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Article

Identification of TCP13 as an Upstream Regulator of ATHB12 during Leaf Development

by
Yoon-Sun Hur
1,
Jiyoung Kim
1,
Sunghan Kim
1,
Ora Son
1,
Woo-Young Kim
2,
Gyung-Tae Kim
3,
Masaru Ohme-Takagi
4,5 and
Choong-Ill Cheon
1,*
1
Department of Biological Science and Research Institute of Women’s Health, Sookmyung Women’s University, Seoul 04310, Korea
2
College of Pharmacy, Sookmyung Women’s University, Seoul 04310, Korea
3
Bioproduction Department of Molecular Biotechnology, Dong-A University, Busan 49315, Korea
4
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan
5
Institute for Environmental Science and Technology (IEST), Saitama University, Saitama 338-8570, Japan
*
Author to whom correspondence should be addressed.
Genes 2019, 10(9), 644; https://doi.org/10.3390/genes10090644
Submission received: 2 August 2019 / Revised: 20 August 2019 / Accepted: 21 August 2019 / Published: 26 August 2019
(This article belongs to the Section Plant Genetics and Genomics)
Browse Figures
Versions Notes

Abstract

:
Leaves grow by distinct phases controlled by gene regulatory networks including many transcription factors. Arabidopsis thaliana homeobox 12 (ATHB12) promotes leaf growth especially during the cell expansion phase. In this study, we identify TCP13, a member of the TCP transcription factor family, as an upstream inhibitor of ATHB12. Yeast one-hybrid screening using a 1.2-kb upstream region of ATHB12 resulted in the isolation of TCP13 as well as other transcription factors. Transgenic plants constitutively expressing TCP13 displays a significant reduction in leaf cell size especially during the cell expansion period, while repression of TCP13 and its paralogs (TCP5 and TCP17) result in enlarged leaf cells, indicating that TCP13 and its paralogs inhibit leaf development, mainly at the cell expansion phase. Its expression pattern during leaf expansion phase is opposite to ATHB12 expression. Consistently, the expression of ATHB12 and its downstream genes decreases when TCP13 was overexpressed, and increases when the expression of TCP13 and its paralogs is repressed. In chromatin immunoprecipitation assays using TCP13-GFP plants, a fragment of the ATHB12 upstream region that contains the consensus sequence for TCP binding is strongly enriched. Taken together, these findings indicate that TCP13 and its paralogs inhibit leaf growth by repressing ATHB12 expression.

1. Introduction

Leaves are plant organs essential for harvesting the light that provides energy for living organisms. The development of leaves involves complicated coordination of several factors including correct spatio-temporal transcriptional regulation of genes, hormonal control, and responses to environmental conditions [1,2,3,4,5]. After the formation of leaf primordia, which are groups of leaf founder cells on the flanks of shoot apical meristems, cell proliferation results in a relatively constant cell size. While cell division continues at the base of the leaf, cell expansion starts at the tip and moves to the base, forming the cell cycle arrest front [1,6,7]. At the same time, differentiation occurs to form specialized cells such as guard cells.
Homeobox genes affect plant development as well as the development of various animals [8,9]. Homeodomain-leucine zipper (HD-Zip) genes, a subset of homeobox genes with tightly linked leucine zipper motifs [10], are critical for plant development. They are classified into four distinct subfamilies (I–IV) of HD-Zips, many of which have been revealed to be critical for various aspects of plant development [11,12]. For example, some HD-Zip III proteins such as PHABULOSA/ATHB14, PHAVOLUTA/ATHB9 and REVOLUTA, regulate the formation of the adaxial domains of leaves [13,14]. Arabidopsis thaliana homeobox 2 (ATHB2) and ATHB4, HD-Zip II proteins, are induced by a low ratio of red to far-red light and contribute to the shade avoidance response, including hypocotyl elongation [15,16]. ATHB2 expression is controlled by basic helix–loop–helix (bHLH) phytochrome-interacting factors PIF4 and PIF5, as Kunihiro et al. [17] showed that PIF5 binds to some of the G-box-rich regions of the ATHB2 promoter. HD-Zip II proteins are also involved in the regulation of adaxial–abaxial patterning through repression of miR165/166 expression, together with HD-Zip III proteins [18]. ATHB12, an HD-Zip I gene, is primarily expressed in leaves and stems and is also inducible by osmotic stress and abscisic acid (ABA) [11,19,20]. During inflorescence stem development, ATHB12 represses the expression of gibberellin 20 oxidase 1 (GA20ox1), thus inhibiting stem elongation [21]. ATHB12 also acts as a regulator of leaf development, promoting cell expansion in leaves as well as elevated ploidy levels [7]. ATHB7 and ATHB12 have high identity of amino acids to each other, displaying overlapping functions including water stress responses [22,23]. How ATHB12 is controlled during leaf development is not known.
To explore the regulation of ATHB12 expression during leaf development, we use the yeast one-hybrid method to identify upstream regulators. One of candidate genes identified as binding to the promoter of ATHB12 in the assay, is TCP13, a member of TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors, the role of which in coordinating cell division and cell differentiation during leaf development has been well established [24,25,26]. Class I TCPs stimulate cell proliferation by promoting the expression of genes involved in cell division, while class II TCPs affect leaf differentiation rather than the mitotic cycle. Increased expression of TCP4, which belongs to the class II, results in reduced leaf size, indicating that CINCINNATA-like TCPs (CIN-TCPs) are negative regulators of leaf growth [27,28]. In addition, TCP4 acts directly on HAT2, an HD-Zip II transcription factor, to control leaf maturation [29]. TCP13 belongs to the class II type TCPs, but its exact function in the leaf development control has not been elucidated.
Our results in this study reveal that TCP13 bound to the ATHB12 promoter and negatively regulated ATHB12 expression. Its overexpression resulted in a reduction of leaf cell size, suggesting that it inhibits cell expansion during leaf growth.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

Arabidopsis thaliana seeds were surface-sterilized and grown on half-strength Murashige and Skoog (MS) medium supplemented with 1% sucrose and 0.8% phytoagar. Seeds were incubated at 4 °C for 2 days and transferred to a growth chamber at 22 °C under long-day condition with a light intensity of 50 μmol m−2 s−1.

2.2. Vector Construction and Plant Transformation

To generate transgenic plants overexpressing TCP13, full-length TCP13 was fused with GFP under the cauliflower mosaic virus (CaMV) 35S promoter. An artificial microRNA (amiR) against three TCPs (TCP13, TCP5 and TCP17) was made according to Efroni et al. [24]. To examine TCP13 expression, an upstream segment (2,969-bp) of TCP13 was amplified by PCR and inserted in front of GUS in pBI121. All the resulting constructs were introduced into Agrobacterium tumefaciens strain GV3101 and transgenic plants were obtained by the floral dip method [30].

2.3. Yeast One-Hybrid (Y1H) Screening and Yeast Two-Hybrid (Y2H) Assays

For yeast one-hybrid screening, a dual reporter consisting of the upstream region of ATHB12 in pHISi-1 (PATHB12::HIS3) and pLacZi (PATHB12::LacZ) was constructed and integrated into S. cerevisae strain YM4271 (MATa, ura3–52, his3-Δ200, ade2–101, ade5, lys2–801, leu2–3,112, trp1–901, tyr1–501, gal4Δ, gal80Δ, ade5::hisG). A yeast strain harboring PATHB12::HIS3 was transformed with a cDNA library of 1500 Arabidopsis transcription factors [31] and grown on SD/-His/-Leu agar medium containing 20 mM or 60 mM 3-amino-1,2,4-triazole (3-AT). To confirm that the isolated transcription factor binds to the upstream region of ATHB12 in yeast, a full-length cDNA of TCP13 was cloned into pGAD424 and the resulting plasmid was introduced into the yeast strain with PATHB12::HIS3. For Y2H assays, the Matchmaker two-hybrid system (Takara, Shiga, Japan) was used as previously reported [32].

2.4. Bimolecular Fluorescence Complementation (BiFC)

The coding regions of TCP13, TCP5, TCP17 and TCP7 were subcloned into p326-YFPC vector, and those of ATHB2, ATHB4, HAT3 and ATHB53 were inserted into p326-YFPN vector. Pairs of TCPs-YFPC and ATHBs-YFPN plasmids were co-introduced into Arabidopsis protoplasts by the PEG-method [33], and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence signals were observed after incubation for 16 h.

2.5. Microscopic Observation

Arabidopsis leaves were fixed in formaldehyde-acetic acid-alcohol (FAA) and cleared in chloral hydrate solution [7,34]. Cells of cleared tissues were observed by differential interference contrast (DIC) microscopy (Carl Zeiss, LSM700, Oberkochen, Germany). Photographs of cells at about a quarter from the bottom of the leaf, and halfway between the leaf margin and the mid-vein [35] were taken and used in measuring the cell areas. Leaves and cell sizes were measured with Image J software (http://rsb.info.nih.gov/ij).

2.6. Real-Time Quantitative PCR

RNA was extracted from leaves or whole seedlings using RNeasy plant mini kit (Qiagen, Germantown, MD, USA). cDNA was synthesized using the isolated RNA by M-MLV reverse transcriptase (Promega, Madison, WI, USA). Real-time quantitative PCR was performed with qPCRBIO SyGreen Blue Mix (PCR Biosystems, London, UK) using a LightCycler 96 (Roche, Mannheim, Germany). mRNA levels were normalized with UBQ5 and PP2A. Some of the gene-specific primers used in real-time PCR were reported previously [7] and others are listed in Table S1.

2.7. GUS Assay

GUS staining was performed as described previously [7]. Seedlings, floral organs and mature leaves were incubated in GUS staining solution containing 0.5 mg/mL 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc) and cleared through acetone and ethanol series. To examine the effect of TCP13 on ATHB12 expression, plasmids containing GFP, TCP13-GFP or ATHB2-GFP under the control of the CaMV 35S promoter were introduced into protoplasts from leaves of PATHB12::GUS plants, and proteins from the transfected protoplasts were prepared in GUS extraction buffer (50 mM sodium phosphate buffer, pH 7.0, 10 mM EDTA, pH 8.0, 0.1% SDS and 0.1% Triton X-100). GUS activity was measured using 1 mM 4-methylumbellyferyl-β-D-glucuronide (4-MUG) in GUS extraction buffer. GUS activity was normalized with protein concentrations measured by the Bradford assay.

2.8. Chromatin Immunoprecipitation (ChIP)-qPCR

Ten-day-old seedlings of P35S::GFP and P35S::TCP13-GFP were used for ChIP-qPCR. After crosslinking in 1% formaldehyde, sheared chromatin complexes were obtained by sonication (Bioruptor TOS-UCD-300; Cosmo Bio, Tokyo, Japan). Protein-DNA complexes were immunoprecipitated with GFP antibody (Abcam, Cambridge, MA, USA), and real-time PCR was performed using the primers listed in Table S1.

2.9. Luciferase Assay

The P35S::GFP or P35S::TCP13-GFP or P35S::ATHB2-GFP were used as effector plasmids, PATHB12::LUC or mutated PATHB12::LUC as reporter genes, and Renilla luciferase (P35S::RLUC) as internal control. Arabidopsis protoplasts were isolated and transfected by the polyethylene glycol (PEG)-method, as previously described [33]. Firefly luciferase (LUC) activity was normalized with Renilla luciferase (RLUC) activity.

3. Results

3.1. TCP13 Is an Upstream Regulator of ATHB12

Leaf growth involves complex regulation by many transcription factors at various stages of development. We showed previously that ATHB12-overexpressing plants had enlarged leaves with enlarged and endoreduplicated cells [7]. We therefore wished to know how ATHB12 expression is regulated. To identify a direct upstream regulator(s) of ATHB12 during leaf development, we used a yeast one-hybrid (Y1H) screen involving a cDNA library of 1500 Arabidopsis transcription factors [31]. A 1.2-kb upstream segment of ATHB12 was cloned into pHISi-1 and pLacZi vectors, and the resulting reporter plasmids were used to screen the cDNA library. Twelve genes were isolated from the screen and regarded as putative upstream regulators of ATHB12 (Table S2). We noted that three of them, TCP13, HB51 and NGA1, are known to be active in leaf development [24,36,37], and TCP13 was chosen first for this study because it is a member of the transcription factor family whose critical roles in leaf formation is well publicized [24,38]. First of all, the specific binding of TCP13 to ATHB12 promoter was confirmed by repeating the yeast one-hybrid assay with two different stringency levels of the 3-amino-1,2,4-triazole (3-AT) selection (Figure 1). TCP13 was fused to GAL4 AD (AD-TCP13), and GAL4 AD alone (AD) was used as negative control. In the presence of 20 mM 3-AT, autoactivation of the reporter construct alone was observed but this background was effectively eliminated with 60 mM 3-AT, showing the ATHB12 expression only in the presence of TCP13 in the assay (Figure 1B). This result strongly suggested that TCP13 is an upstream transcription factor controlling the ATHB12 expression probably via direct binding to its promoter region.

3.2. Alterations of TCP13 Level Affect Leaf Morphology

To understand the role of TCP13 in leaf development, we first analyzed its spatial and temporal expression using transgenic plants expressing a PTCP13::GUS construct composed of a 2969-bp upstream region of TCP13 and the GUS gene. TCP13 expression was detected in cotyledons, leaves, petals and siliques (Figure 2). Intriguingly, TCP13 expression was high in cotyledons, but almost undetectable in actively dividing L3 and L4 leaves and the early expanding L1 and L2 leaves of 10-day-old plants (Figure 2B). This was the exact opposite of ATHB12 expression during the expansion phase of leaf development; thus, in 11-day-old plants ATHB12 was not expressed in cotyledons but highly expressed in true L1 and L2 leaves in the early expansion stage (Figure 4B in Hur et al. [7]). TCP13 expression was also observed in the older leaves such as L1 and L2 of 16-day-old plants (Figure 2C). These observations suggested that TCP13 might inhibit the expression of ATHB12 in leaf development.
Subsequently, we examined the effects of TCP13 on leaf development using transgenic plants either overexpressing TCP13 under the cauliflower mosaic virus (CaMV) 35S promoter (TCP13-GFP) or expressing a construct down-regulating TCP13 and its homologs. For the latter purpose, since a T-DNA insertional mutant of TCP13 did not have a distinctive phenotype and in addition, it has been reported that the sequences of TCP5 and TCP17 are highly homologous to that of TCP13 [39], we made use of an artificial microRNA against TCP5, TCP13, and TCP17 under the control of CaMV 35S promoter (amiR-3TCP; Efroni et al. [24]). We observed that the TCP13 overexpressor had reduced rosette leaf laminas, whereas amiR-3TCP plants had enlarged rosette leaves (Figure 3A,B). Both the lengths and widths of fifth leaves of the TCP13 overexpressor were significantly reduced whereas the leaves of amiR-3TCP plants were longer and wider than normal. Evidently, TCP13 and its paralogs are involved in controlling leaf size.
To examine further the role of TCP13, we measured the sizes of first leaves (L1) at different phases of leaf growth: leaf primordia (6 days after sowing; 6 DAS), cell proliferation (8 DAS) and cell expansion (10 and 14 DAS) phases [6]. The TCP13 overexpressor had the most clear-cut effect in the cell expansion phases (10 and 14 DAS); the leaf areas of TCP13 overexpressor were about half of those of the wild type (0.56-fold at 10 DAS and 0.51-fold at 14 DAS) whereas the amiR-3TCP seedlings had leaves that were about 1.2-fold larger than those of the wild type at the same stages (Figure 3C). These results indicated that TCP13 and its paralogs affected leaf growth, possibly at the cell expansion phase of leaf development.

3.3. TCP13 Controls Leaf Growth Mainly by Repressing Cell Expansion

Leaf growth occurs through cell proliferation followed by cell expansion [6]. To determine whether the effect of TCP13 on leaf size is due to cell division or cell expansion, we measured the areas of the palisade cells of the TCP13 overexpressors and amiR-3TCP plants at different phases of leaf development using differential interference contrast (DIC) microscopy. The palisade cells of the TCP13 overexpressor were slightly smaller than those of the wild-type (0.86-fold) at 8 DAS, while those of the amiR-3TCP plants were slightly larger (Figure 4A,B). At 10 and 14 DAS, the TCP13 overexpressor had 0.72- and 0.66-fold reduced cells, respectively. Conversely, the amiR-3TCP seedlings had 1.5-fold enlarged palisade cells at 10 DAS, and 1.1-fold enlarged palisade cells at 14 DAS. The areas of the palisade cells of the transgenic plants were significantly different from wild type at all the stages of leaf development examined including 8 DAS. The effect of TCP13 on leaf cell number was examined in 14-day-old L1 of TCP13-overexpressing plants (Figure S1). Unexpectedly, cell number was reduced at this stage, implying a complex role of TCP13 in leaf development. Taken together, our data suggested that TCP13 and its paralogs reduce leaf size primarily at the cell expansion phase of leaf development.

3.4. TCP13 Negatively Controls ATHB12 Expression

Phenotypes of TCP13-GFP and amiR-3TCP plants indicated that TCP13 inhibits the leaf growth mainly at the cell expansion phase. Furthermore, it was previously shown that ATHB12 promotes the cell expansion of leaves through endoreduplication [7]. We hypothesized that TCP13, as a transcription factor, binds to the upstream region of ATHB12 and regulates its expression. To test this idea, we measured the expression of TCP13 and ATHB12 at different phases of leaf development. The expression of TCP13 was significantly increased at 10 DAS and the trend continued at 14 DAS as well, although with a much-reduced rate. On the other hand, the ATHB12 expression showed its peak at 10 DAS then fell sharply at 14 DAS to about ¼ of the level observed at 10 DAS (Figure 5A). This seemed to be in agreement with the idea of the ATHB12 expression being negatively regulated by TCP13.
We wanted to verify the negative correlation of expression between ATHB12 and TCP13, so that the expression of ATHB12 was also examined in the aforementioned transgenic plants by real-time PCR. Consistent with what was observed in the wild-type plants, it was found to be reduced in the leaves of transgenic plants overexpressing TCP13 while slightly increased expression level was detected in the leaves of amiR-3TCP plants, supporting our proposed role of TCP13 and its paralogs in suppressing the ATHB12 expression (Figure 5B). In the meantime, there was no significant difference between the levels of ATHB12 expression in the two types of transgenic plants when RNAs from whole seedlings were used (Figure S2), implying that TCP13 appears to affect ATHB12 expression only in leaves. Furthermore, EXPA5 and EXPA10, expansion genes whose expression is induced by ATHB12 [7], were downregulated in TCP13-overexpressing plants (Figure 5B). In addition, the expression of CCS52s, marker genes for endoreduplication [40], were significantly decreased in TCP13-overexpressing plants and increased in amiR-3TCP plants. These patterns of expression were confirmed in TCP13-GR seedlings induced with dexamethasone (DEX) in the presence of cycloheximide for 2 h (Figure S3).
In order to clarify whether the change of the expression of ATHB12 were due to TCP13, we transfected protoplasts from leaves harboring PATHB12::GUS with a plasmid containing GFP alone or TCP13-GFP under the control of CaMV 35S promoter. GUS activities measured 0.5 h or 1 h after transfection indicated that TCP13 overexpression indeed reduced ATHB12 expression in the protoplasts (Figure 5C). We also examined the effect of ATHB2 on the expression of ATHB12 because TCP13 was found to interact with ATHB2 (see Figure 6) and ATHB2 contains an EAR motif, a well-known transcriptional repression motif in plants [41]. Transfection of protoplasts of PATHB12::GUS with a P35S::ATHB2-GFP construct reduced GUS activity more than transfection of a control P35S::GFP construct. Then for examining a possible synergistic effect of TCP13 and ATHB2 on the reduction of ATHB12 expression, protoplasts of PATHB12::GUS were transfected with both P35S::TCP13-GFP and P35S::ATHB2-GFP constructs. However, transfection of both TCP13 and ATHB2 did not result in a synergistic effect on the repression but a slight derepression was observed as compared with that of the ATHB2 alone. Validating these data, when these constructs were used to transfect protoplasts isolated from wild-type Arabidopsis leaves together with the plasmid DNA having luciferase reporter construct under the ATHB12 promoter (PATHB12::LUC), exactly the same results were also obtained (Figure S4). ATHB2 and TCP13 interact with each other, resulting in slight de-repression or more repression of ATHB12, depending on which protein binds to ATHB12 promoter first, but at this point, the exact mechanism on how TCP13 and ATHB2 affect ATHB12 expression requires further research.
To determine if ATHB2 can bind directly to the ATHB12 promoter region, yeast one-hybrid analysis was conducted after fusing full-length ATHB2 coding sequence to the GAL4 activation domain (AD-ATHB2). A specific binding of ATHB2 to the promoter of ATHB12 under high stringency selection condition (60 mM 3-AT) was observed, confirming ATHB2 as another transcription factor participating in the regulation of ATHB12 expression possibly through direct binding to the promoter (Figure S5). All these findings supported the view that TCP13 and its paralogs as well as ATHB2 inhibit ATHB12 expression.

3.5. TCP13 Interacts with ATHB2

The Arabidopsis interactome map provides a comprehensive network of protein interactions in Arabidopsis [42]. The interactome data suggest that TCP13 interacts with ATHB2, a homeodomain-leucine zipper protein (HD-Zip) containing an EAR motif [41]. Thus, possibility of the interaction was tested by yeast two-hybrid assays and the result demonstrated that ATHB2, a HD-Zip class II protein, specifically interacts with TCP13 as well as two other CIN-like TCPs (TCP5 and TCP17) reflecting highly conserved amino acid sequence identities among these three CIN-like TCPs, as ATHB2 did not show any interaction with TCP7, a member of a different class of TCPs, in the same assay (Figure 6A). The interactions were revealed to be mediated with the C-terminal region of ATHB2 which contains a leucine zipper (Figure 6B). In addition to ATHB2, TCP13 was found to interact with other members of HD-Zip class II proteins including ATHB4 and HAT3 (Figure 6A), which were further confirmed in vivo by bimolecular fluorescence complementation (BiFC) analysis (Figure 6C). These results indicate that a close functional tie might exist between CIN-type TCPs and the class II HD-Zip proteins established at structural level. Given that ATHB2, ATHB4 and HAT3 contain an EAR motif, a plant-specific repression domain, functional significance of this interaction between TCP13 and ATHB2 in the negative regulation of ATHB12 expression warrants more in-depth examination.

3.6. TCP13 Binds to the Promoter of ATHB12 in Vivo

Since our yeast one-hybrid analysis results suggested that TCP13 regulates the expression of ATHB12 via direct binding to the promoter region, we attempted to map the binding site on the ATHB12 promoter. Class II TCP proteins are predicted to bind to the consensus binding site (G(T/C)GGNCCC) [25,43]. Sequence analysis revealed that the upstream region of ATHB12 contains two potential TCP binding sites (TBSs) at -254 bp (TBS I; GGTTCC) and -158 bp (TBS II; GGCCC), respectively, from the translational start site (Figure 7A). Chromatin immunoprecipitation (ChIP) assays were employed to examine the binding of TCP13 to the upstream region of ATHB12 including the putative TCP binding sites. ChIP assays using TCP13-GFP plants strongly enriched a fragment of the ATHB12 upstream region that contained TBS I (region D in Figure 7A) whereas ChIP using transgenic plants expressing only GFP did not show enrichment of any ATHB12 upstream region as negative control. These indicated that TCP13 binds to the upstream region of ATHB12 in planta. To further verify that the TBS I and TBS II motifs on ATHB12 promoter are the core elements to which TCP13 binds and regulates the expression of ATHB12, we mutagenized the TBS I and TBS II sequences (Figure 7B) and monitored the effect in protoplasts transiently expressing the luciferase reporter gene under the wild-type ATHB12 promoter or the ones carrying these mutations. The expression of luciferase driven by wild-type ATHB12 promoter was substantially repressed with co-transfection of P35S::TCP13-GFP as in the GUS assay shown in Figure 5C (Figure 7B). However, the luciferase activity measured from the cells transfected with either of the two mutant promoters (m1 or m2) exhibited clear de-repression under the same setting, demonstrating that the TBS I and TBS II sequence motifs are indeed the critical elements to which TCP13 convey its regulatory effect, likely through direct binding. We conclude that TCP13 regulates ATHB12 expression through direct binding to the ATHB12 upstream region containing TBS I and TBS II.

4. Discussion

Leaf development requires a controlled cascade of activities of diverse transcription factors. Previously, ATHB12, a homeodomain transcription factor, was reported to promote leaf growth, especially in the cell expansion phase, by activating several genes related to cell expansion [7]. However, how ATHB12 itself was controlled during leaf development remained unknown. In this report, we isolated and characterized an upstream regulator of ATHB12.
TCP13, a class II TCP transcription factor, was isolated as an upstream regulator of ATHB12 (Figure 1). It seems to affect the specific phase(s) of leaf growth, which is reasonable considering the role of ATHB12 in leaf growth [7]. Expression of TCP13 was mainly observed in cotyledons, and was not detected in the first to fourth leaves of 11-day-old seedlings (Figure 2B). This pattern is exactly the opposite of ATHB12 expression during the expansion phase of leaf development [7]. TCP13 affected the areas of leaves at 8 DAS to 14 DAS—specifically cell areas of leaves at the corresponding stages (Figure 3C and Figure 4). Class II TCP transcription factors are key regulators of leaf growth [24,29,44,45,46,47]. Timing of TCP13 expression and the phenotypes of TCP13 transgenic plants indicate that TCP13, one of the class II TCP family, controls a specific period(s) of leaf development including the cell expansion stage. However, given the significant differences in cell area of 8 DAS transgenic plants (Figure 4B) and in cell numbers of 14-DAS plants (Figure S1), we cannot exclude a possibility that TCP13 and its paralogs may also somehow affect cell division.
The expression of TCP13 was examined by GUS assays of transgenic plant expressing a PTCP13::GUS reporter (Figure 2). In addition, the expression of TCP13 and ATHB12 was further studied by real-time PCRs and transactivation assays using PATHB12::GUS (Figure 5), which suggests that TCP13 regulates ATHB12 expression negatively. Many TCPs control gene transcription negatively in various periods of plant development. TCPs such as TCP2 and TCP3, which are regulated by miR319, bind to the promoter regions of class-I KNOX genes and repress their transcription [46]. TCP target genes have been shown to be repressed during ovule development by epigenetic regulation involving the SPL-TPL-HDA19 complex [48]. Furthermore, TCP5-like proteins including TCP13, control expansion of the cells of petals since overexpression of TCP5 results in petals of reduced area [49]. TCP13, and possibly other CIN-TCPs, regulate the cell area of leaves negatively, but how they repress their target genes, which are involved in cell expansion, needs further examination.
The expressions of ATHB12 and its key downstream target genes were all shown to be significantly down regulated in TCP13-GFP expressing transgenic plants (Figure 5B). On the contrary, some of these downstream target genes (notably CCS52B) did not fully recover their expression levels in the in amiR-3TCP transgenic plants. A possible explanation for this discrepancy could be that many of these are indirect targets of ATHB12, and perhaps other additional factors as well as ATHB12 could be required for their full activation. Or the restored ATHB12 expression in the amiR-3TCP plants was not completely linked to the full activation of the ATHB12 protein for some reasons. At this point, the exact explanation remains unanswered.
Interaction between TCP13 and ATHB2 was confirmed by yeast two-hybrid assays and BiFC analysis (Figure 6). In addition, TCP5 and TCP17, other members of CIN-like TCPs sharing high sequence homology with TCP13, also interact with ATHB2 in the same assays. Besides, other HD-Zip class II proteins including ATHB4 and HAT3 also showed interaction with TCP13/5/17. Therefore, it is probable that the rest of CIN-like TCPs such as TCP3 and TCP4 could also interact with ATHB2, ATHB4 and HAT3 to control the expression of ATHB12 or other genes, considering the highly conserved structural similarity in helices and the loop domain in the CIN-like TCPs [25]. TCP4, as an example, affects leaf morphogenesis as well as other developmental phases of plant [27], so that TCP4 may associate with ATHB2 and other HD-Zip class II proteins for functional overlapping and fine-tuning in a similar fashion to other related TCPs including TCP13. A continuous and extensive effort to unveil detailed mechanisms underlying the interaction between TCPs and ATHBs could provide significant understanding on leaf development.
We isolated several transcription factors in addition to TCP13 from the yeast one-hybrid screen for proteins that bind to the 1.2-kb upstream region of ATHB12 (Table S2, Figure 1). Interestingly, some of them also affected the growth of leaves and flowers. NGA1 is an AtNGA family member that acts as negative regulator of the cell proliferation in lateral organs [36]. Overexpression of NGA resulted in reduced leaf cell numbers. In addition, HB51, also called LMI1, is expressed in expanding leaves and affects leaf morphology [37]. It is quite possible that several of the transcription factors isolated from the yeast one-hybrid screening affect ATHB12 expression/repression, thus influencing particular phases of leaf development. Further research on them in relevance of ATHB12 should clarify their roles in leaf development.

Supplementary Materials

The following are available online at https://www.mdpi.com/2073-4425/10/9/644/s1, Table S1: Primers used in this study, Table S2: List of putative upstream regulators of ATHB12 isolated by yeast one-hybrid screening, Figure S1: Effect of TCP13 on leaf cell number in Arabidopsis, Figure S2: ATHB12 expression in whole seedlings of wild-type, TCP13-GFP and amiR-3TCP plants, Figure S3: Expression of downstream genes of ATHB12 after TCP13 induction Figure S4: Effect of TCP13 and ATHB2 on the expression of ATHB12 examined by luciferase assay. Figure S5: Binding of ATHB2 to the ATHB12 promoter examined by yeast one-hybrid assay.

Author Contributions

Conceptualization, Y.-S.H. and C.-I.C.; methodology, Y.-S.H., J.K., O.S. and M.O.-T; formal analysis, Y.-S.H., J.K., O.S., G.-T.K. and M.O.-T; investigation, Y.-S.H., J.K., S.K., O.S., W.-Y.K. and G.-T.K.; resources, W.-Y.K. and M.O.-T; data curation, Y.-S.H. and G.-T.K; writing—original draft preparation, Y.-S.H.; writing—review and editing, Y.-S.H. and C.-I.C.; supervision, C.-I.C.; project administration, C.-I.C.; funding acquisition, W.-Y.K. and C.-I.C.

Funding

This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2011-0030074), by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1A6A3A11933574) and by Korea Ministry of Environment (MOE) as Graduate School specialized in Climate Change.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Figure 1. Isolation of TCP13 as an upstream regulator of ATHB12 by yeast one-hybrid screening. (A) Schematic diagrams of reporter constructs. A 1.2 kb-upstream region of ATHB12 was inserted in front of HIS3 (pHISi-1; PATHB12::HIS3) or LacZ (pLacZi; PATHB12::LacZ), and they were used as reporter genes. TCP13 cDNA was used as the effector construct. (B) TCP13 binding to the ATHB12 promoter. Yeast cells harboring the indicated constructs were grown in the presence of 20 mM (left) or 60 mM (middle) 3-AT. Filter-lift assays (right) of the yeast cells was performed to determine β-galactosidase activities. AD; GAL4 activation domain, AD-TCP13; a fusion of GAL4 AD to TCP13.
Figure 1. Isolation of TCP13 as an upstream regulator of ATHB12 by yeast one-hybrid screening. (A) Schematic diagrams of reporter constructs. A 1.2 kb-upstream region of ATHB12 was inserted in front of HIS3 (pHISi-1; PATHB12::HIS3) or LacZ (pLacZi; PATHB12::LacZ), and they were used as reporter genes. TCP13 cDNA was used as the effector construct. (B) TCP13 binding to the ATHB12 promoter. Yeast cells harboring the indicated constructs were grown in the presence of 20 mM (left) or 60 mM (middle) 3-AT. Filter-lift assays (right) of the yeast cells was performed to determine β-galactosidase activities. AD; GAL4 activation domain, AD-TCP13; a fusion of GAL4 AD to TCP13.
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Figure 2. Expression of TCP13 in planta. Seven-day-old (A), 10-day-old (B) and 16-day-old (C) seedlings, mature rosette leaves (D), flowers (E), and silique (F) of transgenic plants with a 2969-bp upstream region of TCP13 were stained with X-gluc. Scale bars = 1 mm.
Figure 2. Expression of TCP13 in planta. Seven-day-old (A), 10-day-old (B) and 16-day-old (C) seedlings, mature rosette leaves (D), flowers (E), and silique (F) of transgenic plants with a 2969-bp upstream region of TCP13 were stained with X-gluc. Scale bars = 1 mm.
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Figure 3. Phenotypes of TCP13-GFP (P35S::TCP13-GFP) and amiR-3TCP (P35S::amiR-3TCP) seedlings of Arabidopsis thaliana. (A) Growth phenotypes of four-week-old wild-type, TCP13-GFP and amiR-3TCP plants. (B) Leaves of four-week-old wild-type, TCP13-GFP and amiR-3TCP plants. Scale bars = 1 cm. Right panel indicates lengths and widths of fifth leaves (L5) of wild-type, TCP13-GFP and amiR-3TCP plants. Data shown are means ± SD (n > 8). (C) Area of the L1 of 6-, 8-, 10- and 14-day-old wild-type, TCP13-GFP and amiR-3TCP seedlings. DAS, days after sowing. Data shown are means ± SD (n > 6). Significant differences as evaluated by one-way ANOVA: ***, p < 0.005, **, p < 0.01 and *, p < 0.05.
Figure 3. Phenotypes of TCP13-GFP (P35S::TCP13-GFP) and amiR-3TCP (P35S::amiR-3TCP) seedlings of Arabidopsis thaliana. (A) Growth phenotypes of four-week-old wild-type, TCP13-GFP and amiR-3TCP plants. (B) Leaves of four-week-old wild-type, TCP13-GFP and amiR-3TCP plants. Scale bars = 1 cm. Right panel indicates lengths and widths of fifth leaves (L5) of wild-type, TCP13-GFP and amiR-3TCP plants. Data shown are means ± SD (n > 8). (C) Area of the L1 of 6-, 8-, 10- and 14-day-old wild-type, TCP13-GFP and amiR-3TCP seedlings. DAS, days after sowing. Data shown are means ± SD (n > 6). Significant differences as evaluated by one-way ANOVA: ***, p < 0.005, **, p < 0.01 and *, p < 0.05.
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Figure 4. Effect of TCP13 on cell morphology of A. thaliana. (A) Microscopic observation of the palisade cells of the L1 of 8-, 10-, and 14-day-old wild-type, TCP13-GFP and amiR-3TCP seedlings. DAS, days after sowing. Scale bar = 20 μm. (B) Areas of the palisade cells of the L1 of 8-, 10-, and 14-day-old wild-type, TCP13-GFP and amiR-3TCP seedlings. Areas of more than 180 cells from eight leaves were measured. Data shown are means ± SD (n > 180). Significant differences as evaluated by one-way ANOVA: ***, p < 0.005, **, p < 0.01 and *, p < 0.05.
Figure 4. Effect of TCP13 on cell morphology of A. thaliana. (A) Microscopic observation of the palisade cells of the L1 of 8-, 10-, and 14-day-old wild-type, TCP13-GFP and amiR-3TCP seedlings. DAS, days after sowing. Scale bar = 20 μm. (B) Areas of the palisade cells of the L1 of 8-, 10-, and 14-day-old wild-type, TCP13-GFP and amiR-3TCP seedlings. Areas of more than 180 cells from eight leaves were measured. Data shown are means ± SD (n > 180). Significant differences as evaluated by one-way ANOVA: ***, p < 0.005, **, p < 0.01 and *, p < 0.05.
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Figure 5. Effects of TCP13 on ATHB12 expression. (A) Relative expression of TCP13 and ATHB12 in the L1 and L2 of 7-, 10-, and 14-day-old wild-type seedlings. Results are representative of more than three independent experiments. Data shown are means ± SD (n = 2). (B) Relative expression of ATHB12, EXPA5, EXPA10, and CCS52s in 14-day-old wild-type, TCP13-GFP and amiR-3TCP plants examined by real-time quantitative PCR. Results are representative of more than three independent experiments. Data shown are means ± SD (n = 2). (C) GUS activities in protoplasts isolated from transgenic plants with PATHB12::GUS was measured after transfection with P35S::GFP or P35S::TCP13-GFP or P35S::ATHB2-GFP constructs. Data shown are means ± SD (n = 3). RFU, Relative fluorescence units. Significant differences as evaluated by one-way ANOVA: ***, p < 0.005, **, p < 0.01 and *, p < 0.05.
Figure 5. Effects of TCP13 on ATHB12 expression. (A) Relative expression of TCP13 and ATHB12 in the L1 and L2 of 7-, 10-, and 14-day-old wild-type seedlings. Results are representative of more than three independent experiments. Data shown are means ± SD (n = 2). (B) Relative expression of ATHB12, EXPA5, EXPA10, and CCS52s in 14-day-old wild-type, TCP13-GFP and amiR-3TCP plants examined by real-time quantitative PCR. Results are representative of more than three independent experiments. Data shown are means ± SD (n = 2). (C) GUS activities in protoplasts isolated from transgenic plants with PATHB12::GUS was measured after transfection with P35S::GFP or P35S::TCP13-GFP or P35S::ATHB2-GFP constructs. Data shown are means ± SD (n = 3). RFU, Relative fluorescence units. Significant differences as evaluated by one-way ANOVA: ***, p < 0.005, **, p < 0.01 and *, p < 0.05.
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Figure 6. Interaction of TCP13 with HD-Zip class II proteins. (A) TCP13 and TCP13 homologous CIN-TCPs interact with HD- Zip class II protein in yeast two-hybrid assay. Dilutions of yeast transformed with TCP-BD and ATHB-AD were spotted in medium with the presence or absence of histidine (His). TCP7, a member of TCP class I, fused to BD and ATHB53, a HD- Zip class I protein, fused to AD were used as negative controls. BD, binding domain; AD, activation domain. (B) TCP13 interacts with ATHB2 C-terminal. Top panel shows schematic representation of domain structure of ATHB2. HD, homeodomain; LZ, leucine-zipper. TCP13, TCP5, and TCP17-BD interact with ATHB2-Ct-AD, but not with ATHB2-Nt-AD in yeast two-hybrid assay. (C) Bimolecular fluorescence complementation (BiFC) analysis of interaction between CIN-TCPs and HD- Zip class II proteins. Arabidopsis protoplasts were co-transfected with several combinations of the constructs of P35S::ATHB-YFPN and P35S::TCP-YFPC. DAPI stained the nuclei. Chl, chlorophyll; Bf, bright field. BiFC experiments were replicated three times with similar results. Scale bar = 10 μm.
Figure 6. Interaction of TCP13 with HD-Zip class II proteins. (A) TCP13 and TCP13 homologous CIN-TCPs interact with HD- Zip class II protein in yeast two-hybrid assay. Dilutions of yeast transformed with TCP-BD and ATHB-AD were spotted in medium with the presence or absence of histidine (His). TCP7, a member of TCP class I, fused to BD and ATHB53, a HD- Zip class I protein, fused to AD were used as negative controls. BD, binding domain; AD, activation domain. (B) TCP13 interacts with ATHB2 C-terminal. Top panel shows schematic representation of domain structure of ATHB2. HD, homeodomain; LZ, leucine-zipper. TCP13, TCP5, and TCP17-BD interact with ATHB2-Ct-AD, but not with ATHB2-Nt-AD in yeast two-hybrid assay. (C) Bimolecular fluorescence complementation (BiFC) analysis of interaction between CIN-TCPs and HD- Zip class II proteins. Arabidopsis protoplasts were co-transfected with several combinations of the constructs of P35S::ATHB-YFPN and P35S::TCP-YFPC. DAPI stained the nuclei. Chl, chlorophyll; Bf, bright field. BiFC experiments were replicated three times with similar results. Scale bar = 10 μm.
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Figure 7. (A) Binding of TCP13 to the upstream region of ATHB12. Association of TCP13 with the upstream region of ATHB12 was confirmed by chromatin immunoprecipitation (ChIP) assays. Schematic diagram indicates TCP binding sites (TBS I-II) and ChIP amplicons (A-E) in the upstream region of ATHB12. The enrichment of each DNA fragment was normalized by the level of input DNA and the ChIP enrichment value of the control PP2A promoter. Data shown are means ± SD (n = 2). (B) Luciferase assays using wild-type or mutated ATHB12 promoter. The diagram shows the sequences of wild-type (WT) or mutated (m1 and m2) versions of ATHB12 promoter. Arabidopsis protoplasts were transiently transfected with different reporter genes, PATHB12 (WT)::LUC (pATHB12 (WT) or PATHB12 (m1)::LUC (pATHB12 (m1)) or PATHB12 (m2)::LUC (pATHB12 (m2)) and, together with P35S::RLUC as internal control and effectors such as P35S::GFP or P35S::TCP13-GFP constructs. Firefly luciferase (LUC) activity was normalized with Renilla luciferase (RLUC) activity. Data shown are means ± SD (n = 2).
Figure 7. (A) Binding of TCP13 to the upstream region of ATHB12. Association of TCP13 with the upstream region of ATHB12 was confirmed by chromatin immunoprecipitation (ChIP) assays. Schematic diagram indicates TCP binding sites (TBS I-II) and ChIP amplicons (A-E) in the upstream region of ATHB12. The enrichment of each DNA fragment was normalized by the level of input DNA and the ChIP enrichment value of the control PP2A promoter. Data shown are means ± SD (n = 2). (B) Luciferase assays using wild-type or mutated ATHB12 promoter. The diagram shows the sequences of wild-type (WT) or mutated (m1 and m2) versions of ATHB12 promoter. Arabidopsis protoplasts were transiently transfected with different reporter genes, PATHB12 (WT)::LUC (pATHB12 (WT) or PATHB12 (m1)::LUC (pATHB12 (m1)) or PATHB12 (m2)::LUC (pATHB12 (m2)) and, together with P35S::RLUC as internal control and effectors such as P35S::GFP or P35S::TCP13-GFP constructs. Firefly luciferase (LUC) activity was normalized with Renilla luciferase (RLUC) activity. Data shown are means ± SD (n = 2).
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Hur, Y.-S.; Kim, J.; Kim, S.; Son, O.; Kim, W.-Y.; Kim, G.-T.; Ohme-Takagi, M.; Cheon, C.-I. Identification of TCP13 as an Upstream Regulator of ATHB12 during Leaf Development. Genes 2019, 10, 644. https://doi.org/10.3390/genes10090644

AMA Style

Hur Y-S, Kim J, Kim S, Son O, Kim W-Y, Kim G-T, Ohme-Takagi M, Cheon C-I. Identification of TCP13 as an Upstream Regulator of ATHB12 during Leaf Development. Genes. 2019; 10(9):644. https://doi.org/10.3390/genes10090644

Chicago/Turabian Style

Hur, Yoon-Sun, Jiyoung Kim, Sunghan Kim, Ora Son, Woo-Young Kim, Gyung-Tae Kim, Masaru Ohme-Takagi, and Choong-Ill Cheon. 2019. "Identification of TCP13 as an Upstream Regulator of ATHB12 during Leaf Development" Genes 10, no. 9: 644. https://doi.org/10.3390/genes10090644

APA Style

Hur, Y.-S., Kim, J., Kim, S., Son, O., Kim, W.-Y., Kim, G.-T., Ohme-Takagi, M., & Cheon, C.-I. (2019). Identification of TCP13 as an Upstream Regulator of ATHB12 during Leaf Development. Genes, 10(9), 644. https://doi.org/10.3390/genes10090644

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