miR319a targeting of TCP4 is critical for petal growth and development in Arabidopsis

Abstract

In a genetic screen in a drnl-2 background, we isolated a loss-of-function allele in miR319a (miR319a(129)). Previously, miR319a has been postulated to play a role in leaf development based on the dramatic curled-leaf phenotype of plants that ectopically express miR319a (jaw-D). miR319a(129) mutants exhibit defects in petal and stamen development; petals are narrow and short, and stamens exhibit defects in anther development. The miR319a(129) loss-of-function allele contains a single-base change in the middle of the encoded miRNA, which reduces the ability of miR319a to recognize targets. Analysis of the expression patterns of the three members of the miR319 gene family (miR319a, miR319b, and miR319c) indicates that these genes have largely non-overlapping expression patterns suggesting that these genes have distinct developmental functions. miR319a functions by regulating the TCP transcription factors TCP2, TCP3, TCP4, TCP10, and TCP24; the level of RNA expression of these TCP genes is down-regulated in jaw-D and elevated in miR319a(129). Several lines of evidence demonstrate that TCP4 is a key target of miR319a. First, the tcp4(soj6) mutant, which contains a mutation in the TCP4 miRNA-binding site complementary to the miR319a(129) mutation, suppresses the flower phenotype of miR319a(129). Second, expression of wild-type TCP4 in petals and stamens (i.e., AP3:TCP4) has no effect on flower development; by contrast, a miRNA-resistant version of TCP4, when expressed in petals and stamens (i.e., pAP3:mTCP4) causes these organs not to develop. Surprisingly, when AP3:TCP4 is present in a miR319a(129) background, petal and stamen development is severely disrupted, suggesting that proper regulation by miR319a of TCP4 is critical in these floral organs.

Fig. 1.

miR319a¹²⁹ mutants exhibit dramatic defects in flower development. (A) drnl-2 mutant flower. Whorl 3 organs are either missing or develop as small filaments; whorl 2 petals are irregularly shaped. One petal has been removed. (B) drnl-2 miR319a¹²⁹ flower. Both second whorl petals and third whorl stamens are converted to filamentous organs. One sepal was manually removed. (C) SEM of a drnl-2 miR319a¹²⁹ flower. Two sepals have been removed. (D) miR319a¹²⁹ flower. Sepals, petals and stamens are stunted; petals are narrow. (E) Wild-type anther exhibiting characteristic four-lobed structure. (F) Anthers from miR319a¹²⁹ flowers are misshapen and lack an organized four-lobed structure. (G) Transformation of a T-DNA that contains a 3.7-kb genomic DNA containing the miR319a gene rescues the miR319a¹²⁹ flower mutant phenotype. (H) SEM of a miR319a¹²⁹ flower showing a stamen congenitally fused to the valve of the carpel. Some perianth organs were manually removed. (I) miR319a¹²⁹ flower showing a stamen congenitally fused to the valve of the carpel. The fused anther produces pollen. (J) Floral organ size comparison between wild-type and miR319a¹²⁹. Two-sample unequal variance directional t test was used to test significance of the difference. All of the numbers were significantly different between wild-type and mutant (*, P < 0.05; **, P < 0.005). P values were 0.013, 0.006, 0.000, and 0.020 for sepal length, petal length, petal width and stamen length respectively. Error bars, standard error of mean (SEM). n = 51–121.

Fig. 2.

TCP and miR319 expression. (A) Mature miRNA sequence of three miR319 genes in Arabidopsis: miR319a, miR319b, and miR319c. miR319a and miR319b encode the identical miRNA. The mutation (G to A) in miR319a¹²⁹ is indicated by the arrow above the miR319a sequence. The miR319 genes target a subset of TCP transcription factors such as TCP4. The tcp4^soj6 mutation (C to T) is indicated by the arrow below the TCP4 sequence. The tcp4^soj6 and miR319a¹²⁹ mutations are complementary. (B) Whole-mount miR319c:GUS seedling. The base of young leaf primordia is strongly stained. (C) Section through a miR319c:GUS seedling. The base of young leaf priordia is strongly stained. (D) Whole-mount miR319a:GUS seedling. Stipules are strongly stained. (E) Section through miR319a:GUS seedling. Stipules are strongly stained. (F) Section through two miR319c:GUS flowers. In the stage 5 flower (Right), the pedicel and base of organ primordia are stained. In the stage 8 flower (Left), the petals, stamen filaments, and the base of the carpels and sepals are stained. Flowers staged according to Smyth et al. (25). (G) Section through a stage 10 miR319a:GUS flower. The proximal region of the petals is stained. (H) Section through a stage 8 miR319a:GUS flower. The petals are strongly stained. (I) Section through three miR319a:GUS flowers. In the stage 5 flower (Center) GUS activity is detectable at the base of the sepals and throughout developing second, third, and fourth whorl organs. In the stage 6 (Left) and stage 7 (Right) flowers, GUS activity is detectable at the base of the sepals and carpels, and at high levels throughout developing petals. In stamens, higher levels of GUS activity are detected in the stamen filament primordia compared to anther primordia. (J) Whole-mount miR319b:GUS inflorescence. The sepal and stamen abscission zones of late stage flowers are stained. (K) qRT-PCR demonstrates that TCP2, TCP3, TCP4, TCP10, and TCP24 RNA levels increase between 1.4- and 2.5-fold in inflorescences from miR319¹²⁹ mutants as compared to wild-type. TCP genes are down-regulated in jaw-D inflorescences. Error bars, SEM. All values are significantly different from wild-type. P values for TCP2, TCP3, TCP4, TCP10, and TCP24 are 0.000, 0.028, 0.006, 0.029, and 0.029 respectively for miR319a¹²⁹ and 0.026, 0.003, 0.000, 0.001, and 0.003 respectively for jaw-D. *, P < 0.05, ^‡, P < 0.005.

Fig. 3.

miR319a targets TCP4. (A) AP3:mTCP4 flowers consists of only sepals and carpels; petals and stamens are missing. (B) Scanning electron micrograph (SEM) of a AP3:mTCP4 flower. One sepal has been manually removed to allow visualization of the inner whorls. (C) AP3:mTCP4 flower (Right) is dramatically smaller than wild-type flower (Left). (D) 35S:TCP4 seedlings are normal. (E) 35S:mTCP4 seedlings are seedling lethal. (F) 35S:TCP4 miR319a¹²⁹ seedlings are seedling lethal and resemble 35S:mTCP4 seedlings. (G) AP3:TCP4 miR319a¹²⁹ flowers (Upper Left) and inflorescence (Right). AP3:TCP4 miR319a¹²⁹ flowers resemble AP3:mTCP4 flowers (A–C) and are much smaller than wild-type (Center) or AP3:TCP4 flowers (Inset).

Fig. 4.

tcp4^soj6 suppresses the phenotype of both miR319a¹²⁹ and miR319a¹²⁹ drnl-2. (A) tcp4^soj6 flowers have a wild-type phenotype. (B) miR319a¹²⁹ flowers have several defects including narrow petals and short stamens. (C) The petals of tcp4^soj6 miR319a¹²⁹ double mutants are taller and more lobed than miR319a¹²⁹ single mutants. (D) Wild-type flower. (E) drnl-2 flowers exhibit strongest phenotypic effects in stamens while petals are only mildly affected. (F) drnl-2 miR319a¹²⁹ flowers have dramatically reduced and filamentous petals and stamens. (G) Compared to the drnl-2 miR319a¹²⁹ double mutant (F), the drnl-2 miR319a¹²⁹ tcp4^soj6 triple mutant develops petals in whorl 2. (H) A model for petal development mediated by DRNL, miR319a, and TCP4. drnl-2 single mutants exhibit only minor defects in petal development, whereas miR319a¹²⁹ mutants have narrow petals that are half as wide as wild-type petals. miR319a¹²⁹ drnl-2 double mutants exhibit a very dramatic defect in petal development characterized by filamentous petal formation. The petal defect in the double mutant is rescued by tcp4^soj6 suggesting that the phenotypic enhancement in drnl-2 is mediated through TCP4. Thus, TCP4 (or a TCP4 target) is a critical convergence point in the petal development pathway controlled by both DRNL and miR319a. Although proper regulation of TCP4 is essential for petal development in a drnl-2 background, tcp4^soj6 cannot rescue the filamentous stamen phenotype in whorl 3 of drnl-2 miR319a¹²⁹ double mutants indicating the phenotypic defects in whorl 3 of drnl-2 are not mediated by TCP4.