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Improving the resistance of Saccharomyces cerevisiae to vanillin, derived from lignin, will benefit the design of robust cell factories for lignocellulosic biorefining. The transcription factor Yrr1p mediates S. cerevisiae resistance to various compounds. In this study, eleven predicted phosphorylation sites were mutated, among which 4 mutants of Yrr1p, Y134A/E and T185A/E could improve vanillin resistance. Both dephosphorylated and phosphorylated mutations at Yrr1p 134 and 185 gathered in the nucleus regardless of the presence or absence of vanillin. However, the phosphorylated mutant Yrr1p inhibited target gene expression, while dephosphorylated mutants promoted expression. Transcriptomic analysis showed that the dephosphorylated Yrr1p T185 mutant, under vanillin stress, upregulated ribosome biogenesis and rRNA processing. These results demonstrate the mechanism by which Yrr1p phosphorylation regulates the expression of target genes. The identification of key phosphorylation sites in Yrr1p offers novel targets for the rational construction of Yrr1p mutants to improve resistance to other compounds.

Hypersensitive-induced reaction (HIR) proteins and more specific members of the proliferation, ion and death superfamily participate in response to pathogen attacks and development of spontaneous hypersensitive lesions in maize and barley leaves. In the present study, a full-length TaHIR3 (1,246-bp) was cloned and characterized from wheat near-isogenic line Thatcher-Lr15 infected by Puccinia triticina isolate 05-19-43② through a homology cloning strategy. A 4,203-bp sequence of TaHIR3 including five exons was also obtained from wheat Thatcher-Lr15 genomic DNA. TaHIR3 shared higher similarity with HIR3 isolated from wheat, barley, and maize. Gene expression of TaHIR3 was spatially measured in young wheat leaves, young roots, young stems, mature seeds, and temporally in wheat leaves inoculated with virulent and avirulent P. triticina. TaHIR3 was expressed in all samples except mature seeds and was upregulated in both combinations. More transcripts accumulated in the incompatible than compatible combination, implying a role in wheat growth and resistance to pathogens attack. A polyclonal antibody was prepared with recombinant protein purified from a prokaryotic expression system with the open reading frame of TaHIR3, and it detected the target protein in wheat leaves by Western blotting. Detection results at the protein level also showed that TaHIR3 was upregulated expression in wheat leaves infected with the leaf rust pathogen. Characterization of TaHIR3 and its expression profiles at the DNA and protein levels suggest that TaHIR3 and its deduced protein play a role in wheat HR causing by the leaf rust pathogen.

The quick separation and simultaneous determination of d-amphetamine and diphenhydramine in the quick-acting anti-motion capsules was investigated by capillary zone electrophoresis. The influence of different parameters (internal standard, injection modes, pH, concentration of the running buffer and applied voltage) was systematically studied. The two compounds could be well separated within 2.0 min in a 40.2 cm fused-silica capillary at a separation voltage of 20 kV in a 50 mM phosphate–12.5 mM borate buffer adjusted to pH 5.5. Correlation coefficients for calibration curves in the range 0.50–1.50 μg mL−1 for d-amphetamine and 2.75–8.25 μg mL−1 for diphenhydramine were higher than 0.999. The limits of detection of d-amphetamine and diphenhydramine were 10.0 and 5.5 ng mL−1 and the recoveries of the compounds in the QAAMC were 99.80 and 99.85%, respectively.