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. 2022 Oct 10;190(4):2484–2500. doi: 10.1093/plphys/kiac437

DWARF AND ROBUST PLANT regulates plant height via modulating gibberellin biosynthesis in chrysanthemum

Xue Zhang 1, Lian Ding 2, Aiping Song 3, Song Li 4, Jiayou Liu 5, Wenqian Zhao 6, Diwen Jia 7, Yunxiao Guan 8, Kunkun Zhao 9, Sumei Chen 10, Jiafu Jiang 11, Fadi Chen 12,✉
PMCID: PMC9706434  PMID: 36214637

Abstract

YABBY (YAB) genes are specifically expressed in abaxial cells of lateral organs and determine abaxial cell fate. However, most studies have focused on few model plants, and the molecular mechanisms of YAB genes are not well understood. Here, we identified a YAB transcription factor in chrysanthemum (Chrysanthemum morifolium), Dwarf and Robust Plant (CmDRP), that belongs to a distinct FILAMENTOUS FLOWER (FlL)/YAB3 sub-clade lost in Brassicaceae. CmDRP was expressed in various tissues but did not show any polar distribution in chrysanthemum. Overexpression of CmDRP resulted in a semi-dwarf phenotype with a significantly decreased active GA3 content, while reduced expression generated the opposite phenotype. Furthermore, plant height of transgenic plants was partially rescued through the exogenous application of GA3 and Paclobutrazol, and expression of the GA biosynthesis gene CmGA3ox1 was significantly altered in transgenic plants. Yeast one-hybrid, luciferase, and chromatin immunoprecipitation-qPCR analyses showed that CmDRP could directly bind to the CmGA3ox1 promoter and suppress its expression. Our research reveals a nonpolar expression pattern of a YAB family gene in dicots and demonstrates it regulates plant height through the GA pathway, which will deepen the understanding of the genetic and molecular mechanisms of YAB genes.

A distinct FlL/YAB3 subclade gene DWARF AND ROBUST PLANT regulates plant height by binding to the CmGA3ox1 promoter modulating gibberellin biosynthesis in chrysanthemum.

Introduction

The YABBY (YAB) genes, characterized by a Cys2/Cys2 zinc finger domain in the N-terminal and the YAB domain in the C-terminal, encode a small family specific to plants that are involved in various plant development processes (Bowman and Smyth, 1999). Before the diversification of the angiosperms, the YAB family underwent at least four gene duplication events (Bartholmes et al., 2012). Thus, in angiosperms, they were divided into five monophyletic subfamilies that were represented by the corresponding proteins in Arabidopsis (Arabidopsis thaliana), that is, FILAMENTOUS FLOWER (FIL)/YAB3, YAB2, YAB5, INNER NO OUTER (INO), and CRABS CLAW (CRC) (Toshihiro Yamada et al., 2004; Lee et al., 2005; Yamada et al., 2011; Bartholmes et al., 2012; Romanova et al., 2021). Six Arabidopsis YAB genes were classified into two categories based on their expression patterns. FIL, YAB2, YAB3, and YAB5 are known as “vegetative” genes, which are expressed in the abaxial domain of developing leaves and flower organ primordia produced by the shoot apical meristem (SAM) and floral meristem (FM). CRC and INO are “reproductive” genes because the expression is restricted to the integument of the carpel and nectary or ovule, respectively (Siegfried et al., 1999; Sarojam et al., 2010; Bartholmes et al., 2012; Huang et al., 2013).

In dicots, the primary function of YAB genes is to determine abaxial cell fate and maintain the abaxial–adaxial polarity in lateral organs (Bowman, 2000; Bowman et al., 2002; Stahle et al., 2009; Du et al., 2018; Shchennikova et al., 2018). YABs promote organ polarity through a highly redundant and complex pathway. FIL function loss showed an abnormal string of filamentous structures and even altered the number and shape of floral organs (Chen et al., 1999; Sawa et al., 1999; Siegfried et al., 1999). A fil yab3 double mutant exhibited abnormal SAM and radiated floral organs (Sawa et al., 1999; Siegfried et al., 1999; Kumaran et al., 2002). More severe phenotypes were found in triple (fil yab3 yab5) and quadruple (fil yab2 yab3 yab5) mutants, showing dwarfed and bushy plants without apical dominance and all lateral organs lacked lamina expansion and polarity (Sarojam et al., 2010; Yang et al., 2019). In the process of leaf polarity establishment, the YAB genes are regulated by both adaxial and abaxial genes. The adaxial factors ASYMMETRIC LEAF1 (AS1) and ASYMMETRIC LEAF1 (AS2) form a repressor complex that can negatively regulate FIL and YAB3, and the abaxial factors KANADI1 (KAN1) and AUXIN RESPONSE FACTOR 4 (ARF4) can positively regulate FIL and YAB3 (Lin et al., 2003; Eshed et al., 2004; Fu et al., 2007), which can promote the expression of KAN1 and ARF4 through positive feedback (Bonaccorso et al., 2012). In other dicot plants, FIL/YAB3 clade genes were functionally conserved in polarity establishment. GRAMINIFOLIA, an ortholog of FIL, regulated the adaxial–abaxial asymmetry of leaves and the growth of lateral organs in Antirrhinum (Antirrhinum majus L.) (Golz et al., 2004). Independent ectopic expression of soybean (Glycine max) GmFIL, tea plant (Camellia sinensis) CsFILa/b, and Chinese cabbage (Brassica campestris L. ssp. pekinensis (Lour.) Olsson) BraYAB1-702 in Arabidopsis all resulted in a leaf curling phenotype (Zhang et al., 2013; Yang et al., 2019; Shen et al., 2022). Likewise, CmYAB1 affected the petal curvature and inflorescence morphology in chrysanthemum (Chrysanthemum morifolium) (Ding et al., 2019). Although the YAB transcription factor family has been widely identified in other dicot plants, such as star fruit (Averrhoa carambola) (Li et al., 2022), cotton (Gossypium spp.) (Yang et al., 2018), sunflower (Helianthus annuus) (Wu et al., 2021), cucumber (Cucumis sativus L.) (Liu et al., 2018), tomato (Solanum lycopersicum) (Huang et al., 2013; Han et al., 2015), apple (Malus pumila) (Shao et al., 2017), and pomegranate (Punica granatum) (Zhao et al., 2020), the majority of their functions are still unknown. However, several reports suggested that YAB family genes in monocots have a distinct function in plant height rather than being involved in regulating the polarity of lateral organs. Prominent examples are that OsYAB1 and OsYAB4 in rice (Oryza sativa L) are involved in regulating gibberellin (GA) biosynthesis to inhibit stem elongation. Overexpression of OsYAB1 and OsYAB4 exhibited a pronounced reduction in plant height, resulting in a semi-dwarf phenotype (Dai et al., 2007b; Yang et al., 2016). In contrast, reports on YAB regulation of plant height in dicots are rare.

Plant height is the main determinant of plant architecture and an important agronomic and horticultural trait. It is confined by stem elongation and plays a decisive role in crop yield (Wang and Li, 2008) and natural attractiveness. Plant hormones are one of the main factors affecting plant height, and the roles of GAs in plant height determination have been uncovered (Hedden and Sponsel, 2015; Wang et al., 2017; Hedden, 2020). A substantial alteration in GA biosynthesis and signaling pathways led to the dwarf or semi-dwarf cereals in the famous “Green revolution” (Peng et al., 1999; Spielmeyer et al., 2002; Yamaguchi, 2008). A series of the bioactive GAs were catalyzed in parallel by GA3ox in the final step of the GA biosynthesis (Yamaguchi, 2008; Hedden, 2020). Alteration of GA3ox gene expression is often associated with alterations in bioactive GA content, thereby seriously affecting plant height (Dai et al., 2007b; Yamaguchi, 2008). According to a previous study, the ga4-7 mutant, a null allele of AtGA3ox1, exhibited a semi-dwarf phenotype with reduced bioactive GAs (Chiang et al., 1995). In Medicago truncatula, a GA-deficient mutant that lost the function of MtGA3ox1 resulted in a dwarf phenotype with small lateral organs (Wen et al., 2021). Conversely, in pea (Pisum sativum L.), phenotype with longer internodes and increased GA1 content was observed in overexpressed PsGA3ox1 plants (Reinecke et al., 2013). Ectopic expression of pumpkin (Cucurbita moschata) CmGA3ox1 in A. thaliana led to elevated GA4 levels, elongated internodes, elongated hypocotyls, and earlier flowering (Radi et al., 2006).

In Arabidopsis, YABs are reported to regulate lateral organ development and determine the abaxial cell fate (Sawa et al., 1999; Siegfried et al., 1999; Kumaran et al., 2002; Bartholmes et al., 2012). Asteraceae is the largest family of dicots, and chrysanthemum are important economic crops with high ornamental and economic values. In our previous study, a FIL/YAB3 clade gene, CmYAB1, was demonstrated to regulate inflorescence shape in chrysanthemum (Ding et al., 2019). Here, we isolated a paralog of CmYAB1, named C. morifolium Dwarf and Robust Plant (CmDRP), and investigated the expression pattern and function of CmDRP in chrysanthemum using in situ hybridization and genetic transformation. Surprisingly, we found that CmDRP belongs to a distinct FlL/YAB3 sub-clade lost in Brassicaceae and expresses in lateral organs in a nonpolar manner not reported in dicots. Furthermore, CmDRP is involved in plant height directly via binding to the CmGA3ox1 promoter. These findings provide insights into the role of YAB transcription factor in affecting plant height in chrysanthemum.

Results

CmDRP, a distinct FIL/YAB3 sub-clade member in chrysanthemum

A phylogenetic tree was constructed using YAB amino acid sequences (Supplemental Data Set S1) obtained from angiosperm genomes to determine the phylogenetic relationships of YAB genes in chrysanthemum and other plants. It showed that YAB homologs in angiosperms are resolved into the FIL/YAB3, CRC, YAB2, YAB5, and INO clades. Although unigene18691, CmYAB1, and AtYAB1/3 belong to FIL/YAB3 clade, unigene18691 forms a different sub-clade with CmYAB1 and AtYAB1/3 (Supplemental Figure S1). Based on its plant architecture phenotypes in overexpress-lines, we named unigene18691 CmDRP.

To further explore the evolution of the FIL/YAB3 subfamily, we obtained 31 amino acid sequences (Supplemental Data Set S2) of the FIL/YAB3 subfamily from the genomes of representative species: four classes of core dicots and monocots, including oilseed rape (Brassica napus), Arabidopsis, soybean (G. max), papaya (Carica papaya), cacao (Theobroma cacao), tomato (S. lycopersicum), sunflower (H. annuus), chrysanthemum, rice (O. sativa), and maize (Zea mays) (Figure 1). The phylogenetic tree of the FIL/YAB3 clade contains two main branches: the dicot clade and the monocot clade (Figure 1). Subsequently, the dicot clade diverged into two major sub-clades (sub-clade I and sub-clade II) with substantial support. CmDRP belonged to a distinct sub-clade II independent of sub-clade I containing CmYAB1, AtYAB1, and AtYAB3 (Figure 1). The functions of all homologous proteins in sub-clade II are still unknown. Moreover, sub-clade II was lost in the genera Arabidopsis and Brassica, which belong to the Brassicaceae, although it is still present in Papaveraceae at the base of the Brassicales (Figure 1). To validate whether sub-clade II was lost in the Brassicaceae, we supplemented FIL/YAB3 amino acid sequences (Supplemental Data Set S3) from the genomes of 35 Brassicaceae species to reconstruct the FIL/YAB3 phylogenetic tree. It was demonstrated that all orthologs of sub-clade II were lost in all species of Brassicaceae (Supplemental Figure S2). Therefore, CmDRP is a member of a distinct FIL/YAB3 sub-clade II lost in Brassicaceae, and its function remains unclear.

Figure 1.

Figure 1

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Phylogeny of the FIL/YAB3 clade in angiosperms. The maximum-likelihood tree was constructed using the JTT model with 1,000 bootstrap replicates. Bootstrap values are shown at the nodes. Bar indicates 0.1 amino acid substitutions per site.

To explore the function of CmDRP, it was cloned from the C. morifolium cultivar ‘Jinba’. It encoded 227 amino acids and contained two conserved domains: a Cys2/Cys2 zinc finger domain in the N-terminal and a YAB domain in the C-terminal (Supplemental Figure S3). Subcellular localization analysis indicated that CmDRP was localized in the nucleus (Supplemental Figure S4), and transcriptional activity assays suggested that CmDRP exhibited transcriptional activation activity in Arabidopsis protoplasts (Supplemental Figure S5).

CmDRP is expressed in a nonpolar way in chrysanthemum

In order to investigate the expression pattern of CmDRP in chrysanthemum, various tissues from the vegetative and reproductive stages were used for RT-qPCR analysis (Figure 2A). The results showed that CmDRP was highly expressed in the shoot apex (SA), followed by lateral buds at the vegetative stage (Figure 2A). CmDRP was highly expressed in floral organs at the reproductive stage (Figure 2A). To further detect the spatial and temporal expression pattern of CmDRP, in situ hybridization was applied to the SA, axillary meristem, leaves, stem, and floral buds. CmDRP mRNA was detected in initiating primordium of the SAM (Figure 2B) and axillary meristem (Figure 2C). CmDRP mRNA mainly accumulated in the middle domain of leaf primordia and did not show any adaxial/abaxial polar distribution (Figure 2, B and D). CmDRP was expressed in vascular tissues in the stem (Figure 2, F–H). The CmDRP transcripts were also observed in the middle region of the floral organ primordia, including the petal, stamen, and pistil primordium (Figure 2, J and L). Overall, CmDRP was widely expressed in various tissues but without polar distribution in chrysanthemum.

Figure 2.

Figure 2

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Expression pattern of CmDRP in chrysanthemum. A, Expression analysis of CmDRP in various tissues of chrysanthemum by RT-qPCR. Roots (R), stems (S), leaves (L), SA, lateral buds, (LB), ray flower petal (Rpe), ray flower pistil (Rpi), disc flower petal (Dpe), disc flower pistil (Dpi), and disc flower stamen (Dst). Values are means of three independent biological replicates from different plants. Data represent means ± se, and significant differences were determined by Tukey’s multiple range test (P < 0.05). B–M, In situ hybridization of CmDRP in WT chrysanthemum. B, Initiating primordium of the SAM. C, Axillary meristem. D, CmDRP transcripts are expressed in leaf primordia. E, A SA hybridized with the CmDRP sense probe as a negative control. F–I, CmDRP was expressed in the stem. F, Transverse section of stem. CmDRP was expressed in vascular tissues. G, Enlarged view of the stem in the panel. H, Longitudinal section of stem. I, CmDRP sense probe in the stem. J–M, Longitudinal section of ray flower and disc flower. J, CmDRP was highly expressed in the center of the ray flower petal, ray flower stamen, and ray flower pistil. L, CmDRP transcripts are accumulated in middle domain of a disc flower petal and disc flower stamen. K and M, In situ hybridization with the CmDRP sense probe. The scale bars represent 200 μm. lp, leaf primordia; ip, initiating primordia; am, axillary meristem; vt, vascular tissue; Rpe, ray flower petal; Rst, ray flower stamen; Rpi, ray flower pistil; Dpe, disc flower petal; and Dst, disc flower stamen.

Overexpression of CmDRP caused a semi-dwarf phenotype

To gain insight into CmDRP function in chrysanthemum, overexpressing and artificial microRNA (amiR) transgenic plants were generated through Agrobacterium-mediated transformation method using the chrysanthemum cultivar ‘Jinba’ (Wang et al., 2019). A total of 9 overexpression lines and 13 amiR-lines were obtained, and 3 representative lines of each (named OX-64, -69, -86 and amiR-56, -60, -61) were selected for further analyses (Figure 3A). Expression levels of CmDRP were significantly increased in OX-lines and decreased in amiR-lines (Figure 3B). Compared with wild-type (WT), a semi-dwarf phenotype was observed in the OX-lines, which were approximately 55% the height of the WT, while stem diameter was significantly increased by 33% compared with that of the WT (Figure 3, A, C, D, and E). Moreover, the average length of internodes in the OX-lines is much shorter, with an average reduction of 42.3% (Figure 3F). In contrast, the height of the amiR plants increased by 41%, and the average length of internodes was longer than that of the WT (Figure 3, A, C, and F). The number of internodes was unchanged in the OX-lines and increased in the amiR-lines (Figure 3G).

Figure 3.

Figure 3

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Phenotypic characterization of WT and CmDRP transgenic plants. A, Representative images of 45-day-old WT and CmDRP transgenic plants at the vegetative stage. Scale bar, 2 cm. B, Expression of CmDRP in the SA of WT and CmDRP transgenic plants was measured by RT-qPCR. OX-64, -69, -86, and amiR-56, -60, and -61 correspond to different OX-CmDRP and amiR-CmDRP lines. Data are from three biological replicates. Data represent means ± se and statistical significance was determined by Student’s t test: * represents 0.01 < P < 0.05, ** represents 0.001 < P < 0.01. CmEF1α is a reference gene used as the internal control. C, Measurement of plant height of WT and CmDRP transgenic plants. n ≥ 15, data represent means ± se, and significant differences were determined by Tukey’s multiple range test (P < 0.05). D, Stems of WT and CmDRP transgenic plants, scale bar, 2 cm. E, Measurement of stem diameter from the bottom to the top third of WT and CmDRP transgenic plants. Values are mean ± se, n ≥ 15. Significant differences were determined by Tukey’s multiple range test (P < 0.05). F and G, Measurement of the average length of internodes and the number of internodes of WT and CmDRP transgenic plants. n ≥ 15, data represent means ± se, and significant differences were determined by Tukey’s multiple range test (P < 0.05). H, Quantification of leaf area of WT and CmDRP transgenic plants. All leaves are from the eighth internode from top to bottom, n ≥ 15. Data represent means ± se, and significant differences were determined by Tukey’s multiple range test (P < 0.05). I, Leaves of WT and CmDRP transgenic plants, scale bar, 2 cm.

In addition to the above phenotypes, a phenotype with altered leaf area and lateral bud elongation was observed in transgenic plants. In the OX-lines, leaf area was significantly increased, about twice as much as that in WT plants, while the leaf area of the amiR-lines was reduced (Figure 3, H and I). Reduced expression of CmDRP resulted in greatly increased lateral bud length. Conversely, the elongation of lateral buds was reduced in the overexpression plants (Figure 4). Based on the phenotypes, it is proposed that CmDRP plays an essential role in plant architecture in chrysanthemum.

Figure 4.

Figure 4

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Morphological analysis of the elongated lateral buds of WT and CmDRP transgenic plants. A, Elongated lateral buds of WT and CmDRP transgenic plants, the bottom row is the plant with the leaves removed. Scale bars, 3 cm. B, Diagrammatic representation showing the position of axillary bud in WT and CmDRP transgenic lines. Each column represents a plant and each layer represents a node of the chrysanthemum. Color squares represent a range of bud lengths. Blue squares represent bud length less than 0.1 cm, yellow squares represent bud length from 0.1 to 0.2 cm, deep pink squares represent bud length from 0.2 to 0.6 cm, and purple squares represent bud length longer than 0.6 cm (n = 11–16). The white squares do not represent nodes and are only present for aesthetics.

CmDRP altered the endogenous GA content of chrysanthemum

GAs are well known to play a decisive role in plant height (Yamaguchi, 2008; Hedden and Sponsel, 2015). To determine whether the semi-dwarf phenotypes of CmDRP transgenic plants were due to the alteration of GAs levels, endogenous GAs in transgenic and WT plants were measured using the LC-MS/MS system. Assessing the GAs content showed that GA1 displayed no change, and GA4 and GA7 levels were not detected in any line, although they are the bioactive GA forms (Figure 5A). Intriguingly, another active GA form, GA3, significantly decreased in the OX-lines. In contrast, a significant increase of GA3 occurred in the amiR-lines (Figure 5A). These results suggested that altering the expression of CmDRP affects the content of active GA3.

Figure 5.

Figure 5

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Partially rescued phenotypes of transgenic plants after exogenous GA3 and PAC treatment. Forty-six-day-old treated plants were used for measurement of the plant height, the number of internodes, and the length of average internodes. A, Endogenous GAs content was altered in CmDRP transgenic plants. B, Partially rescued plant height of transgenic plants after exogenous GA3 and PAC application. C–E, Measurement of plant height, number of internodes, and the average length of internodes of CK and CmDRP transgenic plants after exogenous GA3 and PAC application. In this figure, data represent means ± se (n ≥ 15) and significant differences were determined by Tukey’s multiple range test (P < 0.05). Scale bars: 10 cm.

The phenotypes of CmDRP transgenic plants could be partially rescued by exogenous GA3 and paclobutrazol treatment

To further investigate whether changes in GA3 levels had an effect on transgenic plant height, we treated the transgenic plants with exogenous GA3 and paclobutrazol (PAC, an inhibitor of GAs). Twenty-five-day-old OX-line plants were treated with 50 mg L−1 GA3, while plants from the amiR-line were treated with 200 mg L−1 PAC. After 21 days of exogenous GA3 and PAC application, the plant height, internode number, and the average length of internode were measured. The plant height of WT plants increased by 46% after GA treatment. However, plant height decreased by 40% after PAC treatment. The height of OX-plants sprayed with GA3 increased by 78% compared with the height of OX-plants without GA3 application (Figure 5, B and C), internode elongation occurred in GA3-treated OX-plants (Figure 5E), and the number of internodes became comparable with those of untreated OX-plants (Figure 5D). However, the plant height, internode length, and the number of internodes were still lower than those of WT. In contrast, the height of the amiR-plants decreased by 43% compared with CK-amiR plants after the exogenous application of PAC (Figure 5, B and C). Moreover, the length of internodes decreased in the PAC-treated amiR-plants (Figure 5E), and fewer internodes were observed in amiR-plants compared with CK-amiR plants (Figure 5D). GA3 could partially rescue the phenotypes of dwarfed OX-plants that indicated both GA biosynthesis and signaling pathway may be impaired in transgenic plants.

CmDRP inhibits GA biosynthesis gene expression and binds directly to the CmGA3ox1 promoter

To further identify the genes in the GA pathway regulated downstream of CmDRP, the SAs (including two leaves) were collected from WT and OX-69, -86 transgenic plants at the 3-week-old stage for RNA-Seq analysis. We analyzed the differentially expressed genes in GA biosynthesis and signaling pathways including GA 2-oxidase (GA2ox), GA 20-oxidase (GA20ox), GA 3-oxidase (GA3ox), ent-Copalyl Diphosphate Synthase (CPS), ent-Kaurene Oxidase (KO), ent-Kaurenoic Acid Oxidase (KAO), and DELLA (Supplemental Table S1). To further validate the expression levels of those genes, we performed a RT-qPCR analysis in OX-lines and amiR-lines (Supplemental Figure S6). The results showed that only the expression level CmGA3ox1 was significantly decreased in OX-lines and significantly increased in the amiR-lines (Figure 6A). The expression level of CmGA3ox2 was significantly increased both in OX-lines and in one amiR-60 line. GA2ox genes CmGA2ox1, CmGA2ox2, and CmGA2ox8 exhibited similar trends in OX-lines and amiR-lines. Moreover, the expression of the GA signaling pathway DELLA genes REPRESSOR OF GA (CmRGA1) and GIBBERELLIC ACID INSENSITIVE (CmGAI) were decreased in OX-lines and amiR-lines (Supplemental Figure S6).

Figure 6.

Figure 6

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CmDRP represses the expression of CmGA3ox1 via direct binding to its promoter. A, The expression of CmGA3ox1 in the WT and transgenic lines. The SAs (including two leaves) were collected from WT and OX-69, -86 transgenic plants at the 3-week-old stage for RT-qPCR analysis. Error bars represent ± se (n = 3) and statistical significance was determined by Student’s t test: * represents 0.01 < P < 0.05, ** represents 0.001 < P < 0.01. B, Schematic diagram of the putative cis-elements (P1–P4) in proCmGA3ox1 used for CmDRP binding assay and P5 as a negative control. C, Y1H assay indicated binding of CmGA3ox1 promoter. Cells were grown in a liquid medium to OD 600 of 1 (10°) and diluted in a 10 × dilution series (10−2 to 10−3). 2.5 µL of each dilution was spotted on -Trp/-His/-Leu (-T-H-L) selective medium with 60 mM 3-AT. The proCmGA3ox1 + AD-GUS was used as a negative control. D, LUC activity measurement in chrysanthemum mesophyll protoplasts after coexpression of proCmGA3ox1:LUC and 35S::CmDRP. The 35S empty vector and proCmGA3ox1 were used as the control. Error bars represent ± se (n = 6) and statistical significance was determined by Student’s t test: * represents 0.01 < P < 0.05, ** represents 0.001 < P < 0.01. E, ChIP-qPCR assay indicated that CmDRP binds to the P3 and P4 regions of CmGA3ox1. P5 region as a negative control. 35S::GFP transgenic line was used as empty control. Three technical replicates and three biological replicates from different SAs were used. Error bars represent ± se and statistical significance was determined by Student’s t test: * represents 0.01 < P < 0.05 and ** represents 0.001 < P < 0.01.

The altered GA3 content in transgenic plants and the changes in the expression of the GA biosynthesis gene CmGA3ox1 prompted us to investigate the possible transcriptional regulation relationship between CmDRP and CmGA3ox1. Before exploring whether CmDRP binds directly to the CmGA3ox1 promoter, we cloned the 2,100 bp promoter fragment of CmGA3ox1. Subsequent promoter element analysis identified four putative elements (P1–P4) in the CmGA3ox1 genome sequence. P1 element (5′-TCTGGCT) and P4 element (5′-TCTGAGT) had a similar GA-responsive element (GARE) (Dai et al., 2007b); P2 element (5′-CCCCAC) was reported in a previous study (Shamimuzzaman and Vodkin, 2013); and P3 element (5′-CTAATTATGT) was predicted via the PLANT CARE software (Figure 6B). Subsequently, a yeast one-hybrid (Y1H) assay showed that CmDRP bound to the promoter of CmGA3ox1 (Figure 6C). For further analysis, a dual-luciferase (LUC) transaction assay in chrysanthemum protoplasts was performed. The ratio of LUC/REN was significantly reduced upon co-transformation with 35S::CmDRP + ProCmGA3ox1:LUC when compared to the vector control (Figure 6D). It indicated that CmDRP exhibited a negative regulation on the transcription of CmGA3ox1. To validate this interaction in vivo, 35S::CmDRP-GFP and 35S::GFP transgenic lines were used in a chromatin immunoprecipitation (ChIP) assay. ChIP-qPCR results demonstrated that the promoter of CmGA3ox1 was significantly enriched in the P3 and P4 regions through antibodies recognizing CmDRP-GFP protein, but not in the P1, P2, and P5 regions (Figure 6E). Therefore, CmDRP can directly bind to the CmGA3ox1 promoter, inhibiting its expression.

Discussion

The expression pattern and function of the FIL/YAB3 clade in dicots have diverged

The abaxial expression pattern of the YAB genes was confirmed in Cabomba caroliniana, belonging to the family Cabombaceae of the order Nymphaeales and the ancestor of the extant angiosperms (Saarela et al., 2007; Yamada et al., 2011). Moreover, previous work has demonstrated that YAB family members are expressed in the abaxial of lateral organs produced by SAM and FM in some dicot plants (Bowman and Smyth, 1999; Siegfried et al., 1999; Watanabe and Okada, 2003; Golz et al., 2004; Stahle et al., 2009; Sarojam et al., 2010; Yang et al., 2019). FIL and AtYAB3 were expressed in the abaxial side of leaves and floral organs (Siegfried et al., 1999). Similarly, GRAM, an ortholog of FIL in Antirrhinum, was detected in the abaxial margins of leaf primordia (Golz et al., 2004), and GmFILa is expressed in the abaxial region of growing leaf primordia in soybean (Yang et al., 2019). Intriguingly, none of the YAB genes showed adaxial/abaxial polar expression patterns in lateral organs in rice (Dai et al., 2007a, 2007b; Tanaka et al., 2017). In addition, wheat TaYAB1 is a member of the FIL/YAB3 clade, and its mRNA was highly accumulated in lateral organs but not in a polar manner (Zhao et al., 2006). Whereas, in maize, Z. mays YAB 9/14 (ZYB 9/14), members of the FIL/YAB3 subfamily, are detected in the adaxial side of leaf primordia (Juarez et al., 2004). Thus, the spatial expression pattern of the FIL/YAB3 clade probably has diverged prior to angiosperms divergence (Tanaka et al., 2012). Meanwhile, the FIL/YAB3 clade gene was expressed in a nonpolar manner in rice and adaxially in maize, indicating that the expression pattern of the FIL/YAB3 clade in monocots has diverged as well.

A surprising finding from our study is that CmDRP was expressed in all lateral organs but did not show any polar distribution (Figure 2, B–M), which is distinct from the expression pattern of YAB reported in dicots. It shows a divergence in the expression pattern of the FIL/YAB3 clade in dicots. The expression pattern of CmDRP without polarity may be the result of different upstream regulatory pathways. The adaxial transcription factor HD-ZIPIII negatively regulates the expression of AtYABs in Arabidopsis (Kidner and Timmermans, 2007; Fukushima and Hasebe, 2014), while HD-ZIPIII positively regulates the expression of zyb 9 and zyb 14 (homolog of FIL and YAB3) in maize (Juarez et al., 2004), which may contribute to the adaxial expression pattern of YAB in maize (Yamada et al., 2011). In Petrocosmea, alteration in cis-regulatory contributed to the divergent expression of CYC1C, while changes in trans-acting were responsible for the divergence of CYC1D expression (Yang et al., 2015). In addition, it is reported that OsYAB4 obtained a distinct expression pattern in rice than Arabidopsis, probably through changes in upstream regulatory pathway (Liu et al., 2007). Further identification and functional analyses of related cis-elements and trans-regulators would be important to decipher the reason of there is no polarity expression of CmDRP. Moreover, the distinct expression pattern of CmDRP was coincided with the divergence of CmDRP and AtYAB1/3 in the evolutionary process; that is, CmDRP is not in the same sub-clade as AtYAB1/3 but belongs to a distinct sub-clade II (Figure 1). The YAB genes have not been found to regulate plant height in Arabidopsis but affect the abaxial cells polar development. Although there is no polar distribution in chrysanthemum and rice, there are some differences in specific expression domains. For example, CmDRP mRNA was detected at substantial levels in the middle region of leaf primordia and floral organs (Figure 2, D, J, and L). In contrast, TONGARI-BOUSHI1/2/3 (TOB1/2/3) belonging to the FIL/YAB3 subfamily was uniformly expressed throughout the primordia of the lateral organs (Tanaka et al., 2012, 2017). Although both rice OsYAB1/4 and CmDRP affected plant height, the phenotypes of thicker stems and larger leaves were only found in chrysanthemum (Figure 3, D and I). An unexpected result is that the whole sub-clade II was lost in Brassicaceae (Figure 1 and Supplemental Figure S2). Gene loss events occur during genome-wide polyploidy (Tang et al., 2010; Albalat and Cañestro, 2016). Previous research has identified that whole-genome triplication (WGT) occurred in Brassicaceae (Lysak et al., 2005; Tang et al., 2012; Mabry et al., 2020), and loss of sub-clade II in Brassicaceae is probably a result of the WGT event (Mun et al., 2009; Moghe et al., 2014; Ma et al., 2017). In conclusion, the expression pattern and function of the FIL/YAB3 clade in dicots has diverged. As a member of sub-clade II, CmDRP may provide a reference for functional studies of homologous genes of other species in sub-clade II.

CmDRP is an excellent candidate gene for the breeding of semi-dwarf cultivar

In this study, we found that CmDRP, a member of sub-clade II, is involved in regulating chrysanthemum plant height, and overexpression of CmDRP resulted in a semi-dwarf phenotype. The semi-dwarf phenotype is a valuable agronomical trait. It is conducive to the tolerance/resistance of crops to lodging, shattering, and diseases, which improves the harvest index and planting density. Therefore, it is directly related to the yield and quality of crops (Milach and Federizzi, 2001; Hedden, 2003; Saville et al., 2012). In ornamental plants, different cultivated forms require various plant heights; thus, a semi-dwarf phenotype is also indispensable for ornamental plants. Despite the many dwarf phenotypes that have been reported in crops, most of them are simply dwarfed in plant height. For instance, shoot elongation was suppressed in rice mutant ree1-D, and the osmads57 mutant only displayed a semi-dwarf phenotype (Qi et al., 2011; Gao et al., 2016); overexpression of rice OsEATB (for ERF protein associated with tillering and branching) suppressed internode elongation leading to semi-dwarf plants, but no substantial changes in leaf area and stem thickness (Chu et al., 2019); dwarf plant with thick leaves were observed in maize mutant lil1-1 (Castorina et al., 2018); and D8-Mpl, D8-1, D9-1, and three DELLA alleles found in dwarf maize produced dwarf phenotypes with smaller stem diameter in transgenic Arabidopsis (Lawit et al., 2010). CesA was inserted into Nicotiana benthamiana via virus-induced gene silencing resulting in a dwarf phenotype with small leaves (Burton et al., 2000); rice nd1 plants exhibited narrow leaves and thin culms in addition to dwarfism (Li et al., 2009); and ectopic expression of CmHLB led to decreased leaf size and plant height as well as increased stem diameter in chrysanthemum (Zhao et al., 2022). OX-CmDRP plants are not only dwarf but much more robust, with significantly thicker stems and larger leaves (Figure 3, A, D, E, H, and I). These phenotypes help plants withstand unfavorable environments, such as tolerance/resistance to lodging, pests, and diseases, leading them to occupy more beneficial ecological niches. Therefore, our semi-dwarf phenotype could be an excellent candidate trait for breeding.

Lateral bud elongation is also an important target for breeding. In our study, reduced expression of CmDRP resulted in longer lateral buds (Figure 4). Besides, changes in the indole-3-acetic acid (IAA) and methyl-IAA (ME-IAA) levels occurred in transgenic plants (Supplemental Figure S7). Auxin is the main hormone thought to play a key role in suppressing branch formation (Thimann and Skoog, 1933; Barthelemy and Caraglio, 2007; Zhu and Wagner, 2020). The changes in lateral bud elongation of CmDRP transgenic plants indicated that CmDRP might be involved in regulating the biosynthesis of auxin or in complex crosstalk between GA and IAA. However, these specific mechanisms require further research.

CmDRP functions as a negative regulator of the GA biosynthesis pathway

GA1, GA3, GA4, and GA7 are the major bioactive GAs (Yamaguchi, 2008; Hedden, 2020). We detected no GA4 and GA7, only GA1 and GA3, in chrysanthemum. The content of GA3 was significantly altered in the transgenic plants, and the content of GA1 was not significantly different among plants (Figure 5A). Higher GA levels lead to taller plants and vice versa (Hirano et al., 2017; Lo et al., 2017; Gao and Chu, 2020; Liu et al., 2021). Altered GA3 content occurred in CmDRP transgenic plants, resulting in a semi-dwarf phenotype observed in OX-lines and plants with higher height than WT observed in the amiR-lines, which indicated that GA3 plays the main role in regulating plant height in chrysanthemum. Treating transgenic plants with exogenous hormones demonstrated that GA3 could partially rescue the phenotypes of transgenic plants (Figure 5B), indicating that not only GA biosynthesis but also GA signaling may be impaired. Subsequent RNA-Seq and RT-qPCR results confirmed this inference. Only the expression level CmGA3ox1 was significantly decreased in OX-lines and significantly increased in the amiR-lines (Figure 6A), suggesting that CmGA3ox1 may be a direct downstream target gene of CmDRP. In addition, CmDRP may indirectly mediate other GA oxidase genes (e.g. CmGA3ox2 and CmGA2ox1/2/8) and GA signaling genes (e.g. CmRGA1 and CmGAI) to regulate plant height. Furthermore, in vivo experiments suggested that CmDRP could directly bind and inhibit the expression of CmGA3ox1 (Figure 6).

GA3ox is a key enzyme in the final step of GA biosynthesis that determines GA levels (Yamaguchi, 2008; Wang et al., 2017; Hedden, 2020). The ga4-7 mutant, a null allele of AtGA3ox1, exhibited a semi-dwarf phenotype with reduced bioactive GAs (Chiang et al., 1995). In our study, the expression level of CmGA3ox1 was significantly reduced in OX-lines and significantly increased in amiR-plants (Figure 6A). Furthermore, some transcription factors have been reported to affect GA content through binding to the promoter of the GA biosynthesis gene GA3ox. For example, JUB1 in A. thaliana directly suppressed the expression of GA3ox1 and inhibited the biosynthesis of bioactive GAs (Shahnejat-Bushehri et al., 2016). AGAMOUS directly promotes AtGA3ox1 expression in Arabidopsis (Gomez-Mena et al., 2005). MdBZR1 and MdBZR1-2 bind to the MdGA3ox1 promoter to regulate GA biosynthesis in Apple (Wang et al., 2019). In rice, OsYAB1, a member of the YAB2 subfamily, directly binds to the promoter of OsGA3ox2 to alter bioactive GA1 content, resulting in a semi-dwarf phenotype (Dai et al., 2007b). However, little is known about the direct upstream factors of GA biosynthesis genes in ornamental plants. In our study, CmDRP suppressed the expression of the key biosynthesis gene CmGA3ox1. It binds directly to the promoter in chrysanthemum (Figure 6), enhancing the understanding of the genetic and molecular mechanisms of plant height and enriched regulatory network in the GA biosynthesis pathway.

In summary, we identified that CmDRP belongs to a distinct sub-clade of FIL/YAB3 lost in Brassicaceae and determined its nonpolar expression pattern and distinct function in dicots. We present a working model for the involvement of CmDRP in the regulation of chrysanthemum plant height through the GA pathway. In chrysanthemum, CmDRP negatively regulated GA3 biosynthesis by binding to the promoter of CmGA3ox1 and inhibiting its expression. In the OX-CmDRP transgenic plants, the CmDRP protein was substantially increased, binding to the promoter of the downstream GA biosynthesis gene CmGA3ox1 and substantially suppressing its expression, leading to a decreased GA3 content. The reduced GA3 content led to a semi-dwarf and robust plant (Figure 7). These findings enriched and deepened the understanding of plant height genetics and molecular regulatory mechanism and laid the theoretical foundation for breeding new dwarf varieties.

Figure 7.

Figure 7

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A proposed model of CmDRP regulating chrysanthemum plant architecture. In OX-CmDRP plants, CmDRP downregulates the expression of the downstream GA biosynthesis gene CmGA3ox1 through binding to its promoter, inhibiting the synthesis of GA3. Finally, the OX-CmDRP plants display a semi-dwarf and robust plant architecture.

Materials and methods

Plant materials and growth conditions

The chrysanthemum (C. morifolium) cultivar ‘Jinba’ was obtained from the Nanjing Agricultural University Chrysanthemum Germplasm Resource Preservation Center (Nanjing, China). Plants for morphological analysis were cultivated in a greenhouse using standard management practices. Plants for GA3 and PAC treatment were grown in a growth chamber at 16-h day (25°C) and 8-h night (18°C).

The isolation of CmDRP, phylogenetic analysis, and sequence alignment

The complete coding sequence (CDS) of CmDRP was amplified from chrysanthemum cultivar ‘Jinba’ cDNA using gene-specific primers designed according to transcriptome data (Wang et al., 2019). All YABs protein sequences were obtained from various genomes via Phytozome (phytozome-next.jgi.doe.gov) and in the BRAD database except for CmYAB1 (Unigene2623) and CmDRP (Unigene18691) which were from the chrysanthemum transcriptome (Wang et al., 2019). The phylogenetic trees were reconstructed with MEGA X (Kumar et al., 2018). Protein sequences were aligned through the MUSCLE program and JTT matrix-based model (Thorvaldsen et al., 2010). Tree construction employed the maximum-likelihood method with 1,000 bootstrap replicates. Bootstrap support values are indicated next to the branches. Protein alignment of CmDRP and other YABs was performed using Jalview 2.10.3.

Subcellular localization of CmDRP

The full-length CDS of CmDRP without the termination codon was cloned into the plasmid pORE-R4-35sAA and fused with green fluorescent protein (GFP). CmDRP-GFP fusion constructs and the 35S::GFP-CmDRP and free GFP (35S::GFP) plasmids were transiently coexpressed with a nuclear marker (35S::D53-RFP construct) (Zhou et al., 2013). The fusion vector and the control 35S::GFP were separately transformed into onion epidermal cells using the helium-driven PDS-1000 particle accelerator (Bio-Rad Laboratories, Hercules, California, USA). The leaf epidermal cells were incubated in the dark at 22°C for 16 h and the GFP signal was monitored with a Zeiss LSM 780 confocal microscope (Zeiss, Jena, Germany).The samples were observed in the red channel for mRFP-NLS (laser wavelength: 561 nm: 2.00%, detection wavelength: 586–644 nm; detector gain: 609.1 V) and the yellow channel for GFP (laser wavelength: 488 nm: 7.91%, detection wavelength: 493–567 nm; detector gain: 735.4 V), respectively.

Transcriptional activity of CmDRP

The transcriptional activity of CmDRP was examined using a luminescence assay. A 35S::GAL4DB-CmDRP plasmid was constructed using the pENTR1A-CmDRP plasmid (the primers used are listed in Supplemental Table S2 via the LR reaction [Invitrogen]). The transient expression assay was performed in Arabidopsis protoplasts as previously described (Yoo et al., 2007). There were three independent transformations: positive control, 5 μg of 35S::GAL4DB-AtARF5 and 5 μg of 5 × GAL4-LUC plasmid (luminescence reporter); negative control, 5 μg of 35S::GAL4DB and 5 μg of 5 × GAL4-LUC plasmid; and the test, 5 μg of 35S::GAL4DB-CmDRP and 5 μg of 5 × GAL4-LUC plasmid. The LUC activity was measured as previously reported (Song et al., 2013).

RT-qPCR

Temporal and spatial expression characteristics of CmDRP during the vegetative and reproductive stages were analyzed. Samples from the vegetative stage comprised roots, stems, leaves, and the SAs. Lateral buds were collected from rooted WT chrysanthemum seedlings at the ninth leaf stage grown. Samples from the reproductive stage comprised ray floret petals (Rpe), ray floret pistils (Rpil), disc floret petals (Dpe), disc floret pistils (Dpil), and disc floret stamens (Dst), and were collected from WT chrysanthemum and plants at the early bloom stage. Six samples were taken from individual tissues of WT as one replicate. Total RNA was extracted using an RNA isolation kit (Huayueyang) and was then reverse-transcribed into cDNA using the PrimeScript RT reagent kit (Takara Bio) according to the manufacturer’s protocol. The RT-qPCR reactions were conducted on a LightCycler 96 Real-Time PCR System (Roche, Basel, Switzerland) with the SYBR Premix Ex Taq II kit (Takara Bio) following the manufacturer’s protocol. RT-qPCR data obtained from three biological and three technical replicates were analyzed using the 2−ΔΔCT method (Livak and Schmittgen, 2001). CmEF1α (KF305681) was used as the reference for C. morifolium. The primer pairs used for RT-qPCR analyses are listed in Supplemental Table S2.

In situ hybridization

Chrysanthemum SA, axillary buds, and stems of 30-day-old seedlings and floral buds were fixed in 3.7% [v/v] formol–acetic–alcohol (3.7% [v/v] formaldehyde, 5% [v/v] glacial acetic acid, and 50% [v/v] ethanol). Sample fixation, sectioning, and hybridization were performed as described previously (Zhang et al., 2013). In situ probes were synthesized by PCR amplification using DIG RNA Labeling Kit (SP6/T7) (Roche) with gene-specific primers containing T7 and SP6 RNA polymerase binding sites. The primers are listed in Supplemental Table S2.

Genetic transformation of chrysanthemum

The OX (35S::CmDRP) and amiR (amiR-CmDRP) constructs were transformed into Agrobacterium tumefaciens EHA105 strain using the freeze–thaw transformation method, which was then introduced into the chrysanthemum cultivar ‘Jinba’ using the leaf disk infection method (Wang et al., 2019). After regeneration, RNA was extracted from putative transgenic and WT plants, and cDNA was synthesized using the PrimeScript RT reagent kit (Takara Bio). Subsequently, RT-qPCR analyses were performed to calculate the expression level of CmDRP using qCmDRP-F/R primers (Supplemental Table S2). The 45-day-old WT and transgenic plants were analyzed to observe phenotypes and the plant height, the number of leaves and internodes, the length of average internodes, and the leaf area. The 2-month-old WT and transgenic plants were used to observe lateral branches, and the length was measured.

GA3 and PAC treatment

Gibberellic acid (GA3, Huayueyang) and PAC (Huayueyang) were dissolved in absolute ethanol. For this treatment, twenty-five-day-old plants from the OX and amiR transformation and WT plants were used, and the plants used were similar in size. A solution of 50 mg L−1 GA3 and 200 mg L−1 PAC was sprayed on the abaxial and adaxial side of chrysanthemum leaves of the whole plant once a week for 2 weeks. The plants were grown in the same condition. Treated plants were compared with control plants (treated with the same solution without GA3) after 21 days. The plant height, the number of internodes, and the length of average internodes were measured. This experiment was repeated three times with similar results.

GAs, IAA, and ME-IAA content analysis

The SA (including two leaves) material was collected from WT and transgenic plants at the 3-week-old stage under 16-h light/8-h dark photoperiod. Fresh plant materials were harvested, weighted, immediately frozen in liquid nitrogen, and stored at −80°C. Subsequently, 50 mg of fresh material was ground into powder under liquid nitrogen and extracted with 500 μL H2O/ACN. Six SAs were taken from each line as one replicate. GAs, IAA, and ME-IAA measurements were performed by MetWare (Wuhan, China; http://www.metware.cn/), as described previously (Lv et al., 2021).

RNA-Seq sample preparation, sequencing, and bioinformatic analysis

The SA (including two leaves) was collected from WT and OX transgenic plants at the 3-week-old stage for RNA-Seq analysis. Three biological replicates were performed for OX trangenic lines. Samples were subjected to Illumina sequencing at Beijing Novogene (Tianjin, China) following the manufacturer’s protocol. NEB Next Ultra RNA Library Prep Kit (NEB, USA) was used to generate cDNA libraries. An Illumina HiSeq platform was used to generate 150-bp paired-end reads. The C. morifolium genome was used as the reference genome (unpublished data). Samples were subjected to differential expression analysis using the DESeq2 R package (1.16.1). Genes with a P-value <0.05 found by DESeq2 were assigned as differentially expressed genes.

Y1H assays

For the Y1H assay, full-length CDS of CmDRP was inserted into the pGADT7 vector. The CDS of β-glucuronidase (GUS) was inserted into the pGADT7 vector as the negative control, and the promoter fragments of CmGA3ox1 were cloned into the pHIS2 vector. The primer pairs used for gene cloning are listed in Supplemental Table S2. Constructed pGADT7 and pHIS2 vectors were co-transformed into yeast strain Y187. Subsequently, the trans-formants were cultivated on a selective medium lacking Trp, His, and Leu (SD/-Trp/-His/-Leu) for 3 days at 28°C. DNA–protein interactions were determined by the growth of clones on SD/-Trp/-His/-Leu with 60 mM 3-amino-1,2,4-triazole (3-AT).

Dual-LUC assay

For the LUC assay in chrysanthemum mesophyll protoplasts, promoters of CmGA3ox1 (2,100 bp) were cloned into the transient expression vector pGreenII 0800-LUC. The CDSs of CmDRP were cloned into the pORE-R4 under 35S promoter control to generate the effector construct and then transiently co-expressed in chrysanthemum mesophyll protoplasts as previously described (Yoo et al., 2007). The dual-LUC assay reagents (Promega) were used to examine firefly and Renilla LUC expressions. The primers for all constructs are listed in Supplemental Table S2.

ChIP-qPCR assay

The 35S::CmDRP-GFP and 35S::GFP (as the control) transgenic plants were subjected to ChIP-qPCR assays. Briefly, the SA (including two leaves) material was collected from transgenic plants at the 3-week-old stage and crosslinked with 1% [v/v] formaldehyde under vacuum for 30 min and then ground into powder in liquid nitrogen. Pierce ChIP-grade Protein A/G Magnetic Beads (Thermo Fisher Scientific) and GFP recombinant rabbit monoclonal antibody (Thermo Fisher Scientific) were used. Subsequently, the enriched DNA fragments were examined by RT-qPCR using the primers listed in Supplemental Table S2.

Statistical analyses

All statistical analyses were conducted by SPSS v 19.0 software. Student’s t test was performed in the RT-qPCR and ChIP-qPCR experiment to compare each transgenic line with the WT. The other statistical analyses were performed with one-way ANOVA and Tukey’s multiple range test to determine the significant differences. Student’s t test: * represents 0.01 < P < 0.05, ** represents 0.001 < P < 0.01, *** represents 0.0001 < P < 0.001, and **** represents 0.00001 < P < 0.0001.

Accession numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under following accession numbers: CmDRP (OP093551). RNA-seq data of this study have been deposited in the National Center for Biotechnology Information repository (https://www.ncbi.nlm.nih.gov/bioproject/) with the BioProject ID:PRJNA862288.

Supplemental data

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

Supplemental Figure S1. Phylogenetic analysis of YAB-related proteins in angiosperms.

Supplemental Figure S2. Phylogenetic analysis of FIL/YAB3-related proteins in angiosperms, including all FIL/YAB3 in 37 species of Brassicaceae.

Supplemental Figure S3. Protein sequence alignment of YABs from Arabidopsis, G. max, A. majus, and O. sativa.

Supplemental Figure S4. Subcellular localization of CmDRP in onion epidermal cells.

Supplemental Figure S5. Transactivation analysis of CmDRP.

Supplemental Figure S6. The expression of GA biosynthesis pathway and signaling pathway differentially expressed genes in the WT and CmDRP transgenic lines.

Supplemental Figure S7. Measurement of endogenous auxin in WT and CmDRP transgenic plants.

Supplemental Table S1. Differentially expressed genes of GA biosynthesis and signaling pathways in OX-lines.

Supplemental Table S2. Primers used in the study.

Supplemental Data Set S1. Gene sequences for phylogenetic analysis in Supplemental Figure S1.

Supplemental Data Set S2. Gene sequences for phylogenetic analysis in Figure 1.

Supplemental Data Set S3. Gene sequences for phylogenetic analysis in Supplemental Figure S2.

Supplementary Material

kiac437_Supplementary_Data
Click here for additional data file. (2MB, zip)

Acknowledgments

We thank Zhenxing Wang and Likai Wang (State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration) for technical and writing advices. Thanks to Yuehua Ma (Central Laboratory of the School of Horticulture, Nanjing Agricultural University) for assistance in using the high-resolution confocal laser microscope (ZEISS, LSM800).

Funding

This work was financially supported by the National Natural Science Foundation of China (32171855, 31872149, 32001354), China Agriculture Research System (CARS-23-A18), Seed industry project of Jiangsu Province (JBGS[2021]020), the earmarked fund for Jiangsu Agricultural Industry Technology System (JATS[2021]454), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

Conflict of interest statement. The authors declare that they have no conflicts of interest.

Contributor Information

Xue Zhang, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.

Lian Ding, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.

Aiping Song, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.

Song Li, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.

Jiayou Liu, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.

Wenqian Zhao, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.

Diwen Jia, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.

Yunxiao Guan, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.

Kunkun Zhao, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.

Sumei Chen, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.

Jiafu Jiang, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.

Fadi Chen, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Key Laboratory of Landscaping, Ministry of Agriculture and Rural Affairs, Key Laboratory of Biology of Ornamental Plants in East China, National Forestry and Grassland Administration, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China.

 

These authors contributed equally (X.Z. and L.D.)

F.C., L.D., and X.Z. designed the experiments, and wrote and revised the manuscript. X.Z., L.D., J.L., S.L., W.Z., K.Z., D.J., and Y.G. performed the experiments. L.D., A.S., S.C., J.J., and F.C. guided the research.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is: Fadi Chen (chenfd@njau.edu.cn).

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