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

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.