Wei Heng

Molecular Analysis of the Processes of Surface Brown Spot (SBS) Formation in Pear Fruit (Pyrus bretschneideri Rehd. cv. Dangshansuli) by De Novo Transcriptome Assembly

Browning disorder, which usually occurs post-harvest in pears subjected to long-term storage, can cause browning of the pear flesh and/or core. In 2011, investigators in China found a novel type of brown spot (designated as surface brown spot, SBS) in pre-harvest ‘Dangshansuli’ pears (Pyrus bretschneideri Rehd.). SBS has a large impact on the exterior quality of the pears. Interestingly, the brown coloration was only found on the peel and not the flesh or the core. In this paper, de novo transcriptome analysis of the exocarp of pears with SBS using Illumina sequencing showed that SBS up-regulated the expression of genes related to oxidative phosphorylation, phenolic compound synthesis and polyphenoloxidase (PPO), and SBS was associated with inhibition of primary and secondary metabolism genes. Ca2+-sensor proteins might be involved in the signal transduction that occurs during the process of SBS formation, and this signaling is likely to be regulated by H2O2, abscisic acid (ABA) and gibberellic acid (GA3). Phytohormone and mineral element analyses confirmed that GA3, ABA, H2O2 and Ca2+ contribute to SBS formation. In addition to the seasonal characteristics, low levels of O2 and Ca2+ in the fruit are potential causes of the browning response due to exposure to oxidative stress, oxidative-reductive imbalance and the accumulation of reactive oxygen species (ROS), which affected the membrane integrity. Disruption of the membranes allows for PPO and phenolic compounds to come into contact, and the phenolic compounds are oxidized to form the browning pigments.

Differentially expressed genes related to the formation of russet fruit skin in a mutant of ‘Dangshansuli’ pear (Pyrus bretchnederi Rehd.) determined by suppression subtractive hybridization

The mature fruit skin of ‘Dangshansuli’ pear (Pyrus bretchnederi Rehd.) is yellow-green, but the fruit of the mutant, ‘Xiusu’, is russetted. To understand the mechanism of russet formation, the suppression subtractive hybridization libraries were used to screen for differentially expressed genes between the wild type pear and the mutant. Three hundred and twenty nine and 366 unigenes were obtained from 934 and 929 colonies in the forward and reverse libraries, respectively. The results indicated that phenylpropanoid metabolism, ethylene metabolism and secondary metabolism might be involved in the russet formation process, and fruit skin is likely to be regulated by lignin synthesis, polyamine and H2O2 signalling. Furthermore, the accumulation of lignin and H2O2, together with up-regulated POD enzyme genes expression in the exocarp of ‘Xiusu’ compare with ‘Dangshansuli’ pear control, provided a comprehensive transcriptomics view on the coordination of the russet formation of mutant pear.

Characterization and expression analysis of PbEXP genes in the epidermis of pear ( Pyrus bretschneideri Rehd.)

In the present study, “Dangshansuli” (wild type, WT) and its russet mutant “Xiusu” (mutant type, MT) were used to characterize the PbEXPs in “Dangshansuli” as well as the role of IAA in mediating the expression of PbEXPss. Forty-one expansins were identified in WT and divided into EXPA, EXPB, EXLA and EXLB families. In the same EXP family, they shared similar exon–intron structure and phylogenetic characters. Eight of 41 expansin genes, screened from the transcriptome of WT and its russet mutant “Xiusu”, exhibited differential expression patterns in the epidermis of both WT and MT with the development of pear fruit. In addition, the correlation coefficients between IAA content and expression of expansins of PbEXPB3b and PbEXP4a in MT were 0.763 and 0.762, respectively. After treatments with IAA, NPA and AVG, the expression levels of PbEXPB3b and PbEXP4a changed significantly 15, 30, or 45 days after full bloom (DAFB). The longitudinal and vertical diameters of the epidermis of WT and MT at different stages were measured. We found that high IAA levels enhanced the expression of PbEXPB3b and PbEXP4a in epidermis of MT at the russet turning stage of 75–100 DAFB, which promoted cell expansion and microcrack occurrence and led to russet on the pear fruits.

Relationship between H2O2 in Polyamine Metabolism and Lignin in the Exocarp of a Russet Mutant of ‘Dangshansuli’ Pear (Pyrus bretschneideri Rehd.)

The fruit of the ‘Dangshansuli’ pear has a greenish yellow skin, whereas its mutant, the ‘Xiusu’ pear, has a russet skin, which represents a genetic variation. It has been demonstrated that the formation of russet fruit in the ‘Xiusu’ pear is related to lignin accumulation in skin exocarp cells. In this study, we localized hydrogen peroxide (H2O2) to the cell wall using transmission electron microscopy (TEM) and quantified the concentrations of H2O2 and polyamines. In addition, the expression levels of genes involved in polyamine biosynthesis were measured in the exocarps of samples of young fruits of ‘Dangshansuli’, ‘Xiusu’, ‘Xiusu’ treated with methylglyoxal bis(guanylhydrazone), and ‘Xiusu’ treated with ethephon. The results obtained could explain the mechanism by which H2O2 participates in polyamine metabolism in the lignification of exocarp cells in the russet fruit mutant. The TEM results showed that free H2O2 is present near the cell wall, where lignin is primarily synthesized, and the H2O2 concentration was highly positively correlated with the lignin concentration. Although H2O2 related to lignification showed no significant correlation with the putrescine or spermine concentration, it was highly positively correlated with the spermidine (Spd) concentration. Additionally, the Spd concentration was significantly positively correlated with altered expression of the polyamine oxidase gene (PbPAO). Taken together, these results have demonstrated that H2O2 involved in lignification originates from the oxidation of Spd by the enzyme PAO, with high expression of the PbPAO gene, which suggests that H2O2 from polyamine metabolism affects lignification in the exocarp of the russet mutant pear.

Transcriptome sequencing analysis of two different genotypes of Asian pear reveals potential drought stress genes

Abstract‘Huangguan’ (HG) and ‘Whangkeumbae’ (HK) pears are two important Asian pear cultivars in China and South Korea, respectively. In practical production, ‘Huangguan’ pear is a drought-tolerant genotype while ‘Whangkeumbae’ pear is drought-sensitive. To uncover the mechanisms underlying pear tree tolerance to drought stress, a comprehensive transcriptome analysis was performed in this study. The results revealed a total of 1185 and 1667 differently expressed genes (DEGs) between control and treated plants of HG and HK pear, respectively. KEGG pathways enrichment analysis revealed that the DEGs were involved in the metabolism and signal transduction of various phytohormones. In HG, the DEGs annotated as ABA, gibberellic acid (GA), and salicylic acid (SA) were all upregulated, while those DEGs annotated as jasmonic acid (JA) were upregulated or downregulated. In HK, the DEGs annotated as ABA and JA were both upregulated or downregulated, but there were no DEGs annotated as GA and SA. In addition, there were 743 DEGs expressed in HG, but not in HK. Among them, there were 288 DEGs whose absolute values of log2(fold-change ratio) were greater than 2. Eight of the 288 DEGs were selected randomly for validating the reproducibility and accuracy of the transcriptome RNA-Seq data by using quantitative-PCR. Our results will be helpful for breeding drought-tolerant pear cultivars.

Identification of a suspected pathogen of Chinese pear (Pyrus bretshneideri Redh.) ‘Dangshansuli’ and fungicide screening

Summary We have identified a pathogenic fungus that is responsible for fruit rot and leaf abscission in Chinese pear (Pyrus bretshneideri Rehd., cv. Dangshansuli). Six isolates of the fungus were obtained from diseased fruit and leaves and purified from single conidiospore cultures grown on potato-dextrose agar medium. The internal transcribed spacer (ITS) region of their ribosomal DNA was amplified, cloned, and sequenced. The results indicated that the rDNA-ITS sequences of all six isolates were identical to those of the anthracnose pathogens isolated from Taiwanese jujube (FJ233185) and Japanese pear (AB219012). This indicates that the disease observed in Chinese ‘Dangshangsuli’ pear was caused by the pathogen Colletotrichum gloeosporioides Penz, and/or its teleomorph, Glomerella cingulata. To determine an appropriate control strategy, 21 commercial fungicides were screened, and nine were found to be effective at inhibiting mycelial growth and the germination of conidia using controlled in vitro tests. Tests performed in 2009 and in 2010 revealed that three of these fungicides, Mancozeb, Bilu No. 2, and Triram, were efficient at controlling the disease. These results provide evidence for the most appropriate fungicides to use to control anthracnose disease in Chinese ‘Dangshansuli’ pear.