Qian Mu

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.

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.

A characterization of grapevine of GRAS domain transcription factor gene family

GRAS domain genes are a group of important plant-specific transcription factors that have been reported to be involved in plant development. In order to know the roles of GRAS genes in grapevine, a widely cultivated fruit crop, the study on grapevine GRAS (VvGRAS) was carried out, and from which, 43 were identified from 12× assemble grapevine genomic sequences. Further, the genomic structures, synteny, phylogeny, expression profiles in different tissues of these genes, and their roles in response to stress were investigated. Among the genes, two potential target genes (VvSCL15 and VvSCL22) for VvmiR171 were experimentally verified by PPM-RACE and RLM-RACE, in that not only the cleavage sites of miR171 on the target mRNA were mapped but also the cleaved fragments and their expressing patterns were detected. Transgenic Arabidopsis plants over expression VvSCL15 showed lower tolerance to drought and salt treatments.

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.

Genome-Wide Mapping and Analysis of Grapevine MicroRNAs and Their Potential Target Genes

MicroRNAs (miRNAs) are single‐stranded, nonprotein‐coding, endogenously expressed, small RNAs 19 to 25 nucleotides in length. Recognizing the lack of specific and systematic studies on genome‐wide mapping of grapevine (Vitis vinifera L.) miRNAs, we conducted genome‐wide mapping of Vv‐miRNAs (V. vinifera miRNAs), SB‐miRNAs (V. vinifera L. ‘Summer Black’ miRNAs), and Va‐miRNAs (V. amurensis Rupr. miRNAs). The mapping results revealed that many of miRNAs located within the intergenic region had independent transcription units. To further validate the mapping results and existence of miRNAs, 12 randomly selected precursors of miRNAs (pre‐miRNAs) were successfully cloned and sequenced. Subsequently, 15 conserved and 29 nonconserved intragenic (intronic, exonic) Vv‐miRNA genes, 24 nonconserved intragenic SB‐miRNA genes, and 23 nonconserved intragenic Va‐miRNA genes were labeled on the basis of their locations in host genes, and 15 MIRNA clusters were detected. Interestingly, five miRNA pairs, namely, Vv‐MIR395b and Vv‐MIR395c, Vv‐MIR482 and Vv‐MIRC13, Vv‐MIR172a and Va‐MIR057, SB‐MIR024 and Vv‐MIRC35, and Vv‐MIRC36 and Va‐MIR073 were clustered in the host genes GSVIVT01011558001, GSVIVT01008132001, GSVIVT01031524001, GSVIVT01028156001, and GSVIVT01024516001, respectively. To validate the existence of target genes and miRNA‐guided cleavage sites, 3′‐end product of four predicted target messenger RNAs were amplified by RNA ligase‐mediated 5′ rapid amplification of cDNA ends. In addition, we also conducted contrastive analysis on the genomic location of miRNAs and their potential target genes. Results showed that the order of priority of miRNA–target interaction may be less closely related with their genomic location. These findings could benefit some further study on grapevine functional genomics and will provide new insights into the regulatory mechanisms and evolution of miRNAs in Vitis species.

Cloning, expression, and characterization of miR058 and its target PPO during the development of grapevine berry stone.

Polyphenol oxidases catalyzing the oxygen-dependent oxidation of phenols to quinones are ubiquitous among angiosperms. They are key enzymes playing a significant role during the synthesis of lignin. The inhibition of the synthesis of lignin in grapevine can cause seedless grapevine berry development. In this study, grapevine PPO (Vv-PPO) was predicted as the target gene of Vv-miR058 by bioinformatics analysis, and it was further cloned and its homologous conservation in various plants was analyzed. The expression profiles of miR058 and its target Vv-PPO were detected by qRT-PCR in peel, pulp and seeds of three grapevine cultivars and Vv-PPO was expressed in an opposite variation way with Vv-miR058 where both of them could be detected, suggesting that Vv-miR058 can play an important role by regulating the expression of Vv-PPO. In addition, the potential target gene Vv-PPO for Vv-miR058 was verified by RLM-RACE. This result would be helpful in theoretical basis for further research and seedless grapevine berry production.

Employment of a new strategy for identification of lemon (Citrus limon L.) cultivars using RAPD markers

DNA markers are useful tools that have potential application in crop cultivar identification and fingerprinting besides others. A novel analysis approach called CID strategy was developed that can realize the utility of DNA marker in the separation of plant individuals much better, more efficiently, practical, and referable, in which a Cultivar-Identification-Diagram (CID) was constructed for fingerprinting. In this study, a total of 47 important lemon cultivars cultivated in China were successfully separated with random amplified polymorphic DNA (RAPD) marker analysis. With the analysis using the CID strategy, they could be clearly separated by the fingerprints of 10 RAPD primers. The utilization of the CID of these 47 lemon cultivars was also verified by the identification of two randomly chosen groups of cultivars among the 47. This identification showed that some advantages in fewer primers were used, and all the cultivars could be separated by each other by the corresponding primers marked in the right position on the CID, and this lemon CID could provide the information to separate any lemon cultivars of these 47 which may be of help to the lemon industry in China.

RNA-Sequencing Reveals Biological Networks during Table Grapevine (‘Fujiminori’) Fruit Development

Grapevine berry development is a complex and genetically controlled process, with many morphological, biochemical and physiological changes occurring during the maturation process. Research carried out on grapevine berry development has been mainly concerned with wine grape, while barely focusing on table grape. ‘Fujiminori’ is an important table grapevine cultivar, which is cultivated in most provinces of China. In order to uncover the dynamic networks involved in anthocyanin biosynthesis, cell wall development, lipid metabolism and starch-sugar metabolism in ‘Fujiminori’ fruit, we employed RNA-sequencing (RNA-seq) and analyzed the whole transcriptome of grape berry during development at the expanding period (40 days after full bloom, 40DAF), véraison period (65DAF), and mature period (90DAF). The sequencing depth in each sample was greater than 12×, and the expression level of nearly half of the expressed genes were greater than 1. Moreover, greater than 64% of the clean reads were aligned to the Vitis vinifera reference genome, and 5,620, 3,381, and 5,196 differentially expressed genes (DEGs) were identified between different fruit stages, respectively. Results of the analysis of DEGs showed that the most significant changes in various processes occurred from the expanding stage to the véraison stage. The expression patterns of F3’H and F3’5’H were crucial in determining red or blue color of the fruit skin. The dynamic networks of cell wall development, lipid metabolism and starch-sugar metabolism were also constructed. A total of 4,934 SSR loci were also identified from 4,337 grapevine genes, which may be helpful for the development of phylogenetic analysis in grapevine and other fruit trees. Our work provides the foundation for developmental research of grapevine fruit as well as other non-climacteric fruits.

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.