Shenchun Qu

Expression of pathogenesis related genes in response to salicylic acid, methyl jasmonate and 1-aminocyclopropane-1-carboxylic acid in Malus hupehensis (Pamp.) Rehd

BackgroundMany studies have been done to find out the molecular mechanism of systemic acquired resistance (SAR) in plants in the past several decades. Numbers of researches have been carried out in the model plants such as arabidopsis, tobacco, rice and so on, however, with little work done in woody plants especially in fruit trees such as apple. Components of the pathway of SAR seem to be extremely conserved in the variety of species. Malus hupehensis, which is origin in China, is strong resistance with rootstock. In the study, we attempted to make the expression pattern of pathogenesis related (PR) genes which were downstream components of the SAR pathway in response to salicylic acid(SA), methyl jasmonate(MeJA) and 1-aminocyclopropane-1-carboxylic acid(ACC) in Malus hupehensis.FindingsIn order to analyze the expression pattern, the partial sequence of three PR genes from Malus hupehensis, MhPR1, MhPR5 and MhPR8 was isolated. These three PR genes were induced by SA, MeJA and ACC. However, MhPR1, MhPR5 and MhPR8 performed a distinct pattern of expression in different plant organs. MhPR5 and MhPR8 were basal expression in leaves, stems and roots, and MhPR1 was basal expression only in stems. The expression of MhPR1, MhPR5 and MhPR8 was enhanced during the first 48 h post-induced with SA, MeJA and ACC.ConclusionsThe results showed that a distinct pattern of expression of PR genes in Malus hupehensis which differed from the previous reports on model plants arabidopsis, tobacco and rice. MhPR1, MhPR5 and MhPR8 were induced by SA, MeJA and ACC, which were regarded as the marker genes in the SAR response in Malus hupehensis. In contrast with herbal plants, there could be specific signal pathway in response to SA, JA and ET for woody plants.

Genomic variants of genes associated with three horticultural traits in apple revealed by genome re-sequencing.

The apple (Malus × domestica Borkh.) cultivar ‘Su Shuai’ exhibits greater disease resistance, shorter internodes and lighter fruit flavor compared with its parents ‘Golden Delicious’ and ‘Indo’. To obtain a comprehensive overview of the sequence variation in these three horticultural traits, the genomes of ‘Su Shuai’ and ‘Indo’ were resequenced using next-generation sequencing and compared to the genome of ‘Golden Delicious’. A wide range of genetic variations were detected, including 2 454 406 and 18 749 349 single nucleotide polymorphism (SNP) and 59 547 and 50 143 structural variants (SVs) in the ‘Indo’ and ‘Su Shuai’ genomes, respectively. Among the SVs in ‘Su Shuai’, 17 genes related to disease resistance, 10 genes related to Gibberellin (GA) and 19 genes associated with fruit flavor were identified. The expression patterns of eight of the SV genes were examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results of this study illustrate the genomic variation in these cultivars and provide evidence for a genetic basis for the horticultural traits of disease resistance, short internodes and lighter flavor exhibited in these cultivars. These results provide a genetic basis for the phenotypic characteristics of ‘Su Shuai’ and, as such, these SVs could serve as gene-specific molecular markers in maker-assisted breeding of apples.

Analysis of expressed sequence tags from grapevine flower and fruit and development of simple sequence repeat markers

A total of 6,230 EST sequences were produced from 7,561 clones in a cDNA library generated from grapevine (Vitis vinifera cv. ‘Summer Black’) flower and fruit tissues in this study. After cluster and assembly analysis of the datasets, 3,582 unigenes (GenBank accession numbers GW836604–GW840185) were established, among which 381 were new grapevine EST sequences. Out of the 381 new ESTs, 289 could be mapped on the 19 grapevine chromosomes. 913 unique ESTs with known or putative functions were assigned to 11 putative cellular roles. 540 potentially workable grapevine EST-SSRs were developed from 3,582 unigenes and about 42.6% of these unigenes were identified as true-to-type SSR loci and could amplify polymorphic bands from 22 individual plants of V. vinifera L, indicating that grapevine EST datasets are a valuable source for the development of functional simple sequence repeat (SSR) markers.

Overexpression of the Malus hupehensis MhTGA2 Gene, a Novel bZIP Transcription Factor for Increased Tolerance to Salt and Osmotic Stress in Transgenic Tobacco

Basic leucine zipper (bZIP) proteins form one of the largest families of transcription factors, and their individual contributions in a particular regulatory network remain difficult to assess. Here, we identified and functionally characterized the Malus hupehensis bZIP transcription factor designated MhTGA2 from M. hupehensis leaves treated with salicylic acid (SA). The MhTGA2 protein was localized in the nucleus. The expression of MhTGA2 was higher in leaves than in stems and roots. This gene was significantly induced by SA, methyl jasmonate, and 1-aminocyclopropane-1-carboxylic acid and weakly induced by abscisic acid. The gene was clearly induced by low temperature (4°C) and NaCl and weakly induced by polyethylene glycol but not induced by apple ring spot pathogens. Overexpression of the MhTGA2 gene in transgenic tobacco plants conferred enhanced resistance to NaCl at the stage of seed germination and in seedlings, and it conferred resistance to mannitol at the stage of seed germination. Furthermore, overexpression of MhTGA2 led to higher expression levels of pathogenesis-related proteins and osmotic-stress-related genes in transgenic tobacco than in wild-type plants. These results suggest that the MhTGA2 transcription factor might be active in systemic acquired resistance and might function as a possible regulator of enhanced environmental-stress tolerance. MhTGA2 is thus a potentially constructive candidate gene for engineering salt and osmotic stress tolerance in cultivated plants.

Malus hupehensis miR168 Targets to ARGONAUTE1 and Contributes to the Resistance against Botryosphaeria dothidea Infection by Altering Defense Responses

MicroRNA (miRNA)-mediated post-transcriptional regulation plays a fundamental role in various plant physiological processes, including responses to pathogens. MicroRNA168 has been implicated as an essential factor of miRNA pathways by targeting ARGONAUTE1 (AGO1), the core component of the RNA-induced silencing complex (RISC). A fluctuation in AGO1 expression influences various plant-pathogen interactions, and the homeostasis of AGO1 and miR168 accumulation is maintained by a complicated feedback regulatory loop. In this study, the connection between miR168 and the resistance of Malus hupehensis to Botryosphaeria dothidea is revealed. The induction of both the mature miR168 and its precursor in plants subjected to B. dothidea infection indicate the transcriptional activation of MIR168a. MIR168a promoter analysis demonstrates that the promoter can be activated by B. dothidea and salicylic acid (SA). However, the direct target of miR168, M. hupehensis ARGONAUTE1 (MhAGO1), is shown to be induced under the infection. Expression and transcription activity analysis demonstrate the transcriptional activation and the post-transcriptional suppression of MhAGO1 in response to B. dothidea infection. By inhibiting reactive oxygen species (ROS) production and enhancing SA-mediated defense responses, miR168a delays the symptom development of leaves inoculated with B. dothidea and impedes the pathogen growth, while MhAGO1 is found to have the opposite effects. Collectively, these findings suggest that the expression of miR168 and MhAGO1 in M. hupehensis in response to B. dothidea infection is regulated by a complicated mechanism. Targeting to MhAGO1, a negative regulator, miR168 plays a positive role in the resistance by alterations in diverse defense responses.

Genome-Wide Detection of SNP and SV Variations to Reveal Early Ripening-Related Genes in Grape.

Early ripening in grape (Vitis vinifera L.) is a crucial agronomic trait. The fruits of the grape line ‘Summer Black’ (SBBM), which contains a bud mutation, can be harvested approximately one week earlier than the ‘Summer Black’ (SBC)control. To investigate the molecular mechanism of the bud mutation related to early ripening, we detected genome-wide genetic variations based on re-sequencing. In total, 3,692,777 single nucleotide polymorphisms (SNPs) and 81,223 structure variations (SVs) in the SBC genome and 3,823,464 SNPs and 85,801 SVs in the SBBM genome were detected compared with the reference grape sequence. Of these, 635 SBC-specific genes and 665 SBBM-specific genes were screened. Ripening and colour-associated unigenes with non-synonymous mutations (NS), SVs or frame-shift mutations (F) were analysed. The results showed that 90 unigenes in SBC, 76 unigenes in SBBM and 13 genes that mapped to large fragment indels were filtered. The expression patterns of eight genes were confirmed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR).The re-sequencing data showed that 635 SBC-specific genes and 665 SBBM-specific genes associated with early ripening were screened. Among these, NCED6 expression appears to be related to NCED1 and is involved in ABA biosynthesis in grape, which might play a role in the onset of anthocyanin accumulation. The SEP and ERF genes probably play roles in ethylene response.

Identification and expression analysis of WRKY transcription factor genes in response to fungal pathogen and hormone treatments in apple (Malus domestica)

The WRKY family, a large family of transcription factors (TFs) found in higher plants, plays central roles in many aspects of biological processes and adaption to environment. However, little information is available on this family in apple (Malus domestica). In the present study, a total of 119 candidate WRKY genes in apple genome were identified and classified into three main groups (Group I–III) based on the structure of the conserved domains. Each group or subgroup showed similar exon–intron structures and motif compositions. The evolution analysis showed that 44 MdWRKY genes clustered into 20 intensive regions (<100 kb) and 78 MdWRKY formed 85 pairs of collinear relationships, suggesting that both tandem and segmental duplications played an important role in the evolution and diversification of the WRKY gene family in apple. Furthermore, the expression of the MdWRKY genes in apple leaves in response to biotic stress (Alternaria alternate) and hormone treatments [salicylic acid (SA), methyl jasmonate (MeJA) and ethephon] was examined by using RNA-seq and qRT-PCR. The results showed that 63 MdWRKY genes had differential expression in their transcript abundance in response to Alternaria alternata apple pathotype infection. Moreover, most pathogen responsive MdWRKY genes were also changed significantly when apple leaves were treated by SA, MeJA or ethephon plant growth regulations, suggesting an interaction between SA, JA and ethylene (Eth) hormone signaling under biotic stress. This work may provide the basis for future studies of the genetic modification of WRKY genes for pathogen resistance in apple.

Overexpression of the MhTGA2 gene from crab apple ( Malus hupehensis ) confers increased tolerance to salt stress in transgenic apple ( Malus domestica )

Leaves of Nagafu No. 6 were transformed by the Agrobacterium tumefaciens-mediated method, and 158 hyg-resistant buds were obtained. Using fresh leaf tissue, seven positive lines were obtained by polymerase chain reaction (PCR) with total DNA, and four by reverse transcriptase–polymerase chain reaction (RT–PCR) with total RNA. The results showed that the MhTGA2 gene was successfully transformed into Nagafu No. 6 plants and expressed. At 4 months after transplanting, photosynthetic parameters of young leaves were determined using transgenic and control plants prior to the determination of leaf surface characteristics (epidermal structure and stomatal distribution) by scanning electronic microscopy. The results showed that the net photosynthesis rate, the instantaneous carboxylation rate and stomatal conductance of transgenic plants were higher than the control, whereas the internal carbon dioxide (CO2) concentrations of the transgenic plants were lower. Under non-saline conditions, the stomata of the transgenic plants were better distributed than in the control. Under saline conditions, the epidermal structure of the control leaves was severely hypohydrated and the quantity of stomata was significantly decreased. Conversely, the epidermal structure of transgenic leaves was not significantly altered, indicating salt-stress tolerance. The overall results suggested that MhTGA2 may play a crucial role in the response to salt stress in Nagafu No. 6.