microRNA172 down-regulates glossy15 to promote vegetative phase change in maize

Abstract

Shoot development in many higher plant species is characterized by phase change, where meristems and organs transition from one set of identities to another. The transition from a juvenile to adult leaf identity in maize is regulated by the APETALA2-like gene glossy15 (gl15). We demonstrate here that increasing gl15 activity in transgenic maize not only increases the number of leaves expressing juvenile traits, but also delays the onset of reproductive development, indicating that gl15 plays a primary role in the maintenance of the juvenile phase. We also show that the accumulation of a maize microRNA homologous to miR172 increases during shoot development and mediates gl15 mRNA degradation. These data indicate that vegetative phase change in maize is regulated by the opposing actions of gl15 and miR172, with gl15 maintaining the juvenile phase and miR172 promoting the transition to the adult phase by down-regulation of gl15. Our results also suggest that the balance of activities between APETALA2-like genes and miR172 may be a general mechanism for regulating vegetative phase change in higher plants.

Fig. 1.

gl15 transgenic lines. (A) Map of the gl15 genomic DNA fragment used in transformation experiments. Exons are thick black arrows or vertical lines; the promoter region is a white arrow. Positions of miR172 target site, AP2 DNA-binding domain, and primers used in RT-PCR are shown. (B) DNA gel blot of Gl15-TG events and the H99 parental lines hybridized with a gl15 probe. Lanes 1–6 contain genomic DNA of R0 plants; lane 7 contains genomic DNA from line Gl15-TG7 in a homozygous gl15-H background and, thus, lacks the band corresponding to the endogenous gl15 allele present in H99, indicated by the arrowhead. (C) Toluidine blue-stained leaf epidermal peels from the H99 inbred (a, c, and e) or Gl15-TG7 (b, d, and f). Peels are from leaf 4 (a and b), leaf 8 (c and d), and leaf 12 (e and f). Juvenile leaves stain pink, whereas adult leaf tissue stains aqua with macrohairs present within purple-staining files of bulliform cells.

Fig. 2.

gl15 mRNA and miR172 expression during shoot development. (a) Real-time PCR was used on cDNA to assay gl15 mRNA expression in shoot tissue at various DAS. Relative fold expression (2^-(ΔΔcT)) in tissue from all three genotypes is reported by using 12 DAS H99 as the reference sample. (b) Northern analysis of miR172 accumulation in shoot apices from 6 to 24 DAS. The positive control (S) is 0.5 or 1.0 pmol of the 21-nucleotide sense DNA oligo.

Fig. 3.

gl15 mRNA and miR172 expression during shoot and leaf development. (a) Northern analysis of miR172 accumulation in H99 shoot apex only (A) or leaf only (L) tissues harvested between 7 and 14 DAS. The blot (Right) shows 3-cm leaves further divided into middle (Lm) and basal (Lb) thirds. (b) Relative fold expression (2^-(ΔΔcT))of gl15 mRNA in the same samples as a is reported by using 7 DAS apex as the reference sample. (c)5′ RACE mapping of gl15 cDNA termini within the miR172 binding site. The numbers of clones with particular termini are indicated for both the 10 DAS H99 shoot apex sample (top line) and 14 DAS H99: Gl15-TG7/0 shoot apex sample (second line). The arrow pointing to the right indicates a gl15 transcript that terminated 3′ of the miR172 binding site. The sequences of three miR172 genes in Arabidopsis and their alignment to the gl15 mRNA are also shown with lowercase letters indicating mismatches.