Ternary complex formation between HvMYBS3 and other factors involved in transcriptional control in barley seeds.
ABSTRACT The SHAQKYF R1MYB transcription factor (TF) HvMYBS3 from barley is an activator of gene expression both during endosperm development and in aleurone cells upon seed germination. Its mRNA was detected as early as 10 days after flowering in developing barley endosperm, with a peak at 18 days, and in aleurone cells at 8 h after water imbibition, as shown by Northern blot and in situ hybridization analyses. The HvMYBS3 protein expressed in bacteria binds to oligonucleotides containing a GATA core derived from the promoters of: (i) the developing endosperm gene Itr1 (5'-GATAAGATA-3') encoding trypsin inhibitor BTI-CMe, and (ii) the post-germinating aleurone gene Amy6.4 (5'-TATCCAC-3'/5'-GTGGATA-3') encoding a high-pI alpha-amylase. Transient expression experiments in co-bombarded developing endosperms and in barley aleurone layers demonstrated that HvMYBS3 trans-activated transcription both from Itr1 and Amy6.4 promoters, in contrast with a previously reported seed-expressed R1MYB, HvMCB1, which was an activator of Itr1 and a transcriptional repressor of the Amy6.4 gene. In the yeast three-hybrid system, the HvMYBS3 protein formed a ternary complex with BPBF and BLZ2, two important seed TFs. However, no binary interactions could be detected between HvMYBS3 and BLZ2 or between HvMYBS3 and BPBF.
- SourceAvailable from: Chakravarthi Mohan[show abstract] [hide abstract]
ABSTRACT: Ubiquitin promoters are promising candidates for constitutive transgene expression in plants. In this study, we isolated and characterized a novel 5′ regulatory sequence of a ubiquitin gene from Porteresia coarctata, a stress-tolerant wild grass species. Through functional analysis in heterologous plant systems, we have demonstrated that full length (Port Ubi2.3) or truncated sequence (PD2) of the isolated regulatory fragment can drive constitutive expression of GUS in monocots and/or dicots. In silico analysis of Port Ubi2.3 has revealed the presence of a 640 bp core promoter region followed by two exons and two introns with numerous putative cis-acting sites scattered throughout the regulatory region. Transformation and expression studies of six different deletion constructs in rice, tobacco and sugarcane revealed that the proximal intron has an enhancing effect on the activity of the core promoter in both monocots and dicots, whereas, Port Ubi2.3 was able to render strong expression only in monocots. This regulatory sequence is quite distinct from the other reported ubiquitin promoters in structure and performs better in monocots compared to other commonly used promoters—maize Ubi1 and Cauliflower Mosaic Virus 35S.Plant Cell Reports 03/2013; · 2.51 Impact Factor
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ABSTRACT: BACKGROUND: Transcription factors (TFs) are proteins that have played a central role both in evolution and in domestication, and are major regulators of development in living organisms. Plant genome sequences reveal that approximately 7% of all genes encode putative TFs. The DOF (DNA binding with One Finger) TF family has been associated with vital processes exclusive to higher plants and to their close ancestors (algae, mosses and ferns). These are seed maturation and germination, light-mediated regulation, phytohormone and plant responses to biotic and abiotic stresses, etc. In Hordeum vulgare and Oryza sativa, 26 and 30 different Dof genes, respectively, have been annotated. Brachypodium distachyon has been the first Pooideae grass to be sequenced and, due to its genomic, morphological and physiological characteristics, has emerged as the model system for temperate cereals, such as wheat and barley. RESULTS: Through searches in the B. distachyon genome, 27 Dof genes have been identified and a phylogenetic comparison with the Oryza sativa and the Hordeum vulgare DOFs has been performed. To explore the evolutionary relationship among these DOF proteins, a combined phylogenetic tree has been constructed with the Brachypodium DOFs and those from rice and barley. This phylogenetic analysis has classified the DOF proteins into four Major Cluster of Orthologous Groups (MCOGs). Using RT-qPCR analysis the expression profiles of the annotated BdDof genes across four organs (leaves, roots, spikes and seeds) has been investigated. These results have led to a classification of the BdDof genes into two groups, according to their expression levels. The genes highly or preferentially expressed in seeds have been subjected to a more detailed expression analysis (maturation, dry stage and germination). CONCLUSIONS: Comparison of the expression profiles of the Brachypodium Dof genes with the published functions of closely related DOF sequences from the cereal species considered here, deduced from the phylogenetic analysis, indicates that although the expression profile has been conserved in many of the putative orthologs, in some cases duplication followed by subsequent divergence may have occurred (neo-functionalization).BMC Plant Biology 11/2012; 12(1):202. · 4.35 Impact Factor
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ABSTRACT: KEY MESSAGE : GUS analysis in Cryptomeria japonica revealed that the CjMALE1 promoter is activated in the male strobilus of C. japonica. Toward the development of male sterile technology for Cryptomeria japonica, a male strobilus-dominant promoter of C. japonica was isolated. The CjMALE1 gene was isolated from a male strobilus-specific suppression subtractive hybridization (SSH) library, and the promoter was isolated by the TAIL-PCR method. To characterize the CjMALE1 promoter, β-glucuronidase (GUS)-fused genes were constructed and introduced into C. japonica using Agrobacterium tumefaciens. GUS expression from CjMALE1-2.5 K (2,718 bp fragment)::GUS C. japonica and CjMALE1-1 K (1,029 bp fragment)::GUS C. japonica was detected in the tapetum and microspore mother cells. These promoter fragments were comparably active in the pre-meiotic stage of the male strobilus of C. japonica. Our analysis showed that the 1,029 bp promoter had all the cis-elements necessary for male strobilus-dominant expression of CjMALE1. When CjMALE1-1 K::GUS was introduced into Arabidopsis, GUS expression was detected in the same spatiotemporal pattern as in C. japonica. These results suggest that the CjMALE1 promoter is subject to transcriptional regulatory systems consisting of cis- and trans-elements that have been highly conserved during evolution.Plant Cell Reports 11/2012; · 2.51 Impact Factor