14-3-3 Proteins Are Involved in BR-Induced Ray Petal Elongation in Gerbera hybrida

14-3-3 proteins play a major role in the regulation of primary metabolism, protein transport, ion channel activity, signal transduction and biotic/abiotic stress responses. However, their involvement in petal growth and development is largely unknown. Here, we identified and characterized the expression patterns of seven genes of the 14-3-3 family in gerbera. While none of the genes showed any tissue or developmental specificity of spatiotemporal expression, all seven predicted proteins have the nine α-helices typical of 14-3-3 proteins. Following treatment with brassinolide, an endogenous brassinosteroid, the Gh14-3-3 genes displayed various response patterns; for example, Gh14-3-3b and Gh14-3-3f reached their highest expression level at early (2 h) and late (24 h) timepoints, respectively. Further study revealed that overexpression of Gh14-3-3b or Gh14-3-3f promoted cell elongation, leading to an increase in ray petal length. By contrast, silencing of Gh14-3-3b or Gh14-3-3f inhibited petal elongation, which was eliminated partly by brassinolide. Correspondingly, the expression of petal elongation-related and brassinosteroid signaling-related genes was modified in transgenic petals. Taken together, our research suggests that Gh14-3-3b and Gh14-3-3f are positive regulators of brassinosteroid-induced ray petal elongation and thus provides novel insights into the molecular mechanism of petal growth and development.

Brassinosteroids (BRs) are plant steroid hormones that play key roles in regulating a variety of physiological processes, including leaf expansion, flowering, senescence, stress resistance and cell expansion and elongation (Clouse et al., 1996;Clouse and Sasse, 1998;Yang et al., 2011;Kim et al., 2012;Kaneko-Suzuki et al., 2018;Oh et al., 2020). BR signal cascades are well characterized in Arabidopsis, in which BZR1 is the key transcription factor, affecting plant growth and development by modulating thousands of BR target genes and interacting with other hormone signaling components (He et al., 2005;Wang et al., 2012;Qiao et al., 2017;Zheng et al., 2019;Zhang et al., 2020). 14-3-3 proteins are also important regulatory components of the BR signaling pathway: they regulate plant growth by anchoring BZR1 in the cytoplasm (Gampala et al., 2007;Ryu et al., 2007;Kim and Wang, 2010). Mutants of the 14-3-3-binding site of BZR1, with a phenotype similar to that of the bzr1-1D mutant, show a constitutive BR response and an increase in BZR1 nuclear retention (Gampala et al., 2007;Ryu et al., 2007).
The 14-3-3 proteins involved in BR signaling participate in plant growth and flowering processes by modulating cell differentiation and elongation (Pertl et al., 2010;Zhang et al., 2010;Taoka et al., 2011;Zhou et al., 2015;Kaneko-Suzuki et al., 2018;Minami et al., 2019). Analysis of multiple 14-3-3 mutants revealed their specificity and functional redundancy in primary root elongation under different environmental conditions, in which these genes are positive regulators under control conditions and negative regulators during abiotic stress (van Kleeff et al., 2014). In lily (Lilium longiflorum), 14-3-3 proteins were shown to play a role in the germination and elongation of pollen (Pertl et al., 2010). Minami et al. (2019) reported that, during BR-induced hypocotyl elongation, a 14-3-3 protein interacts with the phosphorylated C-terminus, and thereby enhances the catalytic activity, of plasma membrane H + -ATPase. In addition, 14-3-3 proteins have a regulatory role in cotton fiber elongation (Zhou et al., 2015). Thus, overexpression of 14-3-3L promotes fiber elongation in cotton, while gene silencing of 14-3-3L results in a shortening of cotton fiber length (Zhou et al., 2015). Recently, Zuo et al. (2021) identified eighteen 14-3-3 genes in the apple genome and characterized their expression patterns, suggesting that some of them may participate in the regulation of the flowering process. These results all highlight the importance of 14-3-3 proteins in plant growth.
Gerbera hybrida, belonging to the Asteraceae family, is one of the mainstream cut flowers and its commercial and ornamental value depend on petal morphology and color (Bhatia et al., 2009;Mosqueda Frómeta et al., 2017). Thus, it is important to understand the regulatory mechanisms governing gerbera petal morphology. The research team of Prof. Elomaa has focused on the molecular mechanisms of flower development in Asteraceae, including G. hybrida, for many years (Kotilainen et al., 1999;Broholm et al., 2008;Tahtiharju et al., 2012;Juntheikki-Palovaara et al., 2014;Zhao et al., 2020;Zhang et al., 2021). Broholm et al. (2008) found that overexpression of GhCYC2 in gerbera results in conversion of disc florets into ray-like florets with elongated petals, as well as disruption of stamen development. Functional analysis of GhCYC proteins revealed redundant functions of GhCYC2, GhCYC3 and GhCYC4 in regulating ray floret identity and in promoting petal development (Juntheikki-Palovaara et al., 2014). Various hormones (gibberellin, abscisic acid, ethylene, and BRs) are involved in the regulation of late-stage petal development in gerbera (Zhang et al., 2012;Li et al., 2015;Han et al., 2017;Huang et al., 2017Huang et al., , 2020Ren et al., 2018). Li et al. (2015) found that GA 3 stimulates petal elongation in gerbera, while ABA inhibits it. Further research showed that GhWIP2, a WIP-type ZFP transcription factor, represses cell expansion during petal and leaf development by modulating crosstalk between gibberellin, abscisic acid and auxin (Ren et al., 2018). Another study found that exogenous brassinolide (BL) treatment can boost the elongation of ray floret petals, whereas BRZ (a BR synthesis inhibitor) reduces petal length . However, whether 14-3-3 proteins, as one of the BR signaling components, play a regulatory role in BR-induced petal elongation in gerbera, or indeed in any other flowering species, remains unknown.
Here, seven gerbera 14-3-3 genes were identified and their predicted proteins classified. The expression patterns of all seven genes were comprehensively investigated in various tissues and at different developmental stages. Overexpressing two of these genes, Gh14-3-3b and Gh14-3-3f, in ray florets increased petal length by promoting cell elongation, whereas gene silencing of Gh14-3-3b or Gh14-3-3f reduced petal growth. Further analysis found that several BR-related genes, such as BZR1 homologs (GhBEH1 and GhBEH2) and petal elongation-associated genes (like GhEXP1, GhEXP3, GhEXP10, GhXTH1, and GhXET), were modified in transgenic petals. These results demonstrate a positive regulatory role of Gh14-3-3b and Gh14-3-3f in BRinduced ray petal elongation.

Plant Materials and Growth Conditions
A variety of G. hybrida called "Shenzhen No. 5" was used in this work. The plants were cultured under greenhouse conditions at 26/18 • C (day/night temperature) with a 16 h light/8 h dark photocycle and a relative humidity of 65∼80%. Three types of floret (ray floret, trans floret, and disc floret) at stage 6, as well as young leaf (leaf from plants transplanted into the soil for 10∼15 days), old leaf (basal leaf of plants transplanted into the soil for 3 months), young root (root of plants transplanted into the soil for 10∼15 days), old root (root of plants transplanted into the soil for 3 months), calyx, scape, and ray florets at different developmental stages, were sampled for quantitative real-time PCR (qRT-PCR) analysis. The development stages of ray florets (S1∼S6, "S" represents "stage") are defined according to Meng and Wang (2004). Ray florets at stage 3 were used for transient transformation and hormone treatment assays.

Cloning and Sequence Analysis of Gh14-3-3 Genes
Using the sequences of 14-3-3 genes in Arabidopsis thaliana, BLAST was performed against the transcriptome shotgun assembly database (Accession: PRJNA179026) of G. hybrida cultivar "Shenzhen No. 5" (taxid: 18101) (Kuang et al., 2013), and seven Gh14-3-3 genes were identified. Seven full-length Gh14-3-3 cDNA sequences were amplified from a gerbera cDNA library by PCR using PrimeSTAR Max Premix (Takara, Cat. No. R045) with specific primers. Alignment of the deduced amino acid sequences with Gh14-3-3 homolog from different species was performed using DNAMAN 6.0. Conserved domain analysis was executed in the Conserved Domain Database 1 . Protein structure prediction was performed with SWISS-MODEL 2 . Phylogenetic analysis was performed in MEGA 6.0 using a neighbor-joining algorithm with 1,000 bootstrap replicates. The primers for the constructs in each experiment and 14-3-3 protein information for various species are listed in Supplementary Table 1.

RNA Extraction and qRT-PCR Analysis
Total RNA was extracted from the samples using the Easystep R Super Total RNA Extraction Kit (Promega, Code No. LS1040) following the manufacturer's protocol. First-strand cDNA was synthesized from 1 µg total RNA using the ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Code No. FSQ-301). qRT-PCR was performed using RealStar Green Fast Mixture (GenStar, Code No. A301-01). 1 µL cDNA was added as a qPCR template in a total reaction volume of 20 µL. The samples were amplified using the CFX96 Touch TM Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., United States) as follows: melting at 95 • C for 2 min and amplification with 40 cycles of 95 • C for 5 s and 60 • C for 30 s. All analyses used a housekeeping gene (GhACTIN, AJ763915) as a normalization control (Kuang et al., 2013). The expression level was calculated according to the 2 − Ct method. The primers used for qRT-PCR are listed in Supplementary Table 1.
Detached ray petals from fresh inflorescences at stage 3 were cleaned, and then immersed in the various resuspension buffers mentioned above under a vacuum of −0.09 MPa for 5 min. After 2 min, the vacuum was slowly released and the petals were rinsed with sterile distilled water (dH 2 O) and placed in sterile Petri dishes with two layers of Whatman filter paper. After incubation at 4 • C for 3 days, the transformed petals were grown at 23∼25 • C for 9 days at 50∼60% humidity under long-day conditions (16 h light/8 h dark). At least 15 well-grown inflorescences were used for each treatment, and at least three biological replicates were used for each experiment.

Hormone Treatment of Ray Florets
Our previous study showed that detached petals can develop normally. The result of in vitro hormone and inhibitor experiments performed with detached petals were consistent with the result of in vivo experiments using intact inflorescences Huang et al., 2017). In this study, detached ray petals from inflorescences at stage 3 were used for BL treatments as described previously . Transiently transformed petals were placed in sterile Petri dishes with two layers of Whatman filter paper soaked in 10 µM BL or dH 2 O as control. Subsequently, the petals were cultured at 24∼26 • C for 2 days. At least 15 well-grown inflorescences were used for each treatment, and at least three biological replicates were used for each experiment.

Measurement of Ray Petal and Cell Length
A total of 45 petals were selected to measure their length as previously described . Petals were imaged with a Nikon camera D7200 (Japan) and measured using ImageJ software. To measure the petal cell length and number, the top, middle and basal region of each petal were stained with propidium iodide (0.1 mg mL −1 ) for 5 min. Next, images of adaxial epidermal cells were captured using a confocal laser scanning microscope (LSM710, Carl Zeiss, Germany) and more than 50 cells were analyzed using ImageJ software. At least three biological replicates were used for each observation. The elongation rate was calculated according Han et al. (2017).

Statistical Analysis
The data were analyzed with SPSS (version 13.0; IBM Corp., Armonk, NY, United States). Statistical significance between samples was investigated by Duncan's new multiple range test. The data are presented as mean ± standard error (SE). Different lowercase letters above the bars or line charts indicate significantly different groups: * P < 0.05, * * P < 0.01.

Spatiotemporal Expression Patterns and Response of Gh14-3-3 Genes to BR
To explore the spatiotemporal expression patterns of the seven Gh14-3-3 family members in gerbera, qRT-PCR was performed. We first analyzed their expression in different tissues and found that each gene was expressed in various organs or tissues ( Figure 3A). The highest expression levels appeared in young root, young leaf, disc floret, calyx and old leaf, while the lowest expression levels were mostly observed in old root. The different expression profiles in different tissues imply functional diversity in the Gh14-3-3 gene family.
We next evaluated the expression pattern of Gh14-3-3 genes during ray floret developmental stages (S1∼S6, "S" represents "stage") in gerbera. As ray floret petals developed, the expression levels of these genes changed in different ways ( Figure 3B). However, some patterns were comparable: for example, Gh14-3-3b and Gh14-3-3f showed a similar expression pattern, such that their transcription levels declined from the highest level in S1 to the lowest level in S3, followed by a gradual increase. Similarly, the transcript abundance of three genes (Gh14-3-3a, Gh14-3-3c, and Gh14-3-3d) dropped to the lowest level from S1 to S2, and then fluctuated in a related manner. In addition, Gh14-3-3e and Gh14-3-3g showed the highest expression levels in S2 and S1, and the lowest expression levels in S4 and S3, respectively. These results suggest that the expression of Gh14-3-3 genes is developmentally regulated in petal cells of gerbera.
14-3-3 proteins play an essential role in the BR signaling pathway (Gampala et al., 2007;Ryu et al., 2007). To determine whether the expression of any of the seven Gh14-3-3 genes responds to BR, the transcript levels of these genes were evaluated following BL treatment. As shown in Figure 3C, Gh14-3-3a and Gh14-3-3b shared the same expression profile: both genes began to respond at 1 h after BL treatment, rising to the highest expression level at 2 h, and then gradually decreasing to the lowest level at 24 h. Specifically, Gh14-3-3b had the highest peak value (225) among all seven members in response to BL. Three other genes (Gh14-3-3d, Gh14-3-3e, and Gh14-3-3g) had a similar response pattern to BR with two comparable response peaks (2.0∼2.5) at 0.5 h and 4 h. The expression level of Gh14-3-3f increased over the study period to a maximum at 24 h, while Gh14-3-3c expression varied slightly within a narrow range in response to BR. These results indicate that all members of the Gh14-3-3 gene family responded to BR, with Gh14-3-3a and Gh14-3-3b both reaching the highest expression level at an early stage (2 h) after treatment and Gh14-3-3f at a late stage (24 h).

Gh14-3-3b and Gh14-3-3f Promote Ray Petal Elongation in Gerbera
Based on the above results, it is clear that Gh14-3-3b and Gh14-3-3f have the lowest expression level in S3 (the onset of cell elongation), compared to other developmental stages. The two genes reached their highest expression levels at early (2 h) and late stages (24 h) in response to BR, respectively. Thus, we chose to analyze the roles of Gh14-3-3b and Gh14-3-3f in ray petal elongation by transient overexpression and VIGS assays.
On the other hand, the expression levels of the above genes showed, in many cases, a declining trend in Gh14-3-3b-VIGS and Gh14-3-3f -VIGS petals. In the Gh14-3-3f -VIGS petals, the expression of all eight genes was significantly reduced, compared to the mock (Figure 6B), while in Gh14-3-3b-VIGS petals, the expression of six genes (GhBEH1, GhEXP1, GhEXP2, GhEXP10, GhXTH1, and GhXET) was markedly downregulated, with two genes, GhBEH2 and GhBIN2, showing only a slightly decline. Notably, the expression of all eight genes was enhanced, albeit to different degrees, following BL treatment. These results suggest that Gh14-3-3b and Gh14-3-3f regulate BR-induced ray petal elongation by modulating genes associated with BR signaling and petal development.

Gh14-3-3 Proteins, Which Fall Into Two Groups, May Possess Functional Diversity
Since the first plant 14-3-3 protein was cloned from maize (de Vetten et al., 1992), researchers have identified eight 14-3-3 proteins in rice (Chen et al., 2006;Denison et al., 2011), 18 in apple (Zuo et al., 2021), nine in common bean (Li M. et al., 2016) and seven in cotton (Zhou et al., 2015). In the present study, seven 14-3-3 isoforms were identified in the gerbera transcriptome. Sequence analysis showed that all isoforms share the conserved nine α-helical regions typical of the 14-3-3 family and have 254∼336 amino acids ( Figure 1A). Phylogenetics classified the seven gerbera 14-3-3 proteins into two groups, the ε and non-ε groups, consistent with similar groupings in Arabidopsis, rice, and banana (Yaffe et al., 1997;Chevalier et al., 2009;Li M. et al., 2016). Furthermore, they show a high degree of identity with 14-3-3 proteins in Helianthus annuus and Lactuca sativa, both of which belong to Asteraceae family, hinting that these proteins have similar functions across the Asteraceae (Figure 1A).
Previous studies revealed that 14-3-3 proteins can form homodimers or, instead, can form heterodimers with different isoforms, which promotes functional diversity (Liu et al., 1995;Xiao et al., 1995;Aghazadeh et al., 2015; FIGURE 3 | The spatiotemporal expression pattern of Gh14-3-3 genes. (A) The expression pattern of Gh14-3-3 genes in gerbera tissues and organs. Relative mRNA level of the Gh14-3-3 genes in gerbera tissues (ray floret, disc floret, trans floret, calyx, old root, young root, old leaf, young leaf, and scape) were detected by qRT-PCR. GhACTIN (AJ763915) is the reference gene (Kuang et al., 2013). Gene expression levels were set to 1 in ray floret. (B) The expression of Gh14-3-3 genes during different growth stages (S1∼S6, "S" represents "stage") of ray floret in G. bybrida. The development stages of ray florets were defined according to Meng and Wang (2004). Gene expression levels were set to 1 in S1 ray floret petals. (C) The expression level of Gh14-3-3 genes in ray floret of G. bybrida under BL treatments. The expression levels of Gh14-3-3s in the ray floret of gerbera were detected within 0∼24 h after BL treatment. Gene expression levels were set to 1 in "0 h" and were calculated using the 2 − Ct method. Values were the means ± SE from three biological replicates.   Frontiers in Plant Science | www.frontiersin.org Ormancey et al., 2017). Each isoform displays a different propensity to dimerize with others, depending on the highly variable amino acid sequences in their N-terminal helices (Liu et al., 1995;Xiao et al., 1995). Consistent with this, the sequences of the N-terminal helices of gerbera 14-3-3 proteins are less well conserved than their C-terminal helices ( Figure 1A) and only three of the gerbera proteins (Gh14-3-3b, Gh14-3-3c, and Gh14-3-3f) form homodimers (Figure 2 and Supplementary Figure 2). Among the seven Gh14-3-3 proteins, the number of heterodimers formed ranged from one for Gh14-3-3e to six for Gh14-3-3b, which demonstrates the selectivity of each isoform in protein-protein interactions. Wilson et al. (2016) summarized the regulatory mechanisms of 14-3-3 proteins in several plants during the development of multiple organs, including seedling, leaf, root, flower, and developing seed. We found that the seven 14-3-3 isoforms are expressed in various organs in gerbera to different extents ( Figure 3A). This implies a functional diversity among all seven members, similar to the 14-3-3 proteins of other species (Chen et al., 2006;Yao et al., 2007). In addition, we surveyed the expression patterns of the Gh14-3-3 genes during ray floret developmental phases (S1∼S6) ( Figure 3B). As development progresses through stages S1 to S6, the Gh14-3-3 genes are expressed in various patterns. For example, Gh14-3-3b and Gh14-3-3f display a trend of earlier decrease and later increase from S1∼S6, and they both have the lowest expression level at S3 (Figure 3B). These genes also differ in their response to exogenous BL treatment ( Figure 3C): Gh14-3-3b responds rapidly and reaches its highest expression level in the early phase of the experiment (2 h), while Gh14-3-3f shows a slow response pattern. These results suggest functional diversity of the Gh14-3-3 genes during BR-induced gerbera growth and development (Chen et al., 2006;Zhou et al., 2015;Zuo et al., 2021).
As one of the mainstream cut flowers, gerbera has a high demand in the market. However, it has fewer flower types compared to chrysanthemum. Thus, obtaining a variety of flower types is one of the main goals of gerbera breeding, which requires an understanding of the regulatory mechanism of gerbera flower development. In this study, seven Gh14-3-3 protein genes were identified and their expression patterns were characterized. These genes share a conserved structure, but display different dimerization patterns, which implies they are functionally diverse. Transient transformation assays demonstrated that Gh14-3-3b and Gh14-3-3f play a positive regulatory role in BR-induced ray petal elongation. Thus, as well as providing novel insights into the role of 14-3-3 proteins in ray petal elongation, this study also highlights a number of candidate genes for flower type breeding of gerbera.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

AUTHOR CONTRIBUTIONS
XL carried out the experiments, drafted the manuscript, and revised manuscript. SH conducted the experiments, analyzed the data, and prepared the figures. GH participated in part of the experiments. YC and XW revised the manuscript. YW conceived the study, participated in its design, and revised the manuscript. All authors read and approved the final manuscript.