**Roles of ASYMMETRIC LEAVES2 (AS2) and Nucleolar Proteins in the Adaxial–Abaxial Polarity Specification at the Perinucleolar Region in Arabidopsis**

#### **Hidekazu Iwakawa <sup>1</sup> , Hiro Takahashi <sup>2</sup> , Yasunori Machida 3,\* and Chiyoko Machida 1,\***


Received: 21 August 2020; Accepted: 29 September 2020; Published: 3 October 2020

**Abstract:** Leaves of Arabidopsis develop from a shoot apical meristem grow along three (proximal–distal, adaxial–abaxial, and medial–lateral) axes and form a flat symmetric architecture. *ASYMMETRIC LEAVES2* (*AS2*), a key regulator for leaf adaxial–abaxial partitioning, encodes a plant-specific nuclear protein and directly represses the abaxial-determining gene *ETTIN*/*AUXIN RESPONSE FACTOR3* (*ETT*/*ARF3*). How AS2 could act as a critical regulator, however, has yet to be demonstrated, although it might play an epigenetic role. Here, we summarize the current understandings of the genetic, molecular, and cellular functions of AS2. A characteristic genetic feature of *AS2* is the presence of a number of (about 60) modifier genes, mutations of which enhance the leaf abnormalities of *as2*. Although genes for proteins that are involved in diverse cellular processes are known as modifiers, it has recently become clear that many modifier proteins, such as NUCLEOLIN1 (NUC1) and RNA HELICASE10 (RH10), are localized in the nucleolus. Some modifiers including ribosomal proteins are also members of the small subunit processome (SSUP). In addition, AS2 forms perinucleolar bodies partially colocalizing with chromocenters that include the condensed inactive 45S ribosomal RNA genes. AS2 participates in maintaining CpG methylation in specific exons of *ETT*/*ARF3*. *NUC1* and *RH10* genes are also involved in maintaining the CpG methylation levels and repressing *ETT*/*ARF3* transcript levels. AS2 and nucleolus-localizing modifiers might cooperatively repress *ETT*/*ARF3* to develop symmetric flat leaves. These results raise the possibility of a nucleolus-related epigenetic repression system operating for developmental genes unique to plants and predict that AS2 could be a molecule with novel functions that cannot be explained by the conventional concept of transcription factors.

**Keywords:** *ASYMMETRIC LEAVES2*; AS2/LOB domain; adaxial–abaxial polarity; *ETTIN*/*AUXIN RESPONSE FACTOR3* (*ETT*/*ARF3*); AS2 body; nucleolus; gene body methylation; ribosomal DNA (rDNA)

#### **1. Leaf Developments in Arabidopsis**

Leaves develop from a shoot apical meristem (SAM) as lateral organs along three axes: proximal–distal, adaxial–abaxial, and medial–lateral [1–7]. Initially, groups of cells on the peripheral

zone of the SAM are specified in leaf primordia (P0, Figure 1) and grow along the proximal–distal axis (P1). Then, adaxial–abaxial structures are differentiated (P2). Subsequently, cells proliferate along the medial–lateral axis leading to flat and symmetric leaves (Figure 1) [2,3,8]. To date, numerous genes involved in adaxial–abaxial determination have been reported in *Arabidopsis thaliana* [2,9]. The *ASYMMETRIC LEAVES2* (*AS2*) and *ASYMMETRIC LEAVES1* (*AS1*), which encode a protein with the plant-specific AS2/LOB domain and a protein with the MYB (SANT) domain, respectively, were originally identified as factors involved in symmetric leaf lamina formation [10–13]. Recent studies have revealed, however, that AS2 and AS1 regulate proper morphology along all three axes of leaves. The *Rough Sheath2* (*RS2*) gene of maize, an ortholog of *PHANTASTICA* (*PHAN*) of *Antirrhinum majus* and *AS1* of Arabidopsis, is involved in the proximal–distal patterning of maize leaves through the repression of class 1 *KNOX* genes [10,14,15]. The *PHAN* gene is involved in growth and the adaxial–abaxial determination of lateral organs. In addition, its activity is required early in the growth of leaves in the direction of the proximal–distal axis [16,17]. The ectopic expression of class 1 *Knotted1-like homeobox* (*KNOX*) genes in *as1* and *as2* mutant plants results in reductions in the growth of leaf blades and petioles in Arabidopsis, and these phenotypes are suppressed by mutations of the class 1 *KNOX* genes, *brevipedicellus* (*bp*), *knat2,* and *knat6*. These results indicate that the *AS1* and *AS2* genes of Arabidopsis are involved in the establishment of the proximal–distal axis through the repression of the class 1 *KNOX* genes [18]. In addition, the formation of shorter petioles and leaf blades in *as1* and *as2* is due to repression of gibberellin-synthetic genes by the upregulation of *BP*/*KNAT1*, *KNAT2*, and *KNAT6* [18]. AS1, acting together with AS2, directly represses the expression of the *BP* and *KNAT2* genes [19]. In this review, we focus on the establishment of leaf adaxial–abaxial polarity. *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 3 of 19

**Figure 1.** The leaf structure develops along three axes. Developmental compartments in the shoot apex around the apical meristem and the three structural leaf axes are schematically shown on the left and right sides, respectively (see details in text). CZ, central zone; PZ, peripheral zone; p0, primordium 0; p1, primordium 1; p2, primordium 2. Schematic representations are modified from ref. **Figure 1.** The leaf structure develops along three axes. Developmental compartments in the shoot apex around the apical meristem and the three structural leaf axes are schematically shown on the left and right sides, respectively (see details in text). CZ, central zone; PZ, peripheral zone; p0, primordium 0; p1, primordium 1; p2, primordium 2. Schematic representations are modified from ref. [2].

[2]. **2. Roles of AS2–AS1 in the Development of Leaf Polarity** As described above, AS2 and AS1 proteins, which have AS2/LOB and R2R3 MYB (SANT) domains, respectively (Figure 2a), are identified by a yeast two-hybrid system, pull-down and gelshift assays, and subcellular co-localization analyses. Because of their nature, these experiments indicate that AS2 and AS1 are physically associated with each other in vitro [19,33–36], implying also that they form a protein complex in the nucleus. Transcripts of *AS2* and *AS1* genes accumulate throughout the entire leaf primordia at early stages, in which the AS2–AS1 complex might be formed, but the accumulation patterns change as the leaves develop [37]. *AS2* transcripts are detected in the The *PHABULOSA* (*PHB*), *PHAVOLUTA* (*PHV*), and *REVOLUTA* (*REV*) genes encode class III homeodomain-leucine zipper (HD-ZIPIII) proteins, which determine adaxial cell fate [20–22]. Small RNAs play critical roles in specifying adaxial–abaxial polarity [23,24]. Micro RNAs miR165/166 promote the degradation of *HD-ZIPIII* transcripts in the abaxial domain, which results in the accumulation of HD-ZIPIII in the adaxial domain [24]. Members of the *KANADI* (*KAN*) gene family, which encode proteins with the GARP domain, determine abaxial cell fate [22,25]. The Arabidopsis genome contains six *YABBY* genes, which encode transcription factors with a zinc-finger domain and an HMG-related domain with a helix–loop–helix structure. The three *YABBY* genes, *FILAMENTOUS FLOWER* (*FIL*), *YABBY3* (*YAB3*), and *YAB2* are expressed in the abaxial domains of all leaf-derived

zipper-like (LZL) region, and the internal-conserved-glycine (IcG) region between ZF and LZL (Figure 2a). The AS2/LOB domain is highly conserved in the AS2/LOB family, which consists of 42 members including AS2 in Arabidopsis [12,38,39]. Since the amino acid sequences outside of the domains are diverged among members and the transcription patterns of these genes differ for each gene, the roles of these genes in Arabidopsis development seem to be distinct. Members that might retain functions similar to those of the *AS2* gene do not appear to exist in this family, because the substitution of the AS2/LOB domain of AS2 with those of other members disrupts its function [39]. Considering similarities among the AS2/LOB domains, it is, however, undeniable that these family members may retain partially overlapping functions at the molecular level. They have often been described as transcription factors [40–47]. Recent results on AS2, however, suggest that the term "transcription factor" is not appropriate for a member of this family; they are better described as

Transcriptome analyses of *as2* and *as1* mutants reveal that accumulations of *ETT/ARF3*, *KAN2*, and *YAB5* transcripts, all of which are related to the abaxial cell fate, are increased in *as2* and *as1*, whereas those of the adaxial domain-determining *HD-ZIPIII* are not changed [37,48]. A subsequent systematic analysis has revealed that *ETT/ARF3* is a direct target of the AS2–AS1 complex [49,50]. AS2–AS1 directly represses *ETT/ARF3* by binding to the upstream region of *ETT/ARF3*. Furthermore, AS2–AS1 indirectly represses *ETT/ARF3* via the tasiR-ARF pathway. AS2–AS1 induce the accumulation of miR390 involved in the generation of tasiR-ARF. Subsequently, both the *ETT/ARF3*  and *ARF4* transcripts are degraded (Figure 2b). Therefore, the AS2–AS1 complex represses the

novel functional factors that could play a role in gene expression.

organs, including cotyledons, leaves, and floral organs [26–30]. Furthermore, genetic analyses have shown that four *YABBY* genes (*FIL*, *YAB3*, *YAB2*, *YAB5*) govern embryo patterning and the growth of leaf lamina along the abaxial–adaxial boundary [30].

*ETTIN*/*AUXIN RESPONSE FACTOR3* (*ETT*/*ARF3*) and *ARF4* also specify both abaxial cell fate and the lateral growth of leaf lamina [31]. Transcripts of both *ETT*/*ARF3* and *ARF4* are specifically degraded by the small RNA tasiR-ARF, which is generated through a miR390 pathway in the presumptive adaxial domain and contributes to the determination of the adaxial cell fate [23]. Because a loss of adaxial–abaxial polarity is often accompanied by a defect of leaf lamina expansion, it is suggested that the lateral growth of the lamina could be related to the determination of adaxial–abaxial identity, as previously proposed [2,32].

#### **2. Roles of AS2–AS1 in the Development of Leaf Polarity**

As described above, AS2 and AS1 proteins, which have AS2/LOB and R2R3 MYB (SANT) domains, respectively (Figure 2a), are identified by a yeast two-hybrid system, pull-down and gel-shift assays, and subcellular co-localization analyses. Because of their nature, these experiments indicate that AS2 and AS1 are physically associated with each other in vitro [19,33–36], implying also that they form a protein complex in the nucleus. Transcripts of *AS2* and *AS1* genes accumulate throughout the entire leaf primordia at early stages, in which the AS2–AS1 complex might be formed, but the accumulation patterns change as the leaves develop [37]. *AS2* transcripts are detected in the adaxial domain, while *AS1* transcripts are detected in the central region between the adaxial and abaxial domains of leaf primordia and the vasculature regions in more developed leaf primordia [10,37]. The plant-specific AS2/LOB domain includes a CXXC-type zinc-finger (ZF) motif, a leucine-zipper-like (LZL) region, and the internal-conserved-glycine (IcG) region between ZF and LZL (Figure 2a). The AS2/LOB domain is highly conserved in the AS2/LOB family, which consists of 42 members including AS2 in Arabidopsis [12,38,39]. Since the amino acid sequences outside of the domains are diverged among members and the transcription patterns of these genes differ for each gene, the roles of these genes in Arabidopsis development seem to be distinct. Members that might retain functions similar to those of the *AS2* gene do not appear to exist in this family, because the substitution of the AS2/LOB domain of AS2 with those of other members disrupts its function [39]. Considering similarities among the AS2/LOB domains, it is, however, undeniable that these family members may retain partially overlapping functions at the molecular level. They have often been described as transcription factors [40–47]. Recent results on AS2, however, suggest that the term "transcription factor" is not appropriate for a member of this family; they are better described as novel functional factors that could play a role in gene expression.

Transcriptome analyses of *as2* and *as1* mutants reveal that accumulations of *ETT*/*ARF3*, *KAN2*, and *YAB5* transcripts, all of which are related to the abaxial cell fate, are increased in *as2* and *as1*, whereas those of the adaxial domain-determining *HD-ZIPIII* are not changed [37,48]. A subsequent systematic analysis has revealed that *ETT*/*ARF3* is a direct target of the AS2–AS1 complex [49,50]. AS2–AS1 directly represses *ETT*/*ARF3* by binding to the upstream region of *ETT*/*ARF3*. Furthermore, AS2–AS1 indirectly represses *ETT*/*ARF3* via the tasiR-ARF pathway. AS2–AS1 induce the accumulation of miR390 involved in the generation of tasiR-ARF. Subsequently, both the *ETT*/*ARF3* and *ARF4* transcripts are degraded (Figure 2b). Therefore, the AS2–AS1 complex represses the expression of *ETT*/*ARF3* in the dual pathway [49]. Several phenotypes in *as2*, including defects of development along the adaxial–abaxial axis, are suppressed by the *ett arf4* double mutations. Consistent with these results, an overexpression of a tasiR-ARF-insensitive ETT/ARF3 cDNA produces *as2*-like leaves [51]. Similarly, lamina phenotypes of *as1* are also suppressed by the *ett arf4* double mutation. These results suggest that the elevated *ETT*/*ARF3* and *ARF4* expression in *as2* and *as1* cause several leaf phenotypes, including defects of adaxial–abaxial polarity in these mutants. The importance of the repression of these *ARFs* by AS2–AS1 is further confirmed by the analysis of modifier mutations of *as2* and *as1*,

which are described in the next section. Increased expression levels of *KAN2* and *YAB5* in *as2* and *as1* are caused by indirect regulation by AS2–AS1 [49]. importance of the repression of these *ARFs* by AS2–AS1 is further confirmed by the analysis of modifier mutations of *as2* and *as1*, which are described in the next section. Increased expression levels

of *KAN2* and *YAB5* in *as2* and *as1* are caused by indirect regulation by AS2–AS1 [49].

*as1* cause several leaf phenotypes, including defects of adaxial–abaxial polarity in these mutants. The

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 4 of 19

expression of *ETT/ARF3* in the dual pathway [49]. Several phenotypes in *as2*, including defects of development along the adaxial–abaxial axis, are suppressed by the *ett arf4* double mutations. Consistent with these results, an overexpression of a tasiR-ARF-insensitive ETT/ARF3 cDNA

**Figure 2.** (**a**) Motif and domain organization of AS2 and AS1 proteins. The ZF motif, IcG, and LZL regions of AS2 and the MYB domain and coiled-coil structure of AS1 are shown. (**b**) Dual regulation of *ETT/ARF3* gene expression, including that by the possibly epigenetic system of AS2–AS1. The AS2– AS1 complex represses *ETT/ARF3* directly by binding to its promoter and represses *ETT/ARF3* and *ARF4* indirectly via stimulation of the miR390 and tasiR-ARF pathway. In addition, AS1 and AS2 maintain gene body DNA methylation of the *ETT/ARF3* gene. Solid lines indicate direct regulation and dashed black lines indicate indirect regulation. Schemes of (b) are modified from ref. [2]. **Figure 2.** (**a**) Motif and domain organization of AS2 and AS1 proteins. The ZF motif, IcG, and LZL regions of AS2 and the MYB domain and coiled-coil structure of AS1 are shown. (**b**) Dual regulation of *ETT*/*ARF3* gene expression, including that by the possibly epigenetic system of AS2–AS1. The AS2–AS1 complex represses *ETT*/*ARF3* directly by binding to its promoter and represses *ETT*/*ARF3* and *ARF4* indirectly via stimulation of the miR390 and tasiR-ARF pathway. In addition, AS1 and AS2 maintain gene body DNA methylation of the *ETT*/*ARF3* gene. Solid lines indicate direct regulation and dashed black lines indicate indirect regulation. Schemes of (b) are modified from ref. [2].

#### **3. Modifier Mutations That Enhance Defects of** *AS2* **and** *AS1* **in Leaf Adaxial–Abaxial Polarity 3. Modifier Mutations That Enhance Defects of** *AS2* **and** *AS1* **in Leaf Adaxial–Abaxial Polarity**

Various mutations (about 60) that markedly enhance the defects of adaxial leaf development in *as2* or *as1* have been reported [2]. The genes responsible for these mutations are considered as "modifiers" or modifier genes, which affect the phenotypic expression of other genes. Double mutants generate abaxialized filamentous (needle-like, pin-shaped, pointed) leaves that have lost the adaxial domain (Figure 3). Causative mutations occur in genes that are involved in chromatin modification, biogenesis of small RNAs, and DNA replication [2,52]. Mutations in genes encoding ribosomal proteins are also identified as modifiers in *as2* or *as1* [2,53–58]. In addition, mutations in Various mutations (about 60) that markedly enhance the defects of adaxial leaf development in *as2* or *as1* have been reported [2]. The genes responsible for these mutations are considered as "modifiers" or modifier genes, which affect the phenotypic expression of other genes. Double mutants generate abaxialized filamentous (needle-like, pin-shaped, pointed) leaves that have lost the adaxial domain (Figure 3). Causative mutations occur in genes that are involved in chromatin modification, biogenesis of small RNAs, and DNA replication [2,52]. Mutations in genes encoding ribosomal proteins are also identified as modifiers in *as2* or *as1* [2,53–58]. In addition, mutations in genes encoding nucleolar proteins, such as *RNA HELICASE10* (*RH10*), *NUCLEOLIN1* (*NUC1*), *ROOT INITIATION DEFECTIVE2* (*RID2*), and *APUM23* are involved in ribosome biosynthesis, and enhance the phenotypes of *as2* and *as1* [59–63]. Mutations in *HDT1* and *HDT2* for nucleolar histone deacetylases (HDACs), which localize to the nucleolus, also act as modifiers of the *as2* and *as1* phenotypes [35]. We especially focus on the roles of nucleolar proteins in this review (Table 1).

**Figure 3.** Development of leaves along with three axes. Top views of SAM are schematically shown by open circle. Dot indicates the center of the SAM. Adaxial and abaxial domains are shown by green and light green, respectively. AS2-AS1 contributes to the determination of adaxial domain followed by the medial-lateral growth of leaves with vasculature (indicated by dark green rectangles). Modifiers act cooperatively with AS2–AS1 at leaf primordia to develop the adaxial (Ad) domain from abaxialized (Ab) leaf primordia and to generate leaves with a flat and symmetric structure. The double mutation into AS2 (or AS1) and modifiers results in the production of abaxialized filamentous **Figure 3.** Development of leaves along with three axes. Top views of SAM are schematically shown by open circle. Dot indicates the center of the SAM. Adaxial and abaxial domains are shown by green and light green, respectively. AS2-AS1 contributes to the determination of adaxial domain followed by the medial-lateral growth of leaves with vasculature (indicated by dark green rectangles). Modifiers act cooperatively with AS2–AS1 at leaf primordia to develop the adaxial (Ad) domain from abaxialized (Ab) leaf primordia and to generate leaves with a flat and symmetric structure. The double mutation into AS2 (or AS1) and modifiers results in the production of abaxialized filamentous leaves. Photograph of wild-type leaf is modified from ref. [64].

leaves. Photograph of wild-type leaf is modified from ref. [64].

*(pgy3/ae6/oli5/rpl5a)* AT3G25520 *RPL5B (rpl5b/oli7)* AT5G39740 *RPL7B (rpl7b)* AT2G01250

**Table 1.** Gene mutations that act as modifiers to enhance leaf adaxial–abaxial abnormalities in *as2* and *as1*. **1. Gene Name (Mutant Name) 2. AGI Code 3. Protein 4. Cellular Process and Status 5.References** I. Genes involved in rRNA processing *NUCLEOLIN1* (*nuc1*) AT1G48920 NUCLEOLIN rRNA processing and ribosome biogenesis Components of SSUP-like complex [59,61,70,71] *RNA HELICASE10*  AT5G60990 DEAD-box RNA pre-rRNA processing Transcript levels of several abaxial-determining genes (*KAN2*, *YAB5*, *ETT*/*ARF3*, and *ARF4*) are slightly upregulated in the *as2-1* single mutant and each of the modifier single mutants and are markedly increased in the *as2-1* and modifier double mutants (for example, *as2-1 rh10-1*). When the double mutations of *ETT*/*ARF3* and *ARF4* are introduced to double mutants with *as2-1* and one of the modifier mutations, such as *as2-1 nuc1-1* or *as2-1 rh10-1*, the abaxialized filamentous leaves phenotype (e.g., *as2 rh10* leaves in Figure 3) is restored to the expanded shapes [59,64,65]. These results show that the upregulation of these *ARF* genes in the double mutants is responsible for the disappearance of their adaxial specification in their filamentous leaves. These genetic observations suggest that the repression of these *ARF* genes by the synergistic action of AS2–AS1 and modifier proteins is critical for the proper development of the adaxial domain. These results suggest that modifier proteins act cooperatively with AS2–AS1 to generate flat and symmetric leaves (Figure 3). The modifier genes that encode nucleolar proteins are summarized below.

(*rh10*) helicase family protein Components of SSUP-like complex [59] *ROOT INITIATION DEFECTIVE2* (*rid2*) AT5G57280 RNA methyltransferase-like protein pre-rRNA processing [59,60] *APUM23* (*apum23*) AT1G72320 Pumillio protein containing PUF domain pre-rRNA processing and rRNA maturation [63] II. Genes for ribosomal Nucleoli are membrane-less organelles that appear to assemble through the phase separation of their molecular components [66]. The nucleoli contain internal subcompartments of ribosome biogenesis such as rDNA transcription, the processing of the precursor rRNA to generate mature rRNAs, assembly of these rRNAs, and many ribosomal proteins to generate each of small and large subunits of ribosomes. Genomic regions positioned in close proximity to the nucleolus are known as nucleolus-associated domains (NADs). Recent analyses of DNA sequencing that have been purified along with the nucleolus suggests that NADs in both animal and plant cells are enriched in regions displaying heterochromatic signatures [67,68].

proteins *RPL4D (rpl4d)* AT5G02870 Subunits of ribosome; *RPL5A NUCLEOLIN1* **(***NUC1***) gene**: Nucleolin, one of the most abundant non-ribosomal proteins in the nucleolus, has been described in a large variety of organisms [69]. The Arabidopsis genome encodes two

Ribosomal proteins

components of prerRNA-protein complex [53–57]

nucleolin-like proteins—NUC1 and NUC2. Only the NUC1 gene, however, is ubiquitously expressed under normal growth conditions [61].

The single mutant *nuc1-1* exhibits a pointed narrow leaf shape, which is often observed in other modifier mutations [59,61,70,71]. In *nuc1-1* plants, nucleolar disorganization is observed and accumulated levels of pre-rRNA precursors are detected, indicating that NUC1 is involved in the processing of pre-rRNAs [61,72–74]. An analysis of high-throughput sequencing of DNA purified from the nucleoli of the *NUC1* mutant revealed that NUC1 is required for global genomic organization and stability [67,75]. In addition, human nucleolin is reported to be an assembly intermediate of the SSUP and its candidate components [62,76,77]. The *as2-1 nuc1-1* and *as1-1 nuc1-1* double mutant plants generate filamentous leaves. These mutant phenotypes are partially suppressed by the mutation in *ETT*/*ARF3*, indicating a role in the repression of *ETT*/*ARF3* gene expression for the formation of flat symmetric leaves in the wild-type plants [65].

*RNA HELICASE10* **(***RH10***) gene**: The mutation of *rh10* was isolated as a modifier of *as2* and *as1*. Transcript levels of the abaxial genes, such as *ETT*/*ARF3* and *ARF4*, are elevated in *as2-1 rh10-1*, generating abaxialized filamentous leaves. This phenotype is suppressed by the *ett*/*arf3 arf4* double mutation, indicating a role in the repression of *ETT*/*ARF3* and *ARF4* gene expression for the formation of flat symmetric leaves in the wild-type plants [59]. RH10 is localized to the nucleolus in leaf primordia cells and is an ortholog of budding yeast Rrp3 and human DDX47, which belong to the DEAD-box RNA helicase family, a component of the nucleolar protein complex designated as the small subunit (SSUP) involved in 18S rRNA biogenesis [77,78]. It is reported that the DEAD-box RNA helicase family has an indispensable role in gene regulation through RNA metabolism [77–80]. DDX47 is necessary for maintaining the pluripotency of mouse stem cells [81]. In *rh10-1*, various defects are detected in SSUP-related events, such as the accumulation of 35S/33S rRNA precursors and a reduction in the 18S/25S ratio [59]. Nucleoli are enlarged in the *rh10-1* mutant [59]. RH10 may be involved in the early stages of processing reactions of the precursors of ribosomal RNAs.

*ROOT INITIATION DEFECTIVE2* **(***RID2***) gene**: *RID2* encodes an evolutionarily conserved methyltransferase-like protein, an orthologous protein of the budding yeast, Bud23, which exhibits tight functional and physical interactions with some of the SSUP components [82–84]. The RID2 protein is localized in nuclei and accumulates mainly within nucleoli [60]. RID2 is involved in the processing of pre-rRNAs at various early stages [85,86]. Nucleolar enlargement is also observed in the *rid2* mutant. A mutation in the *RID2* gene has an effect on the adaxial–abaxial organization of leaves on the *as2* background, generating filamentous leaves and upregulating *ARF3*/*ETT* and *ARF4* as found in other modifier mutants and *as2-1* [59].

*APUM23* **gene**: *APUM23*, which encodes a protein that is a member of the Pumilio/PUF domain protein family with its pumilio-like RNA-binding repeats, is localized to the nucleolus and is involved in the processing of 35S pre-rRNA [63,87]. The *apum23-1* mutant has enlarged nucleoli [63]. The double mutants *apum23-3 as2-2* and *apum23-3 as1-1* produce filamentous leaves, suggestive of the involvement of APUM23 in leaf development, similarly as with other nucleolar modifiers.

**Ribosomal protein genes**: It is worth noting that Arabidopsis double mutants of the ribosomal protein gene *rps6a-1*, which has a 9 bp deletion in the coding region of *RPS6A*, and *as2-1* exhibit strong adaxial leaf defects, as indicated by the fact that 80% of the double mutant leaves are filamentous [56]. Rps6 of budding yeast is one of five small-ribosomal-subunit proteins (Rps4, Rps6, Rps7, Rps9, and Rps14) that are components of the SSUP, which is a large ribonucleoprotein required for the biogenesis of the 18S rRNA [88]. Genetic interactions between *AS2*/*AS1* and homologues of *Rps4*, *Rps7*, *Rps9*, and Rps14 in Arabidopsis have yet to be examined. Three other *RPS* mutants (*rps23B*, *rps23B*, and *rps23B*) and fourteen *RPL* genes for ribosomal proteins in the large subunit also enhance the leaf phenotype in *as2* and/or *as1* (Table 1). It would also be intriguing to examine the relationships between the adaxial defects and ribosomal protein genes for such ribonucleoprotein complexes as a large subunit processome [89] in the nucleolus of Arabidopsis [53–57]. Therefore, the wild-type *AS2*

gene, which is specific in plants, may appear to attenuate defects resulting from mutations in the ribosomal protein gene.

*HDT1* **and** *HDT2* **genes**: *HDT1*, which encodes plant-specific nucleolar histone deacetylases (HDACs), is one of the factors responsible for gene silencing of megabase-scale rRNA loci and gene dosage control in nucleolar dominance [90,91], which are achieved by a highly condensed heterochromatic state that is associated with H3K9me2 and 5-methylcytosine enrichment in the promoter regions of rDNA genes [91]. Knockdown of the Arabidopsis genes *HDT1* and *HDT2* for nucleolar histone deacetylases (HDACs) enhances the leaf adaxial defects of *as2* and *as1* to generate severely abaxialized filamentous leaves, as seen in *as2-1 rh10-1* [35]. Considering the role of HDT1 in an epigenetic silencing of rDNAs (in nucleolar dominance), such as in the allopolyploid hybrid *Arabidopsis suecica* between *A. thaliana* and *A. arenosa* [91], the cooperative repression of the abaxial genes by AS2 and epigenetic silencing system of rDNAs described above are involved in the development of flat symmetric leaves.

**Table 1.** Gene mutations that act as modifiers to enhance leaf adaxial–abaxial abnormalities in *as2* and *as1*.


\* Rps6 of budding yeast is one of the proteins that was identified as a bona fide component of the SSUP.

questions.

#### **4. AS2 Bodies: Perinucleolar Granules Co-Localized Partially with the Chromocenter** formation and distribution of AS2 bodies might be modulated developmentally in plants and in a

The AS2-fused YFP (Yellow Fluorescent Protein) was used to investigate subnuclear localization of AS2 protein. The AS2 protein is localized to perinucleolar bodies known as AS2 bodies as well as to the nucleoplasm in the leaf cells of Arabidopsis and some interphase cells of a cultured tobacco cell line BY-2 (Figure 4) [35,93]. As mentioned in Section 2, AS2 has the AS2/LOB domain that includes ZF, IcG, and LZL regions (Figure 2a), which are essential for the formation of AS2 bodies at the perinucleolar regions [94]; the carboxyl-terminal half of AS2 is nonessential for the body formation, but essential for the developmental function of AS2 [12,93]; AS1 co-localizes with AS2 in the cell bodies (Figure 4) [35]. cell-cycle-dependent manner. The subcellular localization of AS2 appears to be subject to multiple controls, since AS2 was exported to the cytoplasm via the action of the geminivirus-encoded nuclear shuttle protein [12,94,99]. Although mechanisms for the formation of AS2 bodies and their roles in repressing the target genes for leaf development have yet to be discovered, the identification of AS2 body components and investigations of how these molecules interact within the nucleolus would provide answers for these

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 9 of 19

**Figure 4.** Schematic representation of the nucleus is shown. Chromosomes and AS2 bodies are indicated by blue and yellow, respectively. AS1 and AS2 are co-localized on AS2 bodies. **Figure 4.** Schematic representation of the nucleus is shown. Chromosomes and AS2 bodies are indicated by blue and yellow, respectively. AS1 and AS2 are co-localized on AS2 bodies.

**5. AS2–AS1 Binds to Exon 1 of the Target Gene** *ETT/ARF3***, and Is Involved in Maintaining CpG Methylation in Exon 6** Four mechanisms have been investigated for the repression of target gene *ETT/ARF3* expression by AS2–AS1: (1) direct binding of the AS1–AS2 complex to the 5′-upstream regions of *ETT/ARF3* to reduce the expression activity of *ETT/ARF3* (Figure 5) [49]; (2) indirect activation of miR390 dependent post-transcriptional gene silencing to negatively regulate both *ETT/ARF3* and *ARF4*  (Figure 2b) [49,50]; (3) direct binding of AS2 to the synthetic GCGGCG-containing nucleotides [47,50], and exon 1 of the *ETT/ARF3* gene containing the CGCCGC (Figure 5) [65]; (4) maintenance of the status of gene body (CpG) methylation in exon 6 of ETT/ARF3 (Figure 5) [49,65,100]. In the present review, we focus on the last two topics. In addition, the amino acid residues that are highly conserved within and adjacent to the ZF motifs of all the AS2 family members are critically important for the body formation: four cysteine residues; proline and alanine residues next to the first cysteine residue; the RRK cluster (Figure 2). The RRK sequence is found within proposed nucleolar localization signals (NoLSs) [95–98] and it is likely that this cluster participates in the perinucleolar localization of AS2. These amino acid residues and three regions (ZF, IcG, and LZL) in the AS2/LOB domain are also required for the ability of AS2 to complement the *as2* mutation and to bind to the coding sequence of the target *ARF3*/*ETT* gene, showing that the formation of AS2 bodies is related to the genetic functions of AS2 in leaf formation. The AS2 bodies appear to be located to the peripheral regions of nucleoli and are partially overlapped with perinucleolar chromocenters with condensed chromatin-containing ribosomal RNA genes (45S rDNA repeats), suggesting that AS2 bodies interact with 45S rDNA repeats (Figure 4) [94].

Several protein members of the AS2/LOB family, including AS2, bind synthetic double-stranded DNAs containing the GCGGCG sequence [101]. AS2, specifically, also binds in vitro the doublestranded CGCCGC sequence in exon 1 of the target gene *ETT/ARF3* [65]. The zinc-finger motif containing the RRK (Arg-Arg-Lys) sequence in AS2 is essential for this binding [65], the formation of AS2 bodies and functions in the development of leaves with normal shapes [94]. Modes of molecular interactions between the amino acid residues in RRK and each of the deoxyribonucleotides in GCGGCG have recently been proposed based on the results of SEC–SAXS (size exclusion chromatography–small angle X-ray scattering) experiments [47]. Since 32 out of 42 members of the family harbor the RRK and/or RRR sequence in the ZF motifs [12], it should be informative to investigate the possible roles of the clusters of these basic amino acid residues in other members in plant physiology, development, and growth [102]. It should be noted that the proportions of cells in which AS2 bodies are generated in plants differ from those in cultured cells. AS2 bodies are detected in only a few percentages of interphase cells of the tobacco-cultured cell line BY-2 and the Arabidopsis-cultured cell line MM2d transformed with the AS2-fused YFP constructs [93,94]. AS2 bodies are, however, detected in almost all interphase cells of the adaxial domain in leaf primordia of the Arabidopsis plants with the AS2-fused YFP construct [94]. The average number of AS2 bodies per YFP-positive cells at interphase (and/or the G0 stage) in leaf primordia was calculated as 1.9 [94]. In contrast, AS2 bodies are formed in all M phase cells of both cell lines, MM2d and BY-2 and in all M phase cells of leaf primordia; AS2 bodies are separated into daughter cells during the M phase progression [93,94]. These observations imply that the formation and distribution of AS2 bodies might be modulated developmentally in plants and in a cell-cycle-dependent manner.

AS2 and AS1 play a role in maintaining cytosine methylation mediated by METHYLTRANSFERASE1 (MET1) in six CpG dinucleotides in exon 6 of *ETT/ARF3* (Figure 5) [49]. The subcellular localization of AS2 appears to be subject to multiple controls, since AS2 was exported to the cytoplasm via the action of the geminivirus-encoded nuclear shuttle protein [12,94,99].

Because levels of CpG methylation are inversely related to the *ETT/ARF3* transcript levels, AS2 and AS1 possibly regulate the transcriptional repression of *ETT/ARF3* through CpG methylation in the recruitment of methylation activity and/or inhibition of demethylation activity at exon 6 [103]. As described in Section 3, mutations in the *RH10*, *NUC1*, and *RID2* genes for nucleolar proteins enhance defects in leaf morphology in the *as2* mutant and, in parallel with this observation, result in an Although mechanisms for the formation of AS2 bodies and their roles in repressing the target genes for leaf development have yet to be discovered, the identification of AS2 body components and investigations of how these molecules interact within the nucleolus would provide answers for these questions.

increase in the transcript level of target genes *ETT/ARF3* and *ARF4*. The levels of CpG methylation at some of the CpG dinucleotides in exon 6 of *ETT/ARF3* decrease in *rh10* and *nuc1* mutants, and further decrease in *rh10 as2* and *nuc1 as2*, suggesting that these nucleolar proteins, in addition to AS2, also [65,103].

#### **5. AS2–AS1 Binds to Exon 1 of the Target Gene** *ETT*/*ARF3***, and Is Involved in Maintaining CpG Methylation in Exon 6** repress the class 1 *KNOX* homeobox genes *BREVIPEDICELLUS* (*BP*), *KNAT2*, and *KNAT6* (Figure 1) [18]. The AS2–AS1 complex physically interacts with CURLY LEAF (CLF), the polycomb repressive

The AS2–AS1 complex is also involved in the establishment of leaf proximal–distal polarity to

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 10 of 19

take part in maintaining the cytosine methylation of CpG dinucleotides in exon 6 of *ETT/ARF3*

How can AS2 be involved in maintaining MET1-regulated CpG methylation in exon 6 of the *ETT/ARF3* gene? MET1 is an ortholog of the Dnmt1 of vertebrates and acts as DNA methyltransferase, which methylates hemimethylated CpG, converting it to fully methylated CpG during DNA replication [104,105]. MET1 is part of a putative protein complex involved in the maintenance of DNA methylation in Arabidopsis [106–111]. MET1 is similar to Dnmt1, in terms of the domain organization [109,112], except that MET1 has no amino acid sequence for the ZF-CxxC motif. If AS2 forms a protein complex with MET1, AS2 provides the ZF motif, which has DNA binding activity, as described above in this section, to the MET1-containing putative protein complex. The promoter regions of inactive 45S rDNAs in Arabidopsis are highly methylated by MET1 and their chromatin states are highly condensed at perinucleolar regions [113,114]. MET1 requires NUC1, one of the AS2 modifiers, and nucleolar histone deacetylase HDA6 for this methylation [113,114] and directly interacts with HDA6 [115,116], which is also associated with AS2 and AS1 [36]. The CpG methylation system for the 45S

Four mechanisms have been investigated for the repression of target gene *ETT*/*ARF3* expression by AS2–AS1: (1) direct binding of the AS1–AS2 complex to the 50 -upstream regions of *ETT*/*ARF3* to reduce the expression activity of *ETT*/*ARF3* (Figure 5) [49]; (2) indirect activation of miR390-dependent post-transcriptional gene silencing to negatively regulate both *ETT*/*ARF3* and *ARF4* (Figure 2b) [49,50]; (3) direct binding of AS2 to the synthetic GCGGCG-containing nucleotides [47,50], and exon 1 of the *ETT*/*ARF3* gene containing the CGCCGC (Figure 5) [65]; (4) maintenance of the status of gene body (CpG) methylation in exon 6 of ETT/ARF3 (Figure 5) [49,65,100]. In the present review, we focus on the last two topics. complex 2 (PRC2) core component, and LIKE-HETEROCHROMATIN PROTEIN1 (LHP1), the PRC1 component, and recruits PRC2 to the homeobox genes *BP* and *KNAT2* [117,118]. AS2–AS1 interacts with the *BP* promoter, likely through the recruitment of the chromatin-remodeling factor HIRA (histone-regulator A) and forms a repressive chromatin state [19]. AS2–AS1 also interacts with LEAF FLOWER RELATED (LFR) in the chromatin remodeling complex and is associated with H3K27me3 in the *BP* gene, but not with the *ETT/ARF3* gene [119]. AS2–AS1 is required for the correct temporal repression of *ETT/ARF3*, which involves a PRC2-independent mechanism [50]. Despite their pivotal role, the means by which AS2–AS1 epigenetically represses *ETT/ARF3* in the establishment of leaf adaxial–abaxial polarity remains unsolved.

**Figure 5.** Molecular relationships between AS2–AS1 and the target gene *ETT/ARF3*. AS1–AS2 directly binds the *ETT/ARF3* regulatory region and represses *ETT/ARF3* expression. AS2 binds to the specific CGC repeat sequence in exon 1. AS2–AS1, RH10, and NUC1 are involved in the maintenance of CpG methylation in exon 6. RH10 and NUC1 are proteins localized in the nucleolus. **Figure 5.** Molecular relationships between AS2–AS1 and the target gene *ETT*/*ARF3*. AS1–AS2 directly binds the *ETT*/*ARF3* regulatory region and represses *ETT*/*ARF3* expression. AS2 binds to the specific CGC repeat sequence in exon 1. AS2–AS1, RH10, and NUC1 are involved in the maintenance of CpG methylation in exon 6. RH10 and NUC1 are proteins localized in the nucleolus.

Several protein members of the AS2/LOB family, including AS2, bind synthetic double-stranded DNAs containing the GCGGCG sequence [101]. AS2, specifically, also binds in vitro the double-stranded CGCCGC sequence in exon 1 of the target gene *ETT*/*ARF3* [65]. The zinc-finger motif containing the RRK (Arg-Arg-Lys) sequence in AS2 is essential for this binding [65], the formation of AS2 bodies and functions in the development of leaves with normal shapes [94]. Modes of molecular interactions between the amino acid residues in RRK and each of the deoxyribonucleotides in GCGGCG have recently been proposed based on the results of SEC–SAXS (size exclusion chromatography–small angle X-ray scattering) experiments [47]. Since 32 out of 42 members of the family harbor the RRK and/or RRR sequence in the ZF motifs [12], it should be informative to investigate the possible roles of the clusters of these basic amino acid residues in other members in plant physiology, development, and growth [102].

AS2 and AS1 play a role in maintaining cytosine methylation mediated by METHYLTRANSFERASE1 (MET1) in six CpG dinucleotides in exon 6 of *ETT*/*ARF3* (Figure 5) [49]. Because levels of CpG methylation are inversely related to the *ETT*/*ARF3* transcript levels, AS2 and AS1 possibly regulate the transcriptional repression of *ETT*/*ARF3* through CpG methylation in the recruitment of methylation

activity and/or inhibition of demethylation activity at exon 6 [103]. As described in Section 3, mutations in the *RH10*, *NUC1*, and *RID2* genes for nucleolar proteins enhance defects in leaf morphology in the *as2* mutant and, in parallel with this observation, result in an increase in the transcript level of target genes *ETT*/*ARF3* and *ARF4*. The levels of CpG methylation at some of the CpG dinucleotides in exon 6 of *ETT*/*ARF3* decrease in *rh10* and *nuc1* mutants, and further decrease in *rh10 as2* and *nuc1 as2*, suggesting that these nucleolar proteins, in addition to AS2, also take part in maintaining the cytosine methylation of CpG dinucleotides in exon 6 of *ETT*/*ARF3* [65,103].

How can AS2 be involved in maintaining MET1-regulated CpG methylation in exon 6 of the *ETT*/*ARF3* gene? MET1 is an ortholog of the Dnmt1 of vertebrates and acts as DNA methyltransferase, which methylates hemimethylated CpG, converting it to fully methylated CpG during DNA replication [104,105]. MET1 is part of a putative protein complex involved in the maintenance of DNA methylation in Arabidopsis [106–111]. MET1 is similar to Dnmt1, in terms of the domain organization [109,112], except that MET1 has no amino acid sequence for the ZF-CxxC motif. If AS2 forms a protein complex with MET1, AS2 provides the ZF motif, which has DNA binding activity, as described above in this section, to the MET1-containing putative protein complex. The promoter regions of inactive 45S rDNAs in Arabidopsis are highly methylated by MET1 and their chromatin states are highly condensed at perinucleolar regions [113,114]. MET1 requires NUC1, one of the AS2 modifiers, and nucleolar histone deacetylase HDA6 for this methylation [113,114] and directly interacts with HDA6 [115,116], which is also associated with AS2 and AS1 [36]. The CpG methylation system for the 45S rDNA might be also involved in CpG methylation in the *ETT*/*ARF3* gene around perinucleolar areas; the *ETT*/*ARF3* gene might be recruited to such an area by an action of AS2 (Figure 6). *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 11 of 19

**Figure 6.** Models of the roles of nucleolar proteins in AS2–AS1 involved in epigenetic regulation of the *ETT/ARF3* gene. Nucleolus and surrounding structures are shown. Nucleosomes are indicated by coiled structure composed of red lines (DNA) and light/dark grey circle (histone octamer). Different genomes are distinguished by different darkness of nucleosomes. NUC1 affected the localization patterns of AS2 bodies at the peripheral region of the nucleolus, which are required for leaf development. AS2 bodies are partially overlapped with chromocenters, represented by dense nucleosome at the peripheral region of the nucleolus. The 45S rDNA repeat loci include transcriptionally active and inactive regions, which chromosomal status are loosened in the nucleolus and condensed on the peri-nucleolus (overlapping with chromocenter), respectively, suggestive of an interaction of AS2 bodies with inactive 45S rDNA. RH10 and NUC1 in addition to AS2 are involved in the maintenance of CpG methylation in exon 6 of *ETT/ARF3* in the nucleoplasm (left panel) or in AS2 bodies (right panel). NUC1 is involved in the maintenance of CpG methylation in 45S rDNA. **Figure 6.** Models of the roles of nucleolar proteins in AS2–AS1 involved in epigenetic regulation of the *ETT*/*ARF3* gene. Nucleolus and surrounding structures are shown. Nucleosomes are indicated by coiled structure composed of red lines (DNA) and light/dark grey circle (histone octamer). Different genomes are distinguished by different darkness of nucleosomes. NUC1 affected the localization patterns of AS2 bodies at the peripheral region of the nucleolus, which are required for leaf development. AS2 bodies are partially overlapped with chromocenters, represented by dense nucleosome at the peripheral region of the nucleolus. The 45S rDNA repeat loci include transcriptionally active and inactive regions, which chromosomal status are loosened in the nucleolus and condensed on the peri-nucleolus (overlapping with chromocenter), respectively, suggestive of an interaction of AS2 bodies with inactive 45S rDNA. RH10 and NUC1 in addition to AS2 are involved in the maintenance of CpG methylation in exon 6 of *ETT*/*ARF3* in the nucleoplasm (left panel) or in AS2 bodies (right panel). NUC1 is involved in the maintenance of CpG methylation in 45S rDNA.

**6. Subcellular localization of AS2** Although it is often reported that AS2 and other members of the AS2/LOB family are nuclear The AS2–AS1 complex is also involved in the establishment of leaf proximal–distal polarity to repress the class 1 *KNOX* homeobox genes *BREVIPEDICELLUS* (*BP*), *KNAT2*, and *KNAT6* (Figure 1) [18]. The AS2–AS1 complex physically interacts with CURLY LEAF (CLF), the polycomb repressive complex 2

unknown cellular conditions.

proteins [2,12,35,38,93,120], mechanisms of the nuclear localization of AS2 protein are poorly

cluster, which is thought to be critical for nuclear localization. The examination of subcellular localization of the mutant as2 (as2-RRK/3A) with the alanine replacement at the RRK sequence in the zinc-finger motif (Figure 2a) with the alanine cluster, however, shows that the mutant protein is still present in the nucleoplasm; it is not exported to the cytoplasm and does not form the AS2 bodies at the perinucleolar region [94]. These observations show that the RRK sequence is not involved in the nuclear localization of AS2. This result is consistent with the previous finding [121]: that is, the mutant proteins of ASL18/LBD16, another member of the AS2/LOB family, from which the RRK sequence is deleted are still localized to the nucleus and nuclear localization signals are proposed to be present in the coiled-coil sequence and the carboxyl-terminal region. Furthermore, the mutant as2 protein that lacks the IcG region is exclusively localized to the cytoplasm [94]. It is also reported that AS2 was exported to the cytoplasm via the action of the geminivirus-encoded nuclear shuttle protein and localized to the plant P-body complex [99]. Therefore, the AS2 protein might be subject to multiple subcellular localization controls, depending on its interactions with other proteins and other

As described above, 32 among the 42 members of the AS2/LOB family harbor ZF motifs, the amino acid sequences of which include RRK or RRR sequences [12,38]. The observation that the as2 mutant lacking RRK is localized to the nucleoplasm, but does not form AS2 bodies at the nucleolar periphery, suggests that RRK appears to be involved in the transport of AS2 to the perinucleolar region and/or to the formation of AS2 bodies. The RRK sequence is present within the proposed NoLSs [95–98]. It is intriguing to test whether this cluster of the basic amino acid residues in the zincfinger could be directly involved in the transfer to the peripheral region of the nucleoli from the nucleoplasm and/or the formation of AS2 bodies by a phase separation mechanism, because the (PRC2) core component, and LIKE-HETEROCHROMATIN PROTEIN1 (LHP1), the PRC1 component, and recruits PRC2 to the homeobox genes *BP* and *KNAT2* [117,118]. AS2–AS1 interacts with the *BP* promoter, likely through the recruitment of the chromatin-remodeling factor HIRA (histone-regulator A) and forms a repressive chromatin state [19]. AS2–AS1 also interacts with LEAF FLOWER RELATED (LFR) in the chromatin remodeling complex and is associated with H3K27me3 in the *BP* gene, but not with the *ETT*/*ARF3* gene [119]. AS2–AS1 is required for the correct temporal repression of *ETT*/*ARF3*, which involves a PRC2-independent mechanism [50]. Despite their pivotal role, the means by which AS2–AS1 epigenetically represses *ETT*/*ARF3* in the establishment of leaf adaxial–abaxial polarity remains unsolved.

#### **6. Subcellular localization of AS2**

Although it is often reported that AS2 and other members of the AS2/LOB family are nuclear proteins [2,12,35,38,93,120], mechanisms of the nuclear localization of AS2 protein are poorly understood. The RRK sequence in the zinc-finger motif of AS2 (Figure 2a) is only a basic amino acid cluster, which is thought to be critical for nuclear localization. The examination of subcellular localization of the mutant as2 (as2-RRK/3A) with the alanine replacement at the RRK sequence in the zinc-finger motif (Figure 2a) with the alanine cluster, however, shows that the mutant protein is still present in the nucleoplasm; it is not exported to the cytoplasm and does not form the AS2 bodies at the perinucleolar region [94]. These observations show that the RRK sequence is not involved in the nuclear localization of AS2. This result is consistent with the previous finding [121]: that is, the mutant proteins of ASL18/LBD16, another member of the AS2/LOB family, from which the RRK sequence is deleted are still localized to the nucleus and nuclear localization signals are proposed to be present in the coiled-coil sequence and the carboxyl-terminal region. Furthermore, the mutant as2 protein that lacks the IcG region is exclusively localized to the cytoplasm [94]. It is also reported that AS2 was exported to the cytoplasm via the action of the geminivirus-encoded nuclear shuttle protein and localized to the plant P-body complex [99]. Therefore, the AS2 protein might be subject to multiple subcellular localization controls, depending on its interactions with other proteins and other unknown cellular conditions.

As described above, 32 among the 42 members of the AS2/LOB family harbor ZF motifs, the amino acid sequences of which include RRK or RRR sequences [12,38]. The observation that the as2 mutant lacking RRK is localized to the nucleoplasm, but does not form AS2 bodies at the nucleolar periphery, suggests that RRK appears to be involved in the transport of AS2 to the perinucleolar region and/or to the formation of AS2 bodies. The RRK sequence is present within the proposed NoLSs [95–98]. It is intriguing to test whether this cluster of the basic amino acid residues in the zinc-finger could be directly involved in the transfer to the peripheral region of the nucleoli from the nucleoplasm and/or the formation of AS2 bodies by a phase separation mechanism, because the nucleolus and many nucleolar bodies are proposed to be formed through such a physico-chemical molecular interaction [122].

#### **7. Possible Roles of AS2 Bodies in Epigenetic Repression of** *ETT*/*ARF3*

As described in Section 3, the level of the leaf abaxial gene *ETT*/*ARF3* expression is influenced by modifier proteins that are localized to the nucleolus. For example, the AS2–AS1 complex binds directly to the upstream region of the *ETT*/*ARF3* gene to repress its transcription [49,64]. Furthermore, the *ETT*/*ARF3* transcriptional level is altered by mutations in various genes for nucleolus-localized proteins, such as RH10, RID2, and NUC1, which affect the biogenesis of ribosomal RNAs and the formation of the nucleolus with a properly organized morphology [59–61]. Perturbation of rRNA biogenesis correlates with structural disorders of the nucleolus, such as nucleolar enlargement in plant cellsand in animal cells [60,85,86,123]. It is, however, still unknown how structural disorders of the nucleolus affects leaf development mediated by AS2–AS1.

Perinucleolar regions might provide the molecular architectures, such as nucleolus-associated chromatin domains (NADs), which correspond to regions of low transcriptional levels [67,68,124,125]. In Arabidopsis, many of 45S rRNA genes are condensed as heterochromatin and silenced by

epigenetic mechanisms that include DNA methylation and histone modification at the periphery of the nucleolus [114,126–128]. MET1, HDA6, and chromatin assembly factor (CAF-1) are all involved in the formation of such an epigenetic state in the perinucleolar subdomain [114,126,127]. AS2 and AS1, which are associated with HDA6 [36], are colocalized to AS2 bodies in the peripheral region of the nucleolus (Figures 4 and 6) [35,93,94]. The *ETT*/*ARF3* gene undergoes MET1-dependent CpG methylation in exon 6 [49]. As described in Sections 4 and 6, mutant proteins of AS2 (as2-RRK/3A, Figure 2a) that do not form AS2 bodies are not functional in leaf morphogenesis [94]. Mutations in RH10, RID2, and NUC1 might affect the integrity of nucleolar morphology [59–61], which would then alter the transcriptional patterns of *ETT*/*ARF3*, a target gene of AS2, although the subnucleolar localization of the target gene to the peripheral subdomain (Figure 6) has yet to be demonstrated. It is an interesting problem to elucidate how the *ETT*/*ARF3* gene transcribed by RNA polymerase II is regulated in the nucleolus or its peripheral region.

Recently, AS2 was shown to bind to DNAs other than *ETT*/*ARF3* [129]. Since AS2 is a plant-specific DNA binding protein, elucidation of the interaction between AS2 and these DNA molecules should uncover a more global and novel regulatory system mediated by AS2 and the nucleolus in plant cells [19,101].

**Author Contributions:** H.I. prepared the manuscript related to Sections 1, 2, 4 and 6. H.T. prepared the manuscript related to Section 5. Y.M. prepared the manuscript related to Sections 4 and 6. C.M. supervised all the research in this manuscript and prepared the manuscript related to Sections 1–3, 5 and 7. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (grant numbers JP18H03330, JP18K06297, JP19K22892, JP20K06702 and 16H06279 (PAGS)) and the Research Foundation for the Electrotechnology of Chubu.

**Acknowledgments:** The authors are grateful to Takanori Suzuki at Ishihara Sangyo Kaisha LTD, Yuki Sakamoto at Osaka University, Sachihiro Matsunaga and Tetsuya Higashiyama at University of Tokyo, Daisuke Kurihara and Shogo Matsumoto at Nagoya University, Michiko Sasabe at Hirosaki University, and Shoko Kojima and Sayuri Ando at Chubu University for their helpful discussions.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**


#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **The Regulation of CIN-like TCP Transcription Factors**

#### **Jingqiu Lan <sup>1</sup> and Genji Qin 1,2,\***


Received: 30 May 2020; Accepted: 20 June 2020; Published: 24 June 2020

**Abstract:** TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR 1 and 2 (TCP) family proteins are the plant-specific transcription factors extensively participating in diverse developmental processes by integrating external cues with internal signals. The roles of CINCINNATA (CIN)-like TCPs are conserved in control of the morphology and size of leaves, petal development, trichome formation and plant flowering. The tight regulation of CIN-like TCP activity at transcriptional and post-transcriptional levels are central for plant developmental plasticity in response to the ever-changing environmental conditions. In this review, we summarize recent progresses with regard to the function and regulation of CIN-like TCPs. CIN-like TCPs are regulated by abiotic and biotic cues including light, temperature and pathogens. They are also finely controlled by microRNA319 (miRNA319), chromatin remodeling complexes and auxin homeostasis. The protein degradation plays critical roles in tightly controlling the activity of CIN-like TCPs as well.

**Keywords:** CIN-like TCP transcription factors; regulation; light; high temperature; microRNA319; BRAHMA; TIE1 transcriptional repressors; TEAR1 E3 ligases

#### **1. Introduction**

Developmental plasticity is central for sessile plants in adaptation to the environmental conditions [1]. The molecular bases for plant developmental plasticity or the mechanisms by which plants translate the environmental cues into the internal signals to direct the optimal growth and development in different plant growing conditions are important for plant survival and are useful for crop improvement by molecular breeding. Since the discovery of the founding members of TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR 1 and 2 (TCP) protein family in plants more than twenty years ago [2–4], TCP proteins have emerged as a central hub for integrating the internal and external cues to control plant developmental plasticity.

TCP is an acronym of the name of founding genes isolated from three species, i.e., *TEOSINTE BRANCHED1* (*TB1*) from maize (*Zea mays*) [2,5], *CYCLOIDEA* (*CYC*) from snapdragon (*Antirrhinum majus*) [3], and *PROLIFERATING CELL FACTOR 1* and *2* (*PCF1* and *PCF2*) from rice (*Oryza sativa*) [4]. *TB1* is a famous maize domestication gene. TB1 represses the outgrowth of axillary branches and promotes the formation of female inflorescences in domesticated maize, while in teosinte—which is the wild ancestor of maize—the twice lower expression of *TB1* leads to a decrease of apical dominance and an increase of shoot branches [5]. The *CYC* gene was isolated from snapdragon. *CYC* is specifically expressed in the dorsal primordia and controls the flower zygomorphic trait. Disruption of both *CYC* and its close homolog *DICHOTOMA* (*DICH*) in snapdragon results in radially symmetric flowers [6]. Both TB1 and CYC play pivotal roles in shaping plant key morphologies. The rice PCF proteins were found to directly bind to the promoter region of *PROLIFERATING CELL NUCLEAR ANTIGEN* (*PCNA*) gene which encodes a protein acting as a DNA polymerase sliding clamp implicated in DNA replication

and cell cycle regulation [4]. Further analysis of the protein sequences of TB1, CYC and PCF proteins found that they all contain a conserved region predicted to form a non-canonical basic helix-loop-helix (bHLH) structure named as the TCP domain [7]. Since PCF1 and PCF2 had DNA-binding activity, TCP proteins were deduced to act as transcription factors and the TCP domain was proposed to be responsible for DNA binding and protein-protein interaction [4,7,8]. TCP domain was proposed to be responsible for DNA binding and protein-protein interaction [4,7,8]. According to the sequence differences in the TCP domain, TCPs are classified into class I and class II subfamilies [7] (Figure 1). The TCP domain of class II TCPs contains additional four-amino acid residues in the conserved basic region [7]. The class II TCPs are further divided into CINCINNATA (CIN)-like TCPs and CYC/TB1-like TCPs based on the additional sequence

*Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 2 of 17

proteins were found to directly bind to the promoter region of *PROLIFERATING CELL NUCLEAR ANTIGEN* (*PCNA*) gene which encodes a protein acting as a DNA polymerase sliding clamp implicated in DNA replication and cell cycle regulation [4]. Further analysis of the protein sequences of TB1, CYC and PCF proteins found that they all contain a conserved region predicted to form a

According to the sequence differences in the TCP domain, TCPs are classified into class I and class II subfamilies [7] (Figure 1). The TCP domain of class II TCPs contains additional four-amino acid residues in the conserved basic region [7]. The class II TCPs are further divided into CINCINNATA (CIN)-like TCPs and CYC/TB1-like TCPs based on the additional sequence differences in the TCP domain [9]. The CYC/TB1 TCP subgroup also carries a conserved glutamic acid-cysteine-glutamic acid (ECE) motif outside the TCP domain [10]. The *CIN* gene was isolated from snapdragon by analyzing the *cin* mutant which produces abnormal leaves and petals with undulated edges [11,12] and is the founding member of the CIN-like TCP subgroup (Figure 1). CIN controls leaf flatness by tightly regulating cell proliferation and differentiation in the different areas of leaf blades [11]. In the model plant *Arabidopsis*, the CIN-like TCPs include eight members which are further grouped into two clades based on the existence of microRNA (miRNA) binding site outside the sequence encoding TCP domain. *TCP2*, *TCP3*, *TCP4*, *TCP10,* and *TCP24* have the miRNA binding sites and post-transcriptionally regulated by miR319 [13], while TCP5, TCP13 and TCP17 form a small clade named as TCP5-like CIN-TCPs that were proved to be important for plant thermomorphogenesis (Figure 2) [14]. differences in the TCP domain [9]. The CYC/TB1 TCP subgroup also carries a conserved glutamic acid-cysteine-glutamic acid (ECE) motif outside the TCP domain[10]. The *CIN* gene was isolated from snapdragon by analyzing the *cin* mutant which produces abnormal leaves and petals with undulated edges [11,12] and is the founding member of the CIN-like TCP subgroup (Figure 1). CIN controls leaf flatness by tightly regulating cell proliferation and differentiation in the different areas of leaf blades [11]. In the model plant *Arabidopsis*, the CIN-like TCPs include eight members which are further grouped into two clades based on the existence of microRNA (miRNA) binding site outside the sequence encoding TCP domain. *TCP2*, *TCP3*, *TCP4*, *TCP10,* and *TCP24* have the miRNA binding sites and post-transcriptionally regulated by miR319 [13], while TCP5, TCP13 and TCP17 form a small clade named as TCP5-like CIN-TCPs that were proved to be important for plant thermomorphogenesis (Figure 2) [14].

**Figure 1.** The phylogeny of TCP transcription factors, including all the TCP proteins in *Arabidopsis thaliana*

**Figure 1.** The phylogeny of TCP transcription factors, including all the TCP proteins in *Arabidopsis* 

refinement methods. The phylogenetic tree was constructed with the Maximum Likelihood (ML)

[23,24,28–31].

and the CIN-like TCPs of other species mentioned in this review. Multiple alignments of the full-length TCP proteins were conducted using MAFFT Version 7 [15] with L-INS-i iterative refinement methods. The phylogenetic tree was constructed with the Maximum Likelihood (ML) method using the IQ-tree2 software [16] with the VT+F+R4 model with 1000 bootstrap replications. The subfamilies and subclasses (Class I, Class II, CIN-like TCP and CYC-like TCPs) are indicated above the divergent branches. The proteins in red words are the CIN-like TCPs which are mainly discussed in this review. The prefixes of TCP proteins are indicated the species. At: *Arabidopsis thaliana*; Brr: *Brassica rapa*; Gh: *Gossypium hirsutum*; Ls: *Lactuca sativa*; Cp: *Cyclamen persicum*; Sly: *Solanum lycopersium*; Am: *Antirrhinum majus*; Mp: *Marchantia polymorpha*; Ppa: *Physcomitrella patens*. The bootstrap support is indicated above the branches. The scale bar denotes the branch length. *Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 3 of 17 method using the IQ-tree2 software [16] with the VT+F+R4 model with 1000 bootstrap replications. The subfamilies and subclasses (Class I, Class II, CIN-like TCP and CYC-like TCPs) are indicated above the divergent branches. The proteins in red words are the CIN-like TCPs which are mainly discussed in this review. The prefixes of TCP proteins are indicated the species. At: *Arabidopsis thaliana*; Brr: *Brassica rapa*; Gh: *Gossypium hirsutum*; Ls: *Lactuca sativa*; Cp: *Cyclamen persicum*; Sly: *Solanum lycopersium*; Am: *Antirrhinum majus*; Mp: *Marchantia polymorpha*; Ppa: *Physcomitrella patens*.

The bootstrap support is indicated above the branches. The scale bar denotes the branch length.

**Figure 2.** An overview of the regulation mechanisms of CIN-like TCP transcription factors by light and temperature during cotyledon opening and hypocotyl elongation processes. The external stimuli including light and high temperature are summarized at the left column. The schematic diagram includes the regulation mechanisms of CIN-like TCPs at the transcriptional and the protein levels. The arrows directly pointing on the double helix symbols indicate transcriptional regulations. The arrows pointing to the proteins indicate the regulations of protein stabilities. The proteins related with the "26S" symbols indicate protein degradation through the ubiquitin-26S proteasome pathway. The blue arrows represent the positive regulation, and the red arrows with dash-headed ends indicate the negative regulation. The green arrows and red dash-headed ends at the double-helix icons indicates the activation and repression of gene expression, respectively. All the unknown factors are indicated with question marks. R:FR, red light: far red light ratio; PIFs, PHYTOCHROME-INTERACTION FACTORs; SAURs, SMALL AUXIN UPREGULATED RNAs; YUCs, YUCCAs. **Figure 2.** An overview of the regulation mechanisms of CIN-like TCP transcription factors by light and temperature during cotyledon opening and hypocotyl elongation processes. The external stimuli including light and high temperature are summarized at the left column. The schematic diagram includes the regulation mechanisms of CIN-like TCPs at the transcriptional and the protein levels. The arrows directly pointing on the double helix symbols indicate transcriptional regulations. The arrows pointing to the proteins indicate the regulations of protein stabilities. The proteins related with the "26S" symbols indicate protein degradation through the ubiquitin-26S proteasome pathway. The blue arrows represent the positive regulation, and the red arrows with dash-headed ends indicate the negative regulation. The green arrows and red dash-headed ends at the double-helix icons indicates the activation and repression of gene expression, respectively. All the unknown factors are indicated with question marks. R:FR, red light: far red light ratio; PIFs, PHYTOCHROME-INTERACTION FACTORs; SAURs, SMALL AUXIN UPREGULATED RNAs; YUCs, YUCCAs.

TCP transcription factors constitute a plant-specific protein family which is conserved in plant kingdom. TCP homologs are identified from diverse plant species [7]. It is proved that TCP proteins are existed in the early land plants during evolutionary history [17–20]. However, it is still in dispute whether they are present in pluricellular green algae [17,20]. The TCP protein family is significantly expanded in angiosperm species by gene or whole-genome duplication independently in basal angiosperm, magnoliids, basal eudicot, monocot, and many major groups within eudicot [10,17,20– 25]. It is hard to distinguish whether class I or class II subfamily is the first to appear in plant kingdom, because the genome of liverwort *Marchantia polymorpha* contains the members belonging to both of TCP transcription factors constitute a plant-specific protein family which is conserved in plant kingdom. TCP homologs are identified from diverse plant species [7]. It is proved that TCP proteins are existed in the early land plants during evolutionary history [17–20]. However, it is still in dispute whether they are present in pluricellular green algae [17,20]. The TCP protein family is significantly expanded in angiosperm species by gene or whole-genome duplication independently in basal angiosperm, magnoliids, basal eudicot, monocot, and many major groups within eudicot [10,17,20–25]. It is hard to distinguish whether class I or class II subfamily is the first to appear in plant kingdom, because the genome of liverwort *Marchantia polymorpha* contains the members belonging to both of the

angiosperm species and to evolve independently in basal eudicot groups and monocot species

the two families [19,26]. As for class II TCPs, the CIN-like TCP subgroup is predicted to be more

two families [19,26]. As for class II TCPs, the CIN-like TCP subgroup is predicted to be more ancestral than the CYC/TB1-like TCPs, since the class II TCPs all belong to CIN-like TCP subgroup in the non-vascular plants [18,19,26,27]. The CYC/TB1-like TCP group is proposed to originate in angiosperm species and to evolve independently in basal eudicot groups and monocot species [23,24,28–31].

TCP family transcription factors governs various key developmental processes during the life cycle of plants. TCPs regulate seed germination, leaf development, outgrowth of shoot branches, flowering, flower development, silique and ovule development, photomorphogenesis, thermomorphogenesis, circadian rhythms, defense responses and senescence [11,32–53]. The tight regulation of TCPs is very important for plant development and survival. Plants evolve many ways to tightly regulate TCP activity. The aim of this review is to give a comprehensive overview on current knowledge relevant to the roles of CIN-like TCPs in different species and the fine regulation of CIN-like TCP by external stimuli, miRNA and other proteins. To understand the detailed functions of TCPs in plants, the downstream targets regulated by TCPs, the regulation of CYC/TB1-like TCPs, please refer to the excellent recent reviews [39,54,55].

#### **2. The Functions of CIN-Like TCP Transcription Factors in Di**ff**erent Species**

One of the most prominent roles of CIN-like TCP transcription factors is that they play a conserved and central role in control of leaf flatness, size, shape and complexity. The loss of *CIN* function in snapdragon *cin* mutant disrupted leaf flatness and forms defective simple leaves with larger size and wavy margins [11,12,56]. In *Arabidopsis*, CIN-like TCPs have highly redundant and additive roles in regulating the morphogenesis of simple leaves (Figure 2). The *Arabidopsis tcp* single mutants produced leaves with no obvious differences from wild-type control. However, disruption of *TCP4* and *TCP10* had already led to larger and curled leaves. The high-order multiple *CIN*-like *tcp* mutants caused even severer leaf curvature and wavier leaf margins in a dose-dependent manner [53,57–59], indicating that the activity of CIN-like TCPs is pivotal for shaping leaf forms. The *CIN*-like *TCP* homolog in turnip (*Brassica rapa*), *BrrTCP2*, has conserved function in control of leaf size and morphology. Overexpression of *BrrTCP2* reduced the leaf size of wild-type *Arabidopsis* and restored the leaf morphology of the *Arabidopsis* multiple mutant *tcp2 tcp4 tcp10* [60]. In the regulation of leaf morphology, CIN-like TCPs repress the activity of leaf marginal meristem which determines leaf serrations in simple leaves or complexity of compound leaves in different plants. In lettuce (*Lactuca sativa*), the Empire type cultivars have more serrated leaves than the Salinas type cultivars. The molecular base is that Empire type cultivars carry a retrotransposable element inserted in the upstream of *LsTCP4* gene, causing lower expression level of *LsTCP4* than that in the Salina type cultivars. The downregulation of *LsTCP4* by the insertion led to the severer leaf serration in Empire type cultivars [61]. However, differential expression analysis between broad- and curly-leaved plants of *Cichorium endivia*, a close relative of *L. sativa* that also displayed wavy or serrated leaves, did not identify *TCP4-like* homologous genes as differentially expressed in leaves with different morphologies, and the two transcripts were abundant in both leaf types [62]. Tomato forms compound leaves regulated by *LACEOLATE* (*LA*) homologous to *CIN-*like *TCPs*. Downregulation of *LA* generated more and larger leaflets, causing super-compound leaves. On the contrary, overexpression of *LA* resulted in the compound leaves turning into simple leaves [63–65]. *CpTCP1* in cyclamen (*Cyclamen persicum*) is a homolog of *CIN-*like *TCPs*. Disruption of TCP function by a dominant repressor in which the ethylene-responsive element binding factor-associated amphiphilic repression (EAR) repression domain (SRDX) was fused to CpTCP1 caused irregular protrusions of acicular and branched shapes in the leaf margins [66]. CIN-like TCPs also regulate the leafy head of Chinese cabbage (*Brassica rapa*). Altering the spatio-temporal expression patterns of *BrpTCP4* led to a cylindrical head shape from a round one [67]. Furthermore, the genetic manipulation of CIN-like TCP activity resulted in different sizes and shapes of leaves in both simple and compound leaves [64,68]. These findings indicate that CIN-like TCPs are central regulators of leaf morphology and that the tight control of the spatio-temporal TCP activity is fundamental in determining diverse leaves in different species.

CIN-like TCPs also modulate the development of organs homologous to leaves such as petals. The *Arabidopsis* single mutant *tcp5* produces wider petals than the wild-type control [69]. Moreover, the *35S:miR-3TCP* transgenic plants in which an artificial miRNA targeting to *TCP5*, *TCP13*, and *TCP17* was expressed to knock down the three genes generate petals with even increased width from tip to base [69]. Besides *TCP5-*like *CIN*-*TCPs* which was identified to determine petal size, the other five *CIN*-like *TCP* genes targeted by miRNA319 also played vital roles in control of petal growth. The mutant carrying a loss-of-function mutation in *miR319a* (named as *MiR319a129*) exhibited narrow petals and sterile anthers, indicating that CIN-like TCPs not only inhibit the growth of petal [70], but also play an essential role in plant fertility. The overexpression of the miR319-resistant form of *TCP4* by a petal-specific promoter rescued the narrow petals in *MiR319a<sup>129</sup>* mutant [70,71]. CIN-like TCPs also modify the morphology of petals besides petal sizes. Expression of a dominant repressor in which TCP3 was fused to an EAR motif to disrupt the function of TCPs resulted in curled petals in *Arabidopsis*. Expression of other CIN-like TCP chimeric repressors also caused curled petals [72]. The function of CIN-like TCPs in regulating petal development is conserved among different species. For examples, the introduction of chimeric repressors of *Arabidopsis* CIN-like TCPs in *Chrysanthemum morifolium* or *Ipomoea nil* also led to similar wavy and serrated petals [73,74]. Suppression of TCP functions by expression of chimeric repressors of CpTCP1 homologous to *Arabidopsis* TCP3 in *C. persicum* caused ruffled petals [66].

At the cellular level, CIN-like TCPs regulate cell proliferation, cell elongation or expansion and cell differentiation. During leaf and petal development, CIN-like TCPs inhibit cell proliferation and promote cell differentiation. Disruption of CIN-like TCPs prolong the leaf cell proliferation in the leaf blade with more rapid growth in the margin than in the center of blade, leading to the increased number of pavement cells and wavy margins [11,12,35,58,59,75]. As specialized epidermal cells, trichomes are also regulated by CIN-like TCP transcription factors. CIN-like TCPs suppress the trichome differentiation and subsequent trichome branching. The numbers of trichomes and trichome branches were both significantly increased in *jaw-D* and *tcp2 tcp4 tcp10* mutants, but were decreased in *TCP4* overexpression lines [76]. The function of CIN-like TCPs is also conserved in the regulation of trichome formation. Overexpression of *miR319a* in *Populus tomentosa* resulted in higher density of trichomes on the leaf surface when compared with that of wild-type control. When the functions of CIN-like TCPs were enhanced by inhibiting the roles of miR319, the number of trichomes was largely decreased [77]. Cotton fibers are specific trichome types on the seed epidermis. The constitutive overexpression of *GhTCP4* homologous to *Arabidopsis* TCP4 in upland cotton (*Gossypium hirsutum*) repressed the elongation of cotton fiber [78]. However, CIN-like TCPs positively regulate hypocotyl cell elongation in *Arabidopsis*. Induction of CIN-like TCPs using mTCP4-GR in which TCP4 fusion with rat glucocorticoid receptor (GR) by dexamethasone (DEX) treatment in transgenic lines significantly increased the length of hypocotyl cells (Figure 2) [79]. Overexpression of *TCP5-*like *CIN-TCPs* led to the significant increase of hypocotyl under shade, high temperature or under normal growth conditions (Figure 2) [14]. In consistence with the results, the *tcp5 tcp13 tcp17* triple mutant displayed short hypocotyls [14]. These findings demonstrate that CIN-like TCPs control cell proliferation, elongation and differentiation in a specific cell type-dependent manner at different context.

CIN-like TCPs are reported to be essential for regulating other biological processes. For examples, CIN-like TCPs facilitate the transition from vegetative to reproductive growth. The flowering time of *cin-*like *tcp* multiple mutants was significantly postponed, while overexpression of *TCP4* led to early flowering in *Arabidopsis* [80]. The tomato *LA* gene belonging to *CIN-*like *TCP* group controls flowering as well [81]. In addition, CIN-like TCPs participate in developmental plasticity in response to biotic stresses in *Arabidopsis* and rice. CIN-like TCPs are also implicated in the typical morphological alterations caused by infection of phytopathogens such as phytoplasmas in *Arabidopsis* [43–45]. Rice ragged stunt virus (RRSV) downregulated rice *TCP21* belonging to miR319-targeted CIN-like TCPs by up-regulating the expression of *miR319* gene. Overexpression of *TCP21* increased the rice resistance to RRSV [82].

#### **3. Light Regulates CIN-Like TCP Transcription Factors**

Light is a critical environmental stimulus affecting plant development and growth including cotyledon opening, hypocotyl elongation and flowering [83–86]. When seeds germinate in dark under soil and then the seedlings grow out with exposure to light in nature, plants undergo an important morphological change from skotomorphogenesis to photomorphogenesis including cotyledon opening and inhibition of hypocotyl elongation [83,87,88]. The bHLH transcription factors PHYTOCHROME-INTERACTING FACTORs (PIFs) including PIF3 are central regulators in promoting skotomorphogenesis by suppressing cotyledon opening and the elongation of hypocotyl [89,90]. However, the molecular mechanisms of light-induced cotyledon opening are not well-known. Recently, CIN-like TCPs have been identified to participate in controlling light-induced cotyledon opening during photomorphogenesis (Figure 2). Interestingly, *CIN*-like *TCP* genes including *TCP3*, *TCP4* and *TCP10* are predominantly expressed in cotyledons under both light and dark growth conditions [13]. Why do CIN-like TCPs promote cotyledon opening in the light but not affect cotyledon closing in dark? Chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) analyses showed that TCP4 directly bind to the promoter regions of *SMALL AUXIN UPREGULATED RNA* (*SAUR*) genes including *SAUR16* and *SAUR50*. The promoter regions of the *SAUR* genes are also directly targeted by PHYTOCHROME INTERACTING FACTOR3 (PIF3), a key component inhibiting cotyledon opening. The molecular mechanism is that the accumulated PIF3 in the dark represses the transactivation activity of TCP4 possibly by competing the binding to the promoter regions of *SAUR* genes with TCP4 in the dark, while in the light PIFs are rapidly degraded and causing more TCP4 proteins to bind to the promoters of the *SAUR* genes and to upregulate their expression to promote cotyledon opening (Figure 2) [86]. However, PIF3 does not interact with TCP4 in this process. The exact mechanism by which PIF3 inhibits TCP4 binding to the promoter regions of *SAUR* genes is still an open question.

In addition to controlling the light-regulated cotyledon opening in plant photomorphogenesis, CIN-like TCPs also participate in the regulation of light-regulated hypocotyl elongation under shade. The shade avoidance syndrome (SAS) of plants caused by neighboring shade or low ratio of red light to far red light (R:FR) includes long hypocotyl, elongated leaf petiole, reduced shoot branches and early flowering [91]. It is known that shade or low R:FR upregulates the expression level of *BRC1* or *TB1* belonging to *CYC*/*TB1*-like *TCP* subgroup [37], while recently TCP5-like CIN-TCPs has been reported to regulate the rapid growth of hypocotyl in response to shade (Figure 2) [92]. The hypocotyl elongation of the triple mutant *tcp5 tcp13 tcp17* was insensitive to shade, while overexpression of *TCP17* led to longer hypocotyls under shade or white light. TCP17 is an unstable protein which is stabilized by shade. When plants were transferred from shade to white light, TCP17 was degraded and the degradation were inhibited by treatment with the 26S proteasome inhibitor MG132 [92]. This result indicates that white light promotes the degradation of TCP17 via the 26S proteasome, while shade inhibits the process (Figure 2). Interestingly, the transcriptional level of *TCP17* was rapidly downregulated by shade in reverse, indicating accumulation of TCP17 under shade is dependent on the post-transcriptional regulation [92]. It will be very interesting to identify the E3 ligase mediating the degradation of TCP17 under white light and the molecular mechanisms of suppression of the TCP17 degradation machinery by shade.

#### **4. High Temperature Regulates CIN-Like TCP Transcription Factors**

Ambient temperature is one of the most important environmental factors governing plant behavior. Plants adopt a series of morphological changes called thermomorphogenesis in adaptation to high temperature [93,94]. Thermomorphogenesis includes leaf hyponastic growth, petiole elongation and hypocotyl elongation [93]. TCP5-like CIN-TCPs have recently been identified to act as key factors in positively regulating plant thermomorphogenesis. High temperature not only induces the expression of *TCP5, TCP13* and *TCP17* genes at the transcriptional level, but also stabilizes the protein of TCP5-like CIN-TCPs at the post-transcriptional level in *Arabidopsis* (Figure 2) [14,95].

Interestingly, high temperature treatment regulates both the expression level and the expression pattern of *TCP5*. When TCP5pro-GUS transgenic lines in which *GUS* reporter gene was driven by *TCP5* promoter was treated under high temperature, the GUS staining was strengthened in the hypocotyls and cotyledons, and at the same time was shifted from the leaf blades to petioles, in consistence with the leaf trait of thermomorphogenesis with elongated petioles and reduced areas of blades [14]. High temperature also up-regulates the expression of *PIF4* which is the first key factor identified in control of plant thermomorphogenesis [96,97]. TCP5 protein not only directly bound to the promoter region of *PIF4* gene to increase its expression level [14], but also interacted with PIF4 at the protein level [14]. Moreover, TCP17 protein interacted with the blue light receptor CRYTOCHROME1 (CRY1) at lower temperature to block the activity of TCP17. High ambient temperature increased the protein stability of TCP17 and led to the release of TCP17 from TCP17-CRY1 complex, promoting the interactions between TCP17 and PIF4 [93]. The interactions between PIF4 with TCP5 or TCP17 synergistically promoted the expression of a lot of common downstream genes including *PRE1* and *YUC8*, thus enhancing plant thermomorphogenesis (Figure 2) [14,95]. Accordingly, overexpression of *CIN-like TCP5* gene led to constitutive thermomorphogenesis, while the hypocotyls and petioles of *tcp5 tcp13 tcp17* were shorter than that of wild-type control under normal temperature or high temperature [14,95]. It is worth mentioning that although PIF4 is homologous to PIF3 which is a key regulator in photomorphogenesis [86], they use different mechanisms to regulate the activity of CIN-like TCPs. PIF3 do not interact with TCP4, but inhibits the binding activity of TCP4 to the promoter of their downstream genes in an unknown way under dark [86]. Adversely, PIF4 interacts with TCP5-like CIN-TCPs and obviously strengthened their transactivation activity in activating the downstream genes [14,95]. These results demonstrate that high temperature regulates the function of TCP5-like CIN-TCPs which positively regulate plant thermomorphogenesis by a different mechanism underlying the regulation of cotyledon opening by TCP4 in *Arabidopsis*. However, the transcription factors and E3 ligases that are responsible for regulating the expression of *TCP5*-like *CIN-TCPs* and the stability of their products under different ambient temperatures need to be further identified.

#### **5. Phytoplasmas Regulate CIN-Like TCP Transcription Factors**

Phytoplasmas are phytopathogens transmitted by insects and infect a wide range of plant species, causing great economic losses in agriculture [47,98]. Like the most pathogens, phytoplasmas produce effectors to alter the host-pathogen interface in facilitating their growth during infection [47]. The effectors cause some typical changes of plant morphology including overgrowth of lateral branches, altered leaf shape and sterile flowers [98]. The aster yellows phytoplasma witches' broom (AY-WB) strain infect a wide range of dicot and monocot species [47,48]. The secreted AY-WB protein 11 (SAP11) is a virulence nuclear effector with a nuclear localization signal at its N-terminus. Overexpression of *SAP11* in *Arabidopsis* produced serrate and wavy leaves almost identical to those of *jaw-D* and the multiple *cin*-like *tcp* mutants [48]. SAP11 interacts with CIN-like TCP proteins [48], leading to the TCP degradation which is not inhibited by the 26S proteasome inhibitor epoxomicin or protease inhibitor cocktail (Figure 2). This indicates that the SAP11-mediating TCP protein degradation is not through ubiquitin-26S proteasome pathway [48]. Because CIN-like TCPs positively regulate the expression of *LOX2* gene by directly binding to its promoter [53], the overexpression of *SAP11* caused the downregulation of the *LOX2* gene and reduced the production of jasmonic acid (JA) in both *Arabidopsis* and tobacco (*Nicotiana benthamiana*), facilitating the infection of phytoplasmas [47,49]. The SAP11 protein homologs in different phytoplasmas strains displayed the varied abilities in control of the stability of CIN-like TCPs. These strains include AY-WB, onion yellow strain M (OY-M), peanut pupurea witches' broom (PnWB), *Candidatus* phytoplasmas mali (CaPM) [49]. When SAP11 homologs were co-expressed with CIN-like TCPs in tobacco, the abundance of TCP proteins were measured to determine the abilities of SAP11 proteins in mediating TCP degradation [49]. The results showed that SAP11AYWB had the strongest ability to mediate the degradation of TCP2, TCP3, TCP4, TCP5, TCP10 and TCP24, while SAP11CaPM only mediated the degradation of TCP2 and TCP10 with lower ability

than SAP11AYWB. SAP11PnWB and SAP11OYM only exhibited a weak ability to destabilize TCP2 [45]. The SAP11 homolog from the Maize Bushy Stunt Phytoplasmas (SAPMBSP) have been shown to only interact with CYC/TB1-like TCPs, but not any members of CIN-like TCPs in maize [46]. Accordingly, the MBSP-infected maize showed overgrowth of tillers controlled by the CYC/TB1-like TCPs, but not had any effects on the morphology of leaves [46]. Similarly, SWP1 which is a SAP11-like phytoplasmas effector from wheat blue dwarf phytoplasma interacted with BRC1 and mediate the degradation of BRC1 when *SWP1* was overexpressed in *Arabidopsis* [50]. These findings indicate that the effector SAP11 proteins from different phytoplasmas strains have different specificity in promoting the degradation of TCPs. As the SAP11 protein have no protease activity, the mechanisms underlying SAP11-mediated TCP degradation remains to be further discovered [48].

#### **6. miRNAs Regulate CIN-Like TCP Transcription Factors**

miRNAs are small RNAs that recognize targeting mRNA via base pairing to the highly complementary binding sites and suppress the stability and translation of mRNAs [99,100]. A subset of *CIN-*like *TCP* genes contains a miR319-targeting sequence at the 3'-terminus of transcripts in almost all angiosperm groups [13,63]. The Arabidopsis *jagged and wavy*-*Dominant* (*jaw-D*) mutant was first identified from a collection of activation tagging mutants by forward genetics [13]. The mutant *jaw-D* displayed a predominant phenotype with the serrated and curved leaves [13,101]. Further analysis showed that T-DNA with four cauliflower mosaic virus (CaMV) 35S enhancer was inserted in neighboring region of MIRNA gene *MIR319a* in *jaw-D*. The expression of *miR319a* was activated and the target *CIN*-like *TCP* genes including *TCP2*, *TCP3*, *TCP4*, *TCP10* and *TCP24* were significantly downregulated in the mutant, suggesting that the transcript abundance of the corresponding *TCP* genes was regulated by miR319a (Figure 3) [13]. The overexpression of *miR319* also caused epinastic cotyledons, more trichomes, defective secondary cell wall biosynthesis and venation patterning, a modest delay in flowering, crinkled petals, short stamen, reduced male fertility and crinkled fruits by downregulating *CIN*-like *TCP* genes [42,70,102–104]. *Arabidopsis* genome contains three *MIR319* genes including *MIR319a*, *MIR319b*, and *MIR319c* which have highly redundant function in control of the abundance of *CIN*-like *TCP* transcripts [70]. However, the three *MIR319* genes also showed largely non-overlapping expression patterns revealed by GUS reporter analysis in plants, suggesting that they may have distinct roles in control of *TCP* abundance in a temporal and spatial manner during plant development [70]. During leaf development, the *MIR319a* gene is only expressed at the stipules, which is completely complementary to the expression pattern of *MIR319c* that the highest expression level is detected at the basal region of leaf primordia and young leaves, indicating the functional divergences between the two genes. *MIR319b* is only expressed in the sepal and stamen abscission zones of inflorescences at the reproductive stage [70]. *MIR319a* and *MIR319c* have partially spatiotemporal overlapping expression patterns during early inflorescence development [70]. Though the GUS activity for promoter analysis of *MIR319b* was not detected in leaves, the *mir319b* single mutant moderately reduced the size of leaf serrations, and *mir319a*/*b* double mutant almost entirely suppressed serration formation [59], suggesting that *MIR319b* is essential for leaf development with a possible low expression level in leaves.

miR319 is a conserved and ancient plant miRNA family and plays important roles in plant morphological adaptation to environmental conditions by targeting *TCP* for degradation. The miR319 and miR159 share highly similarity in mature miRNA sequence, secondary structure, conservation pattern and biogenesis in *Arabidopsis*. miR319 and miR159 are proposed to evolve from a common ancestor in land plants [105]. miR159 did not induce the cleavage of *TCP* mRNAs due to the specificity of sequences, while miR319 mediated the cleavage of *MYB33* and *MYB65* mRNA, which are pivotal targets of miR159 [101,105]. Two miR319 copies were identified in the genome of *M. polymorpha,* which also contains two *MpTCP* genes [106]. However, the two *MpTCP* genes have no possible miR319-targeting site and one target of miR319 was identified as *MpMYB33* [106,107]. In *Physcomitrella*

and *Selaginella*, the *TCP* genes also have no miR319-targeting sites [108–110], indicating that miR319 regulation of *CIN*-like *TCP* possibly evolve after the divergence of lycophytes and euphyllophytes. *Int. J. Mol. Sci.* **2019**, *20*, x FOR PEER REVIEW 9 of 17

**Figure 3.** An overview of the regulation mechanisms of CIN-like TCP transcription factors during leaf development. The external stimuli and internal factors are summarized at the left column. The schematic diagram includes the regulation mechanisms of CIN-like TCPs at the transcriptional level, at the post-transcriptional level, and at the protein level. The arrows directly pointing on the double helix symbols indicate transcriptional regulations. The arrows pointing to the proteins indicate the regulations of protein stabilities or antagonistic functions. The proteins related with the "26S" symbols indicate protein degradation through the ubiquitin-26S proteasome pathway. The blue arrows represent the positive regulation, and the red arrows with dash-headed ends indicate the negative regulation. The green arrows and red dash-headed ends at the double-helix icons indicates the activation and repression of gene expression, respectively. All the unknown factors are indicated with question marks. R:FR, red light: far red light ratio; YUCs, YUCCAs; LOX2, LIPOXYGENASE 2; SAP11, SECRETED AY-WB PROTEIN 11; ARR16, ARABIDOPSIS RESPONSE REGULATOR 16; IAA3, INDOLE-3-ACETIC ACID INDUCIBLE 3; BRM, BRAHMA; TIE1, TCP INTERACTOR CONTAINING EAR MOTIF PROTEIN 1; TEAR1, TIE1-ASSOCIATED RING-TYPE E3 LIGASE 1. **Figure 3.** An overview of the regulation mechanisms of CIN-like TCP transcription factors during leaf development. The external stimuli and internal factors are summarized at the left column. The schematic diagram includes the regulation mechanisms of CIN-like TCPs at the transcriptional level, at the post-transcriptional level, and at the protein level. The arrows directly pointing on the double helix symbols indicate transcriptional regulations. The arrows pointing to the proteins indicate the regulations of protein stabilities or antagonistic functions. The proteins related with the "26S" symbols indicate protein degradation through the ubiquitin-26S proteasome pathway. The blue arrows represent the positive regulation, and the red arrows with dash-headed ends indicate the negative regulation. The green arrows and red dash-headed ends at the double-helix icons indicates the activation and repression of gene expression, respectively. All the unknown factors are indicated with question marks. R:FR, red light: far red light ratio; YUCs, YUCCAs; LOX2, LIPOXYGENASE 2; SAP11, SECRETED AY-WB PROTEIN 11; ARR16, ARABIDOPSIS RESPONSE REGULATOR 16; IAA3, INDOLE-3-ACETIC ACID INDUCIBLE 3; BRM, BRAHMA; TIE1, TCP INTERACTOR CONTAINING EAR MOTIF PROTEIN 1; TEAR1, TIE1-ASSOCIATED RING-TYPE E3 LIGASE 1.

#### miR319 is a conserved and ancient plant miRNA family and plays important roles in plant **7. Chromatin Remodeling Complexes Regulate the Activity of CIN-Like TCPs**

morphological adaptation to environmental conditions by targeting *TCP* for degradation. The miR319 and miR159 share highly similarity in mature miRNA sequence, secondary structure, conservation pattern and biogenesis in *Arabidopsis*. miR319 and miR159 are proposed to evolve from a common ancestor in land plants [105]. miR159 did not induce the cleavage of *TCP* mRNAs due to the specificity of sequences, while miR319 mediated the cleavage of *MYB33* and *MYB65* mRNA, which are pivotal targets of miR159 [101,105]. Two miR319 copies were identified in the genome of *M. polymorpha,* which also contains two *MpTCP* genes [106]. However, the two *MpTCP* genes have no possible miR319-targeting site and one target of miR319 was identified as *MpMYB33* [106,107]. In *Physcomitrella* and *Selaginella*, the *TCP* genes also have no miR319-targeting sites [108–110], indicating that miR319 regulation of *CIN*-like *TCP* possibly evolve after the divergence of lycophytes and euphyllophytes. **7. Chromatin Remodeling Complexes Regulate the Activity of CIN-Like TCPs** The activity of CIN-like TCPs is controlled by chromatin remodeling complexes including The activity of CIN-like TCPs is controlled by chromatin remodeling complexes including SWITCH/SUCROSE NONFERMENTING (SWI/SNF) complex and TCP INTERACTOR CONTAINING EAR MOTIF PROTEIN 1 (TIE1)-TOPLESS (TPL)/TOPLESS-RELATED (TPR) complex at the protein level (Figure 3). SWI/SNF complexes use ATPase to provide the energy in deciding the nucleosome position conformation and thus determining the accessibility of chromatin [111]. *BRAHMA* (*BRM*) encodes a SWI/SNF ATPase in *Arabidopsis* [112–114]. The hypomorphic mutations in *BRM* suppressed the phenotypes including fewer trichomes and smooth margins in *TCP4* overexpression lines [114]. And the hypomorphic *brm* mutants produced curled leaves and delayed leaf maturation resembling the multiple *cin*-like *tcp* mutants, indicating that BRM promotes the activity of CIN-like TCPs (Figure 3) [114]. BRM interacts with TCP4 and together bind to the promoter region of type A *ARABIDOPSIS RESPONSE REGULATOR* (*ARR*) gene *ARR16* to promote the expression of *ARR16* (Figure 3) [115]. The modulation of CIN-like TCP activity by BRM provides a fine regulation of leaf sensitivity to the phytohormone cytokinin (CK) during leaf development.

SWITCH/SUCROSE NONFERMENTING (SWI/SNF) complex and TCP INTERACTOR

Compared with the positive regulation of CIN-like TCP activity mediated by BRM, TIE1-TPL/TPR complexes repressed CIN-like TCP activity by recruiting histone deacetylases (HDA) (Figure 3) [116]. TIE1 was identified to regulate TCP activity by analyzing a gain-of-function mutant *tie1-D* obtained by screening a collection of activation tagging mutants for leaf-defective ones. Overexpression of *TIE1* in *tie1-D* or in transgenic plants using CaMV 35S promoter to drive *TIE1* all led to curled and serrated leaves that are observed in the multiple *cin*-like *tcp* mutants [116]. *TIE1* encodes a transcriptional repressor containing a typical EAR motif at the C-terminal end. Indeed, TIE1 has transcriptional repression activity and directly interacts with the corepressor TPL/TPRs through EAR motif. TIE1 also interacts with CIN-like TCPs via the N-terminal domain. Consequently, TIE1 suppresses the activity of CIN-like TCPs by acting as a bridge connecting corepressor TPL/TPRs with CIN-like TCPs during leaf development (Figure 3) [116]. Interestingly, TIE1 also interacted with BRC1 belonging to CYC/TB1-like TCP group [40]. *TIE1* had overlapping expression pattern with *BRC1* in young axillary buds and overexpression of *TIE1* resulted in excessive branches, indicating that *TIE1* also represses the activity of BRC1 during shoot branching [40]. The function of *TIE1* is conserved in controlling shoot branching in cotton (*Gossypium hirsutum*) [117]. GhTIE1 interacted with CYC subclade proteins GhBRC1, GhBRC2, and GhTCP13 *in vivo.* Silencing of *GhTIE1* in cotton seriously decreased shoot branching [117]. A similar mechanism in suppression of CIN-like TCP activity is mediated by SPOROCYTELESS/NOZZLE (SPL/NZZ) during ovule development [118]. SPL/NZZ is a key regulator responsible for promoting the differentiation of megasporocytes. No megasporocytes were formed in the ovules of *spl*/*nzz* mutants. SPL/NZZ also contains a typical EAR repressor motif at the C-terminal domain and has the transcriptional repression activity. SPL/NZZ uses C-terminal EAR motif to interact with TPL/TPRs and uses its N-terminal domain to interact with CIN-like TCPs [118]. Overexpression of *SPL* in T-DNA activation tagging mutant *spl-D* caused the defective ovule arrangement in ovaries resembling to that of the multiple *cin*-like *tcp* mutants. Consistently, overexpression of the *CIN*-like *TCPs* led to no megasporocytes resembling the phenotype of *spl* loss-of-function mutants [118]. These results indicate that SPL inhibits the activity of CIN-like TCPs in a way similar to TIE1 by connecting TPL/TPR corepressors with CIN-like TCPs.

The regulation of CIN-like TCP activity by TIE1, SPL or BRM during leaf or ovule development is parallel to the regulation of key regulators in auxin signaling. The EAR motif-containing AUXIN (AUX)/INDOLE-3-ACETIC ACID (IAA) repressors mediate auxin signaling by recruiting TPL/TPRs to suppress the activity of AUXIN RESPONSE FACTORS (ARFs) [119–121]. Auxin triggers the degradation of AUX/IAA via 26S proteasome, the released ARFs such as MONOPTEROS (MP) bind to SWI/SNF chromatin remodeling ATPases BRM to promote the accessibility of chromatin and the expression of downstream genes. Interestingly, TIE1 is also an unstable protein as AUX/IAA repressors and the degradation of TIE1 is mediated by an E3 ligase TIE1-ASSOCIATED RING-TYPE E3 LIGASE1 (TEAR1) (Figure 3) [122]. Disruption of *TEAR1* leads to serrated and curled leaves similar to that observed in the multiple *cin*-like *tcp* mutants and *tie1-D* [122]. These findings suggest that *TEAR1* indirectly regulates the activity of CIN-like TCPs by switching the interactors of CIN-like TCPs from TIE1 to BRM (Figure 3), thus changing the chromatin state to control leaf development. However, the signals triggering the TIE1 degradation to release the suppression of CIN-like TCPs by TEAR1 need to be further identified.

#### **8. Concluding Remarks and Perspectives**

CIN-like TCPs are key transcription factors essential for plant growth and development in response to environmental cues and internal signals. The temporal and spatial activity of CIN-like TCPs determines cell proliferation, expansion and differentiation of cells in different organs in shaping plant morphology at various developmental stages. Consequently, the fine-tuning of CIN-like TCP activity is critical for plant developmental plasticity. At the transcriptional level, *CIN*-like *TCPs* are dynamically and specifically expressed in organs and also are induced by environmental signals including light and temperature [14,92,95]. However, the upstream regulation which determines the

dynamic expression pattern and induction of *CIN*-like *TCP* genes are insufficient. The transcriptional repressor RABBIT EARS (RBE) has been reported to decreased the expression of *TCP4*, *TCP5*, *TCP13* and *TCP17* in promoting petal growth and *TCP4* and *TCP5* are possibly direct targets of RBE [69,71]. More studies on detailed analysis of the promoter regions of *CIN*-like *TCPs* are necessary for elucidating other upstream regulators, especially the direct regulators. The truncated promoters can be used to drive reporters in determining the minimal regions required for the expression patterns of *CIN*-like *TCPs*. Transcription factors directly interacting with the promoters of *CIN*-like *TCPs* could be identified by yeast-one-hybrid screening.

CIN-like TCPs are central for regulating biosynthesis and signaling of different phytohormones including auxin, JA and brassinosteroid (BR) [53,123,124]. However, little is known about how phytohormones regulate CIN-like TCPs. It has been shown that auxin, gibberellin (GA), strigolactone (SL) and cytokinin (CK) regulate *BRC1* belonging to*CYC*/*TB1*-like *TCP* group of class II *TCPs*[36,125,126]. The decreased auxin level by overexpression of *IAA CARBOXYL METHYLTRANSFERASE1* (*IAMT1*) which converted IAA to methyl-IAA ester led to curly leaves and reduced the expression level of some *CIN*-like *TCPs* [127], indicating that auxin positively regulates *CIN*-like *TCPs* at the transcriptional level. Further studies are needed to determine whether other plant hormones and environmental signals except light and temperature could possibly regulate CIN-like TCPs and how these signals could be integrated to control the activity of CIN-like TCPs.

At the post-transcriptional level, the miR319-*TCP* regulation module is conserved and widely studied in several plant species [13,63]. Could the other miRNAs targeting *CIN*-like *TCPs* exist in different plant species? which are those transcription factors deciding the expression level and pattern of *MIR319* genes? These questions are still open.

At the protein level, we know little about the degradation mechanisms of CIN-like TCPs mediated by 26S proteasome or other protein degradation pathways. The regulation mechanisms of CIN-like TCPs by class I TCP transcription factors and other interacting proteins are still largely unknown. It is still a challenge to thoroughly understand the shaping of plant morphology controlled by the CIN-like TCP-centered network under various environmental and developmental conditions in *Arabidopsis* and the other plant species.

**Author Contributions:** J.L. and G.Q. wrote the manuscript; J.L. and G.Q. have read and agreed to publish the version of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the National Science Fund for Distinguished Young Scholars of China (Grant No. 31725005) and the Science Fund for the Creative Research Groups of the National Natural Science Foundation of China (Grant No. 31621001).

**Acknowledgments:** Because of space constraints, we apologize to all colleagues whose work and publications have not been mentioned and cited.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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