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Hydrogen sulfide (H2 S) is a newly-discovered signaling molecule in plants and has caused increasing attention in recent years, but its function in stomatal movement is unclear. In plants, H2 S is synthesized via cysteine degradation catalyzed by D-/L-cysteine desulfhydrase (D-/L-CDes). AtD-/L-CDes::GUS transgenic Arabidopsis thaliana (L.) Heynh. plants were generated and used to investigate gene expression patterns, and results showed that AtD-/L-CDes can be expressed in guard cells. We also determined the subcellular localization of AtD-/L-CDes using transgenic plants of AtD-/L-CDes::GFP, and the results showed that AtD-CDes and AtL-CDes are located in the chloroplast and in the cytoplasm, respectively. The transcript levels of AtD-CDes and AtL-CDes were affected by the chemicals that cause stomatal closure. Among these factors, ACC, a precursor of ethylene, has the most significant effect, which indicates that the H2 S generated from D-/L-CDes may play an important role in ethylene-induced stomatal closure. Meanwhile, H2 S synthetic inhibitors significantly inhibited ethylene-induced stomatal closure in Arabidopsis. Ethylene treatment caused an increase of H2 S production and of AtD-/L-CDes activity in Arabidopsis leaves. AtD-/L-CDes over-expressing plants exhibited enhanced induction of stomatal closure compared to the wild-type after ethylene treatment; however, the effect was not observed in the Atd-cdes and Atl-cdes mutants. In conclusion, our results suggest that the D-/L-CDes-generated H2 S is involved in the regulation of ethylene-induced stomatal closure in Arabidopsis thaliana.

Summary In rice (Oryza sativa), OsF2H and OsFNSII direct flavanones to independent pathways that form soluble flavone C‐glycosides and tricin‐type metabolites (both soluble and lignin‐bound), respectively. Production of soluble tricin metabolites requires CYP75B4 as a chrysoeriol 5′‐hydroxylase. Meanwhile, the close homologue CYP75B3 is a canonical flavonoid 3′‐hydroxylase (F3′H). However, their precise roles in the biosynthesis of soluble flavone C‐glycosides and tricin–lignins in cell walls remain unknown. We examined CYP75B3 and CYP75B4 expression in vegetative tissues, analyzed extractable flavonoid profiles, cell wall structure and digestibility of their mutants, and investigated catalytic activities of CYP75B4 orthologues in grasses. CYP75B3 and CYP75B4 showed co‐expression patterns with OsF2H and OsFNSII, respectively. CYP75B3 is the sole F3′H in flavone C‐glycosides biosynthesis, whereas CYP75B4 alone provides sufficient 3′,5′‐hydroxylation for tricin–lignin deposition. CYP75B4 mutation results in production of apigenin‐incorporated lignin and enhancement of cell wall digestibility. Moreover, tricin pathway‐specific 3′,5′‐hydroxylation activities are conserved in sorghum CYP75B97 and switchgrass CYP75B11. CYP75B3 and CYP75B4 represent two different pathway‐specific enzymes recruited together with OsF2H and OsFNSII, respectively. Interestingly, the OsF2H‐CYP75B3 and OsFNSII‐CYP75B4 pairs appear to be conserved in grasses. Finally, manipulation of tricin biosynthesis through CYP75B4 orthologues can be a promising strategy to improve digestibility of grass biomass for biofuel and biomaterial production.

Flavonoids are essential for male fertility in some but not all plant species. In rice (Oryza sativa), the chalcone synthase mutant oschs1 produces flavonoid-depleted pollen and is male sterile. The mutant pollen grains are viable with normal structure, but they display reduced germination rate and pollen-tube length. Analysis of oschs1/+ heterozygous lines shows that pollen flavonoid deposition is a paternal effect and fertility is independent of the haploid genotypes (OsCHS1 or oschs1). To understand which classes of flavonoids are involved in male fertility, we conducted detailed analysis of rice mutants for branch-point enzymes of the downstream flavonoid pathways, including flavanone 3-hydroxylase (OsF3H; flavonol pathway entry enzyme), flavone synthase II (CYP93G1; flavone pathway entry enzyme), and flavanone 2-hydroxylase (CYP93G2; flavone C-glycoside pathway entry enzyme). Rice osf3h and cyp93g1 cyp93g2 CRISPR/Cas9 mutants, and cyp93g1 and cyp93g2 T-DNA insertion mutants showed altered flavonoid profiles in anthers, but only the osf3h and cyp93g1 cyp93g2 mutants displayed reduction in seed yield. Our findings indicate that flavonoids are essential for complete male fertility in rice and a combination of different classes (flavanones, flavonols, flavones, and flavone C-glycosides) appears to be important, as opposed to the essential role played primarily by flavonols that has been previously reported in several plant species.

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