Figure 1.

Genetic and Phenotypic Description of the siz1 and mms21 Mutants.

(A) Organization of the SIZ1 and MMS21 genes and proteins. The SAP, PHD, PIIT, SP-RING, and SIM domains are highlighted in blue, green, orange, red, and purple, respectively. Untranslated regions and introns are shown as gray boxes and lines, respectively. Exons are numbered. The red triangles show the positions of the T-DNA insertions. The lines underneath the wild-type and mutant SIZ1 proteins locate the peptides identified during our MS analysis of SUMO1/SUMO2 conjugates. The arrows locate the primers used for RT- PCR in (B). The amino acid (aa) sequence lengths of the siz1-2 and mms21-1 polypeptides that match their wild-type counterparts are shown.

(B) PCR analysis of plants containing wild-type and mutant versions of SIZ1 and MMS21. Panels identified by the green lines represent RT-PCR of the siz1-2 and mms21-1 transcripts. Panels identified by the blue lines represent genomic PCR analyses demonstrating the presence of the 6His-SUMO1(H89-R) transgene and absence of an intact SUMO1 and SUMO2 genes in the respective genotypes.

(C) Alignment of 111 transcripts from siz1-2 plants generated by RNA-seq around the predicted T-DNA insertion site. SIZ1 and T-DNA-related sequences are colored in gray and red/orange, respectively. A poly(A) tract is indicated in blue. The most common junction between the SIZ1 and T-DNA sequences identified the T-DNA insertion site at 1758 bp from the ATG translation start codon (red) with a less prevalent junction upstream (orange). The previously reported insertion site at 1723 bp is also shown (Miura et al., 2005).

(D) Quantification by RT-PCR of SIZ1 transcript levels in wild-type and siz1-2 plants show in (C), using primers that probed the SIZ1 locus either upstream or downstream of the T-DNA insertion. The values were normalized to those for ACT2 and represented as a ratio to the wild type.

(E) Immunoblot detection of SIZ1 protein in 8-d-old unstressed wild-type and siz1-2 seedlings, using anti-SIZ1 antibodies. The arrowhead locates the predicted full-length SIZ1 protein.

(F) and (G) Representative 8-d-old wild-type, siz1-2, and mms21-1 plants with or without the SUMO conjugate purification background [6His-S1(H89-R) sumo1-1 sumo2-1].

(F) Phenotype of young seedlings. Root tips are highlighted by the yellow line. Bar = 0.5 cm.

(G) Plants grown for 20 d (bottom) and 40 d (top) in a long-day photoperiod.