Os09g0306400

The Os09g0306400 was reported asOsbZIP71 which encodes a rice bZIP TF, it plays an important role in the abscisic acid (ABA) signaling pathway of abiotic stress in plants.

Background

• The bZIP transcription factor (TF) family plays an important role in the abscisic acid (ABA) signaling pathway of abiotic stress in plants. We here report the cloning and characterization of OsbZIP71, which encodes a rice bZIP TF. Functional analysis showed that OsbZIP71 is a nuclear-localized protein that specifically binds to the G-box motif, but has no transcriptional activity both in yeast and rice protoplasts. In yeast two-hybrid assays, OsbZIP71 can form both homodimers and heterodimers with Group C members of the bZIP gene family. Expression of OsbZIP71 was strongly induced by drought, polyethylene glycol (PEG), and ABA treatments, but repressed by salt treatment. OsbZIP71 overexpressing (p35S::OsbZIP71) rice significantly improved tolerance to drought, salt and PEG osmotic stresses.
• The bZIP family, which consists of proteins with a DNA-binding domain (BD) rich in basic amino acid residues and adjacent to a leucine zipper dimerization domain, is one of the largest T Ffamilies in higher plants. There are 75b ZIP sin Arabidopsis and 89 or 92 bZIPs in rice. According to DNA binding specificity and sequence similarities of bZIP domains,these bZIP TFs have been classified into 11 groups(I–XI) or 13 groups (A, B, C, D, E, F, G,H,I,J,K,LandS).Group Ab ZIP TFs in Arabidopsis, such as ABI5 and its homologs, bind ABA-responsive elements (ABRE) and act as abscisic acid (ABA) response factors.These Group Amembers are also known as the ABRE binding factors (ABFs/AREBs), and are involved in plant responses to dehydration and salt stress.
• In rice, the function of Group S bZIPs remains poorly understood, although LIP19/OsbZIP38 and OBF1/OsbZIP87 are thought to possibly participate in the cold signaling pathway, and OsbZIP16 positively regulates drought tolerance and plays a positive role in ABA signal transduction. The functional specificities of most individual members in Group S remain obscure, and need to be addressed and characterized to enable a fully understanding of bZIP function. In our microarray analysis of rice Zhonghua 11 (japonica) under drought treatment, we noticed that one gene OsbZIP71 (Group S1) was strongly induced by drought. We here report the identification and functional analysis of OsbZIP71 by investigating ABA sensitivity and stress tolerance of using over expression(p35S::OsbZIP71), inducible expression (RD29A::OsbZIP71) and RNAi knockdown transgenic plants. Expression profiling analyses of the over expression and RNAi plants were also performed to deduce the putative target genes regulated by OsbZIP71. Our results indicate that OsbZIP71 might function in an important role in ABA-mediated drought and salt tolerance in rice.

Annotated Information

Function

Figure 1.Comparison of rice and Arabidopsis Group S bZIP proteins (a) and phylogenetic relationships among plant Group S1 bZIP members (b).(from reference ).

• OsbZIP71(LOC_Os09g13570) encodes a 154 amino acid protein. Amino acids 29–50 contain a typical bZIP basic region domain, and residues 51–112 contain a leucine zipper domain, which may also act as an active domain (Fig. 1a).
• The b-galactosidase activity of pBD71 showed no difference compared with the control construct, indicating that OsbZIP71 had no transactivation activity.
• The bZIP proteins are known to interact with G-box ciselement with an ACGT core sequence. The binding specificity of OsbZIP71 to the G-box element was determined using a one-hybrid system. results indicate that OsbZIP71 can function as a trans-acting factor for the G-box element, but the binding activity was not very high.
• Dimerization is one of the distinguishing characteristics of bZIP TFs. Liu et al. examined the dimerization activity of OsbZIP71 in vivo using a yeast two-hybrid system , showing that OsbZIP71 can form both homodimers as well as heterodimers with Group C proteins in yeast.
• It is proposed that: when plants encounter salt and drought stresses, OsbZIP71 is directly activated by the ABA-dependent pathway, and forms heterodimers with Group C members of the bZIP family or interacts with OsMyb4 to form a complex. These multi-protein complexes may then act together to bind promoters (containing G-box elements) of OsNHX1 and COR413-TM1, thereby enhances rice tolerance to drought and salt stresses.
• OsbZIP71 was strongly induced by ABA, and is involved in ABA sensitivity, which is an important aspect of the ABA-dependent regulation pathway. OsbZIP71 is associated with plant responses to low-oxygen stress.

GO assignment(s): GO:0005634, GO:0043565, GO:0046983

Mutation

• Two OsbZIP71-overexpressing lines (OE-16 and OE-19)
• two RNAi knockdown lines (RNAi-21 and RNAi-23)
• two inducible expression lines (IE-3 and IE-4)
• and wild type They were used for germination tests. All transgenic lines were independent homozygous and single copy transgenic lines identified by Southern blot:
  

 OsbZIP71 IE lines show insensitivity to ABA during seed germination.

 OsbZIP71 RNAi seedlings show hypersensitivity to ABA.

Expression

• Liu et al. checked OsbZIP71 expression profile under different abiotic stresses:
  Realtime PCR analysis revealed that the transcript level of OsbZIP71 was strongly induced by drought, PEG, and ABA treatments, but repressed by salt.
  • OsbZIP71 was up-regulated more strongly in roots than in shoots following 6 h of PEG treatment.
  • In drought and ABA treated seedlings, OsbZIP71 expression was induced only in roots after the 0.5 and 4 h time-points, respectively.
  • In the salt treatments, OsbZIP71 was repressed both in shoots and roots within 0.5–6 h, and then restored to its basal expression level at 12 h.
  • OsbZIP71 was not changed at different time-points under normal condition, which suggesting that OsbZIP71 may function in abiotic stress signal transduction.
• those transgenic lines tolerance to salt and PEG osmotic stresses were positive correlated to relative expression of OsbZIP71 and OsbZIP71 may regulate responses to salt and PEG osmotic stresses. OsbZIP71 overexpression significantly increased rice drought tolerance at both seedling and reproductive stages.
• In a word, Overexpression of OsbZIP71 enhanced drought and salt tolerance in rice. OsbZIP71 RNAi lines was sensitive to drought and salt stress in rice. Inducible expression of OsbZIP71 enhanced drought tolerance but sensitive to salt in rice.

Subcellular localization

• To determine the subcellular localization of OsbZIP71, the ORF of OsbZIP71 was fused to GFP in the pA7-GFP vector OsbZIP71 is a nuclear-localized protein (Fig. 1d–f) and may function as a TF.

Evolution

• NCBI BLASTp results also showed that OsbZIP71 was similar to Group S members of the Arabidopsis bZIP family, such as AtbZIP53, AtbZIP2/GBF5, AtbZIP44, and AtbZIP11/ATB2. Based on homology of the bZIP domains, the 16 members of rice Group S can be divided into three subgroups, designated S1, S2, and S3 (Fig. 1b). Further classification using MEGA4.0 with the known classified bZIP TFs indicated that OsbZIP71 belongs to Group S1 of the bZIP family(Fig. 1b)

Knowledge Extension

Figure 2.Summary of ABA signaling pathways that result in drought stress resistant responses.(from reference ).

• Unlike other cereal species, rice is able to germinate and elongate under anoxia. The regulatory mechanism that configures the transcriptome of rice during anaerobic germination is yet to be established. Although some rice varieties exhibit some degree of tolerance to anaerobic stress that normally occurs during partial (hypoxia) or complete submergence (anoxia), it remains a major factor that limits productivity specially for the rainfed lowland cropping systems in Southeast Asia, where an average family relies on subsistence rice farming.
• The ability of rice to germinate under anoxia by extending the coleoptile is a highly unusual characteristic and a key feature underpinning the ability of rice seeds to establish in such a stressful environment. The process has been a focal point for research for many years. However, the molecular mechanisms underlying the anoxic growth of coleoptile still remain largely unknown.
• Combined with recent findings on ABA signal transduction in Arabidopsis (Fig. 2), investigation into the genetic regulation of core ABA signal transduction components could promote the introduction of drought tolerance responses into various crop species. Since the identification of PYR/PYL/RCARs, a better understanding of signaling networks among the canonical ABA signaling components has shed light on the genetic modification of ABA signal transduction for improving drought tolerance in plants.
• OsbZIP71 is a member of anoxia stressed up-regulated transcription factors with potential significance to the pattern of cis-element enrichment among the upregulated genes.
• Major targets for this purpose include PYR/PYL/RCARs-PP2CSnRK2s complexes as well as their phosphorylation substrates such as transcription factors or channel proteins. A PP2C binds to a SnRK2 in the absence of ABA and mediate dephosphorylation of the SnRK2 (Fig. 2). On the other hand, activation of SnRK2s as a critical positive signal for ABA signaling usually begins with autophosphorylation of SnRK2s.
• It was reported that many genes mediate plant responses to ABA and abiotic stress tolerance, such as OsbZIP23, OsABI5, etc. Osb-ZIP71 overexpression lines have not altered ABA sensitivity; but inducible lines show insensitive to ABA both at seed germination and seedling growth, on the contrary, RNAi lines show hypersensitive to ABA both at seed germination and seedling growth. A reasonable explanation is that OsbZIP71 mediates ABA sensitivity which involves in abiotic stress tolerance. Overexpression of the OsbZIP71 doesn’t induce any ABA insensitive phenotype, because that a complex network of ABA-responsive TFs involved in ABA responses. The abo3 mutant was also shown hypersensitive to ABA at both seed germination and seedling growth, but overexpression ABO3 lines in Arabidopsis were not changed ABA sensitivity .

Labs working on this gene

• State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
• State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center, Changsha 410125, China
• National Center for Molecular Crop Design, Weiming Kaituo Agriculture Biotech Co., Ltd, Beijing 100085, China