The noncoding RNA HIDDEN TREASURE 1 promotes phytochrome B-dependent seed germination by repressing abscisic acid biosynthesis.

Light is a major environmental factor for seed germination. Red light-activated phytochrome B (phyB) promotes seed germination by modulating the dynamic balance of two phytohormones, gibberellic acid (GA) and abscisic acid (ABA). How phyB modulates ABA biosynthesis after perceiving a light signal is not yet well understood. Here, we identified the noncoding RNA HIDDEN TREASURE 1 (HID1) as a repressor of ABA biosynthesis acting downstream of phyB during Arabidopsis thaliana seed germination. Loss of HID1 function led to delayed phyB-dependent seed germination. Photoactivated phyB promoted the accumulation of HID1 in the radicle within 48 h of imbibition. Our transcriptomics analysis showed that HID1 and phyB co-regulate the transcription of a common set of genes involved in ABA and GA metabolism. Through a forward genetic screen, we identified three ABA biosynthesis genes, ABA DEFICIENT 1 (ABA1), ABA2, and ABA3, as suppressors of HID1. We further demonstrated that HID1 directly inhibits the transcription of 9-CIS-EPOXYCAROTENOID DIOXYGENASE (NCED9), a gene encoding a key rate-limiting enzyme of ABA biosynthesis. HID1 interacts with ARABIDOPSIS TRITHORAX-RELATED7 (ATXR7), an H3K4me3 methyltransferase, inhibiting its occupancy and H3K4me3 modification at the NCED9 locus. Our study reveals a nuclear mechanism of phyB signaling transmitted through HID1 to control the internal homeostasis of ABA and GA, which gradually optimizes the transcriptional network during seed germination.

Genome Sequence Resource for Colletotrichum viniferum, the Cause of Grapevine Ripe Rot in China.

Grape ripe rot is an important disease that has seriously damaged the yield and quality of grape worldwide. The disease is caused by Colletotrichum viniferum, a hemibiotrophic fungus that belongs to the Glomerellaceae family of Sordariomycetes class. Here, we present the genome of C. viniferum CvYL2a from grape, based on Illumina HiSeq 2500 and PacBio RS II. The high-quality genome consists of 70 contigs with a 73.41 Mb genome size and encodes 14,668 protein-coding genes. These genes were annotated using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, EuKaryotic Orthologous Groups, Nonredundant Protein, and Swiss-Prot databases. In addition, we identified a series of genes involved in pathogenicity, including 909 carbohydrate-active enzymes, 67 secondary metabolite gene clusters, and 307 cytochrome P450 enzymes. This genome sequence provides a valuable reference for research on grape-C. viniferum interactions, the pathogenesis of C. viniferum, and comparative genome analyses.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

VvHDZ28 positively regulate salicylic acid biosynthesis during seed abortion in Thompson Seedless.

Seedlessness in grapes is one of the features most appreciated by consumers. However, the mechanisms underlying seedlessness in grapes remain obscure. Here, we observe small globular embryos and globular embryos in Pinot Noir and Thompson Seedless from 20 to 30 days after flowering (DAF). From 40 to 50 DAF, we observe torpedo embryos and cotyledon embryos in Pinot Noir but aborted embryos and endosperm in Thompson Seedless. Thus, RNA-Seq analyses of seeds at these stages from Thompson Seedless and Pinot Noir were performed. A total of 6442 differentially expressed genes were identified. Among these, genes involved in SA biosynthesis, VvEDS1 and VvSARD1, were more highly expressed in Thompson Seedless than in Pinot Noir. Moreover, the content of endogenous SA is at least five times higher in Thompson Seedless than in Pinot Noir. Increased trimethylation of H3K27 of VvEDS1 and VvSARD1 may be correlated with lower SA content in Pinot Noir. We also demonstrate that VvHDZ28 positively regulates the expression of VvEDS1. Moreover, over-expression of VvHDZ28 results in seedless fruit and increased SA contents in Solanum lycopersicum. Our results reveal the potential role of SA and feedback regulation of VvHDZ28 in seedless grapes.

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.

Overexpression of VpPR10.1 by an efficient transformation method enhances downy mildew resistance in V. vinifera.

KEY MESSAGE: Putrescine and spermidine increase the transformation efficiency of Vitis vinifera L. cv. Thompson seedless. Accumulation of VpPR10.1 in transgenic V. vinifera Thompson seedless, likely increases its resistance to downy mildew. A more efficient method is described for facilitating Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless somatic embryogenesis using polyamines (PAs). The efficacies of putrescine, spermidine and spermine are identified at a range of concentrations (10 µM, 100 µM and 1 mM) added to the culture medium during somatic embryo growth. Putrescine (PUT) and spermidine (SPD) promote the recovery of proembryonic masses (PEM) and the development of somatic embryos (SE) after co-cultivation. Judging from the importance of the time-frame in genetic transformation, PAs added at the co-cultivation stage have a stronger effect than delayed selection treatments, which are superior to antibiotic treatments in the selection stage. Best embryogenic responses are with 1 mM PUT and 100 µM SPD added to the co-culture medium. Using the above method, a pathogenesis-related gene (VpPR10.1) from Chinese wild Vitis pseudoreticulata was transferred into Thompson Seedless for functional evaluation. The transgenic line, confirmed by western blot analysis, was inoculated with Plasmopara viticola to test for downy mildew resistance. Based on observed restrictions of hyphal growth and increases in H2O2 accumulation in the transgenic plants, the accumulation of VpPR10.1 likely enhanced the transgenic plants resistance to downy mildew.

A stilbene synthase allele from a Chinese wild grapevine confers resistance to powdery mildew by recruiting salicylic acid signalling for efficient defence.

Stilbenes are central phytoalexins in Vitis, and induction of the key enzyme stilbene synthase (STS) is pivotal for disease resistance. Here, we address the potential for breeding resistance using an STS allele isolated from Chinese wild grapevine Vitis pseudoreticulata (VpSTS) by comparison with its homologue from Vitis vinifera cv. 'Carigane' (VvSTS). Although the coding regions of both alleles are very similar (>99% identity on the amino acid level), the promoter regions are significantly different. By expression in Arabidopsis as a heterologous system, we show that the allele from the wild Chinese grapevine can confer accumulation of stilbenes and resistance against the powdery mildew Golovinomyces cichoracearum, whereas the allele from the vinifera cultivar cannot. To dissect the upstream signalling driving the activation of this promoter, we used a dual-luciferase reporter system in a grapevine cell culture. We show elevated responsiveness of the promoter from the wild grape to salicylic acid (SA) and to the pathogen-associated molecular pattern (PAMP) flg22, equal induction of both alleles by jasmonic acid (JA), and a lack of response to the cell death-inducing elicitor Harpin. This elevated SA response of the VpSTS promoter depends on calcium influx, oxidative burst by RboH, mitogen-activated protein kinase (MAPK) signalling, and JA synthesis. We integrate the data in the context of a model where the resistance of V. pseudoreticulata is linked to a more efficient recruitment of SA signalling for phytoalexin synthesis.

Transcriptome profiling of Vitis amurensis, an extremely cold-tolerant Chinese wild Vitis species, reveals candidate genes and events that potentially connected to cold stress.

Vitis amurensis Rupr. is an exceptional wild-growing Vitis (grape) species that can safely survive a wide range of cold conditions, but the underlying cold-adaptive mechanism associated with gene regulation is poorly investigated. We have analyzed the physiochemical and transcriptomic changes caused by cold stress in a cold-tolerant accession, 'Heilongjiang seedling', of Chinese wild V. amurensis. We statistically determined that a total of 6,850 cold-regulated transcripts were involved in cold regulation, including 3,676 up-regulated and 3,174 down-regulated transcripts. A global survey of messenger RNA revealed that skipped exon is the most prevalent form of alternative spicing event. Importantly, we found that the total splicing events increased with the prolonged cold stress. We also identified thirty-eight major TF families that were involved in cold regulation, some of which were previously unknown. Moreover, a large number of candidate pathways for the metabolism or biosynthesis of secondary metabolites were found to be regulated by cold, which is of potential importance in coordinating cold tolerance with growth and development. Several heat shock proteins and heat shock factors were also detected to be intensively cold-regulated. Furthermore, we validated the expression profiles of 16 candidates using qRT-PCR to further confirm the accuracy of the RNA-seq data. Our results provide a genome-wide view of the dynamic changes in the transcriptome of V. amurensis, in which it is evident that various structural and regulatory genes are crucial for cold tolerance/adaptation. Moreover, our robust dataset advances our knowledge of the genes involved in the complex regulatory networks of cold stress and leads to a better understanding of cold tolerance mechanisms in this extremely cold-tolerant Vitis species.

The grapevine basic helix-loop-helix (bHLH) transcription factor positively modulates CBF-pathway and confers tolerance to cold-stress in Arabidopsis.

Basic helix-loop-helix (bHLH)-type transcription factors play diverse roles in plant physiological response and stress-adaptive regulation network. Here, we identified one grapevine bHLH transcription factor from a cold-tolerant accession 'Heilongjiang seedling' of Chinese wild Vitis amurensis (VabHLH1) as a transcriptional activator involved in cold stress. We also compared with its counterpart from a cold-sensitive Vitis vinifera cv. Cabernet Sauvignon (VvbHLH1). These two putative proteins are characterized by the presence of the identically conserved regions of 54 amino acid residues of bHLH signature domain, and shared 99.1% amino acid identity, whereas several stress-related cis-regulatory elements located in both promoter regions differed in types and positions. Expressions of two bHLHs in grapevine leaves were induced by cold stress, but evidently differ between two grapevine genotypes upon cold exposure. Two grapevine bHLH proteins were exclusively localized to the nucleus and exhibited strong transcriptional activation activities in yeast cells. Overexpression of either VabHLH1 or VvbHLH1 transcription factor did not affect the growth and development of transgenic Arabidopsis plants, but enhanced tolerance to cold stress. The improved tolerance in VabHLH1- or VvbHLH1-overexpressing Arabidopsis plants is associated with multiple physiological and biochemical changes that occurred during the time-course cold stress. These most common changes include the evaluated levels of proline, decreased amounts of malondialdehyde and reduced membrane injury as reflected by electrolyte leakage. VabHLH1 and VvbHLH1 displayed overlapping, but not identical, roles in activating the corresponding CBF cold signaling pathway, especially in regulating the expression of CBF3 and RD29A. Our findings demonstrated that two grapevine bHLHs act as positive regulators of the cold stress response, modulating the level of COR gene expression, which in turn confer tolerance to cold stress.

CRISPR/Cas9-mediated VvPR4b editing decreases downy mildew resistance in grapevine (Vitis vinifera L.).

Downy mildew of grapevine (Vitis vinifera L.), caused by the oomycete pathogen Plasmopara viticola, is one of the most serious concerns for grape production worldwide. It has been widely reported that the pathogenesis-related 4 (PR4) protein plays important roles in plant resistance to diseases. However, little is known about the role of PR4 in the defense of grapevine against P. viticola. In this study, we engineered loss-of-function mutations in the VvPR4b gene from the cultivar "Thompson Seedless" using the CRISPR/Cas9 system and evaluated the consequences for downy mildew resistance. Sequencing results showed that deletions were the main type of mutation introduced and that no off-target events occurred. Infection assays using leaf discs showed that, compared to wild-type plants, the VvPR4b knockout lines had increased susceptibility to P. viticola. This was accompanied by reduced accumulation of reactive oxygen species around stomata. Measurement of the relative genomic abundance of P. viticola in VvPR4b knockout lines also demonstrated that the mutants had increased susceptibility to the pathogen. Our results confirm that VvPR4b plays an active role in the defense of grapevine against downy mildew.

Methylphenidate ameliorates hypoxia-induced mitochondrial damage in human neuroblastoma SH-SY5Y cells through inhibition of oxidative stress.

AIMS: Methylphenidate (MPH) is a dopamine-reuptake inhibitor approved for the treatment of attention-deficit/hyperactivity disorder (ADHD). Nonetheless, the cellular and molecular mechanisms of MPH are still unknown. We attempt to determine whether MPH protect neuron cells against oxidative stress by using human neuroblastoma SH-SY5Y cells. MAIN METHODS: The SH-SY5Y cells were cultured in normoxic and hypoxic conditions in the presence of different doses of MPH. Then, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and adenosine triphosphate (ATP) production were quantitatively measured by using flow cytometry or spectrophotometry. The mitochondrial ultrastructure of the cells was observed by electron microscope, and the function of mitochondrial was evaluated by measuring mitochondrial membrane potential (MMP) using flow cytometry. The levels of SOD and heme oxygenase-1 (HO-1) proteins were detected by Western blot. KEY FINDINGS: We found that low doses of MPH treatment (50-500 ng/mL) led to decreased ROS and MDA production (P<0.05), increased GSH and SOD as well as ATP concentration (P<0.05) in hypoxic SH-SY5Y cells. Additionally, low doses of MPH significantly inhibited mitochondrial swelling and decreased the percentage of JC-1 monomer positive cells. However, we did not observe the same effects of MPH in normoxia. SIGNIFICANCE: Our results show that low doses of MPH play protective roles in maintaining mitochondrial homeostasis in response to hypoxia-induced oxidative stress. Our findings may provide novel insight into the mechanisms of MPH in the treatment of ADHD, and shed light on the disease mechanisms of ADHD.

VqMAPKKK38 is essential for stilbene accumulation in grapevine.

Vitis species, including grapevine, produce a class of secondary metabolites called stilbenes that are important for plant disease resistance and can have positive effects on human health. Mitogen-activated protein kinase (MAPK) signaling cascades not only play key roles in plant defense responses but also contribute to stilbene biosynthesis in grapevine. MAPKKKs function at the upper level of the MAPK network and initiate signaling through this pathway. In this study, a Raf-like MAPKKK gene, VqMAPKKK38, was identified and functionally characterized from the Chinese wild grapevine V. quinquangularis accession 'Danfeng-2'. We observed that VqMAPKKK38 transcript levels were elevated by powdery mildew infection, high salinity conditions and chilling stresses, as well as in response to treatments by the hormones salicylic acid (SA), methyl jasmonate (MeJA), ethylene (Eth) and abscisic acid (ABA). In addition, based on both transient overexpression and gene suppression of VqMAPKKK38 in grapevine leaves, we found that VqMAPKKK38 positively regulates stilbene synthase transcription and stilbene accumulation probably by mediating the activation of the transcription factor MYB14. In addition, both hydrogen peroxide (H2O2) and calcium influx activated VqMAPKKK38 expression and stilbene biosynthesis, which suggests that VqMAPKKK38 may be involved in the calcium signaling and ROS signaling pathways.

Chinese wild-growing Vitis amurensis ICE1 and ICE2 encode MYC-type bHLH transcription activators that regulate cold tolerance in Arabidopsis.

Winter hardiness is an important trait for grapevine breeders and producers, so identification of the regulatory mechanisms involved in cold acclimation is of great potential value. The work presented here involves the identification of two grapevine ICE gene homologs, VaICE1 and VaICE2, from an extremely cold-tolerant accession of Chinese wild-growing Vitis amurnensis, which are phylogenetically related to other plant ICE1 genes. These two structurally different ICE proteins contain previously reported ICE-specific amino acid motifs, the bHLH-ZIP domain and the S-rich motif. Expression analysis revealed that VaICE1 is constitutively expressed but affected by cold stress, unlike VaICE2 that shows not such changed expression as a consequence of cold treatment. Both genes serve as transcription factors, potentiating the transactivation activities in yeasts and the corresponding proteins localized to the nucleus following transient expression in onion epidermal cells. Overexpression of either VaICE1 or VaICE2 in Arabidopsis increase freezing tolerance in nonacclimated plants. Moreover, we show that they result in multiple biochemical changes that were associated with cold acclimation: VaICE1/2-overexpressing plants had evaluated levels of proline, reduced contents of malondialdehyde (MDA) and decreased levels of electrolyte leakage. The expression of downstream cold responsive genes of CBF1, COR15A, and COR47 were significantly induced in Arabidopsis transgenically overexpressing VaICE1 or VaICE2 upon cold stress. VaICE2, but not VaICE1 overexpression induced KIN1 expression under cold-acclimation conditions. Our results suggest that VaICE1 and VaICE2 act as key regulators at an early step in the transcriptional cascade controlling freezing tolerance, and modulate the expression levels of various low-temperature associated genes involved in the C-repeat binding factor (CBF) pathway.

A pathogenesis related protein, VpPR-10.1, from Vitis pseudoreticulata: an insight of its mode of antifungal activity.

Previously, VpPR-10.1 was isolated and characterized from a cDNA library of a fungus-resistant accession of Chinese wild grape (Vitis pseudoreticulata). We found that expression of VpPR-10.1 is affected by the fungal pathogen Erysiphe necator. To investigate the biochemical basis of the nuclease activity of VpPR-10.1 and its role in antifungal resistance, we generated recombinant VpPR-10.1 as well as site-directed mutations targeting three conserved amino acid residues among plant PR-10 s: Lys55, Glu149, and Tyr151. We showed that wild-type recombinant VpPR-10.1 exhibits both RNase and DNase activities. Mutant VpPR10.1-Y151H essentially retained all these activities. In contrast, VpPR10.1-K55N, where Lys55 in the P-loop region is mutated to Asn, and VpPR10.1-E149G, where Glu149 is mutated to Gly, lost their nuclease activity, indicating that both residues play a critical role in catalyzing RNA and DNA degradation. Furthermore, VpPR10.1 and VpPR10.1-Y151H inhibited the growth of the cultured fungal pathogen Alternaria alternate. Through transient expression in grapevine, we also demonstrated that VpPR10.1-K55N and VpPR10.1-E149G compromised resistance to E. necator. Finally, we further found that VpPR-10.1 can lead to programmed cell death and DNA degradation when incubated with tobacco BY-2 suspension cells. We show here that Lys55 and Glu149, but not Tyr151, are required for the RNase, DNase and antifungal activities of VpPR-10.1. The strong correlation between the level of VpPR-10.1 nuclease activity and its antifungal property indicates that the former is the biochemical basis for the latter. Taken together, our experiments revealed that VpPR-10.1 is critical in mediating fungal resistance in grape, potentially playing a dual role by degrading pathogen RNA and inducing programmed death of host cells.

The comparative analysis of the potential relationship between resveratrol and stilbene synthase gene family in the development stages of grapes (Vitis quinquangularis and Vitis vinifera).

Resveratrol is positively correlated with grapevine disease resistance and its consumption is also highly beneficial to human health. HPLC analyses showed that resveratrol content was significantly higher in most wild Chinese grapevines than in most European Vitis vinifera grapevine cvs. Fruit of the wild Chinese genotype Vitis quinquangularis Danfeng-2 contains much higher levels of resveratrol than some others. Because stilbene synthase is responsible for resveratrol biosynthesis, 41 full-length stilbene synthase genes were isolated from Danfeng-2 using the RACE method. A neighbor-joining tree of the STS family displayed high similarity between Danfeng-2 and V. vinifera cv. Pinot Noir. The content of the endogenous stilbene synthase family in tissues and the expression levels induced by powdery mildew were both higher in Danfeng-2 than in Pinot Noir. Moreover, expression in the berry was significantly higher than in the leaves. Our results demonstrated that resveratrol accumulation was consistent with endogenous STS gene expressions, and that both were higher in Danfeng-2 than in Pinot Noir. Therefore, STS genes and producing resveratrol from V. quinquangularis played more important role in Vitis resistance. Otherwise, the gene VqSTS6 was markedly higher than the other VqSTS genes in the six tissues/organs assayed by Real-time PCR, which will offer a useful basis for commercial application of resveratrol from Chinese wild grapes.

Subcellular localization and functional analyses of a PR10 protein gene from Vitis pseudoreticulata in response to Plasmopara viticola infection.

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.