Volume 2, Issue 4, Pages 675-687 (July 2009) Arabidopsis MSI1 Is Required for Negative Regulation of the Response to Drought Stress  Alexandre Cristina , Möller-Steinbach Yvonne , Schönrock Nicole , Gruissem Wilhelm , Hennig Lars   Molecular Plant  Volume 2, Issue 4, Pages 675-687 (July 2009) DOI: 10.1093/mp/ssp012 Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 1 Overlapping Gene Sets Are Controlled by MSI1, CAF-1, and the E2F–DP–RBR1 Pathway. (A) Venn diagram representations of overlapping gene sets with altered expression in msi1-cs and in the CAF-1 mutants fas1 and fas2 (Schönrock et al., 2006b). (B) Venn diagram representations of overlapping gene sets with altered expression in msi1-cs and in E2Fa–DPa overexpressing plants (Vandepoele et al., 2005). The upper and lower panels in (A) and (B) represent up-regulated and down-regulated genes, respectively. (C) Increased expression of cell-cycle-phase-specific genes in msi1-cs plants. 9910 genes identified by Menges et al. (2003) were sorted into 14 bins, based on their maximal expression during the cell cycle. Shown are the mean ± SE of the signal log ratios (SLR) between expression signals in wild-type and msi1-cs plants for all genes in a bin. Shaded areas represent periods of S-phase and mitosis (M), respectively. Molecular Plant 2009 2, 675-687DOI: (10.1093/mp/ssp012) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 2 The Expression of Replacement Histone H3.2 and CYCLIN B1;1 Genes Is Up-Regulated in msi1-cs. (A) CYCB1;1 and H3.2 average signals from microarrays. Error bars indicate the range of the measurements. Numbers above bars represent fold change versus wild-type. (B) Confirmation of CYCB1;1 and H3.2 up-regulation in independent samples of the original msi1-cs line MSI1OEc2 and of the independent msi1-cs line MSI1OEa3. RNA was extracted from 26-day-old rosette leaves for all genotypes. GAPDH was used as reference gene. Molecular Plant 2009 2, 675-687DOI: (10.1093/mp/ssp012) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 3 ABA-Responsive Genes Are Up-Regulated in msi1-cs. (A) Functional classification of genes with altered expression in msi1-cs into hormone-response categories (Nemhauser et al., 2006). Shown are p-values for significant enrichment of hormone-responsive genes. (B, D) Average signals from microarrays for four selected genes found up-regulated in msi1-cs (At2g39030, MYB2, LTP3, and LTP4). Error bars indicate the range of the measurements. Numbers above bars represent fold change versus wild-type. (C, E) Confirmation of microarray results for two independent MSI1-co-suppression lines (msi1-cs line and MSI1OEa3). RNA was extracted from 26-day-old rosette leaves. Molecular Plant 2009 2, 675-687DOI: (10.1093/mp/ssp012) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 4 Osmotic and Salt Stress-Responsive Genes Are Up-Regulated in msi1-cs. (A) Cluster analysis of gene expression after ABA treatment and during abiotic stress. Self-organizing tree clustering (SOTA) of the 515 genes up-regulated in msi1-cs. (B) SOTA of the 288 genes from cluster 1 in (A). Gene expression data after ABA treatment and after various stresses was taken from Goda et al. (2008) and Kilian et al. (2007), respectively. Each horizontal line represents the average expression profile of one cluster. The color scale at the top represents the expression level (–2 to +2). The tree structure on the left represents cluster similarities. Numbers on the right are labels for clusters and numbers of probesets in each cluster. (C) Principle component analysis of gene expression at 3 h after ABA treatment, at 24 h after abiotic stress treatment, and in msi1-cs. The applied stresses were cold, osmotic, salt, drought, genotoxic, oxidative, UV-B, wounding, and heat (for details, see Kilian et al., 2007). Molecular Plant 2009 2, 675-687DOI: (10.1093/mp/ssp012) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 5 Analysis of Enriched Promoter Elements for Genes with Altered Expression in msi1-cs. (A) Five enriched promoter elements identified by the WeederWeb algorithm (Pavesi et al., 2006) in cluster 1 from Figure 4A resemble the ABA-responsive element (ABRE, PyACGTGGC) and ABRE-like motifs (C/G/T)ACGTG(G/T) (A/C) that are bound by ABFs. (B) One enriched promoter element identified in cluster 1 from Figure 4A resembles the CACG-like motif that is bound by NAC transcription factors. (C) Three enriched promoter elements identified in cluster 2 from Figure 4A resemble the site II motif (TGGGCC/T) that is bound by TCP-domain transcription factors. Molecular Plant 2009 2, 675-687DOI: (10.1093/mp/ssp012) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 6 RD20 Is a Direct Target of MSI1. (A) Structure of the MSI1–GFP construct introduced into the msi1-1 mutant. 2 kb upstream promoter sequences of the MSI1 gene were used to drive expression of a MSI1 cDNA–GFP fusion. The used MSI1–GFP line is homozygous for the msi1-1 allele. (B) Protein blots of wild-type (WT) and MSI1–GFP transgenic plants using 20 μg of soluble proteins extracted from 14-day-old seedlings. Endogenous MSI1 and the MSI1–GFP fusion protein were both detected by the anti-MSI1 antibody, while the anti-GFP antibody detected only the fusion protein. (C) The binding of MSI1 to possible target genes was tested in 11-day-old seedlings of an MSI1–GFP line in which the MSI1–GFP fusion construct complements the msi1-1 null allele. Shown is one of two independent ChIP experiments. Numbers represent enrichment of signal in MSI1–GFP over wild-type. αGFP, anti-GFP antibody; n.d., not determined due to weak signals. Molecular Plant 2009 2, 675-687DOI: (10.1093/mp/ssp012) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 7 msi1-cs Plants Have Increased Free Proline Levels and Drought Tolerance. (A) Wild-type (top row) and msi1-cs (bottom row) plants after 12 d without watering. Note the wilting rosette leaves of wild-type plants in contrast to the still unstressed msi1-cs plants. (B) Wild-type (top row) and msi1-cs (bottom row) plants after 16 d without watering. Note the more severe wilting of wild-type plants compared to msi1-cs plants. (C) Free proline levels were measured in rosette leaves of non-stressed 7-week-old wild-type and msi1-cs plants. Molecular Plant 2009 2, 675-687DOI: (10.1093/mp/ssp012) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions