Ectopic enhancer-enhancer interactions as causal forces driving RNA-directed DNA methylation in gene regulatory regions.

Cis-regulatory elements (CREs) are integral to the spatiotemporal and quantitative expression dynamics of target genes, thus directly influencing phenotypic variation and evolution. However, many of these CREs become highly susceptible to transcriptional silencing when in a transgenic state, particularly when organised as tandem repeats. We investigated the mechanism of this phenomenon and found that three of the six selected flower-specific CREs were prone to transcriptional silencing when in a transgenic context. We determined that this silencing was caused by the ectopic expression of non-coding RNAs (ncRNAs), which were processed into 24-nt small interfering RNAs (siRNAs) that drove RNA-directed DNA methylation (RdDM). Detailed analyses revealed that aberrant ncRNA transcription within the AGAMOUS enhancer (AGe) in a transgenic context was significantly enhanced by an adjacent CaMV35S enhancer (35Se). This particular enhancer is known to mis-activate the regulatory activities of various CREs, including the AGe. Furthermore, an insertion of 35Se approximately 3.5 kb upstream of the AGe in its genomic locus also resulted in the ectopic induction of ncRNA/siRNA production and de novo methylation specifically in the AGe, but not other regions, as well as the production of mutant flowers. This confirmed that interactions between the 35Se and AGe can induce RdDM activity in both genomic and transgenic states. These findings highlight a novel epigenetic role for CRE-CRE interactions in plants, shedding light on the underlying forces driving hypermethylation in transgenes, duplicate genes/enhancers, and repetitive transposons, in which interactions between CREs are inevitable.

Establishment of an efficient callus transient transformation system for Vitis vinifera cv. 'Chardonnay'.

Grape (Vitis L.), a highly valued fruit crop, poses significant challenges in genetic transformation and functional characterization of genes. Therefore, there is an urgent need for the development of a rapid and effective method for grape transformation and gene function identification. Here, we introduce a streamlined Agrobacterium-mediated transient transformation system for grape calli. Optimal conditions were established with a leaf-derived callus induction medium; chiefly B5 medium supplemented with 0.05 mg/L NAA, 0.5 mg/L 2,4-D, and 2.0 mg/L KT; and a callus proliferation medium (B5 medium supplemented with 0.5 mg/L NAA and 2.0 mg/L 6-BA), respectively. Notably, GUS enzyme activity peaked (352.96 ± 33.95 mol 4-MU/mg/min) by sonication with Agrobacterium tumefaciens EHA105 and 100 µM AS for 4 min, followed by vacuum infection for 5 min, and co-culture at 25 °C in the dark for 1 day using callus as explants at an optical density (OD600) of 0.8. VaCIPK18 gene was transiently transformed into calli, and transcripts of the gene (endogenous and exogenous) were detected at higher levels than in non-transformed calli (endogenous). Moreover, after 10 days of treatment at 4 °C or -4 °C, the callus net weight of transformed callus was significantly higher than that of the untransformed callus, indicating that the VaCIPK18-overexpressing grape callus could improve cold tolerance. Overall, we establish a simple but effective transient transformation approach for grape callus, which could serve as a useful tool for the rapid assessment of gene function in this important crop.

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.

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.

Exogenous Melatonin Promotes Cold Tolerance in Grape Seedlings: Physiological, Transcriptomic, and Functional Evidence.

Melatonin (MEL) is an antioxidant molecule that enhances plant tolerance to environmental stress. However, the mechanisms by which MEL regulates cold signaling pathways in grapes under cold stress remain elusive. Here, we investigated the physiological and transcriptomic changes in grape seedlings treated with exogenous MEL to determine their protective role under cold stress. Results showed that 150 µM MEL effectively attenuated cold-induced cell damage by reducing reactive oxygen species (ROS) and preserving the chloroplast structure and function. MEL also inhibited tannin degradation, which contributed to its protective effect. Exogenous MEL promoted the synthesis of endogenous MEL, abscisic acid, auxin, and cytokinin while inhibiting gibberellin. Transcriptomic profiling revealed 776 differentially expressed transcripts in MEL-treated samples compared to controls. Functional analysis of a candidate hub gene, VvHSFA6b, showed that its overexpression in grape calli enhances cold tolerance by activating jasmonic acid synthesis pathway genes, promoting JA accumulation, and inhibiting JAZ-repressed transcription factors.

Mdm-miR160-MdARF17-MdWRKY33 module mediates freezing tolerance in apple.

Apple (Malus domestica) trees are vulnerable to freezing temperatures. Cold resistance in woody perennial plants can be improved through biotechnological approaches. However, genetic engineering requires a thorough understanding of the molecular mechanisms of the tree's response to cold. In this study, we demonstrated that the Mdm-miR160-MdARF17-MdWRKY33 module is crucial for apple freezing tolerance. Mdm-miR160 plays a negative role in apple freezing tolerance, whereas MdARF17, one of the targets of Mdm-miR160, is a positive regulator of apple freezing tolerance. RNA sequencing analysis revealed that in apple, MdARF17 mediates the cold response by influencing the expression of cold-responsive genes. EMSA and ChIP-qPCR assays demonstrated that MdARF17 can bind to the promoter of MdWRKY33 and promotes its expression. Overexpression of MdWRKY33 enhanced the cold tolerance of the apple calli. In addition, we found that the Mdm-miR160-MdARF17-MdWRKY33 module regulates cold tolerance in apple by regulating reactive oxygen species (ROS) scavenging, as revealed by (i) increased H2 O2 levels and decreased peroxidase (POD) and catalase (CAT) activities in Mdm-miR160e OE plants and MdARF17 RNAi plants and (ii) decreased H2 O2 levels and increased POD and CAT activities in MdmARF17 OE plants and MdWRKY33 OE calli. Taken together, our study uncovered the molecular roles of the Mdm-miR160-MdARF17-MdWRKY33 module in freezing tolerance in apple, thus providing support for breeding of cold-tolerant apple cultivars.

Phenotypically abnormal cotyledonary Vitis vinifera embryos differ in anatomy, endogenous hormone levels and transcriptome profiles.

In many perennial fruit species, including grapevine (Vitis vinifera L.), the highly complex process of somatic embryogenesis (SE) can result in the formation of a deformed embryo, although the underlying reasons are still poorly understood. Here, V. vinifera cv. 'Chardonnay' cotyledonary embryos with distinct morphologies were used to address this issue. Normal cotyledonary embryos (NCEs) and elongated cotyledonary embryos (ECEs) were observed to have better-developed vasculature and shoot meristems than the vitrified cotyledonary embryos (VCEs) and fused cotyledonary embryos (FCEs), but ECEs were less developed. We determined that the morphological differences in these phenotypically abnormal embryos were likely associated with endogenous hormone levels, since concentrations of the phytohormones indoleacetic acid (IAA) and abscisic acid (ABA) in NCEs were higher than in the other three types. Comparative transcriptome analysis revealed large differences in gene expression of the hormone signaling pathways in normal and abnormal cotyledonary embryos. Weighted gene co-expression network analysis of the different cotyledonary types allowed the identification of co-regulated gene modules associated with SE, suggesting a role for ERF family genes and other transcription factors (TFs) in regulating morphology. Moreover, an analysis of morphology-specific gene expression indicated that the activation of a specific protein kinase, small heat shock proteins (sHSPs) and certain TFs was closely associated with the formation of normal cotyledonary embryos. Our comparative analyses provide insights into the gene networks regulating somatic cotyledon development and open new avenues for research into plant regeneration and functional genomic studies of malformed embryos.

Maintaining the long-term accuracy of water distribution models with data assimilation methods: A comparative study.

The upgrading of water supply services is calling for more accurate and adaptive numerical models to give insight into actual water distribution systems (WDSs), which underlines the importance of carefully calibrating model parameters. Due to unavoidable uncertainties in the calibration process such as measurement errors, errors in model parameters assumed to be known, and local-optimum of calibration algorithms, calibrated parameters could still contain non-negligible latent errors, and the calibrated model may not able to maintain its long-term accuracy when operating conditions change. To solve this problem, there is growing interest in adopting data assimilation (DA) methods to utilize more comprehensive information in long-term measurements to reduce the impact of uncertainties and maintain the accuracy and stability of calibrated models. In this study, two traditional calibration methods and four DA methods were tested and compared in two WDSs with different structures, which aims to form a general understanding of the behavior and applicability of different methods. The calibration results show that DA methods perform better than traditional methods and are more robust to different types of uncertainties, which provide an effective way to maintain the long-term accuracy of WDS models to enable better management of WDSs. Ensemble-based DA methods such as Particle Filter (PF) and Inferential-Measurement Kalman Filter (IMKF) performed well in the real-life system. They avoid linear approximation and can better estimate the impact of uncertainties to assimilate accurate correction information of the parameters. Gradient-based DA methods such as Extended Kalman Filter (EKF) and Variational Bayesian Adaptive Kalman Filter (VBAKF) have lower computational demand, but they are found to be less robust when dealing with large system uncertainties and nonlinearities.

Bridging hydraulics and graph signal processing: A new perspective to estimate water distribution network pressures.

The low spatial density of monitored nodal pressures (nodal heads) has already become a bottleneck restricting the development of smart technologies for water distribution networks (WDNs). Inferring unknown nodal heads through available WDN information is an effective way to bypass data limitations, but an accurate and easy-to-implement method is still absent. For general WDNs, the spatial distribution of nodal heads is approximately 'smooth' as there are few dramatic head changes. If heads can be divided into components with different spatial varying speeds, then they can be approximated by a few slow varying components. On this basis, a graph-based head reconstruction (GHR) method is proposed, which employs graph signal processing technologies to reconstruct the slow varying parts to estimate unknown nodal heads. Four metrics are proposed to bridge WDN hydraulics and signal processing to quantify the similarity of adjacent nodal heads, which enhance the smoothness of heads over the graph, and thus increase estimation accuracy. GHR was tested with different parameter settings and compared with other head estimation methods. Results showed that GHR has less restrictive parameter requirements compared with hydraulic simulation, and outperforms traditional data interpolation methods with better accuracy. At a larger looped network under potential model uncertainties and measurement errors, GHR still accurately estimated the heads for more than 10,000 unknown nodes, achieving a mean absolute error of 0.13 m using only 100 pressure meters. Thus the proposed method provides an efficient, robust, and convenient way to estimate unknown nodal heads in WDNs.

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.

Complete chloroplast genomes of Chinese wild-growing Vitis species: molecular structures and comparative and adaptive radiation analysis.

As a basalmost family of Vitaceae, Chinese wild Vitis species offer key insights into the demographic history of grapes. In this study, we obtained 10 complete chloroplast (cp) genomes from Chinese wild-growing Vitis species based on our whole genome re-sequencing data. These chloroplast genomes ranged from 160,838 to 232,020 bp in size and exhibited typical quadripartite structures. Comparative analyses revealed that inverted repeat (IR) regions are especially abundant and contribute to cp genome arrangements. Phylogenetic analysis of the whole Vitis cp genomes supported three clearly partitioned main origins, in keeping with their geographic distributions, among which East Asian species from China were found to be sister species with Eurasian Vitis species but exhibited significant divergence from the North American group. Two well-supported subgroups were observed within the Chinese wild-growing Vitis species. Among these species, Vitis piasezkii and Vitis betulifolia were closely related species, exhibiting a support rate of 100%. The molecular clock-based divergence time suggested that the earliest split subspecies was Vitis pseudoreticulata, which further indicated that the origin and initial gene pool are located in southern China (the habitat of V. pseudoreticulata is located in the region). Coincidentally, the divergence time was during the Pleistocene period (2.6-0.1 Ma). Due to glacial/interglacial temperature fluctuations, cold-adapted subspecies, e.g., Vitis amurensis, could re-colonize new habitats. Our results may help to elucidate the adaptive radiation of Chinese wild Vitis species in different environments.

Transcriptional changes during tomato ripening and influence of brackish water irrigation on fruit transcriptome and sugar content.

Efficient management and utilization of brackish water irrigation help to minimize yield losses and promote fruit quality and sugar content in tomato fruit. However, the functional genes involved in sugar metabolic pathways and potential molecular pathways responsive to brackish water irrigation remain unknown. To this end, physiological responses and comparative transcriptional profiling was used to analyze the tomato fruit during the white-ripe period (CK1) and mature period (CK2) in plants grown under four water management strategies (rotating irrigation with brackish and fresh water during fruit development, T1; fresh water irrigation, T2; mixed brackish and fresh water irrigation, T3; mixed water and fresh water irrigation in sequence, T4). Comparative analysis revealed that during fruit development (CK2 cv CK1) differentially expressed genes (DEGs) involved in photosynthetic pathways and sucrose-starch metabolism were downregulated. However, two DEGs encoding putative beta-fructofuranosidases were significantly upregulated at the mature stage, which promoted the accumulation of glucose and fructose in CK2. Comparing four types of management strategies, rotating irrigation with brackish water and fresh water (T1) led to reprograming of global gene expression. Moreover, the upregulated DEGs in T1 were significantly enriched for signaling, hormone metabolism, and stress tolerance, suggesting the coordination of both stresses signaling as well as the plant hormone. These results provide a valuable reference for rational use of brackish water in the production of high-quality tomato in arid and semi-arid regions.

Deep learning identifies accurate burst locations in water distribution networks.

Pipe bursts in water distribution networks lead to considerable water loss and pose risks of bacteria and pollutant contamination. Pipe burst localisation methods help water service providers repair the burst pipes and restore water supply timely and efficiently. Although methods have been reported on burst detection and localisation, there is a lack of studies on accurate localisation of a burst within a potential district by accessible meters. To address this, a novel Burst Location Identification Framework by Fully-linear DenseNet (BLIFF) is proposed. In this framework, additional pressure meters are placed at limited, optimised places for a short period (minutes to hours) to monitor system behaviour after the burst. The fully-linear DenseNet (FL-DenseNet) newly developed in this study modifies the state-of-the-art deep learning algorithm to effectively extract features in the limited pressure signals for accurate burst localisation. BLIFF was tested on a benchmark network with different parameter settings, which showed that accurate burst localisation results can be achieved even with high model uncertainties. The framework was also applied to a real-life network, in which 57 of the total 58 synthetic bursts in the potential burst district were correctly located when the top five most possible pipes are considered and among them, 37 were successfully located when considering only the top one. Only one failed because of the very small pipe diameter and remote location. Comparisons with DenseNet and the traditional fully linear neural network demonstrate that the framework can effectively narrow the potential burst district to one or several pipes with good robustness and applicability. Codes are available at https://github.com/wizard1203/waternn.

KCa3.1 deficiency attenuates neuroinflammation by regulating an astrocyte phenotype switch involving the PI3K/AKT/GSK3ß pathway.

Neuroinflammation may induce a phenotype switch to reactive astrogliosis in neurodegenerative disorders. The calcium-activated potassium channel (KCa3.1) is active in the phenotypic switch that occurs during astrogliosis in Alzheimer's disease and ischemic stroke. Here, transcriptome sequencing (RNA-Seq), immunohistochemistry, western blotting, pharmacological blockade, and calcium imaging were used to investigate astrocyte KCa3.1 activity in neuroinflammation, Tau accumulation, and insulin signaling deficits in male wild-type C57BL/6 and KCa3.1-/- knockout (KO) mice, and in primary astrocyte cultures. KCa3.1 deficiency in KO mice decreased lipopolysaccharide (LPS)-induced memory deficits, neuronal loss, glial activation, Tau phosphorylation, and insulin signaling deficits in vivo. KCa3.1 expression in astrocytes was associated with LPS-induced upregulation of the Orai1 store-operated Ca2+ channel protein. The KCa3.1 channel was found to regulate store-operated Ca2+ overload through an interaction with Orai1 in LPS-induced reactive astrocytes. The LPS-induced effects on KCa3.1 and Orai1 indirectly promoted astrogliosis-related changes via the PI3K/AKT/GSK3ß and NF-κB signaling pathways in vitro. Unbiased evaluation of RNA-Seq results for actively translated RNAs confirmed that substantial astrocyte diversity was associated with KCa3.1 deficiency. Our results suggest that KCa3.1 regulated astrogliosis-mediated neuroinflammation, Tau accumulation, and insulin signaling deficiency via PI3K/AKT/GSK3ß and NF-κB signaling pathways, and contributing to neuronal loss and memory deficits in this neuroinflammation mouse model.

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.

Genome-wide evolutionary characterization and expression analyses of major latex protein (MLP) family genes in Vitis vinifera.

The major latex protein/ripening-related protein (MLP/RRP) subfamily is known to be involved in a wide range of biological processes of plant development and various stress responses. However, the biological function of MLP/RRP proteins is still far from being clear and identification of them may provide important clues for understanding their roles. Here, we report a genome-wide evolutionary characterization and gene expression analysis of the MLP family in European Vitis species. A total of 14 members, was found in the grape genome, all of which are located on chromosome 1, where are predominantly arranged in tandem clusters. We have noticed, most surprisingly, promoter-sharing by several non-identical but highly similar gene members to a greater extent than expected by chance. Synteny analysis between the grape and Arabidopsis thaliana genomes suggested that 3 grape MLP genes arose before the divergence of the two species. Phylogenetic analysis provided further insights into the evolutionary relationship between the genes, as well as their putative functions, and tissue-specific expression analysis suggested distinct biological roles for different members. Our expression data suggested a couple of candidate genes involved in abiotic stresses and phytohormone responses. The present work provides new insight into the evolution and regulation of Vitis MLP genes, which represent targets for future studies and inclusion in tolerance-related molecular breeding programs.

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.

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.

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.