Mutation of Arabidopsis HY1 causes UV-C hypersensitivity by impairing carotenoid and flavonoid biosynthesis and the down-regulation of antioxidant defence

Previous pharmacological results confirmed that haem oxygenase-1 (HO-1) is involved in protection of cells against ultraviolet (UV)-induced oxidative damage in soybean [Glycine max (L.) Merr.] seedlings, but there remains a lack of genetic evidence. In this study, the link between Arabidopsis thaliana HO-1 (HY1) and UV-C tolerance was investigated at the genetic and molecular levels. The maximum inducible expression of HY1 in wild-type Arabidopsis was observed following UV-C irradiation. UV-C sensitivity was not observed in ho2, ho3, and ho4 single and double mutants. However, the HY1 mutant exhibited UV-C hypersensitivity, consistent with the observed decreases in chlorophyll content, and carotenoid and flavonoid metabolism, as well as the down-regulation of antioxidant defences, thereby resulting in severe oxidative damage. The addition of the carbon monoxide donor carbon monoxide-releasing molecule-2 (CORM-2), in particular, and bilirubin (BR), two catalytic by-products of HY1, partially rescued the UV-C hypersensitivity, and other responses appeared in the hy1 mutant. Transcription factors involved in the synthesis of flavonoid or UV responses were induced by UV-C, but reduced in the hy1 mutant. Overall, the findings showed that mutation of HY1 triggered UV-C hypersensitivity, by impairing carotenoid and flavonoid synthesis and antioxidant defences

Characterization, Expression Profiling, and Biochemical Analyses of the Cinnamoyl-CoA Reductase Gene Family for Lignin Synthesis in Alfalfa Plants

Cinnamoyl-CoA reductase (CCR) is a pivotal enzyme in plant lignin synthesis, which has a role in plant secondary cell wall development and environmental stress defense. Alfalfa is a predominant legume forage with excellent quality, but the lignin content negatively affects fodder digestibility. Currently, there is limited information on CCR characteristics, gene expression, and its role in lignin metabolism in alfalfa. In this study, we identified 30 members in the CCR gene family of Medicago sativa. In addition, gene structure, conserved motif, and evolution analysis suggested MsCCR1–7 presumably functioned as CCR, while the 23 MsCCR-likes fell into three categories. The expression patterns of MsCCRs/MsCCR-likes suggested their role in plant development, response to environmental stresses, and phytohormone treatment. These results were consistent with the cis-elements in their promoters. Histochemical staining showed that lignin accumulation gradually deepened with the development, which was consistent with gene expression results. Furthermore, recombinant MsCCR1 and MsCCR-like1 were purified and the kinetic parameters were tested under four substrates. In addition, three-dimensional structure models of MsCCR1 and MsCCR-like1 proteins showed the difference in the substrate-binding motif H212(X)2K215R263. These results will be useful for further application for legume forage quality modification and biofuels industry engineering in the future

Cadmium-Induced Hydrogen Sulfide Synthesis Is Involved in Cadmium Tolerance in <i>Medicago sativa</i> by Reestablishment of Reduced (Homo)glutathione and Reactive Oxygen Species Homeostases

<div><p>Until now, physiological mechanisms and downstream targets responsible for the cadmium (Cd) tolerance mediated by endogenous hydrogen sulfide (H₂S) have been elusive. To address this gap, a combination of pharmacological, histochemical, biochemical and molecular approaches was applied. The perturbation of reduced (homo)glutathione homeostasis and increased H₂S production as well as the activation of two H₂S-synthetic enzymes activities, including _L-cysteine desulfhydrase (LCD) and _D-cysteine desulfhydrase (DCD), in alfalfa seedling roots were early responses to the exposure of Cd. The application of H₂S donor sodium hydrosulfide (NaHS), not only mimicked intracellular H₂S production triggered by Cd, but also alleviated Cd toxicity in a H₂S-dependent fashion. By contrast, the inhibition of H₂S production caused by the application of its synthetic inhibitor blocked NaHS-induced Cd tolerance, and destroyed reduced (homo)glutathione and reactive oxygen species (ROS) homeostases. Above mentioned inhibitory responses were further rescued by exogenously applied glutathione (GSH). Meanwhile, NaHS responses were sensitive to a (homo)glutathione synthetic inhibitor, but reversed by the cotreatment with GSH. The possible involvement of cyclic AMP (cAMP) signaling in NaHS responses was also suggested. In summary, LCD/DCD-mediated H₂S might be an important signaling molecule in the enhancement of Cd toxicity in alfalfa seedlings mainly by governing reduced (homo)glutathione and ROS homeostases.</p></div

Hydrogen Gas Is Involved in Auxin-Induced Lateral Root Formation by Modulating Nitric Oxide Synthesis

Metabolism of molecular hydrogen (H2) in bacteria and algae has been widely studied, and it has attracted increasing attention in the context of animals and plants. However, the role of endogenous H2 in lateral root (LR) formation is still unclear. Here, our results showed that H2-induced lateral root formation is a universal event. Naphthalene-1-acetic acid (NAA; the auxin analog) was able to trigger endogenous H2 production in tomato seedlings, and a contrasting response was observed in the presence of N-1-naphthyphthalamic acid (NPA), an auxin transport inhibitor. NPA-triggered the inhibition of H2 production and thereafter lateral root development was rescued by exogenously applied H2. Detection of endogenous nitric oxide (NO) by the specific probe 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA) and electron paramagnetic resonance (EPR) analyses revealed that the NO level was increased in both NAA- and H2-treated tomato seedlings. Furthermore, NO production and thereafter LR formation induced by auxin and H2 were prevented by 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO; a specific scavenger of NO) and the inhibitor of nitrate reductase (NR; an important NO synthetic enzyme). Molecular evidence confirmed that some representative NO-targeted cell cycle regulatory genes were also induced by H2, but was impaired by the removal of endogenous NO. Genetic evidence suggested that in the presence of H2, Arabidopsis mutants nia2 (in particular) and nia1 (two nitrate reductases (NR)-defective mutants) exhibited defects in lateral root length. Together, these results demonstrated that auxin-induced H2 production was associated with lateral root formation, at least partially via a NR-dependent NO synthesis

Concentrations of low molecular weight thiols and their disulfides, and hGSH/hGSSGh ratio in root tissues.

<p>Seedlings were pretreated with or without 100 µM NaHS, 2 mM PAG, 1 mM GSH, individual or combination for 6 h, and then exposed to 200 µM CdCl₂ for another 12 h. Values are means ± SD of three independent experiments with three replicates for each. Different letters within columns indicate significant differences (<i>P</i><0.05) according to Duncan's multiple range test.</p><p>Concentrations of low molecular weight thiols and their disulfides, and hGSH/hGSSGh ratio in root tissues.</p

NaHS, GSH and BSO pretreatments differentially regulated seedling growth, TBARS accumulation, (h)GSH content, and (h)GSH/(h)GSSG(h).

<p>Fresh weight of 10 roots (A), TBARS accumulation (B), (h)GSH contents (C), and (h)GSH/(h)GSSG(h) ratio (D) in root tissues were determined after seedlings were pretreated with or without 100 µM NaHS, 1 mM GSH, 1 mM BSO, individual or combination for 6 h, and then exposed to 200 µM CdCl₂ for 72 h (A), 24 h (B) and 12 h (C and D). Values are means ± SD of three independent experiments with three replicates for each. Bars denoted by the same letter did not differ significantly at <i>P</i><0.05 according to Duncan's multiple range test.</p

Time course changes of GSH pool and H₂S synthesis upon Cd stress.

<p>Upon 200 µM CdCl₂ treatment for 12 h, contents of (h)GSH (A), (h)GSSG(h) (B) and H₂S (D), the ratio of (h)GSH/(h)GSSG(h) (C), and the activities of LCD (E) and DCD (F) in root tissues were analyzed. Values are means ± SD of three independent experiments with three replicates for each. Bars denoted by the same letter did not differ significantly at <i>P</i><0.05 according to Duncan's multiple range test.</p

NaHS and GSH pretreatments alleviated Cd-induced ROS production, but blocked by PAG.

<p>LSCM results (A). Seedlings were pretreated with or without 100 µM NaHS, 2 mM PAG, 1 mM GSH, individual or combination for 6 h, and then exposed to 200 µM CdCl₂ for another 6 h. After various treatments, the roots were respectively stained with H₂DCFDA, then washed thoroughly to removal extra dye and immediately photographed by LSCM. Scale bar, 0.5 mm. The relative DCF fluorescence intensity in the corresponding roots (B).</p

NaHS increased endogenous H₂S and (h)GSH contents, and the ratio of (h)GSH/(h)GSSG(h) upon Cd stress.

<p>Endogenous H₂S concentration in root tissues (A) was detected at 3 h after the beginning of 100 µM NaHS pretreatment (−3 h), and 200 µM CdCl₂ or chemical-free control treatments for 12 h (12 h). Meanwhile, contents of (h)GSH (B) and the ratio of (h)GSH/(h)GSSG(h) (C) in root tissues were detected at the indicated time points of treatments. Values are means ± SD of three independent experiments with three replicates for each. Bars denoted by the same letter did not differ significantly at <i>P</i><0.05 according to Duncan's multiple range test.</p