Xiangpeng Leng

Characterization of grapevine microR164 and its target genes.

MicroRNAs (miRNAs) are an extensive class of newly identified small RNAs that regulate gene expression at post-transcription level by mRNA cleavage or translation. In our study, we used qRT-PCR and found that Vv-miR164 is expression in grapevine leaves, stems, tendrils, inflorescences, flowers and fruits. In addition, two potential target genes for Vv-miR164 were also found and verified by PPM-RACE and RLM-RACE. The results not only maps the cleavage site of the target mRNA but allowed for detection the expression pattern of cleaved fragments that can indicate the regulatory function of this miRNA on its target genes. These target genes were explored by qRT-PCR where some exhibited different expression patterns from their corresponding miRNA, indicating the cleavage mode of the miRNA on its target genes. The efficient and powerful approach used in this study can help in further understanding of how miRNAs cleaved their target mRNAs. Results from this study prove the importance of Vv-miR164 in regulating development and growth of grapes, and adds to the existing knowledge of small RNA-mediated regulation in grapes.

Comparative transcriptome analysis of grapevine in response to copper stress

Grapevine is one of the most economically important and widely cultivated fruit crop worldwide. With the industrialization and the popular application of cupric fungicides in grape industry, copper stress and copper pollution are also the factors affecting grape production and berry and wine quality. Here, 3,843 transcripts were significantly differently expressed genes in response to Cu stress by RNA-seq, which included 1,892 up-regulated and 1,951 down-regulated transcripts. During this study we found many known and novel Cu-induced and -repressed genes. Biological analysis of grape samples were indicated that exogenous Cu can influence chlorophylls metabolism and photosynthetic activities of grapevine. Most ROS detoxification systems, including antioxidant enzyme, stress-related proteins and secondary metabolites were strongly induced. Concomitantly, abscisic acid functioned as a negative regulator in Cu stress, in opposite action to ethylene, auxin, jasmonic acid, and brassinolide. This study also identified a set of Cu stress specifically activated genes coding copper transporter, P1B-type ATPase, multidrug transporters. Overall, this work was carried out to gain insights into the copper-regulated and stress-responsive mechanisms in grapevine at transcriptome level. This research can also provide some genetic information that can help us in better vinery management and breeding Cu-resistant grape cultivars.

Grapevine microRNAs responsive to exogenous gibberellin

BackgroundMicroRNAs (miRNAs), involving in various biological and metabolic processes, have been discovered and analyzed in quite a number of plants species, such as Arabidopsis, rice and other plants. However, there have been few reports about grapevine miRNAs in response to gibberelline (GA3).ResultsSolexa technology was used to sequence small RNA libraries constructed from grapevine berries treated with GA3 and the control. A total of 122 known and 90 novel grapevine miRNAs (Vvi-miRNAs) were identified. Totally, 137 ones were found to be clearly responsive to GA3, among which 58 were down-regulated, 51 were up-regulated, 21 could only be detected in the control, and seven were only detected in the treatment. Subsequently, we found that 28 of them were differentially regulated by GA3, with 12 conserved and 16 novel Vvi-miRNAs, based on the analysis of qRT-PCR essays. There existed some consistency in expression levels of GA3-responsive Vvi-miRNAs between high throughput sequencing and qRT-PCR essays. In addition, 117 target genes for 29 novel miRNAs were predicted.ConclusionsDeep sequencing of short RNAs from grapevine berries treated with GA3 and the control identified 137 GA3-responsive miRNAs, among which 28 exhibited different expression profiles of response to GA3 in the diverse developmental stages of grapevine berries. These identified Vvi-miRNAs might be involved in the grapevine berry development and response to environmental stresses.

Identification and bioinformatic analysis of signal responsive/ calmodulin-binding transcription activators gene models in Vitis vinifera

In this study, 10 grapevine (Vitis vinifera)SR/CAMTA (Signal Responsive/Calmodulin-binding Transcription Activators) gene models were identified from three grapevine genome protein datasets. They belong to four gene groups: VvCAMTA1, VvCAMTA3, VvCAMTA4 and VvCAMTA5, which were located on chromosome 5, 7_random, 1 and 5, respectively. Alternative splicing could explain the multiple gene models in one gene group. Subcellular localization using the WoLF tool showed that most of the VvCAMTAs were located in the nucleus, except for VvCAMTA3.1, VvCAMTA3.2 and VvCAMTA5.2, which were located in the chloroplast, chloroplast and cytosol, respectively. Subcellular localization using TargetP showed that most of the VvCAMTAs were not located in the chloroplast, mitochondrion and secretory pathway in cells. VvCAMTA1.1 and VvCAMTA1.2 were located in the mitochondria. The digital gene expression profile showed that VvCAMTAs play important roles in Ca2+ signal transduction. The gene expression patterns of VvCAMTAs were different; for example, VvCAMTA1 was expressed mainly in the bud, while VvCAMTA3 was expressed mainly in fruit and inflorescence, with low expression in the bud. The results of this study make a substantial contribution to our knowledge concerning genes, genome annotation, and cell signal transduction in grapevine.

Transporters, chaperones, and P-type ATPases controlling grapevine copper homeostasis

With more copper and copper-containing compounds used as bactericides and fungicides in viticulture, copper homeostasis in grapevine (Vitis) has become one of the serious environmental crises with great risk. To better understand the regulation of Cu homeostasis in grapevine, grapevine seedlings cultured in vitro with different levels of Cu were utilized to investigate the tolerance mechanisms of grapevine responding to copper availability at physiological and molecular levels. The results indicated that Cu contents in roots and leaves arose with increasing levels of Cu application. With copper concentration increasing, malondialdehyde (MDA) content increased in roots and leaves and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) increased to protect the plant itself from damage. The expression patterns of 19 genes, encoding transporters, chaperones, and P-type ATPases involved in copper homeostasis in root and leaf of grapevine seedling under various levels of Cu2+ were further analyzed. The expression patterns indicated that CTr1, CTr2, and CTr8 transporters were significantly upregulated in response both to Cu excess and deficiency. ZIP2 was downregulated in response to Cu excess and upregulated under Cu-deficient conditions, while ZIP4 had an opposite expression pattern under similar conditions. The expression of chaperones and P-type ATPases in response to Cu availability in grapevine were also briefly studied.

Whole genome identification and analysis of FK506-binding protein family genes in grapevine (Vitis vinifera L.).

In plant and animal species FK506-binding protein (FKBP) family genes are important conserved genes and it is defined as the receptors of FK506 and rapamycin, where they work as PPIase and protein folding chaperones. FKBP have been isolated from Arabidopsis thaliana, Oryza sativa, and Zea mays. In grape, twenty-three genes containing the FK506-binding domain (FKBP_C) were first time identified by HMMER and blast research, they were classified into three groups and 17 out of the 23 genes were located on 11 chromosomes (Chr1, 3, 5, 7, 8, 14, 15, 16, 17, 18, and 19). The predicted gene expression pattern and semi-quantitative RT-PCR results revealed that five VvFKBPs were expressed in all tissues, while seven VvFKBPs were expressed only in some of the tissues, and the remaining VvFKBPs were not expressed in leaf, stem, inflorescences, flowers, and a mixture of fruit tissues (small, medium and big-sized fruits). Most of the VvFKBPs in grapevine ‘Summer Black’ were similar to those predicted one in ‘Pinot Noir’ except for VvFKBP16-4 and VvFKBPa. VvFKBP12, FaFKBP12 and PpFKBP12 were cloned from ‘Summer Black’, ‘Sweet Charlie’ and ‘Xiahui 6’. Protein structure analysis confirmed that homologous genes have some differences during the process of protein structure construction. In this study, we characterized and verified 23 FKBP family genes in grapevine (Vitis vinifera L.) as well as their sub-cellular and chromosome location. The successful cloning of CDS regions and protein structural analysis of VvFKBP12, FaFKBP12, and PpFKBP12 can provide useful information for further study.

Discovery of Conservation and Diversification of miR171 Genes by Phylogenetic Analysis based on Global Genomes

The microRNA171 (miR171) family is widely distributed and highly conserved in a range of species and plays critical roles in regulating plant growth and development through repressing expression of SCARECROW‐LIKE (SCL) transcription factors. However, information on the evolutionary conservation and functional diversification of the miRNA171 family members remains scanty. We reconstructed the phylogenetic relationships among miR171 precursor and mature sequences so as to investigate the extent and degree of evolutionary conservation of miR171 in Arabidopsis thaliana (L.) Heynh. (ath), grape (Vitis vinifera L.) (vvi), poplar (Populus trichocarpa Torr. & A.Gray ex Hook.) (ptc), and rice (Oryza sativa L.) (osa). Despite strong conservation of over 80%, some mature miR171 sequences, such as ptc‐miR171j ‐l, and ‐m and osa‐miR171g, ‐h, and ‐i, have undergone critical sequence variation, leading to functional diversification, since they target non‐SCL gene transcript(s). Phylogenetic analyses revealed a combination of old ancestral relationships and recent lineage‐specific diversification in the miR171 family within the four model plants. The cis‐regulatory motifs on the upstream promoter sequences of miR171 genes were highly divergent and shared some similar elements, indicating their possible contribution to the functional variation observed within the miR171 family. This study will buttress our understanding of the functional differentiation of miRNAs and the relationships of miRNA–target pairs based on the evolutionary history of miRNA genes.

Genome-wide identification and analysis of FK506-binding protein family gene family in strawberry (Fragaria × ananassa)

The FK506 binding proteins (FKBPs) are abundant and ubiquitous proteins belonging to the large peptidyl-prolylcis-trans isomerase superfamily. FKBPs are known to be involved in many biological processes including hormone signaling, plant growth, and stress responses through a chaperone or an isomerization of proline residues during protein folding. The availability of complete strawberry genome sequences allowed the identification of 23 FKBP genes by HMMER and blast analysis. Chromosome scaffold locations of these FKBP genes in the strawberry genome were determined and the protein domain and motif organization of FaFKBPs analyzed. The phylogenetic relationships between strawberry FKBPs were also assessed. The expression profiles of FaFKBPs genes results revealed that most FaFKBPs were expressed in all tissues, while a few FaFKBPs were specifically expressed in some of the tissues. These data not only contribute to some better understanding of the complex regulation of the strawberry FKBP gene family, but also provide valuable information for further research in strawberry functional genomics.

Depiction of Grapevine Phenology by Gene Expression Information and a Test of its Workability in Guiding Fertilization

The development of precision agriculture calls for the emergence of new approaches to more accurately depict plant phenology. Gene expression data can predict and indicate plant growth state and phenological events accurately at the molecular level, and thus could be developed as a novel means of describing crop phenophase. Here, we analyzed the expression profiles of nine genes involved in grapevine flower and berry development, and screened the most informative genes for use in depicting grapevine phenology. Of the genes tested, VvAP1, VvAP3, VvFLC were found to be best suited to depicting grapevine phenology. The feasibility and efficiency of using the genetically depicted grapevine phenology was further tested in fertilization trials. The results showed that fertilization could be used to decrease flower and berry drop ratio and increase berry weight and size to a greater extent when taking into account variations in the activity of specific genes. Thus, phenologies predicted by a knowledge of gene activity can definitely be formed, and can be regarded as “genetic phenology”. A first grapevine genetic phenology profile was completed, and used to pre-depict grapevine phenophases to accurately guide the timing of grapevine farming activities and in the pre-diagnosis of the influence of some stresses on grapevines. Genetic phenology could be developed into a simple, low-cost and highly effective technology for accurate prediction of traditional crop phenology at the molecular level that is well suited to precision agriculture.

A novel and efficient strategy for practical identification of tomato (Solanum lycopersicon) varieties using modified RAPD fingerprints

Tomato breeding and variety development have led to the generation of a large number of varieties in many countries worldwide. This has created a growing and urgent need for an improved strategy for genotyping and identification since the traditional methods based on phenotype are growing unreliable. DNA markers could provide distinct benefits in tomato variety identification; however, DNA fingerprint analyses have not made DNA marker data readily usable for identification of varieties in tomato and other crops. A manual cultivar and/or variety identification diagram (MCID) strategy has been developed and has been found to make DNA markers more usable for the identification of genotyped plant individuals. We adopted this strategy, using modified RAPD markers to identify 42 tomato varieties from different geographical origins and seed merchants. All of the varieties were clearly separated and individually identified by reproducible fingerprints of only 6 RAPD primers. The tomato MCID that is generated is usable for the identification of any two or more tomato varieties. In addition, fewer primers can be used to make a distinction between varieties using this approach, since the selected fingerprints from each primer are used after they have been generated. The information in this first version of the tomato MCID can be enriched through identification and incorporation of more varieties and adaptation to other molecular markers in order to provide a more comprehensive tomato variety identification service for the horticultural industry.