Genetic analysis reveals functional redundancy and the major target genes of the Arabidopsis miR159 family

Abstract

Currently, there are very few loss-of-function mutations in micro-RNA genes. Here, we characterize two members of the Arabidopsis MIR159 family, miR159a and miR159b, that are predicted to regulate the expression of a family of seven transcription factors that includes the two redundant GAMYB-like genes, MYB33 and MYB65. Using transfer DNA (T-DNA) insertional mutants, we show that a mir159ab double mutant has pleiotropic morphological defects, including altered growth habit, curled leaves, small siliques, and small seeds. Neither mir159a nor mir159b single mutants displayed any of these traits, indicating functional redundancy. By using reporter-gene constructs, it appears that MIR159a and MIR159b are transcribed almost exclusively in the cells in which MYB33 is repressed, as had been previously determined by comparison of MYB33 and mMYB33 (an miR159-resistant allele of MYB33) expression patterns. Consistent with these overlapping transcriptional domains, MYB33 and MYB65 expression levels were elevated throughout mir159ab plants. By contrast, the other five GAMYB-like family members are transcribed predominantly in tissues where miR159a and miR159b are absent, and consequently their expression levels are not markedly elevated in mir159ab. Additionally, mMYB33 transgenic plants can phenocopy the mir159ab phenotype, suggesting that its phenotype is explained by deregulated expression of the redundant gene pair MYB33 and MYB65. This prediction was confirmed; the pleiotropic developmental defects of mir159ab are suppressed through the combined mutations of MYB33 and MYB65, demonstrating the narrow and specific target range of miR159a and miR159b.

Fig. 1.

Characterization and structure of the MIR159 loci. Mapping of the pri-MIR159 transcripts and the T-DNA insertion sites for MIR159a (A) and MIR159b (B). LB, left border; RB, right border; B, Basta-resistant gene. Arrows indicate transcriptional start sites with numbers indicating relative positions of the stem–loop regions and the varying polyadenylation sites. In both instances, the T-DNA loci were tandem inverted insertions because both plant–T-DNA junctions were isolated by using left border primers. (C) Relative RNA levels of pri-MIR159 transcripts as determined by quantitative RT-PCR on RNA prepared from seedlings of wild-type, mir159a, and mir159b plants. (D) RNA gel blot analysis of mature miR159 levels in 72-h imbibed seeds. (E) A phylogenetic tree based on the stem–loop sequences of rice and Arabidopsis MIR159 genes.

Fig. 2.

Phenotypic characteristics of mir159ab plants. (A) The smaller growth stature of mir159ab plants. (B) Curled leaf phenotype (left) compared with wild-type (right). (C) Shorter but fatter fruits in mir159ab plants (left). (D) Smaller, irregularly shaped seeds of mir159ab plants (left) compared with wild type (right).

Fig. 3.

RNA levels of the MIR159 genes and their targets. (A) Expression of MIR159a (black bars) and MIR159b (white bars) during seed imbibition. (B) Expression of MIR159a (black bars), MIR159b (white bars), and MIR159c (gray bars) in different plant tissues. SA, shoot apical region; IF, inflorescences; SD, 3-day-old imbibed seeds. (C) Expression of MYB33 (black bars), MYB65 (white bars), and MYB101 (gray bars) during seed imbibition. (D) Expression of the GAMYB-like genes in 3-day-old seedlings of wild type (black bars), mir159a (dark gray bars), mir159b (light gray bars), and mir159ab (white bars). (E) Expression of the GAMYB-like genes in wild-type (black bars) and mir159ab (white bars) rosettes. (F) Expression of the GAMYB-like genes in wild-type (black bars) and mir159ab (white bars) inflorescences. (G) Expression of the GAMYB-like genes in wild-type (black bars) and mir159ab (white bars) siliques. Values listed on the right side of graphs correspond to those of MYB101.

Fig. 4.

Spatial expression analysis of MIR159 and target genes. (A–D and I) GUS staining of MIR159a:GUS transgenic plants in inflorescence (A), root tips (B), emerging lateral roots (C), the shoot apex region (D), and seeds (I) imbibed for 24 h. (E–H and J) GUS staining of MIR159b:GUS transgenic plants in inflorescence (E), roots (F), emerging lateral roots (G), the shoot apex region (H), and 48-h-old seedlings (J). Staining of MIR159b:GUS lines was generally much stronger than that of MIR159a:GUS lines (K), MYB101 promoter:GUS in inflorescences (L), MYB33:GUS in wild-type (M), and MYB33:GUS in mir159ab. AF, anther filament; R, receptacle; S, sepal; A, anther; C, carpels.

Fig. 5.

Phenotypes of mMYB33 and mir159ab/myb33/myb65 plants. (A) Aerial views of rosettes of 5-week-old plants of wild-type, mir159ab, and three mMYB33 lines grown under short days. (B) Aerial views of 3-week-old rosettes of mir159ab and mMYB33 (line 2) grown under long days. Also shown are siliques, seeds, and mature plants from the same lines. (C) Quantitative RT-PCR of 6-week-old mature plants was used to determine the relative expression of total [(T)] MYB33 levels, endogenous [(E)] MYB33 (using a primer to the miR159 target site that solely amplifies the wild-type MYB33 allele), MYB65, pri-MIR159a, and pri-MIR159b transcripts. (D) Aerial view of rosettes of mir159ab/myb33, mir159ab/myb33/myb65, and myb33/myb65.