Figure 4

AtProDH1 gene modulates plant tolerance under combined stress. atprodh1 mutant and wild-type (Wt) plants were exposed to individual drought (D, at 40% FC), pathogen (P, at 10⁴ CFU/mL) and drought recovery (DR) and combined drought recovery-pathogen (DRP) and drought-pathogen (DP) treatments. Heat map showing expression pattern of AtProDH1 gene (AT3G30775) was drawn using linear fold change values (D over C and P, DRP over M) from microarray-based transcriptome profile and is presented here (A). Colour bar in blue and red shows down- and up-regulated expression respectively. AtProDH1 transcript expression is shown in WT plants under D, P, DR, DRP and DP stress at 2, 6 and 24 hpt. Semi-quantitative reverse transcriptase PCR was performed with 50 ng of total RNA for 34 cycles. Ubiquitin (AtUbi5) gene expression was studied as a reference in the respective samples and is presented as loading control (B). Diseased leaves exhibit chlorosis or cell death. Disease phenotype was captured at 4 days post treatment (dpt) in WT and atprodh1 mutant plants under individual and combined stress treatments and is presented for four biological replicates (C). Disease-associated cell death was captured using trypan blue staining at 3 dpt in Wt and atprodh1 plants in three biological replicates. The extent of cell death is directly proportional to the intensity of blue colour and is represented as fold change over absolute control along each image (ImageJ software) (D). Bacterial multiplication in inoculated plants (P, DRP and DP) was monitored at 1 and 3 dpt. Bars represent the mean and SEM for eight biological replicates. Statistical significance for a particular treatment in the mutant plant was determined over respective Wt using student’s t-test. Asterisk represents statistically significant value at P < 0.05 (E). Relative water content (RWC) in Wt and mutant plants under different treatments was assessed at 24 hpt. Data represent the mean and SEM for three biological replicates. Asterisk represents statistically significant value at P < 0.05 based on Student’s t-test between well-watered plants and other treatments (F). Proline content in Wt and atprodh1 plants under different treatments was assessed at 24 hpt. Data are the mean of two biological replicates from one experiment. Bars represent mean ± SEM. Student’s t-test was applied to calculate statistical significance in mutant plants over respective Wt treatment. Asterisk represents statistically significant value at P < 0.05 (G). Wt, wild-type; AC, absolute control; D, drought; P, pathogen; DR, drought-recovery; DRP, drought-recovery-pathogen; DP, drought-pathogen; 1 and 3, 1 and 3 dpt.