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.

Background: Hydrogen gas (H₂) is hypothesised to play a role in plants that are coping with stresses by regulating signal transduction and gene expression. Although the beneficial role of H₂ in plant tolerance to cadmium (Cd) has been investigated previously, the corresponding mechanism has not been elucidated. In this report, the transcriptomes of alfalfa seedling roots under Cd and/or hydrogen-rich water (HRW) treatment were first analysed. Then, the sulfur metabolism pathways were focused on and further investigated by pharmacological and genetic approaches. Results: A total of 1968 differentially expressed genes (DEGs) in alfalfa seedling roots under Cd and/or HRW treatment were identified by RNA-Seq. The DEGs were classified into many clusters, including glutathione (GSH) metabolism, oxidative stress, and ATP-binding cassette (ABC) transporters. The results validated by RT-qPCR showed that the levels of relevant genes involved in sulfur metabolism were enhanced by HRW under Cd treatment, especially the genes involved in (homo)glutathione metabolism. Additional experiments carried out with a glutathione synthesis inhibitor and Arabidopsis thaliana cad2–1 mutant plants suggested the prominent role of glutathione in HRW-induced Cd tolerance. These results were in accordance with the effects of HRW on the contents of (homo)glutathione and (homo)phytochelatins and in alleviating oxidative stress under Cd stress. In addition, the HRW-induced alleviation of Cd toxicity might also be caused by a decrease in available Cd in seedling roots, achieved through ABC transporter-mediated secretion. Conclusions: Taken together, the results of our study indicate that H₂ regulated the expression of genes relevant to sulfur and glutathione metabolism and enhanced glutathione metabolism which resulted in Cd tolerance by activating antioxidation and Cd chelation. These results may help to elucidate the mechanism governing H₂-induced Cd tolerance in alfalfa.

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.

Whether or not hydrogen gas (H₂) can reduce cadmium (Cd) toxicity in Ganoderma lucidum has remained largely unknown. Here, we report that Cd-induced growth inhibition in G. lucidum was significantly alleviated by H₂ fumigation or hydrogen-rich water (HRW), evaluated by lower oxidative damage and Cd accumulation. Moreover, the amelioration effects of H₂ fumigation were better than of HRW in an optimum concentration of H₂ under our experimental conditions. Further results showed that H₂-alleviated growth inhibition in G. lucidum was accompanied by increased nitric oxide (NO) level and nitrate reductase (NR) activity under Cd stress. On the other hand, the mitigation effects were reversed after removing endogenous NO with its scavenger cPTIO or inhibiting H₂-induced NR activity with sodium tungstate. The role of NO in H₂-alleviated growth inhibition under Cd stress was proved to be achieved through a restoration of redox balance, an increase in cysteine and proline contents, and a reduction in Cd accumulation. In summary, these results clearly revealed that NR-dependent NO might be involved in the H₂-alleviated Cd toxicity in G. lucidum through rebuilding redox homeostasis, increasing cysteine and proline levels, and reducing Cd accumulation. These findings may open a new window for H₂ application in Cd-stressed economically important fungi.

Although emerging evidence revealed that molecular hydrogen (H₂) positively regulates numerous physiological responses, understanding the synthesis of H₂ and its functions is a challenge for biology. The correct timing of flowering is controlled by environmental stimuli and endogenous signals. We report that endogenous H₂ facilitates the floral transition in Arabidopsis. The Nuclear Architecture Related 1 (NAR1) gene of Arabidopsis was prokaryotic expressed and purified, and its H₂-synthesizing activity was subsequently detected. Furthermore, mutation experiments were used to investigate the catalytic roles of conserved sequences and Cys sites. Biochemistry and molecular approaches were employed to investigate the role of H₂ in regulating floral transition. Similar to [Fe-Fe] hydrogenase (HYD1) from Chlamydomonas reinhardtii, NAR1 protein is a H₂-synthesizing enzyme in Arabidopsis thaliana. Protein mutation experiments in vitro show that four Cys residues are important for the inducible catalyzing activity upon hypoxia. Accordingly, NAR1-dependent circadian-rhythmic H₂ was observed under light/dark cycles, which was accompanied with contrasting changes in nitric oxide (NO) signal. Knockdown of NAR1 by CRISPR or RNAi reduced H₂ production and delayed flowering. Whereas, exogenous H₂ supply and overexpressing NAR1 or CrHYD1 promoted early flowering. These early flowering phenotypes were aggravated by the removal of endogenous NO, but abolished by NO addition. Since the mutant (mms19) defective in Fe-S cluster assembly function displays early flowering regardless of H₂ addition, we further deduce that NAR1 control of early flowering is largely achieved by H₂. Biochemical and genetic evidences show that NAR1-driven floral transition is functionally linked to the modulation of circadian oscillators via NO signaling. Since H₂ production is modulated by circadian rhythms and constitutively produced, it may integrate external and internal cues into floral transition, and the modulation of its production might be a promising strategy for crop breeding cultivation.