Fig 1.
CLAU and LA function in parallel pathways.
(A-D, F-M) Genetic interactions between genotypes with altered CLAU and LA expression levels. La-2/+: a semi-dominant LA allele with increased and precocious expression due to miR319 resistance. La-6, FIL>>miR319: LA null or LA downregulation, respectively. clausa: CLAU null. FIL>>CLAU: CLAU upregulation. All leaves depicted are fully expanded fifth leaves. Bars = 1 cm. White and red arrowheads represent primary leaflets and missing primary and intercalary leaflets, respectively. (E) Cartoon depicting the expression domain of the FIL promoter. (N-O) Quantification of leaf complexity in genotypes with altered CLAU and LA expression levels. Graphs represent mean ± SE of six independent biological repeats. Different types of leaflets are indicated according to the color code. Statistical significance of differences in the total leaflet number was examined in a one-way ANOVA, p<0.0001. Different letters indicate significant differences between samples in an unpaired two-tailed t-test with Welch’s correction. Statistical significance of differences in quantification of each leaflet type are presented in S1 Fig.
Fig 2.
LA determines the developmental window in which CLAU is expressed.
(A-L) Expression of the CLAUSA promoter CLAU::nYFP in altered LA genotypes. (A-C) Expression of CLAU::nYFP in different developmental stages of WT; (E-G) Expression of CLAU::nYFP in different developmental stages of La-2/+ (LA upregulation); (I-K) Expression of CLAU::nYFP in different developmental stages of FIL>>miR319 (Reduced LA activity). The pattern of CLAU::nYFP expression was detected by a confocal laser scanning microscope (CLSMmodel SP8; Leica), with the solid-state laser set at 514 nm excitation/ 530 nm emission. Chlorophyll expression was detected at 488nm excitation/ 700nm emission. Bars = 100 um. (D, H, L): Cartoon summarizing the expression of CLAU::nYFP throughout development in each genotype. (M) Expression levels of CLAU in altered LA genotypes was determined in successive leaf developmental stages using RT-qPCR. Graphs represent mean ± SE of three independent biological repeats. Asterisks indicate significant differences from CLAU expression in WT in an unpaired two-tailed t-test, p≤0.0387.
Fig 3.
TKN2 mediates the increased morphogenesis in CLAU- and LA-deficient backgrounds.
(A-F) Overexpression of TKN2-SRDX in the background of CLAU and LA deficiency. All leaves depicted are fully expanded fifth leaves. Bars = 1 cm. Red arrowheads represent primary leaflets and missing primary and intercalary leaflets. (G) Quantification of leaf complexity upon overexpression of TKN2-SRDX in the background of CLAU and LA deficiency. Graphs represent mean ± SE of at least three independent biological repeats. Different types of leaflets are indicated according to the color code. Asterisks indicate significant differences of the total leaflet number from the background genotype (without TKN2-SRDX overexpression) in an unpaired two-tailed t-test with Welch’s correction, p≤0.0335. Statistical significance of differences in quantification of each leaflet type are presented in S4 Fig. (H) Cartoon depicting the expression domain of the BLS promoter.
Fig 4.
TKN2 promotes morphogenetic activity in altered CLAU and LA backgrounds.
(A-L) Overexpression of TKN2 in the background of genotypes with altered CLAU and LA expression levels. All leaves depicted are fully expanded fifth leaves. Bars = 1 cm. Red arrowheads represent missing primary and intercalary leaflets. (M) Shaded cartoon of the terminal leaflet of the indicated genotypes. (N) Quantification of leaf complexity of the second left-hand lateral leaflet of leaf No. 5 upon overexpression of TKN2 in genotypes with altered CLAU and LA expression levels. Graphs represent mean ± SE of at least three independent biological repeats. Letters indicate significant differences between samples in a one-way ANOVA with a Tukey post-hoc test, p<0.027.
Fig 5.
clausa has an altered GA profile and reduced sensitivity to GA treatment.
(A-D) Phenotypes of leaves with reduced CK (FIL>>CKX) increased CLAU (FIL>>CLAU), or that received exogenous GA. (E-G) Phenotypes of leaves with reduced GA response (FIL>>PROΔ17), or increased CLAU and reduced GA response (FIL>>CLAU+ PROΔ17), or reduced CK content (FIL>>CKX) treated with GA. (H-J) Effect of GA treatment on WT and clausa. All leaves depicted are fully expanded fifth leaves. Bars = 1 cm. White and red arrowheads represent primary leaflets and missing primary and intercalary leaflets, respectively. (K) Quantification of GAs in WT and clausa. Asterisks indicate significant differences between WT and clausa for each GA in an unpaired two-tailed t-test, *p≤0.05, **p≤0.01, ***p≤0.001. (L) Expression of SlGA20ox-1, the enzyme that converts GA19 to GA20, in WT and clausa, was determined in young shoots (m+6) of 2-week-old plants using RT-qPCR. Graphs represent mean ± SE of five independent biological repeats. Asterisks indicate significant differences in an unpaired two-tailed t-test, p = 0.0078. (M) Depiction of GA biosynthesis pathways arrested in clausa. The compounds GA24, GA53 and GA19 accumulate (red underline) while the active GA4 and the GA1 precursor GA20 (green underline) are reduced, suggesting reduced GA20ox function. (N) Quantification of leaf complexity. Graphs represent mean ± SE of at least three independent biological repeats. Different types of leaflets are indicated according to the color code. Statistical significance of differences in the total leaflet number was examined in a one-way ANOVA, p<0.0001. Different letters indicate significant differences between samples in an unpaired two-tailed t-test with Welch’s correction (p<0.015). Statistical significance of differences in quantification of each leaflet type are presented in S5 Fig. (O) Quantification of leaf complexity following GA treatment in WT and clausa. Graphs represent mean ± SE of at least three independent biological repeats. Different types of leaflets are indicated according to the color code. Statistical significance of differences in the total leaflet number was examined in a one-way ANOVA, p<0.0001. Different letters indicate significant differences between samples in a Dunnett post-hoc test (p<0.0097). Statistical significance of differences in quantification of each leaflet type are presented in S6 Fig.
Fig 6.
LA reduces leaf margin’s sensitivity to CK.
(A-F) Phenotypes of leaves with increased CK (FIL>>IPT) or decreased CK (FIL>>CKX) levels, and increased LA activity (La-2/+) or reduced LA activity (FIL>>miR319). All leaves depicted are fully expanded fifth leaves. Bars = 2 cm. White and red arrowheads represent primary leaflets and missing primary and intercalary leaflets, respectively. (G-I) Confocal micrographs of TCSv2::3XVENUS in successive developmental stages in indicated genotypes. TCSv2 driven signals are reduced in La2/+ in the fifth plastochron. The pattern of VENUS expression was detected by a confocal laser scanning microscope (CLSM model SP8; Leica), with the solid-state laser set at 514 nm for excitation and 530 nm for emission. Chlorophyll expression was detected at 488nm excitation/ 700nm emission. Bars = 100 um.
Fig 7.
Global expression analyses of LA, CLAU and TKN2.
(A-B) Venn diagrams depicting co-regulated genes in LA and TKN2 genotypes. Roughly a third to half of the genes upregulated upon LA activity downregulation (miR319 overexpression), or downregulated upon LA activity upregulation (La-2/+), are co-regulated upon upregulation of TKN2 activity (overexpression of TKN2) and/or downregulation of the activity of TKN2 targets (overexpression of TKN2-SRDX). (C) Hormone signaling pathways co-regulated in the different genotypes. Arrow color corresponds with genotypes provided below, arrow direction indicates up or down regulation. (D-G) CLAU and LA promote differentiation in partially discrete pathways. Morphogenesis and differentiation genes were identified from a published dataset (Ichihashi et al., 2014). 1824 genes fit the condition of m+P3 exp> P4 exp> P5 exp> P6 exp and are considered "Morphogenesis" genes. 1451 genes fit the condition of m+P3 exp< P4 exp< P5 exp< P6 exp and are considered "Differentiation" genes. (D) "Morphogenetic" genes, as a group, are significantly enriched in the upregulated group in clausa (p<0.014) and pFIL>>miR319 (p<0.044). (F) Known morphogenesis genes identified that validate the methodology are listed in blue. Cartoon depicts the pathways significantly enriched in morphogenesis genes, denoting significant upregulation in clausa (pink diamond) and FIL>>miR319 (blue diamond). None of the genes overlap, and both common and distinct pathways are represented in the two genotypes. Fisher’s exact test was used to calculate statistical significance of KEGG pathway enrichment; p-values were corrected by calculating false discovery rate (FDR) using the Benjamini Hochberg test, corrected p-value<0.05. (E) "Differentiation" genes, as a group, are significantly enriched in the downregulated group in clausa (p<0.0001) and pFIL>>miR319 (p<0.0001). (G) Known differentiation genes identified that validate the methodology are listed in red.—Cartoon depicts the pathways significantly enriched in differentiation genes, denoting significant downregulation in clausa (pink diamond) and pFIL>>miR319 (blue diamond). Only common pathways are represented in the two genotypes, and 10–15% of the genes overlap. Fisher’s exact test was used to calculate statistical significance of KEGG pathway enrichment; p-values were corrected by calculating FDR using the Banjamini Hochberg test, corrected p-value<0.05. (H) Cartoon depicting morphogenesis and differentiation pathways. Different morphogenetic genes generate different pathways (blue arrows) that result in different patterning with similar leaf complexity. Different as well as overlapping differentiation genes generate different, partially overlapping pathways (red arrows) that, in the most simple leaved, differentiated form, look similar in addition to having equal complexity. See also Figs 8 and S1, S4 and S6 and S3 and S4.
Fig 8.
Model for the role of CLAU, LA and TKN2 and GA-CK balance in leaf development.
CK promotes morphogenesis, and GA promotes differentiation. Increasing CLAU or LA levels leads to increased GA sensitivity and decreased CK sensitivity, pulls the leaf developmental program towards differentiation, and results in simplified leaf forms. Decreasing CLAU or LA levels leads to increased CK sensitivity and decreased GA sensitivity, pulls the leaf developmental program towards morphogenesis and results in elaborate leaf forms. LA acts in a dose-dependent manner. While only 1 copy of the dominant LA allele in La-2/+ gain-of-function mutants produces some leaflets, adding additional copy of LA allele in the La-2 homozygous plants produces a completely simple leaf. The additional LA copy can be replaced by increasing CLAU levels, by reducing CK levels or by increasing GA levels [39]. Reducing CLAU or LA activity or increasing TKN2 activity leads to increase in leaf complexity, which is further enhanced when combining two of them.