Profiling of sugar transporter genes in grapevine coping with water deficit

The profiling of grapevine (Vitis vinifera L.) genes under water deficit was specifically targeted to sugar transporters. Leaf water status was characterized by physiological parameters and soluble sugars content. The expression analysis provided evidence that VvHT1 hexose transporter gene was strongly down-regulated by the increased sugar content under mild water-deficit. The genes of monosaccharide transporter VvHT5, sucrose carrier VvSUC11, vacuolar invertase VvGIN2 and grape ASR (ABA, stress, ripening) were up-regulated under severe water stress. Their regulation in a drought-ABA signalling network and possible roles in complex interdependence between sugar subcellular partitioning and cell influx/efflux under Grapevine acclimation to dehydration are discussed.     

Role of ABA and Gibberellin A<Subscript>3</Subscript> on gene expression pattern of sugar transporters and invertases in <Emphasis Type="Italic">Vitis vinifera</Emphasis> cv. Malbec during berry ripening

Sugars are key constituents that affect quality of grape berries, and consequently the grape metabolic profile relevant to wine’s industry. However, enzymes and transporter genes expression involved in sugar transport at different phenological stages are scarcely studied. In addition, little is known about the role of the plant hormones ABA and Gibberellin (GA₃) as endogenous regulators, over the expression pattern of the sugars transporters genes in grapevine. The aim of this study was to analyze the expression pattern of the most relevant sugar transporters and invertases in leaves and berries of grapevine plants cv. Malbec during berry ripening stages and its shift after ABA and GA₃ sprays. In leaves, VvHT1 was the sugar transporter highly expressed, whereas VvHT6 was the most abundant in berries throughout berry ripening. Moreover, VvSUC12 and VvSUC27 were expressed at veraison greater in leaves than in berries, suggesting an active phloem loading at the onset of ripening. Applications of ABA and GA₃ enhanced the expression of VvSUC12 and VvSUC27 in pre-veraison leaves. Furthermore, hormones increased the expression of VvHT2, VvHT3 and VvHT6 in berries at different stages of ripening favoring sugar unloading from phloem. In conclusion, ABA and GA₃ are involved in the long-distance sugar transport from leaves to berries in Vitis vinifera L. cv. Malbec, and their exogenous application could be a suitable strategy to improve the process.     

Abscisic acid deficiency antagonizes high-temperature inhibition of disease resistance through enhancing nuclear accumulation of resistance proteins SNC1 and RPS4 in Arabidopsis

Plant defense responses to pathogens are influenced by abiotic factors, including temperature. Elevated temperatures often inhibit the activities of disease resistance proteins and the defense responses they mediate. A mutant screen with an Arabidopsis thaliana temperature-sensitive autoimmune mutant bonzai1 revealed that the abscisic acid (ABA)-deficient mutant aba2 enhances resistance mediated by the resistance (R) gene suppressor of npr1-1 constitutive1 (SNC1) at high temperature. ABA deficiency promoted nuclear accumulation of SNC1, which was essential for it to function at low and high temperatures. Furthermore, the effect of ABA deficiency on SNC1 protein accumulation is independent of salicylic acid, whose effects are often antagonized by ABA. ABA deficiency also promotes the activity and nuclear localization of R protein resistance to Pseudomonas syringae4 at higher temperature, suggesting that the effect of ABA on R protein localization and nuclear activity is rather broad. By contrast, mutations that confer ABA insensitivity did not promote defense responses at high temperature, suggesting either tissue specificity of ABA signaling or a role of ABA in defense regulation independent of the core ABA signaling machinery. Taken together, this study reveals a new intersection between ABA and disease resistance through R protein localization and provides further evidence of antagonism between abiotic and biotic responses.     

Nuclear Import and Dimerization of Tomato ASR1, a Water Stress-Inducible Protein Exclusive to Plants

The ASR (for ABA/water stress/ripening) protein family, first described in tomato as nuclear and involved in adaptation to dry climates, is widespread in the plant kingdom, including crops of high agronomic relevance. We show both nuclear and cytosolic localization for ASR1 (the most studied member of the family) in histological plant samples by immunodetection, typically found in small proteins readily diffusing through nuclear pores. Indeed, a nuclear localization was expected based on sorting prediction software, which also highlight a monopartite nuclear localization signal (NLS) in the primary sequence. However, here we prove that such an "NLS" of ASR1 from tomato is dispensable and non-functional, being the transport of the protein to the nucleus due to simple diffusion across nuclear pores. We attribute such a targeting deficiency to the misplacing in that cryptic NLS of two conserved contiguous lysine residues. Based on previous in vitro experiments regarding quaternary structure, we also carried out live cell imaging assays through confocal microscopy to explore dimer formation in planta. We found homodimers in both the cytosol and the nucleus and demonstrated that assembly of both subunits together can occur in the cytosol, giving rise to translocation of preformed dimers. The presence of dimers was further corroborated by means of in vivo crosslinking of nuclei followed by SDS-PAGE.     

ABA and GA3 increase carbon allocation in different organs of grapevine plants by inducing accumulation of non‐structural carbohydrates in leaves,enhancement of phloem area and expression of sugar transporters

Grape quality for winemaking depends on sugar accumulation and metabolism in berries. Abscisic acid (ABA) and gibberellins (GAs) have been reported to control sugar allocation in economically important crops, although the mechanisms involved are still unknown. The present study tested if ABA and gibberellin A₃ (GA₃) enhance carbon allocation in fruits of grapevines by modifying phloem loading, phloem area and expression of sugar transporters in leaves and berries. Pot‐grown Vitis vinifera cv. Malbec plants were sprayed with ABA and GA₃ solutions. The amount of soluble sugars in leaves and berries related to photosynthesis were examined at three points of berry growth: pre‐veraison, full veraison and post‐veraison. Starch levels and amylase activity in leaves, gene expression of sugar transporters in leaves and berries and phloem anatomy were examined at full veraison. Accumulation of glucose and fructose in berries was hastened in ABA‐treated plants at the stage of full veraison, which was correlated with enhancement of Vitis vinifera HEXOSE TRANSPORTER 2 (VvHT2) and Vitis vinifera HEXOSE TRANSPORTER 6 (VvHT6) gene expression, increases of phloem area and sucrose content in leaves. On the other hand, GA₃ increased the quantity of photoassimilates delivered to the stem thus increasing xylem growth. In conclusion, stimulation of sugar transport by ABA and GA₃ to berries and stems, respectively, was due to build‐up of non‐structural carbohydrates in leaves, modifications in phloem tissue and modulation in gene expression of sugar transporters.     

AKINβ1 is Involved in the Regulation of Nitrogen Metabolism and Sugar Signaling in Arabidopsis

Sucrose non-fermenting-1-related protein kinase 1 (SnRK1) has been located at the heart of the control of metabolism and development in plants. The active SnRK1 form is usually a heterotrimeric complex. Subcellular localization and specific target of the SnRK1 kinase are regulated by specific beta subunits. In Arabidopsis , there are at least seven genes encoding beta subunits, of which the regulatory functions are not yet clear. Here, we tried to study the function of one beta subunit, AKINβ1. It showed that AKINβ1 expression was dramatically induced by ammonia nitrate but not potassium nitrate, and the investigation of AKINβ1 transgenic Arabidopsis and T-DNA insertion lines showed that AKINβ1 negatively regulated the activity of nitrate ruductase and was positively involved in sugar repression in early seedling development. Meanwhile AKINβ1 expression was reduced upon sugar treatment (including mannitol) and did not affect the activity of sucrose phosphate synthase. The results indicate that AKINβ1 is involved in the regulation of nitrogen metabolism and sugar signaling.     

Abscisic acid,sucrose, and auxin coordinately regulate berry ripening process of the Fujiminori grape

The aim of this study was to examine the effect of abscisic acid (ABA), sucrose, and auxin on grape fruit development and to assess the mechanism of these three factors on the grape fruit ripening process. Different concentrations of ABA, sucrose, and auxin were used to treat the grape fruit, and the ripening-related indices, such as physiological and molecular level parameters, were analyzed. The activity of BG protein activity was analyzed during the fruit development. Sucrose, ABA, and auxin influenced the grape fruit sugar accumulation in different ways, as well as the volatile compounds, anthocyanin content, and fruit firmness. ABA and sucrose induced, but auxin blocked, the ripening-related gene expression levels, such as softening genes PE, PG, PL, and CELL, anthocyanin genes DFR, CHI, F3H, GST, CHS, and UFGT, and aroma genes Ecar, QR, and EGS. ABA, sucrose, and glucose induced the fruit dry weight accumulation, and auxin mainly enhanced fruit dry weight through seed weight accumulation. In the early development of grape, starch was the main energy storage; in the later, it was glucose and fructose. Sucrose metabolism pathway-related gene expression levels were significant for glucose and fructose accumulation. BG protein activity was important in the regulation of grape ABA content levels. ABA plays a core role in the grape fruit development; sucrose functions in fruit development through two pathways: one was ABA dependent, the other ABA independent. Auxin blocked ABA accumulation to regulate the fruit development process.     

AKINβ1 is Involved in the Regulation of Nitrogen Metabolism and Sugar Signaling in Arabidopsis

Sucrose non-fermenting-1-related protein kinase 1 (SnRK1) has been located at the heart of the control of metabolism and development in plants. The active SnRK1 form is usually a heterotrimeric complex. Subcellular localization and specific target of the SnRK1 kinase are regulated by specific beta subunits. In Arabidopsis, there are at least seven genes encoding beta subunits, of which the regulatory functions are not yet clear. Here, we tried to study the function of one beta subunit, AKINβ1. It showed that AKINβ1 expression was dramatically induced by ammonia nitrate but not potassium nitrate, and the investigation of AKINβ1 transgenic Arabidopsis and T-DNA insertion lines showed that AKINβ1 negatively regulated the activity of nitrate ruductase and was positively involved in sugar repression in early seedling development. Meanwhile AKINβ1 expression was reduced upon sugar treatment (including mannitol) and did not affect the activity of sucrose phos-phate synthase. The results indicate that AKINβ1 is involved in the regulation of nitrogen metabolism and sugar signaling.     

ABA inhibits myristoylation and induces shuttling of the RGLG1 E3 ligase to promote nuclear degradation of PP2CA

Hormone‐ and stress‐induced shuttling of signaling or regulatory proteins is an important cellular mechanism to modulate hormone signaling and cope with abiotic stress. Hormone‐induced ubiquitination plays a crucial role to determine the half‐life of key negative regulators of hormone signaling. For ABA signaling, the degradation of clade‐A PP 2Cs, such as PP 2 CA or ABI 1, is a complementary mechanism to PYR / PYL / RCAR ‐mediated inhibition of PP 2C activity. ABA promotes the degradation of PP 2 CA through the RGLG 1 E3 ligase, although it is not known how ABA enhances the interaction of RGLG 1 with PP 2 CA given that they are predominantly found in the plasma membrane and the nucleus, respectively. We demonstrate that ABA modifies the subcellular localization of RGLG 1 and promotes nuclear interaction with PP 2 CA . We found RGLG 1 is myristoylated in vivo , which facilitates its attachment to the plasma membrane. ABA inhibits the myristoylation of RGLG 1 through the downregulation of N‐myristoyltransferase 1 ( NMT 1 ) and promotes nuclear translocation of RGLG 1 in a cycloheximide‐insensitive manner. Enhanced nuclear recruitment of the E3 ligase was also promoted by increasing PP 2 CA protein levels and the formation of RGLG 1–receptor–phosphatase complexes. We show that RGLG 1 ^Gly2Ala mutated at the N‐terminal myristoylation site shows constitutive nuclear localization and causes an enhanced response to ABA and salt or osmotic stress. RGLG 1/5 can interact with certain monomeric ABA receptors, which facilitates the formation of nuclear complexes such as RGLG 1– PP 2 CA – PYL 8. In summary, we provide evidence that an E3 ligase can dynamically relocalize in response to both ABA and increased levels of its target, which reveals a mechanism to explain how ABA enhances RGLG 1– PP 2 CA interaction and hence PP 2 CA degradation.