Physiological and molecular responses of 'Hamlin' sweet orange trees expressing the VvmybA1 gene under cold stress conditions.

MAIN CONCLUSION: Overexpression of VvmybA1 transcription factor in 'Hamlin' citrus enhances cold tolerance by increasing anthocyanin accumulation. This results in improved ROS scavenging, altered gene expression, and stomatal regulation, highlighting anthocyanins' essential role in citrus cold acclimation. Cold stress is a significant threat to citrus cultivation, impacting tree health and productivity. Anthocyanins are known for their role as pigments and have emerged as key mediators of plant defense mechanisms against environmental stressors. This study investigated the potential of anthocyanin overexpression regulated by grape (Vitis vinifera) VvmybA1 transcription factor to enhance cold stress tolerance in citrus trees. Transgenic 'Hamlin' citrus trees overexpressing VvmybA1 were exposed to a 30-day cold stress period at 4 °C along with the control wild-type trees. Our findings reveal that anthocyanin accumulation significantly influences chlorophyll content and their fluorescence parameters, affecting leaf responses to cold stress. Additionally, we recorded enhanced ROS scavenging capacity and distinct expression patterns of key transcription factors and antioxidant-related genes in the transgenic leaves. Furthermore, VvmybA1 overexpression affected stomatal aperture regulation by moderating ABA biosynthesis, resulting in differential responses in a stomatal opening between transgenic and wild-type trees under cold stress. Transgenic trees exhibited reduced hydrogen peroxide levels, enhanced flavonoids, radical scavenging activity, and altered phytohormonal profiles. These findings highlighted the role of VvmybA1-mediated anthocyanin accumulation in enhancing cold tolerance. The current study also underlines the potential of anthocyanin overexpression as a critical regulator of the cold acclimation process by scavenging ROS in plant tissues.

Transcriptome and Metabolome Analyses of Aroma Differences between Chardonnay and a Chardonnay Bud Sport.

Chardonnay is one of the most popular white grape wine varieties in the world, but this wine lacks typical aroma, considered a sensory defect. Our research group identified a Chardonnay bud sport with typical muscat characteristics. The goal of this work was to discover the key candidate genes related to muscat characteristics in this Chardonnay bud sport to reveal the mechanism of muscat formation and guide molecular design breeding. To this end, HS-SPME-GC-MS and RNA-Seq were used to analyze volatile organic compounds and the differentially expressed genes in Chardonnay and its aromatic bud sport. Forty-nine volatiles were identified as potential biomarkers, which included mainly aldehydes and terpenes. Geraniol, linalool, and phenylacetaldehyde were identified as the main aroma components of the mutant. The GO, KEGG, GSEA, and correlation analysis revealed HMGR, TPS1, TPS2, TPS5, novel.939, and CYP450 as key genes for terpene synthesis. MAO1 and MAO2 were significantly downregulated, but there was an increased content of phenylacetaldehyde. These key candidate genes provide a reference for the development of functional markers for muscat varieties and also provide insight into the formation mechanism of muscat aroma.

Supplementing with monochromatic blue LED light during the day, rather than at night, increases anthocyanins in the berry skin of grapevine (Vitis vinifera L.).

MAIN CONCLUSION: Supplying monochromatic blue LED light during the day, but not at night, promotes early coloration and improves anthocyanin accumulation in the skin of grape berries. Specific light spectra, such as blue light, are known to promote the biosynthesis and accumulation of anthocyanins in fruit skins. However, research is scarce on whether supplement of blue light during different periods of one day can differ in their effect. Here, we compared the consequences of supplying blue light during the day and night on the accumulation of anthocyanins in pigmented grapevine (Vitis vinifera) berries. Two treatments of supplemented monochromatic blue light were tested, with light emitting diodes (LED) disposed close to the fruit zone, irradiating between 8:00 and 18:00 (Dayblue) or between 20:00 and 6:00 (Nightblue). Under the Dayblue treatment, berry coloration was accelerated and total anthocyanins in berry skins increased faster than the control (CK) and also when compared to the Nightblue condition. In fact, total anthocyanin content was similar between CK and Nightblue. qRT-PCR analysis indicated that Dayblue slightly improved the relative expression of the anthocyanin-structural gene UFGT and its regulator MYBA1. Instead, the expression of the light-reception and -signaling related genes CRY, HY5, HYH, and COP1 rapidly increased under Dayblue. This study provides insights into the effect of supplementing monochromatic LED blue light during the different periods of one day, on anthocyanins accumulation in the berry skin.

Evolution of PIN gene family between monocotyledons and dicotyledons and VvPIN1 negatively regulates freezing tolerance in transgenic Arabidopsis.

The PIN-FORMED (PIN) proteins mediate the auxin flow throughout the plant and have been identified in many species. However, evolution differences in the PIN gene families have not been systematically analyzed, and their functions under abiotic stresses in grape are largely unexplored. In this study, 373 PIN genes were identified from 25 species and divided into 3 subgroups. Physicochemical properties analysis indicated that most of the PIN proteins were unstable alkaline hydrophobic proteins in nature. The synteny analysis showed that the PINs contained strong gene duplication. Motif composition revealed that PIN gene sequence differences between monocotyledons and dicotyledons were due to evolutionary-induced base loss, and the loss was more common in dicotyledonous. Meanwhile, the codon usage bias showed that the PINs showed stronger codon preference in monocotyledons, monocotyledons biased towards C3s and G3s, and dicotyledons biased towards A3s and T3s. In addition, the VvPIN1 can interact with VvCSN5. Significantly, under freezing treatment, the ion leakage, O 2 · - $$ \left({O}_2^{\cdotp -}\right) $$ , H2O2, and malondialdehyde (MDA) were obviously increased, while the proline (Pro) content, peroxidase (POD) activity, and glutathione (GSH) content were decreased in VvPIN1-overexpressing Arabidopsis compared to the wild type (WT). And quantitative real-time PCR (qRT-PCR) showed that AtICE1, AtICE2, AtCBF1, AtCBF2, and AtCBF3 were down-regulated in overexpression lines. These results demonstrated that VvPIN1 negatively regulated the freezing tolerance in transgenic Arabidopsis. Collectively, this study provides a novel insight into the evolution and a basis for further studies on the biological functions of PIN genes in monocotyledons and dicotyledons.

Plasmopara viticola effector PvCRN20 represses the import of VvDEG5 into chloroplasts to suppress immunity in grapevine.

Chloroplasts play a crucial role in plant defense against pathogens, making them primary targets for pathogen effectors that suppress host immunity. This study characterizes the Plasmopara viticola CRN-like effector, PvCRN20, which interacts with DEG5 in the cytoplasm but not with its interacting protein, DEG8, which is located in the chloroplast. By transiently overexpressing in tobacco leaves, we show that PvCRN20 could inhibit INF1- and Bax-triggered cell death. Constitutive expression of PvCRN20 suppresses the accumulation of reactive oxygen species (ROS) and promotes pathogen colonization. PvCRN20 reduces DEG5 entry into chloroplasts, thereby disrupting DEG5 and DEG8 interactions in chloroplasts. Overexpression of VvDEG5 and VvDEG8 induces ROS accumulation and enhances grapevine resistance to P. viticola, whereas knockout of VvDEG8 represses ROS production and promotes P. viticola colonization. Consistently, ectopic expression of VvDEG5 and VvDEG8 in tobacco promotes chloroplast-derived ROS accumulation, whereas co-expression of PvCRN20 counteracted this promotion by VvDEG5. Therefore, DEG5 is essential for the virulence function of PvCRN20. Although PvCRN20 is located in both the nucleus and cytoplasm, only cytoplasmic PvCRN20 suppresses plant immunity and promotes pathogen infection. Our results reveal that PvCRN20 dampens plant defenses by repressing the chloroplast import of DEG5, thus reducing host ROS accumulation and facilitating pathogen colonization.

Integrative genomics reveals the polygenic basis of seedlessness in grapevine.

Seedlessness is a crucial quality trait in table grape (Vitis vinifera L.) breeding. However, the development of seeds involved intricate regulations, and the polygenic basis of seed abortion remains unclear. Here, we combine comparative genomics, population genetics, quantitative genetics, and integrative genomics to unravel the evolution and polygenic basis of seedlessness in grapes. We generated the haplotype-resolved genomes for two seedless grape cultivars, "Thompson Seedless" (TS, syn. "Sultania") and "Black Monukka" (BM). Comparative genomics identified a ∼4.25 Mb hemizygous inversion on Chr10 specific in seedless cultivars, with seedless-associated genes VvTT16 and VvSUS2 located at breakpoints. Population genomic analyses of 548 grapevine accessions revealed two distinct clusters of seedless cultivars, and the identity-by-descent (IBD) results indicated that the origin of the seedlessness trait could be traced back to "Sultania." Introgression, rather than convergent selection, shaped the evolutionary history of seedlessness in grape improvement. Genome-wide association study (GWAS) analysis identified 110 quantitative trait loci (QTLs) associated with 634 candidate genes, including previously unidentified candidate genes, such as three 11S GLOBULIN SEED STORAGE PROTEIN and two CYTOCHROME P450 genes, and well-known genes like VviAGL11. Integrative genomic analyses resulted in 339 core candidate genes categorized into 13 functional categories related to seed development. Machine learning-based genomic selection achieved a remarkable prediction accuracy of 97% for seedlessness in grapevines. Our findings highlight the polygenic nature of seedlessness and provide candidate genes for molecular genetics and an effective prediction for seedlessness in grape genomic breeding.

Multiple deletions of candidate effector genes lead to the breakdown of partial grapevine resistance to downy mildew.

Grapevine downy mildew, caused by the oomycete Plasmopara viticola (P. viticola, Berk. & M. A. Curtis; Berl. & De Toni), is a global threat to Eurasian wine grapes Vitis vinifera. Although resistant grapevine varieties are becoming more accessible, P. viticola populations are rapidly evolving to overcome these resistances. We aimed to uncover avirulence genes related to Rpv3.1-mediated grapevine resistance. We sequenced the genomes and characterized the development of 136 P. viticola strains on resistant and sensitive grapevine cultivars. A genome-wide association study was conducted to identify genomic variations associated with resistant-breaking phenotypes. We identified a genomic region associated with the breakdown of Rpv3.1 grapevine resistance (avrRpv3.1 locus). A diploid-aware reassembly of the P. viticola INRA-Pv221 genome revealed structural variations in this locus, including a 30 kbp deletion. Virulent P. viticola strains displayed multiple deletions on both haplotypes at the avrRpv3.1 locus. These deletions involve two paralog genes coding for proteins with 800-900 amino acids and signal peptides. These proteins exhibited a structure featuring LWY-fold structural modules, common among oomycete effectors. When transiently expressed, these proteins induced cell death in grapevines carrying Rpv3.1 resistance, confirming their avirulence nature. This discovery sheds light on the genetic mechanisms enabling P. viticola to adapt to grapevine resistance, laying a foundation for developing strategies to manage this destructive crop pathogen.

Evolutionary analysis of genes from WOX family and their expression profile in grape (Vitis vinifera) under different stresses.

The WUSCHEL-related homeobox (WOX) transcription factor family plays critical roles in plant growth, development, and stress adaptation, but the biological functions in response to various stress of the WOX gene family have not been extensively researched in grapevine (Vitis vinifera ). In this study, 12 grapevine WOXs were identified from the grapevine genome. Quantitative PCR and microarray expression profiling found that the expression of WOXs had an obvious tissue-specific pattern. Conjoint analysis between various tissues and treated materials indicated VvWUS1 expression is associated with expression of genes from grapevine rupestris stem pitting-associated virus; and VvWOX3 with grapevine fanleaf virus. The gene expression patterns of the WOXs in grape were different under salt stress, with VvWOX8/9 , VvWUS1 , and VvWOX3 responding more strongly to salt stress than control by 18.20-, 9.50-, and 9.19-fold. This study further improves understanding of the evolution and function of the WOX gene family, and offers a theoretical framework and reference for breeding grapevine to better tolerate adversity and permit cultivation of seedlings free of viruses.

Overexpression of a Grape WRKY Transcription Factor VhWRKY44 Improves the Resistance to Cold and Salt of Arabidopsis thaliana.

Plants are often exposed to biotic or abiotic stress, which can seriously impede their growth and development. In recent years, researchers have focused especially on the study of plant responses to biotic and abiotic stress. As one of the most widely planted grapevine rootstocks, 'Beta' has been extensively proven to be highly resistant to stress. However, further research is needed to understand the mechanisms of abiotic stress in 'Beta' rootstocks. In this study, we isolated and cloned a novel WRKY transcription factor, VhWRKY44, from the 'Beta' rootstock. Subcellular localization analysis revealed that VhWRKY44 was a nuclear-localized protein. Tissue-specific expression analysis indicated that VhWRKY44 had higher expression levels in grape roots and mature leaves. Further research demonstrated that the expression level of VhWRKY44 in grape roots and mature leaves was highly induced by salt and cold treatment. Compared with the control, Arabidopsis plants overexpressing VhWRKY44 showed stronger resistance to salt and cold stress. The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were significantly increased, and the contents of proline, malondialdehyde (MDA) and chlorophyll were changed considerably. In addition, significantly higher levels of stress-related genes were detected in the transgenic lines. The results indicated that VhWRKY44 was an important transcription factor in 'Beta' with excellent salt and cold tolerance, providing a new foundation for abiotic stress research.

A single viral amino acid shapes the root system architecture of a plant host upon virus infection.

BACKGROUND: Grapevine fanleaf virus (GFLV) is one of the most detrimental viral pathogens of grapevines worldwide but no information is available on its effect on the root system architecture (RSA) of plant hosts. We used two wildtype GFLV strains and their single amino acid mutants to assess RSA traits in infected Nicotiana benthamiana and evaluate transcriptomic changes in host root gene expression in replicated time course 3'RNA-Seq experiments. Mutations targeted the multi-functional GFLV-encoded protein 1EPol*/Sd, a putative RNA-dependent RNA polymerase and determinant of foliar symptoms in N. benthamiana plants. RESULTS: Plant infection with wildtype GFLV strain GHu and mutant GFLV strain F13 1EPol G802K, both carrying a lysine in position 802 of protein 1EPol*/Sd, resulted in a significantly lower number of root tips (-30%), and a significantly increased average root diameter (+ 20%) at 17 days post inoculation (dpi) in comparison with roots of mock inoculated plants. In contrast, the RSA of plants infected with wildtype GFLV strain F13 and mutant GFLV strain GHu 1EPol K802G, both carrying a glycine in position 802 of protein 1EPol*/Sd, resembled that of mock inoculated plants. Modifications of RSA traits were not associated with GFLV titer. Root tissue transcriptome analysis at 17 dpi indicated dysregulation of pattern recognition receptors, plant hormones, RNA silencing, and genes related to the production of reactive oxygen species (ROS). For wildtype GFLV strain GHu, RSA modifications were correlated with an abundant accumulation of ROS in the pericycle of primary roots at 7 dpi and the duration of vein clearing symptom expression in apical leaves. Dysegulation of a hypersensitive response was an overarching gene ontology found through enrichment analyses of 3'RNA-Seq data. CONCLUSIONS: Our findings revealed the causative role of lysine in position 802 of protein 1EPol*/Sd in a novel RSA phenotype during viral infection and documented GFLV-N. benthamiana interactions at the root level based on (i) antiviral response, (ii) receptor mediated production of ROS, and (iii) hormone regulation. A correlation between above and below ground symptoms was reported for the first time in plants infected with wildtype GFLV strain GHu. Further work is warranted to test whether the modified RSA of a plant host might impact GFLV acquisition and transmission by the ectoparasitic dagger nematode Xiphinema index.

Transition of survival strategies under global climate shifts in the grape family.

Faced with environmental changes, plants may either move to track their ancestral niches or evolve to adapt to new niches. Vitaceae, the grape family, has evolved diverse adaptive traits facilitating a global expansion in wide-ranging habitats, making it ideal for investigating transition between move and evolve strategies and exploring the underlying mechanisms. Here we inferred the patterns of biogeographic diversification and trait evolution in Vitaceae based on a robust phylogeny with dense sampling including 495 species (~52% of Vitaceae species). Vitaceae probably originated from Asia-the diversity centre of extant genera and the major source of dispersals. Boundaries of the Eocene, Oligocene and Miocene were identified as turning points in shifting strategies. A significant decrease in move strategy was identified during the Oligocene, followed by increases in move and evolve. After the Miocene, evolve began to dominate, during which increased niche opportunities and key trait innovations played important roles.

Volatomic differences among Vitis amurensis cultivars and its hybrids with V. vinifera revealed the effects of genotype, region, and vintage on grape aroma.

Vitis amurensis grape, an East Asian Vitis species, has excellent cold and disease resistance and exhibits high winemaking potential. In this study, the aroma compounds in grapes from five V. amurensis cultivars ('Beiguohong', 'Beiguolan', 'Shuangfeng', 'Shuanghong', 'Shuangyou') and three interspecific hybrids ('Beibinghong', 'Xuelanhong', 'Zuoyouhong') from two regions (Zuojia and Ji'an, Jilin, China) were identified via HS-SPME-GC/MS. The results showed that V. amurensis grapes had a greater concentration of aroma compounds than the interspecific hybrid berries. 'Beibinghong' was relatively rich in terpenes, although their concentrations were all lower than the threshold. 'Shuangfeng' contained more concentrations of free C6/C9 compounds, alcohols, aromatics and aldehydes/ketones than the other cultivars. The aroma characteristics of 'Beiguolan' and 'Shuanghong' were relatively similar. The grapes from the lower temperature and more fertile soil of Zuojia contained more C6/C9 compounds, norisoprenoids and alcohols, while aromatics were more abundant in the grapes from Ji'an, which was warmer than the Zuojia region. Herbaceous, floral, fruity and sweet were the main aroma series of V. amurensis grapes. Our study could provide a reference for the development and utilization of V. amurensis grapes and lay a foundation for the development of wild grape cultivars and the production of wines with characteristic styles.

Physical cell-cell contact elicits specific transcriptomic responses in wine yeast species.

Fermenting grape juice provides a habitat for a well-mapped and evolutionarily relevant microbial ecosystem consisting of many natural or inoculated strains of yeasts and bacteria. The molecular nature of many of the ecological interactions within this ecosystem remains poorly understood, with the partial exception of interactions of a metabolic nature such as competition for nutrients and production of toxic metabolites/peptides. Data suggest that physical contact between species plays a significant role in the phenotypic outcome of interspecies interactions. However, the molecular nature of the mechanisms regulating these phenotypes remains unknown. Here, we present a transcriptomic analysis of physical versus metabolic contact between two wine relevant yeast species, Saccharomyces cerevisiae and Lachancea thermotolerans. The data show that these species respond to the physical presence of the other species. In S. cerevisiae, physical contact results in the upregulation of genes involved in maintaining cell wall integrity, cell wall structural components, and genes involved in the production of H2S. In L. thermotolerans, HSP stress response genes were the most significantly upregulated gene family. Both yeasts downregulated genes belonging to the FLO family, some of which play prominent roles in cellular adhesion. qPCR analysis indicates that the expression of some of these genes is regulated in a species-specific manner, suggesting that yeasts adjust gene expression to specific biotic challenges or interspecies interactions. These findings provide fundamental insights into yeast interactions and evolutionary adaptations of these species to the wine ecosystem.IMPORTANCEWithin the wine ecosystem, yeasts are the most relevant contributors to alcoholic fermentation and wine organoleptic characteristics. While some studies have described yeast-yeast interactions during alcoholic fermentation, such interactions remain ill-defined, and little is understood regarding the molecular mechanisms behind many of the phenotypes observed when two or more species are co-cultured. In particular, no study has investigated transcriptional regulation in response to physical interspecies cell-cell contact, as opposed to the generally better understood/characterized metabolic interactions. These data are of direct relevance to our understanding of microbial ecological interactions in general while also creating opportunities to improve ecosystem-based biotechnological applications such as wine fermentation. Furthermore, the presence of competitor species has rarely been considered an evolutionary biotic selection pressure. In this context, the data reveal novel gene functions. This, and further such analysis, is likely to significantly enlarge the genome annotation space.

VlMYB4 and VlCDF3 co-targeted the VlLOG11 promoter to regulate fruit setting in grape (Vitis vinifera L).

KEY MESSAGE: The VlLOG11 mediates the cytokinin signaling pathway to regulate grape fruit setting. Fruit set, as an accepted agronomic trait, is inextricably linked with fruit quality and yield. Previous studies have demonstrated that exogenous treatment with the synthetic cytokinin analog, forchlorfenuron (CPPU), significantly enhances fruit set. In this study, a significant reduction in endogenous cytokinins was found by measuring the content of cytokinins in young grape berries after CPPU treatment. LONELY GUYs (VlLOGs), a key cytokinin-activating enzyme working in the biosynthesis pathway of cytokinins, exhibited differential expression. Some differentially expressed VlLOGs genes were presented by RNA seq data and their functions and regulation patterns were further investigated. The results showed that VlLOG11 was differentially expressed in young grape berries after CPPU treatment. Overexpression of VlLOG11 in tomato increases the amount of fruit set, and upregulated the expression of genes associated with cytokinin signaling including SlHK4, SlHK5, SlHP3, SlHP4, SlPHP1, SlPHP2. VlMYB4 and VlCDF3 could regulate the expression of VlLOG11 by directly binding to its promoter in young grape berries during fruit set. These results strongly demonstrated that VlMYB4/VlCDF3-VlLOG11 regulatory module plays a key role in the process of fruit setting in grape. This provided a basis for the molecular mechanism of VlLOG11-mediated cytokinin biosynthesis in young grape fruit set.

ADP-ribosylation factor 1 (ARF1) protein interacts with elicitor PvNLP7 from Plasmopara viticola to mediate PvNLP7-triggered immunity.

Revealing the effector-host molecular interactions is crucial for understanding the host immunity against Plasmopara viticola and devising innovative disease management strategies. As a pathogenic oomycete causing grapevine downy mildew, Plasmopara viticola employs various effectors to manipulate the defense systems of host plants. One of these P. viticola derived effectors is necrosis- and ethylene-inducing peptide 1 (Nep1) -like protein (PvNLP7), which has been known to elicit cell death and immune responses in plants. However, the underlying molecular mechanisms remain obscure, prompting the focus of this study. Through yeast two-hybrid screening, we have identified the Vitis rotundifolia ADP-ribosylation factor (VrARF1) as a host interactor of PvNLP7. This interaction is corroborated through bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP) assays. Heterologous expression of VrARF1 in Nicotiana benthamiana verifies its accumulation in both the cytoplasm and nucleus, and induction of cell death. Moreover, the VrARF1 gene is strongly induced during early P. viticola infection and upon PvNLP7 transient expression. Overexpression of the VrARF1 gene in grapevine and N. benthamiana enhances resistance to P. viticola and Phytophthora capsici, respectively, via induction of defense related genes PR1 and PR2. Conversely, virus-induced gene silencing (VIGS) of NbARF1 in N. benthamiana, homologous to VrARF1, markedly attenuates PvNLP7-triggered cell death and reduces the expression of four PTI marker genes (PTI5, Acre31, WRKY7 and Cyp71D20) and two defense related genes (PR1 and PR2), rendering plants transiently transformed with PvNLP7 more susceptible to oomycete P. capsici. These findings highlight the role of ARF1 in mediating PvNLP7-induced immunity and indicate its potential as a target for engineering disease-resistant transgenic plants against oomycete pathogens.

Genome-wide identified VvOFP genes family and VvOFP4 functional characterization provide insight into fruit shape in grape.

Ovate Family Proteins (OFPs) are emerging as novel transcriptional regulators of fruit shape. Despite their established role in various species, their involvement in regulating grape fruit shape remains understudied. This study encompassed a comprehensive evaluation of 16 grape OFP genes in total at the whole genome level. Phylogenetic and synteny analyses established a close relationship between grape VvOFP genes and their tomato counterparts. Expression profiling post-treatment with gibberellic acid (GA3) and thidiazuron (TDZ) revealed that certain OFP genes responded to these regulators, with VvOFP4 showing peak expression three days post-anthesis. Functional assays via overexpression of VvOFP4 in tobacco and tomato altered the morphology of both vegetative and reproductive organs, including leaves, stamens, and fruits/pods. Paraffin sections of transgenic tobacco stems and tomato fruits demonstrated that VvOFP4 overexpression modifies cell dimensions, leading to changes in organ morphology. Additionally, treatments with GA3 and TDZ similarly influenced the shape of grape pulp cells and thereby the overall fruit morphology. These findings suggest that the VvOFP4 gene plays a crucial role in fruit shape determination by modulating cell shape and presents a potential target for future grape breeding programs aimed at diversifying fruit shapes.

Trichomes and unique gene expression confer insect herbivory resistance in Vitis labrusca grapevines.

BACKGROUND: Grapevine (Vitis) is one of the world's most valuable fruit crops, but insect herbivory can decrease yields. Understanding insect herbivory resistance is critical to mitigating these losses. Vitis labrusca, a wild North American grapevine species, has been leveraged in breeding programs to generate hybrid grapevines with enhanced abiotic and biotic stress resistance, rendering it a valuable genetic resource for sustainable viticulture. This study assessed the resistance of V. labrusca acc. 'GREM4' and Vitis vinifera cv. 'PN40024' grapevines to Popillia japonica (Japanese beetle) herbivory and identified morphological and genetic adaptations underlying this putative resistance. RESULTS: 'GREM4' displayed greater resistance to beetle herbivory compared to 'PN40024' in both choice and no-choice herbivory assays spanning periods of 30 min to 19 h. 'GREM4' had significantly higher average leaf trichome densities than 'PN40024' and beetles preferred to feed on the side of leaves with fewer trichomes. When leaves from each species that specifically did not differ in trichome densities were fed on by beetles, significantly less leaf area was damaged in 'GREM4' (3.29mm2) compared to 'PN40024' (9.80mm2), suggesting additional factors beyond trichomes contributed to insect herbivory resistance in 'GREM4'. Comparative transcriptomic analyses revealed 'GREM4' exhibited greater constitutive (0 h) expression of defense response and secondary metabolite biosynthesis genes compared to 'PN40024', indicative of heightened constitutive defenses. Upon herbivory, 'GREM4' displayed a greater number of differentially expressed genes (690) compared to 'PN40024' (502), suggesting a broader response. Genes up-regulated in 'GREM4' were enriched in terpene biosynthesis, flavonoid biosynthesis, phytohormone signaling, and disease defense-related functions, likely contributing to heighted insect herbivory defense, while genes differentially expressed in 'PN40024' under herbivory were enriched in xyloglucan, cell wall formation, and calcium ion binding. The majority of genes implicated in insect herbivory defense were orthologs with specific expression patterns in 'GREM4' and 'PN40024', but some paralogous and genome-specific genes also likely contributed to conferring resistance. CONCLUSIONS: Our findings suggest that 'GREM4' insect herbivory resistance was attributed to a combination of factors, including trichomes and unique constitutive and inducible expression of genes implicated in terpene, flavonoid, and phenylpropanoid biosynthesis, as well as pathogen defense.

De Novo Synthesis of Resveratrol from Sucrose by Metabolically Engineered Yarrowia lipolytica.

Resveratrol, a phenylpropanoid compound, exhibits diverse pharmacological properties, making it a valuable candidate for health and disease management. However, the demand for resveratrol exceeds the capacity of plant extraction methods, necessitating alternative production strategies. Microbial synthesis offers several advantages over plant-based approaches and presents a promising alternative. Yarrowia lipolytica stands out among microbial hosts due to its safe nature, abundant acetyl-CoA and malonyl-CoA availability, and robust pentose phosphate pathway. This study aimed to engineer Y. lipolytica for resveratrol production. The resveratrol biosynthetic pathway was integrated into Y. lipolytica by adding genes encoding tyrosine ammonia lyase from Rhodotorula glutinis, 4-coumarate CoA ligase from Nicotiana tabacum, and stilbene synthase from Vitis vinifera. This resulted in the production of 14.3 mg/L resveratrol. A combination of endogenous and exogenous malonyl-CoA biosynthetic modules was introduced to enhance malonyl-CoA availability. This included genes encoding acetyl-CoA carboxylase 2 from Arabidopsis thaliana, malonyl-CoA synthase, and a malonate transporter protein from Bradyrhizobium diazoefficiens. These strategies increased resveratrol production to 51.8 mg/L. The further optimization of fermentation conditions and the utilization of sucrose as an effective carbon source in YP media enhanced the resveratrol concentration to 141 mg/L in flask fermentation. By combining these strategies, we achieved a titer of 400 mg/L resveratrol in a controlled fed-batch bioreactor. These findings demonstrate the efficacy of Y. lipolytica as a platform for the de novo production of resveratrol and highlight the importance of metabolic engineering, enhancing malonyl-CoA availability, and media optimization for improved resveratrol production.

Cytogenetic and Molecular Effects of Kaolin's Foliar Application in Grapevine (Vitis vinifera L.) under Summer's Stressful Growing Conditions.

Grapevine varieties from "Douro Superior" (NE Portugal) experience high temperatures, solar radiation, and water deficit during the summer. This summer's stressful growing conditions induce nucleic acids, lipids, and protein oxidation, which cause cellular, physiological, molecular, and biochemical changes. Cell cycle anomalies, mitosis delay, or cell death may occur at the cellular level, leading to reduced plant productivity. However, the foliar application of kaolin (KL) can mitigate the impact of abiotic stress by decreasing leaf temperature and enhancing antioxidant defence. Hence, this study hypothesised that KL-treated grapevine plants growing in NE Portugal would reveal, under summer stressful growing conditions, higher progression and stability of the leaf mitotic cell cycle than the untreated (control) plants. KL was applied after veraison for two years. Leaves, sampled 3 and 5 weeks later, were cytogenetically, molecularly, and biochemically analysed. Globally, integrating these multidisciplinary data confirmed the decreased leaf temperature and enhanced antioxidant defence of the KL-treated plants, accompanied by an improved regularity and completion of the leaf cell cycle relative to the control plants. Nevertheless, the KL efficacy was significantly influenced by the sampling date and/or variety. In sum, the achieved results confirmed the hypothesis initially proposed.

Chloroplast Genomes of Vitis flexuosa and Vitis amurensis: Molecular Structure, Phylogenetic, and Comparative Analyses for Wild Plant Conservation.

The chloroplast genome plays a crucial role in elucidating genetic diversity and phylogenetic relationships. Vitis vinifera L. (grapevine) is an economically important species, prompting exploration of wild genetic resources to enhance stress resilience. We meticulously assembled the chloroplast genomes of two Korean Vitis L. species, V. flexuosa Thunb. and V. amurensis Rupr., contributing valuable data to the Korea Crop Wild Relatives inventory. Through exhaustive specimen collection spanning diverse ecological niches across South Korea, we ensured comprehensive representation of genetic diversity. Our analysis, which included rigorous codon usage bias assessment and repeat analysis, provides valuable insights into amino acid preferences and facilitates the identification of potential molecular markers. The assembled chloroplast genomes were subjected to meticulous annotation, revealing divergence hotspots enriched with nucleotide diversity, thereby presenting promising candidates for DNA barcodes. Additionally, phylogenetic analysis reaffirmed intra-genus relationships and identified related crops, shedding light on evolutionary patterns within the genus. Comparative examination with chloroplast genomes of other crops uncovered conserved sequences and variable regions, offering critical insights into genetic evolution and adaptation. Our study advances the understanding of chloroplast genomes, genetic diversity, and phylogenetic relationships within Vitis species, thereby laying a foundation for enhancing grapevine genetic diversity and resilience to environmental challenges.