Altered Promoter and G-Box Binding Factor for 1-Deoxy-d-Xylulose-5-Phosphate Synthase Gene Grown from Poa pratensis Seeds after Spaceflight.

In plant cells, the nucleus DNA is considered the primary site of injury by the space environment, which could generate genetic alteration. As the part of genomic mutation, genetic variation in the promoter region could regulate gene expression. In the study, it is observed that there is a deletion in the upstream regulatory region of the 1-deoxy-d-xylulose-5-phosphate synthase 1 gene (PpDXS1) of Poa pratensis dwarf mutant and the PpDXS1 transcript abundance is lower in the dwarf mutant. It is indicated that the deletion in the promoter region between wild type and dwarf mutant could be responsible for the regulation of PpDXS1 gene expression. The PpDXS1 promoter of dwarf mutant shows a lower activity as determined by dual luciferase assay in Poa pratensis protoplast, as well as the GUS activity is lower in transgenic Poa pratensis plant. To further investigate the effect of the deletion in the promoter region on PpDXS1 transcript accumulation, the transient assay and yeast one-hybrid experiment demonstrate that the deletion comprises a motif which is a target of G-box binding factor (GBF1), and the motif correlates with an increase in transactivation by GBF1 protein. Taken together, these results indicate that the deletion in the promoter of PpDXS1 isolated from dwarf mutant is sufficient to account for the decrease in PpDXS1 transcript level and GBF1 can regulate the PpDXS1 gene expression, and subsequently affect accumulation of various isoprenoids throughout the plant.

Transcriptional circuitry underlying seed coat development in Arabidopsis.

We analyzed two sub-regions of the maternal seed coat, chalazal (CZSC) and distal (SC), using transcriptomic and histological analyses in the model plant Arabidopsis thaliana. Hierarchical clustering analysis showed that the CZSC and SC are transcriptionally distinct, though the two sub-regions are more similar during early stages of seed development. Robust statistical and network analysis revealed novel roles for both sub-regions during the course of the seed lifecycle and provides insight into the regulatory circuitry underlying these poorly studied sub-regions of the seed. Data show many of the processes that characterize the SC including starch deposition during the morphogenesis phase, and mucilage deposition and cell wall thickening during the maturation phase, are either absent or expressed to a much lesser extent in the CZSC. We further analyzed the CZSC in detail and show that this sub-region is likely involved in the control of information into the seed from the maternal plant and that some of these processes are predicted to operate through the activity of bZIP transcription factors through the G-box DNA sequence motif.

Tobacco MYC2 regulates jasmonate-inducible nicotine biosynthesis genes directly and by way of the NIC2-locus ERF genes.

In Arabidopsis, the MYC2-family basic helix-loop-helix transcription factors mediate transcriptional regulation of jasmonate-responsive genes, and their transcriptional activities are suppressed by physical interactions with jasmonate-ZIM domain (JAZ) proteins. Jasmonate-inducible nicotine formation in Nicotiana plants has been shown to be suppressed by tobacco JAZ proteins, and be regulated by both MYC2-related and NIC2-locus ethylene response factor (ERF) transcription factors. We here show that tobacco MYC2 (NtMYC2) recognizes the G-box sequences, 5'-CAC(G/A)T(G/T)-3', found in the proximal promoter regions of several nicotine biosynthesis genes, including Putrescine N-Methyltransferase 2 (PMT2) and Quinolinate Phosphoribosyltransferase 2 (QPT2). Transient transactivation assays using cultured tobacco cells showed that NtMYC2 and NIC2-locus ERF189 additively activated the PMT2 and QPT2 promoters depending on their cognate binding sites. RNA interference (RNAi) silencing of NtMYC2 in tobacco hairy roots strongly decreased transcript levels of jasmonate-responsive structural genes, including those involved in nicotine biosynthesis, as well as the NIC2-locus ERF genes. Conversely, ERF189 was not required for the expression of NtMYC2. NtMYC2, but not ERF189, interacted with tobacoo JAZs in a yeast two-hybrid assay. These results indicate that NtMYC2 controls nicotine biosynthesis genes in two combinatorial ways, by directly binding the G-box in the target promoters and by up-regulating the NIC2-locus ERF genes.

Synergy between two transcription factors directs gene expression in Dictyostelium tip-organiser cells.

cotC requires the transcription factor CudA for its expression in the posterior, prespore cells of the slug, while the expL7 gene requires CudA for its expression in the anterior, tip-organiser region. In order to identify additional transcription factors that might mediate tip-organiser specific expression, we performed affinity chromatography on slug nuclear extracts. The affinity matrix bore cap-site distal sequences from region A of the expL7 promoter; an essential region located upstream of the CudA binding domain. One of the proteins purified was G-box binding factor (GBF), a zinc finger transcription factor which binds to G-rich elements, known as G boxes, that are present in the promoters of many developmental genes, including cotC. Previous work identified an essential sequence motif within region A and we show that this element is a G box, that binds recombinant GBF. Moreover, a G box from within the cotC promoter can substitute for region A of expL7 in directing tip-organiser specific expression of expL7. Thus the same two transcription factors, CudA and GBF, seem to co-operate to direct both tip-organiser and prespore gene expression. How then is specificity achieved? Replacing a CudA binding region in the cotC promoter with the CudA binding domain from expL7 strongly represses cotC promoter activity. Hence we suggest that differences in the topology of the multiple CudA half- sites contained within the two different CudA binding regions, coupled with differences in the signalling environment between tip-organiser cells and prespore cells, ensure correct expL7 expression.

Analysis of endocytic pathways in Drosophila cells reveals a conserved role for GBF1 in internalization via GEECs.

In mammalian cells, endocytosis of the fluid phase and glycosylphosphatidylinositol-anchored proteins (GPI-APs) forms GEECs (GPI-AP enriched early endosomal compartments) via an Arf1- and Cdc42-mediated, dynamin independent mechanism. Here we use four different fluorescently labeled probes and several markers in combination with quantitative kinetic assays, RNA interference and high resolution imaging to delineate major endocytic routes in Drosophila cultured cells. We find that the hallmarks of the pinocytic GEEC pathway are conserved in Drosophila and identify garz, the fly ortholog of the GTP exchange factor GBF1, as a novel component of this pathway. Live confocal and TIRF imaging reveals that a fraction of GBF1 GFP dynamically associates with ABD RFP (a sensor for activated Arf1 present on nascent pinosomes). Correspondingly, a GTP exchange mutant of GBF1 has altered ABD RFP localization in the evanescent field and is impaired in fluid phase uptake. Furthermore, GBF1 activation is required for the GEEC pathway even in the presence of Brefeldin A, implying that, like Arf1, it has a role in endocytosis that is separable from its role in secretion.

A carrot G-box binding factor-type basic region/leucine zipper factor DcBZ1 is involved in abscisic acid signal transduction in somatic embryogenesis.

Carrot (Daucus carota) somatic embryogenesis has been extensively used as an experimental system for studying embryogenesis. In maturing zygotic embryos, abscisic acid (ABA) is involved in acquisition of desiccation tolerance and dormancy. On the other hand, somatic embryos contain low levels of endogenous ABA and show desiccation intolerance and lack dormancy, but tolerance and dormancy can be induced by exogenous application of ABA. In ABA-treated carrot embryos, some ABA-inducible genes are expressed. We isolated the Daucus carota bZIP1 (DcBZ1) gene encoding a G-box binding factor-type basic region/leucine zipper (GBF-type bZIP) factor from carrot somatic embryos. The expression of DcBZ1 was detected in embryogenic cells, non-embryogenic cells, somatic embryos, developing seeds, seedlings, and true leaves. Notably, higher expression was detected in embryogenic cells, true leaves, and seedlings. The expression of DcBZ1 increased in seedlings and true leaves after ABA treatment, whereas expression was not affected by differences in light conditions. During the development of zygotic and somatic embryos, increased expression of DcBZ1 was commonly detected in the later phase of development. The recombinant DcBZ1 protein showed specific binding activity to the two ABA-responsive element-like motifs (motif X and motif Y) in the promoter region of the carrot ABA-inducible gene according to results from an electrophoretic mobility shift assay. Our findings suggest that the carrot GBF-type bZIP factor, DcBZ1, is involved in ABA signal transduction in embryogenesis and other vegetative tissues.

AtbZIP16 and AtbZIP68, two new members of GBFs, can interact with other G group bZIPs in Arabidopsis thaliana.

AtbZIP16 and AtbZIP68 are two putative G group bZIP transcription factors in Arabidopsis thaliana, the other three members of G group bZIPs are GBF1-3 which can bind G-box. Members of G group have conservative protein structure: highly homological basic region and a proline-rich domain in the N-terminal region. Here, we report that AtbZIP16 and AtbZIP68 could bind cis elements with ACGT core, such as G-box, Hex, C-box and As-1, but with different binding affinities which from high to low were G-box > Hex > C-box > As-1; AtbZIP16 and AtbZIP68 could form homodimer and form heterodimer with other members of G group; N-terminal proline rich domain of AtbZIP16 had transactivation activity in yeast cells while that of AtbZIP68 did not; AtbZIP16 and AtbZIP68 GFP fusion protein localized in the nucleus of onion epidermal cells. These results indicated that AtbZIP16 and AtbZIP68 were two new members of GBFs. In Arabidopsis, AtbZIP16 and AtbZIP68 may also participate in light-responsive process in which GBF1-3 are involved.

A central integrator of transcription networks in plant stress and energy signalling.

Photosynthetic plants are the principal solar energy converter sustaining life on Earth. Despite its fundamental importance, little is known about how plants sense and adapt to darkness in the daily light-dark cycle, or how they adapt to unpredictable environmental stresses that compromise photosynthesis and respiration and deplete energy supplies. Current models emphasize diverse stress perception and signalling mechanisms. Using a combination of cellular and systems screens, we show here that the evolutionarily conserved Arabidopsis thaliana protein kinases, KIN10 and KIN11 (also known as AKIN10/At3g01090 and AKIN11/At3g29160, respectively), control convergent reprogramming of transcription in response to seemingly unrelated darkness, sugar and stress conditions. Sensing and signalling deprivation of sugar and energy, KIN10 targets a remarkably broad array of genes that orchestrate transcription networks, promote catabolism and suppress anabolism. Specific bZIP transcription factors partially mediate primary KIN10 signalling. Transgenic KIN10 overexpression confers enhanced starvation tolerance and lifespan extension, and alters architecture and developmental transitions. Significantly, double kin10 kin11 deficiency abrogates the transcriptional switch in darkness and stress signalling, and impairs starch mobilization at night and growth. These studies uncover surprisingly pivotal roles of KIN10/11 in linking stress, sugar and developmental signals to globally regulate plant metabolism, energy balance, growth and survival. In contrast to the prevailing view that sucrose activates plant SnRK1s (Snf1-related protein kinases), our functional analyses of Arabidopsis KIN10/11 provide compelling evidence that SnRK1s are inactivated by sugars and share central roles with the orthologous yeast Snf1 and mammalian AMPK in energy signalling.

The SebHLH transcription factor mediates trans-activation of the SeFAD2 gene promoter through binding to E- and G-box elements.

Microsomal oleic acid desaturase (FAD2) catalyzes the first extra-plastidial desaturation in plants, converting oleic acid to linoleic acid, which is a major constituent in all cellular membranes as well as in seed storage oils. Seed-specific FAD2 (SeFAD2) produced 40% of linoleic acids in the total fatty acids of sesame (Sesamum indicum) seeds. The expression of SeFAD2 transcripts was spatially and temporally controlled during seed development. To investigate the regulatory mechanism controlling seed-specific SeFAD2 expression, we isolated a well-matched sequence homologous to the basic region/helix-loop-helix proteins by yeast one-hybrid screening and named it SebHLH. SebHLH transcripts were expressed in developing seeds and roots of sesame. SebHLH:GFP fusion protein localized in the nucleus. Recombinant SebHLH protein bound E-box (CANNTG) and G-box (CACGTG) elements in the region from -179 to -53 of the SeFAD2 gene promoter, and the external C and G nucleotides in the E- and G-box motifs were essential for SebHLH protein binding. The SebHLH gene, under the CaMV35S promoter, and the GUS reporter gene driven by E- and G-box motifs were co-expressed in developing sesame seeds and Arabidopsis transgenic leaves. This co-expression demonstrated that SebHLH protein mediates transactivation of the SeFAD2 gene promoter through binding to E- and G-box elements. E- or G-box elements frequently occur in the 5'-flanking region of genes that are involved in triacylglycerol biosynthesis and that exhibit seed-specific expression in Arabidopsis and other plants, suggesting that bHLH transcription factors play a key role in the transcriptional regulation of genes related to storage lipid biosynthesis and accumulation during seed development.

Identification of a bHLH-type G-box binding factor and its regulation activity with G-box and Box I elements of the PsCHS1 promoter.

With the use of in vivo recombination theory, the screening time of yeast one-hybrid system was decreased in the present study. A basic helix-loop-helix (bHLH) protein PsGBF was successfully obtained from a glutathione (GSH)-induced pea cDNA library using the G-box cis-element of the PsCHS1 promoter as a bait. Electrophoretic mobility shift assay (EMSA) and beta-galactosidase assay results suggested that PsGBF possesses both G-box-specific binding and transcription-activating activities. The specific interaction of PsGBF with G-box was further confirmed by in vivo transient expression assays in tobacco. The current study examined the combination effect of G-box with Box I elements in the interaction with PsGBF or OsMYC. The results indicated that PsGBF bound with the G-box, but not the Box I element. Moreover, this combination effect of G-box and Box I only associated with PsGBF but not with other bHLH-type proteins such as OsMYC.

Identification of a new AT-rich-element binding factor PsATF1 and its combined effect with PsGBF on the activation of PsCHS1 promoter.

Chaltone synthase (CHS) is a key speed-limiting enzyme in the phenylpropanoid pathway which plays an important role in plant defense response against pathogens. In the PsCHS1 promoter, there is an AT-rich element (ATRE) which is required for the maximal elicitor-mediated activation. However, the transcription activator of the ATRE and its regulation mechanism in pea keep unclear. In this paper, a new ATRE-binding factor was isolated from an elicitor-induced pea cDNA expression library and was designated as PsATF1. Electrophoretic mobility shift assay (EMSA) indicated the ATRE-specific binding activity of PsATF1. Beta-galactosidase assays in yeast cells suggested that PsATF1 possessed transcription-activating activity because PsATF1 activated the expression of the reporter gene even without the GAL4 activation domain (AD). The current study also examined the co-activation effects of PsATF1 with another transcription factor PsGBF on ATRE or PsCHS1 promoter through a transient expression system. The present work reports that PsATF1 acts as a complete transcription activator and first indicates that there are combined effects of PsATF1 with PsGBF on the activation of PsCHS1 promoter. These results provide theoretical basis to the plant defense gene expression mechanism regulated by multiple activators.

In vivo binding of hot pepper bZIP transcription factor CabZIP1 to the G-box region of pathogenesis-related protein 1 promoter.

We find that salicylic acid and ethephon treatment in hot pepper increases the expression of a putative basic/leucine zipper (bZIP) transcription factor gene, CabZIP1. CabZIP1 mRNA is expressed ubiquitously in various organs. The green fluorescent protein-fused transcription factor, CabZIP1::GFP, can be specifically localized to the nucleus, an action that is consistent with the presence of a nuclear localization signal in its protein sequence. Transient overexpression of the CabZIP1 transcription factor results in an increase in PR-1 transcripts level in Nicotiana benthamiana leaves. Using chromatin immunoprecipitation, we demonstrate that CabZIP1 binds to the G-box elements in native promoter of the hot pepper pathogenesis-related protein 1 (CaPR-1) gene in vivo. Taken together, our results suggest that CabZIP1 plays a role as a transcriptional regulator of the CaPR-1 gene.

Disruption of SLP-76 interaction with Gads inhibits dynamic clustering of SLP-76 and FcepsilonRI signaling in mast cells.

We developed a confocal real-time imaging approach that allows direct observation of the subcellular localization pattern of proteins involved in proximal FcepsilonRI signaling in RBL cells and primary bone marrow-derived mast cells. The adaptor protein Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is critical for FcepsilonRI-induced calcium flux, degranulation, and cytokine secretion. In this study, we imaged SLP-76 and found it in the cytosol of unstimulated cells. Upon FcepsilonRI cross-linking, SLP-76 translocates to the cell membrane, forming clusters that colocalize with the FcepsilonRI, the tyrosine kinase Syk, the adaptor LAT, and phosphotyrosine. The disruption of the SLP-76 interaction with its constitutive binding partner, Gads, through the mutation of SLP-76 or the expression of the Gads-binding region of SLP-76, inhibits the translocation and clustering of SLP-76, suggesting that the interaction of SLP-76 with Gads is critical for appropriate subcellular localization of SLP-76. We further demonstrated that the expression of the Gads-binding region of SLP-76 in bone marrow-derived mast cells inhibits FcepsilonRI-induced calcium flux, degranulation, and cytokine secretion. These studies revealed, for the first time, that SLP-76 forms signaling clusters following FcepsilonRI stimulation and demonstrated that the Gads-binding region of SLP-76 regulates clustering of SLP-76 and FcepsilonRI-induced mast cell responses.