The Arabidopsis plastid-signalling mutant gun1 (genomes uncoupled1) shows altered sensitivity to sucrose and abscisic acid and alterations in early seedling development

Abstract

Developing seedlings of the Arabidopsis gun1 (genomes uncoupled1) mutant, which is defective in retrograde plastid-to-nucleus signalling, show several previously unrecognized mutant phenotypes. gun1 seedlings accumulated less anthocyanin than wild-type seedlings when grown in the presence of 2% (w/v) sucrose, due to lower amounts of transcripts of early anthocyanin biosynthesis genes in gun1. Norflurazon and lincomycin, which induce retrograde signalling, further decreased the anthocyanin content of sucrose-treated seedlings, and altered the temporal pattern of anthocyanin accumulation. Lincomycin treatment altered the spatial pattern of sucrose-induced anthocyanin accumulation, suggesting that plastids provide information for the regulation of anthocyanin biosynthesis in Arabidopsis seedlings. The temporal pattern of accumulation of LHCB1 transcripts differed between wild-type and gun1 seedlings, and gun1 seedlings were more sensitive to sucrose suppression of LHCB1 transcript accumulation than wild-type seedlings. Growth and development of gun1 seedlings was more sensitive to exogenous 2% sucrose than wild-type seedlings and, in the presence of lincomycin, cotyledon expansion was enhanced in gun1 seedlings compared to the wild type. gun1 seedlings were more sensitive than wild-type seedlings to the inhibition of seedling growth and development by abscisic acid. These observations clearly implicate GUN1 and plastid signalling in the regulation of seedling development and anthocyanin biosynthesis, and indicate a complex interplay between sucrose and plastid signalling pathways.

Fig. 1.

Effect of sucrose, norflurazon, and lincomycin on anthocyanin accumulation in wild-type and gun1 seedlings. The upper part of the figure shows 1 cm × 1 cm images of representative wild-type (Col-0) and gun1 seedlings grown for 4 d on 0.5× MS-agar medium, ±2% sucrose (suc), ±0.5 mM lincomycin (Linc), or ±5 μM norflurazon (NF). The top row of images shows seedlings grown in the presence of sucrose (+suc), with the bottom row showing seedlings grown in the absence of sucrose (–suc). The lower part of the figure shows the anthocyanin content of the seedlings, with the bars numbered corresponding to the numbered images above. Anthocyanins were extracted from five replicate samples of 25 seedlings from each treatment by homogenization in acidified methanol. The anthocyanin content was determined from the absorbance of the extract at 530 nm and 657 nm, and expressed as 1000×A₅₃₀–A₆₃₇ per seedling. The results are shown as mean ±standard errors for each set of five replicates. The experiment was repeated four times with essentially identical results.

Fig. 2.

Cotyledons of wild-type and gun1 seedlings grown in the presence of sucrose and lincomycin. The figure shows representative 4-d-old wild-type (Col-0) and gun1 seedlings grown on 0.5× MS-agar medium containing 2% sucrose and 0.5 mM lincomycin. Size bar=1 mm. The lower part of the figure shows the mean width ±standard error of the mean for 100 4-d-old seedlings.

Fig. 3.

Transcripts of genes encoding anthocyanin biosynthesis enzymes. RT-PCRs were performed on RNA extracted from 4-d-old wild-type (Col-0) and gun1 seedlings grown on 0.5× MS-agar medium containing 2% sucrose. ‘Early’ anthocyanin biosynthesis genes: PAL, phenylalanine ammonia-lyase 1 (At2g37040); CHS, chalcone synthase (At5g13930); CHI, chalcone isomerase (At3g55120), F3H, flavanone 3-hydroxylase (At3g51240). ‘Late’ anthocyanin biosynthesis genes: DFR, dihydroflavonol reductase (At5g42800); LDOX, leucoanthocyanidin dioxygenase (At4g22880). Controls: ACT7, actin2/7 (At5g09810); UBQ10 ubiquitin 10 (At4g05320). The primers used are listed in Table 1.

Fig. 4.

Time-course of anthocyanin accumulation in wild-type and gun1 seedlings. Anthocyanins were extracted in acidified methanol from five replicate samples of 25 seedlings of wild-type (Col-0) and gun1 seedlings grown on 0.5× MS-agar medium, ±2% sucrose (suc), ±5 μM norflurazon or ±0.5 mM lincomycin. Seedlings were harvested daily 2–8 d after germination. Anthocyanin content was determined from the absorbance of the extract at 530 nm and 657 nm, and the results expressed as 1000×A₅₃₀–A₆₃₇ per seedling. The results are shown as mean ±standard errors for each set of five replicates. The experiment was repeated four times with essentially identical results.

Fig. 5.

Anthocyanin content of wild-type and gun1 seedlings grown in the presence of sucrose or sorbitol. Anthocyanins were extracted from five replicate samples of 25 seedlings of wild-type (Col-0) and gun1 seedlings grown for 4 d on 0.5× MS-agar medium, ±2% sucrose (suc) or 58 mM sorbitol (+sorb). Anthocyanin content was determined from the absorbance of the extract at 530 nm and 657 nm, and the results expressed as 1000×A₅₃₀–A₆₃₇ per seedling. The results are shown as mean ±standard errors for each set of five replicates.

Fig. 6.

Effect of sucrose and lincomycin on LHCB1 transcripts in wild-type and gun1 seedlings. RT-PCRs were performed on three replicate samples of RNA extracted from 4-d-old wild-type (Col-0) and gun1 seedlings grown on 0.5× MS-agar medium, ±2% sucrose (suc) and ±0.5 mM lincomycin (Linc). The PCR products are shown in the upper part of the figure. The amounts of LHCB1 PCR product normalized to those of ACT7 are shown in the lower part of the figure. Three independent RT-PCRs were analysed for each treatment.

Fig. 7.

Time-course of LHCB1 transcript abundance in wild-type and gun1 seedlings. RT-PCRs were performed on three replicate samples of RNA extracted from 3–8-d-old wild-type (Col-0) and gun1-1 seedlings grown on 0.5× MS-agar medium, ±2% sucrose (suc), and ±0.5 mM lincomycin (Linc). The PCR products are shown in the upper part of the figure. The amounts of LHCB1 PCR product normalized to those of ACT7 are shown in the lower part of the figure. The ratios are expressed as mean ±standard error for the three independent RT-PCRs analysed for each treatment.

Fig. 8.

Effect of sucrose on development of wild-type and gun1 seedlings. Upper panel: growth stages as defined by Boyes et al. (2001); 0.1, seed imbibition; 0.5, radicle emergence; 0.7, hypocotyl and cotyledon emergence; 1.0, cotyledons fully open. Lower panel: percentage of seedlings having reached each growth stage 54 h after transfer of 4 °C dark-stratified seeds to continuous illumination (120 μmol m⁻² s⁻¹) at 22 °C. Wild-type (Col-0) and gun1-1 seedlings were grown on 0.5× MS-agar medium ±2% sucrose (suc). Three replicate samples of 100 seedlings were examined and the results presented as means ±standard errors.

Fig. 9.

Effect of ABA on development of wild-type and gun1 seedlings. Percentage of 9-d-old seedlings having reached the growth stages defined by Boyes et al. (2001); 0.1, seed imbibition; 0.5, radicle emergence; 0.7, hypocotyl and cotyledon emergence; 1.0, cotyledons fully open. Three replicate samples of 100 wild-type (Col-0) and gun1 seeds were sown on 0.5× MS-agar medium ±0.5 μM ABA, stratified for 48 h in the dark at 4 °C and then transferred to continuous illumination (120 μmol m⁻² s⁻¹) at 22 °C. The number of seedlings reaching, or passing, each growth stage was counted 9 d after transfer to light at 22 °C. Results are expressed as mean ±standard error for three replicate samples.