The Interplay Between Hydrogen Sulfide and Phytohormone Signaling Pathways Under Challenging Environments

Hydrogen sulfide (H2S) serves as an important gaseous signaling molecule that is involved in intra- and intercellular signal transduction in plant–environment interactions. In plants, H2S is formed in sulfate/cysteine reduction pathways. The activation of endogenous H2S and its exogenous application has been found to be highly effective in ameliorating a wide variety of stress conditions in plants. The H2S interferes with the cellular redox regulatory network and prevents the degradation of proteins from oxidative stress via post-translational modifications (PTMs). H2S-mediated persulfidation allows the rapid response of proteins in signaling networks to environmental stimuli. In addition, regulatory crosstalk of H2S with other gaseous signals and plant growth regulators enable the activation of multiple signaling cascades that drive cellular adaptation. In this review, we summarize and discuss the current understanding of the molecular mechanisms of H2S-induced cellular adjustments and the interactions between H2S and various signaling pathways in plants, emphasizing the recent progress in our understanding of the effects of H2S on the PTMs of proteins. We also discuss future directions that would advance our understanding of H2S interactions to ultimately mitigate the impacts of environmental stresses in the plants

The Interplay between Hydrogen Sulfide and Phytohormone Signaling Pathways under Challenging Environments

Hydrogen sulfide (H2S) serves as an important gaseous signaling molecule that is involved in intra- and intercellular signal transduction in plant–environment interactions. In plants, H2S is formed in sulfate/cysteine reduction pathways. The activation of endogenous H2S and its exogenous application has been found to be highly effective in ameliorating a wide variety of stress conditions in plants. The H2S interferes with the cellular redox regulatory network and prevents the degradation of proteins from oxidative stress via post-translational modifications (PTMs). H2S-mediated persulfidation allows the rapid response of proteins in signaling networks to environmental stimuli. In addition, regulatory crosstalk of H2S with other gaseous signals and plant growth regulators enable the activation of multiple signaling cascades that drive cellular adaptation. In this review, we summarize and discuss the current understanding of the molecular mechanisms of H2S-induced cellular adjustments and the interactions between H2S and various signaling pathways in plants, emphasizing the recent progress in our understanding of the effects of H2S on the PTMs of proteins. We also discuss future directions that would advance our understanding of H2S interactions to ultimately mitigate the impacts of environmental stresses in the plants

Triplin, a small molecule, reveals copper ion transport in ethylene signaling from ATX1 to RAN1

<div><p>Copper ions play an important role in ethylene receptor biogenesis and proper function. The copper transporter RESPONSIVE-TO-ANTAGONIST1 (RAN1) is essential for copper ion transport in <i>Arabidopsis thaliana</i>. However it is still unclear how copper ions are delivered to RAN1 and how copper ions affect ethylene receptors. There is not a specific copper chelator which could be used to explore these questions. Here, by chemical genetics, we identified a novel small molecule, triplin, which could cause a triple response phenotype on dark-grown <i>Arabidopsis</i> seedlings through ethylene signaling pathway. <i>ran1-1</i> and <i>ran1-2</i> are hypersensitive to triplin. Adding copper ions in growth medium could partially restore the phenotype on plant caused by triplin. Mass spectrometry analysis showed that triplin could bind copper ion. Compared to the known chelators, triplin acts more specifically to copper ion and it suppresses the toxic effects of excess copper ions on plant root growth. We further showed that mutants of ANTIOXIDANT PROTEIN1 (ATX1) are hypersensitive to tiplin, but with less sensitivity comparing with the ones of <i>ran1-1</i> and <i>ran1-2</i>. Our study provided genetic evidence for the first time that, copper ions necessary for ethylene receptor biogenesis and signaling are transported from ATX1 to RAN1. Considering that triplin could chelate copper ions in <i>Arabidopsis</i>, and copper ions are essential for plant and animal, we believe that, triplin not only could be useful for studying copper ion transport of plants, but also could be useful for copper metabolism study in animal and human.</p></div

Macrophage Raptor Deficiency-Induced Lysosome Dysfunction Exacerbates Nonalcoholic SteatohepatitisSummary

Background & Aims: Nonalcoholic steatohepatitis (NASH) is an increasingly prevalent nonalcoholic fatty liver disease, characterized by inflammatory cell infiltration and hepatocellular damage. Mammalian target of rapamycin complex 1 (mTORC1) has been investigated extensively in the context of cancer, including hepatocellular carcinoma. However, the role of mTORC1 in NASH remains largely unknown. Methods: mTORC1 activity in macrophages in human mild and severe NASH liver was compared. Mice with macrophage-specific deletion of the regulatory-associated protein of mTOR (Raptor) subunit and littermate controls were fed a high-fructose, palmitate, and cholesterol diet for 24 weeks or a methionine- and choline-deficient diet for 4 weeks to develop NASH. Results: We report that in human beings bearing NASH, macrophage mTORC1 activity was lower in livers experiencing severe vs mild NASH liver. Moreover, macrophage mTORC1 disruption exacerbated the inflammatory response in 2 diet-induced NASH mouse models. Mechanistically, in response to apoptotic hepatocytes (AHs), macrophage polarization toward a M2 anti-inflammatory phenotype was inhibited in Raptor-deficient macrophages. During the digestion of AHs, macrophage mTORC1 was activated and coupled with dynamin-related protein 1 to facilitate the latter’s phosphorylation, leading to mitochondrial fission-mediated calcium release. Ionomycin or A23187, calcium ionophores, prevented Raptor deficiency–mediated failure of lysosome acidification and subsequent lipolysis. Blocking dynamin-related protein 1–dependent mitochondria fission impaired lysosome function, resulting in reduced production of anti-inflammatory factors such as interleukins 10 and 13. Conclusions: Persistent mTORC1 deficiency in macrophages contributes to the progression of NASH by causing lysosome dysfunction and subsequently attenuating anti-inflammatory M2-like response in macrophages during clearance of AHs. Keywords: mTORC1, NASH, Lysosome, Drp

<i>ran1-1</i> and <i>ran1-2</i> are hypersensitive to triplin and copper can partially reverse the effects of triplin.

<p>(A) Phenotypes of 3-day-old, dark-grown seedlings of Col-0, <i>ran1-1</i> and <i>ran1-2</i> treated with 10 μM triplin. (B) Triplin dose responses of Col-0, <i>ran1-1</i>, <i>ran1-2</i> and two <i>35S</i>:<i>RAN1-GFP (ran1-2)</i> transgenic lines. Data is the average hypocotyl length under each condition. Each experiment was repeated three times, more than 30 seedlings were used every time. Error bars represent SEM. (C) Phenotypes of 3-day-old, dark-grown seedlings of <i>ran1-2</i>, <i>ran1-2 etr1-1</i> and <i>ran1-2 ein2-5</i> treated with 100 μM triplin. (D) The phenotypes of 3-day-old, dark-grown seedlings of Col-0 treated with 100 μM triplin in the presence of 20 μM ZnSO₄, 20 μM CuSO₄ or H₂O as a control. (E) The hypocotyl length of seedlings as described in (D). Each experiment was repeated three times, more than 30 seedlings were used every time. Error bars represent SEM. **p < 0.01 indicated the difference of the hypocotyl length between the seedlings treated with CuSO₄ compared with H₂O and ZnSO₄ in the presence of 100 μM triplin. Scale bars represent 1 mm.</p

Triplin acts through ethylene signaling pathway to cause a triple response phenotype in <i>Arabidopsis</i> seedlings.

<p>(A) Phenotypes of 3-day-old, dark-grown Col-0 seedlings treated with 100 μM triplin, 50 μM ACC, or 1% (v/v) DMSO as a control. Scale bar represents 1 mm. (B) The chemical structure of triplin. (C) The hypocotyl length of 3-day-old, dark-grown Col-0, <i>etr1-1</i>, <i>etr1-2</i>, <i>ein2-5 and ein3 eil1</i> seedlings treated with 100 μM triplin, 50 μM ACC, or 1% (v/v) DMSO as a control. Each experiment was repeated three times, more than 30 seedlings were used every time. Error bars represent SEM. ***P < 0.0001 (two-tailed Student’s t-test) indicated a significant difference of the hypocotyl length of the mutants compared to Col-0 treated with 100 μM triplin. (D) qRT-PCR analysis the expression of the ethylene response gene <i>ERF1</i> treated by 1% (v/v) DMSO, 100 μM triplin, or 50 μM ACC. Each experiment was repeated three times, and error bars represent SEM.</p

Triplin can affect the transport of copper ions <i>in vivo</i>.

<p>(A) The phenotypes of 3-day-old, dark-grown seedlings of Col-0 on different growth medium with (+) or without (-) 20 μM triplin. -Cu represents the growth medium made of all essential elements needed for plant growth except copper ion. BCS represents the growth medium made of 0.5xMS salt with 500 μM of the copper ion chelator BCS. Scale bar represents 1 mm. (B) The hypocotyl length of 3-day-old seedlings as described in (A). The difference of the hypocotyl lengths represent either the seedlings grown on -Cu or BCS medium compared to the ones grown on 0.5xMS. (C) The hypocotyl length of 3-day-old seedlings of Col-0 and <i>35S</i>: <i>RAN1-GFP</i> lines grown in dark under different dose of triplin. (D) Triplin’s effects on ethylene-binding to ETR1 expressed in yeast. Saturable ethylene binding to intact yeast cells expressing the ethylene binding domain of ETR1 and membranes isolated from these yeast cells was measured. Ethylene binding is indicated as counts per minute (CPM). Each experiment was repeated three times, and error bars represent SEM. In (B) and (C), experiments were repeated three times, more than 30 seedlings were used every time. Error bars represent SEM. *P < 0.05, **P < 0.01, ***P < 0.0001 (two-tailed Student’s t-test) indicate a significant difference between groups of different treatments.</p

The copper chaperone ATX1 interacts with RAN1.

<p>(A) <i>atx1-1</i> and <i>atx1-2</i> are hypersensitive to triplin. The phenotypes of 3-day-old, dark-grown Col-0, <i>atx1-1</i> and <i>atx1-2</i> seedlings treated with 20 μM triplin are shown. Scale bar represents 1 mm. (B) Hypocotyl lengths of 3-day-old, dark-grown seedlings of Col-0, <i>atx1-1</i>, <i>atx1-2</i>, <i>ran1-2</i>, <i>atx1-1 ran1-2</i> and two <i>35S</i>:<i>ATX1-GFP (atx1-1)</i> transgenic lines treated with different doses of triplin are shown. Each experiment was repeated three times, more than 30 seedlings were used every time. Error bars represent SEM. (C) Subcellular co-localization of ATX1 and RAN1. ATX1-RFP and RAN1-GFP were transiently expressed in N. benthamiana leaves and observed and imaged under a confocal microscope. Scale bars represent 20 μM. (D) ATX1 interacted with RAN1 in yeast two-hybrid assay. ATX1 was fused to a GAL4 DNA-binding domain (BD) and RAN1-N (289 amino-terminal amino acids of RAN1) was ligated to a GAL4 activation domain (AD). The protein interactions were examined on cells grown on synthetic dropout (-Leu/-Trp/-His/-Ade) medium plus X-α-Gal (50mg/L) plates for 3 days. (E) Bimolecular fluorescence complementation assays showed interaction between ATX1 and RAN1 using the split luciferase system. Nicotiana benthamiana leaves were infiltrated with agrobacteria containing different construct combinations harboring both the C- and N-terminal of the luciferase fused to either ATX1 and RAN1-N or just one of them (controls). (F) Co-Immunoprecipitation assays showed interaction between ATX1 and RAN1. Nicotiana benthamiana leaves were infiltrated with agrobacteria containing ATX1-GFP/FLAG and RAN1-N-FLAG/GFP or just one of them (controls). The protein extracts were immunoblotted with anti-FLAG antibody or anti-GFP antibody.</p

Triplin can chelate copper ions <i>in vitro</i>.

<p>(A) The phenotypes of 3-day-old, dark-grown seedlings of Col-0 treated with 50 μM CuSO₄ in the presence of 1% (v/v) DMSO or 100 μM triplin. Scale bar represents 1 mm. (B) The root length of the seedlings as described in (A). Each experiment was repeated three times, more than 30 seedlings were used every time. Error bars represent SEM. (C) The relative copper contents of 3-day-old, dark-grown Col-0 seedlings on 0.5xMS growth medium with or without 100 μM triplin and/or 20 μM CuSO₄. D.W represents dry weight of the seedlings. Each experiment was repeated three times (Error bars represent SEM). (D) MALDI-TOF-MS analysis of the mixture of equal volume of 100 μM CuSO₄ and 100 μM triplin. Mr of triplin is 446.0g/mol. Mr of triplin+Cu is 510.0g/mol. *P < 0.05 and ***P < 0.0001 (two-tailed Student’s t-test) indicate a significant difference between groups of different treatments.</p