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The sensor kinase Sucrose Non-fermenting-1-Related Kinase 1 (SnRK1) plays a central role in energy and metabolic homeostasis. KIN10 is a major catalytic (α) kinase subunit of SnRK1 regulated by transcription, posttranslational modification, targeted protein degradation, and its subcellular localization. Geminivirus Rep Interacting Kinase 1 and 2 (GRIK1 and 2) are immediate upstream kinases of KIN10. In the transient protein expression assays carried out in Nicotiana benthamiana (N. benthamiana) leaves, GRIK1 not only phosphorylates KIN10 but also simultaneously initiates its degradation. Posttranslational GRIK-mediated KIN10 degradation is dependent on both GRIK kinase activity and phosphorylation of the KIN10 T-loop. KIN10 proteins are significantly enriched in the grik1-1 grik2-1 double mutant, consistent with the transient assays in N. benthamiana. Interestingly. Among the enriched KIN10 proteins from grik1-1 grik2-1, is a longer isoform, putatively derived by alternative splicing which is barely detectable in wild-type plants. The reduced stability of KIN10 upon phosphorylation and activation by GRIK represents a mechanism that enables the KIN10 activity to be rapidly reduced when the levels of intracellular sugar/energy are restored to their set point, representing an important homeostatic control that prevents a metabolic overreaction to low-sugar conditions. Since GRIKs are activating kinases of KIN10, KIN10s in the grik1 grik2 double null mutant background remain un-phosphorylated, with only their basal level of activity, are more stable, and therefore increase in abundance, which also explains the longer isoform KIN10L which is a minor isoform in wild type is clearly detected in the grik1 grik2 double mutant.

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CYCLIN-DEPENDENT KINASE 8 (CDK8), a component of the kinase module of the Mediator complex in Arabidopsis, is involved in many processes, including flowering, plant defense, drought, and energy stress responses. Here, we investigated cdk8 mutants and CDK8-overexpressing lines to evaluate whether CDK8 also plays a role in regulating lipid synthesis, an energy-demanding anabolism. Quantitative lipid analysis demonstrated significant reductions in lipid synthesis rates and lipid accumulation in developing siliques and seedlings of cdk8, and conversely, elevated lipid contents in wild-type seed overexpressing CDK8. Transactivation assays show that CDK8 is necessary for maximal transactivation of the master seed oil activator WRINKLED1 (WRI1) by the seed maturation transcription factor ABSCISIC ACID INSENSITIVE3, supporting a direct regulatory role of CDK8 in oil synthesis. Thermophoretic studies show GEMINIVIRUS REP INTERACTING KINASE1, an activating kinase of KIN10 (a catalytic subunit of SUCROSE NON-FERMENTING1-RELATED KINASE1), physically interacts with CDK8, resulting in its phosphorylation and degradation in the presence of KIN10. This work defines a mechanism whereby, once activated, KIN10 downregulates WRI1 expression and suppresses lipid synthesis via promoting the degradation of CDK8. The KIN10-CDK8-dependent regulation of lipid synthesis described herein is additional to our previously reported KIN10-dependent phosphorylation and degradation of WRI1.

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Fruit chilling injury is the result of physiological dysfunction due to membrane lipid phase change, oxidative damage of biomacromolecules and respiratory metabolism abnormality. However, the involvement of transcription factors in response to fruits chilling tolerance remains largely unclear. Here, MaMYB13 was identified to participate in banana fruit response to chilling stress. MaMYB13 has transcriptional activation activity. When exposed to low temperature, expression of MaMYB13 was enormously induced. Moreover, MaMYB13 promoter was activated by chilling stress. MaMYB13 bound to the promoters of several important very-long-chain fatty acids (VLCFAs) and phenylpropanoids biosynthesis-related genes, including MaKCS11, Ma4CL6 and MaAAE1, and activated their transcription. Furthermore, MaKIN10 X1/3 interacted with MaMYB13 and enhanced MaMYB13-mediated transcriptional activation possibly via phosphorylation. Altogether, our results unravel the mechanism of MaMYB13-MaKIN10 X1/3 interaction regulating banana fruit chilling tolerance through activating the expression of MaKCS11, Ma4CL6 and MaAAE1, providing new insights into the regulatory network of MYB transcription factor.

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De novo phosphatidylcholine (PC) biosynthesis via the Kennedy pathway involves highly endergonic biochemical reactions that must be fine-tuned with energy homeostasis. Previous studies have shown that CTP:phosphocholine cytidylyltransferase (CCT) is an important regulatory enzyme in this pathway and that its activity can be controlled at both transcriptional and posttranslational levels. Here we identified an important additional mechanism regulating plant CCT1 activity. Comparative analysis revealed that Arabidopsis CCT1 (AtCCT1) contains catalytic and membrane-binding domains that are homologous to those of rat CCT1. In contrast, the C-terminal phosphorylation domain important for stringent regulation of rat CCT1 was apparently missing in AtCCT1. Instead, we found that AtCCT1 contains a putative consensus site (Ser-187) for modification by sucrose nonfermenting 1-related protein kinase 1 (SnRK1 or KIN10/SnRK1.1), involved in energy homeostasis. Phos-tag SDS-PAGE coupled with MS analysis disclosed that SnRK1 indeed phosphorylates AtCCT1 at Ser-187, and we found that AtCCT1 phosphorylation substantially reduces its activity by as much as 70%. An S187A variant exhibited decreased activity, indicating the importance of Ser-187 in catalysis, and this variant was less susceptible to SnRK1-mediated inhibition. Protein truncation and liposome binding studies indicated that SnRK1-mediated AtCCT1 phosphorylation directly affects the catalytic domain rather than interfering with phosphatidate-mediated AtCCT1 activation. Overexpression of the AtCCT1 catalytic domain in Nicotiana benthamiana leaves increased PC content, and SnRK1 co-expression reduced this effect. Taken together, our results suggest that SnRK1 mediates the phosphorylation and concomitant inhibition of AtCCT1, revealing an additional mode of regulation for this key enzyme in plant PC biosynthesis.

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@#Objective To compare the effect of impulse electroacupuncture (impulse-EA) and music electroacupuncture (music-EA) for Tongdu Qishen on spatial learning and memory, and expression of cytokine in the hippocampus of APP/PS1 mice. Methods A total of 32 APP/PS1 double transgenic male mice were randomly divided into model group (n=8), drug group (n=8), impulse-EA group (n=8) and music-EA group (n=8), the same background and age male C57BL/6 mice were observed as normal group (n=8). The impulse-EA group and music-EA group accepted EA at Baihui (GV20) and Yintang (GV29), connected with their own electroacupuncture stimulators, for 20 minuts, then, they were pricked Renzhong (GV26) for a while. The drug group accepted donepezil hydrochloride 0.92 mg/kg intra-gastrically. The normal group, model group and drug group were grabbed and bounded in the same way. After 15 days of treatment, they were assessed with Morris water maze. The expression of high mobility group box pro-tein-1 (HMGB1) and interleukin-10 (IL-10) in hippocampus were measured with immunohistochemistry and Western blotting. Results The escape latency shortened inEA groups compared with that of the model group since the fourth day of Morris water maze training (P<0.05), and it was the least in the music-EA group; and the ratio of swimming across the target quadrant increased (P<0.05), but there was no significant difference between EA groups (P>0.05). The ex-pression of HMGB1 decreased (P<0.05) and the expression of IL-10 increased (P<0.05) in EA groups com-pared with that of the model group, and HMGB1 decreased more and IL-10 increased more in the music-EA group than in the impulse-EA group (P<0.05), according to the measurement in immunohistochemistry, not in Western blotting. Conclusion Both impulse-EA and music-EA based on Tongdu Qishen can promote the recovery of the learning and memory in APP/PS1 mice, and music-EA may do more effectively in inhibition of pro-inflammatory factors and promotion of the anti-inflammatory factors.

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Objective To observe the effect of Saccharomyces boulardii(SB) on interleukin-17, interleukin-10 and transforming growth factor-β1 of inflammatory bowel disease ( IBD ) rats which were induced by trinitro-benzene-sulfonic acid( TNBS) . Methods Balb/c female rats were randomly divided into three groups,normal group,TNBS group and TNBS+SB group. TNBS method was used to set up IBD rat models. TNBS group:the mice were injected by 5％ TNBS 0. 1 ml+50％ ethanol 0. 1 ml per mouse from rectum for 5 days,and then were filled in stomach by normal saline 0. 1 ml on the 2nd day for 2 weeks;TNBS+SB group:the mice were injected by 5％ TNBS 0. 10 ml+50％ ethanol 0. 10 ml per mouse from rectum for 5 days,and then were filled in stomach by SB (SB no less than 109 CFU/ml) 0. 1 ml on the 2nd day for 2 weeks;the normal group:the mice were injected by 50％ ethanol 0. 2 ml per mouse from rectum for 5 days, and then were filled in stomach by normal saline 0. 1 ml on the 2nd day for 2 weeks. At the 15th day,the general situation of mice were observed,serum and colon tissue was collected. Alterations of colon inflamma-tion were observed by means of haematoxylin-eosin ( HE);the level of IL-17,IL-10 and TGF-β1 in serum were evaluated by enzyme linked immunosorbent assay (ELISA). The level of IL-17,IL-10 and TGF-β1 in colon tissue was evaluated by immunohistochemistry. Results The IL-17 level of serum and colon tissue in-creased(P<0. 05),but IL-10(P<0. 05) and TGF-β1(P<0. 05)decreased in TNBS group compared with those in the normal group. The level of IL-17 in TNBS+SB group was lower than that in TNBS group( P<0. 05),but the levels of IL-10(P<0. 05) and TGF-β1(P<0. 05)were higher than those in TNBS group. Compared with the normal group,the level of IL-17 in TNBS+SB group increased ( P<0. 05 ) , but IL-10 (P<0.05) and TGF-β1(P <0.05)decreased. Conclusion The expression of cytokine IL-17 increase, IL-10 and TGF-β1 decrease in peripheral blood and colon tissue of IBD. SB may ameliorate the intestinal inflammatory response of IBD by balancing the expression of IL-17,IL-10 and TGF-β1.

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Autophagy is important for degradation and recycling of cytoplasmic materials in all eukaryotes and is often triggered by environmental stress. How autophagy is activated in plants under different environmental conditions is still poorly understood. Our recent studies show that induction of autophagy by different abiotic stress conditions can occur via either a TOR-dependent or -independent pathway, depending on the stress. The SnRK1 protein kinase complex acts upstream of TOR in regulation of autophagy during nutrient deficiency, salt and osmotic stresses. In contrast, oxidative and ER stress regulate autophagy in a SnRK1-dependent but TOR-independent manner. Here we summarize and discuss these distinct pathways for activation of autophagy under different environmental stress conditions.

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Autophagy is a highly conserved system in eukaryotes for the bulk degradation and recycling of intracellular components. Autophagy is involved in many physiological processes including development, senescence, and responses to abiotic and biotic stress. The adenosine 5'-monophosphate (AMP)-activated protein kinase AMPK positively regulates autophagy in mammals; however, the potential function of AMPK in plant autophagy remains largely unknown. Here, we identified KIN10, a plant ortholog of the mammalian AMPK, as a positive regulator of plant autophagy and showed that it acts by affecting the phosphorylation of ATG1 (AUTOPHAGY-RELATED GENE 1) proteins in Arabidopsis. Transgenic Arabidopsis lines overexpressing KIN10 (KIN10-OE) showed delays in leaf senescence, and increased tolerance to nutrient starvation, these phenotypes required a functional autophagy pathway. Consistent with KIN10 having a potential role in autophagy, the nutrient starvation-induced formation of autophagosomes and cleavage of GFP-ATG8e were accelerated in the KIN10-OE lines compared to the wild type. Moreover, the KIN10-OE lines were less sensitive to drought and hypoxia treatments, compared with wild type. Carbon starvation enhanced the level of phosphorylated YFP-ATG1a in the KIN10-OE lines compared to that of wild type. Together, these findings suggest that KIN10 is involved in positive regulation of autophagy, possibly by affecting the phosphorylation of ATG1s in Arabidopsis.

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Banana cultivars may experience chilling or freezing injury in some of their cultivated regions, where wild banana can still grow very well. The clarification of the cold-resistant mechanism of wild banana is vital for cold-resistant banana breeding. In this study, the central stress integrator gene KIN10 and some cold-acclimation related genes (HOS1 and ICE1s) from the cold-resistant wild banana 'Huanxi' (Musa itinerans) were cloned and their expression patterns under different temperature treatments were analyzed. Thirteen full-length cDNA transcripts including 6 KIN10s, 1 HOS1 and 6 ICE1s were successfully cloned. Quantitative real-time PCR (qRT-PCR) results showed that all these genes had the highest expression levels at the critical temperature of banana (13 °C). Under chilling temperature (4 °C), the expression level of KIN10 reduced significantly but the expression of HOS1 was still higher than that at the optimal temperature (28 °C, control). Both KIN10 and HOS1 showed the lowest expression levels at 0 °C, the expression level of ICE1, however, was higher than control. As sucrose plays role in plant cold-acclimation and in regulation of KIN10 and HOS1 bioactivities, the sucrose contents of wild banana under different temperatures were detected. Results showed that the sucrose content increased as temperature lowered. Our result suggested that KIN10 may participate in cold stress response via regulating sucrose biosynthesis, which is helpful in regulating cold acclimation pathway in wild banana.

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Stem cells are capable of both self-renewal (proliferation) and differentiation. Determining the regulatory mechanisms controlling the balance between stem cell proliferation and differentiation is not only an important biological question, but also holds the key for using stem cells as therapeutic agents. The Caenorhabditis elegans germ line has emerged as a valuable model to study the molecular mechanisms controlling stem cell behavior. In this study, we describe a large-scale RNAi screen that identified kin-10, which encodes the ß subunit of protein kinase CK2, as a novel factor regulating stem cell proliferation in the C. elegans germ line. While a loss of kin-10 in an otherwise wild-type background results in a decrease in the number of proliferative cells, loss of kin-10 in sensitized genetic backgrounds results in a germline tumor. Therefore, kin-10 is not only necessary for robust proliferation, it also inhibits the proliferative fate. We found that kin-10's regulatory role in inhibiting the proliferative fate is carried out through the CK2 holoenzyme, rather than through a holoenzyme-independent function, and that it functions downstream of GLP-1/Notch signaling. We propose that a loss of kin-10 leads to a defect in CK2 phosphorylation of its downstream targets, resulting in abnormal activity of target protein(s) that are involved in the proliferative fate vs. differentiation decision. This eventually causes a shift towards the proliferative fate in the stem cell fate decision.

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