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1.  Development and characterization of low α-linolenic acid Brassica oleracea lines bearing a novel mutation in a ‘class a’ FATTY ACID DESATURASE 3 gene 
BMC Genetics  2014;15:94.
Traditional canola (Brassica napus L.; AACC, 2n = 38) cultivars yield seed oil with a relatively high proportion of α-linolenic acid (ALA; C18:3cis∆9,12,15), which is desirable from a health perspective. Unfortunately, due to the instability of this fatty acid, elevated levels also result in oils that exhibit a short shelf life and problems associated with use at high temperatures. As a result, the development of cultivars bearing reduced amounts of ALA in their seeds is becoming a priority. To date, several low ALA B. napus cultivars (~2-3% ALA of total fatty acids) have been developed and molecular analyses have revealed that the low ALA phenotype of lines tested thus far is a result of mutations within two ‘class b’ FATTY ACID DESATURASE 3 (FAD3) genes. Since B. napus possesses six FAD3 genes (two ‘class a’, two ‘class b’ and two ‘class c’) and ALA levels of approximately 2-3% remain in these low ALA lines, it is likely that the mutation of additional FAD3 genes could further decrease the content of this fatty acid.
In this study, we generated low ALA (≤2%) lines of B. oleracea, which is the C genome progenitor species of B. napus, via ethyl methanesulphonate (EMS) mutagenesis. We identified a novel nonsense mutation within the ‘class a’ FAD3 gene (BoFAD3-2) in these lines, which would result in the production of an encoded protein lacking 110 amino acids at its C terminus. When expressed in Saccharomyces cerevisiae, this mutant protein exhibited a drastic decline in its Δ-15 desaturase activity compared to the wild-type (wt) protein. Furthermore, we demonstrated that the expression of the mutant BoFAD3-2 gene was significantly reduced in developing seeds of low ALA lines when compared to expression in wt plants.
Given the additive nature of FAD3 mutations on ALA content and the ease with which B. napus can be re-synthesized from its progenitor species, the mutant isolated here has the potential to be used for the future development of B. napus cultivars exhibiting further reductions in ALA content.
PMCID: PMC4236532  PMID: 25167929
Low linolenic acid; Brassica oleracea; fatty acid desaturase 3 (FAD3); EMS mutagenesis
2.  Correction: Genome-Wide Identification and Analysis of the TIFY Gene Family in Grape 
PLoS ONE  2013;8(12):10.1371/annotation/c9d9d321-b821-4060-8bf9-ff181229fea7.
PMCID: PMC3865316
3.  Effect of GA3 Treatment on Seed Development and Seed-Related Gene Expression in Grape  
PLoS ONE  2013;8(11):e80044.
The phytohormone gibberellic acid (GA3) is widely used in the table grape industry to induce seedlessness in seeded varieties. However, there is a paucity of information concerning the mechanisms by which GAs induce seedlessness in grapes.
Methodology/Principal Findings
In an effort to systematically analyze the cause of this GA3-induced seed abortion, we conducted an in depth characterization of two seeded grape cultivars (‘Kyoho’ and ‘Red Globe’), along with a seedless cultivar (‘Thompson Seedless’), following treatment with GA3. In a similar fashion to the seedless control, which exhibited GA3-induced abortion of the seeds 9 days after full bloom (DAF), both ‘Kyoho’ and ‘Red Globe’ seeded varieties exhibited complete abortion of the seeds 15 DAF when treated with GA3. Morphological analyses indicated that while fertilization appeared to occur normally following GA3 treatment, as well as in the untreated seedless control cultivar, seed growth eventually ceased. In addition, we found that GA3 application had an effect on redox homeostasis, which could potentially cause cell damage and subsequent seed abortion. Furthermore, we carried out an analysis of antioxidant enzyme activities, as well as transcript levels from various genes believed to be involved in seed development, and found several differences between GA3-treated and untreated controls.
Therefore, it seems that the mechanisms driving GA3-induced seedlessness are similar in both seeded and seedless cultivars, and that the observed abortion of seeds may result at least in part from a GA3-induced increase in cell damage caused by reactive oxygen species, a decrease in antioxidant enzymatic activities, and an alteration of the expression of genes related to seed development.
PMCID: PMC3818301  PMID: 24224035
4.  Genomic Organization, Phylogenetic Comparison and Differential Expression of the SBP-Box Family Genes in Grape 
PLoS ONE  2013;8(3):e59358.
The SBP-box gene family is specific to plants and encodes a class of zinc finger-containing transcription factors with a broad range of functions. Although SBP-box genes have been identified in numerous plants including green algae, moss, silver birch, snapdragon, Arabidopsis, rice and maize, there is little information concerning SBP-box genes, or the corresponding miR156/157, function in grapevine.
Methodology/Principal Findings
Eighteen SBP-box gene family members were identified in Vitis vinifera, twelve of which bore sequences that were complementary to miRNA156/157. Phylogenetic reconstruction demonstrated that plant SBP-domain proteins could be classified into seven subgroups, with the V. vinifera SBP-domain proteins being more closely related to SBP-domain proteins from dicotyledonous angiosperms than those from monocotyledonous angiosperms. In addition, synteny analysis between grape and Arabidopsis demonstrated that homologs of several grape SBP genes were found in corresponding syntenic blocks of Arabidopsis. Expression analysis of the grape SBP-box genes in various organs and at different stages of fruit development in V. quinquangularis ‘Shang-24’ revealed distinct spatiotemporal patterns. While the majority of the grape SBP-box genes lacking a miR156/157 target site were expressed ubiquitously and constitutively, most genes bearing a miR156/157 target site exhibited distinct expression patterns, possibly due to the inhibitory role of the microRNA. Furthermore, microarray data mining and quantitative real-time RT-PCR analysis identified several grape SBP-box genes that are potentially involved in the defense against biotic and abiotic stresses.
The results presented here provide a further understanding of SBP-box gene function in plants, and yields additional insights into the mechanism of stress management in grape, which may have important implications for the future success of this crop.
PMCID: PMC3601960  PMID: 23527172
5.  Genome-Wide Identification and Analysis of the TIFY Gene Family in Grape 
PLoS ONE  2012;7(9):e44465.
The TIFY gene family constitutes a plant-specific group of genes with a broad range of functions. This family encodes four subfamilies of proteins, including ZML, TIFY, PPD and JASMONATE ZIM-Domain (JAZ) proteins. JAZ proteins are targets of the SCFCOI1 complex, and function as negative regulators in the JA signaling pathway. Recently, it has been reported in both Arabidopsis and rice that TIFY genes, and especially JAZ genes, may be involved in plant defense against insect feeding, wounding, pathogens and abiotic stresses. Nonetheless, knowledge concerning the specific expression patterns and evolutionary history of plant TIFY family members is limited, especially in a woody species such as grape.
Methodology/Principal Findings
A total of two TIFY, four ZML, two PPD and 11 JAZ genes were identified in the Vitis vinifera genome. Phylogenetic analysis of TIFY protein sequences from grape, Arabidopsis and rice indicated that the grape TIFY proteins are more closely related to those of Arabidopsis than those of rice. Both segmental and tandem duplication events have been major contributors to the expansion of the grape TIFY family. In addition, synteny analysis between grape and Arabidopsis demonstrated that homologues of several grape TIFY genes were found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes arose before the divergence of lineages that led to grape and Arabidopsis. Analyses of microarray and quantitative real-time RT-PCR expression data revealed that grape TIFY genes are not a major player in the defense against biotrophic pathogens or viruses. However, many of these genes were responsive to JA and ABA, but not SA or ET.
The genome-wide identification, evolutionary and expression analyses of grape TIFY genes should facilitate further research of this gene family and provide new insights regarding their evolutionary history and regulatory control.
PMCID: PMC3439424  PMID: 22984514
6.  Identification of the dehydrin gene family from grapevine species and analysis of their responsiveness to various forms of abiotic and biotic stress 
BMC Plant Biology  2012;12:140.
Dehydrins (DHNs) protect plant cells from desiccation damage during environmental stress, and also participate in host resistance to various pathogens. In this study, we aimed to identify and characterize the DHN gene families from Vitis vinifera and wild V. yeshanensis, which is tolerant to both drought and cold, and moderately resistant to powdery mildew.
Four DHN genes were identified in both V. vinifera and V. yeshanensis, which shared a high sequence identity between the two species but little homology between the genes themselves. These genes were designated DHN1, DHN2, DHN3 and DHN4. All four of the DHN proteins were highly hydrophilic and were predicted to be intrinsically disordered, but they differed in their isoelectric points, kinase selectivities and number of functional motifs. Also, the expression profiles of each gene differed appreciably from one another. Grapevine DHN1 was not expressed in vegetative tissues under normal growth conditions, but was induced by drought, cold, heat, embryogenesis, as well as the application of abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA). It was expressed earlier in V. yeshanensis under drought conditions than in V. vinifera, and also exhibited a second round of up-regulation in V. yeshanensis following inoculation with Erysiphe necator, which was not apparent in V. vinifera. Like DHN1, DHN2 was induced by cold, heat, embryogenesis and ABA; however, it exhibited no responsiveness to drought, E. necator infection, SA or MeJA, and was also expressed constitutively in vegetative tissues under normal growth conditions. Conversely, DHN3 was only expressed during seed development at extremely low levels, and DHN4 was expressed specifically during late embryogenesis. Neither DHN3 nor DHN4 exhibited responsiveness to any of the treatments carried out in this study. Interestingly, the presence of particular cis-elements within the promoter regions of each gene was positively correlated with their expression profiles.
The grapevine DHN family comprises four divergent members. While it is likely that their functions overlap to some extent, it seems that DHN1 provides the main stress-responsive function. In addition, our results suggest a close relationship between expression patterns, physicochemical properties, and cis-regulatory elements in the promoter regions of the DHN genes.
PMCID: PMC3460772  PMID: 22882870
Grapevine; Dehydrin; Stress-induced expression; Powdery mildew; Promoter

Results 1-6 (6)