Identification and sequence analysis of a dreb subfamily transcription factor involved in drought stress tolerance from rice

DRE (dehydration responsive element)/CRT (C-repeat) is a cis-acting element that involves in gene expression responsive to abiotic stress in higher plants. To date, all well known DREBP transcription factors in Arabidopsis, rice, maize and other plants regulate gene expression in response to drought, high-salt and cold stresses by binding specifically to DRE/CRT. Using a target sequence of 50 nucleotides on Glutamate dehydrogenase-like protein (JRC2606) promoter containing the core sequence of DRE cis-acting element (A/GCCGAC) for yeast one-hybrid screening, we have identified two transcription factors:  a completely homology of OsRAP2.4A gene and another is a new sequence. The new sequence contained an ORF (Open Reading Frame) of 1017-bp and 5’ non-coding area of 35-bp and 3’ non-coding area of 341-bp. The deduced amino acid sequence contains an AP2 domain and belongs to  the subgroup  A6  of DREB subfamily, temporarily named OsRAP2.4B. Sequence alignment showed that OsRap2.4B had homology with ZmDBF, a maize transcription factor involved in drought stress tolerance

Evaluation of genetic diversity and DNA fingerprinting of 19 standard reference rice varieties using SSR markers

Molecular markers are advanced-tools for identifying new varieties at DNA levels. According to the International Union for the Protection of New Varieties ofPlants,  new breeded varieties need to be tested for the Distinctness, Uniformity and Stability (DUS), before being recognized as the new ones. Traditional DUS criteria based on 62 - 65 morphological and biochemical characteristics, which evaluated on comparison of new varieties with 19 standard reference varieties for traits of interest.  Study on the genotypic polymorphism of 19 standard reference rice varieties provides genotypic information of these varieties for the evaluation of new rice varieties based on genotyping analysis.  The reference marker set (30 markers) was used to evaluate the genetic diversity and DNA fingerprinting of 19 standard reference rice varieties. The results showed the similarity coefficient of 19 varieties varied from 0.04 to 0.548. At the genetic similarity coefficient of 0.1, the 19 rice varieties divided into two main groups. Group one included 3 varieties: DH1, DH5, DH13. Group 2 included the remaining 16 varieties. Inside group two, phylogenetic tree divided into two main branches at the genetic similarity coefficient of 0.3. Branch 1 includes 5 varieties including DH2, DH6, DH10, DH11 and DH7. The 11 remaining varieties were in the branch 2. The most closely varieties were DH6 and DH10 with the genetic similarity coefficient of 0.548. This study shows that, the standard reference varieties have high uniformity and high genotypic polymorphism, could used for testing new varieties based on genotyping by DNA fingerprinting combining with phenotype

Expression of Oryza sativa galactinol synthase gene in maize (Zea may L.)

Galactinol synthase (GolS) is a key biological catalyst for the synthesis of the raffinose oligosaccharides (RFOs) which play important roles in abiotic stress adaptation of plants, especially drought tolerance. GolS gene has been isolated on a variety of plants in order to create material resources for generating transgenic plants resistant to adverse environmental factors. In our previous research, we have isolated a GolS gene from drought stress cDNA library of Oryza sativa L. Moctuyen (named OsGolS). In this study, the expression vector pCAM-Rd/OsGolS carrying the isolated OsGolS gene under the control of stress-inducible Rd29A promoter was constructed and introduced into Agrobacterium tumefaciens LBA4404, which was used for maize transformation.  PCR and Real-time PCR assay indicated that transgene was integrated in the genome of the regenerated Zea mays plants. Reverse transcription-PCR showed that the OsGolS was transcribed into mRNA in Zea mays and was highly expressed. These results provide a basis for the study of the function of OsGolS in drought responses and for the development of drought stress tolerant crops.

Study on pathogenicity of Colletotrichum gloeosporioides on Coffee in North of Vietnam

Coffee is an important crop in Vietnam and it has recently brought a lot of benefit for the country through export. After coffee leaf rust, anthracnose is the second destructive disease for coffee production and is caused primarily by Colletotrichum gloeosporioides. In order to investigate the pathogenicity of C. gloeosporioides on coffee in the North of Vietnam, we carried out isolation of C. gloeosporioides species from different coffee plantations and found that C. gloeosporioides presents in different parts of coffee including leaf, twig, ripe berry and green berry. The pathogenicity of a total of twenty eight C. gloeosporioides isolates was tested on green berry in laboratory. Interestingly, two most pathogenic isolates were originated from green berry. In detail, the rate of green berry infection by C. gloeosporioides isolates varied from 4.44% to 76.67% and the variation of infection rate was also observed clearly among C. gloeosporioides isolates originating from leaf (14.40% to 45.56%), twig (4.44% to 58.89%), ripe berry (22.20% to 61.10%) and green berry (24.40% to 76.67%). Three selected pathogenic isolates representing for C. gloeosporioides isolates originated from twig, ripe berry and green berry were further tested for pathogenicity on hypocotyl seedling in greenhouse. The rate of hypocotyl seedling infection by C. gloeosporioides isolates originated from twig, green berry and ripe berry is 48.89%, 37.78 and 23.33% respectively. Our data suggests that pathogenicity of C. gloeosporioides is variable and specific among isolates, parts of coffee

Genome-wide association mapping of leaf mass traits in a Vietnamese rice landrace panel

Leaf traits are often strongly correlated with yield, which poses a major challenge in rice breeding. In the present study, using a panel of Vietnamese rice landraces genotyped with 21,623 single-nucleotide polymorphism markers, a genome-wide association study (GWAS) was conducted for several leaf traits during the vegetative stage. Vietnamese landraces are often poorly represented in panels used for GWAS, even though they are adapted to contrasting agrosystems and can contain original, valuable genetic determinants. A panel of 180 rice varieties was grown in pots for four weeks with three replicates under nethouse conditions. Different leaf traits were measured on the second fully expanded leaf of the main tiller, which often plays a major role in determining the photosynthetic capacity of the plant. The leaf fresh weight, turgid weight and dry weight were measured; then, from these measurements, the relative tissue weight and leaf dry matter percentage were computed. The leaf dry matter percentage can be considered a proxy for the photosynthetic efficiency per unit leaf area, which contributes to yield. By a GWAS, thirteen QTLs associated with these leaf traits were identified. Eleven QTLs were identified for fresh weight, eleven for turgid weight, one for dry weight, one for relative tissue weight and one for leaf dry matter percentage. Eleven QTLs presented associations with several traits, suggesting that these traits share common genetic determinants, while one QTL was specific to leaf dry matter percentage and one QTL was specific to relative tissue weight. Interestingly, some of these QTLs colocalize with leaf- or yield-related QTLs previously identified using other material. Several genes within these QTLs with a known function in leaf development or physiology are reviewed

Pea DNA helicase 45 overexpression in tobacco confers high salinity tolerance without affecting yield

Salt tolerance is an important trait that is required to overcome salinity-induced reduction in plant productivity. We have reported previously the isolation of a pea DNA helicase 45 (PDH45) that exhibits striking homology with the eukaryotic translation initiation factor eIF-4A. Here, we report that PDH45 mRNA is induced in pea seedlings in response to high salt, and its overexpression driven by a constitutive cauliflower mosaic virus-35S promoter in tobacco plants confers salinity tolerance, thus suggesting a previously undescribed pathway for manipulating stress tolerance in crop plants. The T0 transgenic plants showed high levels of PDH45 protein in normal and stress conditions, as compared with WT plants. The T0 transgenics also showed tolerance to high salinity as tested by a leaf disk senescence assay. The T1 transgenics were able to grow to maturity and set normal viable seeds under continuous salinity stress without any reduction in plant yield in terms of seed weight. Measurement of Na+ ions in different parts of the plant showed higher accumulation in the old leaves and negligible accumulation in seeds of T1 transgenic lines as compared with the WT plants. The possible mechanism of salinity tolerance is discussed. Overexpression of PDH45 provides a possible example of the exploitation of DNA/RNA unwinding pathways for engineering salinity tolerance without affecting yield in crop plants

A DNA helicase from Pisum sativum is homologous to translation initiation factor and stimulates topoisomerase I activity

DNA helicases play an essential role in all aspects of nucleic acid metabolism, by providing a duplex-unwinding function. This is the first report of the isolation of a cDNA (1.6 kb) clone encoding functional DNA helicase from a plant (pea, Pisum sativum). The deduced amino-acid sequence has eight conserved helicase motifs of the DEAD-box protein family. It is a unique member of this family, containing DESD and SRT motifs instead of DEAD/H and SAT. The encoded 45.5 kDa protein has been overexpressed in bacteria and purified to homogeneity. The purified protein contains ATP-dependent DNA and RNA helicase, DNA-dependent ATPase, and ATP-binding activities. The protein sequence contains striking homology with eIF-4A, which has not so far been reported as DNA helicase. The antibodies against pea helicase inhibit in vitro translation. The gene is expressed as 1.6 kb mRNA in different organs of pea. The enzyme is localized in the nucleus and cytosol, and unwinds DNA in the 3' to 5' direction. The pea helicase interacts with pea topoisomerase I protein and stimulates its activity. These results suggest that pea DNA helicase could be an important multifunctional protein involved in protein synthesis, maintaining the basic activities of the cell, and in upregulation of topoisomerase I activity. The discovery of such a protein with intrinsic multiple activity should make an important contribution to our better understanding of DNA and RNA transactions in plants

Inhibition of unwinding and ATPase activities of pea MCM6 DNA helicase by actinomycin and nogalamycin

Pea mini-chromosome maintenance 6 (MCM6) single subunit (93 kDa) forms homohexamer (560 kDa) and contains an ATP-dependent and replication fork stimulated 3′ to 5′ DNA unwinding activity along with intrinsic DNA-dependent ATPase and ATP-binding activities1 (Plant Mol Biol 2010; DOI: 10.1007/s11103-010-9675-7). Here, we have determined the effect of various DNA-binding agents, such as actinomycin, nogalamycin, daunorubicin, doxorubicin, distamycin, camptothecin, cyclophosphamide, ellipticine, VP-16, novobiocin, netropsin, cisplatin, mitoxantrone and genistein on the DNA unwinding and ATPase activities of the pea MCM6 DNA helicase. The results show that actinomycin and nogalamycin inhibited the DNA helicase (apparent Ki values of 10 and 1 µM, respectively) and ATPase (apparent Ki values of 100 and 17 µM, respectively) activities. Although, daunorubicin and doxorubicin also inhibited the DNA helicase activity of pea MCM6, but with less efficiency; however, these could not inhibit the ATPase activity. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of MCM6, resulting in the inhibitions of the activities. This study could be useful in our better understanding of the mechanism of plant nuclear DNA helicase unwinding

A cluster of Ankyrin and Ankyrin-TPR repeat genes is associated with panicle branching diversity in rice.

The number of grains per panicle is an important yield-related trait in cereals which depends in part on panicle branching complexity. One component of this complexity is the number of secondary branches per panicle. Previously, a GWAS site associated with secondary branch and spikelet numbers per panicle in rice was identified. Here we combined gene capture, bi-parental genetic population analysis, expression profiling and transgenic approaches in order to investigate the functional significance of a cluster of 6 ANK and ANK-TPR genes within the QTL. Four of the ANK and ANK-TPR genes present a differential expression associated with panicle secondary branch number in contrasted accessions. These differential expression patterns correlate in the different alleles of these genes with specific deletions of potential cis-regulatory sequences in their promoters. Two of these genes were confirmed through functional analysis as playing a role in the control of panicle architecture. Our findings indicate that secondary branching diversity in the rice panicle is governed in part by differentially expressed genes within this cluster encoding ANK and ANK-TPR domain proteins that may act as positive or negative regulators of panicle meristem's identity transition from indeterminate to determinate state