Antagonistic interaction of BLADE-ON-PETIOLE1 and 2 with BREVIPEDICELLUS and PENNYWISE regulates Arabidopsis inflorescence architecture

The transition to flowering in many plant species, including Arabidopsis thaliana, is marked by the elongation of internodes to make an inflorescence upon which lateral branches and flowers are arranged in a characteristic pattern. Inflorescence patterning relies in part on the activities of two TALE homeodomain transcription factors: BREVIPEDICELLUS (BP) and PENNYWISE (PNY) whose interacting products also promote meristem function. We examine here the genetic interactions between BP-PNY whose expression is upregulated in stems at the floral transition, and the lateral organ boundary genes BLADE-ON-PETIOLE1 ( BOP1 ) and BOP2 , whose expression is restricted to pedicel axils. Our data show that bp and pny inflorescence defects are caused by BOP1/2 gain-of-function in stems and pedicels. Compatible with this, inactivation of BOP1/2 rescues these defects. BOP expression domains are differentially enlarged in bp and pny mutants, corresponding to the distinctive patterns of growth restriction in these mutants leading to compacted internodes and clustered or downward-oriented fruits. Our data indicate that BOP1/2 are positive regulators of KNOTTED1-LIKE FROM ARABIDOPSIS THALIANA6 ( KNAT6 ) expression and that growth restriction in BOP1/2 gain-of-function plants requires KNAT6. Antagonism between BOP1/2 and BP is explained in part by their reciprocal regulation of gene expression, as evidenced by the identification of lignin biosynthetic genes that are repressed by BP and activated by BOP1/2 in stems. These data reveal BOP1/2 gain-of-function as the basis of bp and pny inflorescence defects and reveal how antagonism between BOP1/2 and BP-PNY contributes to inflorescence patterning in a model plant species. of monolignol subunits may be a key regulatory point in the developmental control of secondary wall formation. Collectively, these data support the model that BOP1/2 and BP have reciprocal functions in the stem and show how antagonistic interactions between BOP1/2 and BP-PNY are important for patterning of cell-type differentiation in stems as well as inflorescence architecture. data not shown). Our data reveals an opposite regulatory pattern in inflorescences with BP and PNY functioning as transcriptional repressors of BOP1/2 and KNAT6 . Co-misexpression of BOP1/2 and KNAT6 permits their opposing function downstream of BP-PNY to restrict growth and promote premature differentiation of the stem. These data are compatible with BOP1/2 gain-of-function studies in moss. In this species, stabilization of BOP1/2 transcripts causes premature gametophore differentiation (Saleh et al., 2011).


INTRODUCTION
Flowering plants display a remarkable variety of inflorescence architectures selected to optimally display flowers for pollination and seed dispersal. Formation of the aerial parts of a plant is controlled by the shoot apical meristem (SAM), a cluster of pleuripotent stem cells located at the apex of the primary shoot. The SAM produces a series of reiterative modules known as phytomers to generate the aerial parts of the plant. Each phytomer comprises an internode (stem) subtending a node, which is a leaf associated with a potential axillary meristem (Steeves and Sussex, 1989). Elaboration of the different parts of a module (leaves, internodes, and axillary meristems) varies according to the phase of development and between species to generate architectural diversity (Sussex and Kerk, 2001).
Arabidopsis thaliana has distinct vegetative and reproductive phases. During vegetative development, the SAM generates leaf primordia on its flanks; both internode and axillary meristem formation are inhibited resulting in a compact rosette of leaves. At the end of the vegetative phase, endogenous and environmental cues promote the transition to flowering. The SAM responds to floral inductive signals by acquiring inflorescence meristem (IM) fate. During reproductive development, internodes elongate and axillary meristems proliferate at the expense of leaves to generate lateral branches and flowers in a regular spiral pattern on the inflorescence (Bowman and Eshed, 2000;Fletcher, 2002;Barton, 2010). Whilst the pathways that promote floral fate of axillary meristems and repress leaf development are well-studied, less is known about the formation and patterning of internodes.
Internode patterning is a key determinant of inflorescence architecture, with variations in the length and pattern of internode elongation contributing to diversity in inflorescence height and organization of secondary branches and flowers on the primary stem. Formation of internodes is associated with the proliferation and elongation of cells in the region underlying the central zone of the meristem, termed the rib zone (Steeves and Sussex, 1989;Fletcher, 2002). Following their elongation, internodes are gradually fortified through the differentiation of interfascicular fibres with secondary thickened cell walls, which provides mechanical support (Nieminen et al., 2004; clusters of siliques due to irregular internode elongation, defects in phyllotaxy, reduced apical dominance, and replumless fruits (Byrne et al., 2003;Roeder et al., 2003;Smith and Hake, 2003;Bhatt et al., 2004). Inactivation of the BOP genes also rescued pny inflorescence defects (Fig.   3AEF). Quantitative phenotypic analyses were performed on 24 plants per genotype to further monitor this rescue, by measuring the average height, internode length, and number of rosette paraclades for WT and mutants. These analyses confirmed that loss-of-function bop1 bop2 restored the stature of pny plants and the number of rosette paraclades to WT (Fig. 5AB).
Whereas pny mutants have a significant number of internodes in the 1-5 mm range, the distribution in bop1 bop2 pny triple mutants was similar to WT (Fig. 5C). To quantify rescue of phyllotactic patterning in bop1 bop2 pny triple mutants, we measured divergence angles between successive floral pedicels on the primary stem ( Fig. 5D; see Peaucelle et al., 2007). Whereas the distribution of divergence angles in pny was largely random (mean of 175°), the distribution in bop1 bop2 pny triple mutants was similar to WT (mean of 142° versus 141°). Surprisingly, partial loss of BOP function was sufficient to rescue the pny phenotype since pny bop1 and pny bop2 mutant inflorescences also resembled WT (data not shown).
A final defining characteristic in pny mutants is a replumless fruit (Roeder et al., 2003).
Scanning electron microscopy (SEM) showed that inactivation of BOP1/2 also rescues replum formation in pny fruits ( Fig. S3A-D), similar to inactivation of KNAT6 and consistent with coexpression of BOP1/2 and KNAT6 in valve margins (Fig. 1F;S4;Ragni et al., 2008). We further examined the pattern of lignin deposition in fruit cross-sections ( Fig. S3E-H). In pny mutants, lignin (pink colour) was detected throughout the junction between the valves reflecting lack of the replum. In bop1 bop2 and bop1 bop2 pny triple mutants, lignin formed only at the valve margins as in WT. Collectively, these data demonstrate complete rescue of pny defects, supporting the model that BOP1/2 antagonize BP and PNY activities in the inflorescence. These data further suggest that BOP1/2 and KNAT6 control similar developmental processes, based on their similar interactions with BP and PNY and their overlapping expression patterns in lateral organ boundaries (Ragni et al., 2008; see also Fig. 1; Fig. S4). Ragni et al. (2008) showed that BP and PNY prevent KNAT2 and KNAT6 expression in stems and pedicels and that loss-of-function knat6 (and knat2 knat6) rescues bp and pny defects. This prompted us to examine if BOP1/2 expression domains are likewise expanded in bp and pny mutants, using the BOP2:GUS reporter gene ( Fig. 6A-O). In bp mutants, BOP2 expression was expanded in stems and pedicels, particularly below nodes. Expression on the abaxial side of nodes is consistent with localized growth restriction causing pedicels to point downward.

BOP1/2 expression domains are expanded in bp and pny mutants
Staining was also seen in stripes of abnormal epidermal tissue that extend below the node and become ectopically lignified in mature bp stems ( Figure 6FGHI; Venglat et al., 2002;Mele et al., 2003). Stem cross-sections from just below the node confirmed BOP2 misexpression in the stem cortex beneath the epidermis and in phloem regions associated with the primary vascular bundle (Fig. 6J). In pny mutants, BOP2 expression was also expanded in stems and pedicels above and below nodes, compatible with growth impairment causing irregular internodes and silique clustering (Fig. 6KLMN). Stem cross-sections near pny nodes confirmed BOP2 misexpression throughout the stem cortex (Fig. 6O). BOP1:GUS expression in bp and pny mutants showed a similar pattern (Fig. S5). In summary, the misexpression patterns of BOP1/2 differ in bp and pny mutants, bearing resemblance to the distinct inflorescence defects that characterize these mutants.

BOP1/2 promote KNAT6 expression
Given that BOP1/2 and KNAT6 are both required for bp and pny phenotypes and BOP1/2 gainof-function produces bp and pny-like phenotypes, we compared KNAT6:GUS expression in various BOP gain-of-function lines: bp, pny, and 35S:BOP2 or bop1-6D. Misexpression of KNAT6:GUS in stems was confirmed for all genotypes ( Fig. 7A-D). However, the reporter gene was not expressed in boundaries of the IM, indicating that some of its control sequences were missing (data not shown). We therefore used in situ hybridization to further examine KNAT6 expression in the inflorescence apex and stem ( Fig. 7E-T). In the bp mutant, KNAT6 transcript was misexpressed in the stem cortex and vascular tissue (Fig. 7JN) and beneath the node in a stripe pattern (Fig. 7R) similar to misexpression of BOP2 (Fig. 6IJ). In the pny mutant, KNAT6 was misexpressed in the vascular tissue of elongated stems similar to bop1-6D mutants (Fig.   7KLOPST). Both mutants formed extra vascular bundles resulting in a dense vascular ring with little interfascicular space (Fig. 7OP;Smith and Hake, 2003). KNAT6 transcript levels were also monitored in internodes and pedicels by qRT-PCR. These data confirmed two-to-three fold higher levels of KNAT6 transcript in bp-2, pny, and bop1-6D plants relative to WT and bop1 bop2 controls (Fig. 7U). Higher levels of KNAT6 transcript are consistent with an expanded domain of KNAT6 expression in bop1-6D/35S:BOP2 stems. We therefore concluded that BOP1/2 promote KNAT6 expression. Consistent with this, KNAT6 transcript levels were slightly lower in bop1 bop2 bp and bop1 bop2 pny internodes and pedicels relative to bp-2 and pny single mutants (Fig. 7U). No similar upregulation was observed for KNAT2 in bop1-6D plants (data not shown).

BOP1/2 exert their function through KNAT6
Given that BOP1/2 promote KNAT6 expression, we reasoned that BOP1/2 may exert all or part of their function through KNAT6. To examine this, we tested the effect of knat6 loss-of-function on the phenotype of a strong 35S:BOP2 gain-of-function line with short compact inflorescences (Norberg et al., 2005). In this experiment, plants that were homozygous for the 35S:BOP2 transgene were crossed to WT or to lines homozygous for knat2, knat6, or knat2 knat6 mutations. The phenotypes of progeny were scored in the F1 generation. To rule out transgene silencing, we took the additional step of confirming BOP2 overexpression in F1 populations (Fig. S6). These experiments revealed that partial knat6 loss-of-function (i.e. knat6/+ or knat2/+ knat6/+) was sufficient to restore internode elongation in 35S:BOP2 plants (Fig. 8ACD). In contrast, no rescue occurred in control crosses to WT or knat2 alone (Fig. 8ABE). Compatible with this, mutations in knat2 alone do not rescue bp or pny inflorescence defects (Ragni et al., 2008). These data indicate that BOP1/2 exert much of their function through KNAT6.
Interestingly however, 35S:KNAT6 plants are not short and mimic 35S:BP plants with lobed leaves ( Fig. S7A; see also Lincoln et al., 1994;Dean et al., 2004) indicating that the functions of BP and KNAT6 are redundant when BOP1/2 is not co-misexpressed. Thus, both BOP1/2 and KNAT6 are required to exert changes in inflorescence architecture.

BOP1/2 and BP/PNY are antagonistic regulators of stem lignification
We next sought to determine how BOP1/2 gain-of-function antagonizes BP and PNY activities in the stem. We initially considered that BOP1/2 might function through ASYMMETRIC LEAVES2 (AS2) to inhibit BP and/or PNY expression in stems. BOP1/2 indirectly repress BP in leaves by promoting AS2 expression, whose product is a direct repressor of BP transcription (Guo et al., 2008;Jun et al., 2010). However, inactivation of AS2 failed to rescue the short  Ha et al., 2007) or bp and pny inflorescence defects (Fig. S7BC).
Moreover, no decrease in BP or PNY expression was detected the stem of BOP1/2 loss-or gainof-function mutants (Fig. S8). These data indicate that BOP1/2 control of stem architecture is largely independent of AS2 and transcriptional repression of BP. We therefore examined the model that BOP1/2 function downstream of BP-PNY and have reciprocal functions in the stem based on their compartmentalized expression domains in the inflorescence.
To examine this, we turned to work showing that BP is negative regulator of lignin deposition in stems (Mele et al., 2003). In the primary inflorescence stem, formation of secondary cell walls is tightly regulated over developmental time (Ehlting et al., 2005). Cross-sections were cut from the base of WT and mutant primary stems at the same developmental age and stained with phloroglucinol, which detects lignin deposition, a hallmark of secondary walls in vessel and fibre cells in the inflorescence stem. As expected, complex patterning changes were seen in bp mutants. Phloem fibres overlying primary vascular bundles were prematurely lignified. In addition, gaps were observed in the ring of interfascicular fibre cells with lignin abnormally deposited the epidermis and cortex of these gaps. This pattern correlates with the position of abnormally differentiated stripes of tissue in bp stems that originate below nodes and extend basipetally ( Fig. 9AC; Douglas et al., 2002;Venglat et al., 2002;Mele et al., 2003). Loss-offunction bop1 bop2 partially rescued bp defects resulting in a pattern similar to WT (Fig.   9ACD). Ectopic stem lignification also occurs in pny stems, albeit in a different pattern than for bp, which may reflect differences in where or when BOP1/2 are misexpressed. In pny stems, vascular bundles were more crowded than in WT, resulting in a dense vascular ring (Fig. 7O and S8;Smith and Hake, 2003). Loss-of-function bop1 bop2 also rescued pny defects, resulting in a pattern similar to WT (Fig. S8AEG). Importantly, stem cross-sections from 35S:BOP2 and bop1-6D plants showed expanded patterns of lignification, similar to bp pny double mutants (Fig. 9E;S8;Smith and Hake, 2003). In BOP1/2 overexpressing lines, the vascular ring was dense (similar to pny mutants) and phloem fibre cells overlying primary vascular bundles were prematurely lignified (similar to bp mutants). However, there were no gaps in the vascular ring, presumably due to uniformity in BOP1/2 misexpression. In bop1-6D mutants, parts of the pith were lignified, never observed in WT stem development. Thus, BOP1/2 gain-of-function induces lignified phloem and interfascicular fibres in a pattern reminiscent of the secondary growth that occurs in trees ( Fig. 9E; S8, Neiminen et al., 2004;Baucher et al., 2007). These data support the model

BOP1/2 activate genes repressed by BP
Microarray and EMSA experiments have previously identified lignin biosynthetic genes that are directly repressed by BP in stems (Mele et al., 2003). Direct targets of PNY have not been identified to our knowledge. Therefore, qRT-PCR was used to examine whether lignin biosynthetic gene transcripts are reciprocally regulated by BP and BOP1/2 in inflorescence stems

DISCUSSION
Internodes are elongated at the transition to flowering as a result of expanded rib meristem activity in the IM (Steves and Sussex, 1989;Fletcher, 2002). The meristem expression of BP diminishes with the floral transition and becomes restricted to the cortex of the inflorescence stem and pedicel, where its activity together with PNY promotes internode elongation and vascular patterning (Lincoln et al., 1994;Douglas et al., 2002;Venglat et al., 2002;Smith and Hake, 2003). In this article, we used a genetics approach to examine how interactions between BP-PNY and the lateral-organ boundary regulators BOP1/2 govern Arabidopsis inflorescence architecture. Our data show that a spectrum of bp and pny-like defects in inflorescence architecture are caused by BOP1/2 gain-of-function. Our key findings are that BP and PNY restrict BOP1/2 expression to the pedicel axil together with KNAT6 to prevent their misexpression in stems and pedicels, which causes altered growth patterns in bp and pny inflorescences. Our data also indicate that BOP1/2 promote KNAT6 expression and that both activities are required to inhibit internode elongation in stems (Fig. 10). Our analysis of gain-offunction mutants demonstrates that BOP1/2 function downstream of BP-PNY in a reciprocal manner, accelerating the final steps of stem differentiation in opposition to BP.

BOP1/2 differentially regulate KNOX activity in leaves and the inflorescences
Previous work has established that BOP1/2 in leaves function together with AS1/2 to maintain repression of the class I KNOX genes BP, KNAT2, and KNAT6 during leaf development (Ori et al., 2000;Semiarti et al., 2000;Ha et al., 2003;Ha et al., 2007;Jun et al., 2010). In this context,

Misexpression of BOP1/2 restricts growth to create variations in inflorescence architecture
Variations in inflorescence architecture are extensive among flowering plants, with the length and pattern of internode elongation and pedicel angle acting as key variables in the display of flowers (Steeves and Sussex, 1989;Sussex and Kirk, 2001). Short internodes and pedicels like those in bp mutants are reminiscent of species with spike-type inflorescences where internodes between successive flowers are short (Bell and Bryan, 2008). Conversely, long internodes separating whorls of flowers on the stem, like those in pny mutants, are reminiscent of species with verticillate-type inflorescences (Bell and Bryan, 2008). Our data show that a spectrum of inflorescence architectures ranging from short internodes, to downward-pointing pedicels, to clusters of flowers may be produced by differentially regulating the pattern and degree of BOP gain-of-function in stems. In bp mutants, ectopic BOP1/2 expression on the abaxial side of nodes leads to growth restriction and downward-pointing pedicels.

BOP1/2 and KNAT6 function in the same genetic pathway
Our genetic assays and expression data show that misexpression of BOP1/2 is the cause of inflorescence patterning defects in bp and pny mutants. For reasons that are unclear, inactivation of BOP1/2 partially suppresses bp defects but completely suppresses pny defects. This difference may be related to the slightly different roles that bp and pny play in internode development (Hake and Smith, 2003;Peaucelle et al., 2011). These data extend the work of Ragni et al. (2008) who showed an identical pattern of rescue for bp and pny defects by inactivation of KNAT6 (and to a lesser extent KNAT2), genes that are misexpressed in an overlapping domain with BOP1/2 in bp and pny stems (Figs. 6 and 7). These studies place BOP1/2 and KNAT6 in the same genetic pathway controlling inflorescence architecture. Compatible with this, BOP1/2 gain-of-function  (Lincoln et al., 1994;Chuck et al., 1996;Dean et al., 2004) they may regulate some of the same genes. However, KNAT6 with BOP1/2 function in opposition to BP. A physical complex between BOP1/2 and KNAT6 was not detected in yeast (data not shown). We therefore favour a model in which BOP1/2 bind independently to the same promoters as KNAT6 or induce the expression of a KNAT6 co-factor to exert their effect.
In fruits, BOP1/2 and KNAT6 likewise function in the same genetic pathway as evidenced by rescue of replum formation in pny mutants by either bop1 bop2 or knat6 loss-of-function (Ragni et al., 2008;this study). BOP1/2 and KNAT6 may also share a role in floral-organ abscission based on recent evidence that IDA-dependent signalling inhibits BP activity allowing KNAT2 and KNAT6 to promote abscission (McKim et al., 2008;Shi et al., 2011). Thus, antagonism between BP-PNY and a genetic pathway involving BOP1/2 and KNAT6 is likely to be a conserved module in plant development.

ATH1 is a potential KNAT6 co-factor
The BELL homeodomain protein encoded by ARABIDOPSIS THALIANA HOMEOBOX1 (ATH1) is another potential member of the BOP1/2 and KNAT6 genetic pathway that will be important to investigate. KNOX homeodomain proteins like KNAT6 perform many of their functions as heterodimers with BELL proteins (e.g. Bhatt et al., 2004;Bryne et al., 2003;Kanrar et al., 2006;Rutjens et al., 2009). These partnerships can influence protein-protein interactions, nuclear localization of the KNOX partner, and/or binding-site selection (Smith et al., 2002;Hackbusch et al., 2005;Cole et al., 2006;Rutjens et al., 2009). Interestingly, loss-of-function ath1-1 rescues pny inflorescence defects (like bop1 bop2 and knat6) whereas ATH1 gain-offunction causes short internodes (Rutjens et al. 2009;Gómez-Mena and Sablowski, 2008). Given that ATH1 transcripts are highly up-regulated in bop1-6D internodes (data not shown), an ATH1- Interestingly, publically available poplar microarray data indicates that two potential BOP orthologs are highly expressed in xylem (http://www.bar.utoronto.ca), which suggests a conserved role for BOP1/2 in promoting secondary growth in trees. In conclusion, our data establish BOP1/2 gain-of-function as the basis of bp and pny inflorescence defects. Our study shows that ectopic BOP1/2 activity in stems both restricts growth and promotes terminal cell differentiation, dramatically altering inflorescence architecture. Future studies will establish the molecular basis of antagonism between BP-PNY and BOP1/2. Ultimately, this work will provide important insight into how changes in the interplay between KNOX-BELL factors in the meristem and BOP1/2 in lateral organ boundaries drives species variation in inflorescence architecture. of North Carolina) to create the intermediate plasmid pBAR/35S. Primer pairs B1-1/B1-2 and B2-1/B2-2 1 incorporating BamHI restriction sites were used to amplify BOP1 and BOP2 coding sequences respectively from cloned cDNA templates. The resulting PCR products were digested with BamHI and ligated into the corresponding site in pBAR to generate pBAR/35S-BOP1 and pBAR/35S-BOP2. The EntCUP4 promoter is an alternative constitutive promoter (Malik et al., 2002). To create ptCUP4:BOP1, a DNA fragment containing the BOP1 coding sequence was amplified by PCR from cloned cDNA template using EcoR1-BOP1-F1 and BOP1-RR as the primers. The resulting fragment was digested with EcoRI and BamHI and ligated into the corresponding sites of pBAR1 to generate the intermediate plasmid pBAR1/BOP1. A 0.5-kb DNA fragment containing the EntCUP4 promoter was then amplified by PCR using pEntCUP4nos-GUS as a plasmid template and EcoR1-tCUP-F1 and EcoR1-tCUP-R1 as the primers. The resulting fragment was digested with EcoR1 and ligated into the corresponding sites of pBAR/BOP1 to create ptCUP4:BOP1. Wild-type plants were transformed by floral dipping (Clough and Bent, 1998) using the Agrobacterium strain C58C1 pGV101 pMP90 (Koncz and Schell, 1986). Basta©-resistant transformants were selected on soil using the herbicide Finale (AgrEvo, Winnipeg, Canada). Phenotypes were scored in the T1 generation.

Phenotypic analysis of inflorescence structure
Quantitative phenotypic analyses of 4-week-old plants were performed as described (Ragni et al., 2008). Phyllotaxy measurements were obtained as previously described (Peaucelle et al., 2007).
The divergence angle between the insertion points of two successive floral pedicels along the main inflorescence was measured. Divergence angles were measured for the first 15 siliques of each inflorescence (counting acropetally) according to the orientation that resulted in the smallest average divergence angle. Angle of pedicel orientation was determined using a protractor to measure the angle of pedicel attachment relative to the stem. Orientation was measured for the first eleven siliques of each inflorescence (counting acropetally).

In situ hybridization and localization of GUS activity
Tissues were fixed and analyzed for GUS activity essentially as described by Seiburth and Meyerowitz (1997 St. Louis, MO). Sections (10 µm) cut with a microtome were affixed to glass slides and dewaxed with tert-butanol and xylene prior to imaging. In situ hybridizations were performed as described (Xu et al., 2010). Primers used to make BP and KNAT6 anti-sense probes are listed in Supplemental Table S1.

Scanning Electron Microscopy (SEM)
Samples were prepared for SEM as described in Hepworth et al. (2005). Images were collected using a Vega-II XMU Variable Pressure SEM (Tescan USA, Cranberry Township, PA).

Lignin Staining
Tissue sections (25 µm) were cut from paraffin-embedded mature green siliques to analyze replum patterning or from elongated internodes between the third and fourth siliques on the primary stem to analyze stem patterning. Tissue sections affixed to glass slides were dewaxed and dehydrated prior to addition of 2% phloroglucinol (in 95% ethanol) followed by 6N HCl for colour development. For the analysis of lignin at stem bases, cross-sections were cut from the base of 32-day-old flowering plants with a razor blade and placed in 3 ml of 2% phloroglucinol solution. After 5 minutes, 5 drops of concentrated HCl were added. Two minutes were allowed for colour development and images were immediately collected.

Quantitative RT-PCR (qPCR)
Total RNA was isolated from leaves, pedicels, internodes, or the base of bolting stems (bottom 2.5-cm of 32-day-old flowering plants) using Trizol© reagent (Invitrogen, Carlsbad, CA). cDNA was generated using 1 µg of total RNA as the template and Superscript III RT (Invitrogen, Carlsbad, CA) as the polymerase. qPCR was performed in triplicate using 2 µl of 10-fold diluted cDNA as the template in reactions containing SYBR ® Green and IQ Supermix (BioRad, Hercules, CA) using a Rotor-Gene 6000 thermocycler (Qiagen, Almeda, CA).
Annealing conditions were optimized for each primer pair and data quality was verified by melting curve analysis. Relative transcript levels were calculated as described (Murmu et al., 2010). Values were normalized to GAPC and then to the wild-type control. For Figure 9G only, cDNA was generated using 2 µg of total RNA as the template and diluted 20-fold. ACTIN2 was used as a normalization control. Reactions were performed in triplicate using an annealing temperature of 55ºC. All experiments were repeated at least twice with independently-isolated RNA with similar results obtained. Primers for the analysis of lignin genes are given in Supplemental Table S2.

Accession Numbers
Sequence data for genes described in this article can be found in the GenBank/EMBL data

Supplemental Data
The following materials are available in the online version of this article.

Supplemental Table 1. List of general primers
Supplemental Table 2              At least 24 plants per genotype were analyzed. A, Average height of primary inflorescence; inactivation of BOP1/2 rescues short stature of pny mutants. B, Average number of rosette paraclades; inactivation of BOP1/2 restores apical dominance in pny mutants. C, Distribution of internode lengths between successive siliques on the primary inflorescence. Internodes between the 1 st and 11 th siliques (counting acropetally) were measured. The distribution of siliques in bop1 bop2 pny mutants was similar to WT. D, Distribution of divergence angles between siliques on the primary inflorescence. At least ten successive angles between the 1 st and 24 th siliques (counting acropetally) were measured for n≥14 plants per genotype. The class containing the theoretical angle of 137°is indicated by a vertical line. Average angle (avg). In pny plants, distribution is uniform across all classes but in bop1 bop2 pny plants, the distribution is similar to WT.