The expression of light-regulated genes in the high-pigment-1 mutant of tomato

Abstract

Three light-regulated genes, chlorophyll a/b-binding protein (CAB), ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit, and chalcone synthase (CHS), are demonstrated to be up-regulated in the high-pigment-1 (hp-1) mutant of tomato (Lycopersicon esculentum Mill. ) compared with wild type (WT). However, the pattern of up-regulation of the three genes depends on the light conditions, stage of development, and tissue studied. Compared with WT, the hp-1 mutant showed higher CAB gene expression in the dark after a single red-light pulse and in the pericarp of immature fruits. However, in vegetative tissues of light-grown seedlings and adult plants, CAB mRNA accumulation did not differ between WT and the hp-1 mutant. The ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit mRNA accumulated to a higher level in the hp-1 mutant than WT under all light conditions and tissues studied, whereas CHS gene expression was up-regulated in de-etiolated vegetative hp-1-mutant tissues only. The CAB and CHS genes were shown to be phytochrome regulated and both phytochrome A and B1 play a role in CAB gene expression. These observations support the hypothesis that the HP-1 protein plays a general repressive role in phytochrome signal transduction.

Figure 1

Effect of a 10-min R pulse on the CAB and RBCS mRNA abundance in etiolated 4-d-old WT and hp-1^w-mutant tomato seedlings. A, For the RNA blots shown, RNA was extracted directly (0 h), 1, 2, 4, 6, and 8 h after onset of the R pulse. The control (lanes C) represents the CAB mRNA amount in seedlings that did not receive a R pulse but were kept in continuous darkness. As a loading control the blots were probed with an 18S rRNA probe. B, The CAB and RBCS mRNA abundance was quantified using a phosphor imager and the mean values (±se) are shown for CAB and RBCS, respectively. A value of 100% on the ordinate represents the maximum steady-state mRNA detected within the experiment.

Figure 2

Effect of no light pulse (lanes D), a 10-min R, 15-min FR, and 10-min R followed by 15-min FR pulse (R/FR) on the CAB mRNA abundance in etiolated 4-d-old tomato seedlings. The genotypes used were WT, hp-1^w mutant, hp-1^w,fri¹, and hp-1^w,tri¹ double mutants. A, For the RNA gel blots shown, RNA was isolated 4 h after the light pulse(s) and probed with a CAB cDNA probe. As a loading control the blots were probed with an 18S rRNA probe. B, The CAB mRNA abundance was quantified using a phosphor imager and the mean values (±se) are shown. A value of 100% on the ordinate represents the maximum steady-state mRNA detected within the experiment.

Figure 3

Effect of a 12-h R or B pretreatment terminated with no light pulse (lanes D), a 10-min R, 15-min FR, and 10-min R followed by 15-min FR pulse (R/FR) on the CHS mRNA abundance in etiolated 4-d-old WT and hp-1^w-mutant seedlings of tomato. A, For the RNA gel blots shown, RNA was isolated 4 h after the light pulse(s) and probed with a CHS1 cDNA probe. As a loading control the blots were probed with an 18S rRNA probe. B, The CHS mRNA abundance was quantified using a phosphor imager and the mean values (±se) are shown. A value of 100% on the ordinate represents the maximum steady-state mRNA detected within the experiment.

Figure 4

Circadian rhythmic CAB mRNA accumulation in WT and hp-1^w-mutant seedlings of tomato. A, For the RNA gel blots shown, seedlings were grown in 16-h WL (WL, 6 am–10 pm), 8-h dark (lanes D, 10 pm–6 am) cycles. To describe the diurnal CAB transcript oscillations (WL/D), samples were collected every 4 h starting with 4-d-old seedlings at 8 am. To study the circadian rhythm of CAB genes (WL/D → D/D), seedlings were transferred to darkness on d 5 at 8 am. As a loading control the blots were probed with an 18S rRNA probe. B, The CAB mRNA abundance was quantified using a phosphor imager and the mean values (±se) are shown. A value of 100% on the ordinate represents the mRNA abundance detected on d 4 at noon. {/ANNT;;;left;top}

Figure 5

CAB, RBCS, and CHS mRNA abundance in cotyledons (lanes Cot) and hypocotyls (lanes Hyp) of 4-d-old WT and hp-1^w-mutant seedlings of tomato. A, For the RNA gel blots shown, RNA was isolated from seedlings grown in dark (D) or 16-h WL, 8-h dark cycles (WL/D). As a loading control the blots were probed with a 18S rRNA probe. B, The CAB, RBCS, and CHS mRNA abundance was quantified using a phosphor imager and the mean values (±se) are shown. A value of 100% on the ordinate represents the maximum steady-state mRNA detected within the experiment.

Figure 6

Abundance of CAB, RBCS, and CHS mRNA in young leaves (lanes Le), stems (lanes St), and roots (lanes Ro) of 8-month-old WT and hp-1^w-mutant tomato plants. A, For the RNA gel blots shown, RNA was isolated from adult plants grown in 16-h WL, 8-h dark cycles. As a loading control the blots were probed with a 18S rRNA probe. B, The CAB, RBCS, and CHS mRNA abundance was quantified using a phosphor imager and the mean values (±se) are shown. A value of 100% on the ordinate represents the maximum steady-state mRNA detected within the experiment.

Figure 7

A, The CAB and RBCS mRNA accumulation in the pericarp of the hp-1^w-mutant and WT tomato fruit during fruit ripening. For both CAB and RBCS mRNA accumulation, the amount of mRNA in the pericarp of the youngest hp-1^w-mutant fruit was set at 100%. As a loading control the blots were probed with a 18S rRNA probe. B, A truss of WT (left) and hp-1^w-mutant (right) immature tomato fruits. The fruits of the hp-1^w mutant are darker green and have an elongated shape when compared with WT.