Figure 5

Effect of MYBR1 on leaf senescence in a detached leaf assay. (A) True leaves numbers 3–6 were harvested from 30 d old soil grown plants and incubated on filter paper wetted with 3 mM MES buffer (pH 5.7). Leaves 3 and 4 were photographed after 6 d treatment and leaves 5 and 6 were photographed after 7 d treatment. Leaves from OxMYBR1 plants of three independent lines (#31-3, 1–7 and 42–6) showed delayed senescence relative to other genotypes and leaves from mybr1 and double mutant plants exhibited premature leaf senescence relative to leaves from WT (leaves 5 and 6) and OxMYBR1 plants (all leaves). Two sets of experiment were carried out as above (A) in four replicates and 12 plants in each replicate. Statistical significance was determined using one-way ANOVA with Tukey using the statistical software ‘R’ (P < 0.05). (B) In one set of experiments, chlorophyll was extracted and measured on 0 d. The chlorophyll content was higher in one line of OxMYBR1 (#42-6) and reciprocal double mutants of mybr1 and mybr2 than other genotypes. (C) In another set, chlorophyll was extracted and measured on 6 d for leaves 3–4 and on 7d for leaves 5–6 and the percentage chlorophyll retention was calculated relative to 0 d from (B). Chlorophyll retention was generally higher in OxMYBR1 genotypes. (D) Using three independent homozygous MYBR1_pro:GUS lines (#5-1, 7–6 and X1-4), experiments were carried out as above (A) in two replicates. All leaves from each plant were harvested. GUS staining was performed on 0 d (untreated) and after 4 d of dark induced senescence. Asterisks indicate yellow leaves before and after GUS staining. After the treatment, GUS staining was higher in senescent leaves, but in green leaves was lower than corresponding control leaves.