PMC

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.

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.

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.

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.

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.