BEL1-LIKE HOMEODOMAIN6 and KNOTTED ARABIDOPSIS THALIANA7 interact and regulate secondary cell wall formation via repression of REVOLUTA.

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BEL1-LIKE HOMEODOMAIN6 and KNOTTED ARABIDOPSIS THALIANA7 Interact and Regulate Secondary Cell Wall Formation via Repression of REVOLUTA CW

Yuanyuan Liu,a Shijun You,a Mallorie Taylor-Teeples,b,c Wenhua L. Li,a Mathias Schuetz,a Siobhan M. Brady,b,c and Carl J. Douglasa,1 a Department

of Botany, University of British Columbia, Vancouver, British Columbia V6T1Z4, Canada of Plant Biology, UC Davis, Davis, California 95616 c Genome Center, UC Davis, Davis, California 95616 b Department

The TALE homeodomain transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) is part of a regulatory network governing the commitment to secondary cell wall biosynthesis of Arabidopsis thaliana, where it contributes to negative regulation of this process. Here, we report that BLH6, a BELL1-LIKE HOMEODOMAIN protein, specifically interacts with KNAT7, and this interaction influences secondary cell wall development. BLH6 is a transcriptional repressor, and BLH6KNAT7 physical interaction enhances KNAT7 and BLH6 repression activities. The overlapping expression patterns of BLH6 and KNAT7 and phenotypes of blh6, knat7, and blh6 knat7 loss-of-function mutants are consistent with the existence of a BLH6-KNAT7 heterodimer that represses commitment to secondary cell wall biosynthesis in interfascicular fibers. BLH6 and KNAT7 overexpression results in thinner interfascicular fiber secondary cell walls, phenotypes that are dependent on the interacting partner. A major impact of the loss of BLH6 and KNAT7 function is enhanced expression of the homeodomainleucine zipper transcription factor REVOLUTA/INTERFASCICULAR FIBERLESS1 (REV/IFL1). BLH6 and KNAT7 bind to the REV promoter and repress REV expression, while blh6 and knat7 interfascicular fiber secondary cell wall phenotypes are suppressed in blh6 rev and knat7 rev double mutants, suggesting that BLH6/KNAT7 signaling acts through REV as a direct target.

INTRODUCTION Lignified secondary cell walls provide structural stiffness and strength to specialized plant cells, such xylem tracheids, vessel elements, and fibers (Roberts and McCann, 2000). The secondary cell wall is composed of a matrix of polymers that includes hemicelluloses, cellulose, lignin, and pectin. A complex network of transcription factors regulating secondary wall biosynthesis has been identified by genetic and reverse genetic analysis in Zinnia elegans and Arabidopsis thaliana (reviewed in Demura and Ye, 2010; Zhong et al., 2010; Wang and Dixon, 2012; Schuetz et al., 2013). These studies indicate that the NAC domain transcription factors SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1), NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1), NST2, VASCULARRELATED NAC-DOMAIN6 (VND6), and VND7 are key regulators of secondary wall biosynthesis in different cell types (Kubo et al., 2005; Mitsuda et al., 2005; Zhong et al., 2006; Yamaguchi et al., 2008), while MYB transcription factors (e.g., MYB46, MYB83,

1 Address

correspondence to [email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Carl J. Douglas (carl. [email protected]). C Some figures in this article are displayed in color online but in black and white in the print edition. W Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.114.128322

MYB58, and MYB63) and a Knotted-like homeobox (KNOX) protein KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) act downstream or in parallel to regulate specific aspects of secondary wall biosynthesis (Zhong et al., 2008; Kim et al., 2012; Ko et al., 2012). Some MYBs are direct targets of NAC domain master regulators (Zhong et al., 2007, 2008; McCarthy et al., 2009), while KNAT7 is a target of MYB46 (Ko et al., 2009) and SND1 (Zhong et al., 2008). These previous studies have thus provided the outlines of the transcriptional network that governs the biosynthesis of the three major components of secondary cell wall. The KNAT family of KNOX proteins in Arabidopsis belongs to the plant-specific three-amino acid loop extension (TALE) superclass of homeodomain proteins (Hake et al., 2004). TALE homeodomain proteins are distinguished from the other homeodomain proteins by possessing three additional amino acids in the region between helices 1 and 2 (Bertolino et al., 1995). Arabidopsis BEL1-LIKE HOMEODOMAIN (BLH) proteins also belong to the TALE class and, like KNOX proteins, are unique to plants. BLH proteins have conserved BELL and SKY domains that comprise a conserved bipartite domain in their N terminus called the MEINOX interacting domain (MID) (Bellaoui et al., 2001; Müller et al., 2001). Several studies indicate that KNOXBLH proteins interact to form heterodimers mediated by the interaction of the KNAT MEINOX and BLH MID domains (Bellaoui et al., 2001; Müller et al., 2001). There are 13 BLH genes in Arabidopsis (Smith et al., 2004). BELL1 (BEL1), the founding member of the group, is involved in

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ovule integument development as revealed by the phenotype of bel1 mutants (Reiser et al., 1995). Some BLH genes, such as BLH1, SAWTOOTH1 (SAW1), SAW2, BELLRINGER (BLR; synonymous with PENNYWISE, REPLUMLESS, VAAMANA, BLH9), POUNDFOOLISH, and ARABIDOPSIS THALIANA HOMEOBOX1 (ATH1), play diverse roles in embryo sac development, leaf development, inflorescence development, shoot development, flower patterning, and cell wall matrix formation (Byrne et al., 2003; Bao et al., 2004; Bhatt et al., 2004; Smith et al., 2004; Kumar et al., 2007; Pagnussat et al., 2007; Yu et al., 2009; Etchells et al., 2012), but the functions of other Arabidopsis BLH genes remain to be determined. Although heterodimeric complexes between TALE homeodomain proteins regulating developmental processes in plants are well characterized, these mainly involve Class 1 KNOX proteins (Hake et al., 2004; Hay and Tsiantis, 2010), whereas the BLH partners of Class 2 KNOX proteins remain poorly understood. KNAT7, a Class 2 KNOX gene, was identified by expression profiling as a candidate transcription factor that might regulate secondary wall synthesis in xylem and interfascicular fibers (IFs) during Arabidopsis inflorescence stem development (Ehlting et al., 2005), and coexpression meta-analysis also suggested a strong correlation between KNAT7 expression and secondary cell wall development in Arabidopsis (Brown et al., 2005; Persson et al., 2005; Schuetz et al., 2013; Hao and Mohnen, 2014). KNAT7 was subsequently shown to act as a transcriptional repressor whose expression helps suppress secondary wall formation (Li et al., 2011, 2012) and appears to play a role in cell wall homeostasis in response to resource availability (Li et al., 2012; Schuetz et al., 2013). Here, we report that BLH6 specifically interacts with KNAT7 to influence secondary cell wall development in the Arabidopsis inflorescence stem. Our data suggest that BLH6 and KNAT7 form a functional heterodimer that represses commitment to secondary cell wall biosynthesis in IF. However, expression of BLH6 under the control of the 35S promoter results in additional pleiotropic phenotypes, suggesting that BLH6 may interact with other partners to regulate other aspects of plant development. A major impact of the loss of BLH6 and KNAT7 function is the enhanced expression of the homeodomain-leucine zipper (HDZIP) transcription factor REVOLUTA/INTERFASCICULAR FIBERLESS1 (REV/IFL1). We present molecular and genetic data indicating that REV is a direct target of BLH6 and KNAT7, that a major function of the BLH6-KNAT7 heterodimer is to repress REV/IFL1 expression, and that BLH6 and KNAT7 at least partially modulate commitment to secondary cell wall biosynthesis in a transcription network that requires REV.

RESULTS BLH6 Interacts with KNAT7 Previous work showed that KNAT7 interacts with BLH proteins based on a large-scale yeast two-hybrid screen (Hackbusch et al., 2005), and several BLH proteins (BLH5, BLH7, and ATH1) are candidates for interaction with KNAT7 as potential regulators of secondary wall development, while BLH6 and BLH10

together with KNAT7 have been shown to be to the direct targets of MYB46 (Ko et al., 2009). Furthermore, BLH5 is coexpressed with KNAT7 during Arabidopsis inflorescence stem development (Ehlting et al., 2005), and within the BLH gene family, BLH1 shows highest similarity to BLH5 (Roeder et al., 2003). Thus, our list of candidate BLH components in a putative KNAT7-BLH complex included BLH1, BLH5, BLH6, BLH7, BLH10, and ATH1. We used bimolecular fluorescence complementation (BiFC) (Hu et al., 2002) to assay protein-protein interactions between BLH candidates and KNAT7 in planta. The BLH candidates and KNAT7 were fused to N- or C-terminal fragments of enhanced yellow fluorescent protein (N/C-EYFP). The split EYFPs were fused to both the C and N termini of each gene (Supplemental Figure 1). Different combinations of fusion constructs were used to transform Arabidopsis mesophyll protoplasts using an Arabidopsis leaf mesophyll protoplast transient expression system. Cotransformation of truncated EYFP fusions to these genes revealed that coexpression of BLH6-N-EYFP with KNAT7-CEYFP (Figure 1A), as well as BLH6-C-EYFP with KNAT7-NEYFP, generated nuclear-localized fluorescence (Supplemental Table 1). However, no fluorescence was detected when N- or Cterminal fusions of other BLH candidates to EYFP were coexpressed with corresponding KNAT7-C-EYFP or KNAT7-N-EYFP fusions (Figure 1A; Supplemental Table 1). In addition, no fluorescence was detected when either the BLH6-N-EYFP or BLH6C-EYFP construct was coexpressed with empty C-EYFP and empty N-EYFP (Figure 1A; Supplemental Table 1). These data indicate that BLH6 can specifically interact with KNAT7 in planta. KNAT7 nuclear localization has been reported (Zhong et al., 2008; Li et al., 2011), and BLH1 was shown to be nuclear localized (but absent from the nucleolus; Hackbusch et al., 2005). Recently, it has been shown that an ATH1-GFP (green fluorescent protein) fusion is found in both the cytosol and the nucleus, while after heterodimerization with STM, a KNOX homeodomain protein, it becomes completely nuclear localized (Rutjens et al., 2009). To test the subcellular localization of BLH6, Arabidopsis protoplasts were transfected with a C-terminal fusion BLH6-fulllength EYFP fusion. This analysis revealed that BLH6-EYFP is primarily localized to the nuclei of transformed protoplasts, similarly to KNAT7-EYFP (Figure 1B). We confirmed BLH6 nuclear localization in root cells of lines transgenic for Pro35S: GFP-BLH6 (Supplemental Figure 2). As BiFC identified BLH6 as the only BLH-KNAT7 interacting partner among those tested, and this interaction was not reported in a previous large-scale yeast two-hybrid assay (Hackbusch et al., 2005), we tested the BLH6-KNAT7 interaction again using a yeast two-hybrid system. We included BLH7 as well, as it is an apparent BLH6 paralog (Roeder et al., 2003). When a KNAT7 activation domain (AD) fusion protein construct and BLH6 and BLH7 DNA binding domain (DBD) fusion protein constructs were cotransformed into yeast strains, BLH6-DBDKNAT7-AD coexpression activated the expression of two reporter genes URA3 and LacZ, indicative of BLH6-KNAT7 protein interaction (Figure 2A), but there was no detectable interaction between BLH7 and KNAT7, confirming the results of the BIFC assay.

KNAT7 and BLH6 Regulate REVOLUTA

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BLH6 Is a Transcriptional Repressor

Figure 1. Bimolecular Fluorescence Assay of KNAT7-BLH6 Interaction and BLH6-EYFP Localization. (A) Left column: bright-field images of representative protoplasts cotransfected with KNAT7-C-EYFP and BLH6-N-EYFP, KNAT7-C-EYFP and BLH5-N-EYFP, and EMPTY-C-EYFP and BLH6-N-EYFP. Right column: images of the same protoplasts obtained by a Leica DM-6000B fluorescent microscope. (B) Top row: bright-field images of representative protoplasts transfected with full-length BLH6-EYFP and KNAT7-EYFP fusions, and full-length EYFP alone under the control of the 35S promoter. Bottom row: images of the same protoplasts obtained by the same microscope to image YFP fluorescence. C-EYFP, C-terminal enhanced YFP protein; N-EYFP, N-terminal enhanced YFP protein; F-EYFP, full-length enhanced YFP protein.

We next used the yeast two-hybrid system to test for a role of the KNAT7 MEINOX domain for the interaction with BLH6, as predicted by work on other KNOX proteins (Bürglin, 1997; Mukherjee and Bürglin, 2007). Four KNAT7 domains, KNOX1, KNOX2, MEINOX (KNOX1 and KNOX2), and the homeodomain were fused to AD and tested for their abilities to interact with a DBD-BLH6 fusion (Figures 2B and 2C). The KNAT7 KNOX2 and MEINOX domains, but no other domains, were able to interact strongly with BLH6, suggesting that the KNOX2 portion of the MEINOX domain is sufficient for KNAT7 interaction with BLH6 proteins. To identify which domain(s) of BLH6 interacts with KNAT7, we generated a set of six BLH6 domain-DBD fusions (Figure 2D), and tested their abilities to interact with ADKNAT7. Figure 2E demonstrates that both the BELL domain including adjacent sequences between the BELL and homeodomain that are absent in the MID (shorter) construct (Figure 2D), and the homeodomain itself, are individually sufficient to mediate interaction of BLH6 with KNAT7.

KNAT7 functions as a transcriptional repressor in protoplast transfection assays (Bhargava et al., 2010; Li et al., 2011). To test whether BLH6 has a similar function, we employed a protoplast transactivation system (Figure 3A; Supplemental Figure 3A; Wang et al., 2007). We used one set of transactivating effector plasmids containing Pro35S:GAL4 DB (GD) or GD fusions to the herpes simplex virus VP16 activation domain (GD-VP16), BLH6 (GDBLH6), and KNAT7 (GD-KNAT7), and a second transactivating effector plasmid containing a Pro35S:LexA DB (LD) fused to VP16 (LD-VP16). Reporter plasmids contained ProGAL4:GUS or ProLexA - ProGAL4:GUS, in which GAL4 or LexA and GAL4 DB binding sites are placed upstream of the b-glucuronidase (GUS) gene. Cotransfection of the reporter plasmid ProGAL4:GUS with effector plasmid GD-VP16 caused strong activation of the GUS reporter gene, while cotransfection with the effector containing the GD alone induced a very low level of GUS reporter gene expression (Supplemental Figure 3B). Cotransfection of ProGAL4:GUS with GD-BLH6 resulted in no activation of GUS expression. This indicates that BLH6 alone cannot activate the GUS reporter gene expression when recruited to the promoter region by GD. We next tested the ability of BLH6 to act as a transcriptional repressor by cotransfecting a plasmid with the ProLexA-ProGAL4:GUS reporter with either transactivating effector plasmids LD-VP16 and GD, or plasmid LD-VP16 and GDBLH6. Transfection of the LD-VP16 transactivator construct in combination with GD alone resulted in strong activation of GUS reporter activity (Figure 3B). However, cotransfection of LD-VP16 with GD-BLH6 resulted in lower GUS activity relative to protoplasts transfected with LD-VP16 with GD alone (Figure 3B). This indicates that BLH6 negatively regulates VP16 activated gene expression and thus functions as a transcriptional repressor. To further confirm if the interaction between BLH6 and KNAT7 affects BLH6 transcriptional activity, we placed KNAT7 under the control of the cauliflower mosaic virus 35S promoter with an N-terminal hemagglutinin (HA) tag (35S:HA-KNAT7; Figure 3A). Figure 3B shows that cotransfection of HA-KNAT7 with GDBHL6 and LexA-VP16 further repressed ProLexA-ProGAL4:GUS reporter gene activity relative to GD-BLH6 repression activity alone. This result was consistent between three independent protoplast transfection assays, supporting a model by which physical interaction between KNAT7 and BLH6 results in greater transcriptional repression activity than that of either protein alone. BLH6-GUS Expression Pattern To obtain information on the BLH6 expression pattern and to determine if it overlaps with that of KNAT7, transgenic Arabidopsis plants containing a ProBLH6:GUS transgene were generated and the expression pattern directed by ProBLH6 was examined. In 6-d-old seedlings, strong GUS activity was observed in the root, at the root-hypocotyl junction, and in cotyledons. In roots, expression was especially strong in the vascular cylinder (stele) and at the root tip, while in cotyledons expression was observed in both veins and other tissues (Supplemental Figures 4A to 4E). In mature leaves, BLH6 promoter activity was

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Figure 2. Yeast Two-Hybrid Assay of BLH and KNAT7 Interactions. (A) Assay of AD-KNAT7 interactions with DBD-BLH6 or DBD-BLH7 using two reporter genes, URA3 (assayed in Ura- media) and LacZ (assayed using X-Gal), with growth controls in Leu- Trp- media. MYB75-TT8 interaction was used as a positive control, and DBD-BLH-empty prey vector (AD-) interaction was used as a negative control. (B) Schematic diagram of KNAT7 with four different fragments used to test interaction with BLH6, KNOX1, KNOX2, MEINOX domain (KNOX1+KNOX2), and homeodomain. (C) Assay of AD-KNAT7 fragments interaction with DBD-BLH6 using two reporter genes, URA3 (assayed in Ura- media) and LacZ (assayed on X-Gal), with growth controls in Leu- Trp- media. MYB75-TT8 interaction was used as a positive control. DBD-BLH6- empty prey vector (AD-) interaction was used as a negative control, shown in (A). (D) Schematic diagram of BLH6 with six different fragments: SKY, BELL, MID (longer), MID (shorter), HM, and HM+ VSLTLGL box (Vbox). Truncation scheme of BLH6 is shown in detail below. (E) Assay of DBD-BLH6 fragments interaction with AD-KNAT7 and empty prey vector (AD-Empty) using two reporter genes, URA3 (assayed in Uramedia) and LacZ (assayed on X-Gal), with growth controls in Leu- Trp- media. MYB75-TT8 interaction was used as a positive control, and DBD-BLH6empty prey vector (AD-) interaction was used as a negative control. [See online article for color version of this figure.]

detected not only in the vascular tissue but also throughout the leaves (Supplemental Figure 4F). In flowers, BLH6 promoter activity was observed in vascular tissue of sepals, petals, stigma, and filaments (Supplemental Figure 4G). At the bases of mature inflorescence stems, the BLH6 promoter directed high GUS activity in the IF, developing xylem, in the phloem and cambial regions adjacent to the metaxylem, and also in cortex adjacent to the IF (Figures 4A and 4C). This expression pattern

was consistent in the inflorescence stem developmental stages from near the stem apex to the stem base (Supplemental Figures 4H and 4I). To assess the extent of overlap of this expression pattern with that of KNAT7, we reanalyzed KNAT7 expression by generating transgenic lines expressing a ProKNAT7-KNAT7:GUS transgene that contains all exons and introns plus 2.5-kb flanking 59 sequence. Analysis of GUS activity at the bases of mature ProKNAT7-KNAT7:GUS inflorescence


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(Supplemental Figure 4M). The GUS expression patterns seen in ProKNAT7-KNAT7:GUS and ProKNAT7:GFP lines are consistent with the KNAT7 expression pattern observed by in situ hybridization (Zhong et al., 2008). These data indicate that the BLH6 and KNAT7 promoters direct similar patterns of expression in the developing xylem, IF, and cortex regions of inflorescence stems and suggest that the corresponding proteins co-occur in these cells. Phenotypes of blh6 and blh6 knat7 Loss-of-Function Mutants

Figure 3. Test of BLH6 Transcriptional Activity. (A) Effectors and reporter constructs used in the transfection assays. (B) GUS activity in protoplasts derived from Arabidopsis rosette leaves cotransfected with an LD-VP16 transactivator plasmid and different combinations of effector plasmids (HA-KNAT7, HA-CAT, GD-BLH6, GD-KNAT7, or GD alone) together with the reporter plasmid ProLexA-ProGAL4:GUS. The HA-CAT (chloramphenicol acetyltransferase) was used in place of the HAKNAT7 effector to control for the amount of effector plasmid DNA (10 mg) introduced into protoplasts. The GUS activity of GD was used to normalize the other four. Error bars represent the standard deviations; n = 3 transfections from one representative biological replicate. Asterisks indicate significant differences from GD-BLH6 and GD-KNAT7 based on Student’s t tests.

stems revealed a very similar GUS expression pattern as that of ProBLH6:GUS lines, with expression in developing xylem and IF (Figures 4B and 4D). This pattern was consistent in younger regions of the stem (Supplemental Figures 4J and 4K). We also examined KNAT7 promoter activity in developing inflorescence stems by analyzing GFP fluorescence in live tissue from freshly prepared inflorescence stems of ProKNAT7:GFP transgenic plants. In these plants, ProKNAT7-directed GFP activity was evident in IF and developing xylem (Supplemental Figure 4L) as well as in cortex cells

To analyze the loss-of-function phenotype of BLH6, two independent T-DNA insertion lines of BLH6 were identified (Salk_011023 and GABI373G12), and the T-DNA insertion sites in the last exon were confirmed (Supplemental Figure 5A). RT-PCR showed that BLH6 mRNA was undetectable in homozygous plants (Supplemental Figures 5B and 5C), and the loss-offunction alleles were named blh6-1 and blh6-2. Plants homozygous for blh6-1 and blh6-2 were used for further analyses. Toluidine bluestained hand cross sections taken at 3 cm from the bases of inflorescence stems of 6-week-old plants grown under standard light conditions (16/8 h light/dark) were used to examine anatomical phenotypes in wild-type and mutant lines. A mild irx phenotype was observed in knat7 mutants (Figures 5C, 5E, and 5F) as previously described (Li et al., 2012), while both blh6-1 and blh6-2 also exhibited mild irx phenotypes with occasional collapsed vessels (Figures 5B, 5E, and 5F; Supplemental Figure 6). We generated blh6 knat7 double mutant plants by crossing a knat7-1 with blh6-1. Double mutants identified in the F2 population had no obvious morphological differences compared with the wild type. However, blh6 knat7 mutants showed an enhanced irx phenotype relative to the single mutants (Figures 5D to 5F). A striking aspect of the knat7 phenotype is increased secondary cell wall thickness in IF relative to wild-type plants (Li et al., 2011, 2012). We confirmed this phenotype in knat7 plants (Figure 6C, Table 1) and observed a similar phenotype in blh6-1 (Figures 6B, 6F, and 6H, Table 1) and blh6-2 (Supplemental Figure 6). Quantification of IF cell wall thicknesses indicated that blh6 and knat7 mutants had similar increases in wall thickness relative to the wild type, but that the blh6 knat7 double mutant did not exhibit an enhanced IF cell wall thickness phenotype relative to the single mutants (Figure 6D, Table 1). BLH6 Overexpression Phenotype To explore the phenotypic consequences of BLH6 overexpression, we placed BLH6 under the control of the parsley (Petroselinium crispum) 4CL1 promoter, which directs expression to cells with developing secondary walls (Hauffe et al., 1991) (Pro4CL1:BLH6). Five independent overexpressor lines were selected for study based on the high expression level of BLH6 (Supplemental Figure 7A). While no obvious morphological and developmental defects were observed in Pro4CL1:BLH6 lines relative to wild-type control plants, cross sections of inflorescence stems taken from 6-week-old plants revealed a striking decrease in the secondary wall thickness of IF relative to wild-type plants grown in parallel (Table 1). Representative results are shown in Figures 7B and 7J. Measurements of cell wall

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in parallel, with decreased rosette leaf size, inflorescence stem diameter, and silique length (Supplemental Figures 8A to 8F). The fertility of siliques was reduced to less than a quarter of the wild type in Pro35S:BLH6 plants (Supplemental Figures 8G and 8H). We also generated five independent Pro35S:KNAT7 transgenic lines overexpressing KNAT7 (Supplemental Figure 7B). Unlike Pro35S:BLH6 lines, the morphology and growth of Pro35S:KNAT7 overexpression lines were similar to the wild type. Cross sections taken 3 cm from the bases of 6-week-old wild-type, Pro35S:BLH6, and Pro35S: KNAT7 inflorescence stems revealed a decrease in cell wall thickness of IF relative to the wild type (Figures 7C, 7D, 7K, and 7L), which were about one-half the thickness of wild-type cells (Table 1).

Figure 4. BLH6 and KNAT7 Promoters Direct GUS Expression in the Interfascicular Fibers and Xylem. (A) Stem cross section of a representative ProBLH6:GUS line showing GUS expression in interfascicular fibers and vascular bundles. (B) Stem cross section of a representative ProKNAT7:KNAT7-GUS line showing GUS expression in interfascicular fibers and vascular bundles. (C) Higher magnification image of the representative ProBLH6:GUS line showing the expression pattern in the cambial region and metaxylem of vascular bundles. (D) Higher magnification image of the representative ProKNAT7:KNAT7GUS line showing the expression pattern in the cambial region and metaxylem of vascular bundles. Hand cross sections were taken from the bottom 5 cm of inflorescence stems of 6-week-old plants. C, cambial region; IF, interfascicular fibers; MX, metaxylem; P, phloem; X, xylem. Bars = 100 mm in (A) and (C) and 50 mm in (B) and (D).

thickness indicated that it was reduced by approximately onethird in the Pro4CL1:BLH6 line shown in Figure 7. This IF cell wall phenotype is opposite to that of blh6 loss-of-function mutants and is similar to that previously reported for Pro4CL1: KNAT7 plants (Li et al., 2012). We generated additional transgenic lines with elevated BLH6 expression under the control of the cauliflower mosaic virus 35S promoter (Pro35S:BLH6), which, in contrast to the 4CL1 promoter, directs expression to many cell and tissue types. Five independent overexpression lines were selected for study based on high expression level of BLH6 (Supplemental Figure 7A). Compared with the wild type, the growth of the primary inflorescence stems of Pro35S:BLH6 plants was significantly repressed (Figure 8), and the plants exhibited a decrease in internode length (Supplemental Figure 8F). Organs of Pro35S:BLH6 plants were generally smaller than those of wild-type plants grown

Figure 5. Assay of blh6, knat7, and blh6 knat7 irx Phenotypes. Stem cross sections were taken from the bases of the inflorescence stems of 6-week-old plants and stained with Toluidine blue. Arrows indicate the irx vessels. Bars = 30 mm. (A) The wild type. (B) blh6-1. (C) knat7. (D) blh6 knat7. (E) and (F) Quantification of the irx phenotype in vascular bundles of the basal internode of 6-week-old stems of the wild type, blh6-1, knat7, and blh6 knat7. Error bars represent the standard deviations; n = 24.


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Figure 6. Anatomy of the Interfascicular Fibers in Wild-Type and Mutant Plants. (A) to (D) Stem cross sections were taken from the bases of the inflorescence stems of 6-week-old plants and stained with Toluidine blue. Bars = 30 mm. (A) The wild type. (B) blh6-1. (C) knat7. (D) blh6 knat7. (E) to (H) Transmission electron micrographs of wild-type and blh6-1 interfascicular fiber secondary walls. The wild type ([E] and [G]); blh6-1 ([F] and [H]). Bars = 10 mm in (E) and (F) and 2 mm in (G) and (H).

KNAT7 and BLH6 Require Each Other’s Functions in IF Secondary Cell Wall Formation The interaction of BLH6 and KNAT7 proteins as well as the similar phenotypes of blh6 and knat7 mutants and BLH6 and KNAT7 overexpression lines suggests that they could work together to regulate commitment to secondary wall formation and could at least partially require each other’s functions as part of a transcriptional complex. To test this, we crossed a blh6-1 mutant line to a Pro35S:KNAT7 overexpression line and a knat71 mutant line to Pro4CL1:BLH6 and Pro35S:BLH6 lines. Plants homozygous for blh6 and knat7 mutations but containing and expressing the corresponding overexpression transgene were identified in F2 populations (Supplemental Figure 7). The examination of IF cell wall thicknesses in the inflorescence stems of Pro35S:KNAT7 blh6 plants revealed that the reduced cell wall thickness phenotype observed in Pro35S:KNAT7 plants was repressed in blh6 mutant backgrounds (Figures 7D, 7H, 7L, and 7P, Table 1). Furthermore, the decreases in IF cell wall thickness conferred by the Pro4CL1:BLH6 transgene were repressed in the knat7 mutant background (Figures 7B, 7F, 7J, and 7N, Table 1). Moreover, the striking thinner IF cell walls induced by Pro35S:BLH6 were absent in the knat7 mutant background (Figures 7C, 7G, 7K, and 7O, Table 1). These data suggest that BLH6 manifests its function in repressing IF cell wall deposition through interaction with KNAT7 and vice versa.

Gene Expression Profiling of blh6, knat7, and blh6 knat7 Plants Previous studies indicated increased expression of several genes encoding enzymes involved in secondary cell wall biosynthesis in

knat7 mutant plants (Li et al., 2012). To gain insight into the role played by BLH6 in regulating expression of such genes, we profiled the expression of genes encoding enzymes involved in the biosynthesis of lignin, cellulose, and hemicellulose components of secondary cell walls in blh6, knat7, and blh6 knat7 mutants relative to wild-type inflorescence stems in the basal parts of inflorescence stems. The results shown in Figure 9 indicate that most genes were upregulated in mutants relative to wild-type plants, and these patterns were generally consistent in blh6-1, knat7, and blh6 knat7 mutant backgrounds. Expression of CELLULOSE SYNTHASE (CESA) genes involved in primary cell wall cellulose biosynthesis (CESA1, CESA3, and CESA6) increased slightly, especially CESA3 in all three mutant backgrounds, while there was an increase in expression of two CESA genes associated with secondary cell wall cellulose biosynthesis (CESA7 and CESA8) in all three mutant backgrounds. Two of four hemicellulose biosynthetic genes tested (IRX8 and IRX9) showed increased transcript levels in knat7 blh6 plants but IRX8 expression decreased in the blh6 mutant. However, IRX10 expression was downregulated in blh6, knat7, and blh6 knat7 mutants (Figure 9A). We also profiled expression of a group of 13 genes involved in phenylpropanoid metabolism, lignin monomer biosynthesis, and lignin polymerization. Some genes, including C4H, CCR1, and HCT, showed modest upregulation (1.5- to 2-fold) in blh6, knat7, and blh6 knat7 mutants, while CCoAOMT and LAC4 were modestly upregulated only in the knat7 and blh6 knat7 mutants, and there was evidence for modest downregulation of CAD5 and C3H1 in blh6. Two genes, F5H and PAL2, showed striking patterns of increased expression in all three mutant backgrounds, with 4- to 15-fold higher expression in the mutants. Since a large-scale yeast one-hybrid screen indicated that BLH6

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Table 1. Interfascicular Fiber Cell Wall Thickness in Wild-Type and Mutant Plants Genotype

Cell Wall Thicknessa

Wild type blh6 knat7 knat7blh6 Pro4CL1:BLH6 Pro35S:BLH6 Pro35S:KNAT7 Pro4CL1:BLH6 knat7 Pro35S:BLH6 knat7 Pro35S:KNAT7 blh6

3.70 5.86 5.47 5.47 2.16 1.75 1.93 3.76 5.09 3.26

a

6 6 6 6 6 6 6 6 6 6

0.48 0.83b 0.93b 0.65b 0.41b 0.35b 0.34b 0.56 0.91b 0.47

Mean cell wall thickness in mm 6 SD; n = 50 cells. Significantly different from the wild type; P < 0.01.

b

can bind to the promoter of REV/IFL1, a Class III HD-ZIP transcription factor that regulates vascular patterning and xylem and IF differentiation in Arabidopsis (Gaudinier et al., 2011), we also assayed REV expression in the mutants relative to the wild type. Interestingly, REV exhibited strongly increased expression in all three mutant backgrounds (Figure 9A). Cell Wall Chemistry To assay for quantitative differences in secondary cell wall polymers in mutants relative to wild-type plants, we sampled the basal parts of inflorescence stems of wild-type, blh6, blh6 knat7, and Pro4CL1:BLH6 mutants, the same regions used as the material for gene expression profiling. Analysis of lignin content using the Klason method showed that knat7 and blh6 knat7 loss-of-function mutants had significantly increased lignin content, while a Pro4CL1:BLH6 overexpression line had significantly decreased lignin content (Table 2). No change in lignin content in blh6 was observed. We assayed the levels of sugars derived from cell wall carbohydrates in blh6-1, knat7, blh6 knat7, and Pro4CL1:BLH6 plants by HPLC analysis but observed no differences in these values relative to wild-type plants (Supplemental Table 2). BLH6 and KNAT7 Bind to the REV Promoter and Repress REV Promoter Activity To test the interactions between BLH6/KNAT7 and REV promoter in vivo, we performed chromatin immunoprecipitation (ChIP) using transgenic Arabidopsis plants expressing GFPtagged BLH6 and KNAT7 proteins. The IF and xylary phenotypes of plants overexpressing these constructs were similar to those of plants overexpressing BLH6 and KNAT7 (Supplemental Figures 10E and 10F), indicating that the GFP-BLH6 and GFPKNAT7 fusion proteins are functional. Formaldehyde crosslinked chromatin from 7-d-old seedlings and 4-week-old mature stems was isolated and fragmented. Chromatin fragments from the wild type were used as a negative control. BLH6-GFP-bound and KNAT7-GFP-bound DNA fragments were immunoprecipitated using GFP antibody and were used as templates in the quantitative real-time PCR analysis of REV promoter sequences (regions

REV1-5; Figure 10A). Three regions of the REV promoter were enriched (around 5-fold) compared with the no GFP antibody treatment in chromatin isolated from Pro35S:GFP-BLH6expressing plants (Figure 10B) that had the same phenotype as Pro35S:BLH6 plants described above. The REV2 region around 500 bp upstream of the REV ATG start codon was also enriched in Pro35S:GFP-KNAT7 plants (which had the same phenotype as Pro35S:KNAT7 plants described above; Figure 10C). However, this enrichment was abolished in the blh6 background, in which KNAT7 was overexpressed (Pro35S:GFP-KNAT7 blh6 plants; Figure 10C; Supplemental Figure 7B). To test the effect of BLH6 and KNAT7 binding on REV promoter activity in planta, we cotransformed Pro35S:GFP-BLH6, Pro35S:GFP-KNAT7, or Pro35S effector plasmids with a ProREV:GUS reporter plasmid in Nicotiana benthamiana leaves and measured transient GUS activity. Pro35S:GFP-BLH6 and Pro35S:GFP-KNAT7 expression caused significant decreases in ProREV-directed GUS activity relative the control promoter-only effector (Figure 11), suggesting that BLH6 and KNAT7 bind to the REV promoter and represses REV promoter activity. Taken together, these data suggest that the expression of REV is negatively regulated by the direct binding of BLH6 and KNAT7 to its promoter. BLH6 and KNAT7 Overexpression Lines Phenocopy the rev IF Phenotype To further test the apparent roles of BLH6 and KNAT7 as REV repressors, we assayed REV expression in Pro35S:GFP-BLH6 and Pro35S:GFP-KNAT7 lines. Supplemental Figure 9 shows that REV expression was repressed in both overexpression backgrounds. Cross sections taken 3 cm from the bases of 6week-old wild-type and rev-5 inflorescence stems revealed a decrease in cell wall thickness of IF relative to the wild type (Supplemental Figures 10A and 10B). This phenotype is similar to previous observations of the rev-9 allele (Lev-Yadun et al., 2005) and to Pro35S:BLH6 and Pro35S:KNAT7 IF cell wall phenotypes (Figures 7C and 7D). Furthermore, ifl1 alleles of rev have been reported to lack xylary fibers (Zhong and Ye, 1999). We observed this phenotype again in rev-5 (Supplemental Figures 10B and 10G). Inspection of vascular bundles of Pro35S:BLH6 and Pro35S:KNAT7 indicated evident decline of xylary fiber number (Supplemental Figures 10C, 10D, and 10G), further supporting a direct role for BLH6 and KNAT7 in repressing REV expression. Phenotypic Analysis of rev blh6 and rev knat7 Double Mutants To confirm that REV works downstream of BLH6 and KNAT7, and to determine which aspects of the blh6 and knat7 phenotypes depend on REV function, we generated blh6 rev and knat7 rev double mutants and compared the inflorescence stem phenotypes of the double mutants relative to each single mutant and the wild type. The IF cell wall thicknesses of blh6 and knat7 stems were both thicker than the wild type, and the IF cell wall thickness of rev stems was thinner than the wild type, as previously observed (Figure 12; Supplemental Table 3). However, both rev blh6 and rev knat7 mutants demonstrated a remarkable


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Figure 7. Inflorescence Stem Secondary Cell Wall Phenotypes of BLH6 and KNAT7 Overexpression Lines in Wild-Type, knat7, and blh6 Mutant Backgrounds. Stem cross sections were taken from the bases of the inflorescence stems of 6-week-old plants, stained with Toluidine blue, and examined for alterations in secondary wall thickness. Bars = 30 mm. (A) to (D) Analysis of BLH6 and KNAT7 overexpression lines relative to wild-type control showing vascular bundles and interfascicular fibers. (I) to (L) Analysis of BLH6 and KNAT7 overexpression lines relative to wild-type control showing interfascicular fiber region. (A) and (I) Cross section from a wild-type control plant. (B) and (J) Cross section from a Pro4CL1:BLH6 plant. (C) and (K) Cross section from a Pro35S:BLH6 plant. (D) and (L) Cross section from a Pro35S:KNAT7 plant. (E) to (H) Analysis of Pro4CL1:BLH6 knat7, Pro35S:BLH6 knat7, and Pro35S:KNAT7 blh6 plants relative to the wild type showing vascular bundles and interfascicular fibers. (M) to (P) Analysis of Pro4CL1:BLH6 knat7, Pro35S:BLH6 knat7, and Pro35S:KNAT7 blh6 plants relative to the wild type showing interfascicular fiber region. (E) and (M) Cross section from a wild-type control plant. (F) and (N) Cross section from a Pro4CL1:BLH6 knat7 plant. (G) and (O) Cross section from a Pro35S:BLH6 knat7 plant. (H) and (P) Cross section from a Pro35S:KNAT7 blh6 plant.

decrease in IF secondary wall thicknesses relative to wild-type, blh6, and knat7 stems, phenocopying the rev single mutant in this regard (Figure 12; Supplemental Table 3). Interestingly, examination of vascular bundles from both rev blh6 and rev knat7

inflorescence stems revealed mild irx phenotypes, phenocopying the blh6 and knat7 single mutants in this regard (Figure 12). In addition, rev blh6 and rev knat7 vascular bundles exhibited the rev phenotype of decreased numbers of xylary fibers relative

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developmental regulator and activator of IF secondary wall biosynthesis, is a direct target of BLH6 and KNAT7. Repression of REV expression by BLH6 and KNAT7 thus appears to constitute a negative feedback loop that can modulate commitment to secondary wall biosynthesis. Interaction between KNAT7 and BLH6

Figure 8. Morphological Differences between Mature Pro35S:BLH6 and Wild-Type Plants. (A) Representative 6-week-old wild-type and Pro35S:BLH6 plants. (B) Representative 4-week-old Pro35S:BLH6 plant.

to wild-type, blh6, and knat7 vascular bundles (Supplemental Figure 11). These data are consistent molecular data indicating REV is a direct target of BLH6 and KNAT7 and support a model by which BLH6 and KNAT7 modulate commitment to IF secondary cell wall synthesis by repression of REV expression. However, functions of BLH6 and KNAT7 in vessel cell secondary wall formation seem to be independent of REV.

DISCUSSION TALE homeodomain transcription factors are important regulators of many developmental processes and are known to form homo- and heterodimer complexes that regulate target gene expression (Bellaoui et al., 2001; Hake et al., 2004; Hackbusch et al., 2005; Hay and Tsiantis, 2010). The functions of several KNOX-BLH transcription factor complexes have been reported (reviewed in Hake et al., 2004; Hay and Tsiantis, 2010), but there are little data on such complexes associated with the regulation of secondary cell wall development. Here, we report that in Arabidopsis inflorescence stems, BLH6 is a regulator of secondary cell wall formation and that BLH6 functions together with KNAT7 to form a KNAT7-BLH6 complex that functions as a repression module in secondary cell wall formation. We also show that the HD-ZIP III transcription factor REV/IFL1, a pleiotropic

It is well established that members of the BLH family of TALE homeodomain proteins in plants selectively interact with KNOX TALE homeodomain proteins to form heterodimers (Bellaoui et al., 2001; Hake et al., 2004; Hackbusch et al., 2005; Hay and Tsiantis, 2010; Li et al., 2011). Functional investigations into the BLH partners of Arabidopsis KNOX proteins have largely focused on Class 1 KNAT proteins, such as STM, BP, KNAT2, and KNAT6, which play roles in developmental regulation of shoot apical meristems and inflorescence architecture (Smith and Hake, 2003; Hay and Tsiantis, 2010). Our findings extend this information to include a BLH6-KNAT7 heterodimer that regulates secondary cell wall biosynthesis. Even though the protein sequence of BLH7 shares 68% similarity with BLH6, we were unable to detect a BLH7-KNAT7 interaction in planta and in yeast. Similarly, BLH10 was unable to interact with KNAT7 in our assays, despite the fact that BLH6 and BLH10 expression is upregulated within 6 h of MYB46 induction, together with KNAT7 (Ko et al., 2009). Given the overlapping expression patterns and sequence similarity of BLH6, BLH7, and BLH10, it is possible that BLH7 and BLH10 act redundantly with BLH6 to regulate other aspects of developmental regulation, via common BLH6, BLH7, and BLH10 interacting KNAT proteins other than KNAT7 in the network of possible interactions (Hackbusch et al., 2005). Previous work showed that KNAT7 functions as a strong transcriptional repressor (Li et al., 2012), and BLH6 behaves in a very similar manner (Figure 3). Furthermore, BLH6-KNAT7 interaction enhances the individual repression activities of each transcription factor (Figure 3B). Thus, while we cannot exclude the possibility that BLH6 functions to activate transcription by heterodimerization with other KNAT proteins, the BLH6-KNAT7 complex appears to have potent repression activity. Such repression activity has been observed in other BLH-KNOX complexes (Chen et al., 2004; Hay and Tsiantis, 2010). Expression data indicate that KNAT7 and BLH6 are coexpressed in the same cell types (Figure 4; Supplemental Figure 4), suggesting that in planta interaction between KNAT7 and BLH6 can occur, as observed in yeast and protoplasts (Figures 1 and 2). Interestingly, ProBLH6:GUS shows an expanded expression pattern relative to that of ProKNAT7:GUS (Li et al., 2012), with expression present throughout cotyledons and mature leaves (Supplemental Figure 4). In contrast, GUS expression specified by the KNAT7 promoter is restricted to the vascular system (Li et al., 2012). This suggests that BLH6 may play additional roles in plant development via interaction with other KNOX partners where KNAT7 is not expressed. Consistent with this, ectopic overexpression of BLH6 under the control of the 35S promoter resulted in pleiotropic growth effects (Figure 8; Supplemental Figure 8) not observed in plants overexpressing BLH6 under the control of the 4CL1 secondary cell wall-specific promoter or in


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Figure 9. Expression of Secondary Wall Biosynthetic Genes blh6-1, knat7, and blh6/knat7 Plants Relative to the Wild Type. RNA was isolated from the basal 8 cm of inflorescence stems taken from 6-week-old plants of the genotypes indicated (blh6, knat7, and blh6 knat7) for quantitative RT-PCR analysis of gene expression. Values are given relative to the wild-type levels, arbitrarily set at 1. (A) Expression of cellulose and hemicellulose wall biosynthetic genes and REV. CELLULOSE SYNTHASE (CESA) genes CESA1, CESA3, and CESA6 have been predominantly associated with primary cell wall cellulose deposition, while CESA4, CESA7, and CESA8 have been associated with secondary cell wall cellulose deposition. The FRAGILE FIBER8 (FRA8) and IRREGULAR XYLEM8 (IRX8), IRX9, IRX10, and IRX9L encode glycosyltransferases implicated in xylan biosynthesis. REV encodes a transcription factor that regulates IF cell wall formation. (B) Expression of lignin biosynthetic genes, including PHENYLALANINE AMMONIA LYASE (PAL1, PAL2, and PAL4); CINNAMATE-4-HYDROXYLASE (C4H); 4-COUMARATE-COA LIGASE (4CL1); HYDROXYCINNAMOYL-COA:SHIKIMATE HYDROXYCINNAMOYLTRANSFERASE (HCT); P-COUMARATE 39-HYDROXYLASE1 (C3H1), CAFFEOYL-COA O-METHYLTRANSFERASE1 (CoAOMT1); CINNAMOYL-COA REDUCTASE1 (CCR1); CAFFEIC ACID OMETHYLTRANSFERASE1 (COMT1); CINNAMYL ALCOHOL REDUCTASE5 (CAD5); and FERULATE-5-HYDROXYLASE (F5H); LACCASE4 (LAC4). Error bars represent standard deviations of three biological replicates.

plants overexpressing KNAT7 under the control of the 35S promoter. Genetic Analysis Reveals Diverse BLH6 Functions Analysis of blh6 mutants revealed thicker IF secondary cell wall and mild irx phenotypes, which phenocopy knat7. A test of

genetic interaction between BLH6 and KNAT7 using a blh6 knat7 double mutant revealed that IF cell wall thickness is similar in both single mutants and the blh6 knat7 double mutant (Figure 7), supporting the interpretation that BLH6 and KNAT7 work as a complex in IF cell wall deposition, in which case the loss of function of either one would lead to a defective complex. However, the blh6 knat7 double mutant also displayed

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Table 2. Lignin Content in the Lower Stems of Wild-Type and Mutant Plants Lignin Contenta Insoluble Wild type blh6 knat7 blh6 knat7 Pro4CL1:BLH6

17.5 17.5 19.7 20.2 14.8

6 6 6 6 6

0.5b 1.5 1.4 0.1 0.9

overexpression could target a similar set of genes involved in cell wall biosynthesis and modification. Gene Expression Phenotypes

Soluble 3.1 2.9 2.4 3.9 2.8

6 6 6 6 6

0.2 0.1 0.1 0.2 0.1

Total 20.6 20.4 22.1 24.1 17.6

6 6 6 6 6

0.1 0.5 0.1c 0.1c 1.0c

a

Klason lignin, mg per 100 mg dry weight. SD of measurements taken from three biological replicates. c Significantly different from the wild type; P < 0.05. b

a strikingly enhanced irx phenotype. This additive phenotype indicates that the roles of BLH6 and KNAT7 are not identical and are only partially overlapping, particularly with respect to vessel secondary cell wall development. Partially distinct roles for the BLH6 and KNAT7 TALE homeodomain proteins could be explained by the complexity of potentially different interacting partners. For example, KNAT7 has been shown to interact with OFP4 to regulate vessel cell wall biosynthesis (Li et al., 2011), and BLH6 also has the potential to form heterodimers with other Arabidopsis KNOX and BLH proteins, as well as OFP proteins (Hackbusch et al., 2005). Thus, loss of both BLH6 and KNAT7 functions could affect both the formation of functional BLH6-KNAT7 heterodimers and the formation of other complexes that play related but distinct roles in vessel development and cell wall formation. Consistent with the partially distinct functions of BLH6 and KNAT7, the lignin content of a blh6 knat7 double mutant is higher than that of the corresponding single mutants (Table 2), and only the knat7 single mutant had higher lignin content than the wild type (Table 2) as previously reported (Li et al., 2012). Collectively, these correlations are consistent with a model in which these two transcription factors act together as a heterodimer to negatively modulate secondary cell wall formation in the interfascicular region during stem vasculature development but have other nonoverlapping roles in other aspects of cell wall formation. A striking effect of ectopic overexpression of BLH6 under the control of the 35S promoter was on rosette and reproductive stage plant size and organ number, which were greatly reduced (Supplemental Figure 8). Interestingly, targeting BLH6 overexpression in cells committed to secondary cell wall development using the 4CL1 promoter (active during the lignification phase of secondary cell wall development) eliminated such pleiotropic growth effects. The most likely explanation for the phenotypic effect of Pro35S:BLH6 expression is its interaction with other partners in a TALE protein interaction network (Hackbusch et al., 2005) leading to activation or repression of target genes involved in growth and development. Interestingly, overexpression of BLR/ PNY/BLH9 under the control of the 35S promoter in Arabidopsis leads to growth defects that share similarity to those we observed in Pro35S:BLH6 plants, as well as defects in xylem cell differentiation (Etchells et al., 2012). Since BLH6 and BLR/PNY/BLH9 may share interaction partners (Hackbusch et al., 2005), their

We profiled gene expression in mutants relative to the wild type to reveal direct or indirect targets of BLH6 and KNAT7. The expression of a suite of secondary cell wall biosynthetic genes was upregulated in inflorescence stems in the blh6 background relative to wild-type plants, and a similar but not identical pattern of gene expression changes was observed in the knat7 and blh6 knat7 backgrounds (Figure 11; Li et al., 2012). This suggests deregulation of secondary cell wall biosynthesis in the mutants, consistent with roles for BLH6 and KNAT7 as repressors of commitment to cell wall biosynthesis. Genes involved in lignin biosynthesis were consistently upregulated in blh6, and expression patterns were generally consistent in the blh6, knat7, and blh6 knat7 backgrounds. However in some cases, the knat7 and/or blh6 knat7 expression levels were higher than blh6 (e.g., F5H and LAC4). While these data are broadly suggestive of

Figure 10. ChIP Analysis of Recruitment of BLH6 and KNAT7 to the REV Promoter. (A) Locations of the PCR fragments. PCR primers were designed to amplify REV promoter regions REV1-REV5 as indicated by dashed lines. (B) Enrichment of BLH6-bound fragments to regions REV1-5 of the REV promoter in Pro35S:GFP-BLH6 plants using anti-GFP antibodies. (C) Enrichment of KNAT7-bound fragments to regions REV1-5 of the REV promoter in Pro35S:GFP-KNAT7 and Pro35S:GFP-KNAT7 blh6 plants. Enrichment was abolished in the blh6 background.


Figure 11. BLH6 and KNAT7 Repress REV Promoter Activity in Tobacco. (A) Diagram of the effector and reporter constructs used for transient expression assays in N. benthamiana. (B) BLH6 and KNAT7 repress REV promoter activity in vivo. BLH6 or KNAT7 was coexpressed with the GUS reporter gene driven by the REV promoter in tobacco leaves shown in (A). Activation of the REV promoter by BLH6 or KNAT7 was measured by assaying GUS activity 2 h after transfection. The expression level of the GUS reporter gene in the leaves transfected with no effector was used as control and was set to 100. Error bars indicate the standard deviations; n = 9 transfections from three independent replicates.

deregulation of cell wall biosynthesis in the blh6, knat7, and blh6 knat7 backgrounds, they also suggest a complex regulatory effect on commitment to cell wall biosynthesis within and between mutants and suggest that in some cases, blh6 and knat7 could play distinct roles, possibly by interacting with alternative KNOX or BLH partners in different cell types. Also, there may be cell- and tissue-type differences in cell wall composition and target gene expression in the mutant backgrounds, which would confound the results obtained using whole stem sections for expression and cell wall biochemistry analysis. REV Is a Direct Target of BLH6 and KNAT7 Of particular note is the strong upregulation of REV/IFL1 expression in blh6, knat7, and blh6 knat7 inflorescence stems (over 20-fold relative to the wild type; Figure 9). REV is one of five Arabidopsis Class III HD-ZIP transcription factors involved in embryo, shoot, root, and vascular patterning (Brandt et al., 2012; reviewed in Schuetz et al., 2013). In addition to playing an important role in regulating IF and xylem cell differentiation in

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Arabidopsis (Zhong et al., 1997; Zhong and Ye, 1999; Ratcliffe et al., 2000; Ohashi-Ito et al., 2005), REV has been associated with radial patterning in the stem (Emery et al., 2003; Schuetz et al., 2013). The known targets of Arabidopsis REV and its homolog in Z. elegans, HB-12, which may be relevant to secondary wall formation, include other HD-ZIP III transcription factor genes, and lignin biosynthesis, auxin biosynthesis, and BR signaling pathway-related genes (Ohashi-Ito et al., 2005; Brandt et al., 2012; Reinhart et al., 2013). In support of REV as a direct target of BLH6, a large-scale yeast one-hybrid assay revealed that BLH6 binds to the REV promoter (Gaudinier et al., 2011). We used ChIP to confirm BLH6 and KNAT7 binding to the REV promoter in vivo. This analysis revealed multiple regions of the REV promoter with evidence for BLH6 binding in vivo (Figure 10), one of which also had evidence for KNAT7 binding. Interestingly, KNAT7 binding to the latter region (REV2; Figure 10) required BLH6, since it was absent in the blh6 mutant background. This provides evidence for KNAT7BLH6 heterodimer binding to this region of the promoter. These data also indicate BLH6 may bind to the REV1 and REV3 regions of the REV promoter with interaction partners other than KNAT7, which could modulate REV transcription levels and affect other phenotypes conditioned by REV, such as leaf and vascular patterning (Brandt et al., 2012; Schuetz et al., 2013). It has been reported that BLH and KNOX proteins bind to similar DNA sequences with a TGAC core motif (Krusell et al., 1997; Smith et al., 2002; Tioni et al., 2005; Viola and Gonzalez, 2009). Within the REV promoter, there are several TGAC sites in the regions to which KNAT7 and/or BLH6 were associated by ChIP assays, representing candidate binding sites for BLH6 and KNAT7. The functional consequences of BLH6 and KNAT7 recruitment to the REV promoter were assessed by transient expression assays in N. benthamiana, which showed that BLH6 and KNAT7 not only bind to the REV promoter but also downregulate REV promoter activity (Figure 11). Consistent with these data, plants overexpressing BLH6 or KNAT7 have reduced REV transcript levels in inflorescence stems (Supplemental Figure 9). Furthermore, the increased IF cell wall thickness phenotype of blh6 and knat7 mutants was suppressed in the rev mutant background (Figure 12; Supplemental Table 3), and BLH6 and KNAT7 expression patterns in inflorescence stems inferred from GUS and GFP reporter gene fusion (Figure 4; Supplemental Figure 4) and in situ hybridization data for KNAT7 (Zhong et al., 2008) indicate that BLH6 and KNAT7 share similar expression patterns that overlap with REV expression in inflorescence stems (described in Zhong and Ye, 1999). The similarity between the inflorescence stem phenotypes of plants overexpressing BLH6 or KNAT7 (Figure 7) and inflorescence stem phenotypes of rev loss-of-function mutants (Supplemental Figure 10; Zhong et al., 1999; Lev-Yadun et al., 2005), including reduced commitment to secondary cell wall deposition in interfascicular fibers and reduced xylary fiber number per vascular bundle, is also striking. Collectively, these data support a model in which BLH6 and KNAT7, both activated as part of the transcriptional network that positively regulates the biosynthesis of secondary cell wall components (Demura and Ye, 2010; Schuetz et al., 2013), directly target REV for negative regulation, thereby generating a negative feedback loop.

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Figure 12. Inflorescence Stem Secondary Cell Wall Phenotypes in blh6 rev and knat7 rev Mutants. Stem cross sections were taken 3 cm from the bases of the inflorescence stems of 6-week-old plants, stained with phloroglucinol, and examined for alterations in anatomy and secondary wall thickness. Arrows indicate irx vessel cells. Bars = 50 mm. (A) and (G) Representative cross sections from wild-type control stems showing vascular bundle (A) and interfascicular fiber (G) regions of the stem. (B) and (H) Representative cross sections from a blh6 stem showing the vascular bundle region with the irx phenotype (B) and interfascicular fiber region with thicker secondary cell walls relative to the wild type (H). (C) and (I) Representative cross sections from a knat7 stem showing the vascular bundle region with the irx phenotype (C) and interfascicular fiber region with thicker secondary cell walls relative to the wild type (I). (D) and (J) Representative cross section from a rev-5 stem showing the vascular bundle region (D) and interfascicular fiber region with thinner secondary cell walls relative to the wild type (J). (E) and (K) Representative cross section from a rev blh6 stem showing the vascular bundle region with the irx phenotype (E) and interfascicular fiber region (K) with thinner secondary cell walls relative to the wild type and blh6. (F) and (L) Representative cross section from a rev knat7 stem showing the vascular bundle region with the irx phenotype (F) and interfascicular fiber region (L) with thinner secondary cell walls relative to the wild type and knat7.

REV plays multiple and pleiotropic roles in plant development, including tissue patterning, secondary wall formation, and vascular cambium development (Robischon et al., 2011; Schuetz et al., 2013; Porth et al., 2014) and has recently been shown to play a role in modulating plant development according to light availability by interacting with light signaling pathways to mediate the shade avoidance response (Brandt et al., 2012). These findings support a role for REV in modulating plant development in response to environmental inputs. While posttranscriptional

regulation of REV expression by miRNAs is well established, there is little information on transcriptional regulation of REV particularly in response to environmental cues (Brandt et al., 2012). Our data showing that BLH6 and KNAT7 target REV for negative transcriptional regulation provide one potential mechanism for fine-tuning of REV transcript levels in accordance with environmental cues. Since REV seems to achieve many of its apparently disparate functions by directly activating auxin biosynthetic, transport, and signaling gene transcription (Brandt


et al., 2012; Reinhart et al., 2013), REV downregulation could temper the auxin response, affecting the commitment to secondary wall formation. While it is clear that BLH6 and KNAT7 are likely to play complex regulatory roles, some of which are independent of REV (such as regulation of xylem vessel wall properties; Figure 12), we favor the hypothesis that one function of these regulators, acting through REV as a direct target, is to temper the strong developmental commitment to secondary wall formation activated by the feed-forward NAC and MYB domain transcription factor network. Such regulation in accordance with the availability of light or other resources may be required to maintain metabolic homeostasis in conjunction with the large metabolic commitment to secondary cell wall biosynthesis. METHODS Plant Material and Growth Conditions Arabidopsis thaliana ecotype Columbia-0 was used as the wild type throughout, and all mutants and transgenic lines are in this background. For seedlings used for phenotypic and genotypic analysis, seeds were surface-sterilized by 75% ethanol and grown on Murashige and Skoog basal salts with minimal organics (Sigma-Aldrich) and 1% sucrose, solidified with 0.7% (w/v) agar (Sigma-Aldrich). Seeds were cold-treated at 4°C in darkness for 48 h, then moved to 22°C under a 16/8-h (light/dark cycle) photoperiod and constant white light at ;120 mmol m22 s21 for seed germination and seedling growth for 7 to 10 d. Plants designated for further analysis were grown on soil under long-day conditions (16/8-h light/dark cycle) at 100 mmol m22 s21, 22°C, and 55% humidity. T-DNA insertion mutant lines for BLH6 were obtained from the ABRC (http://arabidopsis.org). The presence of the T-DNA insertion was examined by PCR using gene-specific primers (59-TGGGGAACAACAAGATGGAT-39 and 59-TCAAGCTACAAAATCATGTACC-39 for blh6-1 and blh6-2) and T-DNA left border LBb1.3 (59-ATTTTGCCGATTTCGGAAC-39). Lines were genotyped to select homozygotes at the insertion site by PCR amplification using a combination of gene-specific primers and T-DNA primer and transcriptional levels were tested by quantitative RT-PCR using oligos (59-ATGGAGAATTATCCAGAAACA-39 and 59-ATTTATGAACCAGTTTGAAAC-39 for the first three exons of blh6 and 59-ATGGAGAATTATCCAGAAACA-39 and 59-TCAAGCTACAAAATCATGTACC-39 for blh6). The knat7-1 allele (Li et al., 2012) was used for all knat7 phenotypic and genetic analyses, and knat7-1, blh6-1, and rev-5 alleles (Otsuga et al., 2001) were used to generate double mutants. Total RNA Isolation and Quantitative RT-PCR Analysis Total RNA from bases of inflorescence stems of 6-week-old Arabidopsis plants of the appropriate genotypes was extracted using TRIZOL reagent (Invitrogen, Life Technologies) following the manufacturer’s instructions. Two micrograms of total RNA from each sample, treated with RNase-free DNase (Qiagen) RNA was used for reverse transcriptase synthesis using the Omniscript RT kit (Qiagen) according to the manufacturer’s instructions. For quantitative RT-PCR of the bases of inflorescence stems, PCR amplification was performed with gene-specific primers (Supplemental Table 5), and ACTIN2 was used as the control gene. Quantitative RT-PCR was performed using IQ SYBR Green Supermix (Bio-Rad) and amplified using a CFX Connect real-time system (Bio-Rad) according to the manufacturer’s protocol. Differences in gene expression, expressed as the fold change relative to the control, were calculated as described previously (Bhargava et al., 2010). Each measurement was performed as three replicates and was repeated in three biological

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replicates (independent pools of inflorescence stems from the appropriate genotypes). Cloning and Plant Transformation To generate BiFC constructs, clones of the complete open reading frames of BLH1, BLH5, BLH6, BLH7, BLH10, ATH1, and KNAT7 were isolated from cDNA prepared from Arabidopsis inflorescence stem mRNA as previously described. The clones of BLH candidates were transferred to the Gateway compatible destination vector pSAT4-DEST-n(174)EYFP-C1 (Citovsky et al., 2006) by LR-mediated recombination, to generate fusions to the N-terminal half of YFP. KNAT7 was cloned into pSAT5-DEST-c(175end) EYFP-C1 (Citovsky et al., 2006) to generate fusions to the C-terminal half of YFP. BLH6 and KNAT7 inserts were also cloned into pSAT6-EYFPN1 (Citovsky et al., 2006) to generate C-terminal fusions to full-length YFP. To generate the Pro35S:BLH6 and Pro35S:KNAT7 constructs, the fulllength open reading frames of BLH6 and KNAT7 were amplified by PCR from cDNA prepared from Arabidopsis inflorescence stem. PCR products were subcloned into pCR8⁄GW⁄TOPO using LR Clonase (Life Technologies) into the binary vector pEarley Gate 100. To generate the Pro35S: GFP-BLH6 and Pro35S:GFP-KNAT7 constructs, the TOPO entry vector BLH6 and KNAT7 open reading frame inserts described above were cloned into the binary vector pEarley Gate 104. The ProKNAT7:GFP construct was generated by recombining a 2.5-kb fragment of the KNAT7 promoter and ER-GFP into the Gateway-compatible vector pK7m24GW using LR Clonase II. KNAT7 promoter sequences and primers are described by Brady et al. (2011). Plant expression constructs were introduced into Agrobacterium tumefaciens strain GV3101 and transformed into Arabidopsis Columbia-0 (Col-0) using the floral dip method (Clough and Bent, 1998) to generate transgenic plants. Yeast Two-Hybrid and BiFC Assays The ProQuest Two-Hybrid System (Invitrogen) was used as described previously (Guo et al., 2009). KNAT7, KNOX1 domain, KNOX2 domain, MEINOX domain, and homeodomain were cloned into prey vector (pDEST22), and BLH6 (and BLH6 domains and BLH7) were cloned into bait vector (pDEST32) (see Supplemental Table 4 for primers used). The known interaction between MYB75 and TT8 (Zimmermann et al., 2004) was used as a positive control. Coexpression of BLH6 and BLH7 and the empty bait vector was used as a negative control. The ability of yeast transformants to grow on minimal SD (synthetic dextrose) medium lacking both leucine and tryptophan is indicative of the presence of both prey and bait constructs. Positive interactions were identified by their ability to activate URA3, or LacZ genes, assayed by the appearance of yeast colonies on triple selection minimal SD medium lacking leucine, tryptophan, and uracil or by blue color when assayed with X-Gal (5- bromo-4chloro-3-indolyl-b-D-galactopyranoside). For BiFC assays, Arabidopsis leaf mesophyll protoplast cells were isolated, transfected, and imaged as described previously (Wang et al., 2007; Li et al., 2011). In brief, protoplasts were isolated from rosette leaves of 3-week-old plants. Constructs prepared as described above were transfected into protoplasts and incubated in the dark for 18 to 20 h to allow expression of the introduced genes. The YFP fluorescence was examined and photographed using an Olympus AX70 light microscope. Microscopy Freshly harvested inflorescence stems were hand-sectioned with the use of razor blades. Sections were stained directly on the slide in a drop of aqueous 0.02% Toluidine blue O (Sigma-Aldrich) for 1 min and rinsed in water or stained with 1% phloroglucinol (w/v) in concentrated HCl for 5 min and mounted in a drop of 50% glycerol beneath a cover slip and examined immediately with an Olympus AX70 light microscope.

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Tissue embedding for light and transmission electron microscopy was performed as described by Li et al. (2011). Samples were taken 3 cm from the bases of inflorescence stems from 8-week-old plants. The measurements of the secondary cell wall thickness were determined by examining 50 separate interfascicular fiber cells (excluding cells at the margins of the interfascicular regions) in light micrograph images from 1-mm inflorescence stem cross sections of wild-type, blh6-1, blh6 knat7, Pro35S:BLH6, Pro35S:KNAT7, Pro4CL1:BLH6, Pro35S:BLH6 knat7, Pro35S:KNAT7 blh6, and Pro4CL1:BLH6 knat7, rev, blh6 rev, and knat7 rev inflorescence stem bases. Using ImageJ (http://imagej.nih.gov/ij/), three measurements per cell were taken across the cell walls from the middle lamellae inwards toward the cell centers. The average of the three measurements was taken as the value for that cell. Confocal Imaging For live-cell imaging, individual stem sections were immersed in a drop of water and sealed beneath a thickness #1.5 cover slip (Fisher Scientific) in a small Petri plate. Intrinsic lignin fluorescence and gene-specific fluorescence (ProKNAT7:GFP) on live stem tissues were imaged using an Olympus FV1000MPE multiphoton laser scanning microscope connected to a Mai Tai Deepsee laser. A GFP excitation/emission filter (488/525) was used to look at the gene-specific fluorescence, and a 49,6-diamidino-2phenylindole excitation/emission filter (350/460) was used to look at the intrinsic lignin fluorescence. Samples were imaged using a Leica water immersion 403 objective (LUMPLFLN40XW) with a numerical aperture of 0.8. Images were captured with Olympus Fluoview version 3.1. Transcription Activity Assays Arabidopsis leaf mesophyll protoplast cells were isolated and transfected as described previously (Wang et al., 2007). Transactivators LD-VP16 and GD-VP16, and reporters LexA-GAL4:GUS and GAL4:GUS were obtained from Shucai Wang (Wang et al., 2007). All reporter and effector plasmids used in transfection assays were prepared using the EndoFree Plasmid Maxi Kit (Qiagen). Ten micrograms of effector and reporter plasmid DNA described above were transfected into protoplasts and incubated in the dark for 20 to 22 h. After incubation, cells were centrifuged at 180g for 3 min, and the supernatant was removed. The cells were resuspended in 100 mL 13 cell culture lysis reagent (Promega) and immediately followed with 4methylumbelliferyl-b-D-glucuronide assay as described previously (Wang et al., 2007). All transfection assays were performed as three replicates, and assays were repeated on at least three separate occasions. GUS activity was assayed after protoplasts had been incubated in darkness for 20 to 22 h. GUS Expression Assays A 2.3-kb Arabidopsis genomic DNA fragment, 59 to the BLH6 translation start site, was amplified by PCR using gene-specific primers and was cloned into the Gateway-compatible binary vector pMDC163 (Curtis and Grossniklaus, 2003) to create ProBLH6:GUS. An Arabidopsis genomic DNA fragment that contains all exons and introns plus 2.5-kb flanking 59 sequence of KNAT7 was amplified by PCR using gene-specific primers and was cloned into pMDC163 to create ProKNAT7-KNAT7:GUS. Following Agrobacterium-mediated transformation, six independent ProBLH6:GUS and ProKNAT7-KNAT7:GUS transgenic lines were analyzed. The GUS activity was assayed histochemically as described by Li et al. (2011). Chemical Analysis Lignin content was determined by a modified Klason method (Coleman et al., 2008), and sugars were analyzed as described previously (Bhargava et al., 2010).

ChIP-RT-PCR Plants expressing Pro35S:GFP-BLH6, Pro35S:GFP-KNAT7, and Pro35S: GFP-KNAT7 blh6 with high GFP fluorescence for BLH6 and KNAT7 were selected for immunoprecipitation. Main stems of 5-week-old plants were fixed in 1% formaldehyde and washed three times. Tissue was frozen in liquid nitrogen and ground, and immunoprecipitation was performed according to Lee et al. (2007). Immunoprecipitation was performed with anti-GFP antibody (Roche). Primers were designed to amplify REV promoter regions (Supplemental Table 4). Primers were tested as described above for the RT- PCR experiments with Col-0 genomic DNA. Immunoprecipitation was performed for three biological replicates with two technical replicates each for each primer set per GFP line and with wildtype plants as a control with the same number of biological and technical replicates. Transient Protein-DNA Interaction Detection in Tobacco Leaves A 2.5-kb Arabidopsis genomic DNA fragment, 59 to the REV translation start site, was amplified by PCR using gene-specific primers and was cloned into the Gateway-compatible binary vector pGWB3 (Nakagawa et al., 2007) to create reporter ProREV:GUS. Agrobacterium strain GV3103 carrying effector (Pro35S:GFP-BLH6 or Pro35S:GFP-KNAT7), ProREV:GUS, and p19 silencing inhibitor were grown in Luria-Bertani media with 25 mg/L rifampicin and 50 mg /L kanamycin and suspended in infiltration buffer (10 mM MES, pH 5.7, containing 10 mM MgCl2 and 200 mM acetosyringone) to an OD600 of 0.7:0.7:1, respectively. The cultures were hand-infiltrated using a 1-mL syringe into 3-week-old Nicotiana benthamiana leaves. Leaf samples were harvested 48 h after infiltration and assayed for GUS activity. Approximately 100 mg of tissue was frozen and ground in liquid nitrogen. Ground tissue was then mixed with 200 mL GUS extraction buffer (50 mM NaHPO4, pH 7.0, 10 mM bmercaptoethanol, 10 mM Na2 EDTA, 0.1% SDS, and 0.1% Triton X-100). After centrifugation for 10 min (12,000g) at 4°C, a quantitative 4methylumbelliferyl-b-D-glucuronide fluorescent assay for GUS determination was performed using 50 mg of protein/sample in 100 mL of GUS assay buffer (1 mM 4-methylumbelliferyl-b-D-glucuronide [SigmaAldrich] in extraction buffer). A 50-mL aliquot was removed immediately and added into 2 mL of stop buffer (0.2 M sodium carbonate) to be used as control. The rest of the mixture was covered in aluminum foil and incubated at 37°C. Reactions were stopped at different time points by transferring 50 mL to a tube with 450 mL of stop buffer (0.2 M Na 2CO3). 4Methylumbelliferone fluorescence was determined using an Infinite 200 Pro-series reader (excitation at 365 nm; emission at 455 nm). Protein concentration of tissue homogenates was determined with Bradford reagent (Bio-Rad). Relative binding of the transcription factors to the promoter bait sequences was determined relative to the GUS control. All transient assays were performed with three biological replicates with three technical replicates each.

Accession Numbers Sequence data from this article can be found in the Arabidopsis Genome Initiative database under the following accession numbers: ACTIN2 (At3g18780), BLH6 (At4Gg34610), KNAT7 (At1g62990), BLH7 (At2g16400), BLH10 (At1g19700), BLH5 (At2g27220), ATH1 (At4g32980), BLH1 (At2g35940), and REV (At5g60690). Supplemental Data The following materials are available in the online version of this article. Supplemental Figure 1. Schematic Diagrams of Fusion Proteins Used for Bimolecular Florescence Complementation.


Supplemental Figure 2. BLH6 Nuclear Localization in Transgenic Lines Expressing Pro35S:GFP-BLH6.

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Received June 4, 2014; revised November 4, 2014; accepted November 17, 2014; published December 9, 2014.

Supplemental Figure 3. Test of the Transcriptional Activity of BLH6 Supplemental Figure 4. Histochemical Localization of BLH6 and KNAT7 Promoter Activity Supplemental Figure 5. Schematic Diagram of BLH6 Gene Structure and the Position of T-DNA Insertions Supplemental Figure 6. Anatomy of the Vascular Bundles and Interfascicular Regions of Wild-Type and blh6-2 Inflorescence Stems. Supplemental Figure 7. Analysis of BLH6 and KNAT7 Expression Driven by 35S or 4CL1 Promoters in Wild-Type and blh6 and knat7 Mutant Backgrounds. Supplemental Figure 8. Quantitative Measurement of Morphological Differences between Pro35S:BLH6 and Wild-Type Plants. Supplemental Figure 9. REV Expression Is Repressed in BLH6 and KNAT7 Overexpression Backgrounds. Supplemental Figure 10. The Vascular Bundles of Pro35S:KNAT7, Pro35S:GFP-KNAT7, Pro35S:BLH6, and Pro35S:GFP-BLH6 Phenocopy Those of the rev-5 Mutant. Supplemental Figure 11. Xylary Fiber Cell Numbers in the Vascular Bundles of Wild-Type (Col-0), rev, rev blh6, and rev knat7 Lines. Supplemental Table 1. Summary of Bimolecular Florescence Complementation Assays with BLH Candidates and KNAT7. Supplemental Table 2. Cell Wall Sugar Composition in the Lower Stems of the Wild-Type and Mutant Plants. Supplemental Table 3. Interfascicular Fiber Cell Wall Thickness in Wild-Type, Single Mutant, rev blh6, and rev knat7 Plants. Supplemental Table 4. Primer Sequences to Generate Truncated KNAT7, BLH6, and BLH7 Clones for Yeast Two-Hybrid Assay. Supplemental Table 5. Primers Used for Quantitative RT-PCR Gene Expression and Chromatin Immunoprecipitation Analysis. ACKNOWLEDGMENTS This work was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to C.J.D. and by the NSERC CREATE Grant “Working on Walls.” S.M.B. is supported by a Katherine Esau Junior Faculty Fellowship and a Hellman Fellowship. We thank Michael Friedmann, Brian Ellis, and Etienne Grienenberger (University of British Columbia) for advice and helpful discussions, Melissa Roach and Shawn Mansfield (University of British Columbia) for assistance and support for cell wall analyses, Shumin Wang (University of British Columbia) for transgenic lines, and Miguel de Lucas (University of California, Davis) for experimental guidance. We also thank the ABRC and The Arabidopsis Information Resource (TAIR; Carnegie Institute of Washington, Stanford, CA) for seeds and biological information. Finally, we also thank Jenny Tran (University of British Columbia), who assisted in this work as an undergraduate research assistant.

AUTHOR CONTRIBUTIONS Y.L., W.L.L., C.J.D., M.T.-T., and S.M.B. participated in research design. Y.L., W.L.L., and M.T.-T. conducted experiments. Y.L., S.Y., S.M.B., and C.J.D. carried out data analysis. Y.L., S.Y., W.L.L., M.S., and C.J.D prepared the article. C.J.D. and S.M.B. provided funding for this study.

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BEL1-LIKE HOMEODOMAIN6 and KNOTTED ARABIDOPSIS THALIANA7 Interact and Regulate Secondary Cell Wall Formation via Repression of REVOLUTA Yuanyuan Liu, Shijun You, Mallorie Taylor-Teeples, Wenhua L. Li, Mathias Schuetz, Siobhan M. Brady and Carl J. Douglas Plant Cell; originally published online December 9, 2014; DOI 10.1105/tpc.114.128322 This information is current as of December 11, 2014 Supplemental Data

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