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Figure
4. Monitoring gene expression during flower development in Arabidopsis thaliana. Confocal imaging of green fluorescentprotein (GFP) reporter genes was used in living plants to monitor the expression patterns of multiple proteins and genes involved in flower primordial developmental processes. The expression and polarity of PINFORMED1 (PIN1), the auxin efflux facilitator, was followed. (A) PIN1 mRNA levels as measured by real-time PCR analysis of dissected wild-type inflorescences immersed in 100 µM IAA show greater than two-fold upregulation after 60 min relative to mock-treated controls. Identical treatments carried out on pin1-1 mutant apices using 5 mM IAA in lanolin paste resulted in approximately three-fold induction after 30 min. (B, C, F, G) show maximum intensity projections of the meristem viewed from above, while (D, E, H, I) show corresponding transverse optical sections below the epidermis, respectively. (B–E) Response of pPIN1::PIN1-GFP (green) to exogenous auxin. (B, D) pPIN1::PIN1-GFP-expressing meristem before NAA treatment. (C) and (E) show the same meristem as in (B) and (D) after treatment with 5 mM NAA for 6 h. Expression becomes delocalized and increases in cells that previously expressed pPIN1::PIN1-GFP at low levels (arrows in (B–E)). This occurs both in the epidermis (compare (B) and (C)) and layers below (compare (D) and (E)). (F–I) Response of pPIN1::PIN1-GFP to treatment with 100 µM NPA for 14 h. In both the epidermis (F, G) and subepidermal layers (H, I), there is a delocalization of expression after 14 h. (J–M) Time lapse of pDR5rev::3XVENUS-N7 (red) and pPIN1::PIN1-GFP (green) expression together (J, K) and pDR5rev::3XVENUS-N7 (red) alone (L, M). At both the initial time point (J, L) and 12 h later (K, M), pDR5rev::3XVENUS-N7 expression initiates when pPIN1::PIN1-GFP expression first marks a new site (arrowheads in (J) and (K)). After PIN1-GFP reverses polarity in cells adaxial to primordia, primordial expression of pDR5rev::3XVENUS-N7 persists and subsequently appears in daughter cells of earlier-expressing cells (encircled by broken line in (M)). Expression in nondaughter cells occurs at a later stage of floral bud development (arrowheads in (M)). (N, O) Recovery of fluorescence after bleaching. Cells located within incipient primordia (I2 in (N)) and more mature primordia (P2 in (N)) that expressed pDR5rev::3XVENUS-N7 were selectively irradiated with 514 nm laser light until expression became undetectable (circled regions in (O)). Seven hours after bleaching, fluorescence could again be detected in the same cells at I2 (arrow in (P)) but not in P2. Scale bars in (B), (F), and (J–N), 30 µm. Adapted and reprinted by permission from Cell Press: Current biology (Heisler et al, 2005), copyright 2005. |
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