Stacy D. Singer

Identification of the dehydrin gene family from grapevine species and analysis of their responsiveness to various forms of abiotic and biotic stress

BackgroundDehydrins (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.ResultsFour 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.ConclusionsThe 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.

Genomic Organization, Phylogenetic Comparison and Differential Expression of the SBP-Box Family Genes in Grape

Background 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. Conclusion 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.

Genome-Wide Identification and Analysis of the TIFY Gene Family in Grape

Background 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. Conclusion 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.

Enhancer–promoter interference and its prevention in transgenic plants

Biotechnology has several advantages over conventional breeding for the precise engineering of gene function and provides a powerful tool for the genetic improvement of agronomically important traits in crops. In particular, it has been exploited for the improvement of multiple traits through the simultaneous introduction or stacking of several genes driven by distinct tissue-specific promoters. Since transcriptional enhancer elements have been shown to override the specificity of nearby promoters in a position- and orientation-independent manner, the co-existence of multiple enhancers/promoters within a single transgenic construct could be problematic as it has the potential to cause the mis-expression of transgene product(s). In order to develop strategies with, which to prevent such interference, a clear understanding of the mechanisms underlying enhancer-mediated activation of target promoters, as well as the identification of DNA sequences that function to block these interactions in plants, will be necessary. To date, little is known concerning enhancer function in plants and only a very limited number of enhancer-blocking insulators that operate in plant species have been identified. In this review, we discuss the current knowledge surrounding enhancer–promoter interactions, as well as possible means of minimizing such interference during plant transformation experiments.

Minimizing the unpredictability of transgene expression in plants: the role of genetic insulators

The genetic transformation of plants has become a necessary tool for fundamental plant biology research, as well as the generation of engineered plants exhibiting improved agronomic and industrial traits. However, this technology is significantly hindered by the fact that transgene expression is often highly variable amongst independent transgenic lines. Two of the major contributing factors to this type of inconsistency are inappropriate enhancer-promoter interactions and chromosomal position effects, which frequently result in mis-expression or silencing of the transgene, respectively. Since the precise, often tissue-specific, expression of the transgene(s) of interest is often a necessity for the successful generation of transgenic plants, these undesirable side effects have the potential to pose a major challenge for the genetic engineering of these organisms. In this review, we discuss strategies for improving foreign gene expression in plants via the inclusion of enhancer-blocking insulators, which function to impede enhancer-promoter communication, and barrier insulators, which block the spread of heterochromatin, in transgenic constructs. While a complete understanding of these elements remains elusive, recent studies regarding their use in genetically engineered plants indicate that they hold great promise for the improvement of transgene expression, and thus the future of plant biotechnology.

Gibberellin-induced changes in the transcriptome of grapevine ( Vitis labrusca × V. vinifera ) cv. Kyoho flowers

BackgroundGibberellins are well known for their growth control function in flower, fruit and seed development, and as such, exogenous gibberellic acid (GA) application plays an important role in viticulture. Unfortunately, the mechanism by which GA3 acts in the regulation of these complicated developmental processes in grape remains unclear.ResultsIn the present study, we demonstrated that application of GA3 to ‘Kyoho’ grapevine inflorescences at pre-bloom promoted flower opening, and induced fruit coloring as well as seed abortion. In an attempt to obtain a deeper understanding of the molecular mechanisms driving these responses to GA3 treatment, we performed large-scale transcriptome sequencing of grape flowers following GA3 treatment using Illumina sequencing technology. Global expression profiles of GA3-treated and untreated grape flowers were compared and a large number of GA3-responsive genes were identified. Gene ontology (GO) term classification and biochemical pathway analyses indicated that GA3 treatment caused changes in the levels of transcripts involved in cellular processes, reproduction, hormone and secondary metabolism, as well as the scavenging and detoxification of reactive oxygen species (ROS). These findings suggest that GA3-induced morphological alterations may be related to the control of hormone biosynthesis and signaling, regulation of transcription factors, alteration of secondary metabolites, and the stability of redox homeostasis.ConclusionsTaken together, this comprehensive inflorescence transcriptome data set provides novel insight into the response of grape flowers to GA3 treatment, and also provides possible candidate genes or markers that could be used to guide future efforts in this field.

Both the constitutive Cauliflower Mosaic Virus 35S and tissue-specific AGAMOUS enhancers activate transcription autonomously in Arabidopsis thaliana

The expression of eukaryotic genes from their cognate promoters is often regulated by the action of transcriptional enhancer elements that function in an orientation-independent manner either locally or at a distance within a genome. This interactive nature often provokes unexpected interference within transgenes in plants, as reflected by misexpression of the introduced gene and undesired phenotypes in transgenic lines. To gain a better understanding of the mechanism underlying enhancer/promoter interactions in a plant system, we analyzed the activation of a β-glucuronidase (GUS) reporter gene by enhancers contained within the AGAMOUS second intron (AGI) and the Cauliflower Mosaic Virus (CaMV) 35S promoter, respectively, in the presence and absence of a target promoter. Our results indicate that both the AGI and 35S enhancers, which differ significantly in their species of origin and in the pattern of expression that they induce, have the capacity to activate the expression of a nearby gene through the promoter-independent initiation of autonomous transcriptional events. Furthermore, we provide evidence that the 35S enhancer utilizes a mechanism resembling animal- and yeast-derived scanning or facilitated tracking models of long-distance enhancer action in its activation of a remote target promoter.

Arabidopsis GPAT9 contributes to synthesis of intracellular glycerolipids but not surface lipids

Highlight Arabidopsis glycerol-3-phosphate acyltransferase 9 (GPAT9) is an sn-1 specific acyl-CoA:GPAT that contributes to intracellular glycerolipid biosynthesis in seeds, developing leaves and pollen grains, but not to extracellular glycerolipid biosynthesis.

Genome-wide identification and analysis of the aldehyde dehydrogenase (ALDH) gene superfamily in apple (Malus × domestica Borkh.)

Aldehyde dehydrogenases (ALDHs) represent a protein superfamily encoding NAD(P)(+)-dependent enzymes that oxidize a wide range of endogenous and exogenous aliphatic and aromatic aldehydes. In plants, they are involved in many biological processes and play a role in the response to environmental stress. In this study, a total of 39 ALDH genes from ten families were identified in the apple (Malus × domestica Borkh.) genome. Synteny analysis of the apple ALDH (MdALDH) genes indicated that segmental and tandem duplications, as well as whole genome duplications, have likely contributed to the expansion and evolution of these gene families in apple. Moreover, synteny analysis between apple and Arabidopsis demonstrated that several MdALDH genes were found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes appeared before the divergence of lineages that led to apple and Arabidopsis. In addition, phylogenetic analysis, as well as comparisons of exon-intron and protein structures, provided further insight into both their evolutionary relationships and their putative functions. Tissue-specific expression analysis of the MdALDH genes demonstrated diverse spatiotemporal expression patterns, while their expression profiles under abiotic stress and various hormone treatments indicated that many MdALDH genes were responsive to high salinity and drought, as well as different plant hormones. This genome-wide identification, as well as characterization of evolutionary relationships and expression profiles, of the apple MdALDH genes will not only be useful for the further analysis of ALDH genes and their roles in stress response, but may also aid in the future improvement of apple stress tolerance.

Petunia AGAMOUS Enhancer-Derived Chimeric Promoters Specify a Carpel-, Stamen-, and Petal-Specific Expression Pattern Sufficient for Engineering Male and Female Sterility in Tobacco

Previous studies have shown that the AtAGIP promoter derived from the Arabidopsis AGAMOUS (AG) second intron/enhancer specifies a carpel- and stamen-specific expression in its native host species, but not in heterologous species such as tobacco, which restricts its application in the engineering of male and female sterility. These findings also imply that the AG regulatory mechanism that has evolved in Arabidopsis may, to some extent, have diverged from that of tobacco. To test whether a similar chimeric promoter created using the AG second intron/enhancer can overcome this barrier of evolutionary divergence in closely related species, we generated forward- and reverse-oriented chimeric promoters, fPtAGIP and rPtAGIP, from the petunia AG second intron/enhancer (PtAGI) fragment and tested them in tobacco, which, like petunia, belongs to the Solanaceae family. Our results demonstrate that both fPtAGIP and rPtAGIP confer similar carpel- and stamen-specific expression without any leaky activity in vegetative tissues in tobacco as revealed by tissue-specific gene expression and tissue ablation. This pattern resembles that driven by the AtAGIP in Arabidopsis and indicates that the AG regulatory mechanism is more conserved between tobacco and petunia than between tobacco and Arabidopsis. The petunia-derived promoters also exhibited petal-specific activity, and their activities in floral organs were substantially influenced by the orientation of the PtAGI enhancer, with reverse-oriented enhancers displaying approximately double the effectiveness of forward-oriented enhancers. These two properties are novel and have not been observed previously with AtAGIP promoters. Furthermore, we found that PtAGIP promoter-driven tissue ablation is effective for engineering complete sterility in plants, and the resulting sterile trait is stable for at least three mitotic generations at various temperature regimes, which is important for the complete containment of seed-, pollen-, and fruit-mediated gene flow in field conditions.