A Hydrogen-Sulfide-Repressed Methionine Synthase SlMS1 Acts as a Positive Regulator for Fruit Ripening in Tomato.

Ethylene is a key phytohormone that regulates the ripening of climacteric fruits, and methionine is an indirect precursor of ethylene. However, whether methionine synthase plays a role in fruit ripening in Solanum lycopersicum (tomato) is still unknown. In this study, we find that a tomato methionine synthase (named SlMS1), which could be repressed at the transcriptional level by hydrogen sulfide (H2S), acts as a positive regulator for tomato fruit ripening. By a bioinformatics analysis, it is found that SlMS1 and SlMS2 in tomato are highly homologous to methionine synthases in Arabidopsis thaliana. The expression pattern of SlMS1 and SlMS2 is analyzed in tomato, and SlMS1 expression is up-regulated during fruit ripening, suggesting its potential role in regulating fruit ripening. A potential bipartite nuclear localization signal is found in the amino acid sequence of SlMS1; thus, SlMS1 is tagged with GFP and observed in the leaves of Nicotiana benthamiana. Consistently, SlMS1-GFP shows strong nuclear localization and also cytoplasmic localization. The role of SlMS1 in regulating fruit ripening is investigated in tomato fruit by transient silencing (virus-induced gene silencing, VIGS) and transient overexpression. The results show that SlMS1 silencing causes delayed fruit ripening, evidenced by more chlorophyll and less carotenoid accumulation, while SlMS1 overexpression accelerates fruit ripening significantly compared with control. Further investigation shows that SlMS1 overexpression could up-regulate the expression of carotenoid-synthesis-related genes (PSY1, PDS, ZDS), chlorophyll-degradation-related genes (NYC1, PAO, PPH, SGR1), cell-wall-metabolism-related genes (CEL2, EXP, PG, TBG4, XTH5) and ethylene-synthesis-pathway-related genes (ACO1, ACO3, ACS2), while SlMS1 silencing causes the opposite results. The correlation analysis indicates that SlMS1 expression is negatively correlated with chlorophyll content and positively correlated with carotenoid and ripening-related gene expressions. Taken together, our data suggest that SlMS1 is a positive regulator of tomato fruit ripening and a possible target gene for the ripening-delaying effect of H2S.

Transcriptomics Reveals the ERF2-bHLH2-CML5 Module Responses to H2S and ROS in Postharvest Calcium Deficiency Apples.

Calcium deficiency usually causes accelerated quality deterioration in postharvest fruit, whereas the underlining mechanism is still unclear. Here, we report that calcium deficiency induced the development of bitter pit on the surface of apple peels compared with the healthy appearance in control apples during postharvest storage. Physiological analysis indicates that calcium-deficient peels contained higher levels of superoxide anion (O2•-), malondialdehyde (MDA), total phenol, flavonoid contents and polyphenol oxidase (PPO) activity, and reduced calcium, H2S production, anthocyanin, soluble protein content, and peroxidase (POD) activity compared with those in calcium-sufficient peels. The principal component analysis (PCA) results show that calcium content, ROS, and H2S production were the main factors between calcium-deficient and calcium-sufficient apple peels. Transcriptome data indicated that four calmodulin-like proteins (CMLs), seven AP2/ERFs, and three bHLHs transcripts were significantly differentially expressed in calcium-deficient apple peels. RT-qPCR and correlation analyses further revealed that CML5 expression was significantly positively correlated with the expression of ERF2/17, bHLH2, and H2S production related genes. In addition, transcriptional co-activation of CML5 by ERF2 and bHLH2 was demonstrated by apple transient expression assays and dual-luciferase reporter system experiments. Therefore, these findings provide a basis for studying the molecular mechanism of postharvest quality decline in calcium-deficient apples and the potential interaction between Ca2+ and endogenous H2S.

Protein phosphatase Pph3 and its regulatory subunit Psy2 regulate Rad53 dephosphorylation and cell morphogenesis during recovery from DNA damage in Candida albicans.

The ability of the pathogenic fungus Candida albicans to switch cellular morphologies is important for infection and virulence. Recent studies have revealed that C. albicans yeast cells can switch to filamentous growth under genotoxic stress in a manner dependent on the DNA replication/damage checkpoint. Here, we have investigated the functions of Pph3 (orf19.4378) and Psy2 (orf19.3685), whose orthologues in Saccharomyces cerevisiae mediate the dephosphorylation of the DNA damage checkpoint kinase Rad53 and the histone variant H2AX during recovery from DNA damage. Deleting PPH3 or PSY2 causes hypersensitivity to DNA-damaging agents, including cisplatin, methylmethane sulfonate (MMS), and UV light. In addition, pph3Δ and psy2Δ cells exhibit strong filamentous growth under genotoxic stress. Flow cytometry analysis shows that the mutant cells have lost the ability to adapt to genotoxic stress and remain arrested even after the stress is withdrawn. Furthermore, we show that Pph3 and Psy2 are required for the dephosphorylation of Rad53, but not H2AX, during DNA damage recovery. Taken together, these results show that C. albicans Pph3 and Psy2 have important roles in mediating genotoxin-induced filamentous growth and regulating Rad53 dephosphorylation.

The F-box protein Grr1 regulates the stability of Ccn1, Cln3 and Hof1 and cell morphogenesis in Candida albicans.

Both G1 and mitotic cyclins have been implicated in regulating Candida albicans filamentous growth. We have investigated the functions of Grr1 whose orthologue in Saccharomyces cerevisiae is known to mediate ubiquitin-dependent degradation of the G1 cyclins Cln1 and Cln2. Here, we report that deleting C. albicans GRR1 causes significant stabilization of two G1 cyclins Ccn1 and Cln3 and pseudohyphal growth. grr1Delta cells are highly heterogeneous in length and many of them fail to separate after cytokinesis. Interestingly, some isolated rod-like G1 cells of similar sizes are present in the grr1Delta culture. Time-lapse microscopy revealed that the rod-shaped G1 cells first grew exclusively in width before budding and then the bud grew exclusively by apical extension until after cytokinesis, yielding rod-like daughter cells. Consistently, actin patches persistently localize to the bud tip until around the time of cytokinesis. Despite the pseudohyphal phenotype, grr1Delta cells respond normally to hyphal induction. Hyperphosphorylated Cln3 isoforms accumulate in grr1Delta cells, indicating that Grr1 selectively mediates their degradation in wild-type cells. grr1Delta pseudohyphal growth requires neither Hgc1 nor Swel, two important regulators of cell morphogenesis. Furthermore, the cellular level of Hof1, a protein having a role in cytokinesis, is also significantly increased in grr1Delta cells.