The Arabidopsis E3 SUMO ligase SIZ1 regulates plant growth and drought responses

Abstract

Posttranslational modifications of proteins by small ubiquitin-like modifiers (SUMOs) regulate protein degradation and localization, protein-protein interaction, and transcriptional activity. SUMO E3 ligase functions are executed by SIZ1/SIZ2 and Mms21 in yeast, the PIAS family members RanBP2, and Pc2 in human. The Arabidopsis thaliana genome contains only one gene, SIZ1, that is orthologous to the yeast SIZ1/SIZ2. Here, we show that Arabidopsis SIZ1 is expressed in all plant tissues. Compared with the wild type, the null mutant siz1-3 is smaller in stature because of reduced expression of genes involved in brassinosteroid biosynthesis and signaling. Drought stress induces the accumulation of SUMO-protein conjugates, which is in part dependent on SIZ1 but not on abscisic acid (ABA). Mutant plants of siz1-3 have significantly lower tolerance to drought stress. A genome-wide expression analysis identified approximately 1700 Arabidopsis genes that are induced by drought, with SIZ1 mediating the expression of 300 of them by a pathway independent of DREB2A and ABA. SIZ1-dependent, drought-responsive genes include those encoding enzymes of the anthocyanin synthesis pathway and jasmonate response. From these results, we conclude that SIZ1 regulates Arabidopsis growth and that this SUMO E3 ligase plays a role in drought stress response likely through the regulation of gene expression.

Figure 1.

Expression Profile of pSIZ1-GUS. (A) Schematic diagrams of SIZ1 promoter fragments used for promoter-GUS fusions. pSIZ1-1 (3133-bp 5′ sequence), pSIZ1-2 (3535-bp 5′ sequence), and pSIZ1-3 (4311-bp 5′ sequence) were fused to a GUS open reading frame. (B) to (J) Expression patterns of pSIZ1-2:GUS in a 3-d-old seedlings (B), lateral root (C), primary root tip (D), lateral root primordia (E), 10-d-old seedlings (F), adult leaf (G), flower (H), young silique (I), and old silique (J) (see Results for details). Bars = 1 mm, except in (C) to (E), where bars = 0.1 mm.

Figure 2.

Functional Characterization of SIZ1. (A) RNA gel blot analysis of SIZ1. Two-week-old wild-type, siz1-3, and C-siz1-3 plants were used. rRNAs in the bottom panel were used as loading controls. (B) Protein gel blot analysis of SIZ1 using affinity-purified anti-SIZ1 antibodies. Two week-old wild-type, siz1-3, and C-siz1-3 plants were used. LS, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit. (C) One-week-old (panels a to c), 5-week-old (d), and 8-week-old (e) wild-type, siz1-3, and C-siz1-3 plants. Bars =10 mm in (a) to (c) and 3 cm in (d) and (e). (D) Leaf lamina length, width, and area in 25-d-old wild-type and siz1-3 mutant plants. Data are average values ± sd (n = 12). (E) Analysis of epidermal (a) and mesophyll (b) cells of the 5th leaf of 25-d-old siz1-3 and wild-type plants by scanning electron microscopy. In a 160,000-μm² area, a siz1-3 leaf contains 114 ± 16.7 epidermal cells and 45 ± 5.6 stomata, whereas a wild-type leaf contains 47.9 ± 5.8 epidermal cells and 17.4 ± 2.8 stomata (n = 6). Note that the mesophyll cell size of siz1-3 leaf is smaller than that of the wild type. Bars = 100 μm. (F) Anthocyanin contents of wild-type, siz1-3, and C-siz1-3 plants. Error bars represent sd (n = 3). Data are shown as relative units. FW, fresh weight. (G) RNA gel blot analysis of total RNAs (10 μg) from 5-week-old wild-type, siz1-3, and C-siz1-3 plants using specific probes for CHS1, CHI, and PAL1. rRNAs in the bottom panel were used as loading controls.

Figure 3.

Accumulation of SUMO-Protein Conjugates Induced by Dehydration Is Partially Dependent on SIZ1. Protein extracts were analyzed by protein gel blots using anti-SUM1 polyclonal antibodies to detect SUMO-protein conjugates. The large subunit (LS) of Rubisco (55 kD) was used as a loading control. Each lane contained 10 μg of protein. (A) Three-week-old wild-type and aba2 mutant plants exposed to drought for 0, 2, and 4 h. (B) Three-week-old wild-type, siz1-3, and C-siz1-3 plants exposed to drought for 0, 2, 4, and 6 h.

Figure 4.

SIZ1 Plays an Important Role in Drought Tolerance. (A) Water loss quantification in percentage of fresh weight lost (0, 5, 10, 20, 30, and 60 min) in 3-week-old wild-type, siz1-3, and C-siz1-3 plants. The percentage of fresh weight (FW) remaining after the treatment was determined. Data represent average values ± sd (n = 6). (B) Survival of plants subjected to water withholding for 3 weeks after resumption of watering for 1 week. (C) Percentage of survival in (B). Data represent average values ± sd (n = 6). (D) Analysis of SIZ1 protein levels in 2-week-old wild-type plants exposed to drought for 0, 2, and 4 h. Protein extracts were analyzed by protein gel blots using affinity-purified anti-SIZ1 antibodies. LS, large subunit of Rubisco.

Figure 5.

SIZ1 Regulates Gene Expression. Relative expression levels (fold difference) of 16 genes in wild-type and siz1-3 plants under control conditions (labeled as WT C/siz1 C), in wild-type plants after 2 h of drought stress with respect to wild-type plants under control conditions (labeled as WT D/C), and in siz1-3 plants after 2 h of drought stress with respect to siz1-3 plants under control conditions (labeled as siz1 D/C). The expression data were obtained by quantitative RT-PCR. Amplification of eIF4a mRNA within the same reactions was performed as a loading control. Error bars represent sd (n = 3). (1) DWF4, (2) SQS2, (3) DET2, (4) DWF1, (5) MYC2, (6) ANNAt4, (7) COR15a, (8) KIN1, (9) HMG1, (10) CHS1, (11) TT5, (12) DFR, (13) FLS, (14) RAB18, (15) DREB2A, and (16) COR47.