A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development

Abstract

Plant microRNAs (miRNAs) show a high degree of sequence complementarity to, and are believed to guide the cleavage of, their target messenger RNAs. Here, I show that miRNA172, which can base-pair with the messenger RNA of a floral homeotic gene, APETALA2, regulates APETALA2 expression primarily through translational inhibition. Elevated miRNA172 accumulation results in floral organ identity defects similar to those in loss-of-function apetala2 mutants. Elevated levels of mutant APETALA2 RNA with disrupted miRNA172 base pairing, but not wild-type APETALA2 RNA, result in elevated levels of APETALA2 protein and severe floral patterning defects. Therefore, miRNA172 likely acts in cell-fate specification as a translational repressor of APETALA2 in Arabidopsis flower development.

Fig. 1

Sequence complementarity between AP2 RNA and miRNA172 and AP2 RNA and protein accumulation in various genotypes. (A) A diagram of the AP2 mRNA showing the AP2 domains (hatched rectangles) and the miRNA172 binding site (black rectangle). The mutant nucleotides in AP2m1 and AP2m3 are circled. Although AP2m1 causes a Phe-to-Leu amino acid substitution (underlined codon), AP2m3 does not change the amino acid sequence. Nucleotides in miRNA172 that can base-pair with AP2 RNA (with G:U pairing allowed) are in bold. The ap2-2 allele is a splice acceptor site mutation that would generate a stop codon at the indicated position (arrowhead). 3′UTR, 3′ untranslated region; 5′UTR, 5′ untranslated region. (B) Accumulation of miRNA172 in inflorescences. The stained gel below indicates the amount of RNA in each lane. wt, wild type. (C) AP2 RNA abundance in inflorescences. The numbers indicate the relative abundance of AP2 RNA in various genotypes. (D) AP2 protein abundance in inflorescences. Although the AP2 antisera reacted with a number of proteins (one indicated by the asterisk), the identity of the AP2 signal was revealed by its absence in the ap2-2 mutant. The numbers indicate the abundance of AP2, as calculated by using phosphoenolpyruvate carboxylase (PEPC) as the loading control.

Fig. 2

Analyses of 35S::MIR172 transgenic lines. (A) Leaf numbers of T1 transgenic lines. (B) Percentage of T1 transgenic lines with phenotypes that resemble plants with ectopic AG expression, such as curly leaves, carpelloid leaves, and ap2-like flowers. The total number of independent lines analyzed in (A) and (B) is in parentheses. The miRNA172 abundance in (C) 20-day seedlings of T2 lines, (D) 30-day T1 lines, and (E) inflorescences of T2 lines. The stained gels in (C) and (E) and the 5S rRNA hybridization in (D) indicate the amount of total RNA used. (F) AP2 RNA accumulation in 20-day seedlings (the five lanes on the left) and in inflorescences (the two lanes on the right) of T2 lines.

Fig. 3

Phenotypes of 35S::MIR172, 35S::AP2, and 35S::AP2m1 lines and in situ localization of miRNA172. (A) A wild-type flower. (B) An ap2-9 flower with first-whorl organs transformed into carpels (arrow). (C) A 35S::MIR172a-1 flower that closely resembles ap2 flowers. A cauline leaf with stigmatic tissues (D, arrows) and a curly rosette leaf (E) from 35S::MIR172a-1 plants. In situ hybridization using an anti-sense [(F) and (H)] or sense [(G) and (I)] miRNA172 probe on longitudinal sections of inflorescence meristems (IM) with two young floral meristems (FM) [(F) and (G)] and stage 7 flowers [(H) and (I)]. Hybridization signals are represented by the blue color. Size bar, 50 μm. The numbers in (H) and (I) represent the positions of the organs, with 1 being the outermost whorl. (J) A 35S::AP2m1 flower with numerous petals and loss of floral determinacy. (K) A 35S::AP2m1 flower with many staminoid organs and an enlarged floral meristem (arrow). (L) A 35S::AP2 flower with more stamens than the wild type.

Fig. 4

Analyses of 35S::AP2 and 35S::AP2m1 transgenic T1 lines. (A) The frequency of floral defects in 35S::AP2 and 35S::AP2m1 lines. (B) Flowering time as represented by the numbers of total leaves produced before flowering or the numbers of days it takes for the stem to reach 0.5 cm in length. (C) AP2 RNA accumulation in inflorescences from T1 transgenic plants containing the vector alone, 35S::AP2 (with normal flowers but flowers extremely late), or 35S::AP2m1 (with severe floral defects and flowers moderately late). Note that the tissues used were the same as the ones used in AP2 protein analysis shown in Fig. 1D. Numbers at the bottom indicate the relative amount of AP2 RNA in the various genotypes.