments, and maintained the expression level for at least 8 hours. It’s expression was induced by heat within 1 h, and rapidly reached the top expression level within 2 h, then declined to initial level.
| |
| − | OsAREB1 was not induced by KT, MeJA, NaCl and cold .These results indicated that OsAREB1was induced by exogenous ABA, water stress and heat. This result
| |
| − | was consistent with the report of Lu et al.<ref name="refB" />.
| |
| − | | |
| | | | |
| | ===Evolution=== | | ===Evolution=== |
| − | Please input evolution information here.
| + | * '''''OsbZIP46''''' has high sequence similarity to ABA-responsive element binding factor (ABF/AREB) transcription factors ABI5 and OsbZIP23, two transcriptional activators positively regulating stress tolerance in Arabidopsis (Arabidopsis thaliana) and rice, respectively. |
| − | | |
| − | Extending Knowledge
| |
| − | ===Binding activity===
| |
| − | OsAREB1 has ABRE-binding activity in yeast
| |
| − | Blast result indicated that OsAREB1 belongs to ABF subfamily.
| |
| − | Most members of this subfamily can bind to the ABRE cis-element
| |
| − | with a core sequence ACGTGCC. Yeast one-hybrid system was
| |
| − | used to determine the DNA-binding activity of OsAREB1 with
| |
| − | ABRE element. The entire coding region of OsAREB1 was fused
| |
| − | to the GAL4 transcription active domain (TA). The construct
| |
| − | was transformed into yeast (EGY48) harboring ABRE sequence
| |
| − | fused upstream of a lacZreporter gene, and the growth status of
| |
| − | transformants was observed. Yeast cells harboring pPC86 and
| |
| − | G222 could grow on SD medium lacking Trp, while cells only
| |
| − | with G222 could not grow on the selection medium (Fig. 1A).
| |
| − | The colony-lift filter assay suggested that OsAREB1 can bind to
| |
| − | the ABRE cis-element. Shown as Fig. 1B, when the colony grew
| |
| − | on X-gal containing plate, only cells with pPC86-OsAREB1 and
| |
| − | G222 turned blue, cells only with G222 or with both G222 and
| |
| − | pPC86 did not turn blue. This result indicated that only
| |
| − | OsAREB1 can bind to the ABRE cis-element and then active the
| |
| − | expression of lacZ gene. Further, quantificational analysis for
| |
| − | β-galactosidase activity was performed. Compared to the negative control, the relative β-galactosidase activity of the transformants was about four (Fig. 1C), which revealed there’s a distinct enhancement for β-galactosidase activity.
| |
| − | | |
| − | [[File:DNA binding assay.jpg]]
| |
| − | | |
| − | ===AREB regulon===
| |
| − | Abscisic Acid acts as a crucial signal molecule in abiotic
| |
| − | stress responses (Fujita et al. 2011). The ABA content is
| |
| − | increased by abiotic stresses, and leads to expression of
| |
| − | numerous genes. Application of exogenous ABA also
| |
| − | stimulates a myriad of genes. ABRE was identified as a
| |
| − | cis-acting element conserved in promoter regions of
| |
| − | ABA-inducible genes.ArabidopsiscDNAs that encode
| |
| − | bZIP-type TFs were screened as ABRE-binding proteins
| |
| − | (Yamaguchi-Shinozaki & Shinozaki 2006). Among these | |
| − | genes,AREB1/ABF2, AREB2/ABF4,andABF3were
| |
| − | reported to be induced by ABA and osmotic stress in
| |
| − | vegetative tissues (Fujita et al. 2011,). Evidence indicates
| |
| − | that activation of AREB1 needs ABA-dependent posttranscriptional modification. The ABA-activated SnRK2
| |
| − | protein kinases phosphorylate the AREB1 protein (Furihata et al. 2006). TransgenicArabidopsisplants overexpressing the phosphorylated active form of AREB1
| |
| − | showed enhanced expression of a number of ABA-inducible genes (Furihata et al. 2006). The ABA-activated
| |
| − | phosphorylation of AREB/ABFs was completely
| |
| − | impaired in the SnRK2 triple mutant, srk2d srk2e srk2i
| |
| − | (Fujii et al. 2009,; Fujii & Zhu 2009). The down-regulated genes in the srk2d srk2e srk2i andareb1 areb2
| |
| − | abf3triple mutants largely overlapped in ABA-dependent expression, which supports the view that SRK2D/
| |
| − | E/I regulate AREBs in ABA signaling in response to
| |
| − | osmotic stress. (Fujita et al. 2009).
| |
| − | The rice TRANSCRIPTION FACTOR RESPONSIBLE
| |
| − | FOR ABA REGULATION1 (TRAB1) shows high homology toArabidopsisAREB2/ABF4. Expression ofTRAB1
| |
| − | was up-regulated by ABA treatment (Hobo et al. 1999,).
| |
| − | TRAB1 is phosphorylated rapidly in response to ABA treatment (Kagaya et al. 2002).
| |
| − | | |
| − | === activation mechanisms of AREB1===
| |
| − | | |
| − | Kanget al. (17) reported that overexpression of ABF3 and
| |
| − | ABF4/AREB2 resulted in ABA-hypersensitive phenotypes in
| |
| − | germination and seedling growth stages in Arabidopsis. These
| |
| − | transgenic plants also showed improvement of drought stress
| |
| − | tolerance, suggesting that AREB/ABF proteins are involved in
| |
| − | ABA response and stress tolerance in plants. However, AREB1
| |
| − | and AREB2 require ABA for their maximum activation, as shown
| |
| − | by their low transactivation abilities in protoplasts prepared from
| |
| − | the ABA-deficient aba2 mutant (7). We have shown that the
| |
| − | ABA-responsive 42-kDa kinase activities phosphorylate conserved
| |
| − | regions of AREBs, suggesting that ABA-dependent phosphorylation may be involved in activation of the AREB subfamily proteins
| |
| − | (7). Phosphorylation/dephosphorylation-regulated events were reported to play important roles in ABA signaling; SNF1-related
| |
| − | protein kinase homologues, ABA-activated protein kinase
| |
| − | (AAPK) inVicia faba(18), and SRK2E/OST1 inArabidopsis(19,
| |
| − | 20) modulate ABA-dependent stomatal closure.ABI1andABI2,of
| |
| − | which dominant-negative mutation causes ABA-insensitive mutantsabi1andabi2, encode a type 2C protein phosphatase (4, 5, 21).
| |
| − | Because null mutations of ABI1 and ABI2 resulted in ABA
| |
| − | hypersensitivity, ABI1 and ABI2 negatively regulate ABAdependent responses (22).
| |
| − | Here, we report that the ABA-activated 42-kDa kinase activity
| |
| − | phosphorylates Ser/Thr residues in the conserved R-X-X-S/Tsites
| |
| − | of AREB1. Amino acid substitution of the Ser/Thr residues to Ala
| |
| − | and Asp resulted in suppression and high transactivation activity,
| |
| − | respectively. A phosphorylated active form of AREB1 was obtained by substitution of Ser/Thr to Asp in all conserved R-X-XS/T sites. We show that transgenic plants overexpressing the | |
| − | phosphorylated active form AREB1 express not only ABA inducible genes, such as RD29B, but also seed-specific gene
| |
| − | without ABA treatment. We also discuss the activation mechanism
| |
| − | of AREB1 by ABA-dependent phosphorylation in plants.
| |
| − | | |
| − | Fig. 2 shows the properties of AREB1 protein fragment phosphorylation.
| |
| − | | |
| − | [[File:Fig. 2. Properties of AREB1 protein fragment phosphorylation.jpg]]
| |
| − | | |
| − | Fig. 2. Properties of AREB1 protein fragment phosphorylation. (A) Effects of
| |
| − | protein kinase inhibitor staurosporine on phosphorylation of the recombinant AREB1a polypeptide. Staurosporine (20 and 100 nM) was added to
| |
| − | reaction mixture. (B) Effects of BAPTA on phosphorylation of the recombinant
| |
| − | AREB1b polypeptide. Na-BAPTA (5 mM final concentration) was added to
| |
| − | reaction buffer and equilibrated for 30 min before addition of radiolabeled
| |
| − | ATP. In the far right lane, additional kinase activity appeared near 60 kDa (see
| |
| − | Resultsfor details). (C) Stress-dependent phosphorylation of the recombinant
| |
| − | AREB1b polypeptide. Protein extracts prepared from T87 cells treated for 30
| |
| − | min with 50/M ABA (Ab), 0.5 M NaCl (Na), and 0.8 M mannitol (Os, high
| |
| − | osmolality) and at low temperature (Lt, 4°C) or untreated (Ct) were used for
| |
| − | in-gel kinase activity assay. The recombinant AREB1b polypeptide was used as
| |
| − | a substrate. (D) ABA-activated SnRK2-type protein kinases phosphorylate the
| |
| − | recombinant AREB1b polypeptide. Protein extracts prepared from transgenic
| |
| − | T87 cells overexpressing each SnRK2-GFP fusion protein under control of the
| |
| − | CaMV 35Spromoter were used for in-gel kinase activity assay. Phosphorylated
| |
| − | bands derived from SnRK2-GFP fusion proteins were indicated by circles.
| |
| − | Arrowheads indicate the position of 42 kDa in A–D.
| |
| | | | |
| | You can also add sub-section(s) at will. | | You can also add sub-section(s) at will. |
| | | | |
| | ==Labs working on this gene== | | ==Labs working on this gene== |
| − | 1
| + | * National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan 430070, China |
| − | Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai,
| |
| − | 2
| |
| − | College of Life Science and Technology, Yangzhou University, Jiangsu, PR China
| |
| − | 3
| |
| − | *Biological Resources Division, Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki 305-8686, Japan;
| |
| − | †
| |
| − | Laboratory of Plant Molecular Biology, RIKEN Tsukuba Institute, Tsukuba, Ibaraki 305-0074, Japan;¶
| |
| − | | |
| − | Graduate School of Agricultural and Life Sciences, University of Tokyo,
| |
| − | Tokyo 113-8657, Japan;
| |
| − | ‡
| |
| − | RIKEN Plant Science Center, Yokohama, Kanagawa 203-0045, Japan; and Core Research for Evolutional Science and Technology,
| |
| − | Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan
| |
| | | | |
| | ==References== | | ==References== |
| − | <ref name="refA" /> Lu, G. J., Gao, C. X., Zheng, X. N. and Han, B. (2009) Identification of OsbZIP72 as a positive regulator of ABA response and drought tolerance in rice. Planta 229, 605-615. | + | <references> |
| − | | + | * <ref name="ref1"> |
| − | <ref name="refC" /> Daisuke Todaka | + | Tang N, Zhang H, Li X, Xiao J, Xiong L. Constitutive activation of |
| − | 1, Kazuo Nakashima1, Kazuo Shinozaki2and Kazuko Yamaguchi-Shinozaki1,3*(2012) Toward understanding transcriptional regulatorynetworks in abiotic stress responses and tolerance in rice Rice 2012,5:6
| + | transcription factor OsbZIP46 improves drought tolerance in rice. Plant Physiol. |
| − | | + | 2012 Apr;158(4):1755-68. doi: 10.1104/pp.111.190389. Epub 2012 Feb 1. PubMed |
| − | <ref name="refD" /> Takashi Furihata*, Kyonoshin Maruyama*, Yasunari Fujita*, Taishi Umezawa†‡, Riichiro Yoshida†, Kazuo Shinozaki†‡§,and Kazuko Yamaguchi-Shinozaki*§¶(2005) Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1.PNAS February 7, 2006 vol. 103 no. 6 | + | PMID: 22301130; PubMed Central PMCID: PMC3320183. |
| | + | </ref> |
| | + | </references> |
| | | | |
| − | <ref name="refB" />Jin XF1, Xiong AS, Peng RH, Liu JG, Gao F, Chen JM, Yao QH. (2010) OsAREB1, an ABRE-binding protein responding to ABA and glucose, has multiple functions in Arabidopsis.BMB Rep 43(1):34-9.
| |
| | | | |
| | | | |
