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. 2017 Aug 25;175(2):874–885. doi: 10.1104/pp.17.00732

Roles of miR319 and TCP Transcription Factors in Leaf Development1,[OPEN]

Tomotsugu Koyama a,2, Fumihiko Sato b, Masaru Ohme-Takagi c,d
PMCID: PMC5619901  PMID: 28842549

Combinations of mutations in miR319 and TCP transcription factor genes in Arabidopsis thaliana generate cotyledon fusion and various leaf forms with a broad range of complexity.

Abstract

Sophisticated regulation of gene expression, including microRNAs (miRNAs) and their target genes, is required for leaf differentiation, growth, and senescence. The impact of miR319 and its target TEOSINTE BRANCHED1, CYCLOIDEA, and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR (TCP) genes on leaf development has been extensively investigated, but the redundancies of these gene families often interfere with the evaluation of their function and regulation in the developmental context. Here, we present the genetic evidence of the involvement of the MIR319 and TCP gene families in Arabidopsis (Arabidopsis thaliana) leaf development. Single mutations in MIR319A and MIR319B genes moderately inhibited the formation of leaf serrations, whereas double mutations increased the extent of this inhibition and resulted in the formation of smooth leaves. Mutations in MIR319 and gain-of-function mutations in the TCP4 gene conferred resistance against miR319 and impaired the cotyledon boundary and leaf serration formation. These mutations functionally associated with CUP-SHAPED COTYLEDON genes, which regulate the cotyledon boundary and leaf serration formation. In contrast, loss-of-function mutations in miR319-targeted and nontargeted TCP genes cooperatively induced the formation of serrated leaves in addition to changes in the levels of their downstream gene transcript. Taken together, these findings demonstrate that the MIR319 and TCP gene families underlie robust and multilayer control of leaf development. This study also provides a framework toward future researches on redundant miRNAs and transcription factors in Arabidopsis and crop plants.

Leaves are differentiated at the flank of shoot meristems, later forming their shapes and senescing at the final stage of their development (Bar and Ori, 2014; Ichihashi and Tsukaya, 2015). The integration of diverse developmental programs during leaf development requires a complex and robust network of gene regulation. This regulation requires the sophisticated balance between microRNAs (miRNAs) and their target genes (Rodriguez et al., 2016; Fouracre and Poethig, 2016).

miR319 and its target genes, TEOSINTE BRANCHED1, CYCLOIDEA, and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR (TCP), play pivotal roles in leaf development. Among 24 TCP genes in Arabidopsis (Arabidopsis thaliana), the phylogenetically related CINCINNATA-like TCP subfamily consists of five miR319-targeted genes, namely, TCP2, TCP3, TCP4, TCP10, and TCP24, as well as three nontargeted genes (Supplemental Fig. S1; Nath et al., 2003; Palatnik et al., 2003; Lopez et al., 2015; Nicolas and Cubas, 2016; named here as TCP for simplicity). Arabidopsis knockout mutants for three or more TCP genes exhibit serrated and wavy leaves (Koyama et al., 2010), indicating that these TCP genes redundantly regulate the margin and surface of leaves. Moreover, chimeric TCP repressor genes, in which the TCP genes are fused with the strong repression domain, generate serrated and wavy leaves in transgenic Arabidopsis, torenia, chrysanthemum, cyclamen, rose, and morning glory plants, suggesting the conserved role of these TCP genes in leaf development (Hiratsu et al., 2003; Koyama et al., 2007, 2011; Narumi et al., 2011; Gion et al., 2011; Tanaka et al., 2011; Ono et al., 2012; Li and Zachgo 2013; Sasaki et al., 2016).

These actions of TCP transcription factors are largely dependent on the negative regulation of the CUP-SHAPED COTYLEDON1 (CUC1), CUC2, and CUC3 genes (Koyama et al., 2007, 2010; Rubio-Somoza et al., 2014). CUC genes are active in the boundary of two cotyledons and are required for the formation of the sinus (Aida et al., 1997; Aida and Tasaka, 2006; Hibara et al., 2006). CUC2 and CUC3 genes are also active in the sinuses of the leaf margin and are required for leaf serration (Nikovics et al., 2006; Hasson et al., 2011; Maugarny et al., 2016). The TCP transcription factors negatively regulate the expression of CUC genes via genes downstream of the TCPs (Koyama et al., 2007, 2010). Moreover, TCP4 directly interacts with CUC2 and CUC3 and inhibits their activities at the posttranslational level (Rubio-Somoza et al., 2014).

miR319 partially but significantly controls the activity of TCP genes. The nearly complementary nucleotide sequences of TCP mRNAs and miR319 underlie the specific mechanism of gene regulation (Palatnik et al., 2003, 2007; Ori et al., 2007; Shleizer-Burko et al., 2011). A single-nucleotide mutation of miR319 reduces its ability to target the five TCP mRNAs (Nag et al., 2009; Supplemental Fig. S2). Mutations in TCP genes, which are resistant against miR319 (mTCP), induce gain-of-function effects and induce impairments in the shoot meristem, cotyledon boundary, leaf margin, and size (Palatnik et al., 2003, 2007; Koyama et al., 2007; Efroni et al., 2008; Sarvepalli and Nath, 2011; Schommer et al., 2014). Conversely, ectopic expression of MIR319 in Arabidopsis decreases the levels of the five TCP transcripts and results in the formation of jagged and wavy leaves (Palatnik et al., 2003, 2007; Koyama et al., 2007; Efroni et al., 2008). miR319 is encoded by three genes in the Arabidopsis genome, and this apparent redundancy may mask the leaf irregularities observed with a loss-of-function allele of miR319 (Nag et al., 2009). Overexpression of an inhibitory RNA against miR319 (target mimicry; MIM319) implies a dominant-negative effect of miR319 (Todesco et al., 2010; Rubio-Somoza and Weigel, 2013); however, the predominant mechanism through which MIR319 genes act during leaf development remains unclear.

Because of the redundancies of TCP and MIR319 genes, the previous studies on these genes had been often dependent on data derived from dominant-negative and active methods (Nicolas and Cubas, 2016). In contrast, generating multiple mutants that include novel alleles for the MIR319 and TCP genes, we here demonstrate that these mutants changed the leaf and cotyledon morphology more gradually than previously thought. In this study, we obtained genetic evidence that miR319 acts in the formation of the cotyledon boundary and leaf serrations in association with CUC genes. We also generated sextuple tcp mutants and transgenic plants, in which the eight TCP genes were almost suppressed, in order to delineate the role of the highly redundant TCP genes in leaf development. Our results provide a framework toward future researches on miRNAs and transcription factors that redundantly regulate important traits such as development, physiology, and reproduction in Arabidopsis and crop plants.

RESULTS

Characterization of Loss-of-Function Alleles for MIR319A and MIR319B

Since the accumulation of miRNAs is usually associated with the transcription of precursor genes, we first examined which of the precursor genes MIR319A, MIR319B, and MIR319C were active during leaf development in Arabidopsis. The results of RT-PCR analysis demonstrated that the transcript level of MIR319A was largely constant during plant development, whereas those of MIR319B and MIR319C differentially increased in vegetative and reproductive organs, respectively (Supplemental Fig. S3). We expected that MIR319A and MIR319B would contribute to the regulation of leaf development.

Next, we generated loss-of-function mutants of MIR319A and MIR319B to investigate the functions of miR319 during leaf development. mir319a123, which carries a nucleotide replacement in the complementary sequence of MIR319A to the TCP genes (Nag et al., 2009; Supplemental Fig. S2), was crossed with Colombia-0 to standardize the genetic background (hereafter, the resultant mutant is referred to as mir319a to discriminate the Colombia-0 background). mir319b was newly identified in this study from the GABI_KAT mutant collection (Rosso et al., 2003), in which the expression of the MIR319B gene was lost due to the insertion of T-DNA (Supplemental Fig. S4, A and B). We further investigated whether the loss-of-function allele of MIR319B could cause inability to target TCP3 mRNA. When the TCP3 gene construct, under the control of the constitutively active cauliflower mosaic virus 35S promoter (Pro-35S), was introduced into wild-type plants, the resultant transgenic plants (Pro-35S:TCP3) formed normal leaves due to the activity of miR319 (Koyama et al., 2007; Supplemental Fig. S4, C and D). In contrast, the TCP3 construct was introduced into mir319b, and the resultant Pro-35S:TCP3 mir319b exhibited cotyledon fusion and smooth leaves (Supplemental Fig. S4, C–E), as observed in Pro-35S:mTCP3 plants (Supplemental Fig. S2; Koyama et al., 2007, 2010). This suggested that the ability to target the ectopically induced TCP3 mRNA was substantially impaired in mir319b.

Impaired Formation of Leaf Serrations, Hypocotyl, and Inflorescence in mir319a/b

The transgenic plants carrying the mTCP2, mTCP3, or mTCP4 gene generate leaves with a smooth margin (Palatnik et al., 2003; Koyama et al., 2007, 2010; Schommer et al., 2014); therefore, we investigated this phenotype of the mir319-related mutants. The sixth leaf of the wild type usually generated a serration on each side of the margin, whereas the leaves of mir319a and mir319b had serrations of reduced size (Fig. 1, A and B). When mir319a was crossed with mir319b, the resultant mir319a mir319b double mutant (mir319a/b) exhibited reduced serrations and, as observed in the severely defected cases, in eight out of 19 mir319a/b plants analyzed, no serrations were observed (Fig. 1, A and B). These results clarified an additive effect of the mir319a and mir319b mutations on the formation of leaf serrations.

Figure 1.

Figure 1.

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The effects of mir319a and mir319b mutations on leaf serration formation, fruit bearing, and hypocotyl elongation. A, Photograph showing the sixth leaves of wild-type, mir319a, mir319b, and mir319a/b plants. Triangles indicate the serration at the leaf margin. Bars = 1 mm. B, Quantification of serration size in the sixth leaves of wild-type, mir319a, mir319b, and mir319a/b plants. The different letters and crosses in the box plot indicate a statistically significant difference (Tukey-Kramer method; P < 0.05) and outliers, respectively. Numbers in parentheses below the genotypes represent biological replicates. C, Photograph showing the hypocotyls of wild-type and mir319a/b seedlings. Triangles indicate the junction of the hypocotyl and root. Bar = 1 mm. D, Quantification of the hypocotyl length of wild-type and mir319a/b seedlings. The means were calculated from 20 hypocotyls of 7-d-old seedlings. Error bars and asterisks indicate sd and significant difference (Student t test; ***P < 0.001). E, The primary inflorescences of wild-type and mir319a/b plants. Bar = 1 cm

In addition, mir319a/b had a longer hypocotyl than wild type under the light condition (Fig. 1, C and D). These changes in mir319a/b are consistent with the moderate phenotype of transgenic plants overexpressing the mTCP genes (Palatnik et al., 2003; Koyama et al., 2007).

After floral organs senesce in the reproductive growth, wild-type plants expand their siliques, which are usually signs of successful pollination and subsequent embryogenesis (Fig. 1E). mir319a and mir319b were also fertile, whereas mir319a/b rarely exhibited silique expansion and less than 10 seeds were produced per plant (Fig. 1E). Similar abnormalities in the Pro-35S:mTCP3 and MIM319 plants suggested that the MIR319A and MIR319B genes functioned in the reproductive organs via the regulation of TCP genes (Supplemental Fig. S6; Rubio-Somoza and Weigel, 2013).

Effects of mir319a and mir319b Mutations on the Expression of TCP Genes

The overexpression of MIR319A reduces the levels of the miR319-targeted TCP genes in leaves (Palatnik et al., 2003, 2007), but effects of the loss-of-function of MIR319A and MIR319B on the TCP transcripts are obscure. We examined the effects of mir319 mutations on the levels of the TCP transcripts. Our RT-PCR analysis demonstrated that transcripts of the five miR319-targeted TCP genes increased in mir319a/b seedlings in comparison to wild-type seedlings (Fig. 2A). In contrast, several TCP transcripts moderately increased in mir319a, but none of them increased in mir319b (Supplemental Fig. S5), suggesting that each mutation alone might cause mild effects on the levels of the TCP transcripts.

Figure 2.

Figure 2.

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Gene expression analysis of wild-type and mir319a/b seedlings. The transcript levels of TCP (A), the TCP target (B), and CUC (C) genes were analyzed by RT-PCR. The values obtained for wild-type samples were each set at 1. Error bars and asterisks indicate the sd and significant difference of six biological replicates (Student’s t test; *P < 0.05, **P < 0.01), respectively.

mir319 Mutations Enhanced Effects of cuc Mutations on Cotyledon Boundary and Leaf Serration

To investigate the effects of the mir319a/b mutation on the expression levels of TCP downstream genes, we conducted RT-PCR analysis of wild type and mir319a/b seedlings. The transcript levels of SHORT HYPOCOTYL2/INDOLE-3-ACETIC ACID3 (SHY2/IAA3), SMALL AUXIN UP RNA (SAUR), miR164A, and LIPOXYGENASE2 (LOX2), which are direct targets of the TCP3, TCP4, and TCP10 transcription factors (Schommer et al., 2008; Koyama et al., 2010; Danisman et al., 2013), were upregulated in mir319a/b seedlings (Fig. 2B). Since TCP transcription factors negatively regulate the expression of the CUC1, CUC2, and CUC3 genes, we examined the transcript levels of the CUC genes and found their decreased expression in mir319a/b seedlings (Fig. 2C). These results demonstrated that the activation of TCP genes caused by the mir319a/b mutations was able to repress CUC gene expression.

The overexpression of mTCP genes often results in the fusion of cotyledons and the dysfunctionality of shoot meristems in the transgenic seedlings (Palatnik et al., 2003; Koyama et al., 2007). However, mir319a, mir319b, and mir319a/b showed normal cotyledon morphology (Fig. 3; Supplemental Table S1). Since mir319a/b seedlings exhibited a moderate reduction in CUC transcript levels, we expected that the mir319a/b mutations could induce cotyledon fusion phenotypes in the absence of either of the CUC genes. The single cuc1, cuc2, and cuc3 mutants are almost normal in their seedling stage due to the functional redundancy of CUC genes (Fig. 3; Supplemental Table S1; Aida et al., 1997; Vroemen et al., 2003; Hibara et al., 2006). As expected, all combinations of the double cuc and mir319 mutations induced the cotyledon fusion phenotype to various extents (Fig. 3; Supplemental Table S1). In the severe case, the cuc and mir319 mutations induced a cup-shaped cotyledon-like phenotype, resulting in the inability of rosette leaf formation owing to dysfunction of the shoot meristem, which is observed in the double mutants of CUC genes. These results demonstrated that the mir319a and mir319b mutations enhanced the effect of each single cuc mutation on cotyledon development.

Figure 3.

Figure 3.

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The genetic interactions of MIR319 and CUC genes during cotyledon morphogenesis. The genotypes of wild type, mir319a, mir319b, cuc1, cuc2, cuc3, and double mutants of various combinations are indicated above the photographs of cotyledons. Triangles show the fusion of cotyledons. Bars = 1 mm

CUC2 and CUC3 are also involved in the formation of serrations at the leaf margin, and mutations in these genes lead to a dramatic reduction in serration of the sixth leaves (Nikovics et al., 2006; Kawamura et al., 2010; Hasson et al., 2011; Bilsborough et al., 2011). Enhanced leaf serration occurs in accordance with the plant’s age and is recognized as a developmental consequence of heteroblasty (Poethig, 1997). We expected that leaves developed at the later growth stage would form obvious serrations in cuc mutants and could be used to investigate the combined effect of cuc and mir319 mutations on serration formation. As expected, we found a substantial increase in the serration size of the 10th and 14th leaves in comparison to that of the sixth leaves in the wild type (Figs. 1B and 4B). cuc2 and cuc3 formed small but obvious serrations in the leaves at the 14th and 10th positions, respectively (Fig. 4, A and B). Under these experimental conditions, mir319a and mir319b mutations severely decreased the serration size in the cuc2 and cuc3 backgrounds. These results demonstrated that mir319 mutations enhanced the defective effects of cuc2 and cuc3 mutations on leaf serration formation.

Figure 4.

Figure 4.

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The genetic interactions of MIR319 and CUC genes in the formation of leaf serrations. A, The genotypes of various mutants are indicated above the photographs of leaf serrations. Triangles show the serration at the leaf margin. Bars = 1 mm. B, Quantification of the size of leaf serrations. A detailed description of the box plots is provided in Figure 1B.

Gain-of-Function Mutations in TCP4 Impaired the Cotyledon Boundary and Leaf Serrations

Regarding the gain-of-function analysis of the TCP genes, previous studies have used transgenic plants in which overexpression constructs of mTCP genes are introduced (Palatnik et al., 2003; Koyama et al., 2007; Efroni et al., 2008; Sarvepalli and Nath, 2011). Since TCP transcription factors form homo- and heterodimers (Kosugi and Ohashi 2002, Giraud, et al., 2010; Danisman et al., 2013; Kubota et al., 2017), these overexpression constructs might simultaneously affect several TCP activities. To obtain novel insights in gain-of-function effects of the TCP gene and genetic interactions between TCP and CUC genes in the absence of the overexpression constructs, we used suppressor of jaw-d (soj) mutants, which possess single-base nucleotide replacements in TCP4 to be resistant against miR319 (Supplemental Fig. S2; Palatnik et al., 2007). In this study, soj mutants were used as gain-of-function mutants for TCP4 after outcrossing to wild type for the elimination of the jaw phenotype due to the enhancer trap (hereafter, the resultant mutants are named tcp4-dsoj6 and tcp4-dsoj8 for discrimination with the outcrossed mutants). As reported previously (Palatnik et al., 2007), we confirmed the mutation in tcp4-dsoj8 at the site corresponding to the reverse position 11 of miR319, where the wild-type TCP4 mRNAs are cleaved, and tcp4-dsoj6at the next position of the one in soj8 (Supplemental Fig. S2). At the seedling stage, tcp4-dsoj6 showed normal morphology whereas tcp4-dsoj8 occasionally induced the cotyledon fusion phenotype (Fig. 5; Supplemental Table S1). Consistent with the role of miR319 in the formation of leaf serrations, tcp4-dsoj6 and tcp4-dsoj8 mutants impaired the formation of leaf serrations (Supplemental Fig. S6A). In inflorescences, the tcp4-d mutants exhibited markedly low fertility, as observed in mir319a/b and Pro-35S:mTCP3 plants (Supplemental Figure S6B; Fig. 1E).

Figure 5.

Figure 5.

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The genetic interactions of TCP4 and CUC genes during cotyledon morphogenesis. The genotypes of mutants are indicated above the photographs of cotyledons. Triangles show the fusion of cotyledons. Bars = 1 mm.

To determine the effect of adding the gain-of-function TCP4 gene to the loss-of-function of MIR319 genes, tcp4-dsoj6 and tcp4-dsoj8 were individually crossed with mir319a and mir319b. tcp4-dsoj6 mir319b and tcp4-dsoj8 mir319b frequently produced the cotyledon fusion phenotype, and the extent of their irregularity was high in comparison to the individual single mutants (Fig. 5; Supplemental Table S1). These results indicated that a synergistic effect existed between the tcp4-d and mir319 mutations. As reported for floral morphogenesis (Nag et al., 2009), tcp4-dsoj6 mir319a seedlings exhibited a wild-type-like morphology (Fig. 5; Supplemental Table S1). Since mir319a has sequence complementary to tcp4-dsoj6 mRNA (Supplemental Fig. S2), the mutated miR319a may target the tcp4-dsoj6 mRNA in plant cells, leading to a wild-type-like phenotype. The homozygous tcp4-dsoj8 mir319a mutant produced no seeds and was a more severely defected phenotype of tcp4-dsoj8. Since tcp4-dsoj8 mir319a mutations led to the replacement of two nucleotides in the complementary sites of miR319 and TCP4 mRNA (Supplemental Fig. S2), the double mutations might severely impair fertility and embryogenesis in comparison with tcp4-dsoj8.

As mir319 mutations enhanced the defective effect of cuc genes on cotyledon boundary formation (Fig. 3), we further examined whether tcp4-d mutations stimulated the cotyledon fusion phenotype in cuc backgrounds. After crossing the tcp4-dsoj6 and tcp4-dsoj8 mutants with the cuc2 and cuc3 mutants, all combinations of double tcp4-d cuc mutants exhibited the cotyledon fusion phenotype, with remarkably high extent (Fig. 5; Supplemental Table S1). These results demonstrated that the tcp4-d mutations enhanced the defective effect of the cuc2 and cuc3 mutations on cotyledon boundary formation. Since the TCP4 and CUC1 loci are located at close positions on chromosome 1, no homozygous tcp4-d cuc1 double mutant plants were obtained in this study.

These effects of the gain-of-function TCP4 gene mutation are consistent with those of the loss-of-function MIR319 gene mutation. Taken together, our results provide genetic evidence that release of the TCP genes from miR319-targeted negative regulation impairs the formation of the cotyledon boundary and leaf serrations.

Mutations in TCP Genes Stimulated the Complexity of Leaf Forms

The phylogenetic analysis divided the eight TCP genes into two clades, comprising the miR319-targeted and nontargeted TCP genes (Supplemental Fig. S1). The involvement of miR319-targeted TCP genes in leaf development has been extensively investigated; however, the role of nontarget TCP genes, namely, TCP5, TCP13, and TCP17, has not been fully clarified. We previously obtained genetic evidence showing the actions of the TCP5 and TCP13 genes with the five miR319-targeted TCP genes in the regulation of leaf development (Koyama et al., 2010). To extend our findings, here, we clarified the involvement of TCP17 in the regulation of leaf development. The T-DNA insertion line of TCP17 (tcp17) was identified and subsequently crossed with tcp5 and tcp13 to generate tcp5 tcp13 tcp17 (tcp5/13/17; Supplemental Fig. S7, A and B; Alonso et al., 2003). The tcp17, tcp5/17, and tcp5/13/17 mutants generated normal leaves with respect to the wild-type-like margin and surface flatness (Supplemental Fig. S7C), indicating the redundancy of these nontarget TCP genes.

In contrast, tcp3 tcp4 tcp5 tcp10 tcp13 tcp17 (tcp3/4/5/10/13/17) formed wavy and serrated leaves, and this abnormality was more severe than that of tcp3 tcp4 tcp10 (tcp3/4/10) leaves (Fig. 6A; Supplemental Fig. S7D). Therefore, tcp5/13/17 mutations enhanced the leaf abnormalities caused by the tcp3 tcp4 tcp10 mutation (Koyama et al., 2010). Consistent with the observation that TCP transcription factors negatively regulate the expression of CUC genes, tcp3/4/10 and tcp3/4/5/10/13/17 leaves had markedly increased CUC transcript levels in comparison with the wild-type leaves (Fig. 6B). In addition, tcp3/4/10 and tcp3/4/5/10/13/17 mutants inhibited hypocotyl elongation (Supplemental Fig. S8). This phenotype was in contrast to enhanced hypocotyl elongation due to the ectopic activation of the miR319-targeted TCP genes in mir319a/b and Pro-35S:mTCP plants (Fig. 1, C and D; Palatnik et al., 2003; Koyama et al., 2007).

Figure 6.

Figure 6.

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Effects of multiple mutants of the TCP gene subfamily on leaf morphogenesis. A, Photographs of rosettes and the sixth leaves of wild-type, tcp3/4/10, and tcp3/4/5/10/13/17 plants. Bars = 1 cm. B, Expression analysis of CUC1, CUC2, and CUC3 genes. The levels of CUC1, CUC2, and CUC3 transcripts determined by RT-PCR analysis were normalized using levels of the UBQ1 transcript, and the values for the wild type were set at 1. Error bars and asterisks indicate the sd and significant difference of six biological replicates (Student’s t test; *P < 0.05, **P < 0.01), respectively. C, Photographs of the rosettes (top) and shoot apexes (bottom) of 15-d-old wild-type and Pro-35S:MIR319A tcp3/4/5/10/13/17 plants. Asterisks in the bottom panels indicate initiating leaves. Bars = 1 mm.

Furthermore, all eight TCP genes were simultaneously downregulated following the introduction of the Pro-35S:miR319A gene, which dramatically reduced TCP2 and TCP24 transcripts, as reported previously (Palatnik et al., 2003; Koyama et al., 2007; Efroni et al., 2008), in tcp3/4/5/10/13/17 plants (Supplemental Fig. S7E). The Pro-35S:miR319A tcp3/4/5/10/13/17 seedlings exhibited ectopic shoot meristem formation and usually produced several leaves with a jagged and wavy shape but later ceased their development (Fig. 6C). The dysfunctionality in shoot meristems of Pro-35S:miR319A tcp3/4/5/10/13/17 seedlings was consistent with the phenotype of the transgenic plants expressing chimeric TCP repressor genes (Koyama et al., 2007). Collectively, the results of our analyses provided genetic evidence that, although TCP5, TCP13, and TCP17 genes were not targets of miR319, these genes acted in concert with the miR319-targeted TCP genes during leaf development, hypocotyl elongation, and shoot meristem formation.

Roles of miR319 and TCP Genes in the Onset of Leaf Senescence

A previous study found that Pro-35S:mTCP4 plants stimulate leaf senescence, whereas ectopic accumulation of miR319 delays leaf senescence (Schommer et al., 2008; Koyama 2014). In line with this, Pro-35S:mTCP3, tcp4-dsoj8 and mir319a/b exhibited precocious yellowing of leaves. mir319a/b reduced the chlorophyll content, a marker of leaf senescence, earlier than in wild type (Fig. 7, A and B). Conversely, the tcp3/4/10 and tcp3/4/5/10/13/17 mutants exhibited delayed yellowing of leaves and sustained the chlorophyll content (Fig. 7, C and D). The tcp3/4/5/10/13/17 mutant leaves remained green for the longest compared with the other genotypes investigated. These results demonstrate that the differential TCP activities caused by mutations in the MIR319 and TCP genes altered the pace of leaf senescence.

Figure 7.

Figure 7.

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Roles of MIR319 and TCP genes in the onset of leaf senescence. A, A photograph showing wild-type, mir319a/b, tcp4-dsoj8, and Pro-35S:mTCP3 leaves 65 d after germination. The oldest 14 leaves from the indicated genotypes are presented. Bar = 1 cm. B, The relative chlorophyll content of the sixth leaves of wild-type and mir319a/b plants at indicated ages. The error bars and asterisks indicate the sd and significant difference of 16 biological replicates (Student’s t test; ***P < 0.001), respectively. C, A photograph showing wild-type, tcp3/4/10, and tcp3/4/5/10/13/17 leaves 75 d after germination. The detailed description is represented in A. D, The relative chlorophyll content of the sixth leaves of wild-type, tcp3/4/10, and tcp3/4/5/10/13/17 leaves at the indicated ages. A detailed description of SPAD values is represented in B.

DISCUSSION

The interplay of miRNA networks, including miR319 and TCP genes, is a central process in the regulation of leaf development (Chitwood and Sinha, 2016; Rodriguez et al., 2016; Fouracre and Poethig, 2016). To obtain genetic insights into the role of miR319 in the regulation of leaf development, we generated loss-of-function alleles for miR319 and conducted a phenotypic analysis of mir319a/b. Since mir319a and mir319b mutations moderately reduced the size of leaf serrations and the mir319a/b mutation almost suppressed their formation, our results indicate that these MIR319 genes act in a largely quantitative manner during leaf serration formation. Consistently, mir319a/b double mutation increased transcripts of the five TCP genes, but each single mutation caused mild effects on the transcript levels (Fig. 2). This quantitative mode of miR319 action is different from that of miR164, which targets a family of transcription factor genes including CUC1 and CUC2, since the MIR164A gene, among the three MIR164 genes, specifically contributes to the regulation of leaf serration formation (Nikovics et al., 2006; Sieber et al., 2007). Since loss-of-function alleles for MIR319C were not available from, e.g., public resource centers, the contribution of MIR319C in leaf development remains to be clarified.

Our results demonstrate that combined mutations in the miR319-mediated regulatory pathway impair the cotyledon boundary. mir319a/b and tcp4-dsoj6 mutations exhibited normal cotyledon morphology, and the tcp4-dsoj8 mutant weakly exhibited a cotyledon fusion phenotype. In contrast, the combination of tcp4-dsoj6 mir319b and tcp4-dsoj8 mir319b mutations induced the cotyledon fusion phenotype. Therefore, these two independent mutations in the miR319-mediated regulatory pathway act synergistically to inhibit cotyledon boundary formation, whereas the contribution of each mutation alone is relatively small. Compared with the leaf serrations, the cotyledon boundary is likely to be tolerant against the genetic mutations in the miR319-mediated regulatory pathway. Consistently, a tomato homozygous lanceolate mutation impairs its cotyledons and shoot meristem, while a heterozygous mutation is sufficient for conversion of the originally complex leaf form into a simple form (Mathan and Jenkins, 1962; Settler, 1964; Ori et al., 2007).

An important finding of this study is that mutations in the miR319-mediated regulatory pathway enhance the defective effects of cuc mutations on the phenotype. The crossing of mir319a, mir319b, tcp4-dsoj6, and tcp4-dsoj8 with one of three cuc mutations remarkably reduced the size of leaf serrations and induced the cotyledon-fusion phenotype to a considerably high degree. In particular, the cuc2 mutation in combination with tcp4-dsoj6 or tcp4-dsoj8 resulted in the highest frequency of the cotyledon-fusion phenotype. Since the contribution of cuc2 to the cotyledon-fusion phenotype is greater than that of cuc1 and cuc3 (Hibara et al., 2006), our results reflect the main contribution of the cuc2 mutation to cotyledon development. Furthermore, a mutation in one of the CUC genes substantially recovers the phenotype of irregularly serrated cotyledons induced by the chimeric TCP3 repressor gene in transgenic plants (Koyama et al., 2007). The cuc2 mutation suppresses the morphology of the serrated leaf margin caused by the overexpression of the MIR319 gene (Hasson et al., 2011). Overall, these observations provide the genetic evidence of the miR319-mediated control of the TCP genes on the regulation of CUC genes without the overexpression constructs of TCP and MIR319 genes and are consistent with the previous reports of the negative actions of TCP transcription factors on the activities of CUC genes (Fig. 8A; Koyama et al., 2007, 2010; Rubio-Somoza et al., 2014).

Figure 8.

Figure 8.

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Roles of miR319 and TCP genes in leaf morphogenesis. A, A schematic representation of the action of MIR319 and TCP genes. T bars and arrows indicate negative and positive relationships, respectively. miR319 posttranscriptionally represses its target TCP genes downstream of developmental inputs. The miR319-taregeted and nontargeted TCP transcription factors cooperatively regulate their downstream genes, such as CUC genes, for the cotyledon boundary and leaf serration formation and for other physiological responses. B, Differentially modified leaf forms induced by TCP genes activated to various degrees. The sixth leaves of the indicated genotypes are arranged in a line. Bars = 1 cm. Furthermore, details on the mutants are as follows: mir319a/b, tcp3/4/5/10/13/17 (this work), Pro-35S:mTCP3 (Koyama et al., 2007), tcp4-d (Palatnik et al., 2007; this work), and tcp3/4/10, tcp3/4/5/10, tcp3/4/5/10/13 (Koyama et al., 2010).

Regarding the function of TCP genes, we demonstrate that, in concert with the miR319-targeted TCP genes, nontargeted TCP genes regulate leaf development in a highly redundant manner. Reasonably, increased expression of the CUC transcripts in triple and sextuple tcp mutants is associated with drastically complex leaf forms. Extending previous reports on the isolation of mir319- and tcp-related single mutants (Koyama et al., 2007, 2010; Palatnik et al., 2007; Nag et al., 2009), we have generated a series of mutants possessing TCP genes with varying levels of activation. Inhibition of TCP genes increases the complexity of leaves, but conversely, their activation simplifies the complexity of leaves (Fig. 8B). In addition to tcp5/13/17, all single and double mutants tested exhibited the wild-type-like form (this work; Koyama et al., 2007, 2010). Therefore, in addition to control by miR319, the extreme redundancy of TCP genes underlies the generation of differentially modified leaf forms. Our results showing the dysfunctionalities of shoot meristems in Pro-35S:miR319A tcp3/4/5/10/13/17 shed light on another important aspect of this redundancy. We can speculate that, if a certain TCP gene centralizes roles played by all eight TCP genes, a genetic mutation in this single gene would be lethal.

In the above context, miR319 finely tunes the expression of its target TCP genes downstream of unidentified developmental inputs. The overlapping but slightly different expression pattern of the miR319-targeted and nontargeted TCP genes in cotyledons and leaves confers robustness to the regulation of leaf development (Fig. 8A; Palatnik et al., 2003, Koyama et al., 2007; Alvarez et al., 2016). With respect to the downstream network that includes CUC genes, TCP transcription factors directly regulate genes related to the functions of miRNA, differentiation, cell cycling, and plant hormones (Fig. 8A; Schommer et al., 2008, 2014; Koyama et al., 2010; Rodriguez et al., 2010; Efroni et al., 2013; Danisman et al., 2013; Rubio-Somoza and Weigel, 2013; Ballester et al., 2015; Challa et al., 2016). Since TCP transcription factors can form homo- and heterodimers within and between the subfamilies (Kosugi and Ohashi, 2002; Giraud, et al., 2010; Danisman et al., 2013; Kubota et al., 2017), it is possible that each TCP shares its downstream genes in an overlapping manner. In addition, TCP-interacting proteins seem to participate in the modification of protein levels (Li et al., 2012; Efroni et al., 2013; Tao et al., 2013; Li and Zachgo 2013; Ho and Weigel 2014; Wei et al., 2015). However, a view of the whole network intertwined by TCP genes remains to be clarified.

Furthermore, miR319 and TCP genes gradually regulate leaf senescence. mir319a/b induced LOX2, whereas tcp3/4/10 and tcp3/4/5/10/13/17 inhibited LOX2. Therefore, LOX2, which encodes a jasmonate biosynthesis enzyme and accelerates leaf senescence may mediate these senescence-related phenotypes (He et al., 2002; Schommer et al., 2008). In addition, large-scale expression analyses illustrate the down-regulation of TCP genes in association with leaf senescence but do not show any difference in the miR319 level during leaf senescence (Breeze et al., 2011; Thatcher et al., 2015). Thus, miR319 appears to finely tune the activity of TCP genes in order to set the appropriate pace of leaf senescence rather than exclusively inhibit their activity in the senescent leaves.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana; Columbia-0) was used throughout the study unless otherwise indicated. mir319a123 (Landsberg erecta; Nag et al., 2009) was crossed three times with Columbia-0 before use for standardization of the genetic background. mir319b was obtained from the GABI_KAT collection (Rosso et al., 2003). soj6 and soj8 (Palatnik et al., 2007) were outcrossed twice with Col-0 to eliminate an enhancer trap construct. cuc1-5, cuc2-3, and cuc3-105 have been described previously (Hibara et al., 2006). To generate the double mutants indicated in the text, the respective single mutants, in appropriate combinations, were crossed. tcp17 was obtained from the Arabidopsis Bioresource Center (Alonso et al., 2003) and crossed with tcp5 and tcp13 (Koyama et al., 2010) for the generation of tcp5 tcp17 and tcp13 tcp17, respectively. These double tcp mutants were crossed for the generation of the tcp5/13/17 mutant. To generate the sextuple tcp mutant, we first established triple mutants of tcp3/5/17, tcp3/4/5, and tcp3/5/13, using the alleles established (this work; Koyama et al., 2007, 2010). Next, independent crossings of tcp3/4/5 with tcp3/5/13 and tcp3/5/17 resulted in the generation of tcp3/4/5/13 and tcp3/4/5/17, respectively. tcp3/4/5/13 and tcp3/4/5/17 were further crossed for the generation of tcp3/4/5/13/17. Finally, tcp3/4/5/13/17 and tcp3/4/5/10/13 (Koyama et al., 2010) were crossed for the generation of tcp3/4/5/10/13/17. To determine plant genotypes, the single-base nucleotide changes corresponding to mir319a123, soj6, and soj8 were determined by sequencing of the respective genomic regions. Mutations due to the T-DNA insertions were identified by genomic PCR using the appropriate sets of T-DNA- and gene-specific primers, and RT-PCR using gene-specific primers (Supplemental Table S2).

For the growth of plants, seeds were sterilized by sodium hypochlorite and grown on a plate containing half-strength Murashige and Skoog salts, 0.5 g/L MES, and 5 g/L Suc under 16-h-light/8-h-dark conditions, unless otherwise indicated. The Pro-35S:MIR319A construct was described previously (Koyama et al., 2007) and was introduced into tcp3/4/5/10/13/17 for the generation of Pro-35S:miR319A tcp3/4/5/10/13/17 plants using Agrobacterium tumefaciens-mediated gene transformation (Koyama et al., 2010).

Gene Expression

Plant tissues were harvested 9 to 11 h after removal from the dark to minimize potential effects of circadian rhythms on the transcriptional profile of TCP and the target genes (Giraud et al., 2010). Aliquots of total RNA were prepared from plant tissues, reverse-transcribed using an oligo(dT) primer, and subjected to real-time PCR analysis with a CFX96 real-time PCR system (Bio-Rad) using appropriate sets of primers (Supplemental Table S2). A standard curve derived from the reference sample was used to confirm the correct amplification efficiency of the primer pairs and to calculate the transcript levels of the genes of interest. The relative values of the transcript levels were normalized to that of UBQ1. Similar patterns of relative values were obtained when the results were normalized using another internal control gene, PP2AA3.

Light Microscopy Analysis

For the observation of cotyledons, seedlings were grown on a plate for 7 d. For observation of leaf serrations, each experiment was conducted using either set of genotypes as indicated in Figures 1A and 4A. Seeds were sowed on soil and seedlings germinated were individually transferred to specialized plastic pots (Arasystem 360; Betatech). The sets of genotypes were arranged side by side in a platter pooled with water. Plants were nourished with 1 mL of liquid fertilizer (HYPONeX) once a week and grown under 12-h-light/12-h-dark conditions, in which plants prolonged the vegetative growth phase. The 10th and 14th leaves were detached at 5 and 6 weeks old, respectively. Detached leaves were covered with a conventional slide grass, and their serration in the margin of the basal end was photographed using a M205 stereomicroscope (Leica Microsystems). The scale of a serration from its tip to base end was measured from using LAS AF software (Leica Microsystems). Statistical analysis and box-plot development were performed using BellCurve_for_Excel software with the default settings (https://bellcurve.jp/ex/). One-way ANOVA was performed, followed by the Tukey-Kramer method for multiple comparisons. P < 0.05 was considered statistically significant.

Senescence Assay

Plants were grown on soil under a 12-h-light/12-h-dark cycle. To calculate the relative chlorophyll contents, the sixth leaves of the individual genotypes were measured using a SPAD 502-devise (Konica-Minolta; Ling et al., 2011), and the SPAD values were averaged from 16 biological replicates of arbitrary units.

Accession Numbers

Sequence data from this article have been deposited in the GenBank/EMBL data libraries under the following accession numbers: TCP2 (AT4G18390), TCP3 (AT1G53230), TCP4 (AT3G15030), TCP5 (AT5G60970), TCP10 (AT2G31070), TCP13 (AT3G02150), TCP17 (AT5G08070), MIR319A (AT4G23713), MIR319B (AT5G41663), MIR319C (AT2G40805), MIR164A (At2g47585), INDOLE-3-ACETIC ACID3/SHORT HYPOCOTYL2 (At1g04240), CUC1 (At3g15170), CUC2 (At5g53950), and CUC3 (At1g76420).

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank Drs. Detlef Weigel, Thomas Jack, Masao Tasaka, and Masamitsu Aida and the stock centers of GABI_KAT and the Arabidopsis Bioresource Center for providing seeds, Akiko Kuwazawa for her excellent technical assistance, and Drs. Shigetada Nakanishi and Honoo Satake for stimulating discussions.

Footnotes

1

This work was supported by the Japan Society for the Promotion of Science KAKENHI (grant no. 26440158 to T.K.).

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