Global Analysis of Direct Targets of Secondary Wall NAC Master Switches in Arabidopsis  Zhong Ruiqin , Lee Chanhui , Ye Zheng-Hua   Molecular Plant  Volume 3, Issue 6, Pages 1087-1103 (November 2010) DOI: 10.1093/mp/ssq062 Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 1 Transactivation and EMSA Analyses of SND1 Binding Sites in the MYB46 Promoter. (A) Diagrams of the reporter and effector constructs used for transactivation analysis. NosT, nopaline synthase terminator. (B) Transactivation analysis showing the SND1-activated expression of the GUS reporter gene driven by the corresponding MYB46 promoter deletions. Left panel illustrates various deletions of the MYB46 promoter (−612 to −150 from the start codon). The two SND1 binding sites (SNBE1 and SNBE2) are boxed in black. The promoter regions covered by MYB46-P6 (−612 to −498) and MYB46-P2 (−284 to −150) are indicated. The effector and reporter constructs were co-transfected into Arabidopsis leaf protoplasts and the transactivation activity was monitored by assaying the GUS activity. The GUS activity in protoplasts transfected with the reporter construct alone was used as a control and taken as 1. Error bars denote SE of three biological replicates. (C) Transactivation analysis showing the SND1-activated expression of the GUS reporter gene driven by various deletions of the MYB46-P6 region (−612 to −498) as shown at left. (D, E) EMSA analysis showing that the 24-bp SNBE2 oligonucleotides but not the 24-bp P257–234 oligonucleotides (competitor) competed with the binding of SND1 to MYB46-P2 (D) and MYB46-P6 (E). P257–234 corresponds to the −257 to −234 sequence (adjacent to SNBE2) in the MYB46 promoter. The competitors were added in 30-fold (+) or 60-fold (++) molar excess relative to the labeled MYB46-P2 and P6 probes. (F) The 24-bp sequences of SNBE1 and SNBE2. Identical nucleotides between SNBE1 and SNBE2 are shaded. Molecular Plant 2010 3, 1087-1103DOI: (10.1093/mp/ssq062) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 2 Identification of the Consensus SND1 Binding Sequence by EMSA Analysis. The MYB46–SNBE1 sequence was mutated systematically by substituting one nucleotide at a time by the three other nucleotides (shown below each wild-type ones) to generate 72 mutated SNBE1 sequences, each of which has a single nucleotide mutation. Each of these unlabeled mutated SNBE1 oligonucleotides was subjected to competition with the binding of SND1 to the labeled MYB46-P6 probe in 30-fold molar excess. The inset at the left of the top panel depicts EMSA of the binding of SND1 to the MYB46-P6 probe and the competition of this binding by the unlabeled wild-type SNBE1 oligonucleotides. Mutations of the nucleotides critical for the binding by SND1 results in a failure of competition, thus little effect on the shifted band shown by EMSA. The consensus SND1 binding sequence was deduced from the EMSA data. Molecular Plant 2010 3, 1087-1103DOI: (10.1093/mp/ssq062) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 3 Transactivation Analysis of the Consensus SND1 Binding Sequence. (A) Diagrams of the reporter and effector constructs used for transactivation analysis. The reporter constructs were made by ligating three copies of the wild-type or mutated MYB46–SNBE1 sequence upstream of the CaMV 35S minimal promoter that is linked with the GUS reporter gene. (B) Transactivation analysis showing the SND1-activated expression of the GUS reporter gene driven by the wild-type or mutated MYB46–SNBE1 sequences as depicted at left. The MYB46–SNBE1 sequence was mutated by altering all the non-critical nucleotides (SNBE1-M1) or changing one or more critical nucleotides (SNBE1-M2 to M5). The consensus nucleotides in the MYB46–SNBE1 sequence are shaded. Dashes in SNBE1-M1 to M5 denote nucleotides without alterations. The GUS activity in the protoplasts transfected with the reporter construct alone was used as a control and taken as 1. Error bars denote SE of three biological replicates. (C) Cross-section of a stem showing GUS staining in the interfascicular fibers (if) and xylem (xy) in the transgenic plants expressing the GUS reporter gene driven by six copies of MYB46–SNBE1. Molecular Plant 2010 3, 1087-1103DOI: (10.1093/mp/ssq062) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 4 Transactivation Analysis of SNBE Sequences from the Five Known SND1 Direct Targets. (A) Putative SNBE sequences identified from the 1.5-kb promoters of SND1 direct targets, including MYB46, MYB83, SND3, MYB103, and KNAT7. The number shown at the left of each sequence is the position of the first nucleotide relative to the start codon. The plus or minus symbol at the right indicates the SNBE sequence from the forward or reverse strand of DNA, respectively. The SNBE sequences used for transactivation analysis are marked with asterisks. The consensus nucleotides in the SNBEs are shaded. (B) Transactivation analysis showing the activation of the SNBE-driven GUS reporter gene by SND1. Three copies of the representative SNBEs linked with the CaMV 35S minimal promoter were ligated upstream of the GUS reporter gene. The reporter gene construct together with the effector construct containing the CaMV 35S promoter-driven SND1 cDNA were co-transfected into Arabidopsis protoplasts. The GUS activity in the protoplasts transfected with the reporter construct alone was used as a control and taken as 1. Error bars denote SE of three biological replicates. Molecular Plant 2010 3, 1087-1103DOI: (10.1093/mp/ssq062) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 5 Transactivation of the SNBE Sequences by Other SWNs, Including VND6, VND7, NST1, and NST2. The GUS reporter gene was driven by three copies of SNBEs linked with the CaMV 35S minimal promoter. The SNBE sequences used are shown in Figures 3B and 4A. The reporter gene construct together with the effector construct containing the CaMV 35S promoter-driven SWN cDNA were co-transfected into Arabidopsis protoplasts, which were subjected to the GUS activity assay. Error bars indicate SE of three biological replicates. (A) SWNs induced the expression of the GUS reporter gene driven by the representative SNBEs from the five known SND1 direct targets. The GUS activity in the protoplasts transfected with the reporter construct alone was used as a control and taken as 1. (B) Transactivation analysis showing that mutations of the consensus nucleotides in the MYB46–SNBE1 sequence resulted in a significant reduction or a complete loss of activation by other SWNs. The expression of the GUS reporter gene driven by the wild-type MYB46–SNBE1 was taken as 100. Molecular Plant 2010 3, 1087-1103DOI: (10.1093/mp/ssq062) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 6 Verification of the SWN-Mediated Direct Activation of 10 Representative Genes Identified from the Global Gene Expression Analysis. (A) Diagram of the constructs containing the fusion of SWNs with the regulatory region of the human estrogen receptor (HER), which were used for the direct target activation analysis. (B) Quantitative PCR analysis showing direct induction of the expression of 10 representative genes by the estradiol-activated SWNs in the presence of the protein synthesis inhibitor cycloheximide (CHX). Arabidopsis protoplasts transfected with the SWN–HER fusion constructs were treated with CHX alone or CHX and estradiol. RNAs isolated from the treated protoplasts were used for gene expression analysis by quantitative PCR. Error bars denote SE of three biological replicates. Molecular Plant 2010 3, 1087-1103DOI: (10.1093/mp/ssq062) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 7 Transactivation Analysis of the SNBE Sites from the Promoters of XCP1 and RNS3. (A) Diagrams of the reporter and effector constructs used for transactivation analysis. The reporter construct was made by ligating three copies of the SNBE sequence upstream of the CaMV 35S minimal promoter that is linked to the GUS reporter gene. (B) Transactivation analysis showing the SWN-activated expression of the GUS reporter gene driven by XCP1–SNBE1 (left panel) or RNS3–SNBE1 (right panel). Arabidopsis protoplasts co-transfected with the reporter and effector constructs were subjected to GUS activity assay. The GUS activity in the protoplasts transfected with the reporter construct alone was used as a control and taken as 1. Error bars indicate SE of three biological replicates. Molecular Plant 2010 3, 1087-1103DOI: (10.1093/mp/ssq062) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 8 Direct Binding of VND6, VND7, and SND1 to the SNBE Sites in the XCP1 Promoter. Purified recombinant VND6, VND7, or SND1 fused with maltose binding protein (MBP) was incubated with the biotin-labeled XCP1 promoter fragment (−216 to −1 relative to the start codon) and subjected to EMSA. (A) EMSA showing a band shift caused by binding of VND7 (lane 3), VND6 (lane 5), and SND1 (lane 7) to the labeled XCP1 promoter fragment. No band shift was seen in the controls without the addition of proteins (lane 1) or with the addition of MBP (lane 2). Addition of the unlabeled XCP1 promoter fragment (+) in 60-fold molar excess relative to the labeled probe eliminated the band shift caused by VND7 (lane 4), VND6 (lane 6), and SND1 (lane 8). (B) XCP1–SNBE and XCP1–TERE sequences used for the EMSA competition analysis. Two SNBE sites were present in the XCP1 promoter fragment used for EMSA. The consensus nucleotides in the SNBE sequences are shaded. mSNBE1 is the mutated version of XCP1–SNBE1 with mutations of all nine consensus nucleotides. The number shown at the left of each sequence is the position of the first nucleotide relative to the start codon. The TERE sequence is underlined. (C) XCP1–SNBE1 and SNBE2 but not mSNBE1 and TERE effectively compete with the binding of VND7 (upper panel), VND6 (middle panel), and SND1 (lower panel) to the labeled XCP1 promoter fragment. The competitors were added in 30-fold (+) or 60-fold (++) molar excess relative to the labeled XCP1 promoter probe. Molecular Plant 2010 3, 1087-1103DOI: (10.1093/mp/ssq062) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 9 Transactivation and GUS Reporter Gene Analyses of the XCP1 Promoter Deletions. (A) Diagrams of the reporter and effector constructs used for transactivation analysis. The reporter constructs contain serial deletions of the XCP1 promoter linked to the GUS reporter gene. The SNBE1 and SNBE2 sites are boxed in black. (B) Transactivation analysis showing the VND6- or VND7-activated expression of the GUS reporter gene driven by various XCP1 promoter deletions. Arabidopsis protoplasts co-transfected with the reporter and effector constructs were subjected to GUS activity assay. The GUS activity in protoplasts transfected with the reporter construct alone was used as a control and taken as 1. Error bars indicate SE of three biological replicates. (C) Cross-section of stems showing vessel-specific expression of the GUS gene driven by the 213-bp fragment of the XCP1 promoter encompassing both SNBE1 and SNBE2 (XCP1P-213). co, cortex; if, interfascicular fiber; ph, phloem; ve, vessel. (D–F) Cross-sections of stems showing the cortex- and phloem-associated expression of the GUS gene driven by XCP1 promoter deletions without SNBE2 (XCP1P-194 and -130) or without both SNBE1 and SNBE2 (XCP1P-111). Molecular Plant 2010 3, 1087-1103DOI: (10.1093/mp/ssq062) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 10 Complementation of the snd1 nst1 Double Mutant by Expression of SWNs. (A) The pendent stem phenotype conferred by snd1 nst1 was complemented by expression of SWNs driven by the SND1 promoter. (B) Measurement of the breaking strength showing that the mechanical strength of snd1 nst1 inflorescence stems was rescued by expression of SWNs. Each bar represents the breaking force of inflorescence stems of individual plants. (C–H) Restoration of the deposition of lignified secondary walls in the interfascicular fibers of snd1 nst1 stems by expression of SWNs. The bottom parts of the stems of 8-week-old plants were cut for lignin staining with phloroglucinol-HCl. if, interfascicular fiber; xy, xylem. Bar in (C) = 156 μm for (C–H). Molecular Plant 2010 3, 1087-1103DOI: (10.1093/mp/ssq062) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions