Integrated metabolome and transcriptome analysis of differences in quality of ripe Lycium barbarum L. fruits harvested at different periods.

BACKGROUND: Wolfberry is well-known for its high nutritional value and medicinal benefits. Due to the continuous ripening nature of Goji berries and the fact that they can be commercially harvested within a few weeks, their phytochemical composition may change during the harvesting process at different periods. RESULT: The involved molecular mechanisms of difference in fruit quality of ripe Lycium barbarum L. harvested at four different periods were investigated by transcriptomic and metabolomics analyses for the first time. According to the results we obtained, it was found that the appearance quality of L. barbarum fruits picked at the beginning of the harvesting season was superior, while the accumulation of sugar substances in L. barbarum fruits picked at the end of the harvesting season was better. At the same time the vitamin C and carotenoids content of wolfberry fruits picked during the summer harvesting season were richer. Ascorbic acid, succinic acid, glutamic acid, and phenolic acids have significant changes in transcription and metabolism levels. Through the network metabolic map, we found that ascorbic acid, glutamic acid, glutamine and related enzyme genes were differentially accumulated and expressed in wolfberry fruits at different harvesting periods. Nevertheless, these metabolites played important roles in the ascorbate-glutathione recycling system. Ascorbic acid, phenolic substances and the ascorbate-glutathione recycling system have antioxidant effects, which makes the L. barbarum fruits harvested in the summer more in line with market demand and health care concepts. CONCLUSION: This study laid the foundation for understanding the molecular regulatory mechanisms of quality differences of ripe wolfberry fruits harvested at different periods, and provides a theoretical basis for enhancing the quality of L. barbarum fruits.

Decoding VaCOLD1 Function in Grapevines: A Membrane Protein Enhancing Cold Stress Tolerance.

In globally cultivated grapevines, low-temperature stress poses a persistent challenge. Although COLD1 is recognized as a cold receptor in rice, its function in grapevine cold signaling is unclear. Here, we identified VaCOLD1, a transmembrane protein from the cold-tolerant Vitis amurensis Rupr, which is primarily located on plasma and endoplasmic reticulum membranes. Broadly expressed across multiple tissues, VaCOLD1 responds to various environmental stresses, particularly to cold. Its promoter contains distinct hormone- and stress-responsive elements, with GUS assays confirming widespread expression in Arabidopsis thaliana. Validation of interaction between VaCOLD1 and VaGPA1, together with their combined expression in yeast and grape calli, notably improved cold endurance. Overexpression of VaCOLD1 enhances cold tolerance in Arabidopsis by strengthening the CBF-COR signaling pathway. This is achieved through shielding against osmotic disturbances and modifying the expression of ABA-mediated genes. These findings emphasize the critical role of the VaCOLD1-VaGPA1 complex in mediating the response to cold stress via the CBF-COR pathway.

Analysis of GATA transcription factors and their expression patterns under abiotic stress in grapevine (Vitis vinifera L.).

BACKGROUND: GATA transcription factors are type IV zinc-finger proteins that play key roles in plant growth and responses to environmental stimuli. Although these proteins have been studied in model plants, the related studies of GATA gene family under abiotic stresses are rarely reported in grapevine (Vitis vinifera L.). RESULTS: In the current study, a total of 23 VviGATA genes were identified in grapevine and classified into four groups (I, II, III, and IV), based on phylogenetic analysis. The proteins in the same group exhibited similar exon-intron structures and conserved motifs and were found to be unevenly distributed among the thirteen grapevine chromosomes. Accordingly, it is likely that segmental and tandem duplication events contributed to the expansion of the VviGATA gene family. Analysis of cis-acting regulatory elements in their promoters suggested that VviGATA genes respond to light and are influenced by multiple hormones and stresses. Organ/tissue expression profiles showed tissue specificity for most of the VviGATA genes, and five were preferentially upregulated in different fruit developmental stages, while others were strongly induced by drought, salt and cold stress treatments. Heterologously expressed VamGATA5a, VamGATA8b, VamGATA24a, VamGATA24c and VamGATA24d from cold-resistant V. amurensis 'Shuangyou' showed nuclear localization and transcriptional activity was shown for VamGATA5a, VamGATA8b and VamGATA24d. CONCLUSIONS: The results of this study provide useful information for GATA gene function analysis and aid in the understanding of stress responses in grapevine for future molecular breeding initiatives.

Comparative Transcriptome and Widely Targeted Metabolome Analysis Reveals the Molecular Mechanism of Powdery Mildew Resistance in Tomato.

Powdery mildew is a serious problem in tomato production; therefore, the PM-resistant tomato inbred line, '63187', and the susceptible tomato variety, 'Moneymaker (MM)', were used as experimental materials for the combined analysis of transcriptome and widely targeted metabolome on tomato leaves at 0 h post inoculation (hpi), 12 hpi, and 48 hpi. The results indicated that 276 genes were expressed in all treatments, and the K-means cluster analysis showed that these genes were divided into eight classes in '63187' and ten classes in 'MM'. KEGG enrichment showed that amino acid metabolism, signal transduction, energy metabolism, and other secondary metabolites biosynthesis pathways were significantly enriched. Interestingly, the analysis of WRKY family transcription factors (TFs) showed that the expression of four TFs in '63187' increased with no obvious change in 'MM'; and the expression of one TF in 'MM' increased with no obvious change in '63187'. The combined analysis revealed that both phenylpropanoid biosynthesis and flavonoid biosynthesis pathways were enriched in '63187' and 'MM'. In '63187', six metabolites involved in this pathway were downregulated, and four genes were highly expressed, while in 'MM', three metabolites were upregulated, four metabolites were downregulated, and ten genes were highly expressed. These metabolites and genes might be candidates for PM resistance or susceptibility in subsequent studies. These results provide favorable molecular information for the study of the different resistances of tomatoes to PM, and they provide a basis for the breeding of tomato varieties resistant to PM.

Comparative analysis of chloroplast genomes of 29 tomato germplasms: genome structures, phylogenetic relationships, and adaptive evolution.

In order to compare and analyze the chloroplast (cp) genomes of tomato germplasms and understand their phylogenetic relationships, the cp genomes of 29 tomato germplasms were sequenced and analyzed in this study. The results showed highly conserved characteristics in structure, number of gene and intron, inverted repeat regions, and repeat sequences among the 29 cp genomes. Moreover, single-nucleotide polymorphism (SNP) loci with high polymorphism located at 17 fragments were selected as candidate SNP markers for future studies. In the phylogenetic tree, the cp genomes of tomatoes were clustered into two major clades, and the genetic relationship between S. pimpinellifolium and S. lycopersicum was very close. In addition, only rps15 showed the highest average K A/K S ratio in the analysis of adaptive evolution, which was strongly positively selected. It may be very important for the study of adaptive evolution and breeding of tomato. In general, this study provides valuable information for further study of phylogenetic relationships, evolution, germplasm identification, and molecular marker-assisted selection breeding of tomato.

Identification of C3H2C3-type RING E3 ubiquitin ligase in grapevine and characterization of drought resistance function of VyRCHC114.

BACKGROUND: RING is one of the largest E3 ubiquitin ligase families and C3H2C3 type is the largest subfamily of RING, which plays an important role in plant growth and development, and growth and responses to biotic and abiotic stresses. RESULTS: A total of 143 RING C3H2C3-type genes (RCHCs) were discovered from the grapevine genome and separated into groups (I-XI) according to their phylogenetic analysis, and these genes named according to their positions on chromosomes. Gene replication analysis showed that tandem duplications play a predominant role in the expansion of VvRCHCs family together. Structural analysis showed that most VvRCHCs (67.13 %) had no more than 2 introns, while genes clustered together based on phylogenetic trees had similar motifs and evolutionarily conserved structures. Cis-acting element analysis showed the diversity of VvRCHCs regulation. The expression profiles of eight DEGs in RNA-Seq after drought stress were like the results of qRT-PCR analysis. In vitro ubiquitin experiment showed that VyRCHC114 had E3 ubiquitin ligase activity, overexpression of VyRCHC114 in Arabidopsis improved drought tolerance. Moreover, the transgenic plant survival rate increased by 30 %, accompanied by electrolyte leakage, chlorophyll content and the activities of SOD, POD, APX and CAT were changed. The quantitative expression of AtCOR15a, AtRD29A, AtERD15 and AtP5CS1 showed that they participated in the response to drought stress may be regulated by the expression of VyRCHC114. CONCLUSIONS: This study provides valuable new information for the evolution of grapevine RCHCs and its relevance for studying the functional characteristics of grapevine VyRCHC114 genes under drought stress.

A novel R3 MYB transcriptional repressor, MaMYBx, finely regulates anthocyanin biosynthesis in grape hyacinth.

R3-MYBs negatively regulate anthocyanin pigmentation in plants. However, how R3-MYB repressors finely modulate anthocyanin biosynthesis in cooperation with R2R3-MYB activators remains unclear in monocots. We previously identified two anthocyanin-related R2R3-MYB activators (MaMybA and MaAN2) in grape hyacinth (Muscari spp.). Here, we isolated a R3-MYB repressor, MaMYBx, and characterized its role in anthocyanin biosynthesis using genetic and biochemical markers. The temporal expression pattern of MaMYBx was similar to that of MaMybA and MaAN2, and it was correlated with anthocyanin accumulation during flower development. MaMYBx could be activated either by MaMybA alone or by MaMybA/MaAN2 and cofactor MabHLH1, and it suppressed its own activation and that of MaMybA promoters mediated by MaMybA/MaAN2 and MabHLH1. Like MaMybA, MaMYBx interacted with MabHLH1. MaDFR and MaANS transcription and anthocyanin accumulation mediated by MaMybA/MaAN2 and MabHLH1 were inhibited by MaMYBx. Overexpression of MaMYBx in tobacco greatly reduced flower pigmentation and repressed the expression of late structural and regulatory anthocyanin pathway genes. Thus, MaMYBx finely regulates anthocyanin biosynthesis by binding to MabHLH1 and disrupting the R2R3 MYB-bHLH complex in grape hyacinth. The regulatory network of transcriptional activators and repressors modulating anthocyanin biosynthesis is conserved within monocots. MaMYBx seems a potentially valuable target for flower color modification in ornamental plants.

The grape ubiquitin ligase VpRH2 is a negative regulator in response to ABA treatment.

MAIN CONCLUSION: Ubiquitin ligase VpRH2 is a negative regulator in the grape ABA pathway by inhibiting ABL1, PYR1 and GRP2A expressions, and its promoter is inhibited by ABA treatment. In higher plants, ubiquitin ligases play key roles in various cellular processes. As in our previous study (Wang et al. in J Exp Bot 68:1669-1687, 2017), grape RING-H2-type ubiquitin ligase gene VpRH2 and its promoter was induced by powdery mildew and showed resistance to the disease. Diverse small-molecule hormones, like salicylic acid (SA), methyl jasmonate (MeJA) or abscisic acid (ABA), play pivotal roles in plant resistance. Here we found that VpRH2 expression could be induced by SA and MeJA treatment, but inhibited by ABA treatment. The promoter of VpRH2 revealed a similar variation trend under exogenous hormone treatments as the gene expression by GUS activity assay. By a series of deletion fragments, the promoter fragment of VpRH2-P656 to VpRH2-P513 was necessary in response to MeJA treatment, and the inhibition of ABA treatment to the VpRH2 promoter was independent of the ABRE motif. Over-expression of VpRH2 in Arabidopsis thaliana plants displayed ABA-insensitive phenotypes at the germination stage compared to wild type plants. In VpRH2 over-expressing Vitis vinifera cv. Thompson Seedless plants after ABA treatments, the expression of the ABA pathway related genes ABL1 and PYR1 showed a suppresive trend. Moreover, VpGRP2A (an VpRH2-interacting protein) also showed a suppresive trend in response to ABA treatment in VpRH2-overexpressing plants. Our results demonstrate that VpRH2 is a negative regulator in the grape ABA signal pathway by inhibiting ABL1, PYR1 and GRP2A expressions, and its promoter was also inhibited by ABA treatment.

VpSTS29/STS2 enhances fungal tolerance in grapevine through a positive feedback loop.

Accumulation of stilbene phytoalexins stimulates resistance mechanisms against the grapevine fungus Uncinula necator. However, the defensive mechanisms triggered by stilbene synthase (STS) genes, remain largely unknown. Here, we report the function and molecular mechanism of the stilbene synthase gene VpSTS29/STS2 from Vitis pseudoreticulata in the regulation of plant responses to powdery mildew. Stilbene synthesis occurred mainly in root tips and mesophyll cells of transgenic grapevines via transport through the vascular bundles. Overexpression of VpSTS29/STS2 in Vitis vinifera increased the abundance of STSs in mesophyll tissue and resulted in the accumulation of biologically active resveratrol derivatives at the invasion site. Similarly, expression of VpSTS29/STS2 in Arabidopsis increased resistance to Golovinomyces cichoracearum. The VpSTS29/STS2-expressing Arabidopsis lines showed increased piceid accumulation together with more local hypersensitive reactions, inhibition of mycelial growth, and a reduced incidence of pathogens. Transcriptome profiling analyses demonstrated that VpSTS29/STS2-induced defences led to reprograming of global gene expression and activation of salicylic acid (SA) signalling, thus increasing expression of WRKY-MYB transcription factors and other defence response genes. We propose a model for resveratrol-mediated coordination of defence responses in which SA participates in a positive feedback loop.

Dynamic translocation of stilbene synthase VpSTS29 from a Chinese wild Vitis species upon UV irradiation.

Stilbene phytoalexins derived from grapevine can be rapidly accumulated when exposed to an artificial UV-C treatment. However, the underlying mechanisms involved in this accumulation and translocation are unclear. Here, we describe an investigation of the influence of UV-C treatment on the dynamic subcellular distribution of a member of a stilbene synthase family VpSTS29 derived from Chinese wild Vitis pseudoreticulata W.T. Wang when over-expressed in V. vinifera L. cv. Thompson Seedless. Our results show that VpSTS29-GFP was accumulated at a relatively high level in roots and mature leaves of transgenic grape lines, and was predominantly distributed in the cytoplasm. When exposed to UV-C irradiation, VpSTS29 displayed UV-induced feature coupled with the accumulation of stilbene compounds. Notably, VpSTS29-GFP can be translocated from the cytoplasm into chloroplasts upon UV-irradiation. Leaves from the two VpSTS29-GFP-expressing lines displayed more serious UV damage, showing withering and marginal scorching phenotype, and decreased content of H2O2, compared to the untransformed plant. Also, overexpression of VpSTS29 altered the expression of genes related to redox regulation, stilbene biosynthesis and light stimulus. Co-expression of VpSTS29-GFP with Glycolate oxidase 1 (myc-VpGLO1) confirmed the ability of stilbenes to decrease the content of H2O2 in Arabidopsis mesophyll protoplasts. These results provide new insight into the biological functions and properties of stilbene synthase and its product in response to environmental stimulus.

Overexpression of VpEIFP1, a novel F-box/Kelch-repeat protein from wild Chinese Vitis pseudoreticulata, confers higher tolerance to powdery mildew by inducing thioredoxin z proteolysis.

An F-box protein (VpEIFP1) induced by Erysiphe necator was isolated from Vitis pseudoreticulata, a wild Chinese grapevine species naturally resistant to powdery mildew (PM). It contains an F-box domain and two Kelch-repeat motifs. Expression profiles indicate the VpEIFP1 is strongly induced at both transcriptional and translational levels by PM infection. A subcellular localisation assay showed that VpEIFP1 is predominantly located in the nucleus and cytoplasm. Overexpression of VpEIFP1 accelerated the accumulation of hydrogen peroxide (H2O2) and up-regulated the expressions of ICS2, NPR1 and PR1 involved in defence responses, resulting in suppression of PM germination and growth. As an F-box protein, VpEIFP1 interacts with thioredoxin z (VpTrxz) in the yeast-two-hybrid (Y2H) assay and in the bimolecular fluorescence complementation (BiFC) assay. Decreased amounts of VpTrxz protein in transgenic grapevine leaves overexpressing VpEIFP1 were restored by proteasome inhibitor MG132, implying that VpEIFP1 mediated VpTrxz for degradation through the SCFVpEIFP1 (Skp1-Cullin-F-box) E3 ubiquitin ligase complex. The RNA interference line of VpTrxz showed increased H2O2 accumulation following PM inoculation. We propose VpEIFP1 positively modulates the grapevine defence response to PM by inducing the degradation of VpTrxz via the ubiquitin/26S proteasome system.

VpPUB24, a novel gene from Chinese grapevine, Vitis pseudoreticulata, targets VpICE1 to enhance cold tolerance.

The ubiquitination system plays important roles in the degradation and modification of substrate proteins. In this study, we characterize a putative U-box type E3 ubiquitin ligase gene, VpPUB24 (plant U-box protein 24), from Chinese wild grapevine, Vitis pseudoreticulata accession Baihe-35-1. We show that VpPUB24 is induced by a number of stresses, especially cold treatment. Real-time PCR analysis indicated that the PUB24 transcripts were increased after cold stress in different grapevine species, although the relative expression level was different. In grapevine protoplasts, we found that VpPUB24 was expressed at a low level at 22 °C but accumulated rapidly following cold treatment. A yeast two-hybrid assay revealed that VpPUB24 interacted physically with VpICE1. Further experiments indicated that VpICE1 is targeted for degradation via the 26S proteasome and that the degradation is accelerated by VpHOS1, and not by VpPUB24. Immunoblot analyses indicated that VpPUB24 promotes the accumulation of VpICE1 and suppresses the expression of VpHOS1 to regulate the abundance of VpICE1. Furthermore, VpICE1 promotes transcription of VpPUB24 at low temperatures. We also found that VpPUB24 interacts with VpHOS1 in a yeast two-hybrid assay. Additionally, over-expression of VpPUB24 in Arabidopsis thaliana enhanced cold tolerance. Collectively, our results suggest that VpPUB24 interacts with VpICE1 to play a role in cold stress.

RING-H2-type E3 gene VpRH2 from Vitis pseudoreticulata improves resistance to powdery mildew by interacting with VpGRP2A.

Grapevine is one of the world's most important fruit crops. European cultivated grape species have the best fruit quality but show almost no resistance to powdery mildew (PM). PM caused by Uncinula necator is a harmful disease that has a significant impact on the economic value of the grape crop. In this study, we examined a RING-H2-type ubiquitin ligase gene VpRH2 that is associated with significant PM-resistance of Chinese wild-growing grape Vitis pseudoreticulata accession Baihe-35-1. The expression of VpRH2 was clearly induced by U. necator inoculation compared with its homologous gene VvRH2 in a PM-susceptible grapevine V. vinifera cv. Thompson Seedless. Using a yeast two-hybrid assay we confirmed that VpRH2 interacted with VpGRP2A, a glycine-rich RNA-binding protein. The degradation of VpGRP2A was inhibited by treatment with the proteasome inhibitor MG132 while VpRH2 did not promote the degradation of VpGRP2A. Instead, the transcripts of VpRH2 were increased by over-expressing VpGRP2A while VpRH2 suppressed the expression of VpGRP2A. Furthermore, VpGRP2A was down-regulated in both Baihe-35-1 and Thompson Seedless after U. necator inoculation. Specifically, we generated VpRH2 overexpression transgenic lines in Thompson Seedless and found that the transgenic plants showed enhanced resistance to powdery mildew compared with the wild-type. In summary, our results indicate that VpRH2 interacts with VpGRP2A and plays a positive role in resistance to powdery mildew.

The Chinese wild grapevine (Vitis pseudoreticulata) E3 ubiquitin ligase Erysiphe necator-induced RING finger protein 1 (EIRP1) activates plant defense responses by inducing proteolysis of the VpWRKY11 transcription factor.

Ubiquitin-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.

A core functional region of the RFP1 promoter from Chinese wild grapevine is activated by powdery mildew pathogen and heat stress.

RING-finger proteins (RFP) function as ubiquitin ligases and play key roles in plant responses to biotic and abiotic stresses. However, little information is available on the regulation of RFP expression. Here, we isolate and characterize the RFP promoter sequence from the disease-resistant Chinese wild grape Vitis pseudoreticulata accession Baihe-35-1. Promoter-GUS fusion assays revealed that defense signaling molecules, powdery mildew infection, and heat stress induce VpRFP1 promoter activity. By contrast, the RFP1 promoter isolated from Vitis vinifera was only slightly induced by pathogen infection and heat treatment. By promoter deletion analysis, we found that the -148 bp region of the VpRFP1 promoter was the core functional promoter region. We also found that, in Arabidopsis, VpRFP1 expressed under its own promoter activated defense-related gene expression and improved disease resistance, but the same construct using the VvRFP1 promoter slightly improve disease resistance. Our results demonstrated that the -148 bp region of the VpRFP1 promoter plays a key role in response to pathogen and heat stress, and suggested that expression differences between VpRFP1 and VvRFP1 may be key for the differing disease resistance phenotypes of the two Vitis genotypes.