Kunsong Chen

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.

Kiwifruit EIL and ERF genes involved in regulating fruit ripening

Kiwifruit (Actinidia deliciosa) is a climacteric fruit sensitive to low concentrations of ethylene. To investigate the transcriptional mechanisms underlying kiwifruit ethylene response, transcription factors encoding four EIN3-Like (EILs) and 14 Ethylene Response Factors (ERFs) were cloned from kiwifruit. Expression of these transcription factors was examined during fruit development. The expression of transcripts of most AdERFs was higher during early fruit development, with the exception of AdERF3, which increased with maturity. Several AdERFs were apparently down-regulated by ethylene, as they were affected by the ethylene inhibitor 1-methylcyclopropene and by antisense suppression of ACO (for 1-aminocyclopropane-1-carboxylic acid oxidase) in the fruit. In contrast, AdEILs were constitutively expressed during fruit development and ripening. The transcription factors AdEIL2 and AdEIL3 activated transcription of the ripening-related genes AdACO1 and AdXET5 (xyloglucan endotransglycosylase gene) and, when overexpressed in Arabidopsis (Arabidopsis thaliana), stimulated ethylene production. The potential repressor AdERF9 suppressed this promoter activity. These results support a role for kiwifruit EILs and ERFs in transcriptional regulation of ripening-related genes and in the regulation of kiwifruit fruit-ripening processes.

Ethylene-induced modulation of genes associated with the ethylene signalling pathway in ripening kiwifruit

Gene families associated with the ethylene signal transduction pathway in ripening kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var. deliciosa cv. Hayward) were isolated from a kiwifruit expressed sequence tag (EST) database, including five ethylene receptor genes, two CTR1-like genes, and an EIN3-like gene AdEIL1. All were differentially expressed among various kiwifruit vine tissues, and none was fruit specific. During fruit development, levels of transcripts of AdERS1a, AdETR3, and the two CTR1-like genes decreased, whereas those of AdERS1b and AdETR2 peaked at 97 d after full bloom. In ripening kiwifruit, there was a diverse response of the ethylene receptor family to internal and external ethylene. AdERS1a, AdETR2, and AdETR3 expression increased at the climacteric stage and transcripts were induced by external ethylene treatment, while AdERS1b showed no response to ethylene. AdETR1 was negatively regulated by internal and external ethylene in ripening fruit. The two CTR1-like genes also had different expression patterns, with AdCTR1 increasing at the climacteric stage and AdCTR2 undergoing little change. 1-Methylcyclopropene treatment prevented the ethylene response of all components, but transient down-regulation was only found with AdETR2 and AdCTR1. Similar gene and ethylene responses were found in both fruit flesh and core tissues. The ethylene-induced down-regulation of AdETR1 suggests that it may have a role in sensing ethylene and transmitting this response to other members of the receptor family, thus activating the signal transduction pathway.

Genetic diversity and similarity of pear (Pyrus L.) cultivars native to East Asia revealed by SSR (simple sequence repeat) markers

Simple sequence repeat (SSR) markers were used to assess genetic diversity and relationship of Pyrus L. cultivars native mainly to East Asia. A total of 168 putative alleles were generated from six primer-pairs (BGA35, KU10, BGT23b, NH004a, NH011b and NH015a). All the SSR markers showed a high level of genetic polymorphism with a mean of 28 putative alleles per locus and the heterozygosity of 0.63. The Dice’s similarity coefficient between cultivars ranged from 0.02 to 0.98 and Occidental pears generally had low affinities to Asian pears. Ten major groups were generated from all the accessions by UPGMA clusters analysis. Chinese sand pears consisted of four groups with Chinese white pears and Japanese pears, of which Chinese sand pears occurred in all four groups, presenting a large genetic diversity, Chinese white pears were included in three groups, and Japanese pears only fell into one group. In the dendrogram, Chinese sand pears and Chinese white pears did not form discrete group, even subgroups. Some Japanese pear cultivars had high affinities to Chinese sand pear cultivars. These findings supports the authors’ previous viewpoints of Chinese white pears as a variety or an ecotype of Chinese sand pears (P. pyrifolia var. sinensis (Lindley) Y. Teng et K. Tanabe) and the progenitor of Japanese pears coming from China. Cultivars of P. ussuriensis Maxim. were clustered together with one clone of P. hondoensis Nakai et Kikuchi, a relative species of P. ussuriensis. Cultivars of P. communis L. and other Occidental species formed three independent groups and were distant from most Asian pears, except for P. betulaefolia Bge.

Anthocyanins from Chinese Bayberry Extract Protect β Cells from Oxidative Stress-Mediated Injury via HO-1 Upregulation

Oxidative stress plays a pivotal role during the islet transplantation procedure, and antioxidant supplementation may protect grafts against oxidative injury. Chinese bayberry is one of six Myrica species native to China, and we demonstrated here that anthocyanins from Chinese bayberry extract (CBE) protect pancreatic β cells (INS-1) against hydrogen peroxide (H(2)O(2))-induced necrosis and apoptosis. Anthocyanins time- and dose-dependently upregulated heme oxygenase-1 (HO-1) gene expression in β cells and primary islets. HO-1 knockdown increased H(2)O(2)-induced cell death and attenuated the cytoprotective effect of anthocyanins. Anthocyanin treatment activated ERK1/2 and PI3K/Akt signaling, and ERK1/2 and PI3K inhibitors partially attenuated anthocyanin-mediated induction of HO-1. Additionally, β cells pretreated with anthocyanins displayed a decreased extent of apoptosis after transplantation. In summary, these results suggest that anthocyanins in CBE protect β cells from H(2)O(2)-induced cell injury via ERK1/2- and PI3K/Akt-mediated HO-1 upregulation.

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.

A 13-lipoxygenase, TomloxC, is essential for synthesis of C5 flavour volatiles in tomato

C5 volatile compounds, derived from fatty acids, are among the most important contributors to consumer liking of fresh tomatoes. Despite their important roles in flavour, the genes responsible for C5 volatile synthesis have yet to be identified. This work shows that their synthesis is catalysed in part by a 13-lipoxygenase (LOX), TomloxC, the same enzyme responsible for synthesis of C6 volatiles. C5 synthesis is independent of hydroperoxide lyase (HPL); moreover, HPL knockdown significantly increased C5 volatile synthesis. This LOX-dependent, HPL-independent pathway functions in both fruits and leaves. Synthesis of C5 volatiles increases in leaves following mechanical wounding but does not increase in response to infection with Xanthomonas campestris pv. vesicatoria. Large reductions in C5 and C6 volatiles in antisense TomloxC knockdown plants were observed but those reductions did not alter the development of disease symptoms, indicating that these volatiles do not have an important defensive function against this bacterial pathogen.

Lipoxygenase gene expression in ripening kiwifruit in relation to ethylene and aroma production.

During postharvest ripening of kiwifruit [ Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson var. deliciosa cv. Bruno] at 20 degrees C, six lipoxygenase (LOX) genes exhibited different expression patterns. AdLox1 and AdLox5 were up-regulated during ripening, and transcript accumulation was delayed by 1-methylcyclopropene (1-MCP), whereas AdLox2, AdLox3, AdLox4, and AdLox6 were down-regulated with ripening. Levels of two volatiles arising from the LOX pathway, that is, n-hexanal and (E)-2-hexenal, were highest after harvest and declined during ripening at 20 degrees C, whereas the characteristic kiwifruit esters ethyl and methyl butanoate levels increased late in the ripening process. Individual fatty acid concentrations underwent little change during ripening, with linoleic (LA) and linolenic (LeA) acids constituting about 40% of the total. Application of LA and LeA to kiwifruit flesh disks promoted LOX activity and n-hexanal and (E)-2-hexenal generation, whereas inhibitors of LOX, n-propyl gallate (n-PG) and nordihydroguariaretic acid (NDGA), caused a parallel reduction in enzyme activity and in the production of C6 aldehydes. The six LOX genes showed different sensitivities to the LOX substrates and inhibitors. The ethylene up-regulated genes AdLox1 and AdLox5 were induced by LA and LeA and inhibited by n-PG and NDGA. Of the LOX genes that were down-regulated by ethylene, only AdLox4 and AdLox6 were stimulated in response to the substrates and retarded by the inhibitors. The possible roles of the six LOX genes in kiwifruit ripening and aroma development are discussed.

Genomic characterization of putative allergen genes in peach/almond and their synteny with apple

BackgroundFruits from several species of the Rosaceae family are reported to cause allergic reactions in certain populations. The allergens identified belong to mainly four protein families: pathogenesis related 10 proteins, thaumatin-like proteins, lipid transfer proteins and profilins. These families of putative allergen genes in apple (Mal d 1 to 4) have been mapped on linkage maps and subsequent genetic study on allelic diversity and hypoallergenic traits has been carried out recently. In peach (Prunus persica), these allergen gene families are denoted as Pru p 1 to 4 and for almond (Prunus dulcis)Pru du 1 to 4. Genetic analysis using current molecular tools may be helpful to establish the cause of allergenicity differences observed among different peach cultivars. This study was to characterize putative peach allergen genes for their genomic sequences and linkage map positions, and to compare them with previously characterized homologous genes in apple (Malus domestica).ResultsEight Pru p/du 1 genes were identified, four of which were new. All the Pru p/du 1 genes were mapped in a single bin on the top of linkage group 1 (G1). Five Pru p/du 2 genes were mapped on four different linkage groups, two very similar Pru p/du 2.01 genes (A and B) were on G3, Pru p/du 2.02 on G7,Pru p/du 2.03 on G8 and Pru p/du 2.04 on G1. There were differences in the intron and exon structure in these Pru p/du 2 genes and in their amino acid composition. Three Pru p/du 3 genes (3.01–3.03) containing an intron and a mini exon of 10 nt were mapped in a cluster on G6. Two Pru p/du 4 genes (Pru p/du 4.01 and 4.02) were located on G1 and G7, respectively. The Pru p/du 1 cluster on G1 aligned to the Mal d 1 clusters on LG16; Pru p/du 2.01A and B on G3 to Mal d 2.01A and B on LG9; the Pru p/du 3 cluster on G6 to Mal d 3.01 on LG12; Pru p/du 4.01 on G1 to Mal d 4.03 on LG2; and Pru p/du 4.02 on G7 to Mal d 4.02 on LG2.ConclusionA total of 18 putative peach/almond allergen genes have been mapped on five linkage groups. Their positions confirm the high macro-synteny between peach/almond and apple. The insight gained will help to identify key genes causing differences in allergenicity among different cultivars of peach and other Prunus species.