Dissecting the effect of ethylene in the transcriptional regulation of chilling treatment in grapevine leaves.

Ethylene (ETH) plays important roles in various development programs and stress responses in plants. In grapevines, ETH increased dramatically under chilling stress and is known to positively regulate cold tolerance. However, the role of ETH in transcriptional regulation during chilling stress of grapevine leaves is still not clear. To address this gap, targeted hormone profiling and transcriptomic analysis were performed on leaves of Vitis amurensis under chilling stress with and without aminoethoxyvinylglycine (AVG, a inhibitor of ETH synthesis) treatment. APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) and WRKY transcription factors (TF) were only the two highly enriched TF families that were consistently up-regulated during chilling stress but inhibited by AVG. The comparison of leaf transcriptomes between chilling treatment and chilling with AVG allowed the identification of potential ETH-regulated genes. Potential genes that are positively regulated by ETH are enriched in solute transport, protein biosynthesis, phytohormone action, antioxidant and carbohydrate metabolism. Conversely, genes related to the synthesis and signaling of ETH, indole-3-acetic acid (IAA), abscisic acid (ABA) were up-regulated by chilling treatment but inhibited by AVG. The contents of ETH, ABA and IAA also paralleled with the transcriptome data, which suggests that the response of ABA and IAA during chilling stress may regulate by ETH signaling, and together may belong to an integrated network of hormonal signaling pathways underpinning chilling stress response in grapevine leaves. Together, these findings provide new clues for further studying the complex regulatory mechanism of ETH under low-temperature stress in plants more generally and new opportunities for breeding cold-resilient grapevines.

A genome for Cissus illustrates features underlying its evolutionary success in dry savannas.

Cissus is the largest genus in Vitaceae and is mainly distributed in the tropics and subtropics. Crassulacean acid metabolism (CAM), a photosynthetic adaptation to the occurrence of succulent leaves or stems, indicates that convergent evolution occurred in response to drought stress during species radiation. Here we provide the chromosomal level assembly of Cissus rotundifolia (an endemic species in Eastern Africa) and a genome-wide comparison with grape to understand genome divergence within an ancient eudicot family. Extensive transcriptome data were produced to illustrate the genetics underpinning C. rotundifolia's ecological adaption to seasonal aridity. The modern karyotype and smaller genome of C. rotundifolia (n = 12, 350.69 Mb/1C), which lack further whole-genome duplication, were mainly derived from gross chromosomal rearrangements such as fusions and segmental duplications, and were sculpted by a very recent burst of retrotransposon activity. Bias in local gene amplification contributed to its remarkable functional divergence from grape, and the specific proliferated genes associated with abiotic and biotic responses (e.g. HSP-20, NBS-LRR) enabled C. rotundifolia to survive in a hostile environment. Reorganization of existing enzymes of CAM characterized as diurnal expression patterns of relevant genes further confer the ability to thrive in dry savannas.

Characterization of Flavonoids and Transcripts Involved in Their Biosynthesis in Different Organs of Cissus rotundifolia Lam.

Cissus rotundifolia Lam. is used as a medicinal herb and vegetable. Flavonoids are the major components for the therapeutic effects. However, flavonoids constituents and expression profiles of related genes in C. rotundifolia organs are unknown. Colorimetric assay showed the highest flavonoid concentration in roots compared to the stem and leaf. Widely target-based metabolome analysis allowed tentative identification of 199 compounds in three organs. Flavonols and flavones were the dominant flavonoids subclasses. Among the metabolites, 171 were common in the three organs. Unique accumulation profile was observed in the root while the stem and leaf exhibited relatively similar patterns. In the root, six unique compounds (jaceosidin, licoagrochalcone D, 8-prenylkaempferol, hesperetin 7-O-(6″malonyl) glucoside, aureusidin, apigenin-4'-O-rhamnoside) that are used for medicinal purposes were detected. In total, 18,427 expressed genes were identified from transcriptome of the three organs covering about 60% of annotated genes in C. rotundifolia genome. Fourteen gene families, including 52 members involved in the main pathway of flavonoids biosynthesis, were identified. Their expression could be found in at least one organ. Most of the genes were highly expressed in roots compared to other organs, coinciding with the metabolites profile. The findings provide fundamental data for exploration of metabolites biosynthesis in C. rotundifolia and diversification of parts used for medicinal purposes.

The transcription factor VaNAC17 from grapevine (Vitis amurensis) enhances drought tolerance by modulating jasmonic acid biosynthesis in transgenic Arabidopsis.

KEY MESSAGE: Expression of VaNAC17 improved drought tolerance in transgenic Arabidopsis by upregulating stress-responsive genes, modulating JA biosynthesis, and enhancing ROS scavenging. Water deficit severely affects the growth and development of plants such as grapevine (Vitis spp.). Members of the NAC (NAM, ATAF1/2, and CUC2) transcription factor (TF) family participate in drought-stress-induced signal transduction in plants, but little is known about the roles of NAC genes in drought tolerance in grapevine. Here, we explored the role of VaNAC17 in Vitis amurensis, a cold-hardy, drought-tolerant species of grapevine. VaNAC17 was strongly induced in grapevine by drought, exogenous abscisic acid (ABA), and methyl jasmonate (MeJA). A transient expression assay in yeast indicated that VaNAC17 functions as a transcriptional activator. Notably, heterologous expression of VaNAC17 in Arabidopsis thaliana enhanced drought tolerance. VaNAC17-expressing Arabidopsis plants showed decreased reactive oxygen species (ROS) accumulation compared to wild-type plants under drought conditions. RNA-seq analysis indicated that VaNAC17 expression increased the transcription of downstream stress-responsive genes after 5 days of drought treatment, especially genes involved in jasmonic acid (JA) biosynthesis (such as LOX3, AOC1 and OPR3) and signaling (such as MYC2, JAZ1, VSP1 and CORI3) pathways. Endogenous JA levels increased in VaNAC17-OE plants under drought stress. Taken together, these results indicate that VaNAC17 plays a positive role in drought tolerance by modulating endogenous JA biosynthesis and ROS scavenging.

Genome size, chromosome number determination, and analysis of the repetitive elements in Cissus quadrangularis.

Cissus quadrangularis (Vitaceae) is a perennial climber endemic to Africa and is characterized by succulent angular stems. The plant grows in arid and semi-arid regions of Africa especially in the African savanna. The stem of C. quadrangularis has a wide range of applications in both human and animal medicine, but there is limited cytogenetic information available for this species. In this study, the chromosome number, genome size, and genome composition for C. quadrangularis were determined. Flow cytometry results indicated that the genome size of C. quadrangularis is approximately 2C = 1.410 pg. Fluorescence microscopy combined with DAPI stain showed the chromosome numbers to be 2n = 48. It is likely that C. quadrangularis has a tetraploid genome after considering the basic chromosome numbers in Cissus genus (n = 10, 11, or 12). A combination of low-throughput genome sequencing and bioinformatics analysis allowed identification and quantification of repetitive elements that make up about 52% of the C. quadrangularis genome, which was dominated by LTR-retrotransposons. Two LTR superfamilies were identified as Copia and Gypsy, with 24% and 15% of the annotated clusters, respectively. The comparison of repeat elements for C. quadrangularis, Vitis vinifera, and four other selected members in the Cissus genus revealed a high diversity in the repetitive element components, which could suggest recent amplification events in the Cissus genus. Our data provides a platform for further studies on the phylogeny and karyotype evolution in this genus and in the family Vitaceae.

The ethylene response factor VaERF092 from Amur grape regulates the transcription factor VaWRKY33, improving cold tolerance.

Cold stress is a major limiting factor in grape (Vitis) productivity. In this study, we characterized a cold-responsive ethylene response factor (ERF) transcription factor, VaERF092, from Amur grape (Vitis amurensis). VaERF092 expression was induced by both low temperatures and the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC), but was suppressed by treatment with the ethylene inhibitor aminoethoxyvinylglycine (AVG) under cold conditions. Ectopic expression of VaERF092 in Arabidopsis thaliana enhanced cold tolerance. Co-expression network analysis of V. vinifera genes indicated that WRKY33 might be a downstream target of VaERF092. This hypothesis was supported by the fact that VaWRKY33 was expressed temporally after VaERF092 expression and could also be induced by cold and ACC, and inhibited by AVG. Yeast one-hybrid, transient ß-glucuronidase (GUS) and dual-luciferase reporter assays provided evidence for an interaction between VaERF092 and a GCC-box element in the VaWRKY33 promoter. In addition, heterologous overexpression of VaWRKY33 in A. thaliana resulted in enhanced cold tolerance. VaERF092- and VaWRKY33 overexpressing grape calli showed lower low-temperature exothermic values than the empty vector (EV) calli, indicating enhanced tolerance to cold. Together, these results indicated that VaERF092 regulates VaWRKY33 through binding to its promoter GCC-box, leading to enhanced cold stress tolerance.

Overexpression of VaWRKY12, a transcription factor from Vitis amurensis with increased nuclear localization under low temperature, enhances cold tolerance of plants.

KEY MESSAGE: Overexpression of VaWRKY12, whose nuclear translocation increased under low temperature, enhanced the cold tolerance of Arabidopsis and grapevine calli and significantly increased the expression of antioxidant-related genes. Low temperature causes injuries to buds during winter and to young shoots during early spring, thereby affecting grapevine quality and yield. Understanding the regulatory mechanisms of cold stress responses is essential for the breeding of new grapevine cultivars with excellent cold tolerance. Previous studies indicated that WRKY family genes are induced by low temperature in grapevine, but their function in cold stress responses was not clear. Here, a cold-induced WRKY gene, named VaWRKY12, was cloned from Vitis amurensis, which displays remarkable cold tolerance. An atypical transmembrane (TM) region was found in its C-terminal region. Transient expression assays showed that VaWRKY12 was localized in the nucleus and cytoplasm at normal temperature but only in the nucleus after cold treatment. By contrast, a truncated version of VaWRKY12 without the TM region was found specifically in the nucleus at normal temperature, and its binding activity to tandem W-box elements in yeast was stronger than that of VaWRKY12, indicating that the TM region might affect the location and function of VaWRKY12. Overexpression of VaWRKY12 enhanced the cold tolerance of transformed Arabidopsis and grapevine calli. Transcriptome data revealed that the expression of genes encoding antioxidant enzymes, including peroxidases and glutathione S-transferases, was upregulated after cold treatment in VaWRKY12-overexpressing grapevine calli compared to the control calli. This study identifies candidate target genes as a basis for further studies on the roles of VaWRKY12 in cold stress responses in grapevine.

Agent-based intelligent medical diagnosis system for patients.

BACKGROUND: According to the analysis of the challenges faced by the current public health circumstances such as the sharp increase in elderly patients, limited medical personnel, resources and technology, the agent-based intelligent medical diagnosis system for patients (AIMDS) is proposed in this research. OBJECTIVE: Based on advanced sensing technology and professional medical knowledge, the AIMDS can output the appropriate medical prescriptions and food prohibition when the physical signs and symptoms of the patient are inputted. METHODS: Three core modules are designed include sensing module, intuition-based fuzzy set theory/medical diagnosis module, and medical knowledge module. RESULTS: The result shows that the optimized prescription can reach the desired level, with great curative effect for patient disease, through a case study simulation. CONCLUSION: The presented AIMDS can integrate sensor technique and intelligent medical diagnosis methods to make an accurate diagnosis, resulting in three-type of optimized descriptions for patient selection.