The 'Candidatus phytoplasma ziziphi' effectors SJP1 and SJP2 destabilise the bifunctional regulator ZjTCP7 to modulate floral transition and shoot branching.

Phytoplasmic SAP11 effectors alter host plant architecture and flowering time. However, the exact mechanisms have yet to be elucidated. Two SAP11-like effectors, SJP1 and SJP2, from 'Candidatus Phytoplasma ziziphi' induce shoot branching proliferation. Here, the transcription factor ZjTCP7 was identified as a central target of these two effectors to regulate floral transition and shoot branching. Ectopic expression of ZjTCP7 resulted in enhanced bolting and earlier flowering than did the control. Interaction and expression assays demonstrated that ZjTCP7 interacted with the ZjFT-ZjFD module, thereby enhancing the ability of these genes to directly bind to the ZjAP1 promoter. The effectors SJP1 and SJP2 unravelled the florigen activation complex by specifically destabilising ZjTCP7 and ZjFD to delay floral initiation. Moreover, the shoot branching of the ZjTCP7-SRDX transgenic Arabidopsis lines were comparable to those of the SJP1/2 lines, suggesting the involvement of ZjTCP7 in the regulation of shoot branching. ZjTCP7 interacted with the branching repressor ZjBRC1 to enhance suppression of the auxin efflux carrier ZjPIN3 expression. ZjTCP7 also directly bound to and upregulated the auxin biosynthesis gene ZjYUCCA2, thereby promoting auxin accumulation. Our findings confirm that ZjTCP7 serves as a bifunctional regulator destabilised by the effectors SJP1 and SJP2 to modulate plant development.

The 'Candidatus Phytoplasma ziziphi' effectors SJP1/2 negatively control leaf size by stabilizing the transcription factor ZjTCP2 in jujube.

Phytoplasmas manipulate host plant development to benefit insect vector colonization and their own invasion. However, the virulence factors and mechanisms underlying small-leaf formation caused by jujube witches' broom (JWB) phytoplasmas remain largely unknown. Here, effectors SJP1 and SJP2 from JWB phytoplasmas were identified to induce small-leaf formation in jujube (Ziziphus jujuba). In vivo interaction and expression assays showed that SJP1 and SJP2 interacted with and stabilized the transcription factor ZjTCP2. Overexpression of SJP1 and SJP2 in jujube induced ZjTCP2 accumulation. In addition, the abundance of miRNA319f_1 was significantly reduced in leaves of SJP1 and SJP2 transgenic jujube plants and showed the opposite pattern to the expression of its target, ZjTCP2, which was consistent with the pattern in diseased leaves. Overexpression of ZjTCP2 in Arabidopsis promoted ectopic leaves arising from the adaxial side of cotyledons and reduced leaf size. Constitutive expression of the miRNA319f_1 precursor in the 35S::ZjTCP2 background reduced the abundance of ZjTCP2 mRNA and reversed the cotyledon and leaf defects in Arabidopsis. Therefore, these observations suggest that effectors SJP1 and SJP2 induced small-leaf formation, at least partly, by interacting with and activating ZjTCP2 expression both at the transcriptional and the protein level, providing new insights into small-leaf formation caused by phytoplasmas in woody plants.

Jujube witches' broom phytoplasmas inhibit ZjBRC1-mediated abscisic acid metabolism to induce shoot proliferation.

Jujube witches' broom (JWB) phytoplasmas parasitize the sieve tubes of diseased phloem and cause an excessive proliferation of axillary shoots from dormant lateral buds to favour their transmission. In previous research, two JWB effectors, SJP1 and SJP2, were identified to induce lateral bud outgrowth by disrupting ZjBRC1-mediated auxin flux. However, the pathogenesis of JWB disease remains largely unknown. Here, tissue-specific transcriptional reprogramming was examined to gain insight into the genetic mechanisms acting inside jujube lateral buds under JWB phytoplasma infection. JWB phytoplasmas modulated a series of plant signalling networks involved in lateral bud development and defence, including auxin, abscisic acid (ABA), ethylene, jasmonic acid, and salicylic acid. JWB-induced bud outgrowth was accompanied by downregulation of ABA synthesis within lateral buds. ABA application rescued the bushy appearances of transgenic Arabidopsis overexpressing SJP1 and SJP2 in Col-0 and ZjBRC1 in the brc1-2 mutant. Furthermore, the expression of ZjBRC1 and ABA-related genes ZjHB40 and ZjNCED3 was negatively correlated with lateral main bud outgrowth in decapitated healthy jujube. Molecular evidence showed that ZjBRC1 interacted with ZjBRC2 via its N-terminus to activate ZjHB40 and ZjNCED3 expression and ABA accumulation in transgenic jujube calli. In addition, ZjBRC1 widely regulated differentially expressed genes related to ABA homeostasis and ABA signalling, especially by binding to and suppressing ABA receptors. Therefore, these results suggest that JWB phytoplasmas hijack the ZjBRC1-mediated ABA pathways to stimulate lateral bud outgrowth and expansion, providing a strategy to engineer plants resistant to JWB phytoplasma disease and regulate woody plant architecture to promote crop yield and quality.

Effects of Coal Metakaolin on the Mechanical Properties and Microstructure of High-belite Sulphoaluminate Cement.

The effects of coal metakaolin on the mechanical properties of high-belite sulphoaluminate cement under compressive loading were investigated. The composition and microstructure of hydration products at different hydration times were analyzed by X-ray diffraction and scanning electronic microscopy. The hydration process of blended cement was studied via electrochemical impedance spectroscopy. In particular, replacing a part of cement with CMK (10%, 20%, and 30%) was found to promote the hydration process, to refine the pore size, and to improve the compressive strength of the composite. The best compressive strength of the cement was achieved at a CMK content of 30% after 28 days hydration, being improved by 20.13 MPa, or 1.44 times relative to that of undoped specimens. Furthermore, the compressive strength is shown to correlate with the impedance parameter RCCP, which allows the latter to be used for nondestructive assessment of the compressive strength of blended cement materials.

Adipokine ZAG Alters Depression-Like Behavior by Regulating Oxidative Stress in Hippocampus.

Zinc-α2-glycoprotein (ZAG) is an adipokine involved in body metabolism, and now it has been shown to be present in the brain and play a role in some neurological diseases such as epilepsy and Alzheimer's disease. In the present study, we employed ZAG knockout (KO) mice to investigate the effects of ZAG on behaviors after fasting and in vitro used overexpression (OV) ZAG in HT-22 cells to further clarify the possibly underlying mechanism. The results showed that ZAG exists widely in the brain tissues of mice and significantly increased during fasting. In ZAG KO group the depression-like behaviors were significantly increased after fasting for 24 hours, meanwhile the hippocampal reactive oxygen species (ROS) content was significantly increased. In vitro, serum deprivation led to the increasing of neuronal death and ROS, the reduced mitochondrial membrane potential and ATP levels, while ZAG overexpression alleviated these negative effects. The ß3 adrenoreceptor (ß3AR)/protein kinase A (PKA)/cAMP response element-binding (CREB) pathway possibly mediated the effects of ZAG on antioxidation. These results proposed a possible target for novel therapeutic approaches to the treatment of depression and provide potential link between adipose tissue and psychiatric disease.

Screening and identification of a novel antidiabetic peptide from collagen hydrolysates of Chinese giant salamander skin: network pharmacology, inhibition kinetics and protection of IR-HepG2 cells.

In this study, a novel peptide GPPGPA was screened from the collagen hydrolysates of Chinese giant salamander (Andrias davidianus) skin, and its anti-diabetes mechanism was predicted by network pharmacology, and an inhibitory effect on α-glycosidase and protective effect on IR (insulin resistance) and oxidative stress of IR-HepG2 cells were detected. Through network pharmacology screening, GPPGPA was found to have good drug-like properties, and 103 targets of GPPGPA overlap with T2DM targets. These targets were mainly enriched in the PI3K-Akt signaling pathway associated with T2DM, the AGE-RAGE signaling pathway in diabetic complications, the TNF signaling pathway, insulin resistance and so on. The core targets were identified as AKT1, MAPK8, MAPK10 and JUN by constructing a "peptide-target-pathway" network. The molecular docking results showed that GPPGPA was well bound to the core targets. These results suggested that GPPGPA had the potential to reduce T2DM. Further in vitro experiments showed that GPPGPA as a competitive inhibitor could effectively inhibit the activity of α-glucosidase. The results of the IR-HepG2 cell model experiments showed that GPPGPA was not toxic to HepG2 cells, and could reduce IR of HepG2 cells induced by high-glucose and high-insulin, improve glucose consumption, increase the activity of superoxide dismutase (SOD), and reduce the content of malondialdehyde (MDA). The above results suggested that GPPGPA could improve T2DM by reducing insulin resistance through a multi-target and multi-pathway mechanism. GPPGPA could be developed and utilized as a novel hyperglycemic inhibitor in functional food.

Jujube witches' broom phytoplasma effectors SJP1 and SJP2 induce lateral bud outgrowth by repressing the ZjBRC1-controlled auxin efflux channel.

Comprehensively controlling phytoplasma-associated jujube witches' broom (JWB) disease is extremely challenging for the jujube industry. Although the pathogenesis of phytoplasma disease has been highlighted in many plant species, the release of lateral buds from dormancy under JWB phytoplasma infection has not been characterized in woody perennial jujube. Here, two 16SrV-B group phytoplasma effectors, SJP1 and SJP2, were experimentally determined to induce witches' broom with increased lateral branches. In vivo interaction and subcellular localization analyses showed that both SJP1 and SJP2 were translocated from the cytoplasm to the nucleus to target the CYC/TB1-TCP transcription factor ZjBRC1. The N- and C-terminal coiled-coil domains of SJP1 and SJP2 were required for the TCP-binding ability. ZjBRC1 bound directly to the auxin efflux carrier ZjPIN1c/3 promoters and down-regulated their expression to promote the accumulation of endogenous auxin indole-3-acetic acid in jujube calli. Furthermore, JWB phytoplasma infection suppressed ZjBRC1 accumulation and induced ZjPIN1c/3 expression to stimulate lateral bud outgrowth. Therefore, SJP1 and SJP2 stimulate lateral bud outgrowth, at least partly, by repressing the ZjBRC1-controlled auxin efflux channel in jujube, representing a potential strategy for comprehensive phytoplasma-associated disease control and a resource for gene editing breeding to create new cultivars with varying degrees of shoot branching.

Zinc-α2-glycoprotein promotes skeletal muscle lipid metabolism in cold-stressed mice.

Skeletal muscle is the most abundant tissue in the adult body and plays an essential role in maintaining heat production for the entire body. Recently, muscle-derived non-shivering thermogenesis under cold conditions has received much attention. Zinc-α2-glycoprotein (ZAG) is an adipokine that was shown to influence energy metabolism in the adipose tissue. We used ZAG knock-out (ZAG KO) and wild-type (WT) mice to investigate the effect of ZAG on the lipid metabolism of skeletal muscle upon exposure to a low temperature (6°C) for one week. The results show that cold stress significantly increases the level of lipolysis, energy metabolism, and fat browning-related proteins in the gastrocnemius muscle of WT mice. In contrast, ZAG KO mice did not show any corresponding changes. Increased expression of ß3-adrenoceptor (ß3-AR) and protein kinase A (PKA) might be involved in the ZAG pathway in mice exposed cold stress. Furthermore, expression of lipolysis-related proteins (ATGL and p-HSL) and energy metabolism-related protein (PGC1α, UCP2, UCP3 and COX1) was significantly enhanced in ZAG KO mice after injection of ZAG-recombinant plasmids. These results indicate that ZAG promotes lipid-related metabolism in the skeletal muscle when the animals are exposed to low temperatures. This finding provides a promising target for the development of new therapeutic approaches to improve skeletal muscle energy metabolism.

Energy transfer and influencing factors in soil during compaction.

In China, large-area excavation and filling engineering has increased rapidly with the expansion of construction land. The quality of filling engineering is the most important guarantee for the stability of building structures. Among all research on fill soil, the compaction characteristics are significant for indicating the strength and stability of filling engineering. In this paper, two layers of loess fill soil were compacted by a self-manufactured test system with three different compaction energies. Based on the variation in the soil bottom pressure obtained in the tests, the influence of the compaction parameters on the soil bottom pressure was investigated. The results show that the compaction curve can be used instead of the curve of the change in soil bottom pressure with water content; as the soil density increases, the soil bottom pressure increases to the maximum. The relation of the energy consumption ratio of the soil bottom (σ/σz) and the number of soil layers is exponential and reveals the stability of the soil skeleton formed during compaction. This paper describes the compaction characteristics of loess fill soil from the perspective of energy transfer, and the conclusions provide a theoretical basis for soil filling engineering.

Migrant adolescents' behavioral problems compared to host adolescents and adolescents in their region of origin: a longitudinal study.

BACKGROUND: Since the 1990s, families from the ecologically hostile mountainous southern areas of Ningxia Province, China, have been migrating to the northern areas of the province. This study compared the prevalence of behavioral problems among migrant adolescents to those among host adolescents (adolescents from the northern areas) and adolescents in the region of origin (adolescents from the southern areas), to determine whether ecological migration is related to adolescent behavioral problems, and possible changes in such problems over time. METHODS: We used the Children and Adolescents Ecological Migration Survey on Mental Health, administered to 4805 students aged 12-16 years and their parents between 2012 and 2014 (W1), of whom 1753 students and their parents completed the follow-up between 2014 and 2017 (W2). Parents answered questions related to adolescent behavioral problems, main source of family income, parents' desire to reverse migrate, improved standard of living, and parents' educational attainment, while children completed the Eysenck Personality Questionnaire and a classroom environment questionnaire. RESULTS: The prevalence of behavioral problems among the migrant adolescents (28.04%) was significantly higher than among host adolescents (21.59%) or adolescents in the region of origin (24.37%; p < 0.001) at W1. After adjusting for gender and age, parents' work outside the home was the main source of family income (OR = 1.42, 95% CI = 1.13-1.78), and adolescents' learning burden (OR = 1.04, 95% CI = 1.01-1.06) in school negatively influenced behavioral problems. Strong student-teacher relationships (OR = 0.97,95% CI = 0.94-0.99) and parents who had no intention to move back to the original residence (OR = 0.70, 95% CI = 0.52-0.94) exerted a protective effect at W1; at W2, a protective effect was associated with improved living conditions (OR = 0.39-0.55, 95% CI = 0.25-0.84). The extent of behavioral problems among migrant adolescents significantly decreased after two years. CONCLUSION: Ecological migration will increase children's behavioral problems in the early stage, with various factors influencing the extent of these problems.

An Electrical Resistivity Method of Characterizing Hydromechanical and Structural Properties of Compacted Loess During Constant Rate of Strain Compression.

Hydromechanical and structural properties of compacted loess have a significant impact on the stability and reliability of subbase and subgrade, which needs to be quickly determined in the field and laboratory. Hence, an electrical resistivity method was used to characterize the hydromechanical and structural properties of compacted loess during constant rate of strain compression. In the present work, compacted loess samples with a dry density of 1.7 g/cm3, a diameter of 64 mm, a height of 10 mm and different water content ranging from 5-25% were prepared. The constant rate of strain (CRS) tests were conducted by a developed oedometer cell equipped with a pair of horizontal circular electrodes (diameter of 20 mm) and vertical rectangular electrodes (width of 3.5 mm) to determine the electrical resistivity of compacted loess. The results showed that as average water content increases, plastic compression indices increase from 0.220 to 0.350 and the elastic compression indices increase from 0.0152 to 0.030, but they decrease to 0.167 and 0.010 and yield stress decreases from 381.28 kPa to 72.35 kPa. Moreover, as vertical strain increases, the variation trend of average formation factor and average shape factor for the lower water content decreases but increases for the maximum water content, and the anisotropy index first decrease and then tend to increase slightly, which indicates that the structural properties of unsaturated and saturated samples during compression exhibits different trend and the anisotropy of samples tend to be stable as vertical strain increases. As the water content increases, the average formation factor and average shape factor decrease, but the anisotropy index first decreases then increases, suggesting that water content has a significant impact on these electrical indices. More important, The coefficients of average formation factor decrease from 33.830 to -1.698 and the coefficients of average shape factor decrease from 8.339 to -0.398 as water content increases, whereas there is less variation for the coefficient of anisotropic index with a value of 2.190. An equation correlating average formation factor and water content and vertical strain is regressed to characterize the hydromechanical properties of compacted loess by measuring its impedance, which can be used to evaluate the stability of compacted loessic ground and subgrade.

Identification, expression and miRNA targeting of auxin response factor genes related to phyllody in the witches' broom disease of jujube.

Jujube witches' broom (JWB) disease, associated with the presence of phytoplasmas, induces huge crop losses in the woody perennial fruit tree Ziziphus jujuba. An imbalance in the phytohormone auxin is thought to be a key factor in the development of the witches' broom symptoms, and in the alteration of floral development into leafy structures, termed phyllody. The Auxin Response Factor (ARF) gene family controls auxin-responsive gene expression during plant growth and development. However, it remains unknown if the ARF genes are involved in the formation of leaf-like flowers. In the present study, sixteen jujube ARF genes were identified bioinformatically and annotated based on the Z. jujuba cv. Dongzao genome. The ZjARFs were homologous to 12 out of the 23 Arabidopsis ARFs and were distributed in 8 jujube chromosomes and 3 unmapped scaffolds. Phylogenetic analysis grouped the ZjARFs into three classes. Spatio-temporal expression analysis revealed that the ZjARF genes were differentially expressed among different tissues during normal development. The expression of seven ZjARF genes was significantly decreased from flower buds to flowering. JWB-infected jujube plants developed the typical phyllody symptoms and showed lower auxin accumulation during floral development. ZjARF1, ZjARF2, ZjARF3, ZjARF4 and ZjARF8 resulted differentially regulated after phytoplasma infection. ZjARF4 was down-regulated before and during floral development in phytoplasma-infected plants, but it was significantly up-regulated before flowering and down-regulated during flowering in the healthy plants. Target site analysis showed that miRNA167, miRNA529 and miRNA2950 could directly target ZjARF4. Together, the data showed that the auxin-controlled ARF4 gene is likely involved in the disruption of floral development in phytoplasma-infected jujube plants.

Expression of stilbene synthase VqSTS6 from wild Chinese Vitis quinquangularis in grapevine enhances resveratrol production and powdery mildew resistance.

MAIN CONCLUSION: In grape (Vitis), stilbene phytoalexins can either be in situ synthesized or transported to the site of response during powdery mildew infection, enhancing disease resistance. Resveratrol is a phytoprotective stilbenoid compound that is synthesized by stilbene synthase (STS) in response to biotic and abiotic stresses, and is also known to have health benefits in the human diet. We have previously shown that transgenic Vitis vinifera cv. Thompson Seedless plants overexpressing a stilbene synthase gene, VqSTS6, from wild Chinese Vitis quinquangularis had a higher stilbenoid content, leading to an enhanced resistance to powdery mildew (Uncinula necator (Schw.) Burr). However, the biosynthesis and transportation in the plant tissue under powdery mildew infection are still unclear. Here, inhibitor and micro-grafting technologies were used to study the accumulation of resveratrol following powdery mildew infection. We observed that the levels of STS expression and stilbenoids increased in response to powdery mildew infection. Powdery mildew and inhibitor treatment on detached grape branches showed that resveratrol was in situ synthesized. Experiments with grafted plantlets showed that the abundance of stilbenoid compounds increased in the shoot during VqSTS6 overexpression in the root, while VqSTS6-Flag fusion was not tranported to the scions and only expressed in the transgenic rootstocks. Compared with wild-type Thompson Seedless plants, the non-transgenic/VqSTS6 transgenic (scion/rootstock) grafted Thompson Seedless plantlets exhibited increased resistance to powdery mildew. In addition, overexpression of VqSTS6 in roots led to increased levels of stilbenoid compounds in five other European grape varieties (V. vinifera cvs. Chardonnay, Perlette, Cabernet Sauvignon, Riesling and Muscat Hamburg). In conclusion, stilbenoid compounds can be either in situ synthesized or transported to the site of powdery mildew infection, and overexpression of VqSTS6 in the root promotes stilbenoids accumulation and disease resistance in European grapevine varieties.

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.

Effect of norepinephrine treatment on Haemonchus contortus and its excretory products.

Haemonchus contortus is a highly pathogenic gastrointestinal nematode of small ruminant animals. In modern intensive farming, livestock often suffer from different types of stress. However, whether host stress hormones influence H. contortus infection is largely unknown. Therefore, we treated H. contortus with norepinephrine (NE) and analyzed the changes in its excretory/secretory products (ESPs). Label-free quantitative proteomic analysis was used to identify differences in body proteins and ESPs between the control and NE-treated groups. We also investigated the changes in ESP action by analyzing cytokine secretion and goat peripheral blood mononuclear cell (PBMC) proliferation after incubation with ESPs secreted by NE-treated H. contortus. Thirty-two proteins in the body samples and 137 in the ESPs were differentially expressed between the groups. Gene ontology (GO) annotation showed that the functions of these different proteins might be involved in energy metabolism, protein metabolism, lipid metabolism, redox homeostasis, ion channel, and cell structure. NE treatment caused oxidative stress in H. contortus and changed the expression levels of some immunogenic proteins, such as the 15-kDa ESP. Meanwhile, the ESPs secreted by NE-treated H. contortus significantly decreased PBMC proliferation and the interleukin (IL)-2, IL-4, and interferon-gamma contents. Thus, NE treatment significantly affected the H. contortus body and ESP expression, and changes in the ESPs influenced PBMC function. The results reveal a relationship between host hormones and parasites and provide new clues to explain some of the variation in individual responses to infection.

Dynamic translocation of stilbene synthase VpSTS29 from a Chinese wild Vitis species upon UV irradiation.

Stilbene phytoalexins derived from grapevine can be rapidly accumulated when exposed to an artificial UV-C treatment. However, the underlying mechanisms involved in this accumulation and translocation are unclear. Here, we describe an investigation of the influence of UV-C treatment on the dynamic subcellular distribution of a member of a stilbene synthase family VpSTS29 derived from Chinese wild Vitis pseudoreticulata W.T. Wang when over-expressed in V. vinifera L. cv. Thompson Seedless. Our results show that VpSTS29-GFP was accumulated at a relatively high level in roots and mature leaves of transgenic grape lines, and was predominantly distributed in the cytoplasm. When exposed to UV-C irradiation, VpSTS29 displayed UV-induced feature coupled with the accumulation of stilbene compounds. Notably, VpSTS29-GFP can be translocated from the cytoplasm into chloroplasts upon UV-irradiation. Leaves from the two VpSTS29-GFP-expressing lines displayed more serious UV damage, showing withering and marginal scorching phenotype, and decreased content of H2O2, compared to the untransformed plant. Also, overexpression of VpSTS29 altered the expression of genes related to redox regulation, stilbene biosynthesis and light stimulus. Co-expression of VpSTS29-GFP with Glycolate oxidase 1 (myc-VpGLO1) confirmed the ability of stilbenes to decrease the content of H2O2 in Arabidopsis mesophyll protoplasts. These results provide new insight into the biological functions and properties of stilbene synthase and its product in response to environmental stimulus.

Synthesis and biological activities of dithiocarbamates containing 2(5H)-furanone-piperazine.

A series of new dithiocarbamates containing a 2(5H)-furanone-piperazine group was synthesized. These compounds show good in vitro cytoxic activity. Among them, compound 6c exhibits the best inhibitory activity against HeLa cell lines with an IC50 of 0.06 ±â€¯0.01 µM for 72 h, and it has good inhibitory activity against SMMC-7721 cell lines with an IC50 of 0.006 ±â€¯0.04 µM for 72 h, but the toxicity was lower against LO2 cell lines with an IC50 of 45.76 ±â€¯0.01 µM. The result showed that compound 6c is far more cytoxic towards cancer cell lines than towards benign cell lines compared with cytosine arabinoside (ARA) in vitro.

Ectopic expression of VpSTS29, a stilbene synthase gene from Vitis pseudoreticulata, indicates STS presence in cytosolic oil bodies.

MAIN CONCLUSION: Stilbene synthase (STS) and its metabolic products are accumulated in senescing grapevine leaves. Ectopic expression of VpSTS29 in Arabidopsis shows the presence of VpSTS29 in oil bodies and increases trans-piceid in developing leaves. Stilbenes are the natural antimicrobial phytoalexins that are synthesised via the phenylpropanoid pathway. STS is the key enzyme catalysing the production of stilbenes. We have previously reported that the VpSTS29 gene plays an important role in powdery mildew resistance in Vitis pseudoreticulata. However, the synthesis and accumulation of these stilbene products in plant cells remain unclear. Here, we demonstrate that VpSTS29 is present in cytosolic oil bodies and can be transported into the vacuole at particular plant-developmental stages. Western blot and high-performance liquid chromatography showed that STS and trans-piceid accumulated in senescent grape leaves and in pVpSTS29::VpSTS29-expressing Arabidopsis during age-dependent leaf senescence. Subcellular localisation analyses indicated VpSTS29-GFP was present in the cytoplasm and in STS-containing bodies in Arabidopsis. Nile red staining, co-localisation and immunohistochemistry analyses of leaves confirmed that the STS-containing bodies were oil bodies and that these moved randomly in the cytoplasm and vacuole. Detection of protein profiles revealed that no free GFP was detected in the pVpSTS29::VpSTS29-GFP-expressing protoplasts or in Arabidopsis during the dark-light cycle, demonstrating that GFP fluorescence distributed in the STS-containing bodies and vacuole was the VpSTS29-GFP fusion protein. Intriguingly, in comparison to the controls, over-expression of VpSTS29 in Arabidopsis resulted in relatively high levels of trans-piceid, chlorophyll content and of photochemical efficiency accompanied by delayed leaf senescence. These results provide exciting new insights into the subcellular localisation of STS in plant cells and information about stilbene synthesis and storage.

Overexpression of VpEIFP1, a novel F-box/Kelch-repeat protein from wild Chinese Vitis pseudoreticulata, confers higher tolerance to powdery mildew by inducing thioredoxin z proteolysis.

An F-box protein (VpEIFP1) induced by Erysiphe necator was isolated from Vitis pseudoreticulata, a wild Chinese grapevine species naturally resistant to powdery mildew (PM). It contains an F-box domain and two Kelch-repeat motifs. Expression profiles indicate the VpEIFP1 is strongly induced at both transcriptional and translational levels by PM infection. A subcellular localisation assay showed that VpEIFP1 is predominantly located in the nucleus and cytoplasm. Overexpression of VpEIFP1 accelerated the accumulation of hydrogen peroxide (H2O2) and up-regulated the expressions of ICS2, NPR1 and PR1 involved in defence responses, resulting in suppression of PM germination and growth. As an F-box protein, VpEIFP1 interacts with thioredoxin z (VpTrxz) in the yeast-two-hybrid (Y2H) assay and in the bimolecular fluorescence complementation (BiFC) assay. Decreased amounts of VpTrxz protein in transgenic grapevine leaves overexpressing VpEIFP1 were restored by proteasome inhibitor MG132, implying that VpEIFP1 mediated VpTrxz for degradation through the SCFVpEIFP1 (Skp1-Cullin-F-box) E3 ubiquitin ligase complex. The RNA interference line of VpTrxz showed increased H2O2 accumulation following PM inoculation. We propose VpEIFP1 positively modulates the grapevine defence response to PM by inducing the degradation of VpTrxz via the ubiquitin/26S proteasome system.