ALTERED MERISTEM PROGRAM1 regulates leaf identity independently of miR156-mediated translational repression

Abstract

In Arabidopsis, loss of the carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) produces an increase in the rate of leaf initiation, an enlarged shoot apical meristem and an increase in the number of juvenile leaves. This phenotype is also observed in plants with reduced levels of miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors, suggesting that AMP1 might promote SPL activity. However, we found that the amp1 mutant phenotype is only partially corrected by elevated SPL gene expression, and that amp1 has no significant effect on SPL transcript levels, or on the level or the activity of miR156. Although AMP1 has been reported to promote miRNA-mediated translational repression, amp1 did not prevent the translational repression of the miR156 target SPL9 or the miR159 target MYB33. These results suggest that AMP1 regulates vegetative phase change downstream of, or in parallel to, the miR156/SPL pathway, and that it is not universally required for miRNA-mediated translational repression.

Keywords: AMP1; SPL; Translational repression; Vegetative phase change; miR156; miR159.

Fig. 1.

Elevated SPL gene activity only partially suppresses the amp1 phenotype. (A) Plants at 16 DAG. (B) Silhouettes of rosette leaves for the lines shown in A. (C) Percentage of individual plants that produced at least one rosette leaf with abaxial trichomes (ab. tri.) (n≥18). (D) Leaf primordia emergence was scored when leaves became visible without manipulation of the rosette. Data represent the mean±s.e.m (n≥18). (E) Total number of rosette leaves. (F) SAM size at 5 DAG. Images were taken using DIC microscopy, measurements were taken at the widest point of the SAM between emerging leaf primordia. (G) Inflorescence bolts were scored the day they visibly protruded from the rosette. Statistically distinct genotypes were identified by one-way ANOVA with post-hoc Tukey's multiple comparison test (letters indicate statistically distinct groups; P<0.05). All phenotypic analyses were carried out in LD. WT, wild type. Scale bars: 5 mm (A); 100 μm (F).

Fig. 2.

The amp1 phenotype is not associated with repressed SPL activity. (A-G) qRT-PCR analyses of gene expression. (A,B) Shoot apices with LP ≥1 mm removed at 8 DAG. (C-G) Isolated LP 0.5-1 mm in size. 8 DAG=LP1-2; 13-14 DAG=LP 4-5 [amp1 LP were harvested at 13 DAG and wild-type (WT) LP at 14 DAG]; 20 DAG=wild-type LP9-10, amp1 LP14-16. Data represent the mean±s.e.m. All plants were grown in SD conditions. Asterisks represent significant differences between genotypes calculated by an unpaired two-tailed Student's t-test (*P<0.05; **P<0.01; ***P<0.001).

Fig. 3.

Enhanced clv juvenility is a consequence of an increased rate of leaf initiation, rather than repressed SPL gene activity. (A) Plants at 21 DAG. Scale bar: 5 mm. (B) Silhouettes of rosette leaves for the lines shown in A. (C) First leaf to produce abaxial trichomes. Statistically distinct genotypes were identified by one-way ANOVA with post-hoc Tukey’s multiple comparison test (letters indicate statistically distinct groups; P<0.05). (D) Leaf emergence was scored when leaves became visible without manipulation of the rosette. The dashed lines indicate the first leaf to produce abaxial trichomes. Data represent the mean±s.e.m. (n≥23). (E,F) qRT-PCR analyses of gene expression of (E) shoot apices with leaf primordia ≥1 mm removed at 10 DAG and (F) isolated LP1 and LP2 0.5-1 mm in size. Asterisks represent significant differences between wild type (WT) versus clv3 or WT versus clv1. Significance was calculated by an unpaired two-tailed Student's t-test (P<0.05). (G) Staining of miR156-sensitive and miR156-resistant SPL-GUS reporter constructs at 14 DAG. Phenotypic analyses were carried out in LD (A-D) and gene expression analyses were carried out in SD (E-G).

Fig. 4.

miRNA-regulated SPL9 and MYB33 proteins accumulate normally in amp1. (A) The relative abundance of uncleaved/cleaved transcripts, normalized to wild type (WT) 8 DAG. See Fig. 2 legend for details of samples. (B) Staining of miR156-sensitive and miR156-resistant SPL-GUS reporter constructs at 21 DAG. (C,D) Quantification of sSPL9-GUS protein levels by image analysis. In C, top panels show RGB color mode and bottom panels hue saturation brightness mode. Red squares indicate where signal intensity was measured. In D, each dot represents an individual primordia. Data represent the mean±s.e.m. (E-G) Staining of GUS reporter constructs in 8 DAG seedlings (staining in two phenotypically distinct amp1 individuals is shown) (E), 7 DAG seedlings (F) and flowers (G). Scale bars: 5 mm (B); 200 µm (C); 1 mm (E-G).