BackgroundDehydrins (DHNs) protect plant cells from desiccation damage during environmental stress, and also participate in host resistance to various pathogens. In this study, we aimed to identify and characterize the DHN gene families from Vitis vinifera and wild V. yeshanensis, which is tolerant to both drought and cold, and moderately resistant to powdery mildew.ResultsFour DHN genes were identified in both V. vinifera and V. yeshanensis, which shared a high sequence identity between the two species but little homology between the genes themselves. These genes were designated DHN1, DHN2, DHN3 and DHN4. All four of the DHN proteins were highly hydrophilic and were predicted to be intrinsically disordered, but they differed in their isoelectric points, kinase selectivities and number of functional motifs. Also, the expression profiles of each gene differed appreciably from one another. Grapevine DHN1 was not expressed in vegetative tissues under normal growth conditions, but was induced by drought, cold, heat, embryogenesis, as well as the application of abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA). It was expressed earlier in V. yeshanensis under drought conditions than in V. vinifera, and also exhibited a second round of up-regulation in V. yeshanensis following inoculation with Erysiphe necator, which was not apparent in V. vinifera. Like DHN1, DHN2 was induced by cold, heat, embryogenesis and ABA; however, it exhibited no responsiveness to drought, E. necator infection, SA or MeJA, and was also expressed constitutively in vegetative tissues under normal growth conditions. Conversely, DHN3 was only expressed during seed development at extremely low levels, and DHN4 was expressed specifically during late embryogenesis. Neither DHN3 nor DHN4 exhibited responsiveness to any of the treatments carried out in this study. Interestingly, the presence of particular cis-elements within the promoter regions of each gene was positively correlated with their expression profiles.ConclusionsThe grapevine DHN family comprises four divergent members. While it is likely that their functions overlap to some extent, it seems that DHN1 provides the main stress-responsive function. In addition, our results suggest a close relationship between expression patterns, physicochemical properties, and cis-regulatory elements in the promoter regions of the DHN genes.
SummaryUbiquitin-mediated regulation responds rapidly to specific stimuli; this rapidity is particularly important for defense responses to pathogen attack. Here, we investigated the role of the E3 ubiquitin ligase Erysiphe necator-induced RING finger protein 1 (EIRP1) in the defense response of Chinese wild grapevine Vitis pseudoreticulata.The regulatory function of E3 ubiquitin ligase EIRP1 was investigated using molecular, genetic and biochemical approaches.EIRP1 encodes a C3HC4-type Really Interesting New Gene (RING) finger protein that harbors E3 ligase activity. This activity requires the conserved RING domain, and VpWRKY11 also interacts with EIRP1 through the RING domain. VpWRKY11 localizes to the nucleus and activates W-box-dependent transcription in planta. EIRP1 targeted VpWRKY11 in vivo, resulting in VpWRKY11 degradation. The expression of EIRP1 and VpWRKY11 responds rapidly to powdery mildew in Vitis pseudoreticulata grapevine; also, overexpression of EIRP1 in Arabidopsis confers enhanced resistance to the pathogens Golovinomyces cichoracearum and Pseudomonas syringae pv tomato DC3000.Our data suggest that the EIRP1 E3 ligase positively regulates plant disease resistance by mediating proteolysis of the negative regulator VpWRKY11 via degradation by the 26S proteasome.
Several matrix-attachment regions (MARs) from animals have been shown to block interactions between an enhancer and promoter when situated between the two. Since a similar function for plant MARs has not been discerned, we tested the Zea mays ADH1 5' MAR, Nicotiana tabacum Rb7 3' MAR and a transformation booster sequence (TBS) MAR from Petunia hybrida for their ability to impede enhancer-promoter interactions in Arabidopsis thaliana. Stable transgenic lines containing vectors in which one of the three MAR elements or a 4 kb control sequence were interposed between the cauliflower mosaic virus 35S enhancer and a flower-specific AGAMOUS second intron-derived promoter (AGIP)::beta-glucuronidase (GUS) fusion were assayed for GUS expression in vegetative tissues. We demonstrate that the TBS MAR element, but not the ADH1 or Rb7 MARs, is able to block interactions between the 35S enhancer and AGIP without compromising the function of either with elements from which they are not insulated.
Bagged fruits of green apple cultivar “Granny Smith” have been found to turn cardinal red after debagging during fruit-ripening in the Loess Plateau region of China. To understand this phenomenon at post-transcriptional level, we have investigated the roles of microRNAs (miRNAs) in response to debagging. Three small RNA libraries were primarily constructed from peels of “Granny Smith” apples subjected to bagging followed by sunlight re-exposure treatments (0, 6 h, 1 day) (debagging), and from peels of apples without any bagging treatments (0, 6 h, 1 day). 201 known miRNAs belonging to 43 miRNA families and 220 novel miRNAs were identified via high-throughput sequencing. Some miRNAs were found to be differentially expressed after debagging, which indicated that miRNAs affected anthocyanin accumulation through their target genes in mature apple. To further explore the effect of debagging on miRNAs regulating the expression of anthocyanin regulatory genes, four miRNAs and their target genes regulating anthocyanin accumulation, miR156, miR828, miR858, and miR5072, were compared between green cultivar “Granny Smith” and red cultivar “Starkrimson.” Results showed that mdm-miR828 and mdm-miR858 regulated anthocyanin contents in both apple cultivars, while mdm-miR156 only affected anthocyanin accumulation in “Granny Smith,” and miR5072 affected anthocyanin accumulation in “Starkrimson.” Additional analysis of gene ontology for the differentially expressed miRNAs after debagging treatments and their predicted target genes showed that they were involved in photo-protective response after debagging from 0 h to 1 day; they might play important roles in fruit development and adaptation to high light stress.
Downy mildew, caused by the oomycete Plasmopara viticola, is a serious fungal disease in the cultivated European grapevines (Vitis vinifera L.). The class 10 of pathogenesis-related (PR) genes in grapevine leaves was reported to be accumulated at mRNA level in response to P. viticola infection. To elucidate the functional roles of PR10 genes during plant-pathogen interactions, a PR10 gene from a fungal-resistant accession of Chinese wild Vitis pseudoreticulata (designated VpPR10.2) was isolated and showed high homology to PR10.2 from susceptible V. vinifera (designated VvPR10.2). Comparative analysis displayed that there were significant differences in the patterns of gene expression between the PR10 genes from the two host species. VpPR10.2 was induced with high level in leaves infected by P. viticola, while VvPR10.2 showed a low response to this inoculation. Recombinant VpPR10.2 protein showed DNase activity against host genomic DNA and RNase activity against yeast total RNA in vitro. Meanwhile, recombinant VpPR10.2 protein inhibited the growth of tobacco fungus Alternaria alternata and over-expression of VpPR10.2 in susceptible V. vinifera enhanced the host resistance to P. viticola. The results from subcellular localization analysis showed that VpPR10.2 proteins were distributed dynamically inside or outside of host cell. Moreover, they were found in haustorium of P. viticola and nucleus of host cell which was associated with a nucleus collapse at 10 days post-inoculation. Taken together, these results suggested that VpPR10.2 might play an important role in host plant defense against P. viticola infection.
BackgroundFruit color in apple (Malus domestica Borkh.) is ascribed mainly to the accumulation of anthocyanin pigments, and is an important trait for determining fruit market acceptance. Bagging is a commonly used treatment to enhance the red pigmentation in apple skin. The MdMYB1 transcription factor gene plays an important role in the biosynthesis of anthocyanin in apple after bag removal, but little is known about how MdMYB1 transcription is regulated.ResultsIn this study, we investigated pigmentation in the non-red skinned cultivars ‘Granny Smith’ and ‘Golden Delicious’ after bag removal. The fruit skins of the two cultivars showed red/pink pigmentation after bag treatment. Transcript levels of MdMYB1, the master regulator of anthocyanin biosynthesis in apple, increased, and showed a correlation with anthocyanin content in both cultivars after bag removal. The MdMYB1 genomic sequences were compared in the two cultivars, which showed that the green-fruited cultivar ‘Granny Smith’ harbors the MdMYB1–1 and MdMYB1–2 alleles, while the yellow-fruited cultivar ‘Golden Delicious’ harbors only MdMYB1–2. A comparison of methylation levels in the 2 kb region upstream of the MdMYB1 ATG between the bag-treated fruits after removal from the bags and the unbagged fruits showed a correlation between hypomethylation and the red-skin phenotype in ‘Granny Smith’. Moreover, ‘Granny Smith’ fruits responded to treatment with 5-aza-2′-deoxycytidine, an inducer of DNA demethylation. An investigation of the MdMYB1 promoter in ‘Granny Smith’ showed reduced methylation in the regions − 2026 to − 1870 bp, − 1898 to − 1633 bp, and − 541 to − 435 bp after bag removal and 5-aza-2′-deoxycytidine treatments.ConclusionsDifferences in anthocyanin levels between ‘Granny Smith’ and ‘Golden Delicious’ can be explained by differential accumulation of MdMYB1-specific mRNA. Different levels of MdMYB1 transcripts in the two cultivars are associated with methylation levels in the promoter region. Hypomethylation of the MdMYB1 promoter is correlated with the formation of red pigmentation in ‘Granny Smith’ fruit skins. As a result, red pigmentation in Granny Smith’ was more intense than in ‘Golden Delicious’ fruits after bag removal.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1320-7) contains supplementary material, which is available to authorized users.
Proanthocyanidins (PAs) have antioxidant properties and are beneficial to human health. The fruit of apple (Malus × domestica Borkh.), especially the peel, is rich in various flavonoids, such as PAs, and thus is an important source of dietary antioxidants. Previous research on the regulation of PAs in apple has mainly focussed on the transcription level, whereas studies conducted at the post-transcriptional level are relatively rare. In this study, we investigated the function of mdm-miR858, a miRNA with multiple functions in plant development, in the peel of apple fruit. We showed that mdm-miR858 negatively regulated PA accumulation by targeting MdMYB9/11/12 in the peel. During fruit development, mdm-miR858 expression was negatively correlated with MdMYB9/11/12 expression and PA accumulation. A 5 0 -RACE experiment, GUS staining assays and transient luminescent assays indicated that mdm-miR858 cleaved and inhibited the expression of MdMYB9/11/12. Overexpression of mdm-miR858 in apple calli, tobacco and Arabidopsis reduced the accumulation of PAs induced by overexpression of MdMYB9/11/12. Furthermore, we found that MdBBX22 bound to the mdm-miR858 promoter and induced its expression. Overexpression of MdBBX22 induced the expression of mdm-miR858 to inhibit the accumulation of PAs in apple calli overexpressing MdMYB9/11/12. Under light stress, MdBBX22 induced mdm-miR858 expression to inhibit PA accumulation and thereby indirectly enhanced anthocyanin synthesis in the peel. The present results revealed that the MdBBX22-miR858-MdMYB9/11/12 module regulates PA accumulation in apple. The findings provide a reference for further studies of the regulatory mechanism of PA accumulation and the relationship between PAs and anthocyanins.
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