Changjie Xu

Transcriptomic analysis of Chinese bayberry (Myrica rubra) fruit development and ripening using RNA-Seq

BackgroundChinese bayberry (Myrica rubra Sieb. and Zucc.) is an important subtropical fruit crop and an ideal species for fruit quality research due to the rapid and substantial changes that occur during development and ripening, including changes in fruit color and taste. However, research at the molecular level is limited by a lack of sequence data. The present study was designed to obtain transcript sequence data and examine gene expression in bayberry developing fruit based on RNA-Seq and bioinformatic analysis, to provide a foundation for understanding the molecular mechanisms controlling fruit quality changes during ripening.ResultsRNA-Seq generated 1.92 G raw data, which was then de novo assembled into 41,239 UniGenes with a mean length of 531 bp. Approximately 80% of the UniGenes (32,805) were annotated against public protein databases, and coding sequences (CDS) of 31,665 UniGenes were determined. Over 3,600 UniGenes were differentially expressed during fruit ripening, with 826 up-regulated and 1,407 down-regulated. GO comparisons between the UniGenes of these two types and interactive pathways (Ipath) analysis found that energy-related metabolism was enhanced, and catalytic activity was increased. All genes involved in anthocyanin biosynthesis were up-regulated during the fruit ripening processes, concurrent with color change. Important changes in carbohydrate and acid metabolism in the ripening fruit are likely associated with expression of sucrose phosphate synthase (SPS) and glutamate decarboxylase (GAD).ConclusionsMass sequence data of Chinese bayberry was obtained and the expression profiles were examined during fruit ripening. The UniGenes were annotated, providing a platform for functional genomic research with this species. Using pathway mapping and expression profiles, the molecular mechanisms for changes in fruit color and taste during ripening were examined. This provides a reference for the study of complicated metabolism in non-model perennial species.

Expression of Genes Associated with Aroma Formation Derived from the Fatty Acid Pathway during Peach Fruit Ripening

Changes in characteristic aroma volatiles, levels of fatty acids as aroma precursors, and expression patterns of related genes, including lipoxygenase (LOX), hydroperoxide lyase (HPL), alcohol dehydrogenase (ADH), alcohol acyltransferase (AAT), and fatty acid desaturase (FAD), were studied in peach ( Prunus persica L. Batsch., cv. Yulu) fruit during postharvest ripening at 20 degrees C. Concentrations of n-hexanal, (E)-2-hexenal, (E)-2-hexenol, and (Z)-3-hexenol decreased, whereas the production of (Z)-3-hexenyl acetate, gamma-hexalactone, gamma-octalactone, gamma-decalactone, and delta-decalactone increased with fruit ripening. Lactones showed a clear pattern concomitant with the climacteric rise in ethylene production, with gamma-decalactone being the principal volatile compound at the late ripening stage. Of the LOX family genes, PpLOX2 and PpLOX3 had relatively high transcript levels initially followed by a decline with fruit ripening, while levels of PpLOX1 and PpLOX4 transcripts were upregulated by accumulated ethylene production. Expression of PpHPL1, PpADH1, PpADH2, and PpADH3 showed similar decreasing patterns during ripening. Expression levels of PpAAT1 showed a rapid increase during the first 2 days of postharvest ripening followed by a gradual decrease. Contents of polyunsaturated linoleic and linolenic acids increased, and saturated palmitic acid levels tended to decline as the fruit ripened. The increased levels of unsaturated fatty acids closely paralleled increasing expression of PpFAD1 and PpFAD2. The significance of gene expression changes in relation to aroma volatile production is discussed.

Plastid structure and carotenogenic gene expression in red- and white-fleshed loquat (Eriobotrya japonica) fruits

Loquat (Eriobotrya japonica Lindl.) can be sorted into red- and white-fleshed cultivars. The flesh of Luoyangqing (LYQ, red-fleshed) appears red-orange because of a high content of carotenoids while the flesh of Baisha (BS, white-fleshed) appears ivory white due to a lack of carotenoid accumulation. The carotenoid content in the peel and flesh of LYQ was approximately 68 μg g−1 and 13 μg g−1 fresh weight (FW), respectively, and for BS 19 μg g−1 and 0.27 μg g−1 FW. The mRNA levels of 15 carotenogenesis-related genes were analysed during fruit development and ripening. After the breaker stage (S4), the mRNA levels of phytoene synthase 1 (PSY1) and chromoplast-specific lycopene β-cyclase (CYCB) were higher in the peel, and CYCB and β-carotene hydroxylase (BCH) mRNAs were higher in the flesh of LYQ, compared with BS. Plastid morphogenesis during fruit ripening was also studied. The ultrastructure of plastids in the peel of BS changed less than in LYQ during fruit development. Two different chromoplast shapes were observed in the cells of LYQ peel and flesh at the fully ripe stage. Carotenoids were incorporated in the globules in chromoplasts of LYQ and BS peel but were in a crystalline form in the chromoplasts of LYQ flesh. However, no chromoplast structure was found in the cells of fully ripe BS fruit flesh. The mRNA level of plastid lipid-associated protein (PAP) in the peel and flesh of LYQ was over five times higher than in BS peel and flesh. In conclusion, the lower carotenoid content in BS fruit was associated with the lower mRNA levels of PSY1, CYCB, and BCH; however, the failure to develop normal chromoplasts in BS flesh is the most convincing explanation for the lack of carotenoid accumulation. The expression of PAP was well correlated with chromoplast numbers and carotenoid accumulation, suggesting its possible role in chromoplast biogenesis or interconversion of loquat fruit.

Regulatory Mechanisms of Textural Changes in Ripening Fruits

Texture changes in ripening fruits influence consumer preference, fruit storability, transportability, shelf-life, and response to pathogen attack. Genetic regulatory factors as well as environmental conditions simultaneously affect texture changes in ripening fruit. Recent physiological and molecular studies provide insights into our knowledge and understanding of events and/or factors that contribute to changes in fruit texture, including softening and lignification. The roles of enzymes involved in modification and/or regulation of cell wall components as well as ethylene signaling components that play key roles in fruit textural changes during fruit ripening and storage will be presented and discussed. In addition, physical as well as chemical regulation of textural changes in ripening fruit will be explored.

Activator- and repressor-type MYB transcription factors are involved in chilling injury induced flesh lignification in loquat via their interactions with the phenylpropanoid pathway

Summary Two novel MYB transcription factors are involved in lignin biosynthesis and flesh lignification in loquat fruit, which are manipulated by temperature condition and treatments.

Ethylene signal transduction elements involved in chilling injury in non-climacteric loquat fruit

Loquat (Eriobotrya japonica Lindl.) is a subtropical fruit, with some cultivars such as ‘Luoyangqing’ (LYQ) susceptible to chilling injury (CI), while others such as ‘Baisha’ (BS) are resistant. Although loquats are non-climacteric, modulation of ethylene has an effect on ripening-related post-harvest CI. Therefore the role of ethylene signalling in the development of CI was investigated in fruit of both the LYQ and BS cultivars. Three ethylene receptor genes, one CTR1-like gene, and one EIN3-like gene were isolated and characterized in ripening fruit. All of these genes were expressed differentially within and between fruit of the two cultivars. Transcripts either declined over fruit development (EjERS1a in both cultivars and EjEIL1 in LYQ) or showed an increase in the middle stages of fruit development before declining (EjETR1, EjERS1b, and EjCTR1 in both cultivars and EjEIL1 in BS). The main cultivar differences were in levels rather than in patterns of expression during post-harvest storage. EjETR1, EjCTR1, and EjEIL1 genes showed increased expression in response to low temperature and this was particularly notable for EjETR1, and EjEIL1 during CI development in LYQ fruit. The genes were also differentially responsive to ethylene treatment, 1-methycyclopropene (1-MCP) and low temperature conditioning, confirming a role for ethylene in regulation of CI in loquat fruit.

Stable transformation of protocorm-like bodies in Phalaenopsis orchid mediated by Agrobacterium tumefaciens

Genetically transformed plantlets of Phalaenopsis were regenerated after co-cultivation of protocorm-like bodies (PLBs) with Agrobacterium tumefaciens strain LBA4404 containing the vector pTOK233 that harbors genes for β-glucuronidase and hygromycin resistance. Four lines of Phalaenopsis orchid (T0, T5, T10 and Hikaru) were tested on three multiplication media for the production of PLBs. Two types of explants, intact and transversely bisected PLBs, were checked for efficiency of multiplication, and it was found that transversely bisected PLBs were much better than intact ones. The effect of hygromycin concentrations on rooting of PLBs was also established. Initial recovery of the PLBs following co-cultivation was allowed without selection for 6 weeks. The first selection was carried out on regeneration medium containing 3 mg/l hygromycin for 2 months. New PLBs, as well as shoots and roots, were formed from original PLBs when they were regenerated under hygromycin selection. Leaves, roots, original and some newly formed PLBs were taken from hygromycin-resistant rooted plantlets and then subjected to GUS assay. Newly formed PLBs from GUS-positive plantlets were then regenerated on the above-mentioned regeneration medium but containing a lower concentration of hygromycin (1.5 mg/l). When the PLBs regenerated into plantlets, original PLBs were subjected to GUS assay, and again newly formed PLBs were regenerated and selected as described above. The procedures were repeated monthly for four cycles and it was found that a long selection period with the low hygromycin concentration yielded stable transformants. Histochemical GUS assay indicated successful transformation, which was further confirmed by PCR analysis and Southern hybridization of transformants.

Postharvest temperature influences volatile lactone production via regulation of acyl‐CoA oxidases in peach fruit

The biosynthesis of volatile compounds in plants is affected by environmental conditions. Lactones are considered to be peach-like aroma volatiles; however, no enzymes or genes associated with their biosynthesis have been characterized. White-fleshed (cv. Hujingmilu) and yellow-fleshed (cv. Jinxiu) melting peach (Prunus persica L. Batsch) fruit were used as materials in two successive seasons and responses measured to four different temperature treatments. Five major lactones accumulated during postharvest peach fruit ripening at 20 °C. Peach fruit at 5 °C, which induces chilling injury (CI), had the lowest lactone content during subsequent shelf life after removal, while 0 °C and a low-temperature conditioning (LTC) treatment alleviated development of CI and maintained significantly higher lactone contents. Expression of PpACX1 and activity of acyl-CoA oxidase (ACX) with C16-CoA tended to increase during postharvest ripening both at 20 °C and during shelf life after removal from cold storage when no CI was developed. There was a positive correlation between ACX and lactones in peach fruit postharvest. Changes in lactone production in response to temperatures are suggested to be a consequence of altered expression of PpACX1 and long-chain ACX activity.

Involvement of multiple phytoene synthase genes in tissue- and cultivar-specific accumulation of carotenoids in loquat

Summary Four phytoene synthase genes and several variants were characterized, and their evolution and function in differential carotenoid accumulation in leaf, peel, and flesh of white- and red-fleshed loquats were established.

The MrWD40-1 Gene of Chinese Bayberry ( Myrica rubra ) Interacts with MYB and bHLH to Enhance Anthocyanin Accumulation

Anthocyanins are important for fruits as they contribute not only to fruit color but also to human health. Anthocyanin biosynthesis is transcriptionally regulated by the MYB–bHLH–WD40 transcription complex. For Chinese bayberry (Myrica rubra), the MYB and bHLH regulating anthocyanin accumulation, named as MrMYB1 and MrbHLH1, respectively, have been isolated previously. In this study, by searching and assembling the sequences available in the RNA-Seq database of Chinese bayberry, 60 WD40 members were obtained. Through phylogenetic analysis of these members with those related to anthocyanin biosynthesis regulation in other plants, unigenes 803 and 11128, designated as MrWD40-1 and MrWD40-2, respectively, have been isolated as the putative WD40 members regulating anthocyanin biosynthesis. However, positive correlation was observed between the anthocyanin accumulation and the expression patterns of MrWD40-1 but not MrWD40-2, both during fruit development, and in different tissues or cultivars of Chinese bayberry. Tobacco transient assays indicated that the ternary expression of MrMYB1–MrbHLH1–MrWD40-1 induced anthocyanin accumulation earlier and stronger than with binary expression of MrMYB1–MrbHLH1 in the absence of MrWD40-1. Compared with the enhancement effect on anthocyanin biosynthesis of MrWD40-1, MrWD40-2 could not improve the anthocyanin accumulation even with MrMYB1 and MrbHLH1, although the highly conserved four WD repeat motifs were also present in MrWD40-2. Moreover, it was observed that MrWD40-1 physically interacted with both MrMYB1 and MrbHLH1 according to yeast two-hybrid analysis. These results indicated that MrWD40-1, but not MrWD40-2, is the member regulating anthocyanin biosynthesis in Chinese bayberry through the formation of a ternary complex with MrMYB1 and MrbHLH1.