Xue-ren Yin

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.

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.

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.

Ethylene-responsive transcription factors interact with promoters of ADH and PDC involved in persimmon (Diospyros kaki) fruit de-astringency

The persimmon fruit is a particularly good model for studying fruit response to hypoxia, in particular, the hypoxia-response ERF (HRE) genes. An anaerobic environment reduces fruit astringency by converting soluble condensed tannins (SCTs) into an insoluble form. Although the physiology of de-astringency has been widely studied, its molecular control is poorly understood. Both CO2 and ethylene treatments efficiently removed the astringency from ‘Mopan’ persimmon fruit, as indicated by a decrease in SCTs. Acetaldehyde, the putative agent for causing de-astringency, accumulated during these treatments, as did activities of the key enzymes of acetaldehyde synthesis, alcohol dehydrogenase (ADH), and pyruvate decarboxylase (PDC). Eight DkADH and DkPDC genes were isolated, and three candidates for a role in de-astringency, DkADH1, DkPDC1, and DkPDC2, were characterized by transcriptional analysis in different tissues. The significance of these specific isoforms was confirmed by principal component analysis. Transient expression in leaf tissue showed that DkPDC2 decreased SCTs. Interactions of six hypoxia-responsive ERF genes and target promoters were tested in transient assays. The results indicated that two hypoxia-responsive ERF genes, DkERF9 and DkERF10, were involved in separately regulating the DkPDC2 and DkADH1 promoters. It is proposed that a DkERF–DkADH/DkPDC cascade is involved in regulating persimmon de-astringency.

EjAP2-1, an AP2/ERF gene, is a novel regulator of fruit lignification induced by chilling injury, via interaction with EjMYB transcription factors.

Lignin biosynthesis is regulated by many transcription factors, such as those of the MYB and NAC families. However, the roles of AP2/ERF transcription factors in lignin biosynthesis have been rarely investigated. Eighteen EjAP2/ERF genes were isolated from loquat fruit (Eriobotrya japonica), which undergoes postharvest lignification during low temperature storage. Among these, expression of EjAP2-1, a transcriptional repressor, was negatively correlated with fruit lignification. The dual-luciferase assay indicated that EjAP2-1 could trans-repress activities of promoters of lignin biosynthesis genes from both Arabidopsis and loquat. However, EjAP2-1 did not interact with the target promoters (Ej4CL1). Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays indicated protein-protein interactions between EjAP2-1 and lignin biosynthesis-related EjMYB1 and EjMYB2. Furthermore, repression effects on the Ej4CL1 promoter were observed with the combination of EjAP2-1 and EjMYB1 or EjMYB2, while EjAP2-1 with the EAR motif mutated (mEjAP2-1) lost such repression, although mEjAP2-1 still interacted with EjMYB protein. Based on these results, it is proposed that EjAP2-1 is an indirect transcriptional repressor on lignin biosynthesis, and the repression effects were manifested by EAR motifs and were conducted via protein-protein interaction with EjMYBs.

Expression of ethylene response genes during persimmon fruit astringency removal

Thirteen ethylene signaling related genes were isolated and studied during ripening of non-astringent ‘Yangfeng’ and astringent ‘Mopan’ persimmon fruit. Some of these genes were characterized as ethylene responsive. Treatments, including ethylene and CO2, had different effects on persimmon ripening, but overlapping roles in astringency removal, such as increasing the reduction in levels of soluble tannins. DkERS1, DkETR2, and DkERF8, may participate in persimmon fruit ripening and softening. The expression patterns of DkETR2, DkERF4, and DkERF5 had significant correlations with decreases in soluble tannins in ‘Mopan’ persimmon fruit, suggesting that these genes might be key components in persimmon fruit astringency removal and be the linkage between different treatments, while DkERF1 and DkERF6 may be specifically involved in CO2 induced astringency removal. The possible roles of ethylene signaling genes in persimmon fruit astringency removal are discussed.

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.