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Article

Genome-Wide Identification and Expression Analysis of the TCP Genes in Jatropha curcas L. Reveals Its Roles in Involvement of Leaf Shape

by
Rong Zou
1,2,3,4,
Yang Peng
1,5,
Yang Zhao
1,2,3,4 and
Xiurong Wang
1,2,3,4,*
1
College of Forestry, Guizhou University, Guiyang 550025, China
2
Institute for Forest Resources & Environment of Guizhou, Guizhou University, Guiyang 550025, China
3
Key Laboratory of Forest Cultivation in Plateau Mountain of Guizhou Province, Guiyang 550025, China
4
Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang 550025, China
5
State-Owned Longli Forestry Farm of Guizhou Province, Duyun 551200, China
*
Author to whom correspondence should be addressed.
Forests 2023, 14(4), 780; https://doi.org/10.3390/f14040780
Submission received: 10 February 2023 / Revised: 31 March 2023 / Accepted: 5 April 2023 / Published: 10 April 2023
(This article belongs to the Special Issue Advances in Tree Germplasm Innovation and High-Efficiency Propagation)
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Abstract

:
Jatropha curcas var. nigroviensrugosus CV Yang (Jn) exhibits wrinkled leaves and higher flowering and fruiting rates compared to Jatropha curcas L. (Jc). Teosinte branched1/Cincinnata/Proliferating cell factors (TCPs) are known to play crucial roles in plant development and physiological processes. However, it remains unknown whether or not the TCP gene family regulates in leaf development of Jc and Jn. Here, we systematically performed a genome-wide analysis of the Jc TCP family and investigated the differences in the expression of TCP in different leaf morphologies. In total, our results showed that 18 TCP members were identified in the whole genome sequence of Jatropha curcas L.; Jc TCP genes were classified into two categories by phylogenetic tree construction, among which there were 11 members in the Class I subfamily, seven members in the Class Ⅱ subfamily. It was shown that 12 members of Jc TCP genes were located at the seven chromosomes, and proteins belonging to the same TCP group exhibit higher similarity than those from different groups. Furthermore, the expression profiles of 15 TCP genes were discovered at different leaf developmental stages of Jc and Jn. Jc TCP 4, Jc TCP 5, Jc TCP 8, Jc TCP 13, Jc TCP 14, and Jc TCP 16 showed significantly different expressions, and can be used as candidate genes for regulating leaf development. Therefore, the TCP genes play important roles in regulating the leaf development in Jc, and the manipulation of Jc TCP genes can potentially be an important tool used for the genetic improvement of the leaf.

1. Introduction

Jatropha curcas L. (Jatropha, Jc), a small perennial shrub plant, has a high oil content (43%–61%) in its seeds and is commonly known as widely adaptable in various agro-climatic conditions of the Euphorbiaceae family [1,2,3]. Given its sustainable production of food and bioenergy, studies related to Jatropha are now attracting extensive attention [4]. Jatropha nigroviensrugosus CV Yang (Jn), a new variant of Jc developed via cross-breeding, has higher seed yield and oil content than Jc, and exhibits wrinkled leaves, sunken veins in cotyledons, and true leaves [5]. By observation of the leaves, significant differences in leaf anatomical and physiological characteristics were found between Jc and Jn, including the shape and size of the leaf cells. The morphological diversity of leaves helps plants adapt to environmental changes, and differences in the shape of leaves lead to different photosynthetic efficiency of plants. Leaf morphological traits often adjust in response to different light environments and may affect the photosynthetic gas exchanges of plants [6,7,8]. Wang et al., 2020 measured photosynthesis and 20 leaf functional traits of two plants of the genus Camellia, and found that leaf morphological traits were an important factor responsible for differences in the light adaptation between Camellia japonica and Camellia reticulata [9]. However, leaf development is regulated by intercellular signaling molecules such as plant hormones, sugars, and transcription factors, which form a complex network of molecules [10]. Investigating the morphological characteristics and molecular mechanisms underlying leaf development in Jc and Jn will provide valuable insights into the regulatory role of Jatropha curcas TCP transcription factors in leaf development. TCP genes exist widely in plants, and a highly conserved non-classical basic helix–loop–helix (bHLH) pattern is designated as the TCP structural domain located at the N terminus [11]. It was originally named from its initial members, Teosinte branched1 (TB1) in maize (Zea mays) [12], Cycoloidea (CYC) in snapdragon (Antirrhinum majus) [13], and Proliferating cell factors 1 and 2 (PCF1 and PCF2) in rice (Oryza sativa) [14]. TCP genes are a group of transcription factors that regulate various biological processes, including plant growth, development, and response to environmental stimuli. In 1999, Cubas et al. conducted one of the earliest studies on TCP genes and characterized their role in the development of leaves and flowers in Arabidopsis thaliana [15]. Since then, numerous studies have been conducted on TCP genes in various plant species, such as cotton [16], rapeseed [17], Cymbidium goeringii [18], and Prunus mume [19]. Recent studies have revealed that TCPs play important roles in many plants biological processes, such as controlling cell growth and proliferation in meristems and lateral organs [20,21], leaf and flower morphogenesis and senescence [22,23], hormone signaling [24], and plant immunity [25]. In Arabidopsis thaliana (At), AtTCP14 and AtTCP15 regulate embryonic growth and influence seed germination [26]. In tea plants, most CsTCP genes in group 1 are expressed broadly and non-specifically, while CsTCP genes in group 2 are predominantly expressed in buds, flowers, and leaves [27]. Zhan et al. demonstrated through yeast two-hybrid and luciferase complementation imaging assays that ScTCP9, which is located in the nucleus, interacts with ScFT (Flowering locus T), indicating their role in regulating flowering time [28]. Moreover, TCP genes have been implicated in the regulation of stress response pathways. For instance, ZmTCP32 and ZmTCP42 RNA levels were induced by ABA, drought, and polyethylene glycol treatments. Overexpression of ZmTCP42 in Arabidopsis led to hypersensitivity to ABA in seed germination and enhanced drought tolerance, validating its function in drought tolerance [29]. In Liriodendron chinense, expression profiling studies revealed that LcTCP genes respond to various external stimuli such as cold, drought, and heat stress, with LcTCP1 showing particularly strong responses to all the stresses examined [30]. In addition to focusing on the function of the entire TCP gene family, many studies have focused on analyzing a specific TCP gene, or the cooperative function of multiple TCP genes, in order to investigate the unique functions of these TCP genes. Researchers used methods such as gene knockout, overexpression, and transcriptome analysis to study the function of TCP genes. For example, under drought conditions, the introduction of the ZmTCP14 gene positively regulated the accumulation of ROS in plants to reduce drought resistance [31]. In Arabidopsis, loss-of-function mutations in miR319-targeted and non-targeted TCP genes cooperatively induced the formation of serrated leaves in addition to changes in downstream gene transcript levels [32]. Zhang et al. conducted a detailed analysis of a dominant-negative mutant of TCP7 and determined that TCP7 plays important roles during leaf and hypocotyl development, redundantly with at least six Class I TCPs, and regulates CYCD1;1 expression to affect endoreplication [33]. Understanding TCP gene family members and their regulatory mechanisms will contribute to our understanding of plant biology and may have significant implications for plant improvement.
The TCP family has been demonstrated to participate in leaf development control by many genetic and molecular studies. Hervé et al. [34] obtained small and wrinkled leaves by overexpressing At TCP 20 protein; lines overexpressing jaw-D microRNA caused reduced expression of five Class II TCP genes, resulting in leaf wrinkling [35], overexpression of miR319 produced large, deeply lobed, and serrated leaves, leading to downregulation of some TCP genes [36]. TCP genes redundantly regulating the margin and surface of the leaf have also been found [11]. Yu et al. [37] found that the number and depth of leaf serrations were regulated by TCP 5, directly promoting the transcription of KNAT3 and indirectly activating the expression of SAW1. To date, genome-wide characterization of many TCP family members has been completed in many dicotyledons and monocotyledons, such as Arabidopsis [38], poplar [39], rice [40], and tomato [41]. TCP genes have been divided into two main subfamilies based on the differences in the TCP domain; Class I (known as PCF or TCP -P subclass) specifically binds to GGNCCCAC, and Class II (known as CIN and CYC/TB1 clades or TCP -C subclass) specifically binds to G(T/C) GGNCCC [42]. Ori et al. [43] found that proteins such as AtTCP 7, At TCP 8, At TCP 22, and At TCP 23 played important roles in the leaf development of Arabidopsis. However, it remains unknown whether TCPs are also involved in leaf morphology regulation in Jc, and more details of TCPs controlling leaf shape in shrub plants remain to be elucidated.
Here, we systematically performed genome-wide identification of TCP transcription factors in Jc, and then expression analysis of TCP genes in two leaf shapes of Jc and Jn to establish the correlation between their expression and various leaf shapes. Furthermore, we investigated the expression profiles of these TCPs of Jc and Jn at different stages of leaf development. Our study will contribute to further studies on the putative functional characterization of the Jc TCP genes and provide a comprehensive analysis revealing the potential functions of the TCP gene in leaf development.

2. Materials and Methods

2.1. Plant Material and Growth Conditions

Seeds were collected from three healthy Jc and Jn in Menglun Town, Mengla County, Xishuangbanna Dai Autonomous Prefecture, Yunnan Province (101°9′ E, 21°32′ N). After soaking in 25 °C water for 24 h and being treated with 0.5% KMnO4 solution for 12 h, the seeds were washed with running water and placed in sealed bags for transport to the laboratory for storage. To cultivate the plants, the seeds were grown in pots filled with a mixture of vermiculite, perlite, soil, and chicken manure (2:2:2:1, v:v), and maintained in a greenhouse at Guizhou University at 25 °C with a 14 h light period and a light intensity of 2000 Lx.

2.2. Identification, Phylogeny, and Classification of TCP Proteins in Jatropha

Using a Hidden Markov Model (HMM) with the TCP domain (PF03634) as a probe, we searched the entire genome protein sequences of Jc [4] and obtained candidate proteins. In Pfam database (http://www.pfam.xfam.org/ (accessed on 15 January 2023)), the full-length protein sequence of the Jc TCP family was then queried [44] to verify the TCP domain (PF03634) [11]. The TCP protein sequence (https://www.arabidopsis.org/ (accessed on 15 January 2023)) of Arabidopsis thaliana was used as the retrieval sequence to perform The Whole Genome Protein Sequence of Plaster; parameter e value was set to 1e-5 to obtain candidate proteins. The candidate proteins obtained in both ways were multi-sequence homology comparisons, and after the redundant sequences were eliminated, the resulting protein sequences were BLAST again using NCBI (https://blast.ncbi.nlm.nih.gov/ (accessed on 15 January 2023)). To ensure the correctness of the resulting sequence, NCBI CDD (https://www.ncbi.nlm.nih.gov/ (accessed on 15 January 2023)) and SMART (http://smart.embl-heidelberg.de/smart/ (accessed on 15 January 2023)) domain identification was performed on the resulting sequences to remove protein sequences without TCP domains. It was named according to its relative position on the chromosome, not on the chromosome according to its scaffold order.

2.3. Sequence Alignment and Phylogenetic Analysis

General characterization information of Jc TCPs was explored by the ExPASy-Protparam tool (http://www.web.expasy.org/protparam/ (accessed on 15 January 2023)) [45]. To explore the evolutionary relationship of TCP genes in the whole genome of Jatropha, sequences of Arabidopsis, rice, and castor TCP proteins were retrieved from (https://www.arabidopsis.org/) and (http://rice.plantbiology.msu.edu/ (accessed on 15 January 2023)), respectively.
Multiple sequence alignment of amino acid sequences of TCP proteins from Jatropha, Arabidopsis, rice, and castor was performed by ClustalX 1.83 [46]. An unrooted phylogenetic tree based on the full-length protein sequence alignments was constructed using MEGA 7.0 [47], and a phylogenetic tree of the JTT + G model was constructed using the maximum likelihood method (ML) [48]. The Jc TCPs motif logos were obtained by submitting the sequences to the MEME website (http://meme.nbcr.net/meme/cgi-bin/meme.cgi (accessed on 15 January 2023)).

2.4. Chromosomal Location, Duplication Analysis, and Prediction of the Cis-Regulatory Elements

The annotation information of the genome, according to Jc, the retrieved TCP gene, was localized to the 11 chromosomes of the Jc. The TBtools tool [49] was used to analyze duplication events among the identified Jc TCP genes.
The Jc TCP gene structure was plotted using TBtools [49]. To analyze Jc TCP exon–intron structure by TBtools, its motif base order was analyzed by MEME 5.3.0 (http://meme-suite.org/tools/meme (accessed on 15 January 2023)). Multiplex alignment analysis of the TCP protein domain was performed using Clustal X, and visual analysis was performed using Jalview 2.11.1.0 software [46,50]. The sequence of 2000 bp upstream of the TCP family member-starting codon ATG in the TCP family member’s genome database was extracted using TBtools as a promoter sequence to retrieve the promoter elements of each Jc TCP gene by PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 15 January 2023)).

2.5. Expression of Jc TCPs in Different Organs and at Different Jc Developmental Stages

The transcriptome of the roots (SRR5974855), stems (SRR5974856), leaves (SRR5974846), female flowers (SRR5974844), male flowers (SRR5974843), immature fruits (IF) (SRR5974841), semi-mature seeds (SRR5974842), mature seeds (yellow, MFI, SRR5974857), and mature seeds Ⅱ (black-brown, MFⅡ, SRR5974858) under the normal growth conditions were downloaded from the NCBI database (BioProject accession numbers PRJNA399212) (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA399212 (accessed on 15 January 2023)) [4]. The abundance of the JcTCP transcripts was quantified using the Kallisto program [28]. Finally, these results were integrated into cluster heatmaps and were drawn using the R package heatmap to represent the gene expression patterns [28].

2.6. Analysis of TCP Gene Expression in Jn and Jc Leaves

In February 2021, leaf samples were collected from one-year-old Jc and Jn trees, including the topmost fully expanded leaves (baby leaves), the second fully expanded leaves (young leaves), the 3rd to 4th fully expanded leaves (intermediate leaves), and the 7th to 8th fully expanded leaves (mature leaves) (Figure 1) [51]. Three biological replicates were prepared for each leaf sample. The collected leaves were rapidly frozen in liquid nitrogen and stored at −80 °C for RNA extraction. Total RNA was extracted from the leaf tissues using an OMEGA Plant Total RNA Extraction Kit (Bioer Technology, Hangzhou, China), and RNA quality was assessed by 1% agarose gel electrophoresis. The concentration of RNA and OD260/280 and OD260/230 ratios were measured using a nucleic acid micrometer to ensure the quality of RNA extraction. Reverse transcription was performed with a PrimeScript RT reagent Kit with gDNA Eraser (47A) (Takara, Dalian, China); the specific reaction conditions are described in Table S1. Quantitative real-time PCR (qRT-PCR) was performed on a Takara Thermal Cycler Dice real-time system (Takara, Kusatsu, Japan) with a GoTaq qPCR Master Mix (Promega). Actin was the internal reference gene (http://icg.big.ac.cn/index.php/Jatopha curcas (accessed on 15 January 2023)), and the specific reaction conditions are in Tables S1 and S2. The primers used in this work are listed in Table S3.

3. Results

3.1. Identification of TCP Gene Family in Jatropha

After removing redundant sequences, 18 non-redundant Jc TCP genes were identified in seven Jatropha chromosomes. In addition, based on their distributions in the genome and relative linear orders in each chromosome, and the naming conventions, these 18 genes were named JcTCP 1 to JcTCP 18. The length of JcTCP proteins varied from 95 amino acid residues for JcTCP 5 to 526 amino acid residues for JcTCP 12. Among these proteins, JcTCP 7 exhibited the highest molecular weight (46.91 kDa), while JcTCP 5 displayed the lowest molecular weight (9.86 kDa). The theoretical isoelectric point (pI) values of the JcTCP proteins ranged from 5.76 to 10.94, with most proteins having a pI higher than 7.58. The aliphatic index values ranged from 31.72 to 63.36, indicating that the Jc TCP proteins were rich in aliphatic amino acids. All Jc TCP proteins had a GRAVY score less than zero, suggesting that they were hydrophilic. WoLF PSORT predicted that the majority of JcTCP proteins are located in the nucleus, while a few may be found in other subcellular compartments (Table 1).

3.2. Conserved Domains and Motif Analysis

The alignment analysis showed that Jc TCPs shared high homology in the TCP domain (Figure 2). The TCP domain in Jc consisted of 55–60 amino acids that were involved in the basic helix–loop–helix (bHLH) structure. A comparison of TCP domains and a dendrogram showed that the 18 Jc TCP proteins could be classified into two subfamilies. In the two subfamilies, Class I contained 11 genes, and Class II contained 7 genes, respectively. In addition, the Class II subfamily was distinguished into two subclades, CYC/TB1 and CIN. The CYC/TB1 subclade contained two Jc genes, Jc TCP 7 and Jc TCP, while CIN included five members, Jc TCP 3, Jc TCP 4, Jc TCP 12, Jc TCP 13, and Jc TCP 14.
Multiple sequence alignment was performed with DNAMAN [52]. Sequence identifiers were created by Weblogo. The height of the stack indicated the sequence preservation at that position. Black boxes indicated the conserved amino acids in two TCP subfamilies: yellow, conserved in Class I; green, conserved in Class II; blue, conserved in the CYC/TB1 clade; and red, conserved in the CIN clade.

3.3. Analysis of Genetic Structure Characteristics

A new phylogenetic tree of Jc TCP genes was constructed, as well as an analysis of their conserved motif and exon/intron structure (Figure 2). Only Jc TCP 3, Jc TCP 12, and Jc TCP 11 contained UTR structure in the Class Ⅱ subfamily, while the others had no UTR (Figure 3a). Among the TCP genes of Jatropha, nine genes contained introns, and the number of introns varied from one to three. In the Class I subfamily, only four TCP genes (Jc TCP 2, Jc TCP 5, Jc TCP 9, Jc TCP 17) contained introns, accounting for 36.36%. In the Class Ⅱ subfamily, two genes (Jc TCP 13 and Jc TCP 14) had no introns, while the others had one to three introns.
Ten conserved motifs were identified in Jc TCP proteins (Figure 3b and Figure S4). Motif 1 was identified as the TCP domain, which was present in all other Jc TCP proteins except Jc TCP 1, and Motif 1 of the proteins was concentrated at the N terminal. Members of the same clade usually had similar motif compositions. Motif 2 was only found in all Class I proteins. Motif 5 was only found in Class II; each member of Class II contained “Motif 1 + Motif 4”. Jc TCP 7 and Jc TCP 11, which belonged to the CYC/TB1 subclade, showed the same motif composition and distribution. Motifs 2, 3, 6, 7, and 9 were unique to the Class I subfamily, while Motif 5 was restricted to the Class II subfamily. Some motifs, such as Motifs 4, 8, and 10, were shared between two different classes. In addition, some of the two classes of proteins had several specific motifs, respectively. For example, five genes in the CIN subclade (Jc TCP 7, Jc TCP 11, Jc TCP 7, Jc TCP 7, and Jc TCP 11) harbored motifs 4 and 1, while some motifs, such as motifs 8 and 10, were shared in two different classes.

3.4. Chromosomal Localization and Collinear Analysis

The 18 identified TCP genes were subjected to chromosome localization analysis, and the results are shown in Figure 4. Except for JcTCP13, JcTCP14, JcTCP15, JcTCP16, JcTCP17, and JcTCP18, all the other genes were accurately located on 10 chromosomes, with the number of TCP genes per chromosome ranging from 0 to 3. JcChr 01 and JcChr 06 contained three genes, JcChr 09 contained two genes, JcChr 03, JcChr 04, JcChr 05, and JcChr 10 contained one gene, while no TCP gene was found on JcChr 02, JcChr 07, JcChr 08, and JcChr 11 (Figure 4).
Additionally, we performed an analytical search for duplication events in the identified Jc TCP genes. Three pairs of duplicated genes (Jc TCP 3/Jc TCP 14, Jc TCP 7/Jc TCP 11, and Jc TCP 8/Jc TCP 10) were identified, accounting for about 33% of the Jc TCP family. Only Jc TCP 17/Jc TCP 18 pairs of tandemly repeated genes were detected on unmapped scaffold134. As the one gene located on scaffold35 also showed high identity to other genes, there could be even more duplication events. Furthermore, in Jc and Arabidopsis, there were fragment replication relationships between 12 pairs of TCP genes, including AtChr01 and AtChr03, AtChr03 and AtChr05, with more replication events than others.
The TCP genes were located according to the Jctropha curcas L. genome, with colored lines indicating possible gene duplication events.

3.5. Analysis of Phylogenetic and Selection Pressures

To understand the evolutionary relationship of TCP genes in the whole genome of Anthracite, 24 Arabidopsis thaliana, 23 rice, and 21 Ricinus communis, as well as Jatropha, 18 TCP family genes were identified to construct a phylogenetic tree (Figure 5 and Table S6). All TCP genes were roughly divided into 11 groups (A–K), of which the A–G groups were of the Class I subfamily with a total of 48 members, of which the proportion of Jc TCP genes was 14.58%. The H–K groups belonged to the Class II subfamily, with a total of 38 members, and the Jc TCP gene accounted for 28.94%. The TCP genes of Arabidopsis thaliana and rice were more evenly distributed in all groups. In the same group, TCP genes of rice were clustered within the same branch except for Os TCP 12, OsPCF1, and Os TCP 20, suggesting that the large amplification of TCP gene might appear before the emergence of monocotyledonous and dicotyledonous plants, and that there was a certain divergence in the evolutionary history of monocotyledonous and dicotyledonous plants. Additionally, the TCP genes of Jc were distributed in various groups, except for the A group. Except for Jc TCP 6, all the TCP genes of Jc were clustered with those of Ricinus communis, indicating that Ricinus communis was more closely related to Jc compared to rice and Arabidopsis thaliana, and the function of Ricinus communis and Jc TCP genes had more similarity than that of the genes of rice and Arabidopsis thaliana.
According to the TCP genes sequence, a total of 59 pairs of homologous genes were identified in Jc and Arabidopsis, Jc and Ricinus communis, and Jc and Jc by the OrthoFinder program (S5). Only 4 pairs (Jc TCP 3/Jc TCP 14, Jc TCP 4/Jc TCP 13, Jc TCP 7/Jc TCP 11, Jc TCP 8/Jc TCP 10) of paralogous genes and 18 pairs (Jc and Arabidopsis thaliana) and 37 pairs (Jc and Ricinus communis) of orthologous genes were identified (Table 2), indicating that the relationship between Jc and Ricinus communis was closer, which further verified the results of a phylogenetic tree and collinearity analysis. The selection pressure value (Ka/Ks) was calculated, and it was found that the Ka/Ks values of the remaining gene pairs were all less than 1, except for 18 pairs that could not be calculated due to the large degree of sequence divergence and long evolutionary distance, indicating that these TCP genes were subjected to purification selection during evolution.

3.6. Promoter Component Analysis

The upstream sequence (2000 bp from the start codon site) of each Jc TCP gene was retrieved from the current Jc genome compilation (Figure 6). Our data indicated that the characterized cis-regulatory elements distributed in the promoter regions of the Jc TCP genes could be classified into several major functional categories, such as tissue growth, light responsiveness and developmental response elements, hormone responsiveness, and stress responsiveness (Figure 7A,B). Numerous light-responsive elements were located in the promoter regions of all identified Jc TCP genes, including BOX-4 (27.48%), G-BOX (16.03%), and GT1-motif (10.69%). We searched for the presence(s) of the hormone, tissue growth and developmental response elements, and/or stress-responsive cis-regulatory elements in the promoter regions of the Jc TCP genes. Interestingly, the promoters of most Jc TCP genes contained at least one tissue growth and developmental response elements; MSA-like and CAT-box elements (10.13%) were involved in cell cycle regulation and cis-acting elements of meristem growth; HD-Zip1, a response element involved in palisade tissue differentiation, and GCN4_motif, an acting element involved in endosperm development, were found along with the anaerobic response element ARE (51.90%). Several regulatory elements involved in hormone responsiveness were identified in the promoter region of Jc TCPs, such as the TCA element (12.90%) related to salicylic acid responsiveness, AuxRR-core, two elements (namely GARE-motif (28.23%) and P-box) associated with auxin and gibberellins responsiveness, respectively, and ethylene-responsive element (ERE (29.84%)), these elements were identified in the Jc TCPs’ promoter regions. Additionally, two methyl jasmonate (MeJA)-responsive motifs (CGTCA-motif and TGACG-motif) and the ABA-responsive element (ABRE (28.23%)) were also found in the examined promoter regions of the Jc TCP genes. In addition, the promoters of most Jc TCP genes contained at least one stress-responsive element, for example, promoters of element(s) (LTR (28.95%)), involved in the low-temperature stress responsiveness, MYB binding site(s) (MBS (52.63%)), involved in drought-inducibility, and TC-rich (15.79%) repeat(s) (Figure 7C).
According to promoter analysis, we found that the number of light-responsive elements was the largest; hormone-responsive elements were second, and organ growth-responsive elements were slightly more than biotic/abiotic stress-responsive elements. This indicated that the functions of TCP genes were mainly involved in the organ development of plants. Among them, only Jc TCP 3 and Jc TCP 7 genes contained MSA-like elements, HD-Zip1 elements were only distributed in Jc TCP 10 and Jc TCP 14, and elements involved in organ growth-responsive elements were distributed in Jc TCP 10, Jc TCP 12, Jc TCP 13, Jc TCP 14, Jc TCP 15, Jc TCP 16, and Jc TCP 17. It was speculated that the nine genes above were involved in regulating the growth and development of Jc leaves.

3.7. Expression Profiles of the Jc TCP Genes in Different Organs during Development

The publicly available transcriptome data were used to explore the expression profiles of 18 Jc TCP genes in various Jatropha organs (roots (SRR5974855), stems (SRR5974856), leaves (SRR5974846), female flowers (SRR5974844), male flowers (SRR5974843), immature fruits (IF) (SRR5974841), semi-mature seeds (SRR5974842), mature seeds (yellow, MFI, SRR5974857), and mature seeds Ⅱ (black-brown, MFⅡ, SRR5974858) under normal growth conditions (Figure 8 and Table S7). According to the retrieved data, two genes, namely Jc TCP 17 and Jc TCP 7, had no available expression information from the transcriptome profiling (Figure 8). In contrast, most of the remaining 18 Jc TCPs were differentially expressed in the examined organs. For example, Jc TCP 2 and Jc TCP 5 showed specific expression in several organs at different developmental stages, as their transcripts were strongly detectable in roots, stems, leaves, female flowers, and immure fruits, weakly expressed in male flowers and gold fruits, and unnoticed in mature fruits. Conversely, Jc TCP 12 was strongly expressed in leaves but very weakly expressed in mature fruits, whereas Jc TCP 8 was strongly expressed in stems but was not detected in mature fruits (Figure 7). Six genes (Jc TCP 1, Jc TCP 4, Jc TCP 12, Jc TCP 13, Jc TCP 14, and Jc TCP 16) were noted to more strongly express in leaves than other organs, suggesting that these genes played important roles in the development of leaves. Taken together, our analysis of these transcriptome data suggested that Jc TCP TFs had different regulatory functions on the growth and development of various organs during the Jc life cycle.
The heatmaps were generated using the Jctropha curcas L. transcriptome data. IF denotes immature fruit, GF denotes golden fruit, MFI denotes mature fruit I, and MF Ⅱ denotes mature fruit Ⅱ. The color gradient indicated the change in transcript levels as expressed in “reads per kilobase per million of mapped reads” (RPKM) (a) and log2 values (b) (color figure online).

3.8. Expression Profiles of TCP Genes at Different Developmental Stages of Two Shapes of Leaves

The extracted RNA bands were clear with a brightness of 28s, and the OD260/280 was higher than 1.8, which indicated that the RNA was used for subsequent experiments.
To investigate the potential roles of the TCP genes in Jc and Jn leaf development, we analyzed the expression of Jc and Jn leaves at different developmental stages (Figure 9). The results showed that a majority of the TCP genes were differentially responsive to different shapes of leaves. Except for Jc TCP 9, Jc TCP 11, and Jc TCP 15, which were not expressed or expressed at very low levels, the other TCP genes were expressed at different developmental stages in the two shapes of leaves (Figure 9). With the development of leaves, Jc TCP 1 and Jc TCP 7 genes were significantly down-regulated, and there was no significant difference between the two shapes of leaves. In contrast, Jc TCP 4 was gradually up-regulated with the development of leaves, Jc TCP 4 in Jn was significantly lower than that in Jc at the IL and YL stages, and Jc TCP 5 was significantly higher in Jn than in Jc. Jc TCP 8 and Jc TCP 13 genes gradually increased with the development of leaves in Jc, but the opposite was true in Jn. In four stages, Jc TCP 13 in Jn was significantly lower than that in Jc, and Jc TCP 16 in Jn was significantly higher than that in Jc. Jc TCP 14 was gradually up-regulated, and Jc TCP 14 in Jn was significantly higher than that in Jc at the BL, YL, and IL stages. Taken together, our analysis of expression profiles of TCP genes suggested that Jc TCP 4, Jc TCP 5, Jc TCP 8, Jc TCP 13, Jc TCP 14, and Jc TCP 16 could be used as candidate genes for regulating leaf development of the two shapes of leaves.
BL denotes baby leaves, YL denotes young leaves, IL denotes intermediate leaves, and FL denotes functional leaves. Actin was the internal reference gene, and in the BL stage, the gene expression in Jc was set as the control at 1. The different lowercase letters indicate that the same TCP gene at the same developmental stage showed a significant difference in gene expression levels between the Jc and Jn leaves.

4. Discussion

In this study, we identified 18 TCP genes from Jc, which belong to the TCP gene family and possess a typical bHLH domain. Additionally, the TCP gene family of Jc was divided into two categories, Class I (11 members of Jc TCP) and Class II (7 members of Jc TCP). Class II was refined into CIN and CYC/TB1 according to their conserved domains, which was different from corn [53]. Currently, based on PCF1/PCF2 in rice [14] and At TCP 20 in Arabidopsis thaliana [24], the expression characteristics of genes in meristems and mutants, many scholars believe that TCP genes promotes the division and proliferation of mesophyll cells in a highly redundant manner and are involved in the regulation of leaf development [24,53,54]. Class I TCPs have regulated leaves, inflorescence, and flower development and have interacted with different hormonal pathways [55,56], but there was no direct evidence of this. Some studies directly demonstrated that the Class II of TCP genes affected leaf growth and morphological formation by inhibiting the growth and proliferation of mesophyll cells [57,58]. In addition, the property characteristic analysis of Jc TCP proteins showed that the amino acid length, molecular weight, and isoelectric point of 18 Jc TCP proteins were quite different. The structure of the Jc TCP genes showed that the number of introns was 0–3, the intron was small, and the genetic structure was simple, which was similar to that of sugarcane [59].
In the genome-wide identification and analysis of 47 different plants, TCP genes originated in land plants [60]. With a phylogenetic analysis of TCP genes between multiple species, Sánchez Moreano et al. [61] suggested that the massive amplification of TCP genes might predate the emergence of monocotyledonous and dicotyledonous plants. In this study, a total of 86 TCP genes of Arabidopsis [62], rice [40], Ricinus communis [63], and Jc were analyzed. Additionally, the Os TCP and At TCP genes were in 11 groups (A–K). Secondly, the Os TCP genes were clustered in the same branch, which indicated that the TCP genes had a certain difference in the evolutionary process of monocotyledonous plants and dicotyledons. In addition, Jc TCP was more closely related to the Rc TCP genes, verifying the reliability of the results of systematic evolution. Chromosomal localization and collinearity results showed the presence of 12 pairs of homologous genes between Jc and Arabidopsis thaliana, suggesting that the TCP genes of the two species might have been differentiated from the same ancestor. A total of 55 pairs of orthologous genes and 4 pairs of paralogous homologous genes were identified for selection pressure analysis. The results showed that the TCP genes in the three species were purified and selected during evolution, indicating that the TCP gene evolved slowly among the three species.
TCP factors acted as downstream mediators of hormone-induced changes or even as modulators of hormone synthesis, transport, and signal transduction for plant growth and development [60,64]. We analyzed the promoter of 18 Jc TCP genes. The cis-acting elements contained in the TCP gene promoters of Jc were divided into four categories, including photoperiod response elements, hormone response elements, plant organ growth and development response elements, and biological/abiotic stress response elements. This suggested that the Jc TCP gene was involved in multiple biological processes in the anointing [65]. TCP proteins modulate auxin signaling via direct transcriptional regulation of auxin biosynthesis-related genes, such as At TCP 3, At TCP 5, At TCP 13, and At TCP 15 [66,67]. The starting elements associated with the meristem were distributed in multiple Jc TCP genes [68]. At the same time, only Jc TCP 10 and Jc TCP 14 contained HD-Zip1, and cell division-related MSA-like initiation elements are distributed only in Jc TCP 3 and Jc TCP 7. Furthermore, using the public transcriptome, the expression of Jc TCP genes in different tissues was verified, and six genes, Jc TCP 1, Jc TCP 4, Jc TCP 12, Jc TCP 13, Jc TCP 14, and Jc TCP 16, were differentially expressed in leaves, indicating that these genes played an important role in leaf development.
By using RT-qPCR, the relative expression characteristics of the TCP gene at different stages of the two shapes of leaf development were verified. The expression of some members of the same subfamily in Jc or Jn was similar, which could be related to the high redundancy of TCP gene function. Jc TCP 4, Jc TCP 5, Jc TCP 8, Jc TCP 13, Jc TCP 14, and Jc TCP 16 showed significantly different expressions in Jc and Jn leaves, and could be used as candidate genes for regulating leaf development of the two shapes of leaves. However, the specific mechanisms through which individual or many TCP gene function remain elusive. The inverted expression pattern of Class I P. euphratica TCP genes could be correlated to the altered length–width ratio of P. euphratica leaves [69]. In Arabidopsis, TCP 5 was involved in repressing the initiation and outgrowth of leaf serrations by activating two key regulators of margin development [37]. In lettuce, overexpression of lettuce APETALA2 (LsAP2) led to small and crinkly leaves, and many bulges were seen on the surface of the leaf bland, inhibiting CIN-like TCP transcription factors [70]. In this study, only the expression of the Jc TCP gene at various stages of development in the middle leaf of two kinds of anointing tree was verified; the regulation of the development of the leaves of the anointed Jc also requires experiments such as gene cloning and silencing.

5. Conclusions

In conclusion, 18 TCP genes were identified in Jc, distributed on 11 chromosomes. These Jc TCP genes were divided into two classes based on their phylogenetic and structural features. Many cis-acting elements were observed in the Jc TCP promoter sequences, implying that the Jc TCP gene was controlled by a complex regulatory network. It will be interesting to further investigate the mechanism of interaction of TCP genes with other genes to regulate leaf development. Jc TCP genes might play important roles in Jc growth and development, as indicated by their spatial and temporal expression patterns. Notably, most Jc TCP genes of Jc are more highly expressed in leaves than in other organs, and Jc TCP 4, Jc TCP 5, Jc TCP 8, Jc TCP 13, Jc TCP 14, and Jc TCP 1s6 showed significantly different expressions in Jc and Jn leaves; they could be candidates for regulating leaf development of the two shapes of leaves. Taken together, all these findings lay a solid foundation to further unravel the functions of TCP genes in Jc for leaf growth and development.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/f14040780/s1. Table S1: Reaction system; Table S2: The specific reaction conditions; Table S3: Primer design; Figure S4: Motif of amino acid sequence diagram; Table S5: Orthologous (Jc-Rc and Jc-At) gene pairs; Table S6: The TCP genes registry numbers of the various species (Jatropha, Arabidopsis thaliana, rice, and Ricinus communis); Table S7: The heatmaps generated using the Jctropha curcas L. transcriptome data.

Author Contributions

R.Z. and Y.P. designed the experiments, performed the experiments, analyzed the data, and prepared figures and tables. X.W. gave good advice on the work. R.Z. wrote the manuscript. Y.Z. and X.W. modified the figures in the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

The work was supported by the Natural Science Foundation of Guizhou Province (Guizhou Science and Technology Cooperation Support [2020] 1Y127: Study on the function of JnCYCD3:1 gene in Jatropha nigroviensrugosus).

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We are very grateful to the teachers in the College of Forestry and the Institute of Forest Resources & Environment of Guizhou for their helpful discussions and technical assistance.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

qRT-PCRQuantitative real-time PCR
ARFAuxin Response Factor
SCFSkp1/Cullin/F-box
AAAmino acid numbers
ORFThe length of the Open Reading Frame
MWThe protein molecular weight
PIIsoelectric point
IIThe instability index
GRAVYAliphatic index and grand average of hydropathicity
WGDWhole-genome duplications
KsSynonymous substitution rate
KaNonsynonymous substitution rate

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Figure 1. Different developmental stages of Jc and Jn leaves.
Figure 1. Different developmental stages of Jc and Jn leaves.
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Figure 2. Multiple sequence alignment of TCP proteins in Jctropha curcas L.
Figure 2. Multiple sequence alignment of TCP proteins in Jctropha curcas L.
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Figure 3. Genomic structure and motif composition of Jc TCPs. (a) Gene structure analysis and (b) motif distribution of TCP genes in Jctropha curcas L. Ten motifs were indicated by MEME. Each motif was represented by a colored block with accompanying numerical values (color figure available online). The lengths and positions of motifs in the protein sequences were determined by the lengths and positions of the blocks.
Figure 3. Genomic structure and motif composition of Jc TCPs. (a) Gene structure analysis and (b) motif distribution of TCP genes in Jctropha curcas L. Ten motifs were indicated by MEME. Each motif was represented by a colored block with accompanying numerical values (color figure available online). The lengths and positions of motifs in the protein sequences were determined by the lengths and positions of the blocks.
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Figure 4. Physical locations of TCP genes on Jctropha curcas L. chromosomes.
Figure 4. Physical locations of TCP genes on Jctropha curcas L. chromosomes.
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Forests 14 00780 g005 550
Figure 5. Phylogenetic tree of TCP genes from Arabidopsis, Rice, Jatropha, and Ricinus communis.
Figure 5. Phylogenetic tree of TCP genes from Arabidopsis, Rice, Jatropha, and Ricinus communis.
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Forests 14 00780 g006 550
Figure 6. Promoter element analysis of TCP genes in Jctropha curcas L.
Figure 6. Promoter element analysis of TCP genes in Jctropha curcas L.
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Forests 14 00780 g007 550
Figure 7. Statistical analysis of promoter components for TCP genes in Jctropha curcas L. (A) Statistical heat map of the number of promoter elements of 18 TCP genes in Jctropha curcas L., (B) Statistical map of the number of classified promoter elements of 18 TCP genes in Jctropha curcas L., (C) The characteristics of several major functional classes of Jc TCP gene promoter region.
Figure 7. Statistical analysis of promoter components for TCP genes in Jctropha curcas L. (A) Statistical heat map of the number of promoter elements of 18 TCP genes in Jctropha curcas L., (B) Statistical map of the number of classified promoter elements of 18 TCP genes in Jctropha curcas L., (C) The characteristics of several major functional classes of Jc TCP gene promoter region.
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Forests 14 00780 g008 550
Figure 8. Expression profiles of the identified Jc TCP genes in various organs during Jctropha curcas L.
Figure 8. Expression profiles of the identified Jc TCP genes in various organs during Jctropha curcas L.
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Forests 14 00780 g009 550
Figure 9. Analysis of TCP gene expression in Jn and Jc leaves. The different lowercase letters indicate that the same TCP gene at the same developmental stage showed a significant difference in gene expression levels between the Jc and Jn leaves.
Figure 9. Analysis of TCP gene expression in Jn and Jc leaves. The different lowercase letters indicate that the same TCP gene at the same developmental stage showed a significant difference in gene expression levels between the Jc and Jn leaves.
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Table
Table 1. Inventory and characteristics of the TCP genes identified in Jctropha curcas L.
Table 1. Inventory and characteristics of the TCP genes identified in Jctropha curcas L.
Gene NameAccession NumberProteinMW(KD)pIGRAVYAliphatic IndexLoc
Jc TCP 1Jatcu.01g00182917718.595.76−0.39947.09chr01,16313129-16313662 −
Jc TCP 2Jatcu.01g00240523924.49.79−3.22249.9chr01,19888124-19888951 −
Jc TCP 3Jatcu.01g00269834438.578.69−0.82842.68chr01,22051531-22053082 +
Jc TCP 4Jatcu.03g00147337040.186.31−0.60151.99chr03,9416180-9417377 −
Jc TCP 5Jatcu.04g002308959.8610.94−0.40948.04chr04,21737538-21737987 +
Jc TCP 6Jatcu.05g00149831333.549.01−0.77657.76chr05,22093625-22094566 +
Jc TCP 7Jatcu.06g00088241846.919.27−0.78636.23chr06,10403642-10405001 −
Jc TCP 8Jatcu.06g00096035538.88.93−0.5268.6chr06,10815277-10816344 +
Jc TCP 9Jatcu.06g00162741243.778.49−0.24851.26chr06,14359430-14361071 −
Jc TCP 10Jatcu.09g00136835638.568.95−0.44963.36chr09,7223018-7224088 −
Jc TCP 11Jatcu.09g00163323826.977.72−0.84159.93chr09,8690821-8692087 −
Jc TCP 12Jatcu.10g00069152657.19.53−0.84748.5chr10,3612342-3616079 −
Jc TCP 13Jatcu.U00015233937.385.79−0.63934.25scaffold35,825831-826850 +
Jc TCP 14Jatcu.U00041326128.989.51−0.72352.35scaffold35:2332822-2333607 +
Jc TCP 15Jatcu.U00119821923.448.54−0.49757.3scaffold41,1007626-1008285 −
Jc TCP 16Jatcu.U00215033335.639.20−0.38355.08scaffold75,678700-679701 −
Jc TCP 17Jatcu.U00302121923.619.120.06831.72scaffold134,14512-15280 +
Jc TCP 18Jatcu.U00302242846.116.81−0.66950.55scaffold134,43667-44953 +
Table
Table 2. Ka/Ks of homologous gene pairs.
Table 2. Ka/Ks of homologous gene pairs.
Gene PairsKaKsKa/KsGene PairsKaKsKa/Ks
Jc TCP 13/Rc TCP 100.41303.3841 0.1220 Jc TCP 15/Rc TCP 200.7093 NaNNaN
Jc TCP 13/Rc TCP 30.13940.7124 0.1956 Jc TCP 15/Rc TCP 80.6850 2.0846 0.3286
Jc TCP 13/Rc TCP 40.21890.7156 0.3059 Jc TCP 6/RcPCF10.9058 NaNNaN
Jc TCP 4/Rc TCP 100.16210.7071 0.2293 Jc TCP 6/Rc TCP 110.6338 NaNNaN
Jc TCP 4/Rc TCP 30.46052.1172 0.2175 Jc TCP 6/Rc TCP 180.0522 0.4826 0.1082
Jc TCP 4/Rc TCP 40.5321NaNNaNJc TCP 6/Rc TCP 200.2489 1.7971 0.1385
Jc TCP 10/Rc TCP 140.14200.9604 0.1478 Jc TCP 6/Rc TCP 80.7873 NaNNaN
Jc TCP 10/Rc TCP 150.30162.5247 0.1195 At TCP 5/Jc TCP 30.4985 3.4374 0.1450
Jc TCP 8/Rc TCP 140.35562.3572 0.1509 At TCP 17/Jc TCP 30.5780 NaNNaN
Jc TCP 8/Rc TCP 150.07491.0617 0.0706 At TCP 4/Jc TCP 40.3458 2.9209 0.1184
Jc TCP 14/Rc TCP 130.17280.6955 0.2484 At TCP 3/Jc TCP 40.3466 NaNNaN
Jc TCP 14/Rc TCP 50.42811.0216 0.4190 At TCP 11/Jc TCP 60.4484 4.3034 0.1042
Jc TCP 2/Rc TCP 210.14392.0347 0.0707 At TCP 20/Jc TCP 60.2611 2.0023 0.1304
Jc TCP 5/Rc TCP 210.3036NaNNaNAt TCP 12/Jc TCP 70.5767 NaNNaN
Jc TCP 12/Rc TCP 20.06180.5854 0.1056 At TCP 15/Jc TCP 80.4612 NaNNaN
Jc TCP 1/Rc TCP 60.42814.0149 0.1066 At TCP 15/Jc TCP 80.4612 NaNNaN
Jc TCP 1/Rc TCP 70.22301.1044 0.2019 At TCP 19/Jc TCP 90.3622 2.2903 0.1581
Jc TCP 17/Rc TCP 60.28060.9262 0.3029 At TCP 14/Jc TCP 100.5362 NaNNaN
Jc TCP 17/Rc TCP 70.49461.6012 0.3089 At TCP 15/Jc TCP 100.4090 NaNNaN
Jc TCP 18/Rc TCP 60.16340.8614 0.1898 At TCP 18/Jc TCP 110.7134 NaNNaN
Jc TCP 18/Rc TCP 70.45291.4916 0.3036 At TCP 24/Jc TCP 120.4573 NaNNaN
Jc TCP 11/Rc TCP 120.56011.5207 0.3683 At TCP 2/Jc TCP 120.4196 1.7702 0.2371
Jc TCP 11/Rc TCP 160.59881.7005 0.3521 At TCP 10/Jc TCP 130.4913 NaNNaN
Jc TCP 11/Rc TCP 170.29481.0868 0.2712 At TCP 13/Jc TCP 140.5941 NaNNaN
Jc TCP 7/Rc TCP 120.25451.3599 0.1872 At TCP 6/Jc TCP 150.4941 3.0264 0.1632
Jc TCP 7/Rc TCP 160.78032.0336 0.3837 Jc TCP 3/Jc TCP 140.4562 1.2004 0.3801
Jc TCP 7/Rc TCP 170.68043.6230 0.1878 Jc TCP 4/Jc TCP 130.5218 2.6840 0.1944
Jc TCP 15/RcPCF10.7057NaNNaNJc TCP 7/Jc TCP 110.5186 1.9782 0.2622
Jc TCP 15/Rc TCP 110.20991.1577 0.1813 Jc TCP 8/Jc TCP 100.3031 2.2030 0.1376
Jc TCP 15/Rc TCP 180.66911.9555 0.3422
Note: NaN represents a large degree of divergence in sequences and cannot be calculated.
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Zou, R.; Peng, Y.; Zhao, Y.; Wang, X. Genome-Wide Identification and Expression Analysis of the TCP Genes in Jatropha curcas L. Reveals Its Roles in Involvement of Leaf Shape. Forests 2023, 14, 780. https://doi.org/10.3390/f14040780

AMA Style

Zou R, Peng Y, Zhao Y, Wang X. Genome-Wide Identification and Expression Analysis of the TCP Genes in Jatropha curcas L. Reveals Its Roles in Involvement of Leaf Shape. Forests. 2023; 14(4):780. https://doi.org/10.3390/f14040780

Chicago/Turabian Style

Zou, Rong, Yang Peng, Yang Zhao, and Xiurong Wang. 2023. "Genome-Wide Identification and Expression Analysis of the TCP Genes in Jatropha curcas L. Reveals Its Roles in Involvement of Leaf Shape" Forests 14, no. 4: 780. https://doi.org/10.3390/f14040780

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