Gravity-induced root curvature has long been considered to be regulated by differential distribution of the plant hormone auxin. However, the cells establishing these gradients, and the transport mechanisms involved, remain to be identified. Here, we describe a GFP-based auxin biosensor to monitor auxin during Arabidopsis root gravitropism at cellular resolution. We identify elevated auxin levels at the root apex in columella cells, the site of gravity perception, and an asymmetric auxin flux from these cells to the lateral root cap (LRC) and toward the elongation zone after gravistimulation. We differentiate between an efflux-dependent lateral auxin transport from columella to LRC cells, and an effluxand influx-dependent basipetal transport from the LRC to the elongation zone. We further demonstrate that endogenous gravitropic auxin gradients develop even in the presence of an exogenous source of auxin. Live-cell auxin imaging provides unprecedented insights into gravity-regulated auxin flux at cellular resolution, and strongly suggests that this flux is a prerequisite for root gravitropism.gravitropic root curvature ͉ polar auxin transport ͉ auxin carrier proteins G ravity plays a major role in plant morphogenesis by determining the directional growth of plant organs (gravitropism). Roots orient at a preferred angle with respect to gravity [their gravitropic set-point angle (GSA); ref. 1], allowing efficient exploration of the soil (root gravitropism). Main roots of Arabidopsis seedlings, for instance, have a GSA of 0°and grow parallel to the gravity vector. Changes in gravity vector orientation (gravistimulation) induce root curvature, resulting in realignment of the root tip to the GSA. Root curvature is a consequence of gravity signal perception, involving amyloplast sedimentation in the columella cells of the root cap (2), and differential growth induced on opposite flanks in the elongation zone (EZ). In the 1920s, the Cholodny-Went hypothesis and various interpretations of it ever since have proposed that this differential growth within the EZ is mediated by an asymmetric distribution of the plant hormone auxin (3). Supportive evidence for an auxin asymmetry in the EZ after gravistimulation has come from the analyses of radio-labeled auxin distribution, or differential induction of auxin-response promoters (4). It has been questioned, however, whether auxin gradients are necessary or sufficient to cause root gravitropism (3, 5). Furthermore, it is not clear as to how the gravisensing events in the columella cells can give rise to changes in auxin concentration in the EZ. Recently, the gravity-dependent relocation of an auxin efflux carrier protein in columella cells suggested gravity-regulated changes of auxin transport right at the site of gravity perception in the root cap (6). However, differential auxin fluxes through the cap cells and their contribution to gravitropic root curvature remain to be demonstrated. In the work presented here, we applied a GFP-based auxin biosensor to study gravity-induced ...
The transient expression of three mutant forms of green fluorescent protein (GFP) genes, GFP4, GFP5ER, and GFP4S65C, under several constitutive and pollen-specific promoters throughout pollen development in Nicotiana tabacum, Arabidopsis thaliana and Antirrhinum majus is described. Immature pollen of tobacco, Arabidopsis and snapdragon, isolated at different developmental stages, were bombarded with plasmids containing the GFP and cultured in vitro for several days until maturity. The expression of GFP was monitored every day during in vitro maturation, germination and pollination, as well as after in situ pollination. The expression pattern of each GFP construct was compared in parallel experiments to that of beta-glucuronidase (GUS) constructs expressed by the same promoters. The results show that the expression level of all three GFP mutant forms was dependent on the strength of the promoter used. The strongest promoter was the DC3 promoter, and no notable differences in the intensity and brightness of all three versions of GFP were observed. GFP-expressing pollen from tobacco and snapdragon developed in vitro for several days until maturity and germinated in vitro as well as on the surface of stigmata, strongly suggesting that all three GFPs are not toxic for the development of functional pollen. Furthermore, stably transformed tobacco plants expressing GFP under the control of the strong pollen-expressed DC3 and LAT52 promoters were not impaired in reproductive function, confirming that GFP can be used as a non-destructive marker for plant reproductive biology and development.
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