Repression of Lateral Organ Boundary Genes by PENNYWISE and POUND-FOOLISH Is Essential for Meristem Maintenance and Flowering in Arabidopsis^,

Two homeobox domain proteins maintain meristem activity essential for flowering by repressing an organ boundary gene module.

Abstract

In the model plant Arabidopsis (Arabidopsis thaliana), endogenous and environmental signals acting on the shoot apical meristem cause acquisition of inflorescence meristem fate. This results in changed patterns of aerial development seen as the transition from making leaves to the production of flowers separated by elongated internodes. Two related BEL1-like homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF), fulfill this transition. Loss of function of these genes impairs stem cell maintenance and blocks internode elongation and flowering. We show here that pny pnf apices misexpress lateral organ boundary genes BLADE-ON-PETIOLE1/2 (BOP1/2) and KNOTTED-LIKE FROM ARABIDOPSIS THALIANA6 (KNAT6) together with ARABIDOPSIS THALIANA HOMEOBOX GENE1 (ATH1). Inactivation of genes in this module fully rescues pny pnf defects. We further show that BOP1 directly activates ATH1, whereas activation of KNAT6 is indirect. The pny pnf restoration correlates with renewed accumulation of transcripts conferring floral meristem identity, including FD, SQUAMOSA PROMOTER-BINDING PROTEIN LIKE genes, LEAFY, and APETALA1. To gain insight into how this module blocks flowering, we analyzed the transcriptome of BOP1-overexpressing plants. Our data suggest a central role for the microRNA156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE-microRNA172 module in integrating stress signals conferred in part by promotion of jasmonic acid biosynthesis. These data reveal a potential mechanism by which repression of lateral organ boundary genes by PNY-PNF is essential for flowering.

Plant development relies on the activity of the shoot apical meristem (SAM) as a continuous source of founder cells for production of new leaves, shoots, and internodes throughout the life cycle (for review, see Aichinger et al., 2012). A tight balance between the allocation of cells to developing primordia and the perpetuation of pluripotent stem cells in the central zone maintains the SAM at a constant size. In Arabidopsis (Arabidopsis thaliana), the vegetative SAM produces leaves in a spiral phyllotaxy with dormant axillary meristems. In conjunction, internode elongation is repressed, resulting in a basal rosette. The transition to flowering is governed by internal and external signals that converge at the SAM to promote acquisition of inflorescence meristem (IM) fate (for review, see Amasino and Michaels, 2010; Srikanth and Schmid, 2011; Andrés and Coupland, 2012). This process, known as floral evocation, results in new patterns of growth at the shoot apex, including production of flowers, and an increase in stem elongation, called bolting. Lateral organ boundaries are specialized domains of restricted growth that separate meristem and organ compartments and produce axillary meristems (for review, see Aida and Tasaka, 2006; Tian et al., 2014). Early in the transition to flowering, the IM produces cauline leaves and axillary meristems that develop as secondary inflorescences. After several nodes, the IM ceases production of leaves, and axillary meristems develop as flowers.

Floral repressors in the SAM block meristem competence to flowering during vegetative stages of development. Major pathways for promotion of flowering work in two ways: by down-regulation of floral repressors in the meristem and by production of factors that promote IM and floral meristem identity (Bernier, 1988; Yant et al., 2010; Srikanth and Schmid, 2011). The switch to flowering is governed by internal signals, including age, Suc content, and GA, in conjunction with external cues based on photoperiod, vernalization, ambient temperature, and responsiveness to light or stress stimuli (for review, see Srikanth and Schmid, 2011; Wang, 2014). Inputs from these different pathways converge to regulate a number of floral integrator genes, including FLOWERING LOCUS T (FT), which is a central component of the photoperiod response (Srikanth and Schmid, 2011; Andrés and Coupland, 2012). FT encodes a small phosphatidylethanolamine-binding protein that is synthesized in leaves and travels through phloem to the SAM (for review, see Corbesier et al., 2007; Jaeger and Wigge, 2007; Mathieu et al., 2007; Andrés and Coupland, 2012), where it interacts with the basic region/leucine zipper motif (bZIP) transcription factor FD to activate genes conferring inflorescence identity, including SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1)/AGAMOUS-LIKE20 (AGL20), AGL24, and FRUITFULL (FUL; Abe et al., 2005; Teper-Bamnolker and Samach, 2005; Wigge et al., 2005). These factors in turn promote the expression of floral meristem identity genes LEAFY (LFY), APETALA1 (AP1), and CAULIFLOWER (CAL), which confer floral fate (Bowman et al., 1993). In parallel, age-regulated down-regulation of microRNA156 (miR156) stabilizes mRNA encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL3), SPL4, and SPL5 transcription factors, which function with FT-FD to specify flower development by directly activating AP1, LFY, and FUL expression (Yamaguchi et al., 2009; Jung et al., 2012; Wang, 2014). The plant hormone GA is a positive regulator of flowering with function that is more pronounced under short days (SDs) when other regulatory pathways are inactive. Under SDs, GAs activate the transcription of SOC1 and LFY in the shoot apex. Under long days (LDs), GA is not required for activation of SOC1 but is important for activation of other transcripts at the shoot apex. Its targets include SPL genes, which are also directly activated by SOC1 and FD (Galvão et al., 2012; Porri et al., 2012). How these various pathways are integrated with stress signals is an area of active study (Yang et al., 2012; Heinrich et al., 2013; Hou et al., 2013; Diallo et al., 2014; Stief et al., 2014).

Members of the THREE-AMINO-ACID-LOOP-EXTENSION (TALE) class of homeodomain transcription factors constitute major regulators of meristematic activity. This family includes KNOTTED1-like (KNOX) and BEL1-like (BELL) or BEL1-LIKE HOMEODOMAIN (BLH) members, which function as heterodimers (for review, see Hamant and Pautot, 2010; Hay and Tsiantis, 2010). SHOOT MERISTEMLESS (STM), which is the founding member of the KNOX family in Arabidopsis, is required for SAM initiation and maintenance (Clark et al., 1996; Endrizzi et al., 1996; Long et al., 1996). Other TALE members, such as BREVIPEDICELLUS (BP)/KNOTTED-LIKE FROM ARABIDOPSIS THALIANA1 (KNAT1), KNAT6, PENNYWISE (PNY; also known as BELLRINGER, REPLUMLESS, VAAMANA, or LARSON), POUND-FOOLISH (PNF), and ARABIDOPSIS THALIANA HOMEOBOX GENE1 (ATH1) are expressed in the SAM and contribute redundantly with STM in meristem initiation and maintenance (Byrne et al., 2000; Belles-Boix et al., 2006; Rutjens et al., 2009).

PNY contributes to meristem maintenance and flowering with its closest relative, PNF (Smith et al., 2004). During vegetative development, the SAM in pny pnf mutants frequently terminates with development resuming from leaf-derived axillary meristems, a phenotype linked to reduced expression of STM (Smith et al., 2004; Ung et al., 2011; Ung and Smith, 2011). The pny pnf double mutant is also nonflowering. The pny pnf meristem changes shape in response to floral inductive signals, and inflorescence identity genes SOC1 and FUL are up-regulated; however, FT levels are reduced, and floral meristem identity genes LFY, AP1, and CAL are not expressed (Smith et al., 2004; Kanrar et al., 2008). The basis of this phenotype is only partly understood. Ectopic expression of LFY in pny pnf mutants partially rescues flowering at axillary meristems, whereas ectopic expression of FT fails to rescue flowering and partially restores internode elongation at length, suggesting that FT requires PNY-PNF to initiate flower development (Kanrar et al., 2008). Additional data show that STM functions in association with PNY-PNF to specify flowers by promotion of LFY expression (Kanrar et al., 2006, 2008). This has led to the proposal that STM and PNY-PNF function together with flowering time products FT-FD and AGL24-SOC1 to initiate development of reproductive structures, flowers, and internodes (Smith et al., 2011). More recently, PNY-PNF were shown to promote the expression of SPL3, SPL4, and SPL5 transcription factors that direct activation of floral meristem identity genes in parallel with FT-FD (Lal et al., 2011). Compatible with this, miR156 is up-regulated in pny pnf apices. Ectopic expression of SPL4 in pny pnf restores accumulation of LFY and AP1 transcripts and partially restores flower formation (Lal et al., 2011). However, none of these mechanisms identified to date fully explain the basis of pny pnf meristem defects.

In addition to roles in the SAM, these factors have distinct functions in establishing inflorescence architecture. Significant reorganization of KNOX-BELL gene expression occurs at the transition to flowering in correlation with new patterns of aerial development (Lincoln et al., 1994; Byrne et al., 2003; Smith and Hake, 2003; Smith et al., 2004; Proveniers et al., 2007; Gómez-Mena and Sablowski, 2008). PNY and BP maintain proper internode patterning through the regulation of cell wall remodeling proteins (Mele et al., 2003; Etchells et al., 2012). Mutations in bp cause short internodes and downward-pointing flowers, whereas mutations in pny cause irregular elongation of internodes, leading to clusters of flowers on the primary stem with phenotypes enhanced in the double mutant. Studies in Arabidopsis have identified the joint activities of BLADE-ON-PETIOLE (BOP) Broad Complex, Tramtrack, and Bric-a-brac (BTB)-ankyrin coactivators and TALE homeodomain transcription factors as important in maintaining lateral organ boundaries (for review, see Hamant and Pautot, 2010; Hay and Tsiantis, 2010; Khan et al., 2014). BP and PNY restrict expression of lateral organ boundary genes BOP1/2, KNAT2, KNAT6, and ATH1 to boundaries at the base of the floral shoot in controlling growth patterns in the inflorescence (Ragni et al., 2008; Khan et al., 2012a, 2012b; Zhao et al., 2015). These studies revealed that BOP1/2 promote ATH1 and KNAT6 which form a module that opposes BP-PNY activity in regulating inflorescence architecture (Rutjens et al., 2009; Khan et al., 2012a, 2012b, 2014; Li et al., 2012).

Here, we investigated the interaction of BOP1/2 with TALE members in flower formation. Our studies reveal that PNY and PNF repress the lateral organ boundary genes BOP1/2 and transcriptional targets ATH1 and KNAT6 to maintain meristem integrity and flowering. Inactivation of genes in this module fully rescues pny pnf defects in meristem maintenance, internode elongation, and flowering. To gain insight into how this module blocks flowering, we analyzed the transcriptome of BOP1-overexpressing plants. Our data indicate a role for stress signaling by promotion of jasmonic acid (JA) as a potential mechanism for counteracting flowering, including responsiveness to GA acting in part through the miR156-SPL-miR172 module.

RESULTS

Inactivation of BOP1/2, KNAT6, or ATH1 Rescues Meristem Maintenance, Internode Elongation, and Flowering Defects in pny pnf

Previously, we showed that misexpression of boundary genes BOP1/2, KNAT6, and ATH1 in bp and pny internodes perturbs inflorescence architecture through localized restriction of growth. Inactivation of genes in this module fully rescues pny defects in internode elongation and phyllotaxy, but inactivation of KNAT2 has no such effect (Ragni et al., 2008; Khan et al., 2012a, 2012b). We anticipated that antagonistic functions of these same genes might cause pny pnf defects. The pnf single mutant has no obvious phenotype. The pny mutant has a functional SAM, but apical dominance is reduced, flowering is delayed, and organs are clustered on the primary stem because of irregular internode elongation. In pny pnf/+ hemizygous plants, these defects are enhanced, and stem-pedicel fusions occur (Smith and Hake, 2003; Supplemental Fig. S1, A–G). In pny pnf double mutants, the SAM terminates after the initiation of three to five leaves in a majority of seedlings (Smith et al., 2004; Rutjens et al., 2009). Lateral meristems in the axil of rosette leaves support the continued production of leaves, but flowering and internode elongation are blocked (Smith et al., 2004; Rutjens et al., 2009; Lal et al., 2011). To determine if BOP1/2, KNAT/6, and ATH1 are required in generating pny pnf defects, we constructed bop1 bop2 pny pnf, ath1 pny pnf, knat2 pny pnf, knat6 pny pnf, and knat6 knat2 pny pnf mutants. We first tested for rescue of pny pnf defects in SAM maintenance. Previous studies using the ath1-1 allele indicated that SAM arrest in triple mutants with pny pnf is markedly enhanced, likely because of the depletion of BELL-STM functional complexes (Rutjens et al., 2009). Here, we repeated the analysis with ath1-3, which unlike ath1-1 and ath1-4 alleles, produces no full or partial mutant transcript (Supplemental Fig. S2). Although 57.7% of pny pnf plants showed a meristem arrest, no such arrest was observed in ath1-3 pny pnf mutants (“Materials and Methods”; Fig. 1). Meristem function was also rescued by bop1 bop2 and knat6 mutations but not by inactivation of KNAT2 (Fig. 1). These data suggest that PNY-PNF/STM antagonizes the activity of lateral organ boundary genes to maintain stem cell identity. Flower formation, internode elongation, and organ fusion defects were also rescued in bop1 bop2 pny pnf and knat6 pny pnf or ath1-3 pny pnf triple mutants compared with pny pnf and/or pny pnf/+ plants (Fig. 2, A–H; Supplemental Fig. S3). Quantitative phenotypic analyses showed that inflorescence architecture of bop1 bop2 pny pnf, ath1 pny pnf, and knat6 pny pnf mutants was similar to that of wild-type plants (Supplemental Fig. S4). In contrast, knat2 pny pnf mutants remained nonflowering (Fig. 2I).

Figure 1.

Inactivation of BOP1/2, ATH1, and KNAT6 rescues pny pnf meristem arrest. Plants were grown under SDs. Numbers of plants showing a meristem arrest on day 25 are indicated at the upper right. A, Col-0 plant. The SAM produces leaves. B, pny pnf mutant showing a meristem arrest; 90 of 156 (57.7%) of expected pny pnf mutants in a pny pnf/+ segregating population (n = 624) showed SAM arrest (arrow). C, knat2 pny pnf triple mutant (identical to pny pnf mutant); 11 of 36.5 (30.1%) of expected knat2 pny pnf triple mutants in a knat2 pny pnf/+ segregating population (n = 146) showed SAM arrest (arrow). D, ath1 pny pnf triple mutant (no meristem arrest). E, bop1 bop2 pny pnf quadruple mutant (no meristem arrest). F, knat6 pny pnf triple mutant (no meristem arrest). Bars = 5 mm.

Figure 2.

Inactivation of BOP1/2, ATH1, and KNAT6 rescues internode and flower formation in pny pnf mutants. Representative 8-week-old plants are shown. A, Col-0 plant. B, pnf mutant showing a wild-type phenotype. C, pny mutant showing partial loss of apical dominance, short stature, and clusters of siliques. D, pny pnf/+ hemi mutant showing partial loss of apical dominance, short stature, clusters of siliques, and stem/pedicel fusion defects (Supplemental Fig. S1). E, pny pnf double mutant (nonflowering). F, bop1 bop2 pny pnf quadruple mutant (similar to bop1 bop2). Inactivation of BOP1 and BOP2 in pny pnf rescues internode elongation and flowering. G, ath1 pny pnf triple mutant (similar to ath1). Inactivation of ATH1 in pny pnf rescues internode elongation and flowering. H, knat6 pny pnf mutant (similar to the wild type). Inactivation of KNAT6 in pny pnf rescues internode elongation and flowering. I, knat2 pny pnf mutant (identical to pny pnf mutant). Bars = 2 cm.

Overexpression studies further support a role for BOP1/2, ATH1, and KNAT6 in the same genetic pathway. Plants that overexpress BOP1/2 are late flowering with shortened internodes and clustered fruits similar to pny and pny pnf/+ mutants (Supplemental Fig. S1, A–C; Norberg et al., 2005; Ha et al., 2007; Khan et al., 2012b). Plants overexpressing ATH1 and occasionally, KNAT6 have similar defects that mimic the inflorescence architecture of pny and pny pnf/+ mutants (Supplemental Fig. S1, B–I; Proveniers et al., 2007; Gómez-Mena and Sablowski, 2008; Shi et al., 2011). The most severe KNAT6 transgenic lines were strongly inhibited in their development and failed to flower (Supplemental Fig. S1, J and K). Collectively, these data indicate that PNY-PNF plays no essential function in meristem/boundary maintenance, internode elongation, and flowering beyond repression of BOP1/2 and ATH1/KNAT6.

BOP1/2, ATH1, and KNAT2/6 Expression Domains Are Expanded in pny pnf Apices

Inflorescence defects in pny mutants correlate with an expanded pattern of expression for BOP1/2, ATH1, and KNAT2/6 in internodes (Ragni et al., 2008; Khan et al., 2012a, 2012b). We therefore examined the expression patterns of these genes in pny pnf apices. In wild-type apices, BOP2 transcripts accumulate in the adaxial domain of floral meristems until late stage 2, when expression shifts to the boundary with the cryptic bract. Expression is found in the boundary domains of older flowers (Fig. 3A; Xu et al., 2010). ATH1 transcripts are expressed in incipient floral primordia and the dome of stage 2 floral primordia in a pattern similar to KNAT2. KNAT6 transcripts are localized to boundary domains flanking the IM and in flowers also overlapping with KNAT2 (Fig. 3, B–D). In pny pnf apices, the domain of expression for all of these genes expands into the central and rib zones of the meristem (Fig. 3, E–H). This was also observed for BOP1 using a BOP1-GUS line (Supplemental Fig. S5). Misexpression of these genes likely begins during the vegetative stage based on analysis of BOP2:GUS lines (data not shown), consistent with SAM structural defects (Ung et al., 2011). Little or no misexpression was observed in pny or pnf control apices (Supplemental Fig. S6). These data confirm that pny pnf defects are caused by misexpression of BOP1/2, ATH1, and KNAT6 in the meristem. We next examined regulatory interactions between these genes in the pathway.

Figure 3.

BOP2, ATH1, KNAT2, and KNAT6 expression in pny pnf apices. Plants were grown for 3 weeks under SDs and transferred to continuous light to induce flowering. Apices were harvested on day 15. Transcript accumulation was monitored by in situ hybridization using longitudinal sections of Col-0 (A–D) and pny pnf (E–H) apices and gene-specific probes. Numbers in panels indicate the stage of floral development (Smyth et al., 1990). A, Col-0 apex showing BOP2 expression in floral meristems (until stage 2) and the boundary domains of older flowers (late stage 2 and stage 3 are shown). B, Col-0 apex showing ATH1 expression in an incipient floral primordium and the dome of a stage 2 flower. C, Col-0 apex showing KNAT2 transcripts localized to boundary domains flanking the IM and older flowers. Expression is also observed in floral primordia and the dome of stage 2 flowers. D, Col-0 apex showing KNAT6 transcripts localized to boundary domains flanking the IM and in a stage 3 flower. E to H, pny pnf apices showing expanded expression of BOP2 (E), ATH1 (F), KNAT2 (G), and KNAT6 (H) in the central and rib zones of the meristem. Bars = 40 µm.

ATH1 Is a Direct Target of BOP1

BOP1/2 was previously shown to promote the expression of ATH1 and KNAT6 and require these activities to exert changes in inflorescence (Khan et al., 2012a, 2012b). To test if ATH1 and/or KNAT6 are immediate transcriptional targets of BOP1/2, we used a transgenic line expressing a translational fusion of BOP1 to the steroid-binding domain of the rat glucocorticoid receptor (GR; Lloyd et al., 1994). This dexamethasone (DEX)-inducible system was used previously to show that BOP1 directly activates the transcription of ASYMMETRIC LEAVES2 in leaves (Jun et al., 2010). Function of the BOP1-GR fusion protein was confirmed by expressing it under the control of a BOP1 native promoter and observing efficient complementation of bop1 bop2 leaf and abscission defects upon addition of DEX (Supplemental Fig. S7). Direct regulation of ATH1 and/or KNAT6 was tested using the BOP1-GR fusion protein expressed in wild-type plants under the control of a double 35S promoter. D35S:BOP1-GR plants treated with DEX for 4 weeks had shortened internodes and clustered fruits similar to bop1-6D mutants, which constitutively overexpress BOP1 (Fig. 4, A–D; Norberg et al., 2005). Transcripts for ATH1 were increased 13.29-fold and transcripts for KNAT6 were increased 2.59-fold in bop1-6D internodes compared with the wild type (Fig. 4E). Similarly, D35:BOP1-GR plants treated with DEX for 4 weeks showed a 6-fold up-regulation of ATH1 transcript (Fig. 4E). After 2 and 4 h of DEX treatment, transcript levels for ATH1 were at least 2-fold higher, but KNAT6 transcript levels showed no increase relative to mock-treated control plants (Fig. 4F; 24-h time point not shown). Rapid activation of ATH1 suggested that its induction by BOP1 may be direct. We tested this by analyzing ATH1 and KNAT6 expression in response to DEX induction in the presence of the protein synthesis inhibitor cycloheximide (CHX). After 2 and 4 h of combined treatment with DEX and CHX, ATH1 transcripts were increased 5- to 7.5-fold relative to CHX-treated control plants. KNAT6 transcripts were increased up to 2-fold after combined DEX and CHX treatment but not after DEX alone. Presumably, this is an indirect effect of BOP1 dependent on repression of protein synthesis. These data are consistent with ATH1 being a direct target of BOP1 and KNAT6 being an indirect target.

Figure 4.

Activation of ATH1 and KNAT6 in the DEX-induced D35S:BOP1-GR line. A, Col-0 plant. B, bop1-6D mutant with shortened internodes and clustered siliques. C and D, D35S:BOP1-GR plants treated with mock or DEX solutions for 4 weeks. C, Mock-treated D35S:BOP1-GR plant showing a wild-type phenotype. D, DEX-induced D35S:BOP1-GR plant showing a phenotype similar to bop1-6D mutant. E, Comparison of KNAT6 and ATH1 transcript levels in the wild type versus bop1-6D mutants and mock- versus DEX-induced D35S:BOP1-GR plants after continuous treatment for 4 weeks. F, Comparison of KNAT6 and ATH1 transcript levels in DEX-induced D35S:BOP1-GR lines with and without protein synthesis inhibitor CHX. Transcripts were measured after 2 and 4 h of treatment. Bars = 2 cm.

To examine tissue specificity of this interaction, 3.3- and 2-kb ATH1p:GUS reporter genes expressed in D35S:BOP1-GR (“Materials and Methods”) were monitored for induction by DEX. Consistent with previous reports (Proveniers et al., 2007; Gómez-Mena and Sablowski, 2008), these reporters were expressed in shoot apices, leaves, and floral organ abscission zones and weakly expressed in the stem. After 4 h of DEX treatment, GUS activity was enhanced relative to mock-treated controls for both promoter lines in all tissues (Fig. 5, A–H). These data confirm that the ATH1 promoter is responsive to BOP1 induction.

Figure 5.

Identification of the genomic region responsible for ATH1 induction by BOP1. A to H, Functional characterization of the ATH1 regulatory region. Representative expression patterns are shown for D35S:BOP1-GR plants containing 2- (A, C, E, and G) or 3.3-kb (B, D, F, and H) ATH1p:GUS reporter genes as diagrammed in I. Promoter activity was monitored by GUS staining after incubation of 10-d-old seedlings or 6-week-old inflorescences for 4 h in mock or 30 µm of DEX solution. Comparison of mock (A–D) and DEX (E–H) shows that expression is up-regulated in the leaves, flowers, and stem of DEX-induced lines for both promoter constructs. Bars = 1 mm. I, Map of the ATH1 promoter and 5′ untranslated region. Black arrowheads mark the 5′ ends of genomic fragments used in construction of 2- and 3.3-kb ATH1p:GUS reporter genes. Predicted consensus binding sites for TGA bZIP factors (Schindler et al., 1992; Izawa et al., 1993; Fode et al., 2008) are shown in relation to fragments amplified by qRT-PCR after ChIP to test for BOP1 localization (horizontal bars). Sites in red (IV and VII) contain A boxes and show enrichment for BOP1. J, Quantification of BOP1-GR enrichment at sites IV and VII in the ATH1 promoter by qRT-PCR. Anti-GR ChIP was performed using leaves from mock- and DEX-treated 35S:BOP1-GR bop1 bop2 plants. Fold enrichment at sites IV and VII is presented as the ratio of DEX versus mock transcript levels after normalization to the unrelated UBQ5 control sequence. Three biological replicates were quantified to show enrichment at site IV. One biological replicate was quantified to show enrichment at site VII. Three technical replicates were performed for each. Error bars indicate sd.

BTB-ankyrin proteins, including BOP1/2, have no DNA-binding domain and interact with TGA bZIP binding factors for recruitment to DNA (Després et al., 2000; Hepworth et al., 2005; Xu et al., 2010; Khan et al., 2014). Direct association of BOP1 with the ATH1 promoter was tested by chromatin immunoprecipitation (ChIP) using an anti-GR antibody followed by quantitative reverse transcription (qRT)-PCR. Leaf material was collected from BOP1p:BOP1-GR bop1 bop2 flowering plants. Assays were performed using eight sets of primers spanning 2,178 bp of genomic sequence upstream of the ATH1 transcription start site based on regions enriched in TGA bZIP binding sites (Fig. 5I; see ”Materials and Methods“). Motifs that match or closely match consensus binding sites for TGA factors are also found in the intragenic and 3′ untranslated regions of the ATH1 genomic sequence (data not shown). Quantitative analysis by qRT-PCR revealed at least one position in the ATH1 promoter (site IV) showing a reproducible 1.77-fold enrichment of BOP1 protein in DEX-treated plants (Fig. 5J). ChIP assays performed using the mock control showed no significant enrichment at this position or the control UBIQUITIN5 (UBQ5) genomic region. Site IV (nucleotides −2,686 to −2,577) is located approximately 1,515 bp upstream of the ATH1 transcription start site and found within the 3.3-kb ATH1p:GUS construct that is responsive to BOP1 induction in leaves and inflorescences (Fig. 5). Site VII (nucleotides −1,529 to −1,416) was identified as a second potential binding site. Taken together, these data support that BOP1 directly associates with the ATH1 promoter in vivo to regulate its transcription.

Restored Accumulation of Flowering Transcripts in pny pnf Apices after Rescue by Inactivation of BOP1/2, KNAT6, and ATH1

Nonflowering pny pnf apices accumulate SOC1 and FUL transcripts markers of inflorescence identity but fail to accumulate FT or LFY, AP1, and CAL markers of floral fate (Smith et al., 2004; Kanrar et al., 2008). Accumulation of SPL3, SPL4, and SPL5 transcripts is also diminished in pny pnf apices (Lal et al., 2011). Flowering time of wild-type plants was compared with those of bop1 bop2 pny pnf, knat6 pny pnf, and ath1 pny pnf mutants to further quantify rescue. Figure 6A shows that flowering time for knat6 pny pnf mutants and wild-type control plants was similar. Flowering time of bop1 bop1 pny pnf mutants was slightly delayed (+3.6 d) and flowering time of ath1 pny pnf mutants was slightly earlier (−6.9 d) than the wild type, consistent with parental controls (Fig. 6A; Xu et al., 2010). To test if inactivation of BOP1/2, ATH1, and KNAT6 correlates with restored expression of meristem identity genes in pny pnf apices, we measured relative transcript abundance in the wild type and mutants; 25-d-old plants grown under SDs were transferred to LDs to induce flowering. Apices were harvested 12 d later. The floral transition was complete for all genotypes at this time point. Figure 6B confirms that SOC1 and FUL transcripts are relatively unchanged in the wild type compared with mutants. Figure 6B also shows that low to undetectable levels of FD, LFY, AP1, and CAL transcripts in pny pnf apices resumed expression in triple and quadruple mutants, except for CAL, which remained low in bop1 bop2 pny pnf apices. Transcripts for FUL, LFY, AP1, and CAL were elevated in ath1 pny pnf apices, consistent with earlier flowering. Figure 6C shows that patterns of miR156 and SPL transcript accumulation in triple and quadruple mutants are likewise restored to resemble the wild type. Collectively, these data show that PNY-PNF is dispensable for flowering when BOP1/2, ATH1, and KNAT6 activities are eliminated.

Figure 6.

Quantification of flowering time and meristem identity transcripts in the wild type and mutants. A, Quantitative analysis of flowering time. Plants were grown under LDs. Date of apex emergence for bop1 bop2 pny pnf, knat6 pny pnf, and ath1 pny pnf mutants is comparable with that of the wild type with minor variations. Lines containing ath1 flowered slightly earlier (−6.7 d) and lines containing bop1 bop2 flowered slightly later (+3.1 d) than the wild type. *, Significant differences (Student’s t test; P < 0.01). B, Quantitative analysis of meristem identity gene expression. Flowering was induced by shifting plants from SDs to LDs. Apices were harvested on day 37 at the end of 12 LDs. IM identity gene transcripts SOC1 and FUL are expressed at similar levels in Col-0 and pny pnf apices. Floral meristem identity gene transcripts FD, LFY, AP1, and CAL are significantly lower in pny pnf compared with Col-0 apices. Transcript accumulation resumes in bop1 bop2 pny pnf, knat6 pny pnf, and ath1 pny pnf apices. *, Significant differences (Student’s t test; P < 0.05). C, Quantitative analysis of miR156 and SPL transcript abundance in wild-type and mutant apices. Nonflowering in pny pnf correlates with a significant increase in miR156 abundance at the expense of SPL3, SPL4, SPL6, SPL9, and SPL15 transcripts relative to Col-0 plants. Transcript accumulation in bop1 bop2 pny pnf, knat6 pny pnf, and ath1 pny pnf mutants follows a pattern similar to the wild type, consistent with restored flowering. *, Significant differences (Student’s t test; P < 0.05).

BOP1 Overexpression Mimics pny pnf Defects in SPL Transcript Accumulation and Responsiveness to GA

Given that pny pnf mutants misexpress BOP1/2, we used transcript profiling to test if dwarfism and late flowering exhibited by the gain-of-function bop1-6D mutant impact similar pathways. We first monitored the accumulation of miR156 and SPL transcripts in bop1-6D internodes for comparison with pny pnf using qRT-PCR (Fig. 7A). These data show that miR156 transcripts in bop1-6D are 1.4-fold up-regulated relative to the wild type. In addition, SPL transcripts in bop1-6D were significantly down-regulated, with the exception of SPL5. These data suggest that bop1-6D partially mimics pny pnf (compare Fig. 6C with Fig. 7A).

Figure 7.

BOP1 overexpression mimics pny pnf defects in SPL transcript accumulation and GA homeostasis. Plants were grown in continuous light. qRT-PCR was used to assess transcript accumulation in apices and/or internodes. A, Accumulation of miR156 and SPL transcripts in Col-0 and bop1-6D internodes. B, Schematic representation of non-13-hydroxylated GA biosynthetic and catabolic pathways in Arabidopsis (Hu et al., 2008; Yamaguchi, 2008). Green lettering indicates GA biosynthetic enzymes monitored for transcript accumulation in C and D. Red lettering indicates GA catabolic enzyme monitored for transcript accumulation in C and D. Bioactive GA₄ is indicated in bold. Inactive GA metabolites shown on right. CDP, ent-Copalyl diphosphate; CPS, ent-copalyl diphosphate synthase; GGDP, geranylgeranyl diphosphate; KAO, ent-kaurenoic acid oxidase; KO, ent-kaurene oxidase; KS, ent-kaurene synthase. C and D, Accumulation of GA pathway transcripts in pny pnf apices and bop1-6D apices and internodes. E, pny pnf and bop1-6D plants treated with 100 µm of GA₃ or a mock solution. F, Flowering time and plant height of Col-0 and bop1-6D plants treated with 100 µm of GA₃ or a mock solution. *, Significant differences (Student’s t test; P < 0.05).

To further explore similarities and differences between these two mutants, we examined transcripts involved in the regulation of GA, which is a positive regulator of internode elongation and flowering (Mutasa-Göttgens and Hedden, 2009; Porri et al., 2012). The expression levels of genes required for GA biosynthesis and catabolism and DELLA repressors of GA signaling were monitored by qRT-PCR in pny pnf apices and bop1-6D apices and internodes and revealed similar patterns (Fig. 7, B–D). In both genotypes, there was little or no change in ent-kaurene synthase (KS) transcript, but GA20ox1 transcripts were significantly increased (Fig. 7, C and D; Yamaguchi, 2008). In bop1-6D, there was a compensatory decrease in GA3ox1 transcripts functioning later in the biosynthetic pathway (Fig. 7D; Yamaguchi, 2008). In internodes, there was also a compensatory increase in GA2ox7 transcripts required in catabolism (Fig. 7, B and D; Yamaguchi, 2008). All five DELLAs encoding repressors of GA signaling were up-regulated in pny pnf, whereas selective up-regulation of REPRESSOR OF GA1-3 LIKE3 (RGL3) was observed in bop1-6D (Fig. 7, C and D). These data indicate that GA homeostasis is disrupted in both mutants. Nevertheless, deficiency alone does not account for phenotypic defects. Spray treatments with GA₃ failed to rescue flowering in pny and did not enhance internode elongation in bop1-6D, although this mutant flowered 4 d earlier than mock-treated control plants (Fig. 7, E and F; Smith et al., 2004). In conclusion, SPL transcript accumulation and responsiveness to GA are blocked in both mutants. We, therefore, used microarray analysis of bop1-6D internodes to identify additional factors that might antagonize flowering and internode elongation in these mutants.

Overexpression of BOP1 Activates Stress Pathways and Promotes Accumulation of JA as a Mechanism for Repression of Growth and Flowering

The transcriptomes of bop1-6D versus wild-type internodes were assessed by microarray (“Materials and Methods”). Gene Ontology (GO) analysis of differentially regulated genes revealed significant enrichment of terms associated with response to biotic and abiotic stress stimuli (Supplemental Table S1). Response to JA stimulus (GO:009753) was at the top of the list, but other hormone pathways associated with stress showed similar enrichment. In descending order, these were response to salicylic acid stimulus (GO:0009751), response to ethylene stimulus (GO:009723), and response to abscisic acid stimulus (GO:0099737). These data suggest that bop1-6D plants have heighted expression of stress-related genes. Trade-offs between plant defense and plant growth are well established in the recent literature (Navarro et al., 2008; Wild et al., 2012; Yang et al., 2012; Wild and Achard, 2013), and therefore, we further explored this mechanism. We specifically examined floral repressors in the microarray using a candidate gene approach (Fig. 8A). This analysis revealed up-regulation of DELLA, FLOWERING LOCUS C (FLC)-LIKE (FLC-like), and AP2-like members. However, the highest fold changes were observed among AP2/ETHYLENE RESPONSE FACTOR (ERF)-like factors that repress growth and flowering under stress conditions (Magome et al., 2004, 2008; Kang et al., 2011; for review, see Licausi et al., 2013). To validate these findings, selected transcripts were quantified by qRT-PCR using independently isolated tissue samples. Floral repressor transcript profiles of bop1-6D and pny pnf apices genotypes showed strong agreement (Fig. 8B). Consistent with the microarray, no significant change was observed for FLC, but transcripts encoding AP2-like repressors TARGET OF EAT2 (TOE2; 1.6- to 4-fold) and SCHLAFMUTZE (SMZ; 8.5- to 21-fold) were highly up-regulated compared with the wild type. The highest fold changes (6.2- to 454-fold) were observed for stress-induced AP2/ERF floral repressor transcripts, including DWARF AND DELAYED FLOWERING1 (DDF1) and DDF2, which encode proteins that inhibit growth by reducing bioactive GA content (Magome et al., 2004, 2008; Kang et al., 2011; for review, see Licausi et al., 2013).

Figure 8.

Transcript profiling of floral repressor genes in bop1-6D and pny pnf mutants. A, Floral repressor genes differentially expressed in bop1-6D compared with Col-0 internodes according to microarray experiment (“Materials and Methods”). B, Repressor transcript profile of bop1-6D and pny pny mutants quantified by qRT-PCR. No differential expression was observed for FLC transcript. Transcripts encoding AP2-like TOE2 and SMZ repressors and AP2/ERF Dehydration-responsive Element (DRE)-binding Protein-like TINY, DDF1, and DDF2 repressors were differentially up-regulated in agreement with A. *, Significant differences (Student’s t test; P < 0.05).

Inspection of the microarray also showed an increase in expression of biosynthetic enzymes for JA (Fig. 9, A and B). Validation of these data by qRT-PCR confirmed significant up-regulation of transcripts involved in JA biosynthesis in bop1-6D and pny pnf tissues (Fig. 9C). To determine if these increases reflect changes in hormone accumulation in plants, JA levels were quantified in internodes and buds from bop1-6D and pny pnf apices (“Materials and Methods”). BOP1-overexpressing plants showed 2.5-fold higher levels of JA relative to wild-type plants (Fig. 9D). Conversely, hormone levels were decreased in bop1 bop2 compared with wild-type control plants. pny pnf apices showed 1.5-fold higher levels of JA relative to wild-type control apices at the same stage of development (Fig. 9D). These data suggest that BOP1/2 promotes JA production.

Figure 9.

BOP1 overexpression increases JA content by transcriptional up-regulation of biosynthetic genes. A, JA-related genes differentially expressed in bop1-6D compared with Col-0 internodes identified by microarray experiment (“Materials and Methods”). B, Schematic representation of the JA biosynthetic pathway in Arabidopsis (Park et al., 2002; Wasternack and Hause, 2013). Red lettering indicates transcripts investigated by qRT-PCR in C. Linolenic acid is released from membrane lipids by a lipolytic enzyme (DEFECTIVE IN ANTHER DEHISCENCE1 [DAD1]) and converted to allene oxide (12,13-epoxy-octadecantrienoic acid) by lipoxygenase (LOX) and AOS. One cyclization, one reduction, and three rounds of β-oxidation steps are required in producing JA, which is conjugated to Ile (JA-Ile) in bioactive form (Wasternack and Kombrink, 2010). ACX, Acetyl-CoA oxidase; AOC, allene oxide cyclase; JAR1, JASMONATE RESISTANT1; KAT, l-3-ketoacyl CoA thiolase; MFP, multifunctional protein; OPR3/DDE1, 12-oxo-phytodienoic acid-10,11-reductase3/DELAYED DEHISCENCE1. C, Quantitative analysis of JA biosynthetic gene transcripts in bop1-6D and pny pnf mutants grown under SDs or LDs. *, Significant differences (Student’s t test; P < 0.05). D, Concentration of JA in wild-type tissues compared with bop1-6D, bop1 bop2, and pny pnf mutants (“Materials and Methods”).

To further examine JA effects on reproductive plant development, methyl jasmonate (MeJA) was applied to wild-type and pny plants grown under LDs (Fig. 10). Plants of both genotypes treated with MeJA developed a compact rosette with small dark green leaves, similar to those of bop1-6D mutants (Fig. 10, A–C). Wild-type plants treated with MeJA showed partial loss of apical dominance similar to pny mutants (Fig. 10, D–G). Plants in both treatment populations were late flowering with short internodes relative to mock-treated control plants (Fig. 10, D–G) and similar to pny pnf/+ mutants (Supplemental Fig. S1, A–G). Organ fusions or clusters were not observed. In both wild-type and pny populations, a small subset of plants developed a disordered rosette phenotype similar to pny pnf mutants and were nonflowering after 10 weeks (data not shown). No such defects were observed in mock-treated control plants. Thus, treatment of wild-type plants with exogenous MeJA mimics the phenotype of bop1-6D and pny or pny pnf/+ plants.

Figure 10.

Effect of loss or gain of JA content on phenotype of the wild type and mutants. A to G, Wild-type and pny plants were sprayed daily until maturity with 100 µm of MeJA or a mock solution. A, Mock-treated Col-0 plant. B, MeJA-treated Col-0 plant showing small dark green leaves. C, bop1-6D mutant showing a compact rosette similar to that in B. D, JA-treated Col-0 plants showing pny-like partial loss of apical dominance and short stature. E, JA-treated pny mutant showing enhancement of defects in internode elongation and apical dominance relative to mock control (see G). F, JA-treated pny mutant showing delayed flowering relative to mock control. G, Quantitative phenotypic analysis of wild-type and pny mutant plants treated with MeJA. Plants were grown under LDs. For both genotypes, treatment with MeJA resulted in additional rosette paraclades, indicating loss of apical dominance, reduced height, and delayed flowering. H to J, Effect of aos loss of function on pny pnf and bop1-6D phenotypes. Representative plants are shown. H, pny pnf aos mutant remains nonflowering. I and J, Phenotype of bop1-6D versus bop1-6D aos mutants. A small but highly significant (P < 0.0001) increase in plant height (+1.26 cm) and earlier flowering (−1.8 d) are measured in bop1-6D aos compared with bop1-6D control plants. Analysis was performed in a bop1-6D/+ aos/+ segregating population (n = 100). Bars = 1.5 cm. *, Significant differences (Student’s t test; P<0.05).

In parallel, we tested if reducing JA content rescues internode elongation or flowering in pny pnf and/or bop1-6D mutants by crossing them to the allene oxide synthase (aos) mutant, which is defective JA synthesis (Park et al., 2002; Figs. 7B and 10). Triple mutants with pny pnf remained nonflowering, even with addition of exogenous GA₃ (Fig. 10H; data not shown). However, quantitative analysis of bop1-6D aos double mutants revealed a small but significant (P ≤ 0.0001) increase in flowering time (+1.8 d) and plant height (+1.5 cm) compared with bop1-6D siblings in a segregating population (Fig. 10, I and J). These data provide evidence that modulation of growth by JA is a potential factor in conditioning bop1-6D and pny pnf phenotypic defects.

DISCUSSION

Floral evocation is dependent on SAM restructuring to form an IM (Bernier, 1988). The TALE homeodomain PNY and PNF transcription factors are essential for this process by permitting responsiveness to floral inductive signals (Smith et al., 2004; Kanrar et al., 2008; Lal et al., 2011; Smith et al., 2011; Ung et al., 2011; Ung and Smith, 2011). In pny pnf mutants, meristems support the production of leaves, but internode elongation and flower initiation are blocked.

In this article, we characterized the interaction of PNY and PNF with lateral organ boundary factor BOP1/2 and a pair of downstream effectors: the KNOX-BELL homeodomain factors KNAT6 and ATH1. We show that misexpression of these genes in pny pnf apices blocks floral evocation (Fig. 11). Inactivation of BOP1/2 and ATH1 or KNAT6 fully restores pny pnf defects in meristem and boundary maintenance and stem elongation and restores expression of floral meristem identity genes to allow flowering. Remarkably, other factors compensate for the loss of these genes in maintaining the SAM and responsiveness to floral inductive signals. Thus, PNY and PNF allow flowering by excluding boundary genes from the meristem. Similar antagonistic interactions for PNY or BP with members of the BOP1/2-ATH1/KNAT6 module function in various other developmental contexts, including abscission, fruit patterning, and inflorescence architecture (Ragni et al., 2008; Shi et al., 2011; Khan et al., 2012a, 2012b; Li et al., 2012).

Figure 11.

Summary and model. PNY-PNF/STM limits expression of BOP1/2 and downstream effector ATH1/KNAT6 to boundary domains flanking the IM. BOP1 acting through an unknown TGA bZIP cofactor directly activates ATH1, whereas promotion of KNAT6 is indirect (red arrow). These products form a module that represses growth, meristem activity, and flowering by increasing JA content by transcriptional promotion of JA biosynthetic genes. Either directly or indirectly (dashed lines), we propose that misexpression of this pathway leads to down-regulation of GA pathway components and repression of the miR156-SPL-miR172 module at one or more nodes in correlation with increased content of associated classes of floral repressors (e.g. DELLA, AP2-like, and AP2/ERF clades). Ultimately, SPL and FD/FT transcripts (not depicted) fail to accumulate, and activation of floral meristem identity genes LFY, AP1, and CAL required for flower initiation is blocked. Internode elongation is also blocked.

We further investigated the organization of this module and its transcriptional targets. Our data show that BOP1 is a direct regulator of ATH1, whereas promotion of KNAT6 is probably indirect. Indeed, DEX and CHX treatment of 35S:ATH1-GR plants produces rapid induction of KNAT6 transcript, and reporter gene expression is missing at boundaries in ath1-3 but not bop1 bop2 mutants, suggesting a direct requirement for ATH1 (data not shown). BOP1/2 coactivators are recruited to DNA through interactions with TGA bZIP transcription factors (Hepworth et al., 2005; Xu et al., 2010). These TGA factors remain unknown in the context of flowering, but several candidates are being investigated (Fig. 11). Transcript profiling was used to probe how this module blocks flowering. Comparison of the gain-of-function bop1-6D mutant and pny pnf showed similar transcriptional defects in core pathways controlling flowering. Our data are consistent with the model that BOP1/2-ATH1/KNAT6 boundary genes activate stress pathways that promote JA biosynthesis, which directly or indirectly interferes with signals integrated by the miR156-SPL-miR172 module to antagonize IM function (Fig. 11). Details of this model are discussed below.

The miR156-SPL-miR172 Module as a Hub for Integration of Flowering Signals

The miR156-SPL-miR172 module is a core pathway for integration of flowering signals, including age, sugar, GA, and stress (Huijser and Schmid, 2011; Cho et al., 2012; Proveniers, 2013; Cui et al., 2014; Stief et al., 2014; Wang, 2014). In brief, miR156 levels decline with age, leading to a concomitant increase in abundance of SPL transcripts with products that act on distinct targets in leaves and the shoot apex to promote flowering (Wu and Poethig, 2006; Wang et al., 2009; Wu et al., 2009). SPL3 and SPL9 members in the SAM directly promote the activation of floral meristem identity genes (Wu et al., 2009; Yamaguchi et al., 2009). SPL9-like members have additional functions in leaves, where they activate the transcription of miR172b, which lowers the abundance AP2-like floral repressor transcripts and allows accumulation of FT mRNA (Zhu and Helliwell, 2011; Matsoukas et al., 2012; Wang, 2014).

Significant reduction of miR156-regulated SPL transcripts was observed in pny pnf and bop1-6D mutants. This reduction is likely driven by multiple factors, including lower levels of FD, which recruits FT to the promoter of SPLs for activation (Jung et al., 2012; Andrés et al., 2015), and higher steady-state levels of miR156 (Lal et al., 2011). An increase in miR156 was less marked in bop1-6D, suggesting that the reduction in SPL transcript is mediated by miR156 and other regulators. These data are consistent with previous work showing that SPL3/4/5 transcripts are reduced in pny pnf apices and partly account for nonflowering (Lal et al., 2011). Transgenic pny pnf plants expressing an miR156-resistant form of SPL4 were restored for LFY and AP1 expression but only partly restored for flowering, suggesting that multiple SPL factors are involved (Lal et al., 2011).

Concomitantly, transcripts encoding miR172-regulated AP2-like repressors of flowering and internode elongation were elevated in bop1-6D and pny pnf mutants. This group of repressors includes AP2, SMZ, TOE1, TOE2, and TOE3 with overlapping functions (Aukerman and Sakai, 2003; Jung et al., 2007; Mathieu et al., 2009; Yant et al., 2010). SMZ and presumably, other members of this family delay flowering through the direct repression of FT and promotion of miR156 (Mathieu et al., 2009; Yant et al., 2010). Of these, TOE2 and SMZ show consistent up-regulation in the transcriptome of bop1-6D and pny pnf apices. Thus, overexpression of AP2-like members in bop1-6D may be a route to restricting internode elongation and flowering.

Integration with Signals for Stress and Carbohydrate Metabolism

Stress and sugar signals are also integrated through the miR156-SPL-miR172 module to control flowering (for review, see Wang, 2014). Recent studies address the mechanism. One study shows that miR156-SPL3 delays flowering under cool ambient temperatures by regulation of FT (Kim et al., 2012). Similarly, plants overexpressing miR156 are late flowering with increased tolerance to stress linked to down-regulation of SPL9 (Cui et al., 2014). Stief et al. (2014) further showed that heat stress induces miR156 isoforms linked to down-regulation of SPL9-like transcripts (SPL2, SPL9, and SPL11) and delayed flowering. Induction of miR156h in this cascade is predicted to target the pectin methylesterase inhibitor At5g38610 , which may affect bolting (Stief et al., 2014). PNY controls inflorescence patterning by regulating cell wall modification enzymes, including pectin methylesterases, which loosen cell walls in the stem to promote internode elongation and in the SAM to facilitate organ initiation (Etchells et al., 2012; Peaucelle et al., 2011). At5g38610 and related genes are up-regulated in the transcriptome of bop1-6D internodes, whereas PNY-regulated PECTIN METHYLESTERASE5 is down-regulated, consistent with dwarf stature (data not shown; Peaucelle et al., 2011).

The miR156-SPL-miR172 module is also a sensor for nutrients. A developmental decline in miR156 is partially mediated by sugars produced by photosynthesis that accumulate with age (Proveniers, 2013; Yang et al., 2013; Yu et al., 2013). Global transcript changes in bop1-6D mutants are characterized in large part by alterations in stress signaling and carbohydrate metabolism (Supplemental Table S1). GO enrichment analysis of the bop1-6D transcriptome identifies significant down-regulation of cellular carbohydrate metabolism, metabolic processes, and nitrogen metabolism, which potentially act to restrict Suc availability at the shoot apex (Supplemental Table S1). Parts of these changes were confirmed in pny pnf mutants, suggesting that resources are allocated toward defense in detriment to flowering.

Integration with GA Pathways

Our study also identifies GA pathway changes in bop1-6D and pny pnf mutants detrimental to flowering. In wild-type plants, bioactive GA content increases 100-fold at the transition (Eriksson et al., 2006), facilitating internode elongation and flowering by lowering the abundance of DELLA repressors (Mutasa-Göttgens and Hedden, 2009; Galvão et al., 2012; Porri et al., 2012; Yu et al., 2012). GA signals are partly integrated through the miR156-SPL-miR172 module based on studies showing that GA/DELLA regulates SPL3/4/6/9 transcription at the shoot apex independent of SOC1 (Galvão et al., 2012; Porri et al., 2012). Physical interaction of REPRESSOR OF GA1-3 DELLA with SPL9 interferes with activation of MADS-box flowering genes at the shoot apex and activation of miR172b in leaves, thereby maintaining AP2 and AP2-like repression of stem elongation and flowering (for review, see Wang, 2014). Other nodes of integration with the miR156-SPL-miR172 module are likely given so that GA treatment does not markedly accelerate flowering in an miR156 overexpression line (Yu et al., 2012). Transcriptional profiling in bop1-6D and pny pnf plants indicates complex changes affecting biosynthesis, catabolism, and/or signaling. Exogenous GA fails to restore flowering in pny pnf apices or internode elongation in bop1-6D, similar to transgenic plants overexpressing ATH1 (Smith et al., 2004; Gómez-Mena and Sablowski, 2008; this study) and consistent with blockage at multiple steps. Four of five DELLA transcripts are significantly up-regulated in pny pnf apices, whereas RGL3 is selectively up-regulated in bop1-6D. Transcript accumulation and steady-state level of protein show strong correlation in previous studies (Wild et al., 2012). Transgenic plants overexpressing DELLAs or DELLA proteins resistant to degradation are dwarf and late flowering, similar to bop1-6D plants (Dill et al., 2004; Hamama et al., 2012). RGL3, in particular, mediates cross talk between GA and JA pathways (Hou et al., 2013; Wild and Achard, 2013). JA selectively up-regulates RGL3, which binds to jasmonate ZIM-domain repressors of JA signaling to boost the immune response at the expense of growth (Wild et al., 2012; Wild and Achard, 2013).

JA Antagonism of Growth and Flowering

Our data raise the interesting possibility that JA antagonism of GA conditions bop1-6D and pny pnf phenotypic defects. GO analysis of differentially regulated genes in the bop1-6D transcriptome revealed significant enrichment of terms related to stress stimuli, including response to JA stimulus and to a lesser extent, responses to salicylic acid, ethylene, and abscisic acid stimuli, leading to the model that BOP1 overexpression reprioritizes the plant for defense at the expense of growth. Higher levels of JA biosynthetic gene transcripts and hormone are found in bop1-6D and pny pnf apices relative to wild-type control plants. These data support the findings by Canet et al. (2012), which identified BOP1/2 as essential for MeJA induced in priming for resistance to Pseudomonas syringae pv tomato DC3000. Plants exposed to high levels of jasmonate are stunted in growth of roots, leaves, and stems (Ellis et al., 2002; Cipollini, 2005; Bonaventure et al., 2007; Hyun et al., 2008; Zhang and Turner, 2008; Heinrich et al., 2013). Arabidopsis plants treated with jasmonate are also late flowering with short internodes and loss of apical dominance, giving an appearance similar to bop1-6D or pny pnf/+ mutants. Inhibitory effects of MeJA on flowering are also reported in Pharbitis nil (Maciejewska and Kopceiwicz, 2002; Maciejewska et al., 2004), Chenopodium rubrum (Albrechtová and Ullmann, 1994), and einkorn wheat (Triticum monococcum; Diallo et al., 2014). JA antagonism of growth or flowering has been linked to repression of GA biosynthesis (Magome et al., 2004; Heinrich et al., 2013), stabilization of DELLAs (Yang et al., 2012), and/or induction of AP2/ERF factors (Magome et al., 2008; Sun et al., 2008; Kang et al., 2011; Licausi et al., 2013). These data are consistent with JA contributing to bop1-6D and pny pnf developmental defects. Although inactivation of jasmonate biosynthesis by mutation of AOS fails to rescue flowering in pny pnf mutants, a small but significant increase in plant height and flowering time in bop1-6D supports this model.

Our data suggest that resources in pny pnf are reallocated toward defense at the expense of flowering and provide evidence for JA as a factor in modulating growth and meristem activity at boundaries.

MATERIALS AND METHODS

Plant Material and Growth Conditions

In the laboratory of S.R.H., Arabidopsis (Arabidopsis thaliana) plants were grown on soil or in vitro on minimal media (Haughn and Somerville, 1986) in growth chambers at 21°C under continuous light (24 h of light; intensity of 100 µmol m⁻² s⁻¹), LD (16 h of light), or SD (8 h of light) conditions. In the laboratory of V.P., plants were grown in LD (16 h of light; 150 μmol m⁻² s⁻¹) or SD (10 h of light; 1 h at 80 μmol m⁻² s⁻¹, 8 h at 130 μmol m⁻² s⁻¹, and 1 h at 80 μmol m⁻² s⁻¹) conditions. The wild type was the Columbia (Col-0) ecotype of Arabidopsis. Mutant lines were obtained from the Arabidopsis Biological Resource Center (https://abrc.osu.edu/) or the Nottingham Arabidopsis Stock Centre (http://arabidopsis.info/). The pny-40126 (SALK_40126), pnf-96116 (SALK_96116), bop1-3 (SALK_012994), bop2-1 SALK_075879), knat6-1 (SALK_047931), knat6-2 (SALK_054482), knat2-5 (SALK_099837), ath1-1 (GABI-KAT_114A12), and ath1-3 (SALK_113353) mutants have been described previously (Smith and Hake, 2003; Smith et al., 2004; Hepworth et al., 2005; Belles-Boix et al., 2006; Proveniers et al., 2007; Gómez-Mena and Sablowski, 2008). The ath1-4 mutant was a gift from Lin Xu (Li et al., 2012). 35S:BOP2 and bop1-6D overexpression lines were described previously (Norberg et al., 2005). The BOP1:GUS and BOP2:GUS reporter lines were described previously (McKim et al., 2008; Xu et al., 2010). The 35S:KNAT6 overexpression line was also described previously (Shi et al., 2011).

Plant Genetics

Primers and strategies used for genotyping bop1-3, bop2-1, knat6-2 (Khan et al., 2012b), pny-40126 (Smith and Hake, 2003), pnf-96116, pnf-33879 (Smith et al., 2004), knat6-1, knat2-5 (Ragni et al., 2008), ath1-1 (Proveniers et al., 2007), and ath1-3 (Gómez-Mena and Sablowski, 2008) have been previously described. For genotyping ath1-4, primers ath1-4dCAPS-F and ath1-4dCAPS-R were used to amplify a 198-bp product from genomic DNA. Only the ath1-4 product is cleaved by SspI to yield a 173-bp fragment. All mutant combinations were generated by crossing and confirmed by PCR genotyping. Primers are listed in Supplemental Table S2.

Phenotypic Analyses

For quantitative analysis of meristem arrest, seedlings were germinated on agar plates under SDs, transferred to soil on day 10, and scored for meristem arrest on day 25. Progenies from a selfed pny pnf/+ plant (n = 624) and a selfed knat2 pny pnf/+ plant (n = 146) were analyzed in parallel with wild-type plants and bop1 bop2 pny pnf, ath1 pny pnf, and knat6 pny pnf mutants (n = 144). Quantitative analyses of inflorescence phenotypes were performed with 8-week-old plants grown under LDs. Average height, internode length, and rosette paraclade number were determined for 10 plants per genotype as previously described (Ragni et al., 2008). Flowering time was scored for at least 24 plants per genotype by monitoring the date of apex emergence, because bop1 bop2 mutants initiate leaves at a reduced rate (Norberg et al., 2005). Seeds were germinated directly on soil under LDs. All phenotypic analyses were performed at least twice under independent growth conditions with similar results.

In Situ Hybridization and Localization of GUS Activity

Plants for analysis were grown under SDs for 3 weeks followed by 15 d in continuous light before harvesting tissue. We used in situ hybridization to monitor gene expression, because control sequences for expression of KNAT2:GUS and KNAT6:GUS reporters in IMs are missing (Khan et al., 2012b). Tissue fixation, embedding, and sectioning were carried out as described (Nikovics et al., 2006) with minor changes. Hybridization was performed overnight using the following buffer: 50% (v/v) formamide, 10% (w/v) dextran sulfate, 1× Denhardts, 0.3 m NaCl, 10 mm Tris HCl, pH 8, 1 mm EDTA, and 5 mg mL⁻¹ transfer RNA. Primers used to make KNAT6, KNAT2, BOP2, and ATH1 antisense probes are as listed in Supplemental Table S2.

Tissues were analyzed for BOP1:GUS activity as described (Sieburth and Meyerowitz, 1997) with minor changes. Stained tissues were embedded in Paraplast Plus (Sigma) processed using tert-butanol instead of xylenes. Sections (10 µm) were cut from embedded tissue, affixed to glass slides, and dewaxed with tert-butanol before imaging.

Construction of D35S:BOP1-GR, BOP1p-BOP1-GR, D35S:ATH1, and ATH1p:GUS Transgenic Lines

A translational fusion of BOP1 to the steroid-binding domain of the rat glucocorticoid receptor was generated. Treatment with DEX leads to translocation of the GR fusion protein from the cytoplasm to the nucleus as a way of controlling transcription factor activity (Lloyd et al., 1994). The BOP1 coding sequence lacking a stop codon was fused in frame to the GR fragment using overlap extension mutagenesis (Heckman and Pease, 2007). The resulting product was cloned into pCR-BluntII-TOPO (Invitrogen) to create B359. For all cloning steps involving amplification by PCR, iProof was used as the polymerase (BioRad), and cloned inserts were sequenced to ensure fidelity.

To create D35S:BOP1-GR, the BOP1-GR fusion gene present in B359 was amplified by PCR using CDS-BOP1-F and GR-R as the primers. The resulting product was modified to contain dATP overhangs and transferred to the Gateway-compatible entry vector pCR8/GW/TOPO (Invitrogen). LR clonase (Invitrogen) was used to move the insert to a pSM-3-based destination vector containing a double 35S Cauliflower mosaic virus (CaMV) promoter (D35S) and Nos terminator (pBAR, gift of C. Douglas). Wild-type plants were transformed by floral dipping (Clough and Bent, 1998) using the Agrobacterium spp. strain C58C1 pGV3101 pMP90 (Koncz and Schell, 1986). Hygromycin-resistant primary transformants were selected on agar plates containing 10 µm of DEX. After transfer to soil, plants were sprayed daily with 10 µm of DEX to induce nuclear localization of the BOP1-GR fusion protein. Homozygous progeny from one DEX-induced D35S:BOP1-GR line with a dwarf phenotype (line 9) was used for all subsequent experiments.

The D35S:BOP1-GR transgene failed to complement bop1 bop2 plants, presumably because the 35S CaMV promoter fails to provide the correct range of tissue expression. To confirm activity of the fusion protein and for use in ChIP experiments, the BOP1-GR fusion gene was expressed under control of the BOP1 native promoter in bop1 bop2 plants. The transgene was created in two steps. The BOP1 promoter present in pBOP1:GUS (McKim et al., 2008) was amplified by PCR using primers 4H-4kb-EcoR1-F1 and 4H-4kb-XmaI-R1 that incorporated restriction sites at their 5′ ends. The resulting product was digested with EcoR1 and XmaI and cloned into the corresponding sites of the binary vector pBAR (gift from laboratory of J. Dangl) to create B149. The BOP1-GR fusion gene present in B359 was amplified by PCR using primers XmaI-BOP1-F and BOP1-XmaI-R. The resulting product was digested with XmaI and cloned into the corresponding site of B149 to create pBAR/BOP1prom:BOP1-GR. The transgene was introduced into bop1 bop2 plants by floral dipping. Primary transformants resistant to glufosinate-ammonium were selected on soil using the herbicide FINALE (Farnam Companies). Three independent lines were used to assess complementation of bop1 bop2 mutant phenotypes. T2 seeds were sown on agar plates containing phosphinothricin with or without 5 µm of DEX. Plants were transferred to soil and sprayed daily with mock or DEX solution until maturity. Complementation of leaf, floral patterning, and floral organ abscission was observed in all DEX-treated lines (Supplemental Fig. S7).

To make the D35S:ATH1 transgene, the ATH1 coding sequence was amplified by PCR from cloned complementary DNA (cDNA) template using ATH1-CDS-F1 and ATH1-CDS-F1 as the primers. The resulting fragment was cloned into the entry vector pCR8/GW/TOPO and transferred into the pSM-3-based destination vector as described above. Wild-type plants were transformed by floral dipping. Transformants were selected on agar plates containing hygromycin. Phenotypes were scored in the T1 generation.

To create ATH1 promoter fusions to a GUS reporter gene, fragments containing 3.3 or 2 kb of sequence upstream of the ATH1 translation start site were amplified by PCR from genomic DNA template (BAC MSD21) and fused to the coding region of the beta-glucuronidase (uidA or GUS) gene. Primers incorporating BamHI and NcoI restriction sites at their 5′ ends facilitated directional cloning. Products were cloned into pCR-BluntII-TOPO for propagation. Inserts were released by digestion with BamHI and NcoI and ligated into the corresponding sites of pGCO:GUS (Hepworth et al., 2002). Agrobacterium spp. was cotransformed with pSOUP (Hellens et al., 2000). Wild-type plants were transformed by floral dipping, and glufosinate-ammonium-resistant primary transformants were selected on soil. Cloning primers are listed in Supplemental Table S2.

ChIP Experiments

ChIP was performed as described (Chakravarthy et al., 2003) using an anti-GR antibody (catalog no. 1002; Santa Cruz Biotechnology) and mock- or DEX-treated BOP1p:BOP1-GR bop1 bop2 plants grown under LDs. Seeds were germinated on agar plates containing phosphinothricin with or without 10 µm of DEX. After transfer to soil, plants were sprayed daily with mock (0.04% ethanol) or DEX solutions. Leaf tissue was collected from 4-week-old flowering plants for analysis. Quantification of immunoprecipitated DNA by qRT-PCR was performed as previously described (Boyle et al., 2009). Primers were as listed in Supplemental Table S3.

Microarray Experimental Design, Hybridization, and Analysis

Tissue for profiling was harvested from the first expanded internodes of wild-type and bop1-6D flowering plants grown under continuous light. RNA was extracted from four biological replicates per genotype using an RNeasy Plant Mini Kit (Qiagen). The mRNA was amplified according to the protocol described in the MessageAmp aRNA Kit (catalog no. 1750; Ambion). To produce incorporated antisense mRNA, aminoallyl-UTP was incorporated into the newly synthesized RNA; 3 µL of aminoallyl-UTP (50 mm) plus 2 µL of UTP (75 mm) instead of 4 µL of UTP were added during the aRNA amplification. Labeling, hybridization, and scanning were performed as described (Xiang et al., 2011). To normalize for bias in dye labeling, two biological replicates were labeled with [5′-³²P] cytosine-3′-P (Cy3), and two were labeled with Cy5. Experiments were carried out using Arabidopsis 70-mer oligo microarray slides (http://ag.arizona.edu/microarray). Two-color microarray data were preprocessed with the marray package (version 1.42.0) implemented in R/BioConductor (R Development Core Team; Gentleman et al., 2004; https://www.bioconductor.org) using the background correction method normexp (offset = 50) and normalize within arrays method loess. Differentially expressed genes were identified by P values, fold changes, and contrasts using linear models for microarrays (Smyth, 2005) and included a dye effect assessment implemented in R/BioConductor.

qRT-PCR

Total RNA was isolated using Trizol Reagent (Invitrogen) from dissected apices of the wild type and mutants. Plants grown under SDs were harvested on day 25 (SD) or transferred to LDs to induce flowering and harvested after 12 d (LD). Dissected apices were <0.5 cm tall, with the majority of surrounding leaves >0.2 cm removed. Tissues were collected in the subjective afternoon for all samples (after 9–12 h of light in a 16-h cycle). cDNA was generated using 1 µg of RNA as the template under following conditions: step 1: 70°C for 5 min; step 2: 50°C for 60 min; and step 3: 70°C for 15 min. qRT-PCR was carried out as described (Khan et al., 2012b) with the following changes. Reactions in triplicate containing 2 µL of 10-fold diluted cDNA, except for LFY and AP1 reactions, which required 4 µL of diluted cDNA, gene-specific primers (Supplemental Table S3), and POWER SYBR Green PCR Mastermix (Invitrogen) were carried out using a StepOnePlus Thermocycler (Applied Biosystems). GLYCERALDE-3-PHOSPHATE DEHYDROGENASE C was used as a normalization control. Quantification of miR156 mRNA was performed as described (Porri et al., 2012). Data shown are the average of three biological replicates conducted using separate growth trials and independently isolated RNA samples. Error bars indicate sem.

For DEX induction experiments, total RNA was prepared from internodes of 4-week-old flowering plants expressing the D35S:BOP1-GR transgene. Internodes were harvested from primary and secondary inflorescences of five to six plants starting at the bottom above the first silique and going all of the way up to where internodes were too small to collect. Tissue was excised with a new razor blade on parafilm, frozen in liquid nitrogen, and stored at −80°C until further analysis. Plants were treated continuously with mock (0.12% ethanol), 30 µm of DEX, 50 µm of CHX, or 30 µm of DEX and 50 µm of CHX for 2, 4, or 24 h by inverting inflorescences into containers of solution. For long-term treatments, seedlings were germinated on agar plates containing 10 µm of DEX. After transplanting to soil, plants were sprayed daily with a solution of mock (0.04% ethanol) or DEX for 4 weeks until tissue was harvested for RNA extraction. Values were normalized to EUKARYOTIC TRANSLATION INITIATION FACTOR 4A1 transcript (At3g13920), the mock control for DEX treatments, and the CHX control for DEX and CHX treatments to correct for negative effects of CHX on the transcription of BOP1 target genes (Jun et al., 2010; Nakamichi et al., 2010). Data shown are the average of three biological replicates conducted using independently isolated RNA samples. Error bars indicate sem.

Hormone Treatments

To analyze the effect of GA on growth, 10-d-old seedlings grown under continuous light were sprayed daily with GA (100 µm of GA₃ and 0.02% Silwett l-77) or a mock (0.02% Silwett l-77) solution until maturity (Hay et al., 2002). To examine the effect of JA on growth, 7-d-old seedlings grown under LDs were sprayed daily with MeJA (100 µm of MeJA and 0.02% Silwett l-77) or a mock (0.02% Silwett L-77) solution until maturity (Canet et al., 2012). MeJA-treated plants were covered with a plastic dome for 1 h after treatments, and solutions were made fresh once a week. Flowering time was determined by scoring the date of apex emergence. At least 24 plants per genotype were monitored.

JA Measurements

For measurement of JA, wild-type, bop1 bop2, and bop1-6D plants were grown for 6 to 7 weeks under LDs. Pools of 30 apices (buds and internodes) were used for each replicate (100 mg of fresh material). Wild-type and pny pnf plants were grown for 4 weeks under SDs. Pools of 30 apices (90 mg of fresh material) were used for each replicate. Three biological replicates were collected for each condition. Tissues were directly harvested in liquid nitrogen. Tissues were ground in liquid nitrogen and lyophilized. For each sample, 10 mg of freeze-dried powder was extracted with 0.8 mL of acetone:water:acetic acid (80:19:1, v/v/v) containing 2 ng of [5-2H] JA (CDN Isotopes CIL Cluzeau; Le Roux et al., 2014). The extract was vigorously shaken for 1 min, sonicated for 1 min at 25 Hz, shaken for 10 min at 4°C in a Thermomixer (Eppendorf), and then centrifuged (8,000g at 4°C for 10 min). The supernatants were collected, and the pellets were reextracted twice with 0.4 mL of the same extraction solution; then, they were vigorously shaken (1 min) and sonicated (1 min; 25 Hz). After the centrifugations, the three supernatants were pooled and dried (final volume of 1.6 mL). Each dry extract was dissolved in 140 µL of acetonitrile:water (50:50, v/v), filtered, and analyzed using a Waters Acquity Ultra Performance Liquid Chromatograph coupled to a Waters Xevo Triple Quadrupole Mass Spectrometer TQS. The compounds were separated on a reverse-phase column (100 mm × 2.1 mm × 3 µm particle size; Uptisphere C18 UP3HDO; Interchim) using a flow rate of 0.4 mL min⁻¹ and a binary gradient: 0.1% (v/v) acetic acid in water and acetonitrile with 0.1% acetic acid. For JA, the following binary gradient (0.1% [v/v] acetic acid in water) was used: 0 min, 98%; 3 min, 70%; 7.5 min, 50%; 8.5 min, 5%; 9.6 min, 0%; 13.2 min, 98%; and 15.7 min, 98%. Mass spectrometry was conducted in electrospray and multiple reaction monitoring scanning mode in negative ion mode. Relevant instrumental parameters were set as follows: capillary, 1.5 kV (negative mode); source block and desolvation gas temperatures, 130°C and 500°C, respectively. Nitrogen was used to assist the cone and desolvation (150 and 800 L h⁻¹, respectively). Argon was used as the collision gas at a flow of 0.18 mL min⁻¹. The parameters used for multiple reaction monitoring quantification of JA are described in Le Roux et al., 2014. Samples were reconstituted in 140 µL of 50:50 (v/v) acetonitrile:water per 1 mL of injected volume. The JA limit of detection and limit of quantification were extrapolated from calibration curves and samples using the Quantify module of MassLynx software (version 4.1). The amount of JA was expressed as a ratio of peak areas (209 > 62/214 > 62) per dry weight because of impurities contained in the D5 JA standard.

Sequence data from this article can be found in the EMBL/GenBank data libraries under accession numbers At1g70510 (KNAT2), At1g23380 (KNAT6), At5g02030 (PNY), At2g27990 (PNF), At3g57130 (BOP1), At2g41370 (BOP2), and At4g32980 (ATH1).

Supplemental Data

The following supplemental materials are available.

• Supplemental Figure S1. Ectopic expression of KNAT6 and ATH1 mimics pny and pny pnf/+ phenotype.

• Supplemental Figure S2. ATH1 map and characterization of mutant alleles.

• Supplemental Figure S3. Phenotypes of other mutant combinations.

• Supplemental Figure S4. Quantitative phenotypic analyses of bop1 bop2 pny pnf, ath1 pny pnf, and knat6 pny pnf mutants.

• Supplemental Figure S5. BOP1:GUS expression in Col-0 and pny pnf apices.

• Supplemental Figure S6. BOP2, ATH1, KNAT2, and KNAT6 expression in pny and pnf apices.

• Supplemental Figure S7. Complementation of bop1 bop2 mutant by BOP1p::BOP1-GR construct.

• Supplemental Table S1. GO classification of differentially expressed genes in bop1-6D versus Col-0 internode microarrays.

• Supplemental Table S2. List of general primers.

• Supplemental Table S3. List of primers for qRT-PCR.

Glossary

cDNA

complementary DNA

ChIP

chromatin immunoprecipitation

CHX

cycloheximide

Col-0

Columbia-0

DEX

dexamethasone

GO

Gene Ontology

IM

inflorescence meristem

JA

jasmonic acid

LD

long day

MeJA

methyl jasmonate

qRT

quantitative reverse transcription

SAM

shoot apical meristem

SD

short day