Cytokinin-Auxin Crosstalk in the Gynoecial Primordium Ensures Correct Domain Patterning

Abstract

The Arabidopsis (Arabidopsis thaliana) gynoecium consists of two congenitally fused carpels made up of two lateral valve domains and two medial domains, which retain meristematic properties and later fuse to produce the female reproductive structures vital for fertilization. Polar auxin transport (PAT) is important for setting up distinct apical auxin signaling domains in the early floral meristem remnants allowing for lateral domain identity and outgrowth. Crosstalk between auxin and cytokinin plays an important role in the development of other meristematic tissues, but hormone interaction studies to date have focused on more accessible later-stage gynoecia and the spatiotemporal interactions pivotal for patterning of early gynoecium primordia remain unknown. Focusing on the earliest stages, we propose a cytokinin-auxin feedback model during early gynoecium patterning and hormone homeostasis. Our results suggest that cytokinin positively regulates auxin signaling in the incipient gynoecial primordium and strengthen the concept that cytokinin regulates auxin homeostasis during gynoecium development. Specifically, medial cytokinin promotes auxin biosynthesis components [YUCCA1/4 (YUC1/4)] in, and PINFORMED7 (PIN7)-mediated auxin efflux from, the medial domain. The resulting laterally focused auxin signaling triggers ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN6 (AHP6), which then represses cytokinin signaling in a PAT-dependent feedback. Cytokinin also down-regulates PIN3, promoting auxin accumulation in the apex. The yuc1, yuc4, and ahp6 mutants are hypersensitive to exogenous cytokinin and 1-napthylphthalamic acid (NPA), highlighting their role in mediolateral gynoecium patterning. In summary, these mechanisms self-regulate cytokinin and auxin signaling domains, ensuring correct domain specification and gynoecium development.

Figure 1.

Coexpression of cytokinin and auxin signaling reporters reveals mutually exclusive response domains in early stage gynoecial primordia. A, Transmitted light images of a stage-5 gynoecial primordia from an apical and medial perspective with domains and scales indicated. B to K and M to S, Confocal microscopy images of wild type plants containing both TCSn::GFP (green) and DR5::RFP (magenta) reporters were used to identify coexpression in early stage gynoecial primordia (A and L) at stage 5 (B to K), stage 6 (M and Q), stage 7 (N, O, R, and S), and stage 8 (P) after 24 h mock (B, D to G, and M to P) or 24-h BAP treatment (C, H to K, and Q to S). L, Transmitted light images indicate the domains for each stage and have artificial coloring for medial (beige) or lateral (blue) domains. Transmitted light images were overlaid for cell clarity (D to K and P). Schematic drawings in each subfigure show the image perspective (red line) and denote either the position from the apex (snap) or that the image is a maximum intensity projection of serial images (stack). Gynoecium periphery (solid white line), stamen (dotted line), sepal (asterisk), and medial domain invagination (arrowhead). Scale bar = 10 µm.

Figure 2.

Cytokinin signaling regulates medial boundaries and directs local auxin response peaks required for maintaining correct cytokinin signaling via AHP6. A to L, Confocal microscopy images of pSHP2::YFP (A to C), pFIL::GFP (D to F), and DR5::3xVENUS (G-L) in early stage gynoecial primordia of 24 h mock-treated wild type Col-0 (A, D, G, and J), 24-h BAP-treated Col-0 (B, E, H, and K), and the ahp6-1 mutant (C, F, I, and L). M to O, Confocal microscopy images of pAHP6::GFP expression in gynoecial primordia at stage 5 (M), stage 7 (N), and in stage 5 after a 24-h NPA treatment (O). P, Heat map of relative signal intensity in arbitrary units applied to (Q to V). Q to V, Confocal microscopy images of stage-5 gynoecial primordia expressing TCSn::GFP in mock-treated Col-0 (Q) and ahp6-1 (T), 24-h NPA-treated Col-0 (R), and ahp6-1 (U), and 72 h after NPA treatment in Col-0 (S) and ahp6-1 (V). Arrows indicate AHP6 expression in medial preprocambium (N), TCSn repression (R), or TCSn peaks (T) compared to controls. Magenta indicates chloroplast autofluorescence (A to F, M, and O). Each image is a maximum intensity projection of serial images (stack) and the schematic drawings in each subfigure indicate the image perspective (red line). Gynoecial primordium periphery (white circle). Scale bar = 10 µm.

Figure 3.

Cytokinin up-regulates YUC1, YUC4, and PIN7 and represses PIN3 in the gynoecial primordium. Confocal microscopy images of YUC1, YUC4, PIN7, and PIN3 reporter lines. A to J, pYUC1::n3GFP expression after 24-h mock treatment in stage 5 (A, A′; n = 15/21) and stage 7 (B, C; n = 8/11), after 24-h BAP treatment in stage 5 (D, D′; n = 4/5 and E; n = 1/5), in stage-7 gynoecial primordia after 24 h (F; n = 6) and 48 h (G) BAP treatment, and after 24 h PI-55 treatment in stage 5 (H, H′; n = 14/16) and stage 7 (I, J; n = 12/14). A, D, and H shown from the medial perspective in (A′), (D′), and (H′), respectively. Magenta indicates chloroplast autofluorescence. K to N′, pPIN7::PIN7::GFP expression in stage 5 gynoecial primordia after 24-h mock (K, K′; n = 25/27), 24-h BAP (L, L′; n = 10/11 and M; n = 1/11), and 24-h PI-55 treatment (N, N′; n = 10/16). O, Chart of pPIN7::PIN7::GFP expression patterns observed in stage-5 gynoecial primordia 24 h after mock, BAP or PI-55 treatment (n = 54). K, L, and N shown from the medial perspective in (K′), (L′), and (N′), respectively. P-U, pYUC4::n3GFP expression in stage-5 (P, R, T) and stage-7 (Q, S, U) gynoecial primordia after 24-h mock (P and Q; n = 14 and 8), 24-h BAP (R and S; n = 10 and 11), and 24-h PI-55 treatment (T and U; n = 10 and 8). V, Heat map of relative signal intensity in arbitrary units to visualize semiquantitative expression levels (in P to U and W to DD). W to DD, pPIN3::PIN3::GFP expression in stage-5 (W and AA), stage-6 (X and BB), stage-7 (Y and CC), and stage-8 (Z and DD) gynoecial primordia after 24-h mock (W to Z; n = 28) and 24-h BAP treatment (AA to DD; n = 35). Each image is a maximum intensity projection of serial images (stack) and the schematic drawings in each subfigure indicate the image perspective (red line). Gynoecial primordium periphery (white circle), border in medial perspective (dashed white line) and sepal (asterisk) are indicated. Scale bar = 10 μm.

Figure 4.

Quantitative expression of YUC1, YUC4, PIN3, and PIN7 in response to increased or decreased cytokinin signaling. A and B, qPCR analyses of YUC1, YUC4, PIN3, and PIN7 expression in early floral buds after mock versus 2-h, 6-h, and 24-h BAP treatment (A) and mock versus 6-h PI-55 treatment (B). Error bars indicate SD from three biological replicates; *, P < 0.05, Student’s t test.

Figure 5.

YUC1, YUC4 and AHP6 are required for valve outgrowth and hormone homeostasis. A, Quantification of valve phenotypes in stage-12 gynoecia from yuc1, yuc4, and ahp6 mutants analyzed 14 d after 1 d transient treatment with mock, BAP, or NPA. B, Quantification of outgrowth severity in Col-0, yuc1, and yuc4 mutants after five consecutive days of mock or BAP treatment, analyzed 14 d after the first spray.

Figure 6.

Model of cytokinin-auxin feedback mechanisms in the earliest stage gynoecial primordium. A stage-5 gynoecial primordium depicted from the medial domain showing regions of cytokinin signaling (green), auxin signaling (blue), auxin transport (arrowheads), and interactions between components (inhibiting lines and activating arrows). We propose a model in which cytokinin in the central medial domain is important to activate auxin biosynthesis via YUC1 and transport via PIN7 in the basal medial domain. PAT is important for setting up auxin peaks in the apical, lateral domains, ensuring the activation of AHP6, which contributes to the inhibition of cytokinin signaling from the lateral domains. Additionally, cytokinin promotes auxin signaling in the apex by promoting biosynthesis through YUC4 and blocking efflux via repression of PIN3, which together likely contribute to the apical accumulation of auxin. Together, these components are important for ensuring lateral and apical auxin accumulation and valve outgrowth.