Identification of microRNAs from Amur grape (vitis amurensis Rupr.) by deep sequencing and analysis of microRNA variations with bioinformatics

<p>Abstract</p> <p>Background</p> <p>MicroRNA (miRNA) is a class of functional non-coding small RNA with 19-25 nucleotides in length while Amur grape (<it>Vitis amurensis Rupr</it>.) is an important wild fruit crop with the strongest cold resistance among the <it>Vitis </it>species, is used as an excellent breeding parent for grapevine, and has elicited growing interest in wine production. To date, there is a relatively large number of grapevine miRNAs (vv-miRNAs) from cultivated grapevine varieties such as <it>Vitis vinifera L</it>. and hybrids of <it>V. vinifera </it>and <it>V. labrusca</it>, but there is no report on miRNAs from <it>Vitis amurensis Rupr</it>, a wild grapevine species.</p> <p>Results</p> <p>A small RNA library from Amur grape was constructed and Solexa technology used to perform deep sequencing of the library followed by subsequent bioinformatics analysis to identify new miRNAs. In total, 126 conserved miRNAs belonging to 27 miRNA families were identified, and 34 known but non-conserved miRNAs were also found. Significantly, 72 new potential Amur grape-specific miRNAs were discovered. The sequences of these new potential va-miRNAs were further validated through miR-RACE, and accumulation of 18 new va-miRNAs in seven tissues of grapevines confirmed by real time RT-PCR (qRT-PCR) analysis. The expression levels of va-miRNAs in flowers and berries were found to be basically consistent in identity to those from deep sequenced sRNAs libraries of combined corresponding tissues. We also describe the conservation and variation of va-miRNAs using miR-SNPs and miR-LDs during plant evolution based on comparison of orthologous sequences, and further reveal that the number and sites of miR-SNP in diverse miRNA families exhibit distinct divergence. Finally, 346 target genes for the new miRNAs were predicted and they include a number of Amur grape stress tolerance genes and many genes regulating anthocyanin synthesis and sugar metabolism.</p> <p>Conclusions</p> <p>Deep sequencing of short RNAs from Amur grape flowers and berries identified 72 new potential miRNAs and 34 known but non-conserved miRNAs, indicating that specific miRNAs exist in Amur grape. These results show that a number of regulatory miRNAs exist in Amur grape and play an important role in Amur grape growth, development, and response to abiotic or biotic stress.</p

Characterization of microRNAs Identified in a Table Grapevine Cultivar with Validation of Computationally Predicted Grapevine miRNAs by miR-RACE

BACKGROUND: Alignment analysis of the Vv-miRNAs identified from various grapevine cultivars indicates that over 30% orthologous Vv-miRNAs exhibit a 1-3 nucleotide discrepancy only at their ends, suggesting that this sequence discrepancy is not a random event, but might mainly derive from divergence of cultivars. With advantages of miR-RACE technology in determining precise sequences of potential miRNAs from bioinformatics prediction, the precise sequences of vv-miRNAs predicted computationally can be verified with miR-RACE in a different grapevine cultivar. This presents itself as a new approach for large scale discovery of precise miRNAs in different grapevine varieties. METHODOLOGY/PRINCIPAL FINDINGS: Among 88 unique sequences of Vv-miRNAs from bioinformatics prediction, 83 (96.3%) were successfully validated with MiR-RACE in grapevine cv. 'Summer Black'. All the validated sequences were identical to their corresponding ones obtained from deep sequencing of the small RNA library of 'Summer Black'. Quantitative RT-PCR analysis of the expressions levels of 10 Vv-miRNA/target gene pairs in grapevine tissues showed some negative correlation trends. Finally, comparison of Vv-miRNA sequences with their orthologs in Arabidopsis and study on the influence of divergent bases of the orthologous miRNAs on their targeting patterns in grapevine were also done. CONCLUSION: The validation of precise sequences of potential Vv-miRNAs from computational prediction in a different grapevine cultivar can be a new way to identify the orthologous Vv-miRNAs. Nucleotide discrepancy of orthologous Vv-miRNAs from different grapevine cultivars normally does not change their target genes. However, sequence variations of some orthologous miRNAs in grapevine and Arabidopsis can change their targeting patterns. These precise Vv-miRNAs sequences validated in our study could benefit some further study on grapevine functional genomics

Drought stress in ‘Shine Muscat’ grapevine: Consequences and a novel mitigation strategy–5-aminolevulinic acid

Drought is a common and serious abiotic stress in viticulture, and it is urgent to select effective measures to alleviate it. The new plant growth regulator 5-aminolevulinic acid (ALA) has been utilized to alleviate abiotic stresses in agriculture in recent years, which provided a novel idea to mitigate drought stress in viticulture. The leaves of ‘Shine Muscat’ grapevine (Vitis vinifera L.) seedlings were treated with drought (Dro), drought plus 5-aminolevulinic acid (ALA, 50 mg/L) (Dro_ALA) and normal watering (Control) to clarify the regulatory network used by ALA to alleviate drought stress in grapevine. Physiological indicators showed that ALA could effectively reduce the accumulation of malondialdehyde (MDA) and increase the activities of peroxidase (POD) and superoxide dismutase (SOD) in grapevine leaves under drought stress. At the end of treatment (day 16), the MDA content in Dro_ALA was reduced by 27.63% compared with that in Dro, while the activities of POD and SOD reached 2.97- and 5.09-fold of those in Dro, respectively. Furthermore, ALA reduces abscisic acid by upregulating CYP707A1, thus, relieving the closure of stomata under drought. The chlorophyll metabolic pathway and photosynthetic system are the major pathways affected by ALA to alleviate drought. Changes in the genes of chlorophyll synthesis, including CHLH, CHLD, POR, and DVR; genes related to degradation, such as CLH, SGR, PPH and PAO; the RCA gene that is related to Rubisco; and the genes AGT1 and GDCSP related to photorespiration form the basis of these pathways. In addition, the antioxidant system and osmotic regulation play important roles that enable ALA to maintain cell homeostasis under drought. The reduction of glutathione, ascorbic acid and betaine after the application of ALA confirmed the alleviation of drought. In summary, this study revealed the mechanism of effects of drought stress on grapevine, and the alleviating effect of ALA, which provides a new concept to alleviate drought stress in grapevine and other plants

Evaluation of Genome Sequencing Quality in Selected Plant Species Using Expressed Sequence Tags

<div><p>Background</p><p>With the completion of genome sequencing projects for more than 30 plant species, large volumes of genome sequences have been produced and stored in online databases. Advancements in sequencing technologies have reduced the cost and time of whole genome sequencing enabling more and more plants to be subjected to genome sequencing. Despite this, genome sequence qualities of multiple plants have not been evaluated.</p><p>Methodology/Principal Finding</p><p>Integrity and accuracy were calculated to evaluate the genome sequence quality of 32 plants. The integrity of a genome sequence is presented by the ratio of chromosome size and genome size (or between scaffold size and genome size), which ranged from 55.31% to nearly 100%. The accuracy of genome sequence was presented by the ratio between matched EST and selected ESTs where 52.93% ∼ 98.28% and 89.02% ∼ 98.85% of the randomly selected clean ESTs could be mapped to chromosome and scaffold sequences, respectively. According to the integrity, accuracy and other analysis of each plant species, thirteen plant species were divided into four levels. <i>Arabidopsis thaliana</i>, <i>Oryza sativa</i> and <i>Zea mays</i> had the highest quality, followed by <i>Brachypodium distachyon</i>, <i>Populus trichocarpa</i>, <i>Vitis vinifera</i> and <i>Glycine max, Sorghum bicolor</i>, <i>Solanum lycopersicum</i> and <i>Fragaria vesca</i>, and <i>Lotus japonicus</i>, <i>Medicago truncatula</i> and <i>Malus</i> × <i>domestica</i> in that order. Assembling the scaffold sequences into chromosome sequences should be the primary task for the remaining nineteen species. Low GC content and repeat DNA influences genome sequence assembly.</p><p>Conclusion</p><p>The quality of plant genome sequences was found to be lower than envisaged and thus the rapid development of genome sequencing projects as well as research on bioinformatics tools and the algorithms of genome sequence assembly should provide increased processing and correction of genome sequences that have already been published.</p></div

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

<div><p>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 <i>Vitis vinifera</i> 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 <i>F3’H</i> and <i>F3’5’H</i> 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.</p></div

A novel hydroxycinnamoyl transferase for synthesis of hydroxycinnamoyl spermine conjugates in plants.

BackgroundHydroxycinnamoyl-spermine conjugates (HCSpm) are a class of hydroxycinnamic acid amides (HCAAs), which not only are instrumental in plant development and stress response, but also benefit human health. However, HCSpm are not commonly produced in plants, and the mechanism of their biosynthesis remains unclear. In previous investigations of phenolics in Solanum fruits related to eggplant (Solanum melongena L.), we discovered that Solanum richardii, an African wild relative of eggplant, was rich in HCSpms in fruits.ResultsThe putative spermine hydroxycinnamoyl transferase (HT) SpmHT was isolated from S. richardii and eggplant. SrSpmHT expression was high in flowers and fruit, and was associated with HCSpm accumulation in S. richardii; however, SpmHT was hardly detected in eggplant cultivars and other wild relatives. Recombinant SpmHT exclusively selected spermine as the acyl acceptor substrate, while showing donor substrate preference in the following order: caffeoyl-CoA, feruloyl-CoA, and p-coumaroyl-CoA. Molecular docking revealed that substrate binding pockets of SpmHT could properly accommodate spermine but not the shorter, more common spermidine.ConclusionSrSpmHT is a novel spermine hydroxycinnamoyl transferase that uses Spm exclusively as the acyl acceptor substrate to produce HCSpms. Our findings shed light on the HCSpm biosynthetic pathway that may allow an increase of health beneficial metabolites in Solanum crops via methods such as introgression or engineering HCAA metabolism