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Article

Bioinformatic and Phenotypic Analysis of AtPCP-Ba Crucial for Silique Development in Arabidopsis

by
Guangxia Chen
1,
Xiaobin Wu
2,
Ziguo Zhu
1,
Tinggang Li
1,
Guiying Tang
3,
Li Liu
1,4,
Yusen Wu
1,
Yujiao Ma
1,
Yan Han
1,
Kai Liu
1,
Zhen Han
1,
Xiujie Li
1,
Guowei Yang
1 and
Bo Li
1,4,*
1
Shandong Academy of Grape, Jinan 250100, China
2
State Key Laboratory of Nutrient Use and Management, Key Laboratory of Agro-Environment of Huang-Huai-Hai Plain, Ministry of Agriculture and Rural Affairs, Institute of Agricultural Resources and Environment, Shandong Academy of Agricultural Sciences, Jinan 250100, China
3
Institute of Crop Germplasm Resources, Shandong Academy of Agricultural Sciences, Jinan 250100, China
4
State Key Laboratory of Nutrient Use and Management, Shandong Academy of Agricultural Sciences, Jinan 250100, China
*
Author to whom correspondence should be addressed.
Plants 2024, 13(18), 2614; https://doi.org/10.3390/plants13182614
Submission received: 14 June 2024 / Revised: 9 September 2024 / Accepted: 17 September 2024 / Published: 19 September 2024
(This article belongs to the Section Plant Development and Morphogenesis)
Browse Figures
Versions Notes

Abstract

:
Silique development exerts significant impacts on crop yield. CRPs (Cysteine-rich peptides) can mediate cell–cell communication during plant reproduction and development. However, the functional characterization and regulatory mechanisms of CRPs in silique development remain unclear. In this study, we identified many CRP genes downstream of the CRP gene TPD1 (TAPETUM DETERMINANT1) during silique development using a microarray assay. The novel Arabidopsis thaliana pollen-borne CRPs, the PCP-Bs (for pollen coat protein B-class) gene AtPCP-Ba, along with TPD1, are essential for silique development. The AtPCP-Ba was significantly down-regulated in tpd1 flower buds but up-regulated in OE-TPD1 flower buds and siliques. The silencing of AtPCP-Ba compromised the wider silique of OE-TPD1 plants and inhibited the morphology of OE-TPD1 siliques to the size observed in the wild type. A total of 258 CRPs were identified with the bioinformatic analysis in Arabidopsis, Brassica napus, Glycine max, Oryza sativa, Sorghum bicolor, and Zea mays. Based on the evolutionary tree classification, all CRP members can be categorized into five subgroups. Notably, 107 CRP genes were predicted to exhibit abundant expression in flowers and fruits. Most cysteine-rich peptides exhibited high expression levels in Arabidopsis and Brassica napus. These findings suggested the involvement of the CRP AtPCP-Ba in the TPD1 signaling pathway, thereby regulating silique development in Arabidopsis.

1. Introduction

Silique morphology plays a crucial role in determining crop yields. In Arabidopsis thaliana, the silique consists of multiple fertilized ovules and three main regions, including the valve, replum, and valve margin [1,2]. The silique development originates from a gynoecium comprising two fused carpels connected by a central tissue named the septum [3]. This developmental process encompasses the initiation, growth, maturation, and ripening of the silique set. Silique growth involves both cell division and expansion [4], which commences shortly after fertilization and determines the final silique size [5].
The genetic networks contributing to silique development in Arabidopsis have also been elucidated. The YABBY transcription factor gene CRABS CLAW [6], the basic helix–loop–helix (bHLH) transcription factor gene SPATULA [7], and the auxin response factor ETTIN [8] have been identified to influence the carpel morphogenesis. Furthermore, the MADS-box family members SHATTERPROOF 1 and 2 (SHP1/SHP2) and FRUITFUL (FUL) can play crucial roles in regulating fruit patterning, lignin deposition, cell expansion, and cell separation processes within the silique [9,10,11]. Mutants of REPLUMLESS (RPL) exhibit a reduced replum width, indicating a pivotal role for RPL in replum development [12]. The downstream regulation of SHP involves INDEHISCENT (IND) and ALCATRAZ (ALC). FUL acts as a repressor of SHP and IND expression at the valves, while RPL functions similarly in the replum region. FUL and RPL can restrict the expression of SHP, ALC, and IND to narrow strips of cells at the valve margins [13]. The FUL–SHP pathway is crucial in silique morphology and evolutionarily conserved across plants [14]. Several signaling molecules, including GA, ethylene, auxin, and cytokinin, along with their intricate interactions, have been recognized as regulators of silique formation and growth [15]. The signals derived from male and female gametophytes and other vegetative tissues are known to promote silique development [16]. Nevertheless, the mechanisms by which the gametophyte-related genes and signals influence silique development remain unknown.
Plant small signaling peptides are a class of small peptides with a protein length of less than 180 amino acids. As novel and important signaling molecules, they play an important role in plant development and responses to stresses [17]. Cysteine-rich peptides (CRPs) are one crucial class of plant small signaling peptides in various aspects of plant defense, growth, development, and reproduction [18]. They typically exhibit three common structural features, namely a small protein length, typically under 160 amino acids, a conserved secretory signal peptide at the N-terminus, and a cysteine-rich domain at the C-terminus, usually containing four to sixteen cysteine residues [19]. Various cysteine-rich peptides, including the RAPID ALKALINISATION FACTOR (RALF), EPIDERMAL PATTERNING FACTOR (EPF), and EPF-LIKE (EPFL), have been reported to function in reproduction and development. The diverse RALF peptides have different cellular functions depending on their interactions with various receptors [20]. Specifically, the RALF coordinates with its receptor BRI1-associated receptor kinase1 (BAK1) to regulate root growth and hair initiation [21]. The RALF–FERONIA signaling pathway involves numerous cellular processes and stress responses, including Ca accumulation, H-ATPase activity, stomatal movement, flowering time, salinity tolerance, and immune response [22,23]. Members of the EPF/EPFL family, such as EPF2 and EPFL9, competitively bind to their co-receptors, ERECTA (ER) and TOO MANY MOUTHS (TMM), modulating stomatal patterning [24]. EPFL2 and EPFL9 are involved in ovule patterns, thereby regulating seed number with gynoecium and fruit growth by binding to ERECTA (ER)-family Leucine-Rich Repeat Receptor-Like Kinases (LRR-RLKs) [25]. However, research on the involvement of CRPs in Arabidopsis silique development remains limited.
TPD1, a small cysteine-rich peptide, and the LRR-RLK EXCESS MICROSPOROCYTES1 (EMS1, also known as EXTRA SPOROGENOUS CELLS, EXS) are essential for cell specification and proliferation during anther development in Arabidopsis. The AtTPD1 mutation results in the absence of tapetal cells and pollen grains, leading to complete male sterility, a phenotype consistent with ems1 and tpd1 ems1 double mutants [26,27]. TPD1, serving as a peptide ligand, directly interacts with EMS1, triggering the phosphorylation of the EMS1 kinase domain to determine the somatic and reproductive cell fate in Arabidopsis anthers [28,29]. Moreover, ectopic overexpression of TPD1 could induce carpel cell division and alter the silique morphology in Arabidopsis [30,31]. The maize MAC1 and rice TDL1A, which are orthologs of TPD1, are also crucial signal peptides in their respective species [32]. Therefore, exploring the TPD1–EMS1 signaling pathway in both monocots and dicots is imperative. However, the downstream signaling components of the TPD1–EMS1 pathway that regulate the cell fate and division during Arabidopsis development remain unclear.
Exploring and identifying the cysteine-rich peptide-encoding genes in siliques may provide a foundation for utilizing genetic engineering techniques to enhance crop growth and yield. Four A. thaliana PCP-B (pollen coat protein B-class) encoding proteins, namely AtPCP-Ba, AtPCP-Bb, AtPCP-Br, and AtPCP-Bꝺ, as important regulatory small peptides, establish a molecular dialog between the stigma and pollen grains during the earliest stages of the pollen–stigma interaction [33]. Phenotypic analysis revealed defective pollen hydration and delayed pollen tube growth in pcp-b mutants compared to wild-type ones. Triple mutant pcp-ba/b/r pollen showed considerably reduced hydration on the stigma surface, a weaker anchorage to the stigma surface, and slow pollen tube growth [33]. In this study, we isolated and identified the cysteine-rich peptide gene AtPCP-Ba from a microarray analysis of the tpd1 mutant and OE-TPD1 plants and investigated the relationship between AtPCP-Ba and TPD1 in silique development. A knock-down of AtPCP-Ba compromised the wider silique of OE-TPD1 plants, resulting in the restoration into wild-type siliques. Additionally, we conducted an evolutionary tree classification of 258 CRPs across Arabidopsis, Brassica napus, Glycine max, Oryza sativa, Sorghum bicolor, and Zea mays. Among them, 107 CRP genes were predicted to be abundant in flowers and fruits, with many showing high expression levels in Arabidopsis and Brassica napus. These findings suggested the potential involvement of the CRP AtPCP-Ba in the TPD1 signaling pathway, thereby regulating silique development in Arabidopsis.

2. Results

2.1. TPD1 Promotes the Expression of AtPCP-Ba Gene during Silique Development

To investigate the TPD1–EMS1 signaling pathway in silique development, the OE-TPD1 plants, tpd1, and ems1 homozygous mutants were used to perform microarray analysis. The wider silique phenotype in OE-TPD1 plants was observed from the 12th stage of flower development (Figure S1). To screen the essential genes downstream of TPD1 that regulate silique width, the flower buds were selected before the 12th stage of flower development for the microarray assays. Three technical RNA replicates were pooled for each genotype per time point for labeling and hybridization, sourced from three independent biological replicates of the flower buds.
A total of 44 small signaling peptide genes were found to be differentially expressed between wild-type plants and each genotype plant with a false discovery rate (FDR) < 0.05, including nine that were up-regulated in OE-TPD1, one that was down-regulated in OE-TPD1, five that were up-regulated in tpd1 and 30 that were down-regulated in tpd1 by at least twofold (Figure 1A, Excel S1). Notably, among these 44 small signaling peptide genes were 21 cysteine-rich peptides (CRPs), including four (19.1%) CRPs up-regulated in OE-TPD1, three (14.3%) CRPs up-regulated in tpd1, and 15 (71.5%) CRPs down-regulated in tpd1 (Figure 1B). Among these candidates, the AtPCP-Ba gene was identified using the microarray analysis of TPD1 overexpressors and tpd1 mutants from flower buds. AtPCP-Ba exhibited significant up-regulation in OE-TPD1 flower buds and more than double the down-regulation in tpd1 and ems1 mutant flower buds (Table S1). The RT-PCR and real-time PCR confirmed the transcriptional changes in AtPCP-Ba and TPD1 during silique development, with AtPCP-Ba notably down-regulated in tpd1 mutants and up-regulated in the OE-TPD1 plant flower buds (Figure 2). These findings indicated the role of TPD1 in promoting AtPCP-Ba expression at the transcriptional level.

2.2. AtPCP-Ba Is Highly Expressed in Developing Flower Buds

The RT-PCR and real-time PCR analyses were conducted using the RNAs extracted from various tissues to examine the spatial and temporal expression of AtPCP-Ba and validate its relationship with TPD1. The findings revealed elevated levels of the AtPCP-Ba transcripts in developing flower buds, contrasting with lower levels detected in mature pollen, green siliques, leaves, and stems, whereas expression was scarce in roots and seedlings (Figure 3A,B).
The AtPCP-Ba promoter activity assay with a 1 kb sequence in the upstream region was conducted to delineate the spatial and temporal expression profiles of AtPCP-Ba (Figure 3C–G). A robust GUS signal was predominantly observed in the flower buds, notably within the anther (Figure 3D). In addition, a weaker GUS signal was detected in siliques, leaves, and stems (Figure 3E–G). Furthermore, the AtPCP-Ba expression was predominantly confined to tapetal cells from anther developmental stages 5 to 11, as evidenced by the sectioning of GUS-stained anthers (Figure S2), distinguishing it from TPD1, which is primarily expressed in microsporocytes [27].

2.3. Silencing of AtPCP-Ba Compromises the Wider Silique of OE-TPD1 Plants

To investigate the role of the AtPCP-Ba gene in silique development, two construct vectors, p35S::AtPCP-Ba and AtPCP-Ba-RNAi, were used to analyze its relationship with TPD1 during silique development. Both constructs were then transformed into wild-type Ler plants. However, no notable phenotypic changes were observed in the transgenic plants. The result showed that some obvious silique discrepancy existed in AtPCP-Ba-RNAi/OE-TPD1-transformed plants when the AtPCP-Ba-RNAi was transformed into OE-TPD1 plants. Some transformed plants showed that the phenotype of OE-TPD1 siliques partially disappeared, and some transformed plants showed that their silique morphologies are even similar to that of wild-type plants (Figure 4A–F). The siliques of AtPCP-Ba-RNAi/OE-TPD1-transformed plants were analyzed in length, width, and ratio (Figure 4G–I). The similar reduction in the wider silique phenotype was also observed in the AtPCP-Ba-amiRNA/OE-TPD1 transgenic plants (Figure 5A–D). To make sure the silique morphologies changed, we also adopted an artificial microRNA technique and transformed the new construct AtPCP-Ba-amiRNA into OE-TPD1 plants. The same silique phenotypic changes were observed in AtPCP-Ba-amiRNA/OE-TPD1-transformed plants. Two lines with different silique morphologies were selected for further analysis (Figure 5B,C). The RNA from the mature siliques was isolated and used for the real-time PCR analysis. The real-time PCR results revealed lower AtPCP-Ba gene expression in plants with completely disappeared morphology than in those with wider siliques (Figure 5E). Consistent changes in expression were observed between AtPCP-Ba and TPD1 in both lines (Figure 5F). However, the AtPCP-Ba homolog gene AtPCP-Bꝺ exhibited no similar changes in expression between the two lines (Figure 5G). These results indicated that AtPCP-Ba-amiRNA selectively targeted its intended gene without affecting its homologous counterpart, AtPCP-Bꝺ.

2.4. Molecular Characterization of AtPCP-Ba in Plants

The AtPCP-Ba protein possessed a typical N-terminal signal peptide domain and a C-terminal sequence featuring seven cysteine residues, confirming its classification in the CRP family (Figure S3). The multiple sequence alignment analysis revealed numerous homologies of AtPCP-Ba in several crops, including Zea mays, Oryza sativa, Sorghum bicolor, Glycine max, Brassica napus, and Arabidopsis (Excel S2). The CRPs from the above plants were categorized into five subgroups according to evolutionary tree classification, represented by clades GI, GII, GIII, GIV, and GV (Figure 6). The clades GII, GIII, and GIV encompassed a larger number of family members, comprising 57, 72, and 112 CRP genes, respectively, whereas the clades GI and GV included fewer members, with 11 Arabidopsis CRP family genes and six other CRP genes, respectively (Figure 6). Furthermore, we analyzed their conserved motifs, revealing that AtPCP-Ba shared a high similarity and identical motif 2 composition with numerous CRPs in Zea mays, Oryza sativa, Sorghum bicolor, and Arabidopsis (Figure S4C). The presence of motifs 1, 2, and 3 in most CRP proteins indicated their involvement in a conserved domain within CRP proteins (Figure S4C). Additionally, we grouped the CRP genes from monocotyledons and dicotyledons into separate clusters, suggesting independent evolution after the divergence of monocotyledons and dicotyledons (Figure S4).
The analysis was conducted on the 107 CRP genes predicted to be abundant in reproductive organs across the following six species: Arabidopsis, Zea mays, Oryza sativa, Sorghum bicolor, Brassica napus, and Glycine max, utilizing the RNA-seq data obtained from the database (Figure 7). These CRP genes are predicted to be expressed broadly in different species with notable variations among individual genes. Some genes exhibited high expression levels in the reproductive organs of Oryza sativa and Zea mays. Some genes showed significantly increased expression levels in the reproductive organs of Arabidopsis and Brassica napus. Notably, AtPCP-Ba showed similar expression levels in the reproductive organs of Arabidopsis and Brassica napus compared to that of other crops (Figure 7B).

2.5. Expression Pattern of AtPCP-Ba Homologies Regulated by TPD1 in Different Plants and Tissues

The expression levels of AtPCP-Ba, AtPCP-Bꝺ, AtPCP-Br, AT2G41415, and ESF1.1 genes were assessed in flower buds of tpd1 mutants and OE-TPD1 plants using real-time PCR (Figure 8). Significant decreases were observed in the expression levels of these homologous genes in tpd1 flower buds compared to those in the wild type. Conversely, the expression levels increased in the flower buds of OE-TPD1 plants. Notably, no significant change was observed in the expression level of ESF1.2 in tpd1 mutant flower buds. However, its expression level was notably higher in OE-TPD1 plant flower buds than in the wild type. Moreover, increased expression levels were detected for ESF1.3, ATlGl27135, and AT4Gl5953 genes in tpd1 mutant flower buds compared to the wild type. Similarly, their expression levels were significantly elevated in siliques from OE-TPD1-overexpressing plants than the wild type (Figure 8A). These alterations were further investigated using siliques from both wild-type and OE-TPD1 plants, revealing significantly higher expression for all genes, except for the down-regulation observed in the ESF1.3 gene within silique tissues from OE-TPD1-overexpressing plants (Figure 8B). Additionally, AtPCP-Ba was also up-regulated in the OE-TPD1 siliques (Figure 8B). These results indicated the AtPCP-Ba and some homologies were indeed regulated by TPD1 at the transcriptional level.
To further explore the biological functions of the AtPCP-Ba homologous genes, we examined their spatial and temporal expression patterns in Arabidopsis using real-time PCR (Figure S5). In addition to the prominently expressed AtPCP-Ba gene in flower buds, we detected the expression of four genes, including AtPCP-Bꝺ, AtPCP-Br, AT2G41415, and ESF1.1, in flower buds, particularly within pollen. ESF1.1 and AtPCP-Bꝺ also exhibited expression levels in siliques (Figure S5). Conversely, the ESF1.3, ESF1.2, and AT4G15953 genes displayed higher expression levels in siliques, and ESF1.3 was also expressed in pollen. Notably, the ESF1.2 gene expression levels were significantly higher in open flowers and flower buds than in other tissue parts (Figure S5).

3. Discussion

Various plant small signaling peptides play vital roles in diverse growth and development processes, including cell proliferation [34,35], mineral element absorption and regulation [36], root development [36,37,38,39,40,41], pollen fertility [42,43,44], stomata aperture regulation [45,46], defense resistance [47,48,49], and environmental adaptation [50]. Cysteine-rich peptides are essential for pivotal reproductive processes, such as self-incompatibility, pollen tube elongation, guidance, and gamete interactions [51]. AtLURE1s and XIUQIUs, cysteine-rich pollen tube attractants, are secreted by synergid cells and diffuse from the micropylar region of the ovule toward the surfaces of the placenta and septum, respectively [52]. Cysteine-rich peptides EPFL2 and EPFL9 regulate ovule patterning and govern seed numbers in gynoecium and fruit growth through shared receptors [25]. Recent research has demonstrated that AtPCP-Ba, AtPCP-Bb, and AtPCP-Br, as important cysteine-rich peptides, establish the dialog between the stigma and pollen grains during the pollen–stigma interaction [33]. The depletion of PCP-Ba/b/r significantly slows the process of pollen hydration and germination [33]. Pollen PCP-Bb/r peptides competitively bind to the ANJ–FER receptor complex, displacing RALF23/33 and effectively reducing stigmatic ROS levels to facilitate pollen hydration [53]. Our research has demonstrated that AtPCP-Ba was expressed in tapetal cells, which persisted from anther developmental stages 5 to 11 during the tapetal development in Arabidopsis (Figure S2). This result suggested that AtPCP-Ba may function in pollen development, which is consistent with previous research.
In this study, we discovered a novel function of AtPCP-Ba as a vital CRP in silique morphology development. AtPCP-Ba, along with TPD1, represented two newly identified cysteine-rich peptide genes implicated in silique development. Our analysis focused on elucidating the relationship between AtPCP-Ba and TPD1. Using a microarray analysis, we isolated and identified the AtPCP-Ba gene by comparing the gene expression in the tpd1 mutant and OE-TPD1. TPD1 is expressed mostly in developing microsporocytes and is required for the specialization of tapetal cells in the Arabidopsis anther [27]. OE-TPD1 altered the pattern of siliques from the 12th stage of flower development (Figure S1C,D). We speculate that AtPCP-Ba with TPD1 are the signals from male gametophytes to promote silique development. The down-regulation of the AtPCP-Ba gene using artificial microRNA and RNAi technology in OE-TPD1 plants led to decreased expression levels of TPD1 and weakened or even abolished the phenotype of OE-TPD1. Our results confirmed the positive correlation between AtPCP-Ba and TPD1 in the regulation of silique morphology development in Arabidopsis. The AtPCP-Ba gene expression was up-regulated with TPD1 up-regulation. Meanwhile, the AtPCP-Ba gene expression was down-regulated with the TPD1 down-regulation. Additionally, AtPCP-Ba may exert the feedback regulation of TPD1. Because TPD1 was also decreased when AtPCP-Ba gene expression was down-regulated, TPD1 might undergo feedback regulation by AtPCP-Ba, in addition to affecting the expression of the AtPCP-Ba gene.
TPD1, a small, secreted cysteine-rich protein ligand, can interact with the LRR (leucine-rich repeat) domain of the EMS1 receptor kinase, influencing tapetum cell fate and carpel development [28]. The ectopic TPD1 expression induces wider siliques by promoting additional cell division, suggesting that the ectopic expression of TPD1–EMS1 signaling affects cell division [30]. Similar to the TPD1 protein, the AtPCP-Ba protein possesses an N-terminal signal peptide (Figure S3B,C) (https://services.healthtech.dtu.dk/service.php?SignalP-5.0 (accessed on 18 March 2011)). LRR-RLKs and MAPKs play crucial roles in regulating cell division and proliferation in plants [54]. Various ligand–receptor pairs frequently share common downstream signaling components, including mitogen-activated protein kinase (MAPK) signaling networks [55]. The TPD1-activated EMS1 pathway can likely play a significant role in cell division by triggering the MAPK cascade. It is hypothesized that AtPCP-Ba may serve a similar function as a signaling molecule, necessitating the identification of its receptor protein in silique development. Our findings indicated that TPD1 primarily regulated the expression of AtPCP-Ba, which could mitigate the widened silique phenotype of OE-TPD1. Notably, the single receptor could perceive multiple peptides, as seen with EPF2 and STOMAGEN/EPF-LIKE 9 (EPFL9), both competitively binding to the ERECTA receptor to regulate stomatal patterning in the leaf epidermis [24,56]. Despite the genetic interaction between TPD1 and AtPCP-Ba, further research is imperative to identify the receptor of AtPCP-Ba, and additional investigation is required to entirely elucidate the relationship between AtPCP-Ba and TPD1, as well as their specific mechanisms of silique development.
The evolutionary relationships among Arabidopsis, Brassica napus, Glycine max, Oryza sativa, Sorghum bicolor, and Zea mays were deduced from the analysis of 258 small cysteine-rich secretory peptides, resulting in the classification of five distinct subgroups. This evolutionary tree was compared using all protein sequences of CRP members, differing from previous alignment data based on predicted mature protein-coding regions [33]. Among these, 107 cysteine-rich peptide genes were predicted to be highly expressed in flowers and fruits. Most cysteine-rich peptides were clustered in Arabidopsis and Brassica napus. Eleven small CRP proteins demonstrated notable similarities to AtPCP-Ba in Arabidopsis. The real-time PCR assay revealed elevated expression levels of AtPCP-Ba, AtPCP-Bꝺ, AtPCP-Br, AT2G41415, and ESF1.1 in flower buds and pollen. AT1G27135 was highly expressed in pollen, and the other three genes, namely ESF1.2, ESF1.3, and AT4G15953, were expressed in siliques. These CRPs are highly expressed in the reproductive organs of Arabidopsis plants. Additionally, the real-time PCR assay demonstrated a significant down-regulation of gene expression levels of AtPCP-Ba, AtPCP-Bꝺ, AtPCP-Br, AT2G41415, and ESF1.1 in tpd1 mutants, but a significant up-regulation in OE-TPD1 plants (Figure 8A). These results suggested that AtPCP-Ba, along with the other four genes AtPCP-Bꝺ, AtPCP-Br, AT2G41415, and ESF1.1, may be associated with the TPD1 gene in silique development. Some studies have indicated that ESF1.1, ESF1.2, and ESF1.3 participate in the membrane-associated receptor-like cytoplasmic kinase SSP (SHORTSUSPENSOR) and the MAPK (mitogen-activated protein kinase) YDA-mediated signal transduction pathway, thereby regulating the early development of Arabidopsis embryos [57].
The absence of T-DNA mutants for AtPCP-Ba led to the generation of both amiR-AtPCP-Ba and RNAi-AtPCP-Ba transgenic plants via transformation into wild-type Ler. However, no identifiable phenotypic differences were observed in Ler transgenic plants. Nonetheless, a significant reduction in pollen hydration was noted in triple mutants of AtPCP-Ba, AtPCP-Br, and AtPCP-Bb [33]. The high similarity in the amino acid sequences of AtPCP-Ba, AtPCP-Bꝺ, and AtPCP-Br suggested potential functional redundancy in plant development. The artificial microRNA and RNAi technology for the down-regulation of the AtPCP-Ba gene in OE-TPD1 plants led to the weakened or even abolished wide silique phenotype of OE-TPD1. The real-time PCR analysis indicated that the expression level of the homologous gene AtPCP-Bꝺ was similar to that observed in amiR-AtPCP-Ba-OE-TPD1 transgenic plants, where morphological changes had completely disappeared compared to wider siliques (Figure 5G). Further investigation revealed an up-regulated expression level of AtCPB-Aꝺ in transgenic plants with wider siliques expressing amiR-AtPCP-Ba-OE-TPD1 as well as in OE-TPD1 transgenic plants (Figure 5G). This observation was consistent with the finding that elevated TPD1 expression levels led to higher relative expression levels of both AtPCP-Ba and its homologous gene AtPCP-Bꝺ in flower buds and siliques from OE-TPD1 plants (Figure 8), suggesting a potential role for PCP-Ba as a ligand to activate distinct effector targets associated with silique width through TPD1 or to enhance activation via synergistic interactions with putative silique targets. A similar complexity was observed when multiple synergid LUREs collectively functioned through various pollen tube receptors to ensure the proper guidance toward embryo sacs, as seen in species such as T. fournier and Arabidopsis [42,58,59]. The CRPs AtPCP-Ba and TPD1 may function with the same or different carpel receptors to determine the initiation of silique morphology. These findings implied a positive correlation between AtPCP-Ba and TPD1 in cell division and growth during silique development. However, further research is necessary to elucidate the role of AtPCP-Ba in silique development. More homologous CRP genes with AtPCP-Ba need to be knocked down or out together to observe the silique morphology. More researches about the characterization and functional analysis of CRPs need to be carried out to explicate more signal pathways in silique development. Precise interactions between AtPCP-Ba with other homologous CRPs and their corresponding receptors also need to be identified in silique development. The concentration of CRP required for their physiological functions is extremely low. Unlike traditional plant hormones, CRPs primarily consist of amino acids, thereby posing no environmental risk upon exogenous application. The application of CRPs in agricultural production has the potential to enhance crop yield, thus serving modern green agriculture.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

The Arabidopsis thaliana plants used in this study originated from a Landsberg erecta (Ler) background. The seeds were surface-sterilized in 8% sodium hypochlorite for 5 min, rinsed three times in sterilized distilled water, and then placed on Murashige and Skoog (MS) salt agar plates before being transferred to a growth chamber at 4 °C for 2 days. Subsequently, the seeds were cultivated in a growth room at 22 °C under a photoperiod of 16 h light and 8 h dark for 7–10 days, then transferred to soil under the same light conditions. Previous studies have documented OE-TPD1, tpd1, and ems1 [31]. AmiRNA-AtPCP-Ba and RNAi-AtPCP-Ba transgenic plants were developed from the Ler and OE-TPD1 backgrounds.

4.2. Construction of amiRNA-AtPCP-Ba

To construct the amiRNA-AtPCP-Ba, RS300 (MIR319a Arabidopsis thaliana) served as the backbone. The sequence of the AtPCP-Ba gene was analyzed to predict the target of amiRNA in the Arabidopsis thaliana genome using Web MicroRNA Designer (http://wmd3.weigelworld.org/cgi-bin/webapp.cgi?page=Designer (accessed on 8 April 2013)). Two separate PCR steps were employed to obtain AtPCP-Ba amiRNA fragments. First, pRS300 was used as a template to generate two DNA fragments containing the target gene. Subsequently, another round of PCR was conducted using these two DNA fragments as templates to amplify the full-length DNA fragment amiRNA-AtPCP-Ba. The primers used to construct amiRNA-AtPCP-Ba are listed in Table S2.

4.3. Construction of RNAi-AtPCP-Ba

For obtaining the AtPCP-Ba plant RNAi expression vector, the partial coding region of AtPCP-Ba was amplified from the pMD18-AtPCP-Ba plasmid containing the full-length AtPCP-Ba cDNA, using the gene-specific primers RNAi-CDS-F and RNAi-CDS-R (Table S3). The forward primer RNAi-F incorporated NcoI and SwaI restriction enzyme sites at its 5′ end, whereas the reverse primer RNAi-R included XbaI and BamHI restriction enzyme sites at its 3′ end. The digested AtPCP-Ba fragments were then inserted into the XbaI/BamHI and NcoI/SwaI enzyme sites of the binary vector pFGC5941 at inverted repeat sequences, resulting in the plant RNAi expression vector pFGC-AtPCP-Ba, which was capable of forming a hairpin RNAi construct. The primers used to construct the AtPCP-Ba-RNAi are listed in Table S3.

4.4. RT-PCR and Real-Time PCR Assays

Total RNAs from various Arabidopsis tissues, including roots, shoots, leaves, flowers, flower buds, mature pollen, and seedlings, were extracted using a TRIzol reagent kit (266412, Invitrogen, Carlsbad, CA, USA). The RNA from the siliques was isolated using a total RNA extraction kit (BioTeke, Beijing, China). The SuperScript III First-Strand Synthesis System (18064-014, Invitrogen, CA, USA) was used to synthesize first-strand cDNA according to the manufacturer’s instructions. The cDNA pools from different tissues were used to analyze expression patterns, whereas those from flower buds in mutant and wild-type plants were compared for AtPCP-Ba expression levels. Amplifications were performed for 35 cycles for TUB, TPD1, and AtPCP-Ba, with Tubulin-8 serving as an internal control for RT-PCR. The real-time PCR assays were conducted using a 2×Power SYBR Green PCR Master Mix (4367659, Applied Biosystems, Waltham, MA, USA, www.appliedbiosystems.com) with gene-specific primers on an ABI 7500 real-time instrument (Applied Biosystems, www.appliedbiosystems.com). The PCR program consisted of an initial denaturation step at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. After each run, a dissociation curve was generated by gradually heating the samples from 60 °C to 95 °C to confirm amplification specificity. The relative expression levels were determined using the ΔΔCt (threshold cycle) method and normalized to that of ACTIN2/8 within the same cDNA samples, which were assessed in triplicate for three biological replicates. All primers used in this study are listed in Table S3.

4.5. Microarray Analysis

The RNA samples used for microarray assays were extracted from flower buds of wild-type, OE-TPD1, tpd1, and ems1-2 homozygous mutants. ATH1 Genome Arrays were employed to compare their transcriptomes. The transcription levels between the wild-type and mutant plants were quantified in triplicate using the ATH genome chips (Affymetrix, http://www.affymetrix.com (accessed on 8 May 2010)). The genes exhibiting expression changes greater than two-fold were categorized as either up-regulated or down-regulated and were selected as candidates for regulation by TPD1. The candidates were identified based on fold changes exceeding two or less than 0.5 compared to the wild type.
The total RNA was extracted for microarray experiments, and the RNA quality was assessed by running a 2 μg RNA aliquot on the agarose gel. Sixty micrograms of total RNAs were purified using the Qiagen RNAeasy Mini Kit (Qiagen, Valencia, CA, USA) and used for subsequent experiments. The cDNA synthesis, sample labeling, array hybridization, scanning, and data processing were performed as previously described [60].

4.6. GUS Assay

The promoter fragments of AtPCP-Ba were amplified using rTaq (DR100A, TaKaRa, Shiga, Japan) and subcloned upstream of the GUS reporter gene in the pCAMBIA1300 Ti-derived binary vector, followed by transformation into wild-type plants. Plant transformation and GUS activity analyses were conducted as previously described [61]. The GUS activity was assessed by staining different transgenic plant tissues in a solution containing 100 mmol/L NaPO4 (pH 7.0), 0.5 mmol/L potassium ferricyanide [K3Fe(CN)6], 0.5 mmol/L potassium ferrocyanide [K4Fe(CN)6], 0.1% Triton X-100, 10 mmol/L EDTA, and 0.5 mg/mL bromochloroindoyl-β-glucuronide [62]. The staining was performed at 37 °C for 2–3 h, followed by overnight incubation in an acetic acid/ethanol solution (1:3 [v/v]). The GUS-stained tissues were examined using a Leica DM2500 microscope equipped with a DIC system and MZ10F stereo microscope (Leica, Wetzlar, Germany). The 1.0 kb promoter region upstream of the start codon of AtPCP-Ba was fused to the glucuronidase (GUS) reporter gene and introduced into wild-type Arabidopsis plants, resulting in the generation of twenty-one independent pAtPCP-Ba::GUS transgenic plants.

4.7. Light Microscope

The flowers of both wild-type and mutant plants were fixed overnight in an FAA solution comprising 90% ethanol, 5.0% glacial acetic acid, and 5.0% formaldehyde, followed by a 30 min exhaustion process. Subsequently, all the tissues were dehydrated in an ethanol series (70%, 80%, 90%, 95%, and 2 × 100%) for 30 min per concentration, followed by clearing in dimethylbenzene and embedding in paraffin. Dewaxed specimens were sectioned (7 mm) using a microtome (LEICA RM2265, Germany). Anther transverse sections were stained with 0.5% safranin at 37 °C for 40 min and 0.5% Fast Green at room temperature for 1 min. All anther cross-sections were photographed using a microscope (LEICA DM2500, Germany).

4.8. Multiple Sequence Alignment and Phylogenetic Analysis

An unrooted phylogenetic tree was constructed using protein sequences from 13 AtCRP proteins in Arabidopsis, 60 ZmCRP proteins in Zea mays, 33 OsCRP proteins in Oryza sativa, 12 SbCRP proteins in Sorghum bicolor, 35 GmCRP proteins in Glycine max, and 105 BnCRP protein sequences from Brassica napus using the neighbor-joining method in MEGA 7.0. The expression patterns of CRP genes in these six species were analyzed using RNA-seq data obtained from relevant databases. The transcription levels of 107 CRP genes across Arabidopsis, Zea mays, Oryza sativa, Sorghum bicolor, Brassica napus, and Glycine max were then analyzed and visualized.
The CRP sequences from various crops were obtained from complete plant genomes using TBLASTN database searches (PHYTOZOME, https://phytozome.jgi.doe.gov (accessed on 22 September 2021); Comparative Genomics, COGE, https://genomevolution.org (accessed on 18 December 2022)), ensuring the completion up to at least the scaffold level. The Arabidopsis CRP protein sequences were sourced from Arabidopsis Information Resource 4, and the alignment of CRP protein sequences from crops and Arabidopsis was performed using the MUSCLE method [63]. Subsequently, an unrooted phylogenetic tree was constructed using the neighbor-joining (NJ) method with a p-distance model and 1000 bootstrap repeats in MEGA 7.0 [64].

5. Conclusions

In summary, this study elucidated a novel role of AtPCP-Ba as a pivotal small signal peptide in the development of silique morphology. The intimate association between AtPCP-Ba and TPD1 in silique development, along with their broad similarity to other CRP regulatory proteins, strongly suggested their involvement in interacting with unknown carpel targets to activate silique morphology development. Furthermore, the maintenance and diversity of CRP proteins in Arabidopsis and Brassicaceae implied their potential contribution to silique development and crop seed yield.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants13182614/s1, Figure S1: The silique phenotypic observation of the OE-TPD1 and wild type plants; Figure S2: AtPCP-Ba was expressed in tapetal cells; Figure S3: AtPCP-Ba protein contains a N-terminal signal peptide; Figure S4: Phylogenetic tree (A), gene structure (B), and conserved motif (C) of flower and silique CRP protein sequences in Arabidopsis, Zea mays, Oryza sativa and Sorghum bicolor; Figure S5: Expression patterns of PCP-B family genes; Table S1: The microarray assay showed that the expression of AtPCP-Ba was significantly affected in OE-TPD1, tpd1, and ems1 mutants; Table S2: The primers used in the artificial microRNA assays; Table S3: The primers used in the related vector construct, real-time PCR and RT-PCR assays; Excel S1: All the small signaling peptide genes were found to be differentially expressed between wild-type plants and each genotype plant in the microarray; Excel S2: All the cysteine-rich peptides (CRPs) for phylogenetic relationship analysis in Arabidopsis, Zea mays, Oryza sativa, Sorghum bicolor, Brassica napus, and Glycine max.

Author Contributions

G.C. carried out the experiments and wrote the manuscript. X.W., Z.Z. and T.L. analyzed the data and prepared figures. G.T., L.L. and Y.W. participated in the data collection and data analysis. Y.M., Y.H., K.L., Z.H. and G.Y. contributed to the consultation. X.L. and B.L. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

The study was supported by grants from the State Key Laboratory of Nutrient Use and Management (grant no. GZ2023003), the International (Regional) Science and Technology Cooperation Project of Shandong Academy of Agricultural Sciences (grant no. CXGC2024G18), the Guide Fund of Shandong Academy of Grape (grant no. SDAG2021B04), the Agricultural Science and Technology Innovation Project of Shandong Academy of Agricultural Sciences (grant no. CXGC2024A07), the Agricultural Improved Variety Project of Shandong Province (grant no. 2022LZGCQY019), and the Shandong Provincial Natural Science Foundation (grant no. ZR2021QC058).

Data Availability Statement

Transcriptome data under the abiotic stress and biotic treatment are available from the corresponding author ([email protected]) upon reasonable request.

Acknowledgments

The authors are thankful to Xueqin Zhang, State Key Laboratory of Plant Environmental Resilience, College of Biological Science, China Agricultural University, for the experiment materials, helpful discussion, and suggestions.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Plant small signaling peptides and cysteine-rich peptides were altered in microarray data. (A) Analysis of plant small signaling peptide genes that were altered in the microarray data. (Purple oval: the number and percentage of plant small signaling peptides that were increased in OE-TPD1 compared with Ler; green oval: the number and percentage of plant small signaling peptides that were decreased in OE-TPD1 compared with Ler; pink oval: the number and percentage of plant small signaling peptides that were increased in tpd1 compared with Ler; yellow oval: the number and percentage of plant small signaling peptides that were decreased in tpd1 compared with Ler). (B) Analysis of cysteine-rich peptide genes, which were altered in the microarray data. Blue circle: the number and percentage of cysteine-rich peptide genes that were increased in OE-TPD1 compared with Ler; yellow circle: the number and percentage of cysteine-rich peptide genes that were increased in tpd1 compared with Ler; Green circle: the number and percentage of cysteine-rich peptide genes that were decreased in tpd1 compared with Ler. I: increased gene number; D: decreased gene number; the number and percentages in the Venny diagram indicate target gene numbers and percentages in the microarray data.
Figure 1. Plant small signaling peptides and cysteine-rich peptides were altered in microarray data. (A) Analysis of plant small signaling peptide genes that were altered in the microarray data. (Purple oval: the number and percentage of plant small signaling peptides that were increased in OE-TPD1 compared with Ler; green oval: the number and percentage of plant small signaling peptides that were decreased in OE-TPD1 compared with Ler; pink oval: the number and percentage of plant small signaling peptides that were increased in tpd1 compared with Ler; yellow oval: the number and percentage of plant small signaling peptides that were decreased in tpd1 compared with Ler). (B) Analysis of cysteine-rich peptide genes, which were altered in the microarray data. Blue circle: the number and percentage of cysteine-rich peptide genes that were increased in OE-TPD1 compared with Ler; yellow circle: the number and percentage of cysteine-rich peptide genes that were increased in tpd1 compared with Ler; Green circle: the number and percentage of cysteine-rich peptide genes that were decreased in tpd1 compared with Ler. I: increased gene number; D: decreased gene number; the number and percentages in the Venny diagram indicate target gene numbers and percentages in the microarray data.
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Figure 2. Transcriptional expression analysis of AtPCP-Ba in Ler, tpd1, and OE-TPD1. (A) RT-PCR analysis of AtPCP-Ba expression in Ler, tpd1, and OE-TPD1; (B) real-time PCR analysis of AtPCP-Ba expression in Ler, tpd1, and OE-TPD1. The RNA was extracted from flower buds including the carpel before the 12th flower development in Ler, tpd1, and OE-TPD1 plants to analyze AtPCP-Ba expression levels. Different letters denote significant differences (LSD, p ≤ 0.05) between AtPCP-Ba expression in three different plants’ flower buds.
Figure 2. Transcriptional expression analysis of AtPCP-Ba in Ler, tpd1, and OE-TPD1. (A) RT-PCR analysis of AtPCP-Ba expression in Ler, tpd1, and OE-TPD1; (B) real-time PCR analysis of AtPCP-Ba expression in Ler, tpd1, and OE-TPD1. The RNA was extracted from flower buds including the carpel before the 12th flower development in Ler, tpd1, and OE-TPD1 plants to analyze AtPCP-Ba expression levels. Different letters denote significant differences (LSD, p ≤ 0.05) between AtPCP-Ba expression in three different plants’ flower buds.
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Figure 3. The spatial and temporal expression of AtPCP-Ba in Arabidopsis. (A) RT-PCR assay for the expression of AtPCP-Ba in different tissues; (B) real-time PCR assay for the expression of AtPCP-Ba in different tissues; (C–G) promoter activity assay in (C) seedlings, (D) inflorescences, (E) leaves, (F) stems, and (G) siliques of pAtPCP-Ba::GUS-transgenic wild plants. Rt, roots (the root of 10 days seedlings); St, stems (the stem of the plants at anthesis); Lv, leaves (the leaves of the plants at anthesis); OF, open flowers (the open flowers of the plants at anthesis); FB, flower buds (the flower buds of the plants at anthesis); Si, siliques (mature siliques); Sd, seedlings (10 day seedlings). Bars = 1 mm (C–G). The data are represented as mean ± standard error (SE) of three replicates. Statistical significance was determined by one-way analysis of variance; significant differences among means (LSD, p ≤ 0.05) are indicated by different lowercase letters.
Figure 3. The spatial and temporal expression of AtPCP-Ba in Arabidopsis. (A) RT-PCR assay for the expression of AtPCP-Ba in different tissues; (B) real-time PCR assay for the expression of AtPCP-Ba in different tissues; (C–G) promoter activity assay in (C) seedlings, (D) inflorescences, (E) leaves, (F) stems, and (G) siliques of pAtPCP-Ba::GUS-transgenic wild plants. Rt, roots (the root of 10 days seedlings); St, stems (the stem of the plants at anthesis); Lv, leaves (the leaves of the plants at anthesis); OF, open flowers (the open flowers of the plants at anthesis); FB, flower buds (the flower buds of the plants at anthesis); Si, siliques (mature siliques); Sd, seedlings (10 day seedlings). Bars = 1 mm (C–G). The data are represented as mean ± standard error (SE) of three replicates. Statistical significance was determined by one-way analysis of variance; significant differences among means (LSD, p ≤ 0.05) are indicated by different lowercase letters.
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Figure 4. Phenotypic observation of the transgenic OE-TPD1 plants with the AtPCP-Ba-RNAi construct and assay for gene expression. (A–C) Phenotype of OE-TPD1 ((A) genetic background Ler), the transgenic OE-TPD1 with the AtPCP-Ba-RNAi construct ((B) genetic background OE-TPD1), and wild type ((C) genetic background Ler); (D–F) siliques of OE-TPD1 (D), the transgenic OE-TPD1 with the AtPCP-Ba-RNAi construct (E), and wild type (F); (G–I) size observation of siliques of OE-TPD1, the transgenic OE-TPD1 with the AtPCP-Ba-RNAi construct, and wild type; (G) length measurement of the siliques of OE-TPD1, the transgenic OE-TPD1 with the AtPCP-Ba-RNAi construct, and wild type; (H) width measurement of the siliques of OE-TPD1, the transgenic OE-TPD1 with the AtPCP-Ba-RNAi construct, and wild type; (I) ratio analysis of length and width of the siliques of OE-TPD1, the transgenic OE-TPD1 with the AtPCP-Ba-RNAi construct, and wild type; (J) relative expression level of AtPCP-Ba in OE-TPD1, the transgenic OE-TPD1 with the AtPCP-Ba-RNAi construct, and wild type; bars = 1 mm (D–F). Different lowercase letters denote significant differences (LSD, p ≤ 0.05) between different silique sizes and gene expression in three different plant mature siliques. The black triangles indicate different siliques from different plants.
Figure 4. Phenotypic observation of the transgenic OE-TPD1 plants with the AtPCP-Ba-RNAi construct and assay for gene expression. (A–C) Phenotype of OE-TPD1 ((A) genetic background Ler), the transgenic OE-TPD1 with the AtPCP-Ba-RNAi construct ((B) genetic background OE-TPD1), and wild type ((C) genetic background Ler); (D–F) siliques of OE-TPD1 (D), the transgenic OE-TPD1 with the AtPCP-Ba-RNAi construct (E), and wild type (F); (G–I) size observation of siliques of OE-TPD1, the transgenic OE-TPD1 with the AtPCP-Ba-RNAi construct, and wild type; (G) length measurement of the siliques of OE-TPD1, the transgenic OE-TPD1 with the AtPCP-Ba-RNAi construct, and wild type; (H) width measurement of the siliques of OE-TPD1, the transgenic OE-TPD1 with the AtPCP-Ba-RNAi construct, and wild type; (I) ratio analysis of length and width of the siliques of OE-TPD1, the transgenic OE-TPD1 with the AtPCP-Ba-RNAi construct, and wild type; (J) relative expression level of AtPCP-Ba in OE-TPD1, the transgenic OE-TPD1 with the AtPCP-Ba-RNAi construct, and wild type; bars = 1 mm (D–F). Different lowercase letters denote significant differences (LSD, p ≤ 0.05) between different silique sizes and gene expression in three different plant mature siliques. The black triangles indicate different siliques from different plants.
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Figure 5. Phenotypic observation of AtPCP-Ba-amiRNA/OE-TPD1 transgenic plants and assay for gene expression. (A–D) Phenotypic observation of AtPCP-Ba-amiRNA/OE-TPD1 transgenic plants; (E–G) relative expression levels of AtPCP-Ba, TPD1, and AtPCP-Bꝺ in the AtPCP-Ba-amiRNA/OE-TPD1 transgenic plants. Different lowercase letters denote significant differences (LSD, p ≤ 0.05) between different gene expressions in different plant mature siliques. The white triangles indicate different siliques from different plants.
Figure 5. Phenotypic observation of AtPCP-Ba-amiRNA/OE-TPD1 transgenic plants and assay for gene expression. (A–D) Phenotypic observation of AtPCP-Ba-amiRNA/OE-TPD1 transgenic plants; (E–G) relative expression levels of AtPCP-Ba, TPD1, and AtPCP-Bꝺ in the AtPCP-Ba-amiRNA/OE-TPD1 transgenic plants. Different lowercase letters denote significant differences (LSD, p ≤ 0.05) between different gene expressions in different plant mature siliques. The white triangles indicate different siliques from different plants.
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Figure 6. Phylogenetic relationship analysis of cysteine-rich peptides (CRPs) in Arabidopsis thaliana, Zea mays, Oryza sativa, Sorghum bicolor, Brassica napus, and Glycine max. A total of 13 AtCRP genes, 60 ZmCRP genes, 33 OsCRP genes, 12 SbCRP genes, 35 GmCRP genes, and 105 BnCRP genes were clustered into GI, GII, GIII, GIV, and GV groups. The GI group contains AtPCP-Ba (AT5G61605), 8 AtCRPs, and 2 BnCRPs. GII, GIII, GIV, and GV groups encompass 57, 72, 112, and 6 CRP genes, respectively. Full-length amino acid sequences were aligned using MUSCLE, and a tree was constructed using the neighbor-joining (NJ) method in MEGA7.0 software. Each species is depicted in a distinct color.
Figure 6. Phylogenetic relationship analysis of cysteine-rich peptides (CRPs) in Arabidopsis thaliana, Zea mays, Oryza sativa, Sorghum bicolor, Brassica napus, and Glycine max. A total of 13 AtCRP genes, 60 ZmCRP genes, 33 OsCRP genes, 12 SbCRP genes, 35 GmCRP genes, and 105 BnCRP genes were clustered into GI, GII, GIII, GIV, and GV groups. The GI group contains AtPCP-Ba (AT5G61605), 8 AtCRPs, and 2 BnCRPs. GII, GIII, GIV, and GV groups encompass 57, 72, 112, and 6 CRP genes, respectively. Full-length amino acid sequences were aligned using MUSCLE, and a tree was constructed using the neighbor-joining (NJ) method in MEGA7.0 software. Each species is depicted in a distinct color.
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Figure 7. In silico expression analysis of 107 CRPs rich in reproductive organs from six species. A heatmap was generated based on the row scale and log2 fold of fragments per kilobase million (FPKM). (A) Phylogenetic tree of 107 CRP genes rich in flowers and fruits from six species; (B) heatmap of gene expression patterns. Expression levels of 107 CRP genes in six species were obtained in FPKM of transcriptome analysis.
Figure 7. In silico expression analysis of 107 CRPs rich in reproductive organs from six species. A heatmap was generated based on the row scale and log2 fold of fragments per kilobase million (FPKM). (A) Phylogenetic tree of 107 CRP genes rich in flowers and fruits from six species; (B) heatmap of gene expression patterns. Expression levels of 107 CRP genes in six species were obtained in FPKM of transcriptome analysis.
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Figure 8. Relative expression levels of AtPCP-Ba homologous genes in flower buds before anthesis and mature siliques. (A) Real-time PCR assay for expression of AtPCP-Ba homologous genes in the wild-type, tpd1, and OE-TPD1 flower buds before anthesis; (B) real-time PCR assay for expression of AtPCP-Ba homologous genes in the wild-type and OE-TPD1 mature siliques. Different letters denote significant differences (LSD, p ≤ 0.05) between different CRP genes expressions in three different plant flower buds (in (A)) or between different CRP genes expressions in three different plants’ mature siliques (in (B)).
Figure 8. Relative expression levels of AtPCP-Ba homologous genes in flower buds before anthesis and mature siliques. (A) Real-time PCR assay for expression of AtPCP-Ba homologous genes in the wild-type, tpd1, and OE-TPD1 flower buds before anthesis; (B) real-time PCR assay for expression of AtPCP-Ba homologous genes in the wild-type and OE-TPD1 mature siliques. Different letters denote significant differences (LSD, p ≤ 0.05) between different CRP genes expressions in three different plant flower buds (in (A)) or between different CRP genes expressions in three different plants’ mature siliques (in (B)).
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MDPI and ACS Style

Chen, G.; Wu, X.; Zhu, Z.; Li, T.; Tang, G.; Liu, L.; Wu, Y.; Ma, Y.; Han, Y.; Liu, K.; et al. Bioinformatic and Phenotypic Analysis of AtPCP-Ba Crucial for Silique Development in Arabidopsis. Plants 2024, 13, 2614. https://doi.org/10.3390/plants13182614

AMA Style

Chen G, Wu X, Zhu Z, Li T, Tang G, Liu L, Wu Y, Ma Y, Han Y, Liu K, et al. Bioinformatic and Phenotypic Analysis of AtPCP-Ba Crucial for Silique Development in Arabidopsis. Plants. 2024; 13(18):2614. https://doi.org/10.3390/plants13182614

Chicago/Turabian Style

Chen, Guangxia, Xiaobin Wu, Ziguo Zhu, Tinggang Li, Guiying Tang, Li Liu, Yusen Wu, Yujiao Ma, Yan Han, Kai Liu, and et al. 2024. "Bioinformatic and Phenotypic Analysis of AtPCP-Ba Crucial for Silique Development in Arabidopsis" Plants 13, no. 18: 2614. https://doi.org/10.3390/plants13182614

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