Ubiquitin ligase VvPUB26 in grapevine promotes proanthocyanidin synthesis and resistance to powdery mildew.

Proanthocyanidins (PAs) are an important group of flavonoids that contribute to astringency, color, and flavor in grape (Vitis vinifera) and wines. They also play a crucial role in enhancing plant resistance to various stresses. However, the underlying regulatory mechanism governing PAs biosynthesis, particularly in relation to conferring resistance to powdery mildew, has not been extensively explored. This study focused on identifying a key player in PAs biosynthesis, namely the plant U-box (PUB) E3 ubiquitin ligase VvPUB26. We discovered that overexpression of VvPUB26 in grape leads to a significant increase in PAs content, whereas interfering with VvPUB26 has the opposite effect. Additionally, our findings demonstrated that overexpression of VvPUB26 in transgenic grapevines enhances defense against powdery mildew, while interfering with VvPUB26 results in increased susceptibility to the pathogen. Interestingly, we observed that VvPUB26 interacts with the WRKY transcription factor VvWRKY24, thereby facilitating ubiquitination and degradation processes. Through RNA-Seq analysis, we found that VvWRKY24 primarily participates in secondary metabolites biosynthesis, metabolic pathways, and plant-pathogen interaction. Notably, VvWRKY24 directly interacts with the promoters of dihydroflavonol-4-reductase (DFR) and leucoanthocyanidin reductase (LAR) to inhibit PAs biosynthesis. Meanwhile, VvWRKY24 also influences the expression of MYB transcription factor genes related to PAs synthesis. In conclusion, our results unveil a regulatory module involving VvPUB26-VvWRKY24-VvDFR/VvLAR that plays a fundamental role in governing PAs biosynthesis in grapevines. These findings enhance our understanding of the relationship between PAs biosynthesis and defense mechanisms against powdery mildew.

VviKFB07 F-box E3 ubiquitin ligase promotes stilbene accumulation by ubiquitinating and degrading VviCHSs protein in grape.

Stilbene and flavonoid are phytochemicals in plants and play an important role in plant disease resistance and human health. The regulation of stilbene and flavonoid synthesis in plants has been extensively studied at the transcriptional level, but translational and post-translational controls of stilbene and flavonoid biosynthesis are still poorly understood. In this study, a grape F-box E3 ubiquitin ligase VviKFB07 associated with the metabolism of stilbene and flavonoid was screened out with transcriptome. Overexpression of VviKFB07 in the Nicotiana tabacum resulted in a decrease in flavonol and anthocyanin content in corolla, and stable overexpression assays of VviKFB07 in grape callus promoted the accumulation of resveratrol. Subsequently, Yeast two-hybrid and bimolecular fluorescence complementation assays identified the physical interaction between VviKFB07 and VviCHSs proteins. In vivo experiments verified that VviKFB07 was involved in the ubiquitination and degradation of VviCHSs protein. Taken together, our findings clarify the role of ubiquitin ligase VviKFB07 in the synthesis of stilbene and flavonoid in grapes.

Preparation and Characterization of Protein Molecularly Imprinted Poly (Ionic Liquid)/Calcium Alginate Composite Cryogel Membrane with High Mechanical Strength for the Separation of Bovine Serum Albumin.

In order to improve the mechanical strength and imprinting efficiency, a novel bovine serum albumin (BSA) molecularly imprinted poly(ionic liquid)/calcium alginate composite cryogel membrane (MICM) was prepared. The results of the tensile test indicated that the MICM had excellent mechanical strength which could reach up to 90.00 KPa, 30.30 times higher than the poly (ionic liquid) membrane without calcium alginate; the elongation of it could reach up to 93.70%, 8.28 times higher than the poly (ionic liquid) membrane without calcium alginate. The MICM had a very high welling ratio of 1026.56% and macropore porosity of 62.29%, which can provide effective mass transport of proteins. More remarkably, it had a very high adsorption capacity of 485.87 mg g-1 at 20 °C and 0.66 mg mL-1 of the initial concentration of BSA. Moreover, MICM also had good selective and competitive recognition toward BSA, exhibiting potential utility in protein separation. This work can provide a potential method to prepare the protein-imprinted cryogel membrane with both high mechanical strength and imprinting efficiency.

BSISTER transcription factors directly binds to the promoter of IAA19 and IAA29 genes to up-regulate gene expression and promote the root development.

Roots play an important role in the growth and development of plants and auxin participates in regulating plant root development. Some studies have shown that BS (BSISTER) gene (the closest gene of class B gene) is involved in plant root development, but whether BS regulates root development via auxin signaling still not clear. To explore VviBS1 and VviBS2 roles in root development, VviBS1 and VviBS2 were overexpressedin Arabidopsis tt16 mutant and we found that they could restore the phenotype of shorter PR (primary roots) and high density of LR (lateral root) of tt16 compared with the wild type Ws Arabidopsis seedlings. However, the addition of exogenous NAA (naphthalene acetic acid) could not significantly promote the PR length of tt16 Arabidopsis, and the auxin signal transduction of tt16 may be blocked. The expression levels of auxin signal transduction pathway genes in Ws, tt16, p35s:VviBS1 in tt16 and p35s:VviBS2 in tt16 seedlings were detected. It was found that the expression of AtARF2, AtARF12, AtARF14, AtARF15, AtARF20, AtGH3, AtGH3-2 and AtSAUR51 genes in tt16 seedlings was higher than that in Ws, while the expression of AtIAA19 and AtIAA29 in Ws seedlings was higher than that of tt16. More importantly, BS may up regulate AtIAA19 and AtIAA29 expression directly by binding to their promoter. In addition, VviBS1 and VviBS2 also affect seed germination and may regulate leaf yellowing by regulating ethylene synthase. Therefore, our findings reveal a molecular mechanism that BS may modulate root system development via Aux/IAA-based auxin signaling, and provide insight into the BS function in regulation of leaf yellowing.

Genome-wide identification, phylogenetic analysis, and expression profiling of glycine-rich RNA-binding protein (GRPs) genes in seeded and seedless grapes (Vitis vinifera).

Glycine-rich RNA-binding proteins (GRPs) are essential for many physiological and biochemical processes in plants, especially the response to environmental stresses. GRPs exist widely in angiosperms and gymnosperms plant species; however, their roles in Vitis vinifera are still poorly understood. To characterize VviGRP gene family, we performed a genomic survey, bioinformatics and expression analysis of VviGRPs in grape. We identified nineteen VviGRPs gene family members. The result of bioinformatics analysis showed their motif distribution, gene structure characteristics and chromosomal locations. Then we carried out synteny and phylogenetic analysis to study the origin and evolutionary relationship of GRPs. Tissue-specific expression analysis showed that VviGRPs have different expression patterns. Meanwhile, we studied expression profiles of seventeen ovule-expressed genes during seed development of stenospermocarpic seedless and seeded grapes, and the result showed that most of them have much higher relative expression levels in stenospermocarpic seedless grapes than that of seeded one before 25 days after full bloom (DAFB). It is suggested that VviGRPs may involve in the seed development process. Taken together, our research indicated that VviGRPs are related to seed development and will be beneficial for further investigations into the seed abortion mechanism under stenospermocarpic grapes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01082-3.