The MADS-box protein SHATTERPROOF 2 regulates TAA1 expression in the gynoecium valve margins

Abstract

SHATTERPROOF 2 regulates TAA1 expression for the establishment of the gynoecium valve margins. Gynoecium development and patterning play a crucial role in determining the ultimate structure of the fruit and, thus, seed production. The MADS-box transcription factor SHATTERPROOF 2 (SHP2) contributes to valve margin differentiation and plays a major role in fruit dehiscence and seed dispersal. Despite the acknowledged contribution of auxin to gynoecium development, its precise role in valve margin establishment remains somewhat enigmatic. Our study addresses this gap by uncovering the role of SHP2 as a positive regulator of key auxin biosynthetic genes, TAA1 and YUCCA 4. Genetic and molecular analyses revealed that SHP2 directly regulates the expression of TAA1 in the valve margins of a stage 12 gynoecium with known regulators of flower and ovule development, such as AGAMOUS, SEEDSTICK, and SEPATALA 3. Collectively, our findings define a previously unrecognized function of SHP2 in the regulation of auxin biosynthetic genes during gynoecium development and raise the possibility that the auxin produced under SHP2 regulation may contribute significantly to the valve margin establishment.

Keywords: Auxin; Gynoecium; SHATTERPROOF 2; TAA1; Valve margins; YUCCA 4.

Fig. 1

The expression of SHP2 at the junction between valve and replum of the gynoecium is required for its specification a, b SHP2 is expressed at the valve-replum junction in stage 12 gynoecium. A gynoecium expressing SHP2-GFP a and a cross-section of a gynoecium expressing pSHP2:GUS b are presented. The black arrowhead marks SHP2 expression in the ovule integuments. c, d SEM images of wild type (Col-0) and shp1 shp2 mutant gynoecium (stage 12). White arrowheads indicate the valve margin (vm). Stage of gynoecium is according to (Smyth et al. 1990). e–g In developing ovules, the expression is detected in the integuments. Young ovule (stage 3-I) e; late-stage ovule (3-V/3-VI) images taken at different focal distance f, g; post fertilization (stage 4-II/4-III) h. SHP2-GFP expression in the outermost layer of the seed coat at the heart stage of seed development i. Stages of ovule development are according to (Schneitz et al. 1995). The fluorescent signal is green. The white signal in e is the Renaissance dye counterstaining cell walls. The GUS signal is blue. Scale bars: 50 μm a, 100 μm b–d, 10 μm e, 20 μm f–i

Fig. 2

TAA1 and YUC4 expression is regulated by SHP2 a–c The expression of SHP2 a, TAA1 b and YUC4 c is reduced in shp1 shp2 gynoecium (stage 12). Relative quantification of transcript levels assessed by RT-qPCR in Col-0 and shp1 shp2 gynoecium. d Schematic of reporter and effector constructs used in the dual luciferase assays. e, f Ren/Luc ratio for TAA1 e and YUC4 f promoters after co-infiltration with 35S:YFP (control) and 35S:SHP2-cMyc. RENILLA (REN) was used as an internal control. The values are represented as the means ± SD from four biological replicates. p-values are calculated with a two-tailed Student’s t-test. *P < 0.05; **P < 0.01

Fig. 3

Interaction of SHP2 on TAA1 promoter regulates its expression in gynoecium a, b pTAA1:GFP-TAA1 expression at the valve-replum junction (white arrow) in wild-type gynoecium (stage 12) a is absent in shp1 shp2 gynoecium b. The signal at the silique surface is presented. The full Z-stacks are presented in supplementary videos S1 and S2. A close-up of TAA1 expression in wild-type is presented in Supplementary Fig. S3. c, d pTAA1:GFP-TAA1 expression in 3-V/3-VI ovules from stage 12 gynoecium of Col-0 c and shp1 shp2 d. e GFP-TAA1 expression profile (fluorescence intensity, arbitrary unit (a.u.)) along the X-axis (in nm) on the surface of Col-0 (black) and shp1 shp2 (grey) siliques (n = 7 for Col-0, n = 5 for shp1 shp2). f–i pYUC4:3xnGFP is expressed in the style of the silique in Col-0 f but not in shp1 shp2 g. The full Z-stacks are presented in supplementary videos S3 and S4. pYUC4:3xnGFP signal is visible in the ovule inner integuments in wild type h. YUC4 expression is reduced in shp1 shp2 ovules i. The fluorescent signal is green a–d, f–i. The Renaissance dye stained the cell membranes a, b, f, g. Scale bars: 50 μm a–d, f, g, 20 μm h, i. j Schematic representation (top) of TAA1 promoter showing the positions of CArG boxes (solid triangle) within the 3 kb region upstream of ATG. P1-5 indicates the location of amplicons used for ChIP-qPCR analysis. The result of ChIP-qPCR (bottom) shows the enrichment of regions P3 and P4 in the SHP2-GFP IP sample. k Schematic representation of YUC4 promoter (top) showing the positions of CArG boxes (solid triangle) within the 3 kb region upstream of ATG. P1-4 indicates the location of amplicons used for ChIP-qPCR analysis. The result of ChIP-qPCR (bottom) shows a reduced level of P2 and P3 amplicons in the SHP2-GFP IP sample. The values are represented as the means ± SD from three independent experiments. p-values are calculated with a two-tailed Student’s t-test. *, p < 0.05; **, p < 0.01