Threshold-dependent repression of SPL gene expression by miR156/miR157 controls vegetative phase change in Arabidopsis thaliana

Abstract

Vegetative phase change is regulated by a decrease in the abundance of the miRNAs, miR156 and miR157, and the resulting increase in the expression of their targets, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. To determine how miR156/miR157 specify the quantitative and qualitative changes in leaf morphology that occur during vegetative phase change, we measured their abundance in successive leaves and characterized the phenotype of mutations in different MIR156 and MIR157 genes. miR156/miR157 decline rapidly between leaf 1&2 and leaf 3 and decrease more slowly after this point. The amount of miR156/miR157 in leaves 1&2 greatly exceeds the threshold required to specify their identity. Subsequent leaves have relatively low levels of miR156/miR157 and are sensitive to small changes in their abundance. In these later-formed leaves, the amount of miR156/miR157 is close to the threshold required to specify juvenile vs. adult identity; a relatively small decrease in the abundance of miR156/157 in these leaves produces a disproportionately large increase in SPL proteins and a significant change in leaf morphology. miR157 is more abundant than miR156 but has a smaller effect on shoot morphology and SPL gene expression than miR156. This may be attributable to the inefficiency with which miR157 is loaded onto AGO1, as well as to the presence of an extra nucleotide at the 5' end of miR157 that is mis-paired in the miR157:SPL13 duplex. miR156 represses different targets by different mechanisms: it regulates SPL9 by a combination of transcript cleavage and translational repression and regulates SPL13 primarily by translational repression. Our results offer a molecular explanation for the changes in leaf morphology that occur during shoot development in Arabidopsis and provide new insights into the mechanism by which miR156 and miR157 regulate gene expression.

Fig 1. Genes contributing to the production of miR156 and miR157 in vegetative shoots.

(A) Northern blot analysis of miR156 and miR157 levels in the shoot apices of 11-day-old wild-type Col and mir156/mir157 mutants grown in LD. In Col, the miR156 and miR157 probes hybridize to 21 and 20 nt bands. A comparison of the effect of different mutations on the intensity of these bands indicates that the 20 nt transcript is miR156 and the 21 band is primarily miR157, with a small contribution from miR156d. The intensity of the 20 nt and 21 nt bands on the blot probed with a 1:1 mixture of the miR156 and miR157 probes was quantified by normalizing the intensity of each band to t-Met, and then to Col; these data are shown in the table below the figure. (B) Northern blot analysis of miR156 and miR157 levels in LP1&2 of wild-type and mutant plants grown in SD. The blots were hybridized with 1:1 mixed miR156 and miR157 probes, and the results were quantified as described above.

Fig 2. miR157 is more abundant than miR156 and declines more slowly during shoot development.

(A) RT-qPCR analysis of miR156 and miR157 levels in successive leaf primordia of plants grown in SD. Values were normalized to the value in LP 1&2, and are the average of 3 biological replicates ± standard deviation. The shape of fully expanded leaves at the corresponding positions is shown at the top of the graph. Grey = no abaxial trichomes; Black = abaxial trichomes. (B) The estimated amount of miR156 and miR157 in successive leaf primordia. These values were calculated using experimental data for LP1&2, and the RT-qPCR data shown in (A).

Fig 3. The phenotype of plants mutant for genes encoding miR156 and miR157.

(A) Rosettes of three-week-old Col, and miR156 and miR157 single and multiple mutant plants grown in SD. (B) Representative scans of fully expanded rosette leaves 1 through 13 from Col and the miR156 and miR157 single and multiple mutant shown in (A). Grey = no abaxial trichomes; Black = abaxial trichomes.

Fig 4. The effect of miR156 and miR157 mutations on leaf morphology.

(A) The number of leaves without abaxial trichomes in single and multiple mir156 and mir157 mutants grown in SD. (B) The angle between lines tangent to leaf base in single and multiple mir156 and mir157 mutants grown in SD. a = significantly different from Col; b = significantly different from mir56a and miR156c; c = significantly different from miR15a and miR157c; d = significantly different from miR156a/c and miR157a/c; e = significantly different from miR156a/c mir157a/c; f = significantly different from mir156a/c/d mir157a/c (Student's t test; p < 0.01; n = 18 to 23). Error bars = standard deviation.

Fig 5. miR156 is more efficiently loaded into AGO1 than miR157.

AGO1-FLAG was immunoprecipitated (IP) from AGO1-FLAG/ago1-36 and Col (negative control) plants using a FLAG antibody. Small RNAs were then extracted from both the IP and non-IP fractions and subjected to Northern analysis using a mixed miR156/miR157 probe. miR157 (21 nt) was more abundant than miR156 (20 nt) in the input fraction, whereas miR156 and miR157 were about equally abundant in the IP fraction from AGO1-FLAG/ago1-36 plants.

Fig 6. miR156d is less effective than miR156a.

(A) The sequence of miR156 and miR157 transcripts and their target site in SPL transcripts. Only the SPL transcripts involved in vegetative phase change [21] are shown. Nucleotides that are mis-paired in the miR156/miR157:SPL duplex are shown in different colors. (B) The phenotype of transgenic lines expressing MIR156A or MIR156D under the regulation of the CaMV 35S promoter. Lines over-expressing miR156a have significantly more rosette leaves without abaxial trichomes, more total rosette leaves, and more cauline leaves than lines over-expressing miR156d. n = 8 to 12, ± SD.

Fig 7. RT-qPCR analysis of SPL transcript levels in 1 mm leaf primordia of Col and mir156/mir157 mutants grown in SD.

Most transcripts increase less than 2-fold between LP1&2 and LP3&4 in Col, and increase 2-fold or less in miR156 and miR157 mutants. Results are relative to the value for Col LP1&2. Error bars represent the SD for 3 biological replicates.

Fig 8. RT-qPCR analysis of SPL transcript levels in successive 1 mm leaf primordia of the mir156a/c mir157a/c mutant.

Error bars represent the SD for 3 biological replicates.

Fig 9. SPL9 and SPL13 contribute to the precocious phenotype of mir156 and mir157 mutants.

The morphology of fully expanded leaves and the number of leaves without abaxial trichomes in Col and mutant plants grown in SD. Grey = no abaxial trichomes; Black = abaxial trichomes. a = significantly different from Col; b = significantly different from mir156a/c, c = significantly different from miR156a/c mir157a/c. Student's T-test, ± SD.

Fig 10. Developmental variation in SPL9 and SPL13 protein levels is mediated primarily by miR156/miR157-directed translational repression.

(A) Developmental variation in miR156, SPL9-GUS mRNA, and SPL9-GUS protein in leaf primordia of Col plants grown in SD. miR156 and SPL9-GUS transcripts were measured by RT-qPCR, while the abundance of the SPL9-GUS protein was determined using the MUG assay for GUS activity. Values were normalized to the level in LP1&2, which was set to 1 for SPL9-GUS, and 10 for miR156. Data are the average of 3 biological replicates, ± SD. (B) Developmental variation in miR156, SPL13-GUS mRNA, and SPL13-GUS protein in the leaf primordia of Col plants grown in SD; data analysis as in (A). (C) Quantitative analysis of the effect of an induced reduction in miR156 on the level of SPL13-GUS mRNA and protein. The SPL13-GUS line was crossed to the Ind-MIM156 line, and these transgenes were then made homozygous. Young leaf primordia from mock-treated and β-estradiol-treated plants were harvested and analyzed by qRT-PCR and the MUG assay, as described in the Materials and Methods. SPL3 transcript levels were measured as a positive control for miR156 knock-down. (D) Quantitative analysis of SPL9-GUS mRNA (RT-qPCR) and protein (MUG assay) in Col, miR156a/c, miR157a/c and mir156a/c mir157a/c. (E) The relative abundance of uncleaved/cleaved SPL9 and SPL13 transcripts in LP1&2 of Col, miR156a/c, miR157a/c and mir156a/c mir157a/c. Values are relative to the value in Col. Results are from a single experiment with 3 technical replicates. In A, B, C and D the error bars represent the SD of 3 biological replicates.

Fig 11. miR156 is 100 times more abundant than its SPL targets.

The absolute abundance of miR156 and SPL transcripts in Col LP3&4. The value shown for miR156 is 0.01 of the actual value.