Isolation and Characterization of a Calcium-Dependent Protein Kinase Gene, FvCDPK1, Responsive to Abiotic Stress in Woodland Strawberry (Fragaria vesca)

Plant calcium-dependent protein kinases (CDPKs) play vital roles in calcium signal transduction during various developmental processes and during responses to biotic and abiotic stresses. Here, we isolated and characterized a CDPK gene designated FvCDPK1 from a wild diploid strawberry accession Heilongjiang-3 (Fragaria vesca L.). The FvCDPK1 gene contains 12 exons and 11 introns, and the sequences of most exons are highly conserved in higher plants. The full-length cDNA of FvCDPK1 contains 1,825 nucleotides with an open reading frame of 1,653 bp encoding a polypeptide of 550 amino acids. The deduced FvCDPK1 protein contains the basic features of typical plant CDPKs: a catalytic kinase domain and a regulatory calmodulin-like domain containing four EF-hand calcium-binding motifs. Phylogenetic analysis confirmed that FvCDPK1 belongs to the plant CDPK family. When transiently expressed in onion epidermal cells, the FvCDPK1-GFP fusion protein was found to be localized in the nucleus. Expression analysis indicated that FvCDPK1 was expressed in fruits at different developmental and ripening stages, as well as in several tissues such as roots, runners, flowers, leaves, and meristems. Moreover, expression levels of FvCDPK1 were higher in meristems than in other vegetative tissues. Under abiotic stress conditions, however, FvCDPK1 was found to be upregulated upon abscisic acid, NaCl, cold-, or high-temperature treatments. Taken together, our data suggest that FvCDPK1 might play a role in various responses to abiotic stresses in strawberry.     

Genome-Wide Investigation and Expression Analyses of WD40 Protein Family in the Model Plant Foxtail Millet (Setaria italica L.)

WD40 proteins play a crucial role in diverse protein-protein interactions by acting as scaffolding molecules and thus assisting in the proper activity of proteins. Hence, systematic characterization and expression profiling of these WD40 genes in foxtail millet would enable us to understand the networks of WD40 proteins and their biological processes and gene functions. In the present study, a genome-wide survey was conducted and 225 potential WD40 genes were identified. Phylogenetic analysis categorized the WD40 proteins into 5 distinct sub-families (I–V). Gene Ontology annotation revealed the biological roles of the WD40 proteins along with its cellular components and molecular functions. In silico comparative mapping with sorghum, maize and rice demonstrated the orthologous relationships and chromosomal rearrangements including duplication, inversion and deletion of WD40 genes. Estimation of synonymous and non-synonymous substitution rates revealed its evolutionary significance in terms of gene-duplication and divergence. Expression profiling against abiotic stresses provided novel insights into specific and/or overlapping expression patterns of SiWD40 genes. Homology modeling enabled three-dimensional structure prediction was performed to understand the molecular functions of WD40 proteins. Although, recent findings had shown the importance of WD40 domains in acting as hubs for cellular networks during many biological processes, it has invited a lesser research attention unlike other common domains. Being a most promiscuous interactors, WD40 domains are versatile in mediating critical cellular functions and hence this genome-wide study especially in the model crop foxtail millet would serve as a blue-print for functional characterization of WD40s in millets and bioenergy grass species. In addition, the present analyses would also assist the research community in choosing the candidate WD40s for comprehensive studies towards crop improvement of millets and biofuel grasses.     

Identification and expression analysis of group 3 LEA family genes in sorghum [Sorghum bicolor (L.) Moench]

Sorghum with its remarkable adaptability to drought and high temperature provides a model system for grass genomics and resource for gene discovery especially for abiotic stress tolerance. Group 3 LEA genes from barley and rice have been shown to play crucial role in abiotic stress tolerance. Here, we present a genome-wide analysis of LEA3 genes in sorghum. We identified four genes encoding LEA3 proteins in the sorghum genome and further classified them into LEA3A and LEA3B subgroups based on the conservation of LEA3 specific motifs. Further, expression pattern of these genes were analyzed in seeds during development and vegetative tissues under abiotic stresses. SbLEA3A group genes showed expression at early stage of seed development and increased significantly at maturity, while SbLEA3B group genes expressed only in matured seeds. Expression of SbLEA3 genes in response to abiotic stresses such as soil moisture deficit (drought), osmotic, salt, and temperature stresses, and exogenous ABA treatments was also studied in the leaves of 2-weeks-old seedlings. ABA and drought induced the expression of all LEA3 genes, while cold and heat stress induced none of them. Promoter analysis revealed the presence of multiple ABRE core cis-elements and a few low temperature response (LTRE)/drought responsive (DRE) cis-elements. This study suggests non-redundant function of LEA3 genes in seed development and stress tolerance in sorghum.     

Molecular Cloning and Expression Analysis of Nine ThTrx Genes in Tamarix hispida

Thioredoxins are small conserved proteins that play key roles in the oxidative stress response. In this study, nine Trx genes, including five Trxhs, three Trxms, and one Trx-like gene, were cloned from Tamarix hispida. The roles of these ThTrx genes were investigated under various abiotic stress conditions. Expression profiles of the nine ThTrx genes in response to different abiotic stresses in leaf and root tissues were constructed using quantitative real time-polymerase chain reaction. Differential expression of all nine ThTrx genes was observed (>2-fold) in response to NaCl, PEG, or CdCl₂ stress in at least one tissue, indicating that all of these genes act in abiotic stress responses. All ThTrx genes were induced (>2-fold) by abscisic acid (ABA) treatment in the leaves and especially in the roots, suggesting that ABA-dependent signaling pathways regulate ThTrxs. These results demonstrate that ThTrx expression constitutes an adaptive response to abiotic stress in T. hispida and plays an important role in abiotic stress tolerance.     

Molecular Cloning and Characterization of a Novel Dehydrin Gene from Ginkgo biloba

A full-length cDNA encoding a dehydrin was cloned from the living fossil plant Ginkgo biloba by rapid amplification of cDNA ends (RACE). The cDNA, designated as GbDHN, was 813 bp long containing an open reading frame of 489 bp. The deduced GbDHN protein had 163 amino acid residues, which formed a 17 kDa polypeptide with a predicted isoelectric point (pI) of 5.75. GbDHN had an S-segment and a K-segment, indicative of dehydrins, but no Y-segments. Homology analysis indicated that the S-segment and K-segment of GbDHN shared identity with those of other reported dehydrins, indicating that GbDHN belonged to dehydrin superfamily. Genomic sequence of GbDHN was also cloned using genomic walker technology. By comparing genomic DNA with the cDNA, it was found that there was a 257-bp intron in this gene. Promoter analysis indicated that it contained six CAAT boxes, one TATA box, one ABRE box and one GC-motif in the 5′-flanking region. Southern blot analysis revealed that GbDHN belonged to a single copy gene family. RT-PCR analysis revealed that GbDHN constitutively expressed in stems and roots. The increased expression of GbDHN was detected when G. biloba seedlings were treated with exogenous abscisic acid (ABA), salt stress and drought stress. These results indicate that the GbDHN has the potential to play a role in response to ABA and environmental stresses that can cause plant dehydration.     

Expression of vacuolar H+-pyrophosphatase (OVP3) is under control of an anoxia-inducible promoter in rice

Vacuolar H⁺-pyrophosphatase (V-PPase) expression increases in a number of abiotic stresses and is thought to play a role in adaptation to abiotic stresses. This paper reports on the regulation of six V-PPase genes in rice (Oryza sativa L.) coleoptiles under anoxia, using flood tolerant and intolerant cultivars to test our hypothesis. Quantitative PCR analysis showed that one vacuolar H⁺-pyrophosphatase (OVP3) was induced by anoxia, particularly in flood-tolerant rice. Regulation of OVP3 expression under anoxia was investigated by analysing putative OVP promoters. The putative OVP3 promoter contained more previously identified anoxia-inducible motifs than the putative promoters of the other five OVP genes. GUS activity in transgenic rice plants containing the OVP3 promoter region linked to the GUS reporter gene was induced only by anoxia. Salt and cold treatments had little effect on OVP3 promoter-driven GUS expression when compared to the anoxic treatment.     

Induction Kinetics of a Novel Stress-related LEA Gene in Wheat

Drought, high-salt, and low-temperature are major constraints to yield and quality of crops. Late embryogenesis abundant proteins (LEAs), characterized by high hydrophilic and thermal stabilities, stabilize the cell membrane structure and prevent oxidation. LEA genes mediate responses to abiotic stresses such as drought, salt, low-temperature, or ultraviolet radiation. In this study, TaLEA4, a Group III member from the LEA family, was cloned from a cDNA library of stress-treated wheat seedlings by in situ phage hybridization. The full length clone of TaLEA4 is 1,084?bp and contains a 570?bp open reading frame (ORF) encoding a 189-amino-acid protein. Multiple sequence alignment indicated that TaLEA4 had three incompletely repetitive 11-mer amino acid motifs and ??-helix domains. The prediction of protein-sorting signals and localization sites in amino acid sequences (PSORT) showed that TaLEA4 has a nuclear localization signal (NLS) in the amino acid C-terminal sequence. A subcellular localization assay showed that the TaLEA4 protein accumulates in the cytoplasm and the nucleus. Specific expression in various wheat organs indicated that TaLEA4 mRNAs accumulates in abundance in stems under normal growing conditions. Expression profile analysis showed that TaLEA4 was highly induced by drought, and low and high temperatures. Isolation of the TaLEA4 promoter revealed a core promoter element and some cis-acting elements responding to abiotic stresses. This study provides a basis for more detailed functional analyses of LEA proteins, and suggests ways of improving wheat resistance by molecular breeding.     

The overexpression in Arabidopsis thaliana of a Trichoderma harzianum gene that modulates glucosidase activity, and enhances tolerance to salt and osmotic stresses

Using the TrichoEST database, generated in a previous functional genomics project from the beneficial filamentous fungus Trichoderma harzianum, a gene named Thkel1, which codes for a putative kelch-repeat protein, was isolated and characterized. Silencing of this gene in T. harzianum leads to a reduction of glucosidase activity and mycelial growth under abiotic stress conditions. Expression of this gene in Arabidopsis enhances plant tolerance to salt and osmotic stresses, accompanied by an increase in glucosidase activity and a reduction of abscisic acid levels compared to those observed in wild-type plants. Data presented throughout this article suggest the high value of T. harzianum as a source of genes able to facilitate the achievement of producing plants resistant to abiotic stresses without alteration of their phenotype.