Arabidopsis Leaf Flatness Is Regulated by PPD2 and NINJA through Repression of CYCLIN D3 Genes

Abstract

In Arabidopsis (Arabidopsis thaliana), reduced expression of the transcriptional regulator PEAPOD2 (PPD2) results in propeller-like rosettes with enlarged and dome-shaped leaves. However, the molecular and cellular processes underlying this peculiar phenotype remain elusive. Here, we studied the interaction between PPD2 and NOVEL INTERACTOR OF JAZ (NINJA) and demonstrated that ninja loss-of-function plants produce rosettes with dome-shaped leaves similar to those of ppd mutants but without the increase in size. We showed that ninja mutants have a convex-shaped primary cell cycle arrest front, putatively leading to excessive cell division in the central leaf blade region. Furthermore, ppd and ninja mutants have a similar increase in the expression of CYCLIN D3;2 (CYCD3;2), and ectopic overexpression of CYCD3;2 phenocopies the ppd and ninja rosette and leaf shape phenotypes without affecting the size. Our results reveal a pivotal contribution of NINJA in leaf development, in addition to its well-studied function in jasmonate signaling, and imply a new function for D3-type cyclins in, at least partially, uncoupling the size and shape phenotypes of ppd leaves.

Figure 1.

NINJA interacts with PPD2 in vitro and in N. benthamiana leaves. A, In a pull-down (PD) experiment, GST-PPD2 was immobilized on glutathione-Sepharose and the presence of His-NINJA was verified by immunoblotting (IB) using an anti-His antibody. GST-GUS was used as a negative control. The amount of GST-GUS or GST-PPD2 was visualized by Coomassie Brilliant Blue (CBB) staining. B, nYFP-NINJA and cYFP-PPD2 or cYFP (as a negative control) were coexpressed in N. benthamiana leaves. 4′,6-Diamino-phenylindole (DAPI) staining indicates the nuclei. The DAPI-stained and bright-field microscopic images are merged in the bottom panels. Bars = 20 µm.

Figure 2.

PPD2 interacts with the NINJA C domain. A and B, PPD2 (A) and NINJA (B) truncations were tested in Y2H assays to identify the specific domains for the PPD2-NINJA interaction. Transformants containing bait and prey constructs were grown on medium lacking Leu and Trp (-L-T) or Leu, Trp, and His (-L-T-H). Protein domains are represented, and numbers indicate terminal amino acid residues. C, Columbia-0 (Col-0), ppd2, ninja-1, and ninja-2 plants grown in soil for 25 d. Side views of the seventh leaf (L7) are presented below the photographs. Bar = 1 cm. D, Area of the individual leaves of ppd2 and ninja plants grown in soil for 25 d (n = 3 biological replicates with approximately eight plants per replicate), analyzed using mixed models in the SAS Enterprise Guide. Statistically significant differences (P < 0.05) relative to the wild type are marked in boldface italic type.

Figure 3.

The ninja leaf phenotype is not affected by decreased JA biosynthesis. A, Col-0, ninja-1, ninja-2, aos, ninja-1 aos, and ninja-2 aos plants grown in soil for 25 d. Side views of the seventh leaf (L7) are presented below the photographs. Bar = 1 cm. B, Area of leaves 1/2, 3, and 4 of Col-0, ninja-1, ninja-2, aos, ninja-1 aos, and ninja-2 aos plants grown in soil for 25 d (n = 3 biological replicates with approximately eight plants per replicate). Error bars represent se. Statistical significance was evaluated by ANOVA followed by Tukey’s posthoc analysis. Significant differences (P < 0.05) relative to Col-0 (a), ninja-1 (b), and aos (c) are indicated with lowercase letters.

Figure 4.

The ninja mutants have an increased expression of CYCD3;2 and CYCD3;3, and CYCD3;2-OE plants have dome-shaped leaves. A and B, The primary cell cycle arrest front and the perpendicular distance between the highest point of the GUS signal in the center of the leaf and a horizontal line connecting the GUS signal at the leaf margins of leaf 9 or 10 of 21-d-old soil-grown Col-0, ami-ppd, and ninja-2 plants expressing the pCYCB1;1-DB::GUS construct (n = 3 biological replicates with ∼20 leaves per replicate). Error bars represent se. Statistical significance was evaluated by ANOVA followed by Tukey’s posthoc analysis. Significant differences (P < 0.05) relative to pCYCB1;1-DB::GUS are indicated with lowercase letter a. C and D, Col-0, ninja-1, and ninja-2 plants were grown in vitro, and the first leaf pair (L1/2) was harvested after 11, 13, and 15 d for RNA extraction and qRT-PCR analysis to verify the expression of CYCD3;2 and CYCD3;3 (n = 3 biological replicates with approximately five leaves per replicate). Error bars represent se. Statistical significance was evaluated by ANOVA followed by Tukey’s posthoc analysis. Significant differences (P < 0.05) between Col-0 and ninja-1 (a) or ninja-2 (b) are indicated with lowercase letters. E, Col-0, CYCD3;2-OE^#1-#3, and CYCD3;3-OE^#1-#2 plants grown in soil for 25 d. Fold change (FC) values of transgene expression compared with the wild type are also provided. Side views of the seventh leaf (L7) are presented below the photographs. Bar = 1 cm. F, Area of individual leaves of CYCD3;2-OE plants grown in soil for 25 d (n = 3 biological replicates with approximately eight plants per replicate), analyzed using mixed models in the SAS Enterprise Guide. Statistically significant differences (P < 0.05) relative to the wild type are marked in boldface italic type.

Figure 5.

CYCD3;2-OE leaves have a higher frequency of small cells but their meristemoid asymmetric divisions are not changed drastically. A, Average guard cell number (GCN), pavement cell number (PCN), total cell number (TCN), pavement cell area (PCA), and stomatal index (SI) in the lower (abaxial) epidermis of the first leaf pair (L1/2) of CYCD3;2-OE^#1 and CYCD3;2-OE^#2 mutants relative to Col-0 (n = 3 biological replicates with four leaves per replicate). Error bars represent se. Statistical significance was evaluated by ANOVA followed by Tukey’s posthoc analysis. Significant differences (P < 0.05) relative to Col-0 (a) and CYCD3;2-OE^#1 (b) are indicated with lowercase letters. B, For the pavement cell size distribution, cells are divided into bins of 4,000 μm² according to their area. C, Individual meristemoids in the abaxial epidermis of L1/2 of Col-0 and CYCD3;2-OE^#2 mutants were followed for three successive days (D12–D14), and asymmetric division, guard mother cell, and stoma formation events were scored. Arrows, To score the recurrent asymmetric events, only meristemoids that divided asymmetrically in the D12-D13 transition were taken into account.

Figure 6.

PPD2 signaling mutants have narrow and dome-shaped leaves. A, The leaf area, length, and width were measured before (projected) and after (real) leaf 7 (L7) was cut to flatten. B and C, Leaf 7 length-to-width ratio (B) and projected-to-real leaf area, length, and width (C) of Col-0, CYCD3;2-OE^#1, CYCD3;2-OE^#2, ninja-1, and ppd2 plants grown in soil for 25 d (n = 3 biological replicates with approximately eight plants per replicate). Error bars represent se. Statistical significance was evaluated by ANOVA followed by Tukey’s posthoc analysis. Significant differences (P < 0.05) relative to Col-0 (a), CYCD3;2-OE^#1 (b), CYCD3;2-OE^#2 (c), and ninja-1 (d) are indicated with lowercase letters.

Figure 7.

Inactivation of CYCD3;1 and CYCD3;2 decreases the extent of ami-ppd leaf curvature. A, Col-0, ami-ppd, cycd3;1 cycd3;2, cycd3;1 cycd3;2 ami-ppd^#1, and cycd3;1 cycd3;2 ami-ppd^#2 plants grown in soil for 25 d. Side views of the seventh leaf (L7) are presented below the photographs. Bar = 1 cm. B and C, Leaf 7 length-to-width ratio (B) and projected-to-real leaf area, length, and width (C) of Col-0, ami-ppd, cycd3;1 cycd3;2, cycd3;1 cycd3;2 ami-ppd^#1, and cycd3;1 cycd3;2 ami-ppd^#2 plants grown in soil for 25 d (n = 3 biological replicates with approximately eight plants per replicate). Error bars represent se. Statistical significance was evaluated by ANOVA followed by Tukey’s posthoc analysis. D, Area of leaves 1/2, 3, and 4 of Col-0, ami-ppd, cycd3;1 cycd3;2, cycd3;1 cycd3;2 ami-ppd^#1, and cycd3;1 cycd3;2 ami-ppd^#2 plants grown in soil for 25 d relative to Col-0 (n = 3 biological replicates with approximately eight plants per replicate), analyzed using mixed models in the SAS Enterprise Guide. In C and D, statistically significant differences (P < 0.05) relative to Col-0 (a), ami-ppd (b), cycd3;1 cycd3;2 (c), and cycd3;1 cycd3;2 ami-ppd^#1 (d) are indicated with lowercase letters.

Figure 8.

Schematic overview of the involvement of the PPD complex in leaf growth and shape control. In wild-type leaves, the PPD2 protein interacts with the adaptor proteins KIX8/9 and NINJA to recruit TPL, generating a transcriptional repressor complex. The complex inhibits the expression of downstream target genes, including CYCD3s, which results in a relatively straight primary cell cycle arrest front and a flat-leaf phenotype. Upon SAP-mediated proteosomal degradation or down-regulation of members of the repressor complex, the complex is inactive. This results in the overexpression of the CYCD3 cell cycle genes, the formation of a convex primary cell cycle arrest front, and a dome-shaped leaf phenotype.