Genome-Wide Identification of TCP Transcription Factors Family in Sweet Potato Reveals Significant Roles of miR319-Targeted TCPs in Leaf Anatomical Morphology

Plant-specific TCP transcription factors play vital roles in the controlling of growth, development, and the stress response processes. Extensive researches have been carried out in numerous species, however, there hasn’t been any information available about TCP genes in sweet potato (Ipomoea batatas L.). In this study, a genome-wide analysis of TCP genes was carried out to explore the evolution and function in sweet potato. Altogether, 18 IbTCPs were identified and cloned. The expression profiles of the IbTCPs differed dramatically in different organs or different stages of leaf development. Furthermore, four CIN-clade IbTCP genes contained miR319-binding sites. Blocking IbmiR319 significantly increased the expression level of IbTCP11/17 and resulted in a decreased photosynthetic rate due to the change in leaf submicroscopic structure, indicating the significance of IbmiR319-targeted IbTCPs in leaf anatomical morphology. A systematic analyzation on the characterization of the IbTCPs together with the primary functions in leaf anatomical morphology were conducted to afford a basis for further study of the IbmiR319/IbTCP module in association with leaf anatomical morphology in sweet potato.


INTRODUCTION
TCP transcription factors (TFs) belong to a small family of plant-specific TFs (Yin et al., 2018) named after its initial members TEOSINTE BRANCHED1 (TB1) in Zea mays L. (Doebley et al., 1997), CYCLOIDEA (CYC) in Antirrhinum majus L. (Luo et al., 1996), and PROLIFERATING CELL FACTORS 1 and 2 (PCF1 and PCF2) in Oryza sativa L. (Kosugi and Ohashi, 1997). TCPs have been ascertained in numerous plant species, such as Medicago truncatula Gaertn. , Gossypium barbadense L. (Zheng et al., 2018), Citrullus lanatus (Thunb.) Matsum. and Nakai (Shi et al., 2016), Malus domestica Borkh. (Xu et al., 2014), Cucumis sativus L. (Wei et al., 2014), Solanum tuberosum L. (Bao et al., 2019;Wang et al., 2019b), Solanum lycopersicum L. (Parapunova et al., 2014), Populus euphratica Oliv. (Ma et al., 2016), Z. mays (Chai et al., 2017), and Panicum virgatum L. (Zheng et al., 2019). Despite the wide-ranging identification of TCPs, just a few of them have been intensively studied. A non-canonical basic-helix-loop-helix (bHLH) motif was at the N-terminal (about 59 amino acids in length) of all the TCP TFs, commonly referred to as the TCP domain. The TCP domain probably associated with the modulation of DNA binding (Kosugi and Ohashi, 2002) as well as protein-protein interactions (Dhaka et al., 2017). Based on the amino acid residue sequences of the TCP domain, TCPs can be categorized into two classes. For class I, it also referred to as the PCF or TCP-P class, while class II referred to the TCP-C class. The consensus DNAbinding site of class I TCPs is GGNCCCAC, however, which of class II is GTGGNCCC (Kosugi and Ohashi, 2002). Furthermore, class II was categorized into two different subclasses: CIN and CYC/TB1. The R domain rich in arginine with unknown function is present in all members of CYC/TB1 and probably mediating protein interactions through a coiled coil (Lupas et al., 1991;Cubas et al., 1999).
MicroRNAs (miRNAs) are small single-stranded, non-coding RNAs that are typically 20-24 nucleotides (nt) in length. Usually, miRNAs regulate target gene expression through mRNA cleavage at the post-transcriptional level or translational repression (Bartel, 2009;Taylor et al., 2014;Cui et al., 2017). Since most of the target genes of miRNAs are TFs, which can realize the cascade amplification of signals through transcriptional regulation of their target genes, miRNAs have gradually become a potential target gene for crop improvement due to their function in plant growth, development (Axtell et al., 2007), and stress response (Silvestri et al., 2019;Wang et al., 2019a;Kang et al., 2020). In Arabidopsis, eight CIN-like TCP genes out of 24 AtTCPs, including miRJAW-targeted AtTCP2, AtTCP3, AtTCP4, AtTCP10, and AtTCP24, as well as miRJAW-resistant AtTCP5, AtTCP13, and AtTCP17, were found to chord with different pathways to regulate leaf development (Palatnik et al., 2003;Koyama et al., 2007). The down-regulated expression of AtTCP2/4 caused by miR319 target-cleavage resulted in serrated leaves (Palatnik et al., 2003). In switchgrass, miR319 negatively regulated PvTCPs to promote ethylene accumulation and enhance salt tolerance (Liu et al., 2019). In cotton, high levels of GhTCP4, the target of miR319, repressed a homeoboxcontaining factor, GhHOX3 transcriptional activity, to promote the secondary cell wall biosynthetic pathway in fiber cells, resulting in shorter fibers and thicker walls (Cao et al., 2020).
Allohexaploid sweet potato, a major global root and tuber crop, is a significant component as to subsistence agriculture because of its capability to guarantee food security and improving nutrition status regionally (Liu, 2014). However, due to the largeness and complexity of the genome, research on the molecular genetics of sweet potato has lagged behind that of other crops, such as rice and potato. Recently, its whole genome was sequenced . Additionally, its diploid relative wild species Ipomoea trifida (Kunth) G. Don and Ipomoea triloba L. have also been sequenced . These genomic sequences can serve as a reference for the hexaploid sweet potato genome. As we still know little about the TCPs family in sweet potato, an analysis concerning TCP gene family in sweet potato was performed globally in the present study. Eighteen IbTCP genes were ascertained and cloned, and phylogenetic relationships, chromosomal locations, and tissue-specific expression were carried out. Furthermore, we found that the transcription patterns of IbTCPs discrepant during leaf development. IbTCP11 and IbTCP17 were significantly up-regulated in the mature leaves. Blocking IbmiR319 led to an elongated chloroplast and decreased net photosynthetic rate, which further confirmed the critical roles of IbmiR319targeted IbTCPs in leaf anatomical morphology. As a result, our data offers detailed information of IbTCPs category and helps elucidate the function of IbTCPs in leaf anatomical morphology in sweet potato.

Plant Materials
The wild-type (WT) sweet potato (Ipomoea batatas L.) cultivar "Xushu 22" (Xu22), developed by the Sweet Potato Research Institute of the China Agriculture Academy of Science, was used for IbTCP gene cloning. MIM319, overexpressing an artificial IbmiR319 target mimicry to sequester the normal expression of native IbmiR319, was generated using an Agrobacteriummediated embryogenic calli transformation. Untransformed (WT) and transgenic plants transplanted and planted as described before (Yang et al., 2011). The cuttings of 3-4 cm height plants were transplanted grown into the field at the experimental station of Jiangsu Normal University (E 117 • 17.48 ,N 34 • 16.95 ,Jiangsu,China) were used for the assessment of the phenotype and agronomic traits. The plants grown in greenhouses were used for RNA extraction, and those grown in the field were used for morphological observations and photosynthesis parameter measurements.

Identification and Evolutionary Analysis
The TCP protein sequences of Arabidopsis and rice retrieved from the database PlantTFDB 1 or TAIR 2 were used to perform protein to protein BLASTP searching with the e-value of 10 −5 in the I. batatas genome database 3 and its two wild ancestors (I. trifida and I. triloba) genomics database. 4 All sequences were further validated by the conserved domains database (CDD). 5 We named IbTCP1 to IbTCP18 in the light of their allocations in the genome and relative orders on each chromosome. To study the evolutionary relationships of IbTCPs, Clustal X 2.0 was used to align the entire protein sequences of TCPs from sweet potato, Arabidopsis, and rice, which were then used to construct an evolutionary tree in MEGA 6.0 as described before (Tamura et al., 2013).

Sequence Analysis of IbTCP Genes
Using the ExPASy proteomics server, 6 the molecular weight (MW) and isoelectric points (pI) of the IbTCPs were predicted. 7 The conserved motifs of the IbTCPs were ascertained with CDD and the ExPASy proteomics server.

Subcellular Localization of IbTCPs
The coding sequence (CDS) without the termination codon of the IbTCP11 and IbTCP17 were amplified through PCR, and then purified and cloned into pCAMBIA2300-35S-eGFP to obtain subcellular localization vectors 35S: IbTCP11-GFP and 35S:GFP-IbTCP17. The Agrobacterium tumefaciens EHA105 strain harboring 35S: IbTCP11-GFP or 35S:GFP-IbTCP17 was infected into leaf of N. benthamiana. The IbTCP-GFP fusion proteins were detected as described before . The sequences of primers were also listed in Supplementary Table 1.

Identification of IbmiR319 and Prediction of IbmiR319 Target Genes
The sequence of IbmiR319 was acquired from our microRNA library of sweet potato based on high-throughput sequencing (Xie et al., 2017;Tang et al., 2020). To forecast the target sites of IbmiR319, the CDS of the IbTCPs was analyzed using the psRNATarget online tool. 8

Plasmid and Agrobacterium-Mediated Sweet Potato Transformation
The miR319 target mimicry vector p35S-MIM319 was constructed as previously described (Franco-Zorrilla et al., 2007). Genetic transformation of sweet potato was implement as previously described by Yang et al. (2011) to obtained the transgenic plant MIM319.

Gene Expression Analysis
qRT-PCR was carried out to check the expression profiles of genes in different tissues, including the shoot buds (Sb), young leaf (YL), mature leaf (ML), stem (S), fibrous roots (FR), pencil roots (PR), and developing storage roots (DR), or leaves at the different developmental stages (L1-L10: the 1st through 10th leaves counted from the stem tip). Total RNAs was extracted from the above samples using TRIzol (Invitrogen) as described before (Meng et al., 2018). The inverse transcription and the qRT-PCR were performed as described before (Meng et al., 2018). The IbActin gene was used as an internal control. Data from three biological samples were collected, and the mean values were normalized to IbActin. NRT (no reverse transcription control) and NTC (no template control) were also implemented for each gene analysis. The 2 − Ct method was used to judge the relative expression level of IbTCPs (Livak and Schmittgen, 2001). The sequences for the primers are listed in Supplementary Table 1.

Leaf Anatomical Morphology
After 2 months of growth, the third fully-expanded leaf counted from the terminal bud was removed and cross sectioned by hand for optical microscopic examinations and measurement of leaf thickness.
For transmission electron microscopy (TEM) observation, the first fully-expanded fresh leaves were resected and cut into small pieces immediately, fixed with electron microscope fixator (Servicebio, G1102, Wuhan Sevier Biotechnology Co. Ltd.). All the procedure were performed according the described online. 9

Chlorophyll Content and Photosynthesis Parameter Measurements
The cuttings of WT and MIM319 with four to six expanded leaves transplanted to the field for 4 weeks were used for analysis. The chlorophyll content assay was performed using the third fully-expanded leaves of WT and MIM319 according to the described before (Kuo et al., 2019). The total chlorophyll content was figured out according to the formula in C T = 20.29A 645 −8.05A 663 . C T : total chlorophyll content. A 645 , A 663 : the absorbance of the chlorophyll solution at 645 nm and 663 nm, respectively.
Leaf photosynthesis parameters on the third fully-expanded leaves were scaled using a Li-6400 portable photosynthetic system (Ll-6400, Li-COR) on a sunny day. Ten seedlings per line were scaled.
Chlorophyll fluorescence was measured as described (Kuo et al., 2019). Briefly, after adapting to the dark for 30 min, the F v /F m values of the third fully-expanded leaves were measured using a Li-6400 system. Ten seedlings per line were measured in total.

Identification and Cloning of the IbTCP Gene Family in Sweet Potato
To obtain TCP genes in sweet potato, the TCP amino acid sequences of Arabidopsis and O. sativa were utilized as a reference to BLAST against the hexaploid sweet potato Genome Database, its diploid wild ancestor Sweetpotato Genomics Resource database, and PlantTFDB. After removing the redundant sequences, a total of 18 predicted non-redundant IbTCP sequences were identified and further cloned, all of which comprised the conserved TCP domain (Table 1 and Figures 1A,B). Their sequences were listed in Supplementary  Table 2. The CDS lengths of the IbTCPs varied from 396 bp (IbTCP8) to 1527 bp (IbTCP5). IbTCP8, with 131 amino acid residues (aa), was regarded as the shortest IbTCP, whereas the largest was IbTCP5 with 508 amino acid residues. The MW was in the range of 14.703-52.774 kDa, and the variation of pI is from 5.53 to 9.83 (Table 1).
To determine gene allocation pattern on chromosome, investigation of the exact genomic positions of them were carried out. These 18 IbTCPs were scattered across 11 chromosomes and two scaffolds of sweet potato (Table 1 and Figure 1C). Among them, the IbTCPs on chromosomes 2, 10, and 13 were tightly located on the upper end of the arm, while on chromosome 11, they were located tightly on the lower end of the arm. Chromosome 7 contained the highest number of IbTCPs, including IbTCP7, IbTCP8, IbTCP9, and IbTCP10. Chromosomes 1 and 14 contained two loci each.

Phylogenetic Analysis and Category of the IbTCP Family
A phylogenetic tree was structured aimed to investigating about the categorization and evolutionary relationships of sweet potato TCP proteins. A total of 88 TCP full-length amino acid sequences, including 18 IbTCPs, 31 AtTCPs, 23 ZmTCP, seven OsTCPs, and nine SlTCPs, were assembled to structure a phylogenetic tree using the NJ method with 1000 bootstrap replicates (Figure 2 and  Supplementary Table 3). In accordance to the categorization in Arabidopsis, the IbTCPs may be divided into two TCP classes as well. Nine IbTCPs comprised Class I, while the other nine pertain to Class II, which could be classified into two subclasses: four members of the CYC/TB1 clade and the five members of the CIN clade (Figures 1A, 2). The results suggested that all the TCPs were evolutionarily conserved.

Conserved Domain Analysis and Recognition Sequence of miR319
To further comprehend the evolutionary relationships of IbTCPs in sweet potato, the domains of the IbTCPs were confirmed using the ScanProsite tool. 10 As expected, all 18 IbTCPs displayed a highly conserved TCP domain that incorporated a bHLH-type motif located near the N-terminal (Figures 1B, 3A). There's a large difference in the components of the loop and helixes I and II between the class I and II. By analyzing the phylogenetic tree and aligning the TCP domains, the IbTCP proteins are anticipated to be categorized into two classes (Figure 1A), as has been indicated for all species analyzed up to now. The conserved R domain was only found in IbTCP2, IbTCP7, IbTCP10, IbTCP14, and IbTCP17, all of which are members of Class II (Figures 1B, 3B).
MicroRNA319 (miR319) pertains to one of the most ancient and conserved miRNA families. Previous studies have verified that miR319, targeting TF TCP genes, plays important roles in plant growth, morphogenesis, and reproduction (Palatnik et al., 2003;Sun et al., 2017;Cao et al., 2020). The IbmiR319 target sites among the 18 IbTCPs were analyzed using psRNATarget online. The putative recognition sites of IbmiR319 were found in IbTCP1, IbTCP11, IbTCP16, and IbTCP17 ( Figure 3C). These four IbTCP genes all belonged to class II, which corroborates a previous study in Arabidopsis. Although mismatches existed at the 3 end of the IbmiR319 and 5 end of the targeted IbTCP, kernel sequences (3 -GGGAAGUCAGGU-5 ) were conserved ( Figure 3C). These data illustrate that miR319 maintained homologous target interactions in time of the evolution and diversification of plants.

Expression Profiles of IbTCP Genes in Different Tissues
To obtain credible information of the growth and developmental functions of IbTCP in sweet potato, their organic-specific expression patterns, containing the shoot buds, young leaf, mature leaf, stem, fibrous roots, pencil roots, and developing storage roots, were analyzed by qRT-PCR. As indicated in Figure 4, even though all of IbTCPs were expressed in all seven tissues tested, there's considerable variation in transcription levels of different genes among different tissues. Generally speaking, the expression levels of IbTCPs were relatively low in the belowground organs, whereas constitutively high expression in the aboveground organs examined, especially ten IbTCPs (IbTCP2, IbTCP3, IbTCP5, IbTCP7, IbTCP8, IbTCP9, IbTCP10, IbTCP12, IbTCP17, and IbTCP19) showed highly expression levels in the mature leaves. This finding implies that these IbTCP genes probably perform different functions during growth and development. Interestingly, the relatively higher expression of all the IbmiR319-targeted CIN subclass IbTCPs (IbTCP1, IbTCP11, IbTCP16, and IbTCP17) were detected in the shoot bud, young leaf, and mature leaf, while expression levels of the other IbTCPs were high mainly in the mature leaf, suggesting that they may play similar or different roles in leaf development.

IbmiR319-Targeted IbTCPs Play Vital Roles in Leaf Anatomical Morphology
Leaves, as the major photosynthetic organs of plants, take up a crucial position in plant growth and development. Leaves   are also important for tuber crops, since the photosynthetic rate directly affects dry matter accumulation and storage root swelling (Sawada et al., 1999). To understand the function of IbTCPs in leaf development, their transcription levels of 18 IbTCPs at the different developmental stages of the leaves were investigated (Figure 5). The expression levels of most IbTCPs exhibited no signification changes at different developmental stages (Figures 5A,B). Yet, the transcription levels of IbmiR319-targeted IbTCPs, especially IbTCP11 and IbTCP17, presented a significant increase ( Figure 5C). These data indicate that the IbmiR319-IbTCPs module is likely to associate with leaf development. For a more thorough evaluation of the duty of the IbmiR319-IbTCPs module, the subcellular localization of the IbmiR319targeted IbTCPs was performed. IbTCP11 and IbTCP17 were fused with green fluorescence protein and infiltrated into N. tabacum epidermal cells. According to the distribution of green fluorescence signal, these two IbmiR319-target IbTCPs were specifically lay within the nuclei, implying that they are functional TFs (Figure 6). The IbmiR319 target mimicry vector p35S-MIM319 was then constructed and introduced into WT, and the stable transgenic line MIM319, which blocked IbmiR319, was generated (Supplementary Figure 1A). The expression levels of IbTCP11 and IbTCP17 were dramatically up-regulated in MIM319 compared with WT (Supplementary Figure 1B), indicating that these two IbTCPs were the targets of IbmiR319.
The up-regulation of IbTCP11 and IbTCP17 caused by blocking IbmiR319 in sweet potato led to dramatic changes in leaf size and leaf shape (Figures 7A,B). Quantitative analysis revealed that the width of the third fully-expanded leaves of MIM319 transgenic sweet potato ranged from 14.7 to 18.0 cm, which was dramatically less than that of WT (Figure 7C), and the leaf area ranged from 55.50 to 63.25 cm 2 , which was also significantly less than that of WT ( Figure 7E). Conversely, there was no obvious difference in the length of the fully-expanded leaves between MIM319 and WT (Supplementary Figure 2). As a result, the ratio of the length to width of MIM319 was greater than that of WT ( Figure 7D). Taken together, the data indicated that IbmiR319-targeted IbTCPs may have conserved functions during leaf development, similar to their functions in Arabidopsis and rice (Palatnik et al., 2003;Nag et al., 2009;Yang et al., 2013). To elucidate the cellular basis of the morphological changes, microstructure and ultrastructure observations of the leaves were carried out by electron microscopy using hand-sliced paraffin sections. Microscopic analysis of the leaf blade cross-sections revealed fewer mesophyll cells (including the palisade tissue and spongy tissue) and thinner layers of mesophyll cells in MIM319 than in WT (Figure 7F). The thickness of MIM319 leaves (0.264 mm on average) was significantly thinner than that of WT (0.464 mm on average) (Figure 7G). The chloroplasts were flatter, more elongated, and contained less starch grains compared with WT ( Figure 7H). In combination, the data indicated that IbmiR319-target IbTCPs also play vital roles in leaf anatomical morphology.
Leaf size and structure are important since they impact photosynthetic efficiency (Marcotrigiano, 2010). Thinner leaves with less chlorophyll content are considered to have a detriment for the efficient usage of light energy, and lead to less photosynthetic efficiency (Higuchi et al., 1999). To address whether the changes in leaf anatomical morphology of MIM319 affect photosynthesis, some photosynthetic parameters were quantitatively measured. The total chlorophyll content of the MIM319 transgenic plants was 0.0199 mg g −1 FW on average, while that of WT was 0.0331 mg g −1 FW. The total chlorophyll contents of MIM319 were dramatically lower than that in WT ( Figure 7I). Furthermore, the photosynthetic rate of the MIM319 transgenic sweet potato was 22.33 µmol CO 2 m −2 s −1 on average, which was dramatically lower than 31.24 µmol CO 2 m −2 s −1 in the WT ( Figure 7J). Meanwhile, the efficiency of PSII in MIM319 transgenic sweet potato leaves was also examined based on the F v /F m values. However, there were no significant differences between MIM319 and WT ( Figure 7K). Taken together, these data suggested that IbmiR319-targeted IbTCPs may play key roles in the anatomical morphology of the leaves to affect photosynthesis.

DISCUSSION
As plant-specific TFs, TCPs have major functions during growth and development, such as leaf (including single leaf and compound leaf) development, shoot development, flower development (Ori et al., 2007;Broholm et al., 2008;Koyama et al., 2011;Bai et al., 2012), and root development (Hao et al., 2012), and are also involved in phytohormone biosynthesis (Danisman et al., 2012), lignin biosynthesis (Sun et al., 2017;Chahel et al., 2019) and the circadian clock (Giraud et al., 2010;Zhang et al., 2018). Nevertheless, there's scarcely any precise exhaustive information of the TCP TF family in sweet potato which is the seventh largest food crop in terms of production globally and a vital tuber crop. In this study, 18 IbTCPs were identified and cloned in sweet potato and were discovered to be distributed on 11 chromosomes and two scaffolds with different densities (Figure 1C). Phylogenetic analysis revealed that 18 IbTCPs clustered into two classes according to their evolutionary relationships (Figures 1A, 2). These findings are consistent with previous classifications of TCPs from Arabidopsis, Z. mays, and S. lycopersicum (Kim et al., 2014;Parapunova et al., 2014;Chai et al., 2017). Conserved motif analysis showed that TCP domain with a bHLH-type motif existed in both class I and class II of IbTCPs, while the R domain only existed in class II (Table 1 and Figures 1B, 3A,B). Collectively, this evidence supports the classification and conservation of the sweet potato TCP TF family.
It is well-known that class II TCPs precisely regulate the transition from division to expansion (Tsukaya, 2010;Qi et al., 2017). For the leaf lamina, the timing of the transition is a significant determinant of the final size, shape, flatness, and complexity. Class II CYC/TB1 clade TCPs basically participate in the developmental regulation of axillary meristems, which results in the growth of either lateral branches or flowers. This clade included AtTCP2/3/4/5/10 and IbTCP11/17 (Figure 2). We therefore examined the expression patterns of IbTCPs to investigate whether they have essential functions in the growth and development of these organs or tissues. The expression in different tissues and different organs varied widely among IbTCP genes and different development stages of the leaf for individual TCP genes (Figures 4, 5). This states the functional divergence of IbTCPs at different stages of developmental processes, especially leaf development in sweet potato. In our study, all 18 IbTCPs showed relatively weak expression in the belowground organs, but high expression in the aboveground organs, with even IbTCP11 and IbTCP17 showing higher expression in the mature leaves ( Figure 5C). AtTCP3 and AtTCP4, orthologs of IbTCP11, act as decisive modulators of de novo shoot organogenesis (Woorim et al., 2020). A previous study demonstrated that the overexpression of AtTCP4 led to in miniature cotyledons and shoot apical-meristem termination (Efroni et al., 2008), and the ectopic expression of AtTCP3 caused the failure of shoot-meristem formation (Koyama et al., 2007). Double, triple, or even quadruple knockouts for the miR319-regulated TCP genes, AtTCP2, AtTCP3, AtTCP4, and AtTCP10, demonstrate leaf crinkling to varying degrees, yet single knockouts present only slight effects on leaf morphology (Schommer et al., 2008;Bresso et al., 2018). AtTCP5 represses the initiation and outgrowth of leaf serrations by directly promoting the expression of KNAT3 and indirectly activating the transcription of SAW1 . Together, these data reveal the tissue expression diversity of TCPs in various plants. Compared with other plants, the great number of highly-expressed IbTCPs in the aboveground organs indicates their important function in sweet potato development, especially leaf development.
Previous study recorded that TCPs are fundamental regulators of plant growth and development, especially leaf development and senescence (Schippers, 2015). The leaf is the pivotal organ of photosynthesis, and its size and anatomical morphology directly affect photosynthetic efficiency (Marcotrigiano, 2010;Pan et al., 2018). Many genes and pathways can regulate leaf morphogenesis so as to generate organs of a wide variety of sizes and shapes (Rodriguez et al., 2014). Meanwhile, more and more experimental data show that miRNAs regulate the TFs which involved in plant development. The MiR164 family includes post-transcriptionally regulated CUC1, CUC2, and other NAC TFs. The overexpression of the miR164 family in Arabidopsis compromised organ separation, causing fusion between cotyledons and leaves, and also between the inflorescence stem and cauline leaves (Laufs et al., 2004;Mallory et al., 2004;Nikovics et al., 2006). MiR165/166 precisely regulated the expression pattern of polarity genes during leaf development in maize (Juarez et al., 2004). Increased miR408 expression leads to enhanced photosynthesis by elevating the abundance of plastocyanin to improve irradiation utilization efficiency in Arabidopsis, tobacco, and rice (Pan et al., 2018). This study indicates that blocking miR319 in sweet potato increases TCP gene expression to limit photosynthetic performance through thinner and flatter chloroplasts, a lower chlorophyll content, and the development of thinner and smaller leaves.
Our findings provide evidence for the connection between structure and function in the IbTCPs as well as lay a basis for FIGURE 6 | Subcellular localization of two GFP-fused sweet potato TCP proteins. The two IbTCP-GFP fusion proteins (IbTCP11-GFP and IbTCP17-GFP) were transiently expressed in tobacco leaves and observed by confocal microscopy 72 h later. GFP driven by the 35S promoter was used as a control. The left, middle-left, middle-right, and right panels stand for photoes taken in dark, chloroplast, bright, and merged views, respectively. Bars are 20 µm.

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

AUTHOR CONTRIBUTIONS
LR, LZ, DM, and AW designed the research. LR, HW, and TZ implemented most of the research and analysis. XG, WZ, and TW implemented IbTCPs identification, cloning, and bioinformatics analysis. LZ contributed analytical tools. LR, LZ, and AW wrote the manuscript. LZ and DM modified the manuscript. All authors contributed to the article and approved the submitted version.