Molecular interaction of bZIP domains of GBF1, HY5 and HYH in Arabidopsis seedling development

The bZIP proteins, GBF1, HY5 and HYH, play important regulatory roles in Arabidopsis seedling development. Whereas GBF1 plays a dual regulatory role, HY5 and HYH act as positive regulators of photomorphogenesis. The molecular and functional relations of GBF1 with HY5 and HYH in photomorphogenesis have recently been demonstrated. However, the possible interaction of bZIP domain of each of these proteins remains to be investigated. In this study, our results suggest that bZIP domains of HY5 and HYH are able to interact with the bZIP domain of GBF1. Taken together with the earlier study,⁹ these results suggest that the N-terminal domain of GBF1 has an inhibitory effect on its interaction with HY5 and HYH.     

Light regulated modulation of Z-box containing promoters by photoreceptors and downstream regulatory components,COP1 and HY5, in Arabidopsis

The Z-box is one of the light-responsive elements (LREs) found in the promoters of light inducible genes. We have studied the light responsive characteristics of Z-box containing synthetic as well as native promoters. We show that promoters with Z-box as a single LRE or paired with another LRE can respond to a broad spectrum of light. The response is primarily mediated by phyA, phyB and CRY1 photoreceptors at their respective wavelengths of light. We have demonstrated that CAB1 and Z-GATA containing promoters are down-regulated in hy5 mutants in the light. On the other hand, a promoter with Z-box alone is down-regulated in hy5 mutants both in dark and in light conditions, suggesting involvement of a similar regulatory system in the regulation of the promoter in two distinct developmental pathways: skotomorphogenesis and photomorphogenesis. Furthermore, similar to the CAB1 promoter, a Z-GATA containing promoter is derepressed in cop1 mutants in the dark. DNA-protein interaction studies reveal the presence of a DNA-binding activity that is specific to Z-box. These results provide insights into the regulation of the Z-box LRE mediated by various light signaling components.     

Arabidopsis HY5 protein functions as a DNA-binding tag for purification and functional immobilization of proteins on agarose/DNA microplate

Protein microarray is considered to be one of the key analytical tools for high-throughput protein function analysis. Here, we report that the Arabidopsis HY5 functions as a novel DNA-binding tag (DBtag) for proteins. We also demonstrate that the DBtagged proteins could be immobilized and purified on a newly designed agarose/DNA microplate. Furthermore, we show three applications using the microarray: (1) detection of autophosphorylation activity of DBtagged human protein kinases and inhibition of their activity by staurosporine, (2) specific cleavage of DBtagged proteins by a virus protease and caspase 3, and (3) detection of a protein-protein interaction between the DBtagged UBE2N and UBE2v1. Thus, this method may facilitate rapid functional analysis of a wide range of proteins.     

Sulfurtransferases 1 and 2 play essential roles in embryo and seed development in Arabidopsis thaliana

Sulfurtransferases (STRs) catalyze the transfer of a sulfur atom from a donor to a suitable acceptor molecule. The Arabidopsis thaliana genome encodes 20 putative STR proteins. The biological functions of most are unclear. We found that STR1 and STR2 play important roles in embryo/seed development. Mutation of STR1 alone resulted in a shrunken seed phenotype, although growth and development of vegetative and reproductive organs were not affected. The shrunken seed phenotype was associated with the delayed/arrested embryo development, in most cases, at the heart stage. The embryo defect of str1 mutant is not fully penetrant. Approximately 12.5% of embryos developed further and formed normal looking seeds. In severely shrunken seeds, no embryo could be identified after seed collection. Partially shrunken seeds that contained viable embryos could still germinate. However, cotyledons of the seedlings from such seeds were abnormal. An STR1-GUS fusion reporter revealed that the STR1 gene was universally expressed, with high levels of expression in specific tissues/organs including embryos. The incomplete penetrance of str1 embryo/seed phenotype is a result of functional STR2. Single str2 mutant had no phenotype. However, no str1(-/-)/str2(-/-) double mutant embryos were able to develop past the heart stage. Furthermore, STR2 is haplo-insufficient in str1 mutant background, and str1(-/-)/str2(+/-) embryos were 100% lethal. These data provide new insights into the biological functions of the ubiquitous sulfurtransferase in Arabidopsis embryogenesis and seed development.     

ethylene receptor 1 (etr1) Is Sufficient and Has the Predominant Role in Mediating Inhibition of Ethylene Responses by Silver in Arabidopsis thaliana

Ethylene influences many processes in Arabidopsis thaliana through the action of five receptor isoforms. All five isoforms use copper as a cofactor for binding ethylene. Previous research showed that silver can substitute for copper as a cofactor for ethylene binding activity in the ETR1 ethylene receptor yet also inhibit ethylene responses in plants. End-point and rapid kinetic analyses of dark-grown seedling growth revealed that the effects of silver are mostly dependent upon ETR1, and ETR1 alone is sufficient for the effects of silver. Ethylene responses in etr1-6 etr2-3 ein4-4 triple mutants were not blocked by silver. Transformation of these triple mutants with cDNA for each receptor isoform under the promoter control of ETR1 revealed that the cETR1 transgene completely rescued responses to silver while the cETR2 transgene failed to rescue these responses. The other three isoforms partially rescued responses to silver. Ethylene binding assays on the binding domains of the five receptor isoforms expressed in yeast showed that silver supports ethylene binding to ETR1 and ERS1 but not the other isoforms. Thus, silver may have an effect on ethylene signaling outside of the ethylene binding pocket of the receptors. Ethylene binding to ETR1 with silver was ～30% of binding with copper. However, alterations in the K(d) for ethylene binding to ETR1 and the half-time of ethylene dissociation from ETR1 do not underlie this lower binding. Thus, it is likely that the lower ethylene binding activity of ETR1 with silver is due to fewer ethylene binding sites generated with silver versus copper.     

Mapping of interaction domains of putative telomere-binding proteins AtTRB1 and AtPOT1b from Arabidopsis thaliana

We previously searched for interactions between plant telomere-binding proteins and found that AtTRB1, from the single-myb-histone (Smh) family, interacts with the Arabidopsis POT1-like-protein, AtPOT1b, involved in telomere capping. Here we identify domains responsible for that interaction. We also map domains in AtTRB1 responsible for interactions with other Smh-family-members. Our results show that the N-terminal OB-fold-domain of AtPOT1b mediates the interaction with AtTRB1. This domain is characteristic for POT1- proteins and is involved with binding the G-rich-strand of telomeric DNA. AtPOT1b also interacts with AtTRB2 and AtTRB3. The central histone-globular-domain of AtTRB1 is involved with binding to AtTRB2 and 3, as well as to AtPOT1b. AtTRB1-heterodimers with other Smh-family-members are more stable than AtTRB1-homodimers. Our results reveal interaction networks of plant telomeres.