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SUMMARY: The mechanism regulating proteasomal activity under proteotoxic stress conditions remains unclear. Here, we showed that arsenite‐induced proteotoxic stress resulted in upregulation of Arabidopsis homologous PUB22 and PUB23 U‐box E3 ubiquitin ligases and that pub22pub23 double mutants displayed arsenite‐insensitive seed germination and root growth phenotypes. PUB22/PUB23 downregulated 26S proteasome activity by promoting the dissociation of the 19S regulatory particle from the holo‐proteasome complex, resulting in intracellular accumulation of UbG76V‐GFP, an artificial substrate of the proteasome complex, and insoluble poly‐ubiquitinated proteins. These results suggest that PUB22/PUB23 play a critical role in arsenite‐induced proteotoxic stress response via negative regulation of 26S proteasome integrity. [ABSTRACT FROM AUTHOR]

Summary: Drought stress has detrimental effects on plants. Although the abscisic acid (ABA)‐mediated drought response is well established, defensive mechanisms to cope with dehydration‐induced proteotoxicity have been rarely studied. DRR1 was identified as an Arabidopsis drought‐induced gene encoding an ER‐localized RING‐type E3 Ub ligase. Suppression of DRR1 markedly reduced tolerance to drought and proteotoxic stress without altering ABA‐mediated germination and stomatal movement. Proteotoxicity‐ and dehydration‐induced insoluble ubiquitinated protein accumulation was more obvious in DRR1 loss‐of‐function plants than in wild‐type plants. These results suggest that DRR1 is involved in an ABA‐independent drought stress response possibly through the mitigation of dehydration‐induced proteotoxic stress. [ABSTRACT FROM AUTHOR]

SUMMARY: Ubiquitination is a critical post‐translational protein modification that has been implicated in diverse cellular processes, including abiotic stress responses, in plants. In the present study, we identified and characterized a T‐DNA insertion mutant in the At5g10650 locus. Compared to wild‐type Arabidopsis plants, at5g10650 progeny were hyposensitive to ABA at the germination stage. At5g10650 possessed a single C‐terminal C3HC4‐type Really Interesting New Gene (RING) motif, which was essential for ABA‐mediated germination and E3 ligase activity in vitro. At5g10650 was closely associated with microtubules and microtubule‐associated proteins in Arabidopsis and tobacco leaf cells. Localization of At5g10650 to the nucleus was frequently observed. Unexpectedly, At5g10650 was identified as JAV1‐ASSOCIATED UBIQUITIN LIGASE1 (JUL1), which was recently reported to participate in the jasmonate signaling pathway. The jul1 knockout plants exhibited impaired ABA‐promoted stomatal closure. In addition, stomatal closure could not be induced by hydrogen peroxide and calcium in jul1 plants. jul1 guard cells accumulated wild‐type levels of H2O2 after ABA treatment. These findings indicated that JUL1 acts downstream of H2O2 and calcium in the ABA‐mediated stomatal closure pathway. Typical radial arrays of microtubules were maintained in jul1 guard cells after exposure to ABA, H2O2, and calcium, which in turn resulted in ABA‐hyposensitive stomatal movements. Finally, jul1 plants were markedly more susceptible to drought stress than wild‐type plants. Overall, our results suggest that the Arabidopsis RING E3 ligase JUL1 plays a critical role in ABA‐mediated microtubule disorganization, stomatal closure, and tolerance to drought stress. Significance Statement: Ubiquitination is a universal post‐translational protein modification in eukaryotic organisms. We show that Arabidopsis RING E3 ubiquitin ligase JUL1 plays a critical role in ABA‐mediated microtubule disorganization, stomatal closure, and tolerance to drought stress. The present study demonstrates that the ubiquitin‐mediated microtubule structure in guard cell is functionally correlated with ABA‐dependent drought stress response in Arabidopsis. [ABSTRACT FROM AUTHOR]

Summary: Oryza sativa BRASSINAZOLE RESISTANT 1 (OsBZR1) is the closest rice homolog of the Arabidopsis BZR1 and bri1‐EMS‐SUPPRESSOR 1 (BES1)/BZR2 transcription factors. OsBZR1 plays a central role in the rice brassinosteroid signaling pathway. Despite its functional importance, the control mechanism by which the cellular stability of OsBZR1 is regulated has not yet been fully elucidated. Here, we report that a rice U‐box E3 ubiquitin (Ub) ligase OsPUB24 acts as a negative regulator in the BR signaling pathway via the 26S proteasome‐dependent degradation of OsBZR1. The ospub24 T‐DNA knock‐out mutant and Ubi:RNAi‐OsPUB24 knock‐down rice plants displayed enhanced seedling growth, increased lamina joint bending, and hypersensitivity to brassinolide (BL). The expressions of the BR biosynthetic genes suppressed by BR in a negative feedback loop were lower in the mutant progeny than in the wild‐type rice plants, which indicated increased BR responses in the mutant line. OsPUB24 ubiquitinated OsBZR1, resulting in the proteasomal degradation of OsBZR1. In addition, the stability of OsPUB24 was downregulated by BL and bikinin, an inhibitor of Oryza sativa Shaggy/GSK3‐like kinase 22 (OsSK22). OsSK22, the homolog of Arabidopsis BRASSINOSTEROID INSENSITIVE 2 (BIN2) protein kinase, phosphorylated OsPUB24 and elevated the cellular stability of OsPUB24. Our findings suggest that OsPUB24 participates in OsBZR1 turnover, and that the regulatory networks of OsPUB24, OsSK22 and OsBZR1 are crucial for fine‐tuning the BR response in rice. Significance Statement: OsBZR1 transcription factor is a key component of the rice brassinosteroid signaling pathway. Here, we show that OsBZR1 is subject to control by two negative factors, OsSK22 protein kinase and OsPUB24 E3 ubiquitin ligase, in the rice BR signaling pathway. This study illustrates that the signaling process of BR is cooperatively regulated by two distinct types of post‐translational modifications, phosphorylation and ubiquitination. [ABSTRACT FROM AUTHOR]

A highly coordinated complex known as the microprocessor precisely processes primary transcripts of MIRNA genes into mature mi RNAs. In plants, the microprocessor minimally consists of three components: Dicer-like protein 1 ( DCL1), HYPONASTIC LEAF 1 ( HYL1), and SERRATE ( SE). To precisely modulate mi RNA maturation, the microprocessor cooperates with at least 12 proteins in plants. In addition, we here show the involvement of a novel gene, HYL1-interacting GIY- YIG-like endonuclease ( HIGLE). The encoded protein has a GIY- YIG domain that is generally found within a class of homing endonucleases. HIGLE directly interacts with the microprocessor components HYL1 and SE. Unlike the functions of other GIY- YIG endonucleases, the catalytic core of HIGLE has both DNase and RNase activities that sufficiently processes mi RNA precursors into short fragments in vitro. [ABSTRACT FROM AUTHOR]

Extracellular superoxide dismutase ( EC- SOD) is an enzyme that catalyses the dismutation of superoxide anions. It has multiple functions, such as reactive oxygen species scavenging, anti-angiogenic, anti-inflammatory, antichemotatic and antitumor activities. Recently, we demonstrated that EC- SOD inhibits ovalbumin-induced allergic airway inflammation in mice. However, the anti-allergic effect of EC- SOD on skin tissue and the role of EC- SOD in mast cells, which are important for allergic responses, have not been well studied. In this study, we investigated whether EC- SOD can alleviate atopic dermatitis in mice and inhibit mast cell activation. Treatment with human recombinant EC- SOD ameliorated house dust mite-induced atopic dermatitis in mice. Furthermore, the levels of pro-allergic cytokine gene expression and histamine release increased in EC- SOD KO mast cells and decreased in EC- SOD overexpressing mast cells, suggesting that EC- SOD inhibits mast cell activation. Consistently, a passive cutaneous anaphylaxis experiment showed more blood leakage from EC- SOD KO mouse ear skin, implying that the lack of EC- SOD increases allergic responses. These results suggest that EC- SOD inhibits mast cell activation and atopic dermatitis and that the loss of EC- SOD causes more severe allergic responses, implying that EC- SOD might be a good drug candidate for treatment of allergic disorders, such as atopic dermatitis. [ABSTRACT FROM AUTHOR]

The cellular roles of RAD51 paralogs in somatic and reproductive growth have been extensively described in a wide range of animal systems and, to a lesser extent, in Arabidopsis, a dicot model plant. Here, the OsRAD51D gene was identified and characterized in rice ( Oryza sativa L.), a monocot model crop. In the rice genome, three alternative OsRAD51D mRNA splicing variants, OsRAD51D.1, OsRAD51D.2, and OsRAD51D.3, were predicted. Yeast two-hybrid studies, however, showed that only OsRAD51D.1 interacted with OsRAD51B and OsRAD51C paralogs, suggesting that OsRAD51D.1 is a functional OsRAD51D protein in rice. Loss-of-function osrad51d mutant rice plants displayed normal vegetative growth. However, the mutant plants were defective in reproductive growth, resulting in sterile flowers. Homozygous osrad51d mutant flowers exhibited impaired development of lemma and palea and contained unusual numbers of stamens and stigmas. During early meiosis, osrad51d pollen mother cells (PMCs) failed to form normal homologous chromosome pairings. In subsequent meiotic progression, mutant PMCs represented fragmented chromosomes. The osrad51d pollen cells contained numerous abnormal micro-nuclei that resulted in malfunctioning pollen. The abnormalities of heterozygous mutant and T2 Ubi:RNAi-OsRAD51D RNAi-knock-down transgenic plants were intermediate between those of wild type and homozygous mutant plants. The osrad51d and Ubi:RNAi-OsRAD51D plants contained longer telomeres compared with wild type plants, indicating that OsRAD51D is a negative factor for telomere lengthening. Overall, these results suggest that OsRAD51D plays a critical role in reproductive growth in rice. This essential function of OsRAD51D is distinct from Arabidopsis, in which AtRAD51D is not an essential factor for meiosis or reproductive development. [ABSTRACT FROM AUTHOR]

Highlights: [•] AtATL78 is an Arabidopsis plasma-membrane-localized RING E3 ubiquitin ligase. [•] AtATL78 was up-regulated by cold stress and, in contrast, down-regulated by drought. [•] Suppression of AtATL78 increases tolerance to cold stress. [•] Suppression of AtATL78 decreases tolerance to drought stress. [•] AtATL78 plays opposing roles in cold and drought stress responses in Arabidopsis. [ABSTRACT FROM AUTHOR]

The plant U-box (PUB) protein functions as an E3 ligase to poly-ubiquitinate a target protein for its degradation or post-translational modification. Here, we report functional roles for OsPUB15, which encodes a cytosolic U-box protein in the class-II PUB family. Self-ubiquitination assays showed that bacterially expressed MBP-OsPUB15 protein has E3 ubiquitin ligase activity. A T-DNA insertional mutation in OsPUB15 caused severe growth retardation and a seedling-lethal phenotype. Mutant seeds did not produce primary roots, and their shoot development was significantly delayed. Transgenic plants expressing the OsPUB15 antisense transcript phenocopied these mutant characters. The abnormal phenotypes were partially rescued by two antioxidants, catechin and ascorbic acid. Germinating seeds in the dark also recovered the rootless defect. Levels of HO and oxidized proteins were higher in the knock-out mutant compared with the wild type. OsPUB15 transcript levels were increased upon HO, salt and drought stresses; plants overexpressing the gene grew better than the wild type under high salinity. These results indicate that PUB15 is a regulator that reduces reactive oxygen species (ROS) stress and cell death. [ABSTRACT FROM AUTHOR]

Abstract: In Arabidopsis, there are at least seven class I acylhydrolase members, which have a putative N-terminal chloroplast-targeting signal. Here, we show that all seven class I proteins are localized to the chloroplasts and hydrolyze phosphatidylcholine at the sn-1 position. However, based on their activities toward various lipids, Arabidopsis class I enzymes could be further divided into three sub-groups by substrate specificity, one with phospholipase-specific activity, another with phospholipase and galactolipase activities, and the other with broad lipolytic activity toward phosphatidylcholine, galactolipids, and triacylglycerol. These results suggest that the three sub-groups of class I acylhydrolases have specific roles in chloroplasts. [Copyright &y& Elsevier]

Abstract: Xyloglucan endotransglucosylase/hydrolase (XTH) has been recognized as a cell wall-modifying enzyme, participating in the diverse physiological roles. From water-stressed hot pepper plants, we isolated three different cDNA clones (pCaXTH1, pCaXTH2, and pCaXTH3) that encode XTH homologs. RT-PCR analysis showed that three CaXTH mRNAs were concomitantly induced by a broad spectrum of abiotic stresses, including drought, high salinity and cold temperature, and in response to stress hormone ethylene, suggesting their role in the early events in the abiotic-related defense response. Transgenic Arabidopsis plants that constitutively expressed the CaXTH3 gene under the control of the CaMV 35S promoter exhibited abnormal leaf morphology; the transgenic leaves showed variable degrees of twisting and bending along the edges, resulting in a severely wrinkled leaf shape. Microscopic analysis showed that 35S-CaXTH3 leaves had increased numbers of small-sized cells, resulting in disordered, highly populated mesophyll cells in each dorsoventral layer, and appeared to contain a limited amount of starch. In addition, the 35S-CaXTH3 transgenic plants displayed markedly improved tolerance to severe water deficit, and to lesser extent to high salinity in comparison with the wild-type plants. These results indicate that CaXTH3 is functional in heterologous Arabidopsis cells, thereby effectively altering cell growth and also the response to abiotic stresses. Although the physiological function of CaXTHs is not yet clear, there are several possibilities for their involvement in a subset of physiological responses to counteract dehydration and high salinity stresses in transgenic Arabidopsis plants. [Copyright &y& Elsevier]

Abstract: The very long chain fatty acids (VLCFAs) are synthesized by the microsomal fatty acid elongation system in plants. We investigated cellular function of NbECR putatively encoding enoyl-CoA reductase that catalyzes the last step of VLCFA elongation in Nicotiana benthamiana. Virus-induced gene silencing of NbECR produced necrotic lesions with typical cell death symptoms in leaves. In the affected tissues, ablation of the epidermal cell layer preceded disintegration of the whole leaf cell layers, and disorganized cellular membrane structure was evident. The amount of VLCFAs was reduced in the NbECR VIGS lines, suggesting NbECR function in elongation of VLCFAs. The results demonstrate that NbECR encodes a putative enoyl-CoA reductase and that the NbECR activity is essential for membrane biogenesis in N. benthamiana. [Copyright &y& Elsevier]

Retinoblastoma protein (Rb) plays a key role in cell cycle control, cell differentiation, and apoptosis in animals. In this study, we used virus-induced gene silencing (VIGS) to investigate the cellular functions of Rb in higher plants. VIGS ofNbRBR1, which encodes theNicotiana benthamianaRb homolog, resulted in growth retardation and abnormal organ development. At the cellular level, Rb suppression caused prolonged cell proliferation in tissues that are normally differentiated, which indicates that Rb is a negative regulator of plant cell division. Furthermore, differentiation of the epidermal pavement cells and trichomes was partially retarded, and stomatal clusters formed in the epidermis, likely due to uncontrolled cell division of stomata precursor cells. Rb suppression also caused extra DNA replication in endoreduplicating leaf cells, suggesting a role of Rb in the endocycle. These Rb phenotypes were accompanied by stimulated transcription ofE2Fand E2F-regulated S-phase genes. Thus, disruption of Rb function in plants leads to ectopic cell division in major organs that correlates with a delay in cell differentiation as well as increased endoreduplication, which indicates that Rb coordinates these processes in plant organ development. [ABSTRACT FROM AUTHOR]

Abstract: Although fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 3 (FGFR3) both inhibit longitudinal bone growth, little is known about the relationship between FGF2 and FGFR3. Accordingly, the current study examined the expression of FGFR3 mRNA after the administration of FGF2 using cultured chondrocytes from day 17 chick embryos to evaluate the relationship between FGF2 and FGFR3. The chondrocytes were isolated from the caudal one-third portion (LS) of sterna, peripheral regions (USP) and central core regions (USC) of the cephalic portion of the sterna, and lower portion of the proximal tibial growth plate (Ti) of day 17 chick embryo. The expression of FGFR1, FGFR3, and type II and X collagen mRNA in the chondrocytes from the LS, USP, USC, and Ti was determined. FGFR1 was not expressed in the LS and USP chondrocytes, yet strongly expressed in the USC and Ti chondrocytes. With a treatment of FGF2, the expression of FGFR1 slightly increased in the USC chondrocytes and was not related with the concentration of FGF2 in the Ti chondrocytes. FGFR3 was expressed in all the chondrocyte types, yet strongly increased in the LS, USC, USP, and Ti in that order according to the concentration of FGF2. For the LS and USP chondrocytes, the expression of FGFR3 with FGF2 increased in a 4-day culture, yet decreased in a 6-day culture, whereas for the USC chondrocytes, the expression of FGFR3 mRNA with FGF2 increased in a 2-day culture, yet decreased in a 4-day culture, suggesting that the hypertrophic chondrocytes were more numerous and sensitive compared to the proliferative chondrocytes. For all the chondrocyte types, FGF2 appeared to be up-regulated to FGFR3, as the expression of FGFR3 mRNA increased with a higher concentration of FGF2 until a peak level. In conclusion, FGF2 was found to up-regulate to FGFR3 until the peak level of FGFR3 mRNA expression, while in hypertrophic chondrocytes, FGFR3 appeared to cause the differentiaton of chondrocytes, resulting in the inhibition of longitudinal bone growth after the peak level of FGFR3 mRNA expression. [Copyright &y& Elsevier]

Phosphoinositide-specific phospholipase C (PI-PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate inositol 1,4,5-trisphosphate and diacylglycerol, both of which act as secondary messengers in animal cells. In this report, we identified in Vigna radiata L. (mung bean) three distinct partial cDNAs (pVr-PLC1, pVr-PLC2, and pVr-PLC3), which encode forms of putative PI-PLC. All three Vr-PLC genes were transcriptionally active and displayed unique patterns of expression. The Vr-PLC1 and Vr-PLC2 transcripts were constitutively expressed to varying degrees in every tissue of mung bean plants examined. In contrast, the Vr-PLC3 mRNA level was very low under normal growth conditions and was rapidly induced in an abscisic acid-independent manner under environmental stress conditions (drought and high salinity). An isolated genomic clone, about 8.2 kb in length, showed that Vr-PLC1 and Vr-PLC3 are in tandem array in the mung bean genome. The predicted primary sequence of Vr-PLC3 (Mr=67.4 kDa) is reminiscent of the δ-isoform of animal enzymes which contain core sequences found in typical PI-PLCs, such as the catalytic domain comprising X and Y motifs, a lipid-binding C2 domain, and the less conserved EF-hand domain. Results of in vivo targeting experiment using a green fluorescent protein (GFP) showed that the GFP-Vr-PLC3 fusion protein was localized primarily to the plasma membrane of the Arabidopsis protoplast. The C2 domain was essential for Vr-PLC3 to be targeted to the plasma membrane. The possible biological functions of stress-responsive Vr-PLC3 in mung bean plants are discussed. [Copyright &y& Elsevier]

Summary Telomerase is a specialized RNA-directed DNA polymerase that adds telomeric repeats onto the ends of linear eukaryotic chromosomes. It was recently reported that the low, basal level of telomerase activity markedly increased at early S-phase of the cell cycle, and auxin further increased the S-phase-specific telomerase activity in tobacco BY-2 cells. In this study we show that abscisic acid (ABA), a phytohormone known to induce the cyclin-dependent protein kinase inhibitor, effectively abolished both the auxin- and S-phase-specific activation of telomerase in a concentration- and time-dependent fashion in synchronized tobacco BY-2 cells. These results suggest that there exists a hormonal cross-talk between auxin and ABA for the regulation of telomerase activity during the cell cycle of tobacco cells. Treatment of synchronized BY-2 cells with the protein kinase inhibitor staurosporine or H-7 effectively prevented the S-phase-specific activation of telomerase activity. By contrast, when okadaic acid or cantharidin, potent inhibitors of protein phosphatase 2A (PP2A), was applied to the cells, the S-phase-specific high level of telomerase activity was continuously maintained in the cell cycle for at least 14 h after release from M-phase arrest. Incubation of tobacco cell extracts with exogenous PP2A rapidly abrogated in vitro telomerase activity, while okadaic acid and cantharidin blocked the action of PP2A, effectively restoring in vitro telomerase activity. Taken together, these findings are discussed in the light of the suggestion that antagonistic functions of auxin and ABA, and reciprocal phosphorylation and dephosphorylation of telomerase complex, are necessarily involved in the cell cycle-dependent modulation of telomerase activity in tobacco cells. [ABSTRACT FROM AUTHOR]

Summary Marchantia polymorpha, occupying a basal position in the monophyletic assemblage of land plants, displays a notable expansion of plant U‐box (PUB) proteins compared with those in animals. We elucidated the roles of MpPUB9 in regulating salt stress tolerance in M. polymorpha. MpPUB9 expression was rapidly induced by high salinity and dehydration. MpPUB9 possessed an intact U‐box domain in the N‐terminus. MpPUB9‐Citrine localized to punctate structures and was peripherally associated with microsomal membranes. Phenotypic analyses demonstrate that the hyponastic and epinastic thallus growth phenotypes, which were induced by the overexpression and suppression of MpPUB9, may provoke salt stress‐resistant and ‐susceptible phenotypes, respectively. MpPUB9 was also found to directly interact with the exocyst protein MpEXO70.1, leading to its ubiquitination. Under high‐salinity conditions, though the stability of MpPUB9 was dramatically increased, MpEXO70.1 showed slightly faster turnover rates. Transcriptome analyses showed that salt treatment and the overexpression of MpPUB9 co‐upregulated the genes related to the modulation of H2O2 and cell wall organization. Overall, our results suggest that MpPUB9 plays a crucial role in the positive regulation of salt stress tolerance, resulting from its interaction with MpEXO70.1 and modulating turnover of the protein under high‐salt conditions via the coordination of UPS with autophagy. [ABSTRACT FROM AUTHOR]