The role of annexin 1 in drought stress in Arabidopsis

Abstract

Annexins act as targets of calcium signals in eukaryotic cells, and recent results suggest that they play an important role in plant stress responses. We found that in Arabidopsis (Arabidopsis thaliana), AnnAt1 (for annexin 1) mRNA levels were up-regulated in leaves by most of the stress treatments applied. Plants overexpressing AnnAt1 protein were more drought tolerant and knockout plants were more drought sensitive than ecotype Columbia plants. We also observed that hydrogen peroxide accumulation in guard cells was reduced in overexpressing plants and increased in knockout plants both before and after treatment with abscisic acid. Oxidative protection resulting from AnnAt1 overexpression could be due to the low level of intrinsic peroxidase activity exhibited by this protein in vitro, previously linked to a conserved histidine residue found in a peroxidase-like motif. However, analyses of a mutant H40A AnnAt1 protein in a bacterial complementation test and in peroxidase activity assays indicate that this residue is not critical to the ability of AnnAt1 to confer oxidative protection. To further examine the mechanism(s) linking AnnAt1 expression to stress resistance, we analyzed the reactive S3 cluster to determine if it plays a role in AnnAt1 oligomerization and/or is the site for posttranslational modification. We found that the two cysteine residues in this cluster do not form intramolecular or intermolecular bonds but are highly susceptible to oxidation-driven S-glutathionylation, which decreases the Ca(2+) affinity of AnnAt1 in vitro. Moreover, S-glutathionylation of AnnAt1 occurs in planta after abscisic acid treatment, which suggests that this modification could be important in regulating the cellular function of AnnAt1 during stress responses.

Figure 1.

Northern-blot analysis of AnnAt1 mRNA level after different treatments in Arabidopsis Col-0 plants. A, Detached, fully expanded leaves of 6-week-old plants were incubated on the surface of the following solutions (water [mock], 100 μm ABA, 150 mm NaCl, 1 mm salicylic acid [SA], 10 mm H₂O₂, and 100 mm Cd²⁺). To wound them, leaves were squeezed with grooved forceps five times per leaf. Total RNAs were isolated and hybridized with cDNA probes derived from the 3# untranslated region (764–1,116 nucleotides of the ORF) of AnnAt1 and actin 2 cDNA (1,062–1,283 nucleotides of the ORF). This experiment was performed twice and gave similar results. B, Detached, fully expanded leaves were subjected to drought (as described in “Materials and Methods”). Total RNAs were isolated and hybridized with AnnAt1 probe, as in A. As a loading control, 28S rRNA after staining with 0.2% (w/v) methylene blue is shown. The percentage of water loss at each time point is indicated at bottom. This experiment was performed twice and gave similar results.

Figure 2.

Tolerance to dehydration stress of Arabidopsis with different AnnAt1 levels. A, AnnAt1 expression confers increased tolerance to short-term drought. Twenty-five plants (Col-0, ΔannAt1-3, and 35S∷AnnAt1-12 [OE]) were grown in a single pot under short-day conditions. After 4 weeks, water was withdrawn. The photograph showing the differences in the reactions of plants to the short-lasting drought was taken after 5 d of water withdrawal. B, AnnAt1 expression confers increased tolerance to long-term drought. Arabidopsis plants (Col-0, ΔannAt1-1, and 35S∷AnnAt1-5 [OE]) were grown under short-day conditions. After 8 weeks, water was withdrawn for 3 weeks. The photograph showing the differences in each plant's ability to survive the period of total desiccation was taken 2 weeks after reinitiation of watering. These experiments were repeated twice and gave comparable results.

Figure 3.

The role of AnnAt1 in oxidative stress in Arabidopsis cells. A, Confocal images of representative epidermal strips from Arabidopsis plants (Col-0, ΔannAt1-1, ΔannAt1-2, and OE-12) following treatment either with buffer [mock] or 10 μM ABA and staining with an H₂O₂-specific dye, H₂DCFDA. Bright-field images of Col-0 and transgenic lines are also shown. This experiment was repeated four times with similar results, and all experiments were done double blind. B, Quantification of guard cell pigmentation from experiment A. The graph includes an n value of 30 cells from the epidermal strip. Error bars represent sd. C, In vitro peroxidase activity of recombinant AnnAt1 protein expressed in N. benthamiana. Approximately 10 μg of wild-type AnnAt1-His(6) (A) and H40A AnnAt1-His(6) (M) and 10 ng of horseradish peroxidase (HRP; positive control) and bovine serum albumin (BSA; negative control) were dot blotted onto PVDF and developed using enhanced chemiluminescence substrate without antibodies. As a control, heat denatured-samples (d) were spotted.

Figure 4.

Determination of the in vitro redox status of Cys groups in AnnAt1-His(6) protein. A schematic diagram showing the step-by-step procedure for this experiment is shown. Ten micrograms of AnnAt1-His(6) protein was analyzed. N-ethylmaleimide (NEM) was used to block free SH groups. Dithiothreitol (DTT) was the chemical reducing agent used. Protein SHs were labeled with MPB. After electrophoresis and blotting, one of the replicate filters was probed with avidin conjugated with horseradish peroxidase. To confirm equal loading, the second filter was immunoprobed with anti-annexin antibody (data not shown). This experiment was performed three times and gave comparable results.

Figure 5.

The role of Cys residues in the formation of AnnAt1 oligomers. Ten micrograms of purified recombinant proteins, wild type or mutated AnnAt1-His(6) (with one or both Cys residues replaced by Ala), was subjected to chemical cross-linking with DSS. Control samples were subjected to the same procedure except that instead of incubation with DSS the equivalent amount of solvent, DMSO, was added. After electrophoresis and blotting, the AnnAt1 protein was visualized with the enhanced chemiluminescence system. This experiment was performed three times and gave comparable results.

Figure 6.

In vitro and in planta S-glutathionylation of AnnAt1 protein. A, HPLC C18 reverse-phase trace of 280 nm of nonmodified AnnAt1-His(6) protein (solid line) and AnnAt1-His(6) protein after incubation with oxidized glutathione (dashed line). The shift of the main peak protein testifies that incubation results in an increase in protein hydrophilicity. The molecular masses of collected samples determined by quadrupole time-of-flight mass spectrometer were 37,202 D for nonmodified protein and 37,814 D for glutathionylated protein, which suggests the incorporation of 2 mol of GSH per mol of AnnAt1. This experiment was performed three times and gave comparable results. B, Fragmentation spectrum of the Cys-111 glutathionylated peptide (WTSSNQVLMEVACTR), derived from AnnAt1 isolated from N. benthamiana. Major peaks on the spectrum can be annotated, accordingly, with the theoretical collision-induced dissociation fragmentation chart of the analyzed peptide (Supplemental Table S3), to b- and y-series daughter ions resulting from peptide fragmentation. Almost all theoretical daughter ions can be found on the spectrum, unequivocally confirming addition of the glutathione moiety. The most abundant peak on the spectrum (*) corresponds to cysteinylglycine-annexin-modified peptide. Apparently, the γ-peptide bond in the glutathione is the most labile in the whole structure and can be assumed as a neutral loss marker of glutathione-modified peptides. All major peaks on the spectrum are assigned to b- and y-series daughter ions. Peaks that originated from subsequent fragmentation of the glutathione moiety from the glutathione-y3 daughter ion are also indicated (•).

Figure 7.

S-Glutathionylation of AnnAt1-His(6) affects its Ca²⁺-binding affinity. Ca²⁺ titration of the relative fluorescence signal of AnnAt1-His(6) reduced (black squares, AnnAt1r, solid line) and its glutathione derivative (white squares, GS=AnnAt1, dashed line). Protein concentrations were 0.55 μm. Error bars represent sd. The calcium-binding constant K_a calculated for reduced protein was 2.0 ± 0.2 × 10⁴ m⁻¹. S-Glutathionylation results in a 50% decrease in the K_a, or 1.0 ± 0.2 × 10⁴ m⁻¹. This experiment was performed twice and gave comparable results.