Regulation of the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE genes/microRNA156 Module by the Homeodomain Proteins PENNYWISE and POUND- FOOLISH in Arabidopsis  Lal Shruti , Pacis Leo Bryan , Smith Harley M.S.   Molecular Plant  Volume 4, Issue 6, Pages 1123-1132 (November 2011) DOI: 10.1093/mp/ssr041 Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 1 Analysis of SPL and AP1 Transcript Levels by Q–PCR in Wild-Type and pny pnf Shoot Apices. After 30 d of growth in short days, an environmental condition that delays flowering, Q–PCR was used to determine the transcript levels of (A, B) SPL3, (C, D) SPL4, (E, F) SPL5, and (G, H) AP1 in wild-type and pny pnf vegetative shoot apices. After 30 of growth in non-photoinductive conditions, wild-type and pny pnf plants were shifted to long-day growth conditions to induce flowering. In these plants, Q–PCR was utilized to determine the transcript levels for (A, B) SPL3, (C, D) SPL4, (E, F) SPL5, and (G, H) AP1 in wild-type and pny pnf florally induced apices. Transcript levels for SPLs and AP1 were normalized against the mRNA levels for UBQUITIN5. Numbers on the x-axis refer to the number of days that the 30 short-day-grown wild-type and pny pnf plants were grown in floral-inductive conditions. Numbers on the y-axis refer to the relative gene expression. Each time point is a mean of the relative gene expression (± standard error of the mean). Molecular Plant 2011 4, 1123-1132DOI: (10.1093/mp/ssr041) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 2 Expression Levels of miR156 in the Apices of pny pnf Plants. (A) Northern blot analysis was used to determine the levels of miRNA156 in wild-type (WT) and pny pnf apices. The levels of miR156 were examined during vegetative growth, after 20 d of growth in short days (SD) and long days (LD). The levels for miR156 were determined after 30 d of growth in long days for wild-type and pny pnf apices. At this time point, wild-type plants were initiating flowers. In pny pnf plants, apices were harvested after 40 d of growth in long days. A doublet was detected after probing blots with miR156, in which the upper and lower bands correspond to miR157 and miR156, respectively (Wu and Poethig, 2006). The sequences of miR156 and miR157 are similar; therefore, miR157 may also play a role in post-transcriptional regulation of SPLs (Rhoades et al., 2002; Gandikota et al., 2007). The U6 small nuclear RNA was used as a loading control. The numbers at the base of the miR156 blots correspond to the expression levels of miR156 for each experiment. (B) The levels of miR156 were quantified by scanning three Northern blots derived from three biological replicates. The levels of U6 were quantified and used to normalize the levels of miR156. Molecular Plant 2011 4, 1123-1132DOI: (10.1093/mp/ssr041) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 3 Ectopic Expression of SPL4 in pny pnf Partially Restored Flower Formation. The inflorescences shoots for (A) wild-type. (B) An image of a pny pnf plant. (C) Close-up of the leaf-bearing pny pnf apex. (D) 35S:rSPL4. (E) An image of a 35S:rSPL4 pny pnf plant (arrow points at the inflorescence shoots). (F) An inflorescence shoot was removed from a 35S:rSPL4 pny pnf plant (arrows point at terminal flowers). (G) Close-up of a 35S:rSPL4 pny pnf terminal flower composed of two partially fused carpels. (H) All plants were grown in long-day growth conditions. Transcript levels for SPL4, LFY, AP1, and ACTIN 2 were determined in wild-type (WT), pny pnf, and 35S:rSPL4 pny pnf shoot apices after 30 d of growth. Since 35S:rSPL4 flowered early, inflorescence apices were collected after 15 d of growth. Molecular Plant 2011 4, 1123-1132DOI: (10.1093/mp/ssr041) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 4 Ectopic Expression of SPL5 in pny pnf Alters the Identity of the Leaf. (A) An image of a 35S:rSPL5 inflorescence and (B) a 35S:rSPL5 pny pnf plant. (C, D) Close-up images of the 35S:rSPL5 pny pnf terminal leaf-like structures. (E, F) Scanning electron microscopy (SEM) image of the terminal leaf-like organs with (F) a structure protruding from the margin (arrow). Cell types found on the surfaces of (G, H) the terminal leaf-like organs in 35S:rSPL5 pny pnf, (I) a pny pnf leaf and wild-type (J) sepals and (K) carpels. (L) All plants were grown in long-day growth conditions. Transcript levels for SPL5, LFY, AP1, and ACTIN2 were determined in wild-type (WT), pny pnf, and 35S:rSPL5 pny pnf shoot apices after 30 d of growth. Since 35S:rSPL5 flowered early, inflorescence apices were collected after 15 d of growth. (E) Scale bar = 500 μm; (F) Scale bar = 100 μm; (G–K) Scale bars = 50 μm. Molecular Plant 2011 4, 1123-1132DOI: (10.1093/mp/ssr041) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 5 Ectopic Expression of 35S:rSPL3 in pny pnf Plants. (A) An image of a 35S:rSPL3 inflorescence and (B) a 35S:rSPL3 pny pnf plant. (C) A close-up of 35S:rSPL3 pny pnf shoot apex (arrow points at the apex). (D) SEM image of a leaf initiated late in development in 35S:rSPL3 pny pnf. Scale bar = 100 μm. (E) SEM image of the surface of a pny pnf leaf. Scale bar = 100 μm. (F) All plants were grown in long-day growth conditions. Transcript levels for SPL3, LFY, AP1, and ACTIN2 were determined in wild-type (WT), pny pnf, and 35S:rSPL3 pny pnf shoot apices after 30 d of growth. Since 35S:rSPL3 flowered early, inflorescence apices were collected after 15 d of growth. Molecular Plant 2011 4, 1123-1132DOI: (10.1093/mp/ssr041) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 6 Role of PNY and PNF in the Flower Specification Network. A model is shown that displays the photoperiod and endogenous flower specification pathways. FT–FD activate SPL3/SPL4/SPL5 (collectively referred to as SPLs) and act in parallel to induce SOC1 and FUL as well as flower meristem identity genes. FT requires PNY and PNF for specifying flower meristem identity and regulating SPL3, SPL4, and SPL5. In addition, PNY and PNF control the accumulation of SPL3, SPL4, and SPL5 transcripts by repressing miR156 during shoot development. Based on the results in this manuscript, PNY and PNF may function in parallel with SPL3, SPL4, and SPL5. The dashed line between PNY/PNF and miR156 indicates that the nature of this regulation is not known. Molecular Plant 2011 4, 1123-1132DOI: (10.1093/mp/ssr041) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions