Roles of miR319 and TCP Transcription Factors in Leaf Development

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.

Figure 1.

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

Figure 2.

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.

Figure 3.

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

Figure 4.

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.

Figure 5.

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.

Figure 6.

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.

Figure 7.

Roles of MIR319 and TCP genes in the onset of leaf senescence. A, A photograph showing wild-type, mir319a/b, tcp4-d^soj8, 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.

Figure 8.

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).