Edlings grown in soil were treated with no water to induce drought get Quisinostat stress for additional 10 days. We noticed that the wilting levels of rd20 mutant plants were more obvious than those of the wildtype or RD20/rd20 plants (Fig 4C). Only 20 of rd20 plants survived, Quinoline-Val-Asp-Difluorophenoxymethylketone manufacturer whereas the corresponding survival rates were 82?5 for wild-type and heterozygous plants after 3 days of rewatering preceded by 10 days of water-deficit stress treatment (Fig 4D). Seedlings of all genotypesPLOS ONE | DOI:10.1371/journal.pone.0125666 May 1,11 /Microarray Analysis of Arabidopsis-Stressed PlantsFig 4. Responses of the Arabidopsis rd20 mutant to B. cinerea infection and drought. Disease symptoms in leaves after drop-inoculation with B. cinerea (A); and fungal growth in plants after spray-inoculation with B. cinerea (B). Drought sensitivity assay on plants 10 days after stopping irrigation (C); and quantitative analysis of survival on plants continued to be not watered for 10 days and then re-irrigated for 3 days (D). In (B), qPCR amplification of Bc ActinA relative to the At Actin2 gene. In (B) and (D), mean values followed by an asterisk are significantly different from the corresponding control (P < 0.05). All assays were repeated at least three times with similar results. Wt, wild-type; RD20/rd20, heterozygous line; rd20, homozygous Bc ActinA, B. cinerea ActinA gene; At Actin2, Arabidopsis Actin2 gene; dpi, days post-inoculation. doi:10.1371/journal.pone.0125666.gshowed no death when water was applied. Altogether, this suggests that RD20 plays an important role in plant defense to B. cinerea infection and drought stress.Regulation of differentially expressed genes through electrophilic oxylipinAll oxylipins, 12-oxo-phytodienoic acid (OPDA), phytoprostane A1 (PPA1) and jasmonate (JA) are regulators of stress responses [11, 27, 28]. The cyclopentenones, OPDA and PPA1, activate gene expression independently from the cyclopentanone, JA. We investigated whether the regulation of OPDA or PPA1 respondents [11, 27] was also regulated by B. cinerea, heat, salinity and osmotic stress. Previously, it was shown that the OPDA/B. cinerea upregulated genes (OBUGs), DREB2A, REF, UGT73B5, HSP17.4 and PDR12, and PPA1/B. cinerea upregulated genes (PBUGs), GSTU25, GSTU4, PDR12 and ELI3-2, were also induced by cold, drought or oxidative stress [20]. Except of GSTU25, the rest of the commonly expressed genes were also upregulated by osmotic stress (Table 4). Conversely, HSP17.4 was induced by salinity as well, suggesting that plant responses to osmotic stress can share common respondents with OBUGs and PBUGs and other abiotic stresses. Some of the OBUGs (At5g25930, MLO6, At3g04640,PLOS ONE | DOI:10.1371/journal.pone.0125666 May 1,12 /Microarray Analysis of Arabidopsis-Stressed PlantsTable 4. Regulation of genes by OPDA or PPA1 treatment, B. cinerea infection, heat, salinity and osmotic stress. Description Gene locus OPDA/PPA1b OBUGs Receptor-related protein kinase like DRE-binding protein (DREB2A) Mildew resistance locus O6 (MLO6) Gly-rich protein Rubber elongation factor (REF) UDP-glucose transferase (UGT73B5) Cinnamyl-alcohol dehydrogenase (CAD) Class I heat-shock protein(HSP17.4) FAD-linked oxidoreductase family ABC transporter (PDR12) -glucosidase 30; Dark inducible 2 (DIN2) Nitrilase 4 (NIT4) PBUGs CYP89A9 GSTU25 GST22/GSTU4 PDR12 HSF4 ELI3-2 Cyclin, putative SYP111 ACTaNormalized Fold inductiona B. cinereac 4.6 3.4 4.2 8.1 3.5 3.1 17.5 3.3 16.5 22.6 18.3 4 5.9 10.8 9.3 22.6 4.Edlings grown in soil were treated with no water to induce drought stress for additional 10 days. We noticed that the wilting levels of rd20 mutant plants were more obvious than those of the wildtype or RD20/rd20 plants (Fig 4C). Only 20 of rd20 plants survived, whereas the corresponding survival rates were 82?5 for wild-type and heterozygous plants after 3 days of rewatering preceded by 10 days of water-deficit stress treatment (Fig 4D). Seedlings of all genotypesPLOS ONE | DOI:10.1371/journal.pone.0125666 May 1,11 /Microarray Analysis of Arabidopsis-Stressed PlantsFig 4. Responses of the Arabidopsis rd20 mutant to B. cinerea infection and drought. Disease symptoms in leaves after drop-inoculation with B. cinerea (A); and fungal growth in plants after spray-inoculation with B. cinerea (B). Drought sensitivity assay on plants 10 days after stopping irrigation (C); and quantitative analysis of survival on plants continued to be not watered for 10 days and then re-irrigated for 3 days (D). In (B), qPCR amplification of Bc ActinA relative to the At Actin2 gene. In (B) and (D), mean values followed by an asterisk are significantly different from the corresponding control (P < 0.05). All assays were repeated at least three times with similar results. Wt, wild-type; RD20/rd20, heterozygous line; rd20, homozygous Bc ActinA, B. cinerea ActinA gene; At Actin2, Arabidopsis Actin2 gene; dpi, days post-inoculation. doi:10.1371/journal.pone.0125666.gshowed no death when water was applied. Altogether, this suggests that RD20 plays an important role in plant defense to B. cinerea infection and drought stress.Regulation of differentially expressed genes through electrophilic oxylipinAll oxylipins, 12-oxo-phytodienoic acid (OPDA), phytoprostane A1 (PPA1) and jasmonate (JA) are regulators of stress responses [11, 27, 28]. The cyclopentenones, OPDA and PPA1, activate gene expression independently from the cyclopentanone, JA. We investigated whether the regulation of OPDA or PPA1 respondents [11, 27] was also regulated by B. cinerea, heat, salinity and osmotic stress. Previously, it was shown that the OPDA/B. cinerea upregulated genes (OBUGs), DREB2A, REF, UGT73B5, HSP17.4 and PDR12, and PPA1/B. cinerea upregulated genes (PBUGs), GSTU25, GSTU4, PDR12 and ELI3-2, were also induced by cold, drought or oxidative stress [20]. Except of GSTU25, the rest of the commonly expressed genes were also upregulated by osmotic stress (Table 4). Conversely, HSP17.4 was induced by salinity as well, suggesting that plant responses to osmotic stress can share common respondents with OBUGs and PBUGs and other abiotic stresses. Some of the OBUGs (At5g25930, MLO6, At3g04640,PLOS ONE | DOI:10.1371/journal.pone.0125666 May 1,12 /Microarray Analysis of Arabidopsis-Stressed PlantsTable 4. Regulation of genes by OPDA or PPA1 treatment, B. cinerea infection, heat, salinity and osmotic stress. Description Gene locus OPDA/PPA1b OBUGs Receptor-related protein kinase like DRE-binding protein (DREB2A) Mildew resistance locus O6 (MLO6) Gly-rich protein Rubber elongation factor (REF) UDP-glucose transferase (UGT73B5) Cinnamyl-alcohol dehydrogenase (CAD) Class I heat-shock protein(HSP17.4) FAD-linked oxidoreductase family ABC transporter (PDR12) -glucosidase 30; Dark inducible 2 (DIN2) Nitrilase 4 (NIT4) PBUGs CYP89A9 GSTU25 GST22/GSTU4 PDR12 HSF4 ELI3-2 Cyclin, putative SYP111 ACTaNormalized Fold inductiona B. cinereac 4.6 3.4 4.2 8.1 3.5 3.1 17.5 3.3 16.5 22.6 18.3 4 5.9 10.8 9.3 22.6 4.