A novel stress-associated protein 'AtSAP10' from Arabidopsis thaliana confers tolerance to nickel, manganese, zinc, and high temperature stress

Abstract

We describe here the functional characterization of a novel AtSAP10, a member of the Stress Associated Protein (SAP) gene family, from Arabidopsis thaliana ecotype Columbia. AtSAP10 contains an A20 and AN1 zinc-finger domain at the N- and C-terminal, respectively. Arabidopsis SAP10 showed differential regulation by various abiotic stresses such as heavy metals and metalloids (Ni, Cd, Mn, Zn, and As), high and low temperatures, cold, and ABA. Overexpression of AtSAP10 in Arabidopsis conferred strong tolerance to heavy metals such as Ni, Mn, and Zn and to high temperature stress. AtSAP10 transgenic plants under these stress conditions grew green and healthy, attained several-fold more biomass, and had longer roots as compared to wild type plants. Further, while these transgenic plants accumulated significantly greater amounts of Ni and Mn in both shoots and root tissues, there was no significant difference in the accumulation of Zn. AtSAP10 promoter-GUS fusion studies revealed a root and floral organ-specific expression of AtSAP10. Overexpression of AtSAP10-GFP fusion protein showed the localization in both nucleus and cytoplasm. Taken together, these results showed that AtSAP10 is a potentially useful candidate gene for engineering tolerance to heavy metals and to abiotic stress in cultivated plants.

Figure 1. Semi-quantitative RT-PCR analysis of AtSAP10 expression in response to various stress treatments.

Expression analysis was performed with AtSAP10 specific primers from the RNA isolated from the Arabidopsis seedlings subjected to As(III) and As (V) (A), Cd and Zn (B), Ni (C), Mn (D), ABA (E), Heat (F), Salt (G), and Cold (H) treatments. All upper panels represent AtSAP10 and lower panel represent ACT2 used as internal loading control. Numbers on each upper panel represents time intervals in hours for which the stress treatments were given. The presented results are the representative of at least three independent experiments.

Figure 2. Ni resistance phenotype of Arabidopsis AtSAP10 overexpression lines.

(A) Ni resistance phenotypes, (B) Fresh shoot weight, and (C) root length of three transgenic lines AtSAP10–23, AtSAP10–30, and AtSAP10–42 overexpressing AtSAP10 from ACT2pt expression cassette and wild type (WT) plants grown on 90 µM NiCl₂ in half-strength MS medium for three weeks. The average and standard deviation (SD) values are represented for four replicates of 12 seedlings each for WT and all AtSAP10 lines. The asterisks represent the significant difference in biomass accumulation and root length compared with wild type (WT) plants, (*) P<0.05, (**) P<0.01.

Figure 3. Mn resistance phenotype of Arabidopsis AtSAP10 overexpression lines.

(A) Mn resistance phenotypes and (B) Fresh shoot weight of three transgenic lines AtSAP10–23, AtSAP10–30, and AtSAP10–42 overexpressing AtSAP10 from ACT2pt expression cassette and wild type (WT) plants grown on 1 mM MnCl₂ in half-strength MS medium for three weeks. The average and standard deviation (SD) values are represented for four replicates of 12 seedlings each for WT and AtSAP10 lines. The asterisks represent the significant difference in biomass accumulation compared with wild type (WT) plants, (*) P<0.05, (**) P<0.01.

Figure 4. Zn resistance phenotype of Arabidopsis AtSAP10 overexpression lines.

(A) Zn resistance phenotypes, (B) Fresh shoot weight, and (C) root length of three transgenic lines AtSAP10–23, AtSAP10–30, and AtSAP10–42 overexpressing AtSAP10 from ACT2pt expression cassette and wild type (WT) plants grown on 500 µM ZnSO₄ in half-strength MS medium for three weeks. The average and standard deviation (SD) values are represented for four replicates of 12 seedlings each for WT and all AtSAP10 lines. The asterisks represent the significant difference in biomass accumulation and root length compared with wild type (WT) plants, (*) P<0.05, (**) P<0.01.

Figure 5. Analysis of total Ni, Mn, and Zn accumulation in Arabidopsis SAP10 overexpression lines.

The total Ni concentration in shoots (A) and roots (B) of wild type (WT) and four overexpression lines of AtSAP10 grown on hydroponics medium containing 90 µM NiCl₂. Total Mn accumulation in shoots (C) and roots (D) of wild type (WT) and four overexpression transgenic lines of AtSAP10 grown on hydroponics medium containing 1 mM MnCl₂. Total Zn accumulation in shoots (E) and roots (F) of wild type (WT) and four overexpression transgenic lines of AtSAP10 grown on hydroponics medium containing 500 µM ZnSO₄. The average and standard deviation (SD) values are shown for four replicates of 25 plants each for WT and all AtSAP10 lines. Asterisk represents the significant difference in Ni, Mn, or Zn accumulation as compared to wild type (WT), (*) <0.05, (**) <0.01.

Figure 6. Effect of high temperature stress on wild type and AtSAP10 overexpression transgenic lines.

12-day old seedlings were heat stressed (HS) as discussed in the ‘Materials and Methods’ section. Photographs of representative plates containing wild type (WT) and AtSAP10 transgenic lines were taken after 5 days of recovery.

Figure 7. Tissue-specific expression pattern of AtSAP10 as a transcriptional fusion of the GUS reporter gene to the promoter of AtSAP10.

Two days old seedlings of AtSAP10p-GUS (A); inflorescence of AtSAP10p-GUS (B); a flower of AtSAP10p-GUS (C). Scale bar, 1 mm.

Figure 8. Localization of GFP-tagged AtSAP10 in Arabidopsis.

(A) GFP expression in protoplasts: Upper Panel (i, ii, and iii) and middle panel (iv, v, and vi) showing the GFP fluorescence in protoplasts from plants overexpressing AtSAP10-GFP fusion protein in two different planes; lower panel showing the GFP fluorescence in protoplasts transformed with ACT2pt-eGFP as control. Scale bar, 10 µm. (B) Confocal laser scanning microscopy image of AtSAP10-GFP fusion protein of the root tissues: panel (i, ii, and iii) showing the GFP fluorescence in the roots of AtSAP10-GFP fusion plants. Scale bar, 20 µm.