Transcriptomic analysis of formic acid stress response in Saccharomyces cerevisiae.

Formic acid is a representative small molecule acid in lignocellulosic hydrolysate that can inhibit the growth of Saccharomyces cerevisiae cells during alcohol fermentation. However, the mechanism of formic acid cytotoxicity remains largely unknown. In this study, RNA-Seq technology was used to study the response of S. cerevisiae to formic acid stress at the transcriptional level. Scanning electron microscopy and Fourier transform infrared spectroscopy were conducted to observe the surface morphology of yeast cells. A total of 1504 genes were identified as being differentially expressed, with 797 upregulated and 707 downregulated genes. Transcriptomic analysis showed that most genes related to glycolysis, glycogen synthesis, protein degradation, the cell cycle, the MAPK signaling pathway, and redox regulation were significantly induced under formic acid stress and were involved in protein translation and synthesis amino acid synthesis genes were significantly suppressed. Formic acid stress can induce oxidative stress, inhibit protein biosynthesis, cause cells to undergo autophagy, and activate the intracellular metabolic pathways of energy production. The increase of glycogen and the decrease of energy consumption metabolism may be important in the adaptation of S. cerevisiae to formic acid. In addition, formic acid can also induce sexual reproduction and spore formation. This study through transcriptome analysis has preliminarily reveal the molecular response mechanism of S. cerevisiae to formic acid stress and has provided a basis for further research on methods used to improve the tolerance to cell inhibitors in lignocellulose hydrolysate.

A Methionine Sulfoxide Reductase B Is Required for the Establishment of Astragalus sinicus-Mesorhizobium Symbiosis.

Methionine sulfoxide reductase B (MsrB) is involved in oxidative stress or defense responses in plants. However, little is known about its role in legume-rhizobium symbiosis. In this study, an MsrB gene was identified from Astragalus sinicus and its function in symbiosis was characterized. AsMsrB was induced under phosphorus starvation and displayed different expression patterns under symbiotic and nonsymbiotic conditions. Hydrogen peroxide or methyl viologen treatment enhanced the transcript level of AsMsrB in roots and nodules. Subcellular localization showed that AsMsrB was localized in the cytoplasm of onion epidermal cells and co-localized with rhizobia in nodules. Plants with AsMsrB-RNAi hairy roots exhibited significant decreases in nodule number, nodule nitrogenase activity and fresh weight of the aerial part, as well as an abnormal nodule and symbiosome development. Statistical analysis of infection events showed that plants with AsMsrB-RNAi hairy roots had significant decreases in the number of root hair curling events, infection threads and nodule primordia compared with the control. The content of hydrogen peroxide increased in AsMsrB-RNAi roots but decreased in AsMsrB overexpression roots at the early stage of infection. The transcriptome analysis showed synergistic modulations of the expression of genes involved in reactive oxygen species generation and scavenging, defense and pathogenesis and early nodulation. In addition, a candidate protein interacting with AsMsrB was identified and confirmed by bimolecular fluorescence complementation. Taken together, our results indicate that AsMsrB plays an essential role in nodule development and symbiotic nitrogen fixation by affecting the redox homeostasis in roots and nodules.

Digalactosyldiacylglycerol Synthase Gene MtDGD1 Plays an Essential Role in Nodule Development and Nitrogen Fixation.

Little is known about the genes participating in digalactosyldiacylglycerol (DGDG) synthesis during nodule symbiosis. Here, we identified full-length MtDGD1, a synthase of DGDG, and characterized its effect on symbiotic nitrogen fixation in Medicago truncatula. Immunofluorescence and immunoelectron microscopy showed that MtDGD1 was located on the symbiosome membranes in the infected cells. ß-Glucuronidase histochemical staining revealed that MtDGD1 was highly expressed in the infection zone of young nodules as well as in the whole mature nodules. Compared with the control, MtDGD1-RNA interference transgenic plants exhibited significant decreases in nodule number, symbiotic nitrogen fixation activity, and DGDG abundance in the nodules, as well as abnormal nodule and symbiosome development. Overexpression of MtDGD1 resulted in enhancement of nodule number and nitrogen fixation activity. In response to phosphorus starvation, the MtDGD1 expression level was substantially upregulated and the abundance of nonphospholipid DGDG was significantly increased in the roots and nodules, accompanied by corresponding decreases in the abundance of phospholipids such as phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol. Overall, our results indicate that DGD1 contributes to effective nodule organogenesis and nitrogen fixation by affecting the synthesis and content of DGDG during symbiosis.

A purple acid phosphatase plays a role in nodule formation and nitrogen fixation in Astragalus sinicus.

The AsPPD1 gene from Astragalus sinicus encodes a purple acid phosphatase. To address the functions of AsPPD1 in legume-rhizobium symbiosis, its expression patterns, enzyme activity, subcellular localization, and phenotypes associated with its over-expression and RNA interference (RNAi) were investigated. The expression of AsPPD1 was up-regulated in roots and nodules after inoculation with rhizobia. Phosphate starvation reduced the levels of AsPPD1 transcripts in roots while increased those levels in nodules. We confirmed the acid phosphatase and phosphodiesterase activities of recombinant AsPPD1 purified from Pichia pastoris, and demonstrated its ability to hydrolyze ADP and ATP in vitro. Subcellular localization showed that AsPPD1 located on the plasma membranes in hairy roots and on the symbiosomes membranes in root nodules. Over-expression of AsPPD1 in hairy roots inhibited nodulation, while its silencing resulted in nodules early senescence and significantly decreased nitrogenase activity. Furthermore, HPLC measurement showed that AsPPD1 overexpression affects the ADP levels in the infected roots and nodules, AsPPD1 silencing affects the ratio of ATP/ADP and the energy charge in nodules, and quantitative observation demonstrated the changes of AsPPD1 transcripts level affected nodule primordia formation. Taken together, it is speculated that AsPPD1 contributes to symbiotic ADP levels and energy charge control, and this is required for effective nodule organogenesis and nitrogen fixation.

Metabolome analysis of the response and tolerance mechanisms of Saccharomyces cerevisiae to formic acid stress.

Various inhibitors are produced during the hydrolysis of lignocellulosic biomass that can interfere with the growth of yeast cells and the production of bioethanol. Formic acid is a common weak acid inhibitor present in lignocellulosic hydrolysate that has toxic effects on yeast cells. However, the mechanism of the response of Saccharomyces cerevisiae to formic acid is not fully understood. In this study, liquid chromatography-mass spectrometry (LC-MS) was used to investigate the effects of formic acid treatment on cell metabolites of S. cerevisiae. Treatment with different concentrations of formic acid significantly inhibited the growth of yeast cells, reduced the yield of ethanol, prolonged the cell fermentation cycle, and increased the content of malondialdehyde. Principal component analysis and orthogonal partial least squares discriminant analysis showed that 55 metabolites were significantly altered in S. cerevisiae after formic acid treatment. The metabolic relevance of these compounds in the response of S. cerevisiae to formic acid stress was investigated. Formic acid can cause oxidative stress, inhibit protein synthesis, and damage DNA in S. cerevisiae, and these are possible reasons for the inhibition of S. cerevisiae cell growth. In addition, the levels of several aromatic amino acids identified in the cells of formic acid-treated yeast were increased; the biosynthesis of nucleotides was slowed, and energy consumption was reduced. These mechanisms may help to improve the tolerance of yeast cells to formic acid. The results described herein highlight our current understanding of the molecular mechanism of the response of S. cerevisiae to formic acid. The study will provide a theoretical basis for research on the tolerance mechanisms of S. cerevisiae.