Skip to main content
NCBI home page
The Plant Cell logoLink to The Plant Cell
. 2020 Apr 2;32(6):1919–1932. doi: 10.1105/tpc.20.00012

Control of Plant Branching by the CUC2/CUC3-DA1-UBP15 Regulatory Module

Yu Li a,b,1, Tian Xia a,b,1, Fan Gao a,b, Yunhai Li a,b,2
PMCID: PMC7268791  PMID: 32245753

CUC2/CUC3, DA1, and UBP15 function in a common pathway to control the initiation of axillary meristems, thereby determining plant branching in Arabidopsis.

Abstract

Lateral branches are important for plant architecture and production, but how plants determine their lateral branches remains to be further understood. Here, we report that the CUP-SHAPED COTYLEDON2 (CUC2)/CUC3-DA1-UBIQUITIN-SPECIFIC PROTEASE15 (UBP15) regulatory module controls the initiation of axillary meristems, thereby determining the number of lateral branches in Arabidopsis (Arabidopsis thaliana). Mutation in the ubiquitin-dependent peptidase DA1 causes fewer lateral branches due to defects in the initiation of axillary meristems. The transcription factors CUC2 and CUC3, which regulate the axillary meristem initiation, directly bind to the DA1 promoter and activate its expression. Further results show that UBP15, which is a direct substrate of DA1 peptidase, represses the initiation of axillary meristems. Genetic analyses support that CUC2/CUC3, DA1, and UBP15 function, at least in part, in a common pathway to regulate the initiation of axillary meristems. Therefore, our findings establish a genetic and molecular framework by which the CUC2/CUC3-DA1-UBP15 regulatory module controls the initiation of axillary meristems, thereby determining plant architecture.

INTRODUCTION

The diversity of forms in flowering plants is mainly decided by the number, position, and shape of lateral organs in the aerial part of plants; these lateral organs arise from the shoot apical meristem (SAM) and other secondary meristems (Pautler et al., 2013). Branches are important lateral organs that affect plant architecture and plant yield (Wang and Li, 2008; Wang et al., 2018). A branch originates from a secondary meristem in the axil of leaves, referred to as the axillary meristem (AM; Grbic and Bleecker, 2000). The AM can develop into an axillary bud and then form a branch (Schmitz and Theres, 2005; Domagalska and Leyser, 2011). In some conditions, the AMs develop into axillary buds but remain dormant (Napoli et al., 1999). The axillary bud can later develop into a complete branch, depending on environmental and developmental cues. Therefore, the development of branches can be divided into two main steps: the initiation of AMs and the outgrowth of axillary buds (Tantikanjana et al., 2001). Strigolactones act as a major player in regulating bud outgrowth (Domagalska and Leyser, 2011; Waters et al., 2012; Brewer et al., 2013; Smith and Li, 2014; Flematti et al., 2016). Auxins and strigolactones negatively regulate the outgrowth of axillary buds (McSteen and Leyser, 2005; Brewer et al., 2015), and cytokinins function antagonistically with auxins and strigolactones to promote the outgrowth of axillary buds (Müller and Leyser, 2011; Janssen et al., 2014; Barbier et al., 2019).

graphic file with name TPC_202000012DR1_fx1.jpg

Open in a new tab

Several transcription factors influence the initiation of AMs, thereby influencing the formation of plant branches. LATERAL SUPPRESSOR (LS) in tomato (Solanum lycopersicum), LATERAL SUPPRESSOR in Arabidopsis (Arabidopsis thaliana; LAS), and MONOCULM1 (MOC1) in rice (Oryza sativa), which encode the GRAS transcription factors (Greb et al., 2003), regulate AM initiation. The tomato BLIND gene, which encodes an MYB transcription factor, promotes the AM initiation (Schmitz et al., 2002). Its Arabidopsis homologs REGULATORS OF AXILLARY MERISTEMS act redundantly to promote the establishment of AMs during both vegetative and reproductive growth phases (Keller et al., 2006; Müller et al., 2006). NAC transcriptional factors CUP-SHAPED COTYLEDON (CUC) regulate the AM initiation in leaf axils of rosette leaves in Arabidopsis (Hibara et al., 2006; Raman et al., 2008). In addition, MYB transcription factors LATERAL ORGAN FUSION1 (LOF1) and LOF2 and classIII homeodomain/leucine zipper transcription factor REVOLUTA affect multiple growth and developmental processes including the AM initiation (Otsuga et al., 2001; Lee et al., 2009). Recent studies further uncovered the gene regulatory network in the AM initiation through genome-scale analyses (Tian et al., 2014). CUC2 and LAS work as two hubs of this gene regulatory network. CUC2 proteins directly bind to the promoter of LAS and activate its expression (Hibara et al., 2006; Raman et al., 2008; Tian et al., 2014). An auxin minimum has been proposed to be required for the AM initiation (Wang et al., 2014a, 2014b). Cytokinin biosynthesis and signaling also affect normal AM initiation (Wang et al., 2014b; Müller et al., 2015). Therefore, these studies show that multiple factors and signaling pathways influence the initiation of AMs.

We have previously reported that DA1, a ubiquitin-dependent peptidase, regulates seed and organ size in Arabidopsis (Li et al., 2008; Xia et al., 2013; Du et al., 2014; Dong et al., 2017). Here, we uncover a function for DA1 in the regulation of the AM initiation. The da1-1 single mutant forms fewer lateral branches due to defects in the initiation of AMs. The transcription factors CUC2 and CUC3 associate with the promoter of DA1 and activate the expression of DA1. The mutation in the ubiquitin-specific protease UBP15, which is a direct substrate of DA1 peptidase, promotes the AM initiation. Genetic analyses demonstrate that CUC2/CUC3, DA1, and UBP15 function, at least in part, in a common pathway to regulate the initiation of AMs. Therefore, our findings reveal that the CUC2/CUC3-DA1-UBP15 regulatory module controls the initiation of AMs, thereby influencing plant architecture in Arabidopsis.

RESULTS

The da1-1 Mutant Produces Fewer Branches

We have previously shown that DA1 regulates seed and organ size by influencing cell proliferation in Arabidopsis (Li et al., 2008). The da1-1 mutant also produced fewer branches compared with the wild type (Columbia [Col-0]) in long-day conditions (Supplemental Figure 1A). We therefore investigated the development of axillary buds in 45-d-old Col-0 and da1-1 rosette leaf axils in long-day conditions (Supplemental Figure 1B). In most Col-0 plants, the axils of the first pair of rosette leaves contained axillary buds. By contrast, in most da1-1 plants, we did not observe axillary buds in the leaf axils of the first pair of rosette leaves. In addition, the development of axillary buds was delayed in da1-1 (Supplemental Figure 1B).

When plants were first grown in short-day conditions for 28 d and then moved to long-day conditions, the axillary branches in da1-1 were strongly reduced compared with those in the wild type (Figure 1A). Most wild-type plants contained axillary buds in the axils of the third to late rosette leaves, although a majority of the wild-type plants had no axillary buds in the axils of the first pair of leaves (Figure 1D). By contrast, most da1-1 plants had no axillary buds in the axils of the first to sixth rosette leaves (Figure 1D). We frequently observed that da1-1 lacked axillary buds in the axils of late rosette leaves (Figures 1B and 1D). Axillary buds were also absent from a few cauline leaf axils in da1-1 (Figures 1C and 1D). These results indicate that DA1 regulates the formation of axillary branches.

Figure 1.

Figure 1.

Open in a new tab

da1-1 Mutant Produces Fewer Branches.

(A) Plants of Col-0, da1-1, da1-ko1 dar1-1, DA1COM, and 35S:DA1R/K#3. The da1-1 mutant produces fewer branches than the wild type (Col-0). Plants were grown for 28 d in short photoperiods and subsequently grown for 60 d under long-day conditions.

(B) Close-up views of rosette leaf branches in Col-0, da1-1, da1-ko1 dar1-1, DA1COM, and 35S:DA1R/K#3 plants. The da1-1, da1-ko1 dar1-1, and 35S:DA1R/K produce fewer branches than Col-0.

(C) Close-up views of cauline leaf branches in Col-0, da1-1, da1-ko1 dar1-1, DA1COM, and 35S:DA1R/K#3 plants. Cauline leaves of Col-0 form branches in their axils, while da1-1, da1-ko1 dar1-1, and 35S:DA1R/K#3 frequently failed to produce branches.

(D) Schematic representation of axillary branch or bud formation in individual leaf axils of Col-0, da1-1, da1-ko1 dar1-1, DA1COM, and 35S:DA1R/K#3 plants. The thick black horizontal line represents the border between the youngest rosette leaf and the oldest cauline leaf, with positions of progressively younger cauline leaves above the line, and positions of progressively older rosette leaves below it. Each column represents a single plant, and each square within a column represents an individual leaf axil shown from youngest (top) to oldest. Green indicates the presence of an axillary bud, and yellow indicates the absence of an axillary bud in any particular leaf axil. Plants were grown for 28 days in short photoperiods and subsequently shifted to long days (n ≥ 13).

Bar in (A) and (B) = 1 cm; bar in (C) = 1 mm.

The da1-1 allele encodes a mutant protein DA1R358K, which negatively affects the function of DA1 and its closest homolog DAR1, although da1-1/+ heterozygous plants do not show obvious alterations in seed- and organ-size phenotypes (Li et al., 2008). Double da1-knockout1 (da1-ko1) dar1-1 T-DNA insertion mutants exhibit large seeds and organs, like those observed in the da1-1 mutant, while da1-ko1 and dar1-1 single mutants do not show an obvious large seed phenotype (Li et al., 2008). We examined whether da1-ko1, dar1-1, and da1-ko1 dar1-1 double mutations affected the formation of axillary branches. The da1-ko1 dar1-1 double mutant also exhibited fewer lateral branches compared to the wild type (Figures 1A and 1D). Axillary buds were absent from a few rosette and cauline leaf axils in da1-ko1 dar1-1 (Figures 1B and 1C). The da1-ko1 and dar1-1 single mutants did not show an obvious branch-defect phenotype (Supplemental Figures 1C to 1E). As overexpression of the DA1R358K protein (35S:DA1R/K) in the wild type has a dominant-negative effect on organ growth (Li et al., 2008), we investigated whether 35S:DA1R/K plants had the defects in the formation of axillary buds. Compared with the wild-type plants, 35S:DA1R/K plants exhibited fewer branches and axillary buds (Figures 1A to 1D; Supplemental Figure 2). By contrast, overexpression of DA1 (35S:DA1) very slightly increased the branch number compared with the Col-0 plants (Supplemental Figure 3). A genomic fragment of DA1 (DA1COM) complemented the reduced axillary bud phenotype of da1-1 (Figures 1A to 1D; Li et al., 2008). These results demonstrate that DA1 and its closest homolog DAR1 regulate the formation of lateral branches.

da1-1 Represses the AM Initiation

AMs are formed in rosette and cauline leaf axils and develop into axillary buds and branches in response to environmental and internal signals. Considering that da1-1 represses the formation of axillary buds, DA1 could regulate the AM initiation. To test this hypothesis, we examined leaf axils in da1-1 using scanning electron microscopy. The meristem-like structure was not observed in da1-1 rosette leaf axils that did not produce axillary buds (Figure 2A). These results demonstrate that DA1 and its closest homolog DAR1 are involved in the regulation of the AM initiation.

Figure 2.

Figure 2.

Open in a new tab

DA1 Regulates the Initiation of Axillary Meristems.

(A) Scanning electron micrographs of a Col-0 rosette leaf axil showing an axillary bud (left, arrow) and a barren da1-1 rosette leaf axil (right, arrow).

(B) DA1 expression activity was monitored using pDA1:GUS transgenic plants. GUS activity is detected in longitudinal section through shoot apex during vegetative development (left, plants grown for 28 d under short-day conditions) and reproductive development (right, plants grown for 28 d under short-day conditions and subsequently shifted to long-day conditions for 6 d).

(C) pSTM:GUS expression in longitudinal sections of Col-0 and da1-1. GUS signal is reduced in leaf axils of da1-1 plants. Plants were grown for 28 d in short photoperiods.

Bar in (A) = 200 μm; bar in (B) and (C) = 20 μm.

The AM is marked by the expression of the SHOOT MERSTEMLESS (STM) gene (Long and Barton, 2000). Considering that DA1 influences the AM initiation, we asked whether the expression of STM could be affected by the da1-1 mutation. We crossed da1-1 with the pSTM:GUS reporter and generated da1-1;pSTM:GUS plants. β-Glucuronidase (GUS) activity in the pSTM:GUS line was detected in the boundary zone between the SAM and leaf primordia (Figure 2C; Kirch et al., 2003; Lee et al., 2009). By contrast, in da1-1;pSTM:GUS plants, GUS activity was reduced in the center of the leaf axils that failed to produce axillary buds (Figure 2C). These results further demonstrate that DA1 controls the initiation of AMs. We then examined whether DA1 could be expressed in the boundary regions using the pDA1:GUS transgenic line (Li et al., 2008). GUS activity in the pDA1:GUS line was detected in the shoot meristem, young leaves, the region between the shoot meristem and leaf primordia, and the axils of cauline leaves (Figure 2B). As the region between leaf primordia and the shoot meristem will later form leaf axils, expression of DA1 is consistent with its role in the AM initiation.

DA1 Acts, at Least in Part, in a Common Pathway with CUC2/CUC3 to Control the Initiation of AMs

Several genes have been described to regulate AM initiation in Arabidopsis, such as LAS, REGULATORS OF AXILLARY MERISTEMS (RAXs), and CUCs (Greb et al., 2003; Hibara et al., 2006; Keller et al., 2006; Müller et al., 2006; Raman et al., 2008). The las mutants generate almost no branches during vegetative development (Greb et al., 2003). We obtained the las-101 mutant and observed that nearly all axils of rosette leaves in las-101 lacked axillary buds (Figure 3; Supplemental Figure 4B; Hibara et al., 2006). The axils of cauline leaves in las-101 can form branches normally, and most of these branches were fused with main stems (Figure 3; Supplemental Figure 4C), consistent with a previous study (Hibara et al., 2006). We crossed las-101 with da1-1 to generate the da1-1 las-101 double mutant. All axils of rosette leaves in da1-1 las-101 failed to form the axillary buds, and most axils of cauline leaves in da1-1 las-101 lacked axillary buds, although the las-101 single mutation did not affect the AM initiation in axils of cauline leaves (Figure 3; Supplemental Figures 4A to 4C), suggesting that the las-101 mutation synergistically enhanced the AM initiation-defect phenotype of da1-1 in both rosette leaf axils and cauline leaf axils. These data also suggest that DA1 may act redundantly with LAS to control the AM initiation during vegetative and reproductive development.

Figure 3.

Figure 3.

Open in a new tab

DA1 Acts Genetically with CUC2 and CUC3 to Control the Initiation of Axillary Meristems.

(A) Schematic representation of axillary branch or bud formation in individual leaf axils of Col-0, da1-1, las-101, da1-1 las-101, rax1-3, da1-1 rax1-3, cuc2-3, da1-1 cuc2-3, cuc3-105, and da1-1 cuc3-105 plants. The thick black horizontal line represents the border between the youngest rosette leaf and the oldest cauline leaf, with positions of progressively younger cauline leaves above the line and positions of progressively older rosette leaves below the line. Each column represents a single plant, and each square within a column represents an individual leaf axil shown from youngest (top) to oldest. Green indicates the presence of an axillary bud, yellow indicates the absence of an axillary bud in any particular leaf axil, and brown indicates branches fused to the main stem. Plants were grown for 28 d in short photoperiods and subsequently shifted to long days (n ≥ 19).

(B) Frequency of the lack of axillary shoots in the Col-0, da1-1, cuc2-3, da1-1 cuc2-3, cuc3-105, da1-1 cuc3-105, las-101, da1-1 las-101, rax1-3, and da1-1 rax1-3 plants (n ≥ 19). Values are given as mean ± sd. **P < 0.01, compared with da1-1 plants (Student’s t test; Supplemental Data Set). ns, no significance.

In Arabidopsis, MYB transcription factors RAX1, RAX2, and RAX3 act redundantly to control the initiation of AMs (Keller et al., 2006; Müller et al., 2006). The rax1 mutant exhibits a moderate defect in the AM initiation of rosette leaf axils, whereas rax2 and rax3 single mutants do not show a clear defect (Keller et al., 2006), indicating that RAX1 plays a predominant role in determining the initiation of AMs. We therefore crossed da1-1 with rax1-3 to generate the da1-1 rax1-3 double mutant. Consistent with previous results, the rax1-3 mutant frequently failed to initiate axillary meristems in the rosette leaf axils (Figure 3; Supplemental Figure 4E), while rax1-3 showed normal AM initiation in the axils of cauline leaves (Figure 3; Supplemental Figure 4F; Keller et al., 2006). By contrast, the da1-1 rax1-3 double mutant completely lacked axillary buds in rosette leaf axils, and da1-1 rax1-3 also failed to initiate AMs in the axils of most cauline leaves (Figure 3; Supplemental Figures 4D to 4F). These genetic analyses reveal a synergistic interaction between DA1 and RAX1 in the regulation of the AM initiation during vegetative and reproductive development.

NAC transcription factors CUC1, CUC2, and CUC3 have been reported to participate in diverse plant developmental processes, such as boundary formation and AM initiation during postembryonic development (Aida et al., 1997; Hibara et al., 2006). The cuc2-3 mutation moderately represses the AM initiation in rosette leaf axils, while cuc3-105 strongly suppresses the AM initiation in rosette leaf axils and also causes branches to be fused to main stems (Figure 3; Hibara et al., 2006; Raman et al., 2008). By contrast, cuc1 single mutations do not affect AM initiation (Hibara et al., 2006). To understand genetic interactions of DA1 with CUC genes, we crossed da1-1 with cuc2-3 and cuc3-105 and generated da1-1 cuc2-3 and da1-1 cuc3-105 double mutants, respectively. The phenotype of da1-1 cuc2-3 in the AM initiation was more like that observed in the da1-1 single mutant during vegetative and reproductive development (Figure 3). The phenotype of the da1-1 cuc3-105 mutant in AM initiation during vegetative development was more like that observed in the cuc3-105 single mutant, although the da1-1 cuc3-105 double mutant had a slightly stronger phenotype than the cuc3-105 single mutant (Figure 3). The da1-1 cuc3-105 double mutant also lacked AMs in a few cauline leaf axils and had branches fused to main stems (Figure 3). In addition, overexpression of DA1 obviously repressed the AM initiation defect of cuc2-3 and cuc3-105 (Supplemental Figures 5B to 5D). These genetic analyses suggest that DA1 functions, at least in part, with CUC2 and CUC3 in a common pathway to regulate the initiation of AMs during vegetative growth stage.

CUC2 and CUC3 Directly Bind to the Promoter of DA1 and Activate Its Expression

DA1 functions genetically with CUC2 and CUC3 to control the AM initiation, and CUC2 and CUC3 are two NAC transcription factors. We therefore asked whether CUC2 and CUC3 could regulate the expression of DA1. We generated the chemically inducible CUC2 (pER8:CUC2) and CUC3 (pER8:CUC3) transgenic plants. The expression levels of DA1 were examined after treating pER8:CUC2 and pER8:CUC3 transgenic plants with β-estradiol (Figure 4A). The expression of DA1 was significantly elevated after β-estradiol induction. In addition, the expression of DA1 in cuc2-3, cuc3-105, and cuc2-3 cuc3-105 mutants was decreased compared with that in the wild type (Figure 4B). The expression of DAR1 in cuc2-3, cuc3-105, and cuc2-3 cuc3-105 mutants was also decreased compared with that in the wild type (Supplemental Figures 5A). These results indicate that CUC2 and CUC3 regulate the expression of DA1.

Figure 4.

Figure 4.

Open in a new tab

CUC2 and CUC3 Bind to the Promoter of DA1 and Promote Its Expression.

(A) RT-qPCR analysis of DA1 expression levels in pER8:CUC2, pER8:CUC3, and pER8:CUC2;pER8:CUC3 seedlings before (–) and after β-estradiol (+) treatment. ACTIN2 mRNA was used as an internal control. Means were calculated from four biological samples. Values are given as mean ± sd relative to the values before β-estradiol treatment, set at 1. **P < 0.01 (Student’s t test; Supplemental Data Set).

(B) RT-qPCR analysis of DA1 expression level in Col-0, cuc2-3, cuc3-105, and cuc2-3 cuc3-105 rosette leaf axils. Plants were grown for 28 d in short photoperiods. All rosette leaves were removed, and the base region of petioles and the nonelongated stems were used to isolate total mRNA. ACTIN2 mRNA was used as an internal control. Means were calculated from four biological samples. Values are given as mean ± sd relative to Col-0 values, set at 1. *P < 0.05, **P < 0.01 compared with Col-0 plants (Student’s t test; Supplemental Data Set).

(C) A 2.5-kb promoter region of DA1 upstream of its ATG codon contains five predicted binding sequences named A to E. A, B, C, D, and E and A-m, B-m, C-m, D-m, and E-m indicate the wild-type probes and the mutated probe used in the EMSA, respectively (detailed sequences are listed in the Supplemental Table). PFs represent PCR fragments used for ChIP-qPCR analysis. As the sequence C is very close to sequence D, these two sequences were contained in one fragment PF4. PF2, PF3, and PF5 contain the predicted binding sequence A, B, and E, respectively.

(D) ChIP-qPCR analysis shows that CUC2 and CUC3 bind to the promoter fragments of DA1. Chromatin from 35S:myc, 35S:myc-CUC2, and 35S:myc-CUC3 transgenic plants were immunoprecipitated by anti-myc, and the enrichment of the fragments was determined by qPCR. Means were calculated from three biological samples. Values are given as mean ± sd. *P < 0.05, **P < 0.01 compared with 35S:myc transgenic plants (Student’s t test; Supplemental Data Set).

(E) EMSA experiments showed that CUC2 directly binds to the promoter of DA1. The biotin-labeled probe A and MBP-CUC2 formed a DNA–protein complex, but the mutated probe A-m and MBP-CUC2 did not. The retarded DNA–protein complex was reduced by the competition using the unlabeled probe A, but not reduced by the competition using the unlabeled mutated probe A-m.

(F) EMSA experiments showed that CUC3 directly binds to the promoter of DA1. The biotin-labeled probe A and MBP-CUC3 formed a DNA–protein complex, but the mutated probe A-m and MBP-CUC3 did not. The retarded DNA–protein complex was reduced by the competition using the unlabeled probe A, but not reduced by the competition using the unlabeled mutated probe A-m.

To determine whether CUC2 and CUC3 can directly associate with the DA1 promoter, a chromatin immunoprecipitation (ChIP) assay using 35S:myc-CUC2, 35S:myc-CUC3, and 35S:myc transgenic plants was performed. The core sequence CGT[GA] is the DNA binding site of NAC domain transcription factors (Olsen et al., 2005). We identified five candidate binding sequences (A to E) of NAC transcription factors in the 2-kb region of the DA1 promoter (Figure 4C). A ChIP assay showed that the fragments (PF2, PF3, and PF4) containing the CGT[GA] motif were enriched in the chromatin-immunoprecipitated DNA generated with an anti-myc antibody (Figure 4D). However, the fragment PF5 was not enriched in the chromatin-immunoprecipitated DNA. We also did not detect significant enrichment of the fragments PF1 and PF6, which did not contain the CGT[GA] sequence. Thus, CUC2 and CUC3 associate with the promoter region of DA1 in vivo.

To investigate whether CUC2 and CUC3 could directly bind to DA1 promoter, we expressed CUC2 and CUC3 as MBP fusion proteins MBP-CUC2 and MBP-CUC3 and performed DNA electrophoretic mobility shift assays (EMSAs). MBP-CUC2 and MBP-CUC3 could bind to four biotin-labeled probes (A to D) containing the CGT[GA] sequence (Figures 4E and 4F; Supplemental Figures 6A and 6B). However, the DNA–protein association was decreased in a dosage-dependent manner when adding the corresponding unlabeled probes. On the contrary, MBP-CUC2 and MBP-CUC3 failed to bind versions of the probes with mutations (A-m to D-m) in the predicted binding sequence (Figures 4E and 4F; Supplemental Figures 6A and 6B). These results demonstrate that CUC2 and CUC3 directly bind to the promoter of DA1 and promote the expression of DA1.

UBP15 Acts Downstream of DA1 to Control the AM Initiation

To identify downstream components of DA1 in the control of the AM formation, a genetic screen was conducted to isolate the suppressors of da1-1 in the formation of branches (sdb). One of the suppressors, sdb1-1, repressed the reduced axillary-branch phenotype of da1-1 (Figure 5A). The sdb1-1 da1-1 plants produced more axillary branches than the da1-1 single mutant (Figure 5A). The axillary bud formation was further examined in axils of sdb1-1 da1-1 cauline and rosette leaves. As shown in Figure 5B, the pattern of axillary bud formation in the sdb1-1 da1-1 plants was similar to that in wild-type plants, indicating that the sdb1-1 mutation suppresses the defect of da1-1 in the AM initiation.

Figure 5.

Figure 5.

Open in a new tab

UBP15 Acts Downstream of DA1 to Repress the Initiation of Axillary Meristems.

(A) Plants of Col-0, da1-1, and sdb1-1 da1-1. Plants were cultivated in short-day conditions for 28 d and then transferred to long-day conditions.

(B) Schematic representation of axillary branch or bud formation in individual leaf axils of Col-0, da1-1, and sdb1-1 da1-1 plants. Green and yellow boxes show the presence or absence of an axillary bud in leaf axils along the main shoot, respectively. Plants were grown for 28 d in short photoperiods and subsequently shifted to long days (n ≥ 24).

(C) UBP15 gene structure. The start codon (ATG) and the stop codon (TAG) are indicated. Closed boxes indicate the coding sequence, open boxes indicate the 5′ and 3′ untranslated regions, and lines between boxes indicate introns. The mutation site of sdb1-1 is shown.

(D) Fifty-five-day-old plants of Col-0, da1-1, ubp15-1, and da1-1 ubp15-1. Plants were cultivated in short-day conditions for 28 d and then transferred to long-day conditions.

(E) Close-up views of rosette leaf branches in rosette leaf axils of Col-0, da1-1, ubp15-1, and da1-1 ubp15-1.

(F) Close-up views of cauline leaf branches in cauline leaf axils of Col-0, UBP15OE#1, and UBP15OE#2.

(G) Schematic representation of axillary branch or bud formation in individual leaf axils of Col-0, da1-1, ubp15-1, da1-1 ubp15-1, UBP15OE#1, and UBP15OE#2 (n ≥ 15).

(H) pSTM:GUS expression in longitudinal sections of Col-0 and UBP15OE. GUS signal is reduced in leaf axils of UBP15OE plants. Plants were grown for 28 d in short photoperiods.

(I) UBP15 expression activity was monitored using pUBP15:GUS transgenic plants. GUS activity was detected in longitudinal sections through the vegetative shoot apex. Plants were grown for 28 d in short-day conditions.

Bar in (A), (D), and (E) = 1 cm; bar in (F) = 1 mm; bar in (H) and (I) = 20 μm.

We mapped the sdb1-1 mutation and observed that the primary mapping region contained UBIQUITIN-SPECIFIC PROTEASE15 (UBP15)/SUPPRESSOR2 OF DA1 (SOD2; Figure 5C; Supplemental Figure 7A). We previously showed that the organ growth phenotype of da1-1 can be suppressed by mutations in UBP15 (Du et al., 2014). DA1 mediates the degradation of UBP15 by physically interacting with UBP15 and cleaving UBP15 (Dong et al., 2017). These results imply that SDB1 could be the UBP15 gene. Indeed, sequence analyses revealed that sdb1-1 contained a Gly-to-Ala substitution in the 3′ intron–exon boundary of exon 9 in UBP15, resulting in altered splicing of UBP15 mRNA (Figure 5C; Supplemental Figures 7B to 7D).

We then asked whether the T-DNA insertion mutant ubp15-1 could influence the formation of axillary buds. As shown in Figures 5D and 5G, the number of axillary buds in ubp15-1 was only slightly more than that in Col-0, indicating that the ubp15-1 single mutant very slightly affects the AM initiation. We further investigated the axillary buds of plants overexpressing UBP15 (UBP15OE; Du et al., 2014). As shown in Figure 5G, overexpression of UBP15 caused the reduced number of axillary buds in both rosette and cauline leaf axils, as observed in da1-1, indicating that UBP15 represses the initiation of AMs. STM expression was not clearly detected in the leaf axils of UBP15OE that failed to produce axillary buds (Figure 5H), further supporting the role of UBP15 in the initiation of AMs. Consistent with the role of UBP15 in the formation of AMs, UBP15 expression was detected in the shoot meristem, young leaves, and the region between the shoot meristem and leaf primordia (Figure 5I; Du et al., 2014). We further investigated whether ubp15-1 could suppress the AM initiation phenotype of da1-1. As expected, ubp15-1 completely suppressed the axillary meristem–absence phenotype of da1-1 (Figures 5D and 5G), indicating that ubp15-1 was epistatic to da1-1 with respect to the AM initiation. These results indicate that UBP15 acts downstream of DA1 to control the AM initiation.

UBP15 Acts Genetically with CUC2/CUC3 and LAS to Control the Initiation of AMs

Considering that CUC2 and CUC3 associate with the promoter of DA1 and promote its expression (Figure 4) and DA1 acts upstream of UBP15 to influence the formation of AMs (Figure 5), we presumed that CUC2 and CUC3 could act in a common pathway with UBP15 to regulate the formation of AMs. To test this, we crossed ubp15-1 with cuc2-3 and cuc3-105 and identified ubp15-1 cuc2-3 and ubp15-1 cuc3-105 double mutants, respectively. As shown in Figure 6, ubp15-1 repressed the AM initiation defect of cuc2-3 and cuc3-105, respectively. The ubp15-1 cuc2-3 and ubp15-1 cuc3-105 double mutants produced normal axillary buds like those observed in the wild type. In addition, cuc3-105 plants showed branches that were fused to the main stems during reproductive development, whereas the ubp15-1 cuc3-105 double mutants did not show the phenotype of branches fused to main stems during reproductive development (Figure 6). These genetic results imply that UBP15 may function in the same pathway with CUC2 and CUC3 to regulate the initiation of AMs.

Figure 6.

Figure 6.

Open in a new tab

UBP15 Acts in a Common Pathway with CUC2 and CUC3.

(A) Schematic representation of axillary branch or bud formation in individual leaf axils of Col-0, ubp15-1, cuc2-3, ubp15-1 cuc2-3, cuc3-105, ubp15-1 cuc3-105, rax1-3, rax1-3 ubp15-1, las-101, and las-101 ubp15-1 plants (n ≥ 13). The thick black horizontal lines represent the border between the youngest rosette leaf and the oldest cauline leaf, with positions of progressively younger cauline leaves above the line, and positions of progressively older rosette leaves below the line. Each column represents a single plant, and each square within a column represents an individual leaf axil shown from youngest (top) to oldest. Green indicates the presence of an axillary bud, and yellow indicates the absence of an axillary bud in any particular leaf axil. Plants were grown for 28 d in short photoperiods and subsequently shifted to long days (n ≥ 13).

(B) Frequency of the lack of axillary shoots in the Col-0, ubp15-1, cuc2-3, ubp15-1 cuc2-3, cuc3-105, ubp15-1 cuc3-105, rax1-3, rax1-3 ubp15-1, las-101, and las-101 ubp15-1 plants (n ≥ 13). Values are given as mean ± sd. **P < 0.01 compared with da1-1 plants (Student’s t test). ns, no significance.

DA1 acts synergistically with LAS and RAX1 to promote the initiation of AMs, suggesting that DA1 may act redundantly with LAS and RAX1 to control AM formation. We then asked whether UBP15 could genetically interact with LAS and RAX1 to affect AM initiation. To test this, we crossed ubp15-1 with las-101 and rax1-3 and isolated las-101 ubp15-1 and rax1-3 ubp15-1 double mutants, respectively. The ubp15-1 mutation repressed the AM initiation defect of las-101 during vegetative development (Figure 6). By contrast, the ubp15-1 mutation did not obviously repress the AM initiation defect of rax1-3. The las-101 plants showed branches that were fused to main stems during reproductive development. Interestingly, las-101 ubp15-1 double mutants did not exhibit the phenotype of branches fused to main stems during reproductive development (Figure 6), indicating that ubp15-1 suppresses the concaulescence phenotype of las-101. These findings suggest that UBP15 genetically interacts, at least in part, with LAS to influence the boundary formation.

DISCUSSION

Lateral branches of plants arise from AMs in the region between the SAMs and leaf primordia and are important for plant architecture. The initiation of AMs is a crucial step for generating axillary branches. Several genes have been identified to affect the AM initiation in Arabidopsis (Greb et al., 2003; Hibara et al., 2006; Keller et al., 2006; Müller et al., 2006; Raman et al., 2008), but the mechanisms of AM initiation are still largely unknown. Here, we uncover a genetic and molecular framework in which a CUC2/CUC3-DA1-UBP15 regulatory module controls the formation of AMs, thereby determining plant architecture.

The ubiquitin-dependent peptidase DA1 has been described to negatively control organ growth in Arabidopsis (Li et al., 2008; Dong et al., 2017). Here, we observed that the da1-1 mutation disrupted AM initiation in the axils of rosette and cauline leaves, resulting in fewer axillary branches. Supporting the function of DA1 in the initiation of AMs, expression of the meristem marker gene STM in da1-1 was undetectable in the axils of leaves that did not produce axillary buds (Figure 2C). DA1 is expressed in the SAM and the region between SAM and leaf primordium (Figure 2B). These results demonstrate that DA1 positively regulates AM initiation. DA1 has been reported to control organ growth by influencing cell proliferation (Li et al., 2008). How DA1 regulates both cell proliferation and the AM establishment is an interesting question. Considering that DA1 limits cell proliferation and promotes cell differentiation, it is possible that the balance between cell proliferation and cell differentiation might be crucial for the initiation of AMs in the leaf axils. It will be a difficult but worthwhile challenge to investigate this possibility in the future.

NAC transcription factors CUC2 and CUC3 are positive regulators of the AM initiation (Hibara et al., 2006). The genetic analyses suggest that DA1 functions, at least in part, in a common pathway with CUC2 and CUC3 to regulate the AM initiation (Figure 3). Consistent with this, we showed that CUC2 and CUC3 directly associate with the promoter region of DA1 and activate its expression (Figure 4). We further investigated the genetic interaction of DA1 with LAS and RAX1 and observed that DA1 functions synergistically with LAS and RAX1 in the regulation of AM initiation (Figure 3; Supplemental Figure 4), suggesting that DA1 may function redundantly with LAS and RAX1 to regulate the formation of AMs. To identify downstream components of DA1 in the AM formation, we isolated suppressors of da1-1 in the formation of branches and concluded that one of them is a novel allele of the ubiquitin-specific protease UBP15.

We have previously demonstrated that DA1 interacts with and cleaves UBP15, resulting in its degradation (Du et al., 2014; Dong et al., 2017). In accordance with this biochemical data, ubp15-1 completely suppressed the defect of the AM initiation of da1-1, indicating that UBP15 acts downstream of DA1 to control the initiation of AMs (Figure 5). upb15-1 also suppressed the AM initiation defect of cuc2-3 and cuc3-105, respectively (Figure 6). These results suggest that CUC2/CUC3, DA1, and UBP15 function in a common pathway to control the initiation of AMs. By contrast, upb15-1 only slightly suppressed the AM initiation defect of las-101. Interestingly, ubp15-1 completely suppressed the phenotype of branches fused to main stems in las-101 (Figure 6). These findings suggest that UBP15 might have partially overlapping function with LAS in the regulation of the boundary formation. It is also possible that UBP15 may slightly mediate the effect of LAS on the initiation of AMs.

Based on our data, we propose a working model that CUC2 and CUC3 directly associate with the promoter of DA1 and activate its expression and that DA1 then interacts with UBP15 and promotes the degradation of UBP15 to regulate the initiation of AMs (Figure 7). Considering that the cuc2 cuc3 double mutant has stronger defects in the initiation of AMs than da1-1 (Hibara et al., 2006), it is plausible that CUC2 and CUC3 may have other downstream targets. Supporting this notion, LAS has been proposed to act downstream of CUC2 (Hibara et al., 2006; Raman et al., 2008; Tian et al., 2014). DA1 functions redundantly with LAS to control the initiation of AMs. Taken together, our findings point to a genetic and molecular framework in which CUC2/CUC3-DA1-UBP15 regulatory module–mediated control of the AM initiation influences plant lateral branch number.

Figure 7.

Figure 7.

Open in a new tab

CUC2/CUC3-DA1-UBP15 Regulatory Module-Mediated Control of Axillary Meristem Initiation.

CUC2 and CUC3 directly bind to the promoter region of DA1 and activate its expression. DA1 is a positive regulator of the initiation of AMs. DA1 promotes the degradation of UBP15 that represses the initiation of AMs. DA1 functions redundantly with LAS to promote the initiation of AMs. UBP15 also partially mediates the effect of LAS on the initiation of AMs.

The number of lateral branches is an important component of plant architecture, which has been recognized as a target for breeding. It is believed that crops with ideal plant architecture will be highly efficient in their use of light, nutrients, and space and should produce high yields (Wang and Li, 2008). Although the detailed standards of ideal plant architecture are different among different kinds of crops, all models contain the requirement for an appropriate number of lateral branches. In this study, we have identified CUC2/CUC3-DA1-UBP15 as a regulatory module for lateral branches. Therefore, it will be worthwhile to investigate whether homologs of CUC2, CUC3, DA1, and UBP15 could be used to improve plant architecture in key crops.

METHODS

Plant Material and Growth Conditions

The las-101 (SALK_000896), cuc3-105 (ABI_302G09), cuc2-3 (SAIL_605_C09), rax1-3 (SALK_071748), and ubp15-1 (SALK_018601) mutants were obtained from Nottingham Arabidopsis Stock Centre and Arabidopsis Biological Resources Center (Hibara et al., 2006). The da1-1, da1-ko1 dar1-1, DA1COM, 35S:DA1R/K, 35S:DA1, pDA1:GUS, pUBP15:GUS, pSTM:GUS, UBP15OE, and da1-1 ubp15-1 mutants were described previously (Li et al., 2008; Du et al., 2014). The da1-1 las-101, da1-1 rax1-3, da1-1 cuc2-3, and da1-1 cuc3-105 double mutants were generated by crossing da1-1 with las-101, rax1-3, cuc2-3, and cuc3-105, respectively. The ubp15-1 cuc2-3, ubp15-1 cuc3-105, rax1-3 ubp15-1, and las-101 ubp15-1 double mutants were generated by crossing ubp15-1 with cuc2-3, cuc3-105, rax1-3, and las-101, respectively. The sdb1-1 da1-1 double mutant was isolated from an M2 population of da1-1 treated with ethyl methanesulfonate. T-DNA insertion mutants were identified by PCR with primers listed in the Supplemental Table. Seeds were sterilized using 10% (v/v) household bleach for 10 min, washed with sterile water at least three times, stratified for 3 d in dark at 4°C before plating on half-strength Murashige and Skoog (MS) medium with 1% Glu, and germinated at 22°C. For short-day conditions, seedlings were grown at 22°C with 70% RH, and an 8-h-light/16-h-dark cycle in a controlled environmental chamber. For long-day conditions, seedlings were grown at 22°C and a 16-h-light/8-h-dark cycle in a controlled environmental chamber or in a greenhouse. The light intensity was ∼120 μmol m−2·s−1 provided by white fluorescent tubes.

Constructs and Transformation

To generate 35S:myc-CUC2 and 35S:myc-CUC3, the coding sequences of CUC2/CUC3 were amplified with primers listed in the Supplemental Table (myc-CUC2-F/R and myc-CUC3-F/R). The products were then cloned into the BamHI/SacI sites of pCAMBIA1300-221-myc. 35S:myc-CUC2 and 35S:myc-CUC3 plasmids were transformed into Arabidopsis cuc2-3 and cuc3-105 mutants plants using Agrobacterium tumefaciens GV3101. To generate pER8:CUC2 and pER8:CUC3, the coding sequences of CUC2/CUC3 were amplified with primers listed in the Supplemental Table (pER8-CUC2-F/R and pER8-CUC3-F/R). The products were then cloned into the XhoI/PacI sites of pER8 vector. The plasmids were transformed into Arabidopsis (Arabidopsis thaliana) Col-0 plants using A. tumefaciens GV3101. Transgenic plants were grown on half-strength MS medium with 1% Glu containing hygromycin (30 μg/mL).

RNA Isolation and RT-qPCR Analysis

RNAprep pure plant kit was used to extract the total RNA (Tiangen). Total mRNA was reverse transcribed into cDNA using SuperScript III reverse transcriptase (Invitrogen). The Bio-Rad CFX96 machine was used for RT-qPCR analysis using RealStar Green Fast Mixture (A301-05, GenStar). The relative expression level of gene was evaluated using the cycle threshold method. ACTIN2 was used as the reference gene. Seedlings (28 d old) were grown under short-day photoperiods. After all rosette leaves of seedlings were removed, the base region of petioles and the nonelongated stems were used for RNA extraction. Gene-specific primers (Supplemental Table) were used for RT-PCR and RT-qPCR. Error bars are derived from four independent biological experiments, each run in triplicate.

Analysis of Axillary Shoot and Bud Formation

For the analysis of shoot formation, plants were grown under short-day photoperiods for 28 d before being transferred to the long-day conditions. The formation of bud or branch in leaf axils was analyzed at 14 d after the onset of flowering. The shoot or bud formation was investigated by stereomicroscope. For each experiment, at least 13 plants were investigated. Each experiment was repeated at least three times.

Scanning Electron Microscopy

Scanning electron microscopy was performed using a Hitachi S-3000N variable pressure scanning electron microscope. Samples were fixed in formalin–acetic acid–alcohol solution by vacuum treatment for 30 min. Fixed samples were dehydrated with a gradual ethanol series, dried by critical-point drying, and subsequently coated with a gold layer.

GUS Staining

For the GUS activity analysis of pDA1:GUS and pUBP15:GUS transgenic plants, seedlings were grown for 28 d in short-day conditions. The method for GUS staining was used as previously described by Sessions et al. (1999).

ChIP Assay

ChIP assay was performed according to protocols previously described by Gendrel et al. (2005), with minor modifications. The transgenic seeds of 35S:myc-CUC2, 35S:myc-CUC3, and 35S:myc were grown on GM medium for 10 d. The plants were harvested and cross-linked with 1% (v/v) formaldehyde. The chromatin complexes were isolated and sonicated. The chromatin was immunoprecipitated by protein A+G magnetic beads (16-663, Merck Millipore) and anti-myc (ab32, lot no. GR310953-4, Abcam). The precipitated DNA was purified and dissolved in water. Primers used for the ChIP assay are listed in the Supplemental Table (PF1-F, PF1-R, PF2-F, PF2-R, PF3-F, PF3-R, PF4-F, PF4-R, PF5-F, PF5-R, PF6-F, PF6-R, ACTIN7 -F, and ACTIN7 -R).

EMSA

The coding sequences of CUC2 and CUC3 were amplified using primers (MBP-CUC2-F/R and MBP-CUC3-F/R) and then cloned into the EcoRI/PstI sites of pMAL-c2 to generate the MBP-CUC2/CUC3 constructs. The primers are listed in the Supplemental Table. Bacterial lysates expressing MBP-CUC2/CUC3 were extracted from Escherichia coli BL21 (DE3). Bacterial cells were lysed using the PBS buffer (2.7 mM KCl, 137 mM NaCl, 1.4 mM KH2PO4, and 4.3 mM Na2HPO4, pH 7.4) and then sonicated. The MBP-CUC2 and MBP-CUC3 proteins were purified using amylose resin (E8021L, New England Biolabs). EMSAs were performed according to the method previously described by Zhang et al. (2015). The biotin-labeled probes were synthesized. MBP-fusion proteins were incubated with biotin-labeled probes for 30 min at 22°C. The competition experiments were performed by adding 10- to 1000-fold unlabeled DNA.

Accession Numbers

The accession numbers are as follows: CUC2 (At5g53950), CUC3 (At1g76420), DA1 (At1g19270), DAR1 (At4g36860), SOD2/UBP15 (At1g17110), LAS (At1g55580), and RAX1 (At5g23000).

Supplemental Data

DIVE Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

Acknowledgments

We thank Jiayang Li for his critical reading of this article. We thank Masao Tasaka, Nottingham Arabidopsis Stock Centre, and Arabidopsis Biological Resources Center for las-101, cuc2-3, cuc3-105, and rax1-3 seeds and Nam-Hai Chua for the pER8 vector. This work was supported by the National Natural Science Foundation of China (grants 3181101602, 31961133001, and 31425004) and the strategic priority research program of the Chinese Academy of Sciences (grant XDB27010102).

AUTHOR CONTRIBUTIONS

Yu.L., T.X., and F.G. performed experiments. Yu.L., T.X., and Y.L. analyzed data. Yu.L. and Y.L. wrote the article.

References

  1. Aida M., Ishida T., Fukaki H., Fujisawa H., Tasaka M.(1997). Genes involved in organ separation in Arabidopsis: An analysis of the cup-shaped cotyledon mutant. Plant Cell 9: 841–857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barbier F.F., Dun E.A., Kerr S.C., Chabikwa T.G., Beveridge C.A.(2019). An update on the signals controlling shoot branching. Trends Plant Sci. 24: 220–236. [DOI] [PubMed] [Google Scholar]
  3. Brewer P.B., Dun E.A., Gui R., Mason M.G., Beveridge C.A.(2015). Strigolactone inhibition of branching independent of polar auxin transport. Plant Physiol. 168: 1820–1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brewer P.B., Koltai H., Beveridge C.A.(2013). Diverse roles of strigolactones in plant development. Mol. Plant 6: 18–28. [DOI] [PubMed] [Google Scholar]
  5. Domagalska M.A., Leyser O.(2011). Signal integration in the control of shoot branching. Nat. Rev. Mol. Cell Biol. 12: 211–221. [DOI] [PubMed] [Google Scholar]
  6. Dong H., et al. (2017). Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis. Genes Dev. 31: 197–208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Du L., Li N., Chen L., Xu Y., Li Y., Zhang Y., Li C., Li Y.(2014). The ubiquitin receptor DA1 regulates seed and organ size by modulating the stability of the ubiquitin-specific protease UBP15/SOD2 in Arabidopsis. Plant Cell 26: 665–677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Flematti G.R., Scaffidi A., Waters M.T., Smith S.M.(2016). Stereospecificity in strigolactone biosynthesis and perception. Planta 243: 1361–1373. [DOI] [PubMed] [Google Scholar]
  9. Gendrel A.V., Lippman Z., Martienssen R., Colot V.(2005). Profiling histone modification patterns in plants using genomic tiling microarrays. Nat. Methods 2: 213–218. [DOI] [PubMed] [Google Scholar]
  10. Grbic V., Bleecker A.B.(2000). Axillary meristem development in Arabidopsis thaliana. Plant J. 21: 215–223. [DOI] [PubMed] [Google Scholar]
  11. Greb T., Clarenz O., Schafer E., Muller D., Herrero R., Schmitz G., Theres K.(2003). Molecular analysis of the LATERAL SUPPRESSOR gene in Arabidopsis reveals a conserved control mechanism for axillary meristem formation. Genes Dev. 17: 1175–1187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hibara K., Karim M.R., Takada S., Taoka K., Furutani M., Aida M., Tasaka M.(2006). Arabidopsis CUP-SHAPED COTYLEDON3 regulates postembryonic shoot meristem and organ boundary formation. Plant Cell 18: 2946–2957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Janssen B.J., Drummond R.S., Snowden K.C.(2014). Regulation of axillary shoot development. Curr. Opin. Plant Biol. 17: 28–35. [DOI] [PubMed] [Google Scholar]
  14. Keller T., Abbott J., Moritz T., Doerner P.(2006). Arabidopsis REGULATOR OF AXILLARY MERISTEMS1 controls a leaf axil stem cell niche and modulates vegetative development. Plant Cell 18: 598–611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kirch T., Simon R., Grünewald M., Werr W.(2003). The DORNROSCHEN/ENHANCER OF SHOOT REGENERATION1 gene of Arabidopsis acts in the control of meristem cell fate and lateral organ development. Plant Cell 15: 694–705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lee D.K., Geisler M., Springer P.S.(2009). LATERAL ORGAN FUSION1 and LATERAL ORGAN FUSION2 function in lateral organ separation and axillary meristem formation in Arabidopsis. Development 136: 2423–2432. [DOI] [PubMed] [Google Scholar]
  17. Li Y., Zheng L., Corke F., Smith C., Bevan M.W.(2008). Control of final seed and organ size by the DA1 gene family in Arabidopsis thaliana. Genes Dev. 22: 1331–1336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Long J., Barton M.K.(2000). Initiation of axillary and floral meristems in Arabidopsis. Dev. Biol. 218: 341–353. [DOI] [PubMed] [Google Scholar]
  19. McSteen P., Leyser O.(2005). Shoot branching. Annu. Rev. Plant Biol. 56: 353–374. [DOI] [PubMed] [Google Scholar]
  20. Müller D., Leyser O.(2011). Auxin, cytokinin and the control of shoot branching. Ann. Bot. 107: 1203–1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Müller D., Schmitz G., Theres K.(2006). Blind homologous R2R3 Myb genes control the pattern of lateral meristem initiation in Arabidopsis. Plant Cell 18: 586–597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Müller D., Waldie T., Miyawaki K., To J.P.C., Melnyk C.W., Kieber J.J., Kakimoto T., Leyser O.(2015). Cytokinin is required for escape but not release from auxin mediated apical dominance. Plant J. 82: 874–886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Napoli C.A., Beveridge C.A., Snowden K.C.(1999). Reevaluating concepts of apical dominance and the control of axillary bud outgrowth. Curr. Top. Dev. Biol. 44: 127–169. [DOI] [PubMed] [Google Scholar]
  24. Olsen A.N., Ernst H.A., Lo Leggio L., Skriver K.(2005). DNA-binding specificity and molecular functions of NAC transcription factors. Plant Sci. 169: 785–797. [Google Scholar]
  25. Otsuga D., DeGuzman B., Prigge M.J., Drews G.N., Clark S.E.(2001). REVOLUTA regulates meristem initiation at lateral positions. Plant J. 25: 223–236. [DOI] [PubMed] [Google Scholar]
  26. Pautler M., Tanaka W., Hirano H.Y., Jackson D.(2013). Grass meristems I: Shoot apical meristem maintenance, axillary meristem determinacy and the floral transition. Plant Cell Physiol. 54: 302–312. [DOI] [PubMed] [Google Scholar]
  27. Raman S., Greb T., Peaucelle A., Blein T., Laufs P., Theres K.(2008). Interplay of miR164, CUP-SHAPED COTYLEDON genes and LATERAL SUPPRESSOR controls axillary meristem formation in Arabidopsis thaliana. Plant J. 55: 65–76. [DOI] [PubMed] [Google Scholar]
  28. Schmitz G., Theres K.(2005). Shoot and inflorescence branching. Curr. Opin. Plant Biol. 8: 506–511. [DOI] [PubMed] [Google Scholar]
  29. Schmitz G., Tillmann E., Carriero F., Fiore C., Cellini F., Theres K.(2002). The tomato Blind gene encodes a MYB transcription factor that controls the formation of lateral meristems. Proc. Natl. Acad. Sci. USA 99: 1064–1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sessions A., Weigel D., Yanofsky M.F.(1999). The Arabidopsis thaliana MERISTEM LAYER 1 promoter specifies epidermal expression in meristems and young primordia. Plant J. 20: 259–263. [DOI] [PubMed] [Google Scholar]
  31. Smith S.M., Li J.(2014). Signalling and responses to strigolactones and karrikins. Curr. Opin. Plant Biol. 21: 23–29. [DOI] [PubMed] [Google Scholar]
  32. Tantikanjana T., Yong J.W., Letham D.S., Griffith M., Hussain M., Ljung K., Sandberg G., Sundaresan V.(2001). Control of axillary bud initiation and shoot architecture in Arabidopsis through the SUPERSHOOT gene. Genes Dev. 15: 1577–1588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tian C., et al. (2014). An organ boundary-enriched gene regulatory network uncovers regulatory hierarchies underlying axillary meristem initiation. Mol. Syst. Biol. 10: 755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wang B., Smith S.M., Li J.(2018). Genetic regulation of shoot architecture. Annu. Rev. Plant Biol. 69: 437–468. [DOI] [PubMed] [Google Scholar]
  35. Wang Q., Kohlen W., Rossmann S., Vernoux T., Theres K.(2014a). Auxin depletion from the leaf axil conditions competence for axillary meristem formation in Arabidopsis and tomato. Plant Cell 26: 2068–2079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wang Y., Li J.(2008). Molecular basis of plant architecture. Annu. Rev. Plant Biol. 59: 253–279. [DOI] [PubMed] [Google Scholar]
  37. Wang Y., Wang J., Shi B., Yu T., Qi J., Meyerowitz E.M., Jiao Y.(2014b). The stem cell niche in leaf axils is established by auxin and cytokinin in Arabidopsis. Plant Cell 26: 2055–2067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Waters M.T., Brewer P.B., Bussell J.D., Smith S.M., Beveridge C.A.(2012). The Arabidopsis ortholog of rice DWARF27 acts upstream of MAX1 in the control of plant development by strigolactones. Plant Physiol. 159: 1073–1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Xia T., Li N., Dumenil J., Li J., Kamenski A., Bevan M.W., Gao F., Li Y.(2013). The ubiquitin receptor DA1 interacts with the E3 ubiquitin ligase DA2 to regulate seed and organ size in Arabidopsis. Plant Cell 25: 3347–3359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhang Y., Du L., Xu R., Cui R., Hao J., Sun C., Li Y.(2015). Transcription factors SOD7/NGAL2 and DPA4/NGAL3 act redundantly to regulate seed size by directly repressing KLU expression in Arabidopsis thaliana. Plant Cell 27: 620–632. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Plant Cell are provided here courtesy of Oxford University Press

RESOURCES