How to activate a plant gravireceptor. Early mechanisms of gravity sensing studied in characean rhizoids during parabolic flights

Abstract

Early processes underlying plant gravity sensing were investigated in rhizoids of Chara globularis under microgravity conditions provided by parabolic flights of the A300-Zero-G aircraft and of sounding rockets. By applying centrifugal forces during the microgravity phases of sounding rocket flights, lateral accelerations of 0.14 g, but not of 0.05 g, resulted in a displacement of statoliths. Settling of statoliths onto the subapical plasma membrane initiated the gravitropic response. Since actin controls the positioning of statoliths and restricts sedimentation of statoliths in these cells, it can be calculated that lateral actomyosin forces in a range of 2 x 10(-14) n act on statoliths to keep them in place. These forces represent the threshold value that has to be exceeded by any lateral acceleration stimulus for statolith sedimentation and gravisensing to occur. When rhizoids were gravistimulated during parabolic plane flights, the curvature angles of the flight samples, whose sedimented statoliths became weightless for 22 s during the 31 microgravity phases, were not different from those of in-flight 1g controls. However, in ground control experiments, curvature responses were drastically reduced when the contact of statoliths with the plasma membrane was intermittently interrupted by inverting gravistimulated cells for less than 10 s. Increasing the weight of sedimented statoliths by lateral centrifugation did not enhance the gravitropic response. These results provide evidence that graviperception in characean rhizoids requires contact of statoliths with membrane-bound receptor molecules rather than pressure or tension exerted by the weight of statoliths.

Figure 1.

Position of statoliths in chemically fixed rhizoids that were laterally centrifuged with 0.05g (A, D, and G), 0.14g (B, E, and H), and 0.25g (C, F, and I) for 13 min during the microgravity phase of the MAXUS-5 sounding rocket flight. Centrifugation with 0.25g and 0.14g, but not with 0.05g, induced a displacement of statoliths and settlement of some statoliths onto the centrifugal cell flank. A to C, Micrographs showing the position of statoliths in representative samples of each acceleration level. D to F, Geometric centers of the statolith complexes (black circles represent mean ± se in lateral direction, n ≥ 4). The shape of the statolith complex in one representative rhizoid of each acceleration level (rhizoids are different from those displayed in A–C) is shown in gray. G to I, Distribution of statolith frequency across the cell diameter (n ≥ 4). Arrows indicate direction of centrifugal force. Dashed lines represent median axes of the cells. Diameter of rhizoids, 30 μm.

Figure 2.

Distribution of statoliths in characean rhizoids before lift off (−250 s) of the MAXUS-5 sounding rocket and during lateral centrifugation in microgravity (indicated in seconds after lift off). A, Series of micrographs of a representative rhizoid exhibiting symmetrical distribution of statoliths across the cell diameter before lift off. Centrifugal displacement of statoliths caused by the 0.14g acceleration in microgravity was first detectable at +429 s. Individual statoliths settled onto the cell flank toward the end of the microgravity phase (+692 s and +827 s). Arrows indicate the direction of gravitational and centrifugal forces. Diameter of rhizoid, 30 μm. B to D, Distribution of statolith frequency across the cell diameter of four rhizoids at indicated times of the rocket flight demonstrating that statoliths were displaced from a symmetrical arrangement before lift off (B) to an asymmetrical distribution during lateral centrifugation (C and D).

Figure 3.

Graph showing curvature angles of control cells, which were continuously gravistimulated for 120 min at 90° on ground (white bar; n = 69) and of rhizoids that were horizontally positioned for 120 min on ground and intermittently centrifuged 62 times for 22 s at 2g (gray bar; n = 59). Not significantly different curvature values of controls and centrifuged samples (Student's t test, P = 0.8136) provide evidence that increasing the weight of sedimented statoliths by repeated short-term centrifugation does not affect gravitropic curvature. Data represent means ± se.

Figure 4.

Curvature angles of rhizoids after 10-min prestimulation at 90° on ground and subsequent stimulation at 1g, 2g, 3g, 4g, or 5g for 15 min in the direction of the initial gravistimulus. 1g controls (white bar) and centrifuged samples (gray bars) exhibited curvature angles that were all in the same range as revealed by pairwise Student's t test (P > 0.05), indicating that enhanced pressure on the gravisensitive site of the plasma membrane due to an increased weight of statoliths had no impact on gravitropic curvature. Data represent means ± se (n ≥ 11).

Figure 5.

Mean curvature angles of rhizoids that were prestimulated on ground for 10 min at 90° and subsequently inverted 31 times to 270° (gray bars) relative to the mean curvature angles of the corresponding control samples that were set to 100% (C, white bar). Rhizoids were repeatedly inverted for 5, 10, 22, or 30 s during a total experiment duration of 120 min, whereas control samples were stimulated at 90° for 120 min under continuous 1g conditions. Significantly reduced curvature angles of the inverted samples (Student's t test, P < 0.01; indicated by an asterisk) were observed when the duration of the inversion phases was longer than 5 s (n ≥ 44 for each sample). Complete data of all inversion experiments, including the absolute curvature values, are summarized in Table I.

Figure 6.

Displacement of statoliths after the inversion of gravistimulated rhizoids from 90° to 270° on ground. The graph shows the mean distances (±se, n = 18) of statoliths from the upper cell flank at the indicated times after inversion. Only those statoliths were selected for the measurements that were regarded as fully sedimented on the plasma membrane (PM) after 10 min of gravistimulation at 90°. A dashed line was drawn as a reference to indicate the position of the sedimented statoliths on the upper plasma membrane and the subsequent measurements of the distances were referred to this line. The series of micrographs shows that all statoliths have sedimented away from the upper plasma membrane already 5 s after inverting the representative rhizoid. Bar, 1 μm.

Figure 7.

Mean curvature angles of flight samples (gray bars) relative to the mean curvature angles of the corresponding in-flight control samples that were set to 100% (C, white bar). The experiments were conducted during the 36th ESA (sample nos. 1–3) and during the sixth DLR parabolic flight campaign (nos. 4–6). On flight day 2, two flights with reduced flight profiles were conducted (nos. 2A and 2B; for details of flight profiles, see Table II). All samples were tilted into a horizontal position 10 min prior to the first parabola and tilted back after the last parabola of the flight profile. In-flight control samples were laterally centrifuged at 1g during the microgravity phases. No significant differences in curvature angles were observed between flight samples and in-flight controls on any of the flights (Student's t test, P > 0.05; n ≥ 32 for each sample), indicating that graviperception was not interrupted when statoliths became weightless during microgravity. For complete data and absolute curvature values of all flight experiments, see Table II.