Two TGA Transcription Factor Members from Hyper-Susceptible Soybean Exhibiting Significant Basal Resistance to Soybean mosaic virus.

TGA transcription factors (TFs) exhibit basal resistance in Arabidopsis, but susceptibility to a pathogen attack in tomatoes; however, their roles in soybean (Glycine max) to Soybean mosaic virus (SMV) are unknown. In this study, 27 TGA genes were isolated from a SMV hyper-susceptible soybean NN1138-2, designated GmTGA1~GmTGA27, which were clustered into seven phylogenetic groups. The expression profiles of GmTGAs showed that the highly expressed genes were mainly in Groups I, II, and VII under non-induction conditions, while out of the 27 GmTGAs, 19 responded to SMV-induction. Interestingly, in further transient N. benthamiana-SMV pathosystem assay, all the 19 GmTGAs overexpressed did not promote SMV infection in inoculated leaves, but they exhibited basal resistance except one without function. Among the 18 functional ones, GmTGA8 and GmTGA19, with similar motif distribution, nuclear localization sequence and interaction proteins, showed a rapid response to SMV infection and performed better than the others in inhibiting SMV multiplication. This finding suggested that GmTGA TFs may support basal resistance to SMV even from a hyper-susceptible source. What the mechanism of the genes (GmTGA8, GmTGA19, etc.) with basal resistance to SMV is and what their potential for the future improvement of resistance to SMV in soybeans is, are to be explored.

Genome-wide analysis of the bZIP gene family in Chinese jujube (Ziziphus jujuba Mill.).

BACKGROUND: Among several TF families unique to eukaryotes, the basic leucine zipper (bZIP) family is one of the most important. Chinese jujube (Ziziphus jujuba Mill.) is a popular fruit tree species in Asia, and its fruits are rich in sugar, vitamin C and so on. Analysis of the bZIP gene family of jujube has not yet been reported. In this study, ZjbZIPs were identified firstly, their expression patterns were further studied in different tissues and in response to various abiotic and phytoplasma stresses, and their protein-protein interactions were also analyzed. RESULTS: At the whole genome level, 45 ZjbZIPs were identified and classified into 14 classes. The members of each class of bZIP subfamily contain a specific conserved domain in addition to the core bZIP conserved domain, which may be related to its biological function. Relative Synonymous Codon Usage (RSCU) analysis displayed low values of NTA and NCG codons in ZjbZIPs, which would be beneficial to increase the protein production and also indicated that ZjbZIPs were at a relative high methylation level. The paralogous and orthologous events occurred during the evolutionary process of ZjbZIPs. Thirty-four ZjbZIPs were mapped to but not evenly distributed among 10 pseudo- chromosomes. 30 of ZjbZIP genes showed diverse tissue-specific expression in jujube and wild jujube trees, indicating that these genes may have multiple functions. Some ZjbZIP genes were specifically analyzed and found to play important roles in the early stage of fruit development. Moreover, some ZjbZIPs that respond to phytoplasma invasion and abiotic stress environmental conditions, such as salt and low temperature, were found. Based on homology comparisons, prediction analysis and yeast two-hybrid, a protein interaction network including 42 ZjbZIPs was constructed. CONCLUSIONS: The bioinformatics analyses of 45 ZjbZIPs were implemented systematically, and their expression profiles in jujube and wild jujube showed that many genes might play crucial roles during fruit ripening and in the response to phytoplasma and abiotic stresses. The protein interaction networks among ZjbZIPs could provide useful information for further functional studies.

The RCC1 family protein SAB1 negatively regulates ABI5 through multidimensional mechanisms during postgermination in Arabidopsis.

Abscisic acid-insensitive 5 (ABI5) is an essential and conserved plant basic leucine zipper transcription factor whose level controls seed germination and postgerminative development. It has been demonstrated that activity of ABI5 is transcriptionally and post-translationally regulated. However, transcriptional regulation of ABI5 is not fully understood. Here, we identified SAB1 (Sensitive to ABA 1) as a novel negative regulator of ABI5 that simultaneously regulates its stability, promoter binding activity and histone methylation-mediated gene silencing of ABI5. SAB1 encodes a Regulator of Chromatin Condensation 1 (RCC1) family protein and is expressed in an opposite pattern to that of ABI5 during early seedling growth in response to abscisic acid (ABA). SAB1 mutation results in enhanced ABA sensitivity and acts upstream of ABI5. SAB1 physically interacts with ABI5 at phosphoamino acid Ser-145, and reduces the phosphorylation of ABI5 and the protein stability. SAB1 reduces ABI5 binding activity to its own promoter, leading to reduced transcriptional level of ABI5. SAB1 inactivates ABI5 transcription by increasing the level of histone H3K27me2 in the ABI5 promoter. Our findings have identified SAB1 as a crucial new component of ABA signaling which modulates early development of plant by precisely controlling ABI5 activity through multiple mechanisms.

Methods for the expression, purification, preparation, and biophysical characterization of constructs of the c-Myc and Max b-HLH-LZs.

Specific heterodimerization and DNA binding by the b-HLH-LZ transcription factors c-Myc and Max is central to the activation and repression activities of c-Myc that lead to cell growth, proliferation, and tumorigenesis (Adhikary and Eilers, Nat Rev Mol Cell Biol 6:635-645, 2005; Eilers and Eisenman, Genes Dev 22:2755-2766, 2008; Grandori et al., Annu Rev Cell Dev Biol 16:653-699, 2000; Whitfield and Soucek, Cell Mol Life Sci 69:931-934, 2011). Although many c-Myc-interacting partner proteins are known to interact through their HLH domain (Adhikary and Eilers, Nat Rev Mol Cell Biol 6:635-645, 2005), current knowledge regarding the structure and the determinants of molecular recognition of these complexes is still very limited. Moreover, recent advances in the development and use of b-HLH-LZ dominant negatives (Soucek et al., Nature 455:679-683, 2008) and inhibitors of c-Myc interaction with its protein partners (Bidwell et al., J Control Release 135:2-10, 2009; Mustata et al., J Med Chem 52:1247-1250, 2009; Prochownik and Vogt, Genes Cancer 1:650-659, 2010) or DNA highlight the importance of efficient protocols to prepare such constructs and variants. Here, we provide methods to produce and purify high quantities of pure and untagged b-HLH-LZ constructs of c-Myc and Max as well as specific c-Myc/Max heterodimers for their biophysical and structural characterization by CD, NMR, or crystallography. Moreover, biochemical methods to analyze the homodimers and heterodimers as well as DNA binding of these constructs by native electrophoresis are presented. In addition to enable the investigation of the c-Myc/Max b-HLH-LZ complexes, the protocols described herein can be applied to the biochemical characterization of various mutants of either partner, as well as to ternary complexes with other partner proteins.

Isolation and functional characterization of a salt responsive transcriptional factor, LrbZIP from lotus root (Nelumbo nucifera Gaertn).

Basic leucine zipper transcription factor (bZIP) is involved in signaling transduction for various stress responses. Here we reported a bZIP transcription factor (accession: JX887153) isolated from a salt-resistant lotus root using cDNA-AFLP approach with RT-PCR and RACE-PCR method. Full-length cDNA which consisted of a single open reading frame encoded a putative polypeptide of 488 amino acids. On the basis of 78, 76, and 75 % sequence similarity with the bZIPs from Medicago truncatula (XP_003596814.1), Carica papaya (ABS01351.1) and Arabidopsis thaliana (NP_563810.2), we designed it as LrbZIP. Semi quantitative RT-PCR results, performed on the total RNA extracted from tips of lotus root, showed that LrbZIP expression was increased with 250 mM NaCl treatment for 18 h. Effects of low temperature on the expression of LrbZIP was also studied, and its expression was significantly enhanced with a 4 °C treatment for 12 h. In addition, LrbZIP expression was strongly induced by treatment with exogenous 100 µM ABA. To evaluate its function across the species, tobacco (Nicotiana tabacum L.) was transformed with LrbZIP in a binary vector construct. Transgenic plants exhibited higher resistance as compared with the control according to the results of the root growth, chlorophyll content and electrolyte leakage when exposed to NaCl treatment. In addition, LrCDPK2, LrLEA, and TPP also showed enhanced expression in the transgenic plants. Overall, expression of LrbZIP was probably very important for salt-resistant lotus root to survive through salt stress.

Basic leucine zipper transcription factor OsbZIP16 positively regulates drought resistance in rice.

Abiotic stress has been shown to limit the growth, development, and productivity of crops. Here, we characterized the function of a rice bZIP transcription factor OsbZIP16 in drought stress. Expression of OsbZIP16 was dramatically induced under drought conditions. Transient expression and transactivation assays demonstrated that OsbZIP16 was localized in the nucleus and had transactivation activity. At both the seedling and tillering stages, transgenic rice plants overexpressing OsbZIP16 exhibited significantly improved drought resistance, which was positively correlated with the observed expression levels of OsbZIP16. Representative downstream drought-inducible genes were observed to have significantly higher expression levels in transgenic rice plants than in the wild type plants under drought conditions. OsbZIP16 was shown to be induced by exogenous ABA treatment, while overexpression of OsbZIP16 was observed to make transgenic plants more sensitive to ABA than wild type plants were. Transcriptome analysis identified a number of differentially expressed genes between wild type plants and plants overexpressing OsbZIP16, many of which are involved in stress response according to their gene ontologies. Overall, our findings suggest that OsbZIP16 positively regulates drought resistance in rice.

Purification, crystallization and preliminary X-ray analysis of OsAREB8 from rice, a member of the AREB/ABF family of bZIP transcription factors, in complex with its cognate DNA.

The AREB/ABF family of bZIP transcription factors play a key role in drought stress response and tolerance during the vegetative stage in plants. To reveal the DNA-recognition mechanism of the AREB/ABF family of proteins, the bZIP domain of OsAREB8, an AREB/ABF-family protein from Oryza sativa, was expressed in Escherichia coli, purified and crystallized with its cognate DNA. Crystals of the OsAREB8-DNA complex were obtained by the sitting-drop vapour-diffusion method at 277 K with a reservoir solution consisting of 50 mM MES pH 6.4, 29% MPD, 2 mM spermidine, 20 mM magnesium acetate and 100 mM sodium chloride. A crystal diffracted X-rays to 3.65 Å resolution and belonged to space group C222, with unit-cell parameters a = 155.1, b = 206.7, c = 38.5 Å. The crystal contained one OsAREB8-DNA complex in the asymmetric unit.

Cloning and characterization of the stress-induced bZIP gene ZmbZIP60 from maize.

ZmbZIP60 is a member of the bZIP transcription factor family in maize. Expression of ZmbZIP60 is strongly induced by a wide spectrum of stresses, including dehydration, high salinity, abscisic acid and tunicamycin treatments. A truncated form of ZmbZIP60, without a transmembrane domain (ZmbZIP60ΔC) and fused with GFP, is localized in the nucleus, suggesting the translocation of the native protein to the nucleus by release from the membrane. Yeast one-hybrid analysis showed that both ZmbZIP60 and ZmbZIP60ΔC had transcriptional activity. The expression of ZmbZIP60 in Arabidopsis bzip60 mutant partially restored the induction of BiP3 transcription under TM treatment, which indicated that ZmbZIP60 may play a role in the signal transduction of endoplasmic reticulum stress. Overexpression of ZmbZIP60 in wild-type Arabidopsis displayed enhanced bolting trends when subjected to dithiothreitol stress. Real-time PCR analysis revealed that some key genes in floral transition, including CO, FT, and AP1, were up- or down-regulated in ZmbZIP60-overexpressing plants, which may reveal the functional difference of ZmbZIP60 from AtbZIP60.

Transcriptional repression by a bZIP protein regulates Dictyostelium prespore differentiation.

In response to the signaling polyketide DIF-1 DimB directly activates transcription of the ecmB gene in pstB cells; a subset of the prestalk cells that are the precursors of the basal disc. We show that the promoter of pspA, a prespore-specific gene, also contains a DimB binding site. Mutation of this site causes ectopic expression in the prestalk region and ChIP analysis shows that DIF-1 induces binding of DimB to the pspA promoter. DIF-1 represses pspA gene expression in a suspension cell assay but this repression is abrogated in a dimB null strain. These results suggest a coupled control mechanism, whereby the same DIF-DimB signaling pathway that directly activates ecmB gene expression directly represses pspA gene expression.

The ABRE-binding bZIP transcription factor OsABF2 is a positive regulator of abiotic stress and ABA signaling in rice.

Abscisic acid (ABA) is an important phytohormone involved in abiotic stress tolerance in plants. The group A bZIP transcription factors play important roles in the ABA signaling pathway in Arabidopsis but little is known about their functions in rice. In our current study, we have isolated and characterized a group A bZIP transcription factor in rice, OsABF2 (Oryza sativa ABA-responsive element binding factor 2). It was found to be expressed in various tissues in rice and induced by different types of abiotic stress treatments such as drought, salinity, cold, oxidative stress, and ABA. Subcellular localization analysis in maize protoplasts using a GFP fusion vector indicated that OsABF2 is a nuclear protein. In yeast experiments, OsABF2 was shown to bind to ABA-responsive elements (ABREs) and its N-terminal region found to be necessary to transactivate a downstream reporter gene. A homozygous T-DNA insertional mutant of OsABF2 is more sensitive to salinity, drought, and oxidative stress compared with wild type plants. In addition, this Osabf2 mutant showed a significantly decreased sensitivity to high levels of ABA at germination and post-germination. Collectively, our present results indicate that OsABF2 functions as a transcriptional regulator that modulates the expression of abiotic stress-responsive genes through an ABA-dependent pathway.

MTERF2 is a nucleoid component in mammalian mitochondria.

The mammalian MTERF family of proteins has four members, named MTERF1 to MTERF4, which were identified in homology searches using the mitochondrial transcription termination factor, mTERF (here denoted MTERF1) as query. MTERF1 and MTERF3 are known to participate in the control of mitochondrial DNA transcription, but the function of the other two proteins is not known. We here investigate the structure and function of MTERF2. Protein import experiments using isolated organelles confirm that MTERF2 is a mitochondrial protein. Edman degradation of MTERF2 isolated from stably transfected HeLa cells demonstrates that mature MTERF2 lacks a targeting peptide (amino acids 1-35) present in the precursor form of the protein. MTERF2 is a monomer in isolation and displays a non sequence-specific DNA-binding activity. In vivo quantification experiments demonstrate that MTERF2 is relatively abundant, with one monomer present per approximately 265 bp of mtDNA. In comparison, the mtDNA packaging factor TFAM is present at a ratio of one molecule per approximately 10-12 bp of mtDNA. Using formaldehyde cross-linking we demonstrate that MTERF2 is present in nucleoids, and therefore must be located in close proximity to mtDNA. Taken together, our work provides a basic biochemical characterization of MTERF2, paving the way for future functional studies.

Intrinsically unstructured N-terminal domain of bZIP transcription factor HY5.

The Arabidopsis HY5 protein is a basic leucine zipper (bZIP) transcription factor that promotes photomorphogenesis. HY5 binds directly to the promoters of light responsible element containing the G-box and thus regulates their transcriptional activity. The level and activity of HY5 are negatively regulated, in a light-dependent manner, by interaction with the COP1 protein, which targets HY5 for proteasome-mediated degradation in the nucleus. Despite its essential roles in plant development, no structural information exists for HY5. In this article, we report the first structural and biophysical characterization of HY5. Using limited proteolysis in combination with mass spectrometry, circular dichroism, and nuclear magnetic resonance spectroscopy, we have deduced that the N-terminal 77 amino acids of HY5 form a premolten globular structure, while amino acids 78-110, which constitute the basic region (BR) of the protein, exist in a molten globule state. Our studies also revealed that the overall structural features of full-length HY5 are dominated largely by the disordered N-terminal domain, despite the existence of a bZIP domain at its C-terminus. We propose that HY5 is a member of the intrinsically unstructured protein (IUP) family, and that HY5 functions as an unstructured protein and benefits from being the same, in vivo.

Thyroid hormone-induced expression of a bZip-containing transcription factor activates epithelial cell proliferation during Xenopus larval-to-adult intestinal remodeling.

In the intestine during amphibian metamorphosis, stem cells appear, actively proliferate, and differentiate into an adult epithelium analogous to the mammalian counterpart. To clarify the molecular mechanisms regulating this process, we focused on a bZip-containing transcription factor (TH/bZip). We previously isolated TH/bZip from the Xenopus intestine as one of the candidate genes involved in adult epithelial development. Northern blot and in situ hybridization analyses showed that the transient and region-dependent expression of TH/bZip mRNA correlates well with the growth of adult epithelial primordia originating from the stem cells throughout the Xenopus intestine. To investigate its role in the adult epithelial development, we established an in vitro gene transfer system by using electroporation and organ culture techniques, and we overexpressed TH/bZip in the epithelium of Xenopus tadpole intestines. In the presence of thyroid hormone (TH) where the adult epithelial primordia appeared after 3 days of cultivation, overexpression of TH/bZip significantly increased their proliferating activity. On the other hand, in the absence of TH where the epithelium remained as larval-type without any metamorphic changes, ectopic expression of TH/bZip significantly increased the proliferating activity of the larval epithelium but had no effects on its differentiated state. These results indicate that TH/bZip functions as a growth activator during amphibian intestinal remodeling, although TH/bZip expression in the epithelium alone is not sufficient for inducing the stem cells.

TGA1 and G-box binding factors: two distinct classes of Arabidopsis leucine zipper proteins compete for the G-box-like element TGACGTGG.

Regulatory elements containing the sequence ACGT are found in several plant promoters and are recognized by various basic/leucine zipper (bZIP) proteins. The Arabidopsis G-box binding factor 1 (GBF1), initially identified by its ability to bind to the palindromic G-box (CCACGTGG), also interacts with the TGACGT motif if this hexamer sequence is followed by either the dinucleotide GG--as found in the Hex motif of the wheat histone 3 promoter--or GT. Here we describe the isolation of an Arabidopsis bZIP protein, denoted TGA1, that also recognizes ACGT-containing sequences. However, TGA1 differs from members of the GBF family in the spectrum of base pair permutations flanking the ACGT sequence that are required for DNA binding. TGA1 primarily requires a TGACG motif and preferentially binds to those pentamers that are followed by a T residue. We show that although both TGA1 and GBF1 bind to the Hex motif (TGACGTGG), this binding can be distinguished on the basis of their specific DNA-protein contacts. Furthermore, TGA1 also differs from members of the GBF family in that it apparently does not form heterodimers with any member of this family.