SIZ1 small ubiquitin-like modifier E3 ligase facilitates basal thermotolerance in Arabidopsis independent of salicylic acid

Abstract

Small ubiquitin-like modifier (SUMO) conjugation/deconjugation to heat shock transcription factors regulates DNA binding of the peptides and activation of heat shock protein gene expression that modulates thermal adaptation in metazoans. SIZ1 is a SUMO E3 ligase that facilitates SUMO conjugation to substrate target proteins (sumoylation) in Arabidopsis (Arabidopsis thaliana). siz1 T-DNA insertional mutations (siz1-2 and siz1-3; Miura et al., 2005) cause basal, but not acquired, thermosensitivity that occurs in conjunction with hyperaccumulation of salicylic acid (SA). NahG encodes a salicylate hydroxylase, and expression in siz1-2 seedlings reduces endogenous SA accumulation to that of wild-type levels and further increases thermosensitivity. High temperature induces SUMO1/2 conjugation to peptides in wild type but to a substantially lesser degree in siz1 mutants. However, heat shock-induced expression of genes, including heat shock proteins, ascorbate peroxidase 1 and 2, is similar in siz1 and wild-type seedlings. Together, these results indicate that SIZ1 and, by inference, sumoylation facilitate basal thermotolerance through processes that are SA independent.

Figure 1.

siz1-2 and siz1-3 seedlings are thermosensitive. A and B, Ten-day-old wild-type (Col-0), siz1-2, and siz1-3 seedlings were subjected to a heat shock treatment of 39°C for 4 h in the dark at 60% relative humidity. Untreated wild-type, siz1-2, and siz1-3 seedlings (normal) were maintained in light at 22°C for the 4-h period, and all remained viable (100% survival) throughout the duration of the experiment. A, Photograph of representative seedlings 4 d after completion of heat shock treatment and B, seedling survival determined for the same experiment as in A 4 d after treatment, mean with 95% confidence intervals, n = 24 to 25. C, Wild-type, siz1-2, and siz1-3 seedlings were subjected to a heat shock treatment of 39°C in the dark for the time indicated and returned to the dark at 22°C/18°C (16 h/8 h). Hypocotyl growth was determined 2.5 d after treatment, mean ± se and n ≥ 18.

Figure 2.

siz1 increases thermosensitivity during seed germination. Stratified wild-type, siz1-2, siz1-3, NahG siz1-2, NahG, and hot1-3 seeds (3 d in the dark at 4°C) were immediately subjected to heat shock treatment of 45°C for 4 h or incubated at 24°C (normal), sown onto plates, and then maintained under a 16-h daily photoperiod at 24°C. Germination was assessed at the indicated intervals. A, Illustration of representative seeds/seedlings 6 d after heat treatment and B, seed germination data from three independent experiments, mean ± se, n = 21.

Figure 3.

siz1-2 and siz1-3 seedlings exhibit reduced basal thermotolerance but not acquired thermotolerance. A, Ten-day-old wild-type, siz1 (siz1-2 and siz1-3), NahG siz1-2, NahG, and hot1-3 seedlings were not acclimated (normal, 22°C) or high-temperature acclimated by exposure to 39°C for 90 min. After a recovery period of 2 h at 22°C, seedlings were exposed to a heat shock of 45°C for 2 h under the conditions described in the Figure 1 legend. Illustrated are representative seedlings 4 d after heat shock treatment. B, Stratified seeds of genotypes described in A were incubated in the dark at 22°C/18°C (16 h/8 h). Three days thereafter, seedlings were high-temperature acclimated by exposure to 39°C for 90 min. After a recovery period of 2 h at 22°C, seedlings were exposed to a heat shock of 45°C for 2 h under conditions described in the Figure 1 legend and then grown for 2.5 d at 22°C/18°C (16 h/8 h). Illustrated are relative growth determinations (100% indicates the growth of genotypes at 22°C) from three independent experiments, mean ± se, n ≥ 18 seedlings/experiment.

Figure 4.

siz1 hyperaccumulates high levels of SA. Ten-day-old wild-type, siz1 (siz1-2 and siz1-3), NahG siz1-2, NahG, and snc1 seedlings grown at 24°C on medium were harvested. SA content was determined by HPLC analysis. Illustrated are data from three independent experiments, mean ± se (micrograms of SA per gram fresh weight). Experiments were repeated four times in the same condition. snc1 seedlings result hyperaccumulation of SA (Zhang et al., 2003).

Figure 5.

Heat shock-induced SUMO1/2 conjugation is suppressed by siz1-2 and siz1-3. Ten-day-old wild-type, siz1-2, siz1-3, and hot1-3 seedlings were exposed to a 30-min heat shock treatment (39°C, dark, 60% relative humidity). Total protein was extracted from untreated or heat shock-treated seedlings. Twenty micrograms of protein were loaded onto an SDS-PAGE, and the immunoblot was probed with anti-SUMO1, which detects both SUMO1 and SUMO2 (Kurepa et al., 2003).

Figure 6.

Heat shock-induced SUMO1/2 conjugation in NahG seedlings requires SIZ1. Ten-day-old wild-type, siz1-2, NahG siz1-2, and NahG seedlings were exposed to a 30-min heat shock at 39°C in the dark. Total protein was extracted from seedlings as described in Figure 5. Ten micrograms of protein were separated by SDS-PAGE and the immunoblot was probed with anti-SUMO1.

Figure 7.

Heat stress-induced SUMO1/2 conjugation/deconjugation is impaired in NahG and hot1-3 seedlings. Ten-day-old wild-type, NahG, and hot1-3 seedlings were exposed to a 39°C heat stress for 15 or 30 min, returned to 24°C, and collected at indicated time points. Black arrow indicates the serial of heat shock. Total protein was extracted as described in Figure 5. Ten micrograms of protein were separated by SDS-PAGE, and the immunoblot was probed with anti-SUMO1.

Figure 8.

HSF and heat shock-induced gene expression is similar in wild-type and siz1 seedlings. Ten-day-old wild-type and siz1-2 seedlings were exposed to 37°C for the times indicated. Transcript abundance was determined by semiquantitative reverse transcription-PCR for four major HSFs (HSF1, 3, 4, and 7), and five different HSPs (HSP17.6A [class II sHSP], HSP18.2 [class I sHSP], HSP70, HSP83.1, HSP101) and APX1 and APX2. Black arrows indicate the period of heat shock.