Hydrogen sulfide improves salt tolerance through persulfidation of PMA1 in Arabidopsis.

KEY MESSAGE: A new interaction was found between PMA1 and GRF4. H2S promotes the interaction through persulfidated Cys446 of PMA1. H2S activates PMA1 to maintain K+/Na+ homeostasis through persulfidation under salt stress. Plasma membrane H+-ATPase (PMA) is a transmembrane transporter responsible for pumping protons, and its contribution to salt resistance is indispensable in plants. Hydrogen sulfide (H2S), a small signaling gas molecule, plays the important roles in facilitating adaptation of plants to salt stress. However, how H2S regulates PMA activity remains largely unclear. Here, we show a possible original mechanism for H2S to regulate PMA activity. PMA1, a predominant member in the PMA family of Arabidopsis, has a non-conservative persulfidated cysteine (Cys) residue (Cys446), which is exposed on the surface of PMA1 and located in cation transporter/ATPase domain. A new interaction of PMA1 and GENERAL REGULATORY FACTOR 4 (GRF4, belongs to the 14-3-3 protein family) was found by chemical crosslinking coupled with mass spectrometry (CXMS) in vivo. H2S-mediated persulfidation promoted the binding of PMA1 to GRF4. Further studies showed that H2S enhanced instantaneous H+ efflux and maintained K+/Na+ homeostasis under salt stress. In light of these findings, we suggest that H2S promotes the binding of PMA1 to GRF4 through persulfidation, and then activating PMA, thus improving the salt tolerance of Arabidopsis.

Quantitative Proteomic and Phosphoproteomic Profiling of Lung Tissues from Pulmonary Arterial Hypertension Rat Model.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease characterized by elevated pulmonary vascular resistance and increased pressure in the distal pulmonary arteries. Systematic analysis of the proteins and pathways involved in the progression of PAH is crucial for understanding the underlying molecular mechanism. In this study, we performed tandem mass tags (TMT)-based relative quantitative proteomic profiling of lung tissues from rats treated with monocrotaline (MCT) for 1, 2, 3 and 4 weeks. A total of 6759 proteins were quantified, among which 2660 proteins exhibited significant changes (p-value < 0.05, fold change < 0.83 or >1.2). Notably, these changes included several known PAH-related proteins, such as Retnla (resistin-like alpha) and arginase-1. Furthermore, the expression of potential PAH-related proteins, including Aurora kinase B and Cyclin-A2, was verified via Western blot analysis. In addition, we performed quantitative phosphoproteomic analysis on the lungs from MCT-induced PAH rats and identified 1412 upregulated phosphopeptides and 390 downregulated phosphopeptides. Pathway enrichment analysis revealed significant involvement of pathways such as complement and coagulation cascades and the signaling pathway of vascular smooth muscle contraction. Overall, this comprehensive analysis of proteins and phosphoproteins involved in the development and progression of PAH in lung tissues provides valuable insights for the development of potential diagnostic and treatment targets for PAH.

One-dimensional nitrogen doped porous carbon nano-array arranged by carbon nanotubes for electrochemical sensing ascorbic acid, dopamine and uric acid simultaneously.

In this work, one-dimensional nitrogen doped porous carbon nano-arrays arranged by carbon nanotube (1D CNTs@NPC) were first constructed, using a coating technology at room temperature and followed by high temperature carbonization. It was expected that the resulting glassy carbon electrodes modified by 1D CNTs@NPC (CNTs@NPC/GCE) could express different electrochemical responses to ascorbic acid (AA), dopamine (DA), uric acid (UA), by virtue of the synergistic-improved effect between CNTs and NPC. Under the optimized conditions, there were excellent analytical parameters for CNTs@NPC/GCE to detect AA, DA and UA, i.e. a wide linear range of 40-2100µM for AA, 0.5-49µM for DA and 3-50µM for AA with low detection limits of 0.36µM, 0.02µmol l-1and 0.57µM respectively. Importantly, the proposed CNTs@NPC/GCE was efficiently applied to determine AA, DA and UA in some real samples with high stability, reproducibility and selectivity. This work will offer an efficient potential for diagnosing ascorbic acid, dopamine or uric acid-related diseases on clinical testing in future.

Nitric oxide induces S-nitrosylation of CESA1 and CESA9 and increases cellulose content in Arabidopsis hypocotyls.

Nitric oxide (NO), a small signaling gas molecule, participates in several growth and developmental processes in plants. However, how NO regulates cell wall biosynthesis remains unclear. Here, we demonstrate a positive effect of NO on cellulose content that may be related to S-nitrosylation of cellulose synthase 1 (CESA1) and CESA9. Two S-nitrosylated cysteine (Cys) residues, Cys562 and Cys641, which are exposed on the surface of CESA1 and CESA9 and located in the cellulose synthase catalytic domain, were identified to be S-nitrosylated. Meanwhile, Cys641 was located on the binding surface of CESA1 and CESA9, and Cys562 was very close to the binding surface. Cellulose synthase complexes (CSCs) dynamics are closely associated with cellulose content. S-nitrosylation of CESA1 and CESA9 improved particles mobility and thus increased the accumulation of cellulose in Arabidopsis hypocotyl cells. An increase in hemicellulose content as well as an alteration in pectin content facilitated cell wall extension and contributed to cell growth, finally promoting elongation of Arabidopsis hypocotyls. Overall, our work provides a path to investigate the way NO affects the cellulose content of plants.

Hydrogen Sulfide Positively Regulates Abscisic Acid Signaling through Persulfidation of SnRK2.6 in Guard Cells.

The phytohormone abscisic acid (ABA) plays pivotal roles in triggering stomatal closure and facilitating adaptation of plants to drought stress. Hydrogen sulfide (H2S), a small signaling gas molecule, is involved in ABA-dependent stomatal closure. However, how H2S regulates ABA signaling remains largely unclear. Here, we show that ABA induces the production of H2S catalyzed by L-CYSTEINE DESULFHYDRASE1 (DES1) in guard cells, and H2S in turn positively regulates ABA signaling through persulfidation of Open Stomata 1 (OST1)/SNF1-RELATED PROTEIN KINASE2.6 (SnRK2.6). Two cysteine (Cys) sites, Cys131 and Cys137, which are exposed on the surface of SnRK2.6 and close to the activation loop, were identified to be persulfidated, which promotes the activity of SnRK2.6 and its interaction with ABA response element-binding factor 2 (ABF2), a transcription factor acting downstream of ABA signaling. When Cys131, Cys137, or both residues in SnRK2.6 were substituted with serine (S), H2S-induced SnRK2.6 activity and SnRK2.6-ABF2 interaction were partially (SnRK2.6C131S and SnRK2.6C137S) or completely (SnRK2.6C131SC137S) compromised. Introduction of SnRK2.6C131S, SnRK2.6C137S, or SnRK2.6C131SC137S into the ost1-3 mutant could not rescue the mutant phenotype: less sensitivity to ABA- and H2S-induced stomatal closure and Ca2+ influx as well as increased water loss and decreased drought tolerance. Taken together, our study reveals a novel post-translational regulatory mechanism of ABA signaling whereby H2S persulfidates SnRK2.6 to promote ABA signaling and ABA-induced stomatal closure.

Expression, Localization, and Effect of High Salt Intake on Electroneutral Na+/HCO3 - Cotransporter NBCn2 in Rat Small Intestine: Implication in Intestinal NaCl Absorption.

The electroneutral Na+/HCO3 - cotransporter NBCn2 (SLC4A10) of solute carrier family 4 (SLC4) plays important physiological and pathological roles in the body. Our previous study showed that NBCn2 is expressed on the protein level in the small intestine of rat. Here, by reverse-transcription polymerase chain reaction (PCR), we identified a novel full-length NBCn2 variant, i.e., NBCn2-K, from rat small intestine. By pHi measurement with Xenopus oocytes, the activity of NBCn2-K is not significantly different from NBCn2-G. By western blotting, NBCn2 and the Na+/H+ exchanger NHE3 (SLC9A3) are predominantly expressed in the jejunum of rat small intestine. By immunofluorescence, NBCn2 and NHE3 are localized at the apical domain of the jejunum. NaCl overload decreases the expression of NBCn2 by 56% and that of NHE3 by 40% in the small intestine. We propose that NBCn2 is involved in the transepithelial NaCl absorption in the small intestine, and that the down-regulation of NBCn2 by NaCl represents an adaptive response to high salt intake in rat.

Ethylene-Induced Hydrogen Sulfide Negatively Regulates Ethylene Biosynthesis by Persulfidation of ACO in Tomato Under Osmotic Stress.

A number of recent studies identified hydrogen sulfide (H2S) as an important signal in plant development and adaptation to environmental stress. H2S has been proven to participate in ethylene-induced stomatal closure, but how the signaling pathways of H2S and ethylene interact is still unclear. Here, we reveal how H2S controls the feedback-regulation of ethylene biosynthesis in tomato (Solanum lycopersicum) under osmotic stress. We found that ethylene induced the production of H2S in guard cells. The supply of hypotaurine (HT; a H2S scavenger) or DL-pro-pargylglycine (PAG; a synthetic inhibitor of H2S) removed the effect of ethylene or osmotic stress on stomatal closure. This suggests that ethylene-induced H2S is a downstream component of osmotic stress signaling, which is required for ethylene-induced stomatal closure under osmotic stress. We further found that H2S inhibited ethylene synthesis through inhibiting the activity of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidases (ACOs) by persulfidation. A modified biotin-switch method (MBST) showed that H2S can induce persulfidation of LeACO1 and LeACO2 in a dose-dependent manner, and that persulfidation inhibits the activity of LeACO1 and LeACO2. We also found that LeACO1 is persulfidated at cysteine 60. These data suggested that ethylene-induced H2S negatively regulates ethylene biosynthesis by persulfidation of LeACOs. In addition, H2S was also found to inhibit the expression of LeACO genes. The results provide insight on the general mode of action of H2S and contribute to a better understanding of a plant's response to osmotic stress.

The Balance of [Formula: see text] Secretion vs. Reabsorption in the Endometrial Epithelium Regulates Uterine Fluid pH.

Uterine fluid contains a high concentration of [Formula: see text] which plays an essential role in sperm capacitation and fertilization. In addition, the [Formula: see text] concentration in uterine fluid changes periodically during the estrous cycle. It is well-known that the endometrial epithelium contains machineries involving the apical SLC26 family anion exchangers for secreting [Formula: see text] into the uterine fluid. In the present study, we find for the first time that the electroneutral Na+/[Formula: see text] cotransporter NBCn1 is expressed at the apical membrane of the endometrial epithelium. The protein abundance of the apical NBCn1 and that of the apical SLC26A4 and SLC26A6 are reciprocally regulated during the estrous cycle in the uterus. NBCn1 is most abundant at diestrus, whereas SLC26A4/A6 are most abundant at proestrus/estrus. In the ovariectomized mice, the expression of uterine NBCn1 is inhibited by ß-estradiol, but stimulated by progesterone, whereas that of uterine SLC26A4/A6 is stimulated by ß-estradiol. In vivo perfusion studies show that the endometrial epithelium is capable of both secreting and reabsorbing [Formula: see text]. Moreover, the activity for [Formula: see text] secretion by the endometrial epithelium is significantly higher at estrus than it is at diestrus. The opposite is true for [Formula: see text] reabsorption. We conclude that the endometrial epithelium simultaneously contains the activity for [Formula: see text] secretion involving the apical SLC26A4/A6 and the activity for [Formula: see text] reabsorption involving the apical NBCn1, and that the acid-base homeostasis in the uterine fluid is regulated by the finely-tuned balance of the two activities.