Transcriptome Analysis of Genes Involved in Cold Hardiness of Peach Tree (Prunus persica) Shoots during Cold Acclimation and Deacclimation.

We analyzed the transcriptomes in the shoots of five-year-old 'Soomee' peach trees (Prunus persica) during cold acclimation (CA), from early CA (end of October) to late CA (middle of January), and deacclimation (DA), from late CA to late DA (middle of March), to identify the genes involved in cold hardiness. Cold hardiness of the shoots increased from early to late CA, but decreased from late CA to late DA, as indicated by decreased and increased the median lethal temperature (LT50), respectively. Transcriptome analysis identified 17,208 assembled transcripts during all three stages. In total, 1891 and 3008 transcripts were differentially expressed with a |fold change| > 2 (p < 0.05) between early and late CA, and between late CA and late DA, respectively. Among them, 1522 and 2830, respectively, were functionally annotated with gene ontology (GO) terms having a greater proportion of differentially expressed genes (DEGs) associated with molecular function than biological process or cellular component categories. The biochemical pathways best represented both periods from early to late CA and from late CA to late DA were 'metabolic pathway' and 'biosynthesis of secondary metabolites'. We validated these transcriptomic results by performing reverse transcription quantitative polymerase chain reaction on the selected DEGs showing significant fold changes. The relative expressions of the selected DEGs were closely related to the LT50 values of the peach tree shoots: 'Soomee' shoots exhibited higher relative expressions of the selected DEGs than shoots of the less cold-hardy 'Odoroki' peach trees. Irrespective of the cultivar, the relative expressions of the DEGs that were up- and then down-regulated during CA, from early to late CA, and DA, from late CA to late DA, were more closely correlated with cold hardiness than those of the DEGs that were down- and then up-regulated. Therefore, our results suggest that the significantly up- and then down-regulated DEGs are associated with cold hardiness in peach tree shoots. These DEGs, including early light-induced protein 1, chloroplastic, 14-kDa proline-rich protein DC2.15, glutamate dehydrogenase 2, and triacylglycerol lipase 2, could be candidate genes to determine cold hardiness.

Transcriptional regulation of abscisic acid biosynthesis and signal transduction, and anthocyanin biosynthesis in 'Bluecrop' highbush blueberry fruit during ripening.

Highbush blueberry (Vaccinium corymbosum) fruit accumulate high levels of anthocyanins during ripening, which might be controlled by abscisic acid (ABA), a signal molecule in non-climacteric fruits. For an integrated view of the ripening process from ABA to anthocyanin biosynthesis, we analyzed the transcriptomes of 'Bluecrop' highbush blueberry fruit using RNA-Seq at three ripening stages, categorized based on fruit skin coloration: pale green at ca. 30 days after full bloom (DAFB), reddish purple at ca. 40 DAFB, and dark purple at ca. 50 DAFB. Mapping the trimmed reads against the reference sequences yielded 25,766 transcripts. Of these, 143 transcripts were annotated to encode five ABA biosynthesis enzymes, four ABA signal transduction regulators, four ABA-responsive transcription factors, and 12 anthocyanin biosynthesis enzymes. The analysis of differentially expressed genes between the ripening stages revealed that 11 transcripts, including those encoding nine-cis-epoxycarotenoid dioxygenase, SQUAMOSA-class MADS box transcription factor, and flavonoid 3',5'-hydroxylase, were significantly up-regulated throughout the entire ripening stages. In fruit treated with 1 g L-1 ABA, at least nine transcripts of these 11 transcripts as well as one transcript encoding flavonoid 3'-hydroxylase were up-regulated, presumably promoting anthocyanin accumulation and fruit skin coloration. These results will provide fundamental information demonstrating that ABA biosynthesis and signal transduction, and anthocyanin biosynthesis are closely associated with anthocyanin accumulation and skin coloration in highbush blueberry fruit during ripening.

Abscisic acid stimulates anthocyanin accumulation in 'Jersey' highbush blueberry fruits during ripening.

Non-climacteric blueberry (Vaccinium spp.) fruits accumulate high levels of anthocyanins during ripening, which are a good source of dietary antioxidants. This study examined the effects of exogenous abscisic acid (ABA) application on fruit characteristics and anthocyanin accumulation in a northern highbush blueberry (V. corymbosum 'Jersey') during development. Fruits on shrubs were treated with 1gL-1 ABA before the initiation of fruit colouration. Application of ABA temporarily increased the level of ABA in the fruits during development. Exogenous ABA had no obvious effect on fruit growth, but stimulated fruit colouration by accelerating the accumulation of individual anthocyanins, mainly malvidin, delphinidin and petunidin glycosides. This is the first report to show that ABA promotes the accumulation of anthocyanins in blueberry fruits. However, exogenous ABA also promoted fruit softening, which is undesirable during harvest and shelf life.

Intraspecies differences in cold hardiness, carbohydrate content and ß-amylase gene expression of Vaccinium corymbosum during cold acclimation and deacclimation.

Changes in cold hardiness, carbohydrate content and ß-amylase gene expression were monitored in the shoots of the highbush blueberry (Vaccinium corymbosum L.) cultivars 'Sharpblue' and 'Jersey' during cold acclimation (CA) and deacclimation (DA). The seasonal patterns were similar in both cultivars, but the levels of cold hardiness determined by electrolyte leakage analysis were significantly different; 'Jersey' was hardier than 'Sharpblue'. Cold hardiness was closely related to total soluble sugar content (r = -0.98** and -0.99** for 'Sharpblue' and 'Jersey', respectively). In 'Jersey', more soluble sugars accumulated during CA. Of the detected soluble sugars, glucose, fructose and raffinose contents were significantly associated with cold hardiness in both cultivars. Sucrose was abundant in both cultivars, and stachyose content changed significantly during CA and DA. However, they were not associated with cold hardiness. A sharp decrease in starch contents in the middle of CA coincided with ß-amylase gene (VcBMY) expression, indicating the conversion of starch into soluble sugars. During CA, VcBMY was expressed up to twofold higher in 'Jersey' than in 'Sharpblue'. These results suggest that intraspecies differences in the cold hardiness of highbush blueberries are associated with total soluble sugar content, which is driven partly by differential expression of VcBMY.