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Cuticular wax plays crucial roles in protecting plants from environmental stresses, particularly drought stress. Many enzymeencoding genes and transcription factors involved in wax biosynthesis have been identified, but the underlying posttranslational regulatory mechanisms are poorly understood. Here, we demonstrate that DROUGHT HYPERSENSITIVE (DHS), encoding a Really Interesting New Gene (RING)-type protein, is a critical regulator of wax biosynthesis in rice (Oryza sativa). The cuticular wax contents were significantly reduced in DHS overexpression plants but increased in dhs mutants compared with the wild type, which resulted in a response opposite that of drought stress. DHS exhibited E3 ubiquitin ligase activity and interacted with the homeodomain-leucine zipper IV protein ROC4. Analysis of ROC4 overexpression plants and roc4 mutants indicated that ROC4 positively regulates cuticular wax biosynthesis and the drought stress response. ROC4 is ubiquitinated in vivo and subjected to ubiquitin/26S proteasome-mediated degradation. ROC4 degradation was promoted by DHS but delayed in dhs mutants. ROC4 acts downstream of DHS, and Os-BDG is a direct downstream target of the DHS-ROC4 cascade. These results suggest a mechanism whereby DHS negatively regulates wax biosynthesis by promoting the degradation of ROC4, and they suggest that DHS and ROC4 are valuable targets for the engineering of drought-tolerant rice cultivars.

Background Ubiquitination is essential for many cellular processes in eukaryotes, including 26S proteasome-dependent protein degradation, cell cycle progression, transcriptional regulation, and signal transduction. Although numerous ubiquitinated proteins have been empirically identified, their cognate ubiquitin E3 ligases remain largely unknown. Results Here, we generate a complete ubiquitin E3 ligase-encoding open reading frames (UbE3-ORFeome) library containing 98.94% of the 1515 E3 ligase genes in the rice ( Oryza sativa L . ) genome. In the test screens with four known ubiquitinated proteins, we identify both known and new E3s. The interaction and degradation between several E3s and their substrates are confirmed in vitro and in vivo. In addition, we identify the F-box E3 ligase OsFBK16 as a hub-interacting protein of the phenylalanine ammonia lyase family OsPAL1–OsPAL7. We demonstrate that OsFBK16 promotes the degradation of OsPAL1, OsPAL5, and OsPAL6. Remarkably, we find that overexpression of OsPAL1 or OsPAL6 as well as loss-of-function of OsFBK16 in rice displayed enhanced blast resistance, indicating that OsFBK16 degrades OsPALs to negatively regulate rice immunity. Conclusions The rice UbE3-ORFeome is the first complete E3 ligase library in plants and represents a powerful proteomic resource for rapid identification of the cognate E3 ligases of ubiquitinated proteins and establishment of functional E3–substrate interactome in plants.

Although a coal block in the Qinshui Basin is a key location for coalbed methane (CBM) exploration and development, the productivity of various vertical wells varies significantly. The productivity characteristics of 636 vertical wells were analyzed better to understand the effects of different variables on CBM productivity. Grey correlation analysis was used to identify the key determinants of CBM well productivity. The findings indicate that vertical well production is below average, with more than 50% serving as water wells. The production of the central well area is high in the west and south. Highly productive wells are significantly influenced by geological conditions, primarily located in areas with high gas content, moderate cover depth, high permeability, and elevated water head. The engineering factor that exerts the most significant influence on productivity is the rate of liquid level drop. Based on grey correlation analysis, the primary determinants of CBM vertical well production are permeability and cover depth.

Ubiquitin-regulated protein degradation is a critical regulatory mechanism that controls a wide range of biological processes in plants. Here, we report that OsDISl (for Oryza sativa drought-induced SINA protein 1), a C3HC4 RING finger E3 ligase, is involved in drought-stress signal transduction in rice (O.sativa).The expression of OsDISl was up-regulated by drought treatment. In vitro ubiquitination assays showed that OsDISl possessed E3 ubiquitin ligase activity and that the conserved region of the RING finger was required for the activity. Transient expression assays in Nicotiana benthamiana leaves and rice protoplasts indicated that OsDISl was localized predominantly in the nucleus. Overexpression of OsDISl reduced drought tolerance in transgenic rice plants, while RNA interference silencing of OsDISl enhanced drought tolerance. Microarray analysis revealed that a large number of drought-responsive genes were induced or suppressed in the OsDISl overexpression plants under normal and drought conditions. Yeast two-hybrid screening showed that OsDISl interacted with OsNek6 (for O. sativa NIMA-related kinase 6), a tubulin complex-related serine/threonine protein kinase. Coexpression assays in N. benthamiana leaves indicated that OsNek6 was degraded by OsDISl via the 26S proteasome-dependent pathway and that this degradation was abolished by the OsDISl (H71Y) mutation, which is essential for its E3 ligase activity. Together, these results demonstrate that OsDISl plays a negative role in drought stress tolerance through transcriptional regulation of diverse stressrelated genes and possibly through posttranslational regulation of OsNek6 in rice.

Plant viruses are excellent tools for studying microbial-plant interactions as well as the complexities of host activities. Our study focuses on the role of C2 encoded by Beet severe curly top virus (BSCTV) in the virus-plant interaction. Using BSCTV C2 as bait in a yeast two-hybrid screen, a C2-interacting protein, S-adenosyl-methionine decarboxylase 1 (SAMDC1), was identified from an Arabidopsis thaliana cDNA library. The interaction was confirmed by an in vitro pull-down assay and a firefly luciferase complemention imaging assay in planta. Biochemical analysis further showed that the degradation of the SAMDC1 protein was inhibited by MG132, a 26S proteasome inhibitor, and that C2 could attenuate the degradation of the SAMDC1 protein. Genetic analysis showed that loss of function of SAMDC1 resulted in reduced susceptibility to BSCTV infection and reduced viral DNA accumulation, similar to the effect of BSCTV C2 deficiency. Bisulfite sequencing analysis further showed that C2 deficiency caused enhanced DNA methylation of the viral genome in infected plants. We also showed that C2 can suppress de novo methylation in the FWA transgenic assay in the C2 transgene background. Overexpression of SAMDC1 can mimic the suppressive activity of C2 against green fluorescent protein-directed silencing. These results suggest that C2 interferes with the host defense mechanism of DNA methylation-mediated gene silencing by attenuating the 26S proteasome-mediated degradation of SAMDC1.

We have previously shown that the Arabidopsis (Arabidopsis thaliana) RING-H2 E3 ligase RHA2a positively regulates abscisic acid (ABA) signaling during seed germination and postgerminative growth. Here, we report that RHA2b, the closest homolog of RHA2a, is also an active E3 ligase and plays an important role in ABA signaling. We show that RHA2b expression is induced by ABA and that overexpression of RHA2b leads to ABA-associated phenotypes such as ABA hypersensitivity in seed germination and seedling growth, enhanced stomatal closure, reduced water loss, and, therefore, increased drought tolerance. On the contrary, the rha2b-1 mutant shows ABA-insensitive phenotypes and reduced drought tolerance. We provide evidence showing that a rha2a rha2b-1 double mutant generally enhances ABA insensitivity of rha2b-1 in seed germination, seedling growth, and stomatal closure, suggesting that RHA2b and RHA2a act redundantly in regulating ABA responses. Genetic analyses support that, like RHA2a, the RHA2b action in ABA signaling is downstream of a protein phosphatase 2C, ABA-INSENSITIVE2 (ABI2), and in parallel with that of the ABI transcription factors ABI3/4/5. We speculate that RHA2b and RHA2a may have redundant yet distinguishable functions in the regulation of ABA responses.

Background This study investigated the impact of right heart volume overload on renal perfusion in patients with heart failure (HF). We retrospectively analyzed 304 ambulatory HF patients enrolled between October 2017 and August 2022. Echocardiographic parameters—including left atrial diameter (LAD), left ventricular end-diastolic diameter (LVEDD), right atrial diameter (RAD), right ventricular diameter (RVD), and left ventricular ejection fraction (LVEF)—were measured and adjusted for body surface area (BSA). Renal perfusion was assessed via time-to-peak of renal blood flow (TTPr) using renal scintigraphy. Relationships between echocardiographic measures and TTPr were evaluated using Spearman correlation and multivariable ordinal logistic regression analyses. Results In the overall cohort, RAD/BSA showed the strongest correlation with TTPr (rs = 0.608, P  < 0.001), which remained significant after multivariable adjustment. LVEDD/BSA and LVEF showed weak or no associations. Subgroup analyses demonstrated that RAD/BSA had the strongest correlation in HF with reduced ejection fraction (rs = 0.602, P  < 0.001) and HF with preserved ejection fraction (rs = 0.496, P  < 0.001), while LAD/BSA was most strongly correlated in HF with mildly reduced ejection fraction (rs = 0.586, P  < 0.001), all remaining significant after adjustment. Conclusions Echocardiographic parameters of the right heart volume overload were associated with TTPr, suggesting a connection between right heart overload and renal perfusion in cardiorenal syndrome. This points to potential therapeutic targets to improve renal perfusion and outcomes in CHF patients.

The components in plant signal transduction pathways are intertwined and affect each other to coordinate plant growth, development, and defenses to stresses. The role of ubiquitination in connecting these pathways, particularly plant innate immunity and flowering, is largely unknown. Here, we report the dual roles for the Arabidopsis (Arabidopsis thaliana) Plant U-box protein13 (PUB13) in defense and flowering time control. In vitro ubiquitination assays indicated that PUB13 is an active E3 ubiquitin ligase and that the intact U-box domain is required for the E3 ligase activity. Disruption of the PUB13 gene by T-DNA insertion results in spontaneous cell death, the accumulation of hydrogen peroxide and salicylic acid (SA), and elevated resistance to biotrophic pathogens but increased susceptibility to necrotrophic pathogens. The cell death, hydrogen peroxide accumulation, and resistance to necrotrophic pathogens in publ3 are enhanced when plants are pretreated with high humidity. Importantly, publ3 also shows early flowering under middle-and long-day conditions, in which the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 and FLOWERING LOCUS T is induced while FLOWERING LOCUS C expression is suppressed. Finally, we found that two components involved in the Sa-mediated signaling pathway, SID2 and PAD4, are required for the defense and flowering-time phenotypes caused by the loss of function of PUB13. Taken together, our data demonstrate that PUB13 acts as an important node connecting SA-dependent defense signaling and flowering time regulation in Arabidopsis.

Objective This study aimed to explore the characteristics of abdominal aortic blood flow in patients with heart failure (HF) using 99m Tc-diethylenetriaminepentaacetic acid (DTPA) renal scintigraphy. We investigated the ability of renal scintigraphy to measure the cardiopulmonary transit time and assessed whether the time-to-peak of the abdominal aorta (TTPa) can distinguish between individuals with and without HF. Methods We conducted a retrospective study that included 304 and 37 patients with and without HF (controls), respectively. All participants underwent 99m Tc-DTPA renal scintigraphy. The time to peak from the abdominal aorta’s first-pass time–activity curve was noted and compared between the groups. The diagnostic significance of TTPa for HF was ascertained through receiver operating characteristic (ROC) analysis and logistic regression. Factors influencing the TTPa were assessed using ordered logistic regression. Results The HF group displayed a significantly prolonged TTPa than controls (18.5 [14, 27] s vs. 11 [11, 13] s). Among the HF categories, HF with reduced ejection fraction (HFrEF) exhibited the longest TTPa compared with HF with mildly reduced (HFmrEF) and preserved EF (HFpEF) (25 [17, 36.5] s vs. 17 [15, 23] s vs. 15 [11, 17] s) ( P  < 0.001). The ROC analysis had an area under the curve of 0.831, which underscored TTPa’s independent diagnostic relevance for HF. The diagnostic precision was enhanced as left ventricular ejection fraction (LVEF) declined and HF worsened. Independent factors for TTPa included the left atrium diameter, LVEF, right atrium diameter, velocity of tricuspid regurgitation, and moderate to severe aortic regurgitation. Conclusions Based on 99m Tc-DTPA renal scintigraphy, TTPa may be used as a straightforward and non-invasive tool that can effectively distinguish patients with and without HF.

The phytohormone abscisic acid (ABA) is well known for its regulatory roles in integrating environmental constraints with the developmental programs of plants. Here, we characterize the biological function of the Arabidopsis (Arabidopsis thaliana) RING-H2 protein RHA2a in ABA signaling. The rha2a mutant is less sensitive to ABA than the wild type during seed germination and early seedling development, whereas transgenic plants overexpressing RHA2a are hypersensitive, indicating that RHA2a positively regulates ABA-mediated control of seed germination and early seedling development. Double mutant analyses of rha2a with several known ABA-insensitive mutants suggest that the action of RHA2a in ABA signaling is independent of that of the transcription factors ABI3, ABI4, and ABI5. We provide evidence showing that RHA2a also positively regulates plant responses to salt and osmotic stresses during seed germination and early seedling development. RHA2a is a functional E3 ubiquitin ligase, and its conserved RING domain is likely important for the biological function of RHA2a in ABA signaling. Together, these results suggest that the E3 ligase RHA2a is an important regulator of ABA signaling during seed germination and early seedling development.