Arabidopsis thaliana DOF6 negatively affects germination in non-after ripened seeds and interacts with TCP14

Seed dormancy prevents seeds from germinating under environmental conditions unfavourable for plant growth and development and constitutes an evolutionary advantage. Dry storage, also known as after-ripening, gradually decreases seed dormancy by mechanisms not well understood. An Arabidopsis thaliana DOF transcription factor gene (DOF6) affecting seed germination has been characterized. The transcript levels of this gene accumulate in dry seeds and decay gradually during after-ripening and also upon seed imbibition. While constitutive over-expression of DOF6 produced aberrant growth and sterility in the plant, its over-expression induced upon seed imbibition triggered delayed germination, abscisic acid (ABA)-hypersensitive phenotypes and increased expression of the ABA biosynthetic gene ABA1 and ABA-related stress genes. Wild-type germination and gene expression were gradually restored during seed after-ripening, despite of DOF6-induced over-expression. DOF6 was found to interact in a yeast two-hybrid system andin planta with TCP14, a previously described positive regulator of seed germination. The expression of ABA1 and ABA-related stress genes was also enhanced in tcp14 knock-out mutants. Taken together, these results indicate that DOF6 negatively affects seed germination and opposes TCP14 function in the regulation of a specific set of ABA-related gene

Green Fluorescent Protein (GFP) in Vector Systems Played Sense Role of Epigenetic in Plants

The green fluorescent protein (GFP) of jellyfish (_Aequorea victoria_) has significant advantages over other reporter genes, because expression can be detected in living cells without any substrates. Recently, epigenetic phenomena are important to consider in plant biotechnology experiments for elucidate unknown mechanism. Therefore, soybean immature cotyledons were generated embryogenesis cells and engineered with two different gene constructs (pHV and pHVS) using gene gun method. Both constructs contain a gene conferring resistance to hygromycin (_hpt_) as a selective marker and a modified glycinin (11S globulin) gene (_V3-1_) as a target. However, sGFP(_S65T_) as a reporter gene was used only in pHVS as a reporter gene for study the relation between using sGFP(_S65T_) and gene silencing phenomena. Fluorescence microscopic was used for screening after the selection of hygromycin, identified clearly the expression of sGFP(_S65T_) in the transformed soybean embryos bombarded with the pHVS construct. Protein analysis was used to detect gene expression overall seeds using SDS-PAGE. Percentage of gene down regulation was highly in pHV construct compared with pHVS. Thus, sGFP(_S65T_) as a reporter gene in vector system may be play useful role for transgenic evaluation and avoid gene silencing in plants for the benefit of plant transformation system

HnRNP-like proteins as post-transcriptional regulators

Plant cells contain a diverse repertoire of RNA-binding proteins (RBPs) that coordinate a network of post-transcriptional regulation. RBPs govern diverse developmental processes by modulating the gene expression of specific transcripts. Recent gene annotation and RNA sequencing clearly showed that heterogeneous nuclear ribonucleoprotein (hnRNP)-like proteins which form a family of RBPs, are also expressed in higher plants and serve specific plant functions. In addition to their involvement in post-transcriptional regulation from mRNA capping to translation, they are also involved in telomere regulation, gene silencing and regulation in chloroplast. Here, we review the involvement of plant hnRNP-like proteins in post-transcription regulation of RNA processes and their functional roles in control of plant developmental processes especially plant-specific functions including flowering, chloroplastic-specific mRNA regulation, long-distance phloem transportation and plant responses to environmental stresses

miRNA/phasiRNA mediated regulation of plant defense response against P. syringae

Gene silencing is a mechanism of regulation of gene expression where the small RNAs (sRNAs) are key components for giving specificity to the system. In plants, two main types of noncoding small RNA molecules have been found: microRNAs (miRNAs) and small interfering RNAs (siRNAs). DCL proteins acting on large RNA precursors produce the mature forms of sRNAs (20-24nt) that can act as negative regulators of gene expression. In recent years, the role of miRNAs in regulation of gene expression in plant responses against bacterial pathogens is becoming clearer. Comparisons carried out in our lab between expression profiles of different Arabidopsis thaliana mutants affected in gene silencing, and plants challenged with Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, as differentially expressed in these conditions. Through the use of bioinformatics tools, we found a miRNA* of 22 nt putatively responsible for down-regulating expression of these R genes. We have validated this regulation, and have also established that the corresponding pri-miRNA is down-regulated upon PAMPs or bacteria perception. Using GUS reporters, we have characterized the expression pattern of both pri-miRNA and its best target R genes. We demonstrate that plants with altered levels of miRNA* (knockdown or overexpression lines) exhibit altered PTI-associated phenotypes, supporting a role for this miRNA* in the defence response against this bacterial pathogen. Finally, we identify phasiRNAs that arise from the transcript of one of the R target genes in a miRNA*-RDR6-DCL4-dependent manner.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

PAMP-triggered immunity against Pseudomonas syringae involves microRNA-mediated regulation of several uncharacterized R genes

Two main types of noncoding small RNA molecules have been found in plants: microRNAs (miRNAs) and small interfering RNAs (siRNAs). They differ in their biogenesis and mode of action, but share similar sizes (20-24 nt). Their precursors are processed by Dicer-Like RNase III (dcl) proteins present in Arabidopsis thaliana, and in their mature form can act as negative regulators of gene expression, being involved in a vast array of plant processes, including plant development, genomic integrity or response to stress. Small-RNA mediated regulation can occurs at transcriptional level (TGS) or at post-transcriptional level (PTGS). In recent years, the role of gene silencing in the regulation of expression of genes related to plant defence responses against bacterial pathogens is becoming clearer. Comparisons carried out in our lab between the expression profiles of different mutants affected in gene silencing, and plants challenged with Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, differentially expressed in these conditions. Through the use of bioinformatics tools, we found a miRNA* of 22 nt putatively responsible for down-regulating expression of these R genes through the generation of siRNAs. We have also found that the corresponding pri-miRNA is down-regulated after PAMP-perception in a SA-dependent manner. We also demonstrate that plants with altered levels of miRNA* (knockdown lines or overexpression lines) exhibit altered PTI-associated phenotypes, suggesting a role for this miRNA* in this defence response against bacteria. In addition we identify one of the target genes as a negative regulator of defence response against Pseudomonas syringae.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. MINECO, FEDE

Emerging connections between small RNAs and phytohormones

Small RNAs (sRNAs), mainly including miRNAs and siRNAs, are ubiquitous in eukaryotes. sRNAs mostly negatively regulate gene expression via (post-)transcriptional gene silencing through DNA methylation, mRNA cleavage, or translation inhibition. The mechanisms of sRNA biogenesis and function in diverse biological processes, as well as the interactions between sRNAs and environmental factors, like (a)biotic stress, have been deeply explored. Phytohormones are central in the plant’s response to stress, and multiple recent studies highlight an emerging role for sRNAs in the direct response to, or the regulation of, plant hormonal pathways. In this review, we discuss recent progress on the unraveling of crossregulation between sRNAs and nine plant hormones

Modulation of iron responsive gene expression and enzymatic activities in response to changes of the iron nutritional status in _Cucumis sativus_ L.

Regulation exerted by the iron status of the plant on the iron deficiency responses was investigated in cucumber roots (_Cucumis sativus_ L.) both at the biochemical and molecular level. Absence of iron induced the expression of the CsFRO1, CsIRT1, CsHA1 and the Cspepc1 transcripts that was followed by an increase in the corresponding enzymatic activities. Supply of iron repressed gene expression, in particular those of the Fe(III)-chelate reductase and for the high affinity iron transporter and reduce the enzymatic activities. Our results confirm and extend the hypothesis of a coordinate regulation of these responses. Besides these two activities strictly correlated with iron deficiency adaptation, we considered also the H+-ATPase and the phosphoenolpyruvate carboxylase, that have been shown to be involved in this response

Analysis of a conserved cellulase transcriptional regulator reveals inducer-independent production of cellulolytic enzymes in Neurospora crassa.

Cellulose is recalcitrant to deconstruction to glucose for use in fermentation strategies for biofuels and chemicals derived from lignocellulose. In Neurospora crassa, the transcriptional regulator, CLR-2, is required for cellulolytic gene expression and cellulose deconstruction. To assess conservation and divergence of cellulase gene regulation between fungi from different ecological niches, we compared clr-2 function with its ortholog (clrB) in the distantly related species, Aspergillus nidulans. Transcriptional profiles induced by exposure to crystalline cellulose were similar in both species. Approximately 50% of the cellulose-responsive genes showed strict dependence on functional clr-2/clrB, with a subset of 28 genes encoding plant biomass degrading enzymes that were conserved between N. crassa and A. nidulans. Importantly, misexpression of clr-2 under noninducing conditions was sufficient to drive cellulase gene expression, secretion, and activity in N. crassa, to a level comparable to wild type exposed to Avicel. However, misexpression of clrB in A. nidulans was not sufficient to drive cellulase gene expression under noninducing conditions, although an increase in cellulase activity was observed under crystalline cellulose conditions. Manipulation of clr-2 orthologs among filamentous fungi may enable regulated cellulosic enzyme production in a wide array of culture conditions and host strains, potentially reducing costs associated with enzyme production for plant cell wall deconstruction. However, this functionality may require additional engineering in some species

Transposon variants and their effects on gene expression in arabidopsis

Transposable elements (TEs) make up the majority of many plant genomes. Their transcription and transposition is controlled through siRNAs and epigenetic marks including DNA methylation. To dissect the interplay of siRNA–mediated regulation and TE evolution, and to examine how TE differences affect nearby gene expression, we investigated genome-wide differences in TEs, siRNAs, and gene expression among three Arabidopsis thaliana accessions. Both TE sequence polymorphisms and presence of linked TEs are positively correlated with intraspecific variation in gene expression. The expression of genes within 2 kb of conserved TEs is more stable than that of genes next to variant TEs harboring sequence polymorphisms. Polymorphism levels of TEs and closely linked adjacent genes are positively correlated as well. We also investigated the distribution of 24-nt-long siRNAs, which mediate TE repression. TEs targeted by uniquely mapping siRNAs are on average farther from coding genes, apparently because they more strongly suppress expression of adjacent genes. Furthermore, siRNAs, and especially uniquely mapping siRNAs, are enriched in TE regions missing in other accessions. Thus, targeting by uniquely mapping siRNAs appears to promote sequence deletions in TEs. Overall, our work indicates that siRNA–targeting of TEs may influence removal of sequences from the genome and hence evolution of gene expression in plants