Transgenic Analysis Reveals 5′ Abbreviated <Emphasis Type="Italic">Os</Emphasis>RGLP2 Promoter(s) as Responsive to Abiotic Stresses

Germins and germin-like proteins are ubiquitous, expressed at various developmental stages and in response to various abiotic and biotic stresses. In this study, to functionally validate the OsRGLP2 promoter, 5′ deletion analysis of the promoter sequences was performed and the deletion fragments fused with the β-glucuronidase (GUS) and green fluorescent protein reporter genes were used for transient expression in tobacco as well as for generating stable transgenic Arabidopsis plants. Very high level of GUS activity was observed in agroinfiltrated tobacco leaves by the construct carrying the P-1063 and P-565 when subjected to abiotic stresses. Histochemical analysis of transgenic Arabidopsis plants revealed expression of reporter gene in root, leaf and stem sections of plants harboring P-1063 and P-565. Real-time qPCR analysis of transiently expressed tobacco leaves and transgenic Arabidopsis plants subjected to several abiotic stresses supported histochemical data and showed that P-565 responded to all the stresses to which the full-length promoter was responsive. The data suggest that P-565 may be a good alternative to full-length promoter region that harbors the necessary cis-elements in providing stable and high level of expression in response to wound, salt and temperature stresses.     

Expression of a rice <Emphasis Type="Italic">GLP</Emphasis> in <Emphasis Type="Italic">Medicago truncatula</Emphasis> exerting pleiotropic effects on resistance against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> through enhancing FeSOD-like activity

To evaluate the effectiveness of a germin-like protein (GLP) in legumes against the serious soil-borne pathogen Fusarium oxysporum f. sp. lentis, an Oryza sativa root-expressed GLP (OsRGLP1) was expressed in the model legume Medicago truncatula using the recombinant vector pCOsRGLP1. The transgene was highly expressed in M. truncatula transformed lines as assessed by RT-qPCR. Consistent with the active status of the transgene there was an elevated accumulation of H₂O₂ in transformed progeny. Enzymatic characterization of T₁ transgenic progeny showed increased superoxide dismutase (SOD) activity. The additional SOD activity in transgenic lines was insensitive to potassium cyanide and sensitive to H₂O₂ indicating its resemblance to FeSOD. The effectiveness of the OsRGLP1 gene was tested by monitoring the root disease after infection of wild-type and transgenic lines. Wild-type plants were greatly affected by the pathogen infection showing a percent disease index value of 50 compared to 10–18 for the transgenic lines. The tolerance of the transgenic lines leads to recovery in fresh weight and pod production to an almost normal level. Analysis of defense-related genes downstream of hydrogen peroxide (H₂O₂) in transgenic plants showed induction of salicylic acid and jasmonate signaling pathways and increased expression of some pathogenesis-related-1 (PR-1) genes and a plant defensin gene. Overall, the findings suggest that OsRGLP1 provides protection against the fungal pathogen F. oxysporum that may involve the direct influence of H₂O₂ on signaling pathways leading to the activation of defense-related genes.     

Identification and Evolution of FYVE Domain-Containing Proteins and Their Expression Patterns in Response to Abiotic Stresses in Rice

The FYVE domain is a typical zinc finger motif containing four conserved CxxC pairs and has been shown to specifically binds to PtdIns(3)P on the surface of cell membrane. FYVE domain-containing proteins are commonly distributed in eukaryotic cells and have been implicated in diverse functions like signal transduction, membrane trafficking, exocytosis and endocytosis, phosphoinositides (PIs) metabolism, and cytoskeletal regulation. Analysis of the rice genome using comprehensive online databases and research tools resulted in the identification of 19 putative rice FYVE (OsFVYE) proteins. Based on domain architectural and phylogenetic analyses, these OsFYVEs were further classified into six groups. Groups I–V were conserved in Arabidopsis (dicots) and rice (monocot) at both the genetic and protein levels, while group VI was widely present in plants but truncated in Arabidopsis and species of Brassicaceae. Comprehensive and comparative investigation of their expression profiles showed that FYVE genes in plant exhibited a variety of expression patterns during different developmental stages and in response to phytohormones and abiotic stresses. These findings indicated that both OsFYVE and AtFYVE genes may played potential roles in normal plant growth, hormone signal transduction, and abiotic stress tolerance. Results from our study shed light on the potential roles of FYVE proteins in plant growth, development, and stress responses.     

Genome-wide survey and expression analysis of the stress-associated protein gene family in desert poplar, <Emphasis Type="Italic">Populus euphratica</Emphasis>

Stress-associated proteins (SAPs) are a novel class of zinc finger proteins that extensively participate in abiotic stress responses. To date, no overall analysis and expression profiling of SAP genes in woody plants have been reported. Populus euphratica is distributed in desert regions and is extraordinarily adaptable to abiotic stresses. Thus, it is regarded as a promising candidate for studying abiotic stress resistance mechanisms of woody plants. In this study, 18 non-redundant SAP genes were identified from the genome of P. euphratica using basic local alignment search tool algorithms and functional domain verification. Among these 18 PeuSAP genes, 15 were intronless. To investigate the evolutionary relationships of SAP genes in P. euphratica and other Salicaceae plants, phylogenetic analyses were performed. Subsequently, the expression profiles of the 18 PeuSAP genes were analyzed in different tissues and under various stresses (drought, salt, heat, cold, and abscisic acid (ABA) treatment) using quantitative real-time PCR. Tissue expression analysis indicated that PeuSAPs showed no tissue specificity. PeuSAPs were induced by multiple abiotic stresses, especially drought, salt, and heat stresses, perhaps because of abundant cis-acting heat shock elements and drought-inducible elements in the promoter regions of the PeuSAPs. Moreover, single nucleotide polymorphisms (SNPs) variant analysis revealed many synonymous and non-synonymous SNPs in PeuSAP genes, but the zinc finger structure was conserved during evolution. These results provide an overview of the SAP gene family in P. euphratica and a reference for further functional research on PeuSAP genes.     

Regulatory <Emphasis Type="Italic">cis</Emphasis>-elements on citrus peel-specific expressed gene, <Emphasis Type="Italic">CuCRTISO-like</Emphasis>, promoter respond to hormones and abiotic stresses in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

We identified a peel-specific expressed gene in Citrus unshiu fruits by differentially expressed gene (DEG) analysis, which showed a homology with carotenoid isomerase-like genes identified from other plants and, therefore, designated as CuCRTISO-like. Here we determined the promoter sequence of CuCRTISO-like and analyzed histochemical GUS activity using transgenic Arabidopsis plants harboring CuCRTISO-like promoter-GUS gene constructs (pCRTL-Prom1~pCRTL-Prom5 lines). The promoter activity of CuCRTISO-like was detected in the cotyledon at 5 and 10 days after germination (DAG), young leaf, and anther, but not in the cotyledon at 15 DAG and mature leaf. Several cis-acting elements involved in hormones and abiotic stresses are located on the CuCRTISO-like promoter. Salicylic acid and ethylene treatments induced the GUS activity in the pCRTL-prom1 and pCRTL-Prom4 line, respectively. Treatment of drought and wounding stress induced the GUS activity in the pCRTL-Prom4 and pCRTL-Prom3 line, respectively. Heat stress treatment induced GUS activity more strongly as the promoter length decreased except for no GUS activity in the pCRTL-Prom5 line. The CuCRTISO-like expression during fruit maturation of C. unshiu showed a peel-specific expression pattern. Our results suggest that CuCRTISO-like promoter activity is regulated in a developmental and organ-specific manner, and responds to hormones and abiotic stresses.