PCA22 acts as a suppressor of atrzf1 to mediate proline accumulation in response to abiotic stress in Arabidopsis

Abstract

Proline metabolism is important for environmental responses, plant growth, and development. However, its precise roles in plant abiotic stress tolerance are not well understood. Mutants are valuable for the identification of new genes and for elucidating their roles in physiological mechanisms. We applied a suppressor mutation approach to identify novel genes involved in the regulation of proline metabolism in Arabidopsis. Using the atrzf1 (Arabidopsis thaliana ring zinc finger 1) mutant as a parental line for activation tagging mutagenesis, we selected several mutants with suppressed induction of proline accumulation under dehydration conditions. One of the selected mutants [proline content alterative 22 (pca22)] appeared to have reduced proline contents compared with the atrzf1 mutant under drought stress. Generally, pca22 mutant plants displayed suppressed atrzf1 insensitivity to dehydration and abscisic acid during early seedling growth. Additionally, the pca22 mutant exhibited shorter pollen tube length than wild-type (WT) and atrzf1 plants. Furthermore, PCA22-overexpressing plants were more sensitive to dehydration stress than the WT and RNAi lines. Green fluorescent protein-tagged PCA22 was localized to the cytoplasm of transgenic Arabidopsis cells. Collectively, these results suggest that pca22 acts as dominant suppressor mutant of atrzf1 in the abiotic stress response.

Keywords: Abiotic stress response; AtRZF1; pca mutant; pollen tube length; proline metabolism; suppressor..

Fig. 1.

Proline content in the leaves of WT, atrzf1, and pca22 plants. Light-grown 5-week-old plants were grown for 7 d without watering. Leaf tissues were carefully excised after drought treatment, and their proline content was measured. Error bars represent the SD. Differences among WT, atrzf1, and pca22 plants grown under the same conditions are significant at 0.01<P<0.05 (*).

Fig. 2.

Effect of the pca22 mutant on sensitivity to dehydration stress and ABA sensitivity. Effect of osmotic stress and ABA on cotyledon greening. Seeds were sown on MS agar plates supplemented with mannitol, PEG, or ABA and permitted to grow for 8 d; seedlings with green cotyledons were then counted (triplicates, n=50 each). Error bars represent the SD. Differences among WT, atrzf1, and pca22 plants grown under the same conditions are significant at 0.01<P<0.05 (*) or P<0.01 (**).

Fig. 3.

Identification of 35S enhancer elements containing the T-DNA insertion region in pca22. (A) A map of the T-DNA insertion of pca22 on chromosome 2. The structure of the activation-tagged locus of pca22 is shown in the flanking region containing At2g28625 and At2g28630. The arrows mark the positions of gene-specific primers and a T-DNA LB-specific primer used for PCR amplification. (B) T-DNA linkage analysis of the pca22 phenotype. Genomic PCR using the gene-specific primers F1 and R1 yielded a DNA band specific for the WT and atrzf1 lines at the PCA22 locus, whereas PCR using primers F1 and LB3 produced a pca22-specific band. (C) RT–PCR to compare the transcript levels of AtRZF1 and ACT1 control genes in WT, atrzf1, and pca22 mutants. (D) Expression of the three neighboring genes (At2g28620, At2g28625, and At2g28630) encoding a functional protein near the activation-tagged locus was analyzed in pca22 plants and compared with those in atrzf1 by RT–PCR. ACT1 was used as an internal control for RT–PCR.

Fig. 4.

Alignment of the full-length deduced amino acid sequences of PCA22 and PCA22 orthologs from different phylogenetic origins. (A) Shown are the sequences of PCA22 (At2g28625), Camelina sativa XP010510600, Camelina sativa XP010414496, Camelina sativa XP010470060, Capsella rubella XP006295935, Brassica rapa XP009140926, and Eutrema salsugineum XP006409894. Black and gray shading indicate identical and similar amino acids, respectively. Gaps were used to optimize the alignment. (B) Phylogenetic tree depicting homology relationships among Arabidopsis thaliana, C. sativa, C. rubella, B. rapa, and E. salsugineum PCA22 members. Numbers at branch points indicate bootstrap values after 1000 replications. (C) The structure of the conserved regions of the PCA22 protein. The primary structure contains a serine-rich box (9–70) within its central region, which is indicated by the gray box.

Fig. 5.

Mannitol, PEG, and ABA sensitivity as a result of PCA22 overexpression in atrzf1 mutants. (A–C) The effects of mannitol, PEG, and ABA treatment on cotyledon greening. Seeds were sown on MS agar plates supplemented with 400 mM mannitol (A), 10% PEG (B), or 1 μM ABA (C), and permitted to grow for 6, 7, 8, and 9 days after germination (DAG). Seedlings with green cotyledons were counted (triplicates, n=50 each). Error bars represent the SD. Differences between wild-type and transgenic plants grown under the same conditions are significant at 0.05>P>0.01 (*) or P<0.01 (**).

Fig. 6.

PCA22 expression in Arabidopsis plants grown under abiotic stress conditions. (A–D) qPCR analyses showing the expression of PCA22, RAB18, and RD29A in response to abiotic stress conditions. All quantifications were made in three independent RNA samples obtained from plants treated with 400 mM mannitol (A), 10% PEG (B), drought (C), and 100 μM ABA (D) for the indicated times. Error bars indicate the SD of three independent biological samples. Differences among the expression of PCA22, RAB18, or RD29A in Arabidopsis seedlings with and without treatment with various abiotic stresses are significant at 0.05>P>0.01 (*) or P<0.01 (**).

Fig. 7.

Cytoplasmic localization of PCA22. Five-day-old transgenic plants grown on half-strength MS agar medium were analyzed for GFP expression by confocal microscopy. The PCA22–GFP signal was mainly observed in the cytoplasm of the root cells. GFP, green fluorescent protein; B/W, black and white. Scale bars=100 μm. (This figure is available in colour at JXB online.)

Fig. 8.

Abiotic stress sensitivity of PCA22 transgenic plants. (A–C) Effect of mannitol, PEG, and ABA treatment on cotyledon greening. Seeds were sown on MS agar plates supplemented with 400 mM mannitol (A), 10% PEG (B), or 1 μM ABA (C), and were permitted to grow for 7, 8, and 9 days after germination (DAG). Seedlings with green cotyledons were counted (triplicates; n=50 each). Error bars represent the SD. Differences between WT and transgenic plants grown under the same conditions are significant at 0.05>P>0.01 (*) or P<0.01 (**).

Fig. 9.

Length of in vitro grown WT, atrzf1, pca22, and PCA22 transgenic pollen tubes. After 12 h pollen germination, the lengths of 150 pollen tubes from each line were measured. pca22 and PCA22-overexpressing (OX12 and OX16) pollen tubes were significantly shorter than WT, atrzf1, and pca22ri (ri14 and ri27) pollen tubes. Error bars represent the SD. Differences among WT, atrzf1, pca22, PCA22-overexpressing (OX12, OX16), and pca22ri (ri14, ri27) lines grown under the same conditions are significant at 0.05>P>0.01 (*) or P<0.01 (**).