AtPPRT1 negatively regulates salt stress response in Arabidopsis seedlings

Abstract

Salt stress is one of the environmental factors that negatively affect plant growth and development. We have previously reported a putative C3HC4 zinc-finger ubiquitin E3 ligase (AtPPRT1) negatively regulates Abscisic acid (ABA) and drought stress response. According to previous studies, the accumulation of ABA in plants can further regulate the salt stress response. Therefore, in this study, we further analyzed whether AtPPRT1 negatively regulates the salt stress response. The results showed that AtPPRT1 expression was induced by salt stress. Furthermore, under salt stress, the β-glucuronidase (GUS) gene driven by the AtPPRT1 promoter has shown increased activity in the hypocotyl and petioles of Arabidopsis seedlings. Additionally, seedlings of the T-DNA insertion mutant atpprt1 showed significant growth advantage under salt stress, whereas overexpressing AtPPRT1 (OE lines) in Arabidopsis seedlings displayed hypersensitive under salt stress. Etiolated atpprt1 seedlings also demonstrated significantly elongated hypocotyl lengths in salt stress. The elevated or reduced salt tolerance of atpprt1 and AtPPRT1 overexpressing lines was confirmed by the changes in chlorophyll content and 3,3'-Diaminobenzidine (DAB) staining. The above data suggest that AtPPRT1 has a negative effect on salt tolerance in Arabidopsis seedlings.

Keywords: Arabidopsis; AtPPRT1; salt tolerance.

Figure 1.

Bioinformatics analysis of the AtPPRT1 promoter and the transcriptional expression levels of AtPPRT1 under abiotic stresses. (a) The region of 3 kbp upstream of the AtPPRT1 gene was analyzed for the cis-acting elements according to the plant cis-acting regulatory element database (PlantCARE). The promoter region of 1492 bp used in this study is shown in bold line. The numbers in the boxes indicate different binding sites. 1, MYC binding-site motif; 2, ACE binding-site motif; 3, ABRE binding-site motif; 4, O2 site motif; 5, BOX4 promoter motif; 6, ARE binding-site motif; 7, LTR binding-site motif; 8, AE-box promoter motif; 9, W-box promoter motif; 10, MYB binding-site motif; 11, TGA-element; 12, G-Box promoter motif; 13, MBS binding-site motif; 14, P-box promoter motif; 15, TCA-element. (b) the qRT-PCR analysis of AtPPRT1 transcriptional expression levels induced by 120 mM NaCl and 300 mM mannitol. These experiments were repeated three times with similar results. Error bars represent ± SD (n = 3, *p < .05, **p < .01, t-test).

Figure 2.

GUS activity in different tissues and its transcriptional expression levels in ProAtPPRT1:: GUS transformant lines. (a) 7-day-old and (d) 3-day-old homozygous transformed plants’ seedlings and (e) etiolated seedlings were used for GUS staining under non-stress conditions and 120 mM NaCl treatment; (b) the transcriptional expression levels of the GUS gene were analyzed by qRT-PCR. These experiments were repeated three times with similar results. Error bars represent ± SD (n = 3, *p < .05, **p < .01, t-test); (c) the flowers and leaves of 4-week-old ProAtPPRT1:: GUS transformed plants (#21) were used for GUS staining under non-stress conditions and 120 mM NaCl treatment. Scale bar = 1 mm. #5, #8 and #21 indicate different transformant lines.

Figure 3.

The germination and cotyledon greening rates of each line under different NaCl concentrations. (a) Col-0, atpprt1, OE2 and OE10 were monitored for 7 days on MS medium plates with and without salt; the germination (b) and cotyledon greening (c) rates of Col-0, atpprt1, OE2 and OE10 on MS medium plates supplemented with different concentrations of NaCl. Error bars represent ± SD (n = 50, * p < .05 and ** p < .01, t-test).

Figure 4.

AtPPRT1 negatively affects salt stress tolerance in Arabidopsis seedlings. (a) Col-0, atpprt1, OE2, and OE10 grown vertically on 1/2 MS for 3 days were transferred to 1/2 MS without NaCl or supplemented with 120 mM NaCl or 300 mM mannitol for another 7 days; (b) measurement of root lengths under 120 mM NaCl treatment or 300 mM mannitol treatment; measurement of (c) chlorophyll content, (d) fresh weight and (e)) first leaf pair span under 120 mM NaCl in Col-0, atpprt1, OE2, and OE10. The values are the average of three individual biological replications. Error bars represent ± SD (n = 21, *p < .05 and **p < .01, t-test).

Figure 5.

Seedlings survival rate and DAB staining under high salinity. (a) The survival rates of Col-0, atpprt1, OE2 and OE10 under 150 mM NaCl; (b) DAB staining assay of seedlings of Col-0, atpprt1, OE2 and OE10 after 150 mM NaCl treatment.

Figure 6.

AtPPRT1 affects the elongation of hypocotyl in etiolated seedlings under salt stress. (a) Col-0, atpprt1, OE2, and OE10 were grown for 2 days on 1/2 MS medium plates and then transferred to 1/2 MS supplemented with 120 mM NaCl and grown for 5 days. All the seedlings were grown in the dark; (b) the hypocotyl lengths of samples used in (a) were measured. These experiments were repeated three times with similar results. Error bars represent ± SD (n = 21, * p < .05 and ** p < .01, t-test); (c) qRT-PCR analysis of the transcriptional expression levels of AtMAP18 and AtBSK1 in Col-0, atpprt1, OE2, and OE10 in 120 mM NaCl. These experiments were repeated three times with similar results. Error bars represent ± SD (n = 3, *p < .05, **p < .01, t-test).