Elevated <i>SPL</i> expression inhibits adventitious root production.

<p>(A) Roots originating from the hypocotyl of Col, transgenic, and <i>spl</i> mutant seedlings after removal of the primary root. (B) The number of adventitious roots produced by the genotypes illustrated in (A). * Significantly different from Col, p<0.05.</p

<i>spl</i> mutations reduce the expression of genes involved in floral induction and floral meristem identity.

<p>qRT-PCR analysis of transcripts isolated from shoot apices of 11 day-old plants. Values are normalized to Col, and represent the mean from 2 biological replicates ± SE.</p

The effect of <i>SPL</i> mutations on vegetative and reproductive development.

<p>The effect of <i>SPL</i> mutations on vegetative and reproductive development.</p

Developmental Functions of miR156-Regulated <i>SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL)</i> Genes in <i>Arabidopsis thaliana</i>

<div><p>Correct developmental timing is essential for plant fitness and reproductive success. Two important transitions in shoot development—the juvenile-to-adult vegetative transition and the vegetative-to-reproductive transition—are mediated by a group of genes targeted by miR156, SQUAMOSA PROMOTER BINDING PROTEIN (SBP) genes. To determine the developmental functions of these genes in <i>Arabidopsis thaliana</i>, we characterized their expression patterns, and their gain-of-function and loss-of-function phenotypes. Our results reveal that <i>SBP-LIKE</i> (<i>SPL</i>) genes in <i>Arabidopsis</i> can be divided into three functionally distinct groups: 1) <i>SPL2</i>, <i>SPL9</i>, <i>SPL10</i>, <i>SPL11</i>, <i>SPL13</i> and <i>SPL15</i> contribute to both the juvenile-to-adult vegetative transition and the vegetative-to-reproductive transition, with <i>SPL9</i>, <i>SP13</i> and <i>SPL15</i> being more important for these processes than <i>SPL2</i>, <i>SPL10</i> and <i>SPL11</i>; 2) <i>SPL3</i>, <i>SPL4</i> and <i>SPL5</i> do not play a major role in vegetative phase change or floral induction, but promote the floral meristem identity transition; 3) <i>SPL6</i> does not have a major function in shoot morphogenesis, but may be important for certain physiological processes. We also found that miR156-regulated <i>SPL</i> genes repress adventitious root development, providing an explanation for the observation that the capacity for adventitious root production declines as the shoot ages. miR156 is expressed at very high levels in young seedlings, and declines in abundance as the shoot develops. It completely blocks the expression of its <i>SPL</i> targets in the first two leaves of the rosette, and represses these genes to different degrees at later stages of development, primarily by promoting their translational repression. These results provide a framework for future studies of this multifunctional family of transcription factors, and offer new insights into the role of miR156 in <i>Arabidopsis</i> development.</p></div

Expression of miR156-sensitive (sSPL) and miR156-resistant (rSPL) SPL-GUS fusion proteins in transgenic plants.

<p>Plants grown in SD were harvested 1, 2 and 3 weeks after planting; plants grown in LD were harvested at 3 weeks. The inserts in the 3w LD panels are a magnification of the inflorescence. Scale bars represent 2 mm for 1w and 2w, 5 mm for 3w, and 1 mm for the inserts.</p

Loss-of-function alleles of <i>SPL</i> genes.

<p>White box = UTR, Grey box = transcribed region, Black box = SBP DNA binding domain. Genes in the same clade are indicated by vertical lines. The nucleotide and amino acid positions of point mutations are numbered from the translation start site.</p

The abundance of miR156-regulated <i>SPL</i> transcripts in the shoot apices of wild-type Col grown in SD.

<p>(A) Relative abundance of miR156, miR156-regulated <i>SPL</i> and <i>AP1</i> transcripts in 1, 2, 3, 4 and 5 week-old shoot apices of wild-type Col. Values are normalized to the value for 1w and represent the mean ± SE from four biological replicates. The initial abundance of miR156 was arbitrarily set to 10. *Difference between 1w and 3w is significant, p<0.05. **Difference between 3w and 5w is significant, p<0.05. Student's T test. (B) <i>in situ</i> hybridization of miR156-regulated <i>SPL</i> transcripts in shoot apices of 3-week old wild-type Col. Samples were incubated for the same amount of time during the color reaction. <i>In situ</i> hybridization of the SPL3, SPL9, SPL13, and SPL15 probes to shoot apices of 3-week-old <i>sp3-1</i>, <i>spl9-4</i>, <i>spl13-1</i>, and <i>spl15-1</i> plants is shown in the small inserts, and demonstrates the lack of significant background hybridization. Scale bar = 50 μm.</p

miR172 levels are reduced in <i>spl</i> mutants.

<p>The abundance of (A) pri-miR172b, (B) miR172 and (C) <i>SPL3</i> and <i>SPL5</i> mRNA in the shoot apices of 16-day-old <i>spl</i> mutants. Values are the average of 3 biological replicates, ± SE.</p

The phenotypes of <i>spl</i> mutant lines.

<p>(A) Rosettes of 21-day-old Col and <i>spl</i> mutants (SD). (B) The morphology of fully expanded rosette leaves of wild-type Col and <i>spl</i> mutants (SD). Leaves are numbered starting from the base of the rosette. (C) 5-week-old wild-type Col and <i>spl</i> mutants (LD). The inflorescence of an <i>spl2/9/10/11/13/15</i> plant is shown to demonstrate that the cauline leaves in this genotype subtend co-florescence buds, as is also the case for the <i>35S</i>::<i>MIR156A</i> line.</p

The vegetative phenotype of transgenic plants expressing sSPL-GUS and rSPL-GUS fusion proteins.

<p>(A) 17-day-old plants grown in SD. The first two rosette leaves are labelled. Scale bar = 10mm. (B) First 7 fully-expanded rosette leaves of Col, rSPL reporter lines, and a transgenic plant expressing a <i>35S</i>::<i>MIM156</i> target site mimic (SD). (C) The number of leaves without abaxial trichomes in Col and rSPL reporter lines.</p