Estimate of Gravisensitivity

Gravis ens itivity represents the ability to respond to a gravistimulus and can be estimated by different stimulation thresholds (Volkmann and Sievers 1979):

a. The minimal angle (rm) with respect to gravity that induces a reorientation of the extremity of the organ;

b. The presentation time (tp), which is the minimum duration of gravistimulus at 1 g to provoke a slight but significant gravitropic response;

c. The threshold acceleration (at) that can be sensed by the plant organs.

The first parameter (rm) can be determined on the ground by stimulating the organs at small angles with respect to gravity and by following their gravitropic response. Although simple in its principle, this experiment was not carried out very often (see Volkmann and Sievers 1979) since the growth of the plant organs is not straight, their tip being subjected to oscillations (Johnsson 1997). A displacement of a few degrees from the vertical position seems to be perceived (see Volkmann and Sievers 1979). However, it has recently been shown that 15 deg is the minimal angle which induces a response in Arabidopsis roots (Mullen et al. 2000).

For determining the two other parameters (tp, am) experiments should be carried out in the absence of sensed Earth's gravity. Plant physiologists have therefore used clinostats for decades in order to simulate weightlessness by rotating the plant about a horizontal axis at about 1-2 rpm (Figure 6-08A). The unilateral effect of gravity is thus compensated by the rotation on the one axis clinostat and the plants do not show any gravitropic response. However, it is clear that clinorotating does not nullify gravity, but can induce a slight and continuous stimulation (Aarrouf et al. 1999).

The presentation time is determined by stopping the clinostat for various periods and clinorotating the plants again to follow the gravitropic response resulting from gravistimulus (stop of the clinostat). It is thus possible to draw a dose-response curve of the gravitropic reaction. By hypothesizing that the gravitropic response varies as a function of the logarithm of the dose of stimulus:

Response = a x log (Stimulation time) + b where a and b are constants. One can estimate the presentation time, which corresponds to the intercept of the curve with the x-axis (see Figure 6-05B). In theory, it should be possible to submit plants to very short periods of stimulation and to analyze their gravitropic response after clinorotation in order to determine directly the presentation time without any extrapolation. However, this is made difficult because the extremity of the organs oscillates. Direct measurements often lead to longer presentation times (see Larsen 1962), whereas by extrapolation they were estimated in general at about 10-30 sec for roots and 20-80 sec for shoots.

The presentation dose (dp, expressed in g x s) can be measured when stimulus varies as a function of time and as a function of the acceleration. The presentation dose dp, equals the acceleration (a) times the presentation time for a given acceleration (tpa). Thus, dp = ax tpa.

Horizontal

Vertical rotation (2 rpmi<

Horizontal

Figure 6-08. One-axis (in A) and two-axes (in B) clinostats. In A, Pfeffer's clinostat. The plant is rotated about a horizontal axis with a rotation speed of 1-2 rpm. Thus, the unilateral effect of gravity on plant in the vertical position becomes an omnilateral action of gravity. In no case gravity is nullified. The one-axis clinostat is a simulation o f weightlessness in the way that no gravi tropic curvature is observed although the main axis of the plant is horizontal. In B, the plants are rotated about a horizontal axis (1-2 rpm) to simulate weightlessness. They are also centrifuged about a vertical axis to subject them to gravistimulus. The dose of stimulus depends upon the amplitude of the centrifugal acceleration and the duration of the centrifugation. The two-axes clinostat is veiy useful to determine the threshold of gravisensing. Adapted from Shen-Miller et al (1968).

To analyze the presentation dose, a two-axis clinostat is needed on the ground. The plants are rotated about a horizontal axis to simulate weightlessness and centrifuged about a vertical axis (Figure 6-08B). This device permits to determine the threshold acceleration by exposing the plants to very low centrifugal acceleration for long periods. It has been demonstrated that plant organs are able to sense about 10~3 g, but roots seem to be more sensitive than shoots, since tpa is always lower for roots than for shoots (Shen-Miller et al. 1968).

It is well known that gravitropic response can be induced by stimulation periods shorter than the presentation time if gravistimulus is repeated intermittently (Volkmann and Sievers 1979). The minimal duration of stimulation which, repeated, leads to a gravitropic response is called perception time and should be about or even less than 1 sec (0.5 sec for A vena coleoptiles, according to Pickard 1973; or 1 sec for Cress roots, according to Hejnowicz et al. 1998).

Authors

Species

Organ

t„

dB

a,

Merkys and Laurinavicius (1990)

Lactuca

R H

1.5 x Iff4g 2.9x Iff3g

Perbal and Driss-Ecole (1994)

Lens culinaris

R

27 sec

Johnsson et al. (1995)

A vena sativa 1 g tall 1 g short

C C

55 gx s 120 gx s

Volkman and Tewinkel (1996a)

Lepidium sat.

0 g grown

1 g grown

R R

20-30 gxs 50-60 gxs

0 g grown

1 g grown

R R

10-24 gxs 18-31 gxs

Table 6-01. Presentation time (tp, in sec), presentation dose (dp, in g x s) and threshold acceleration (af, in g) of the gravitropic reaction of roots (R), coleoptiles (C) or hypocotyls (H) estimated in microgravity.

Table 6-01. Presentation time (tp, in sec), presentation dose (dp, in g x s) and threshold acceleration (af, in g) of the gravitropic reaction of roots (R), coleoptiles (C) or hypocotyls (H) estimated in microgravity.

Space represents a unique opportunity to study graviperception since it offers the possibility to carry out experiments on presentation time or threshold acceleration (Table 6-01). Although only few experiments have been done in space, the results obtained in microgravity confirmed to a certain extent those obtained with clinostats at least for roots. Thus, tp was found to be about 27 sec in lentil roots grown in microgravity (Perbal and Driss-Ecole 1994) and dp was 20-30 g x s in Cress seedling roots grown in the same condition (Volkmann and Tewinkel 1996a, 1996b). However, it was found with Cress roots grown on a 1-g centrifuge in space before stimulation, that dp was 50-60 g x s which indicated that the sensitivity was different when the roots were grown in 1 g or in microgravity. A similar result was observed by

Perbal et al. (1997) in lentil grown on a clinostat (in simulated microgravity) or in 1 g (in the vertical position) where tpa were 25 and 60 sec, respectively.

The fact that tpa seems to be almost equal when determined by the mean of a clinostat or in microgravity shows that a clinostat can to some extent simulate weightlessness. The difference in gravisensitivity between 0-g grown or 1-g grown seedlings roots was also observed recently on lentil seedlings (Perbal et al. 2004). This finding opened a new way of investigation for analyzing graviperception.

Figure 6-09. Dose response curve of the gravitropic reaction of lentil roots. The curvature observed after 90 min on the clinostat is reported as a function of the time of stimulation. In this example, the lentil seedlings were stimulated by gravity (1 g) from 1 to 20 min and placed on a clinostat (1 rpm). The two curves represent two different mathematical models to fit the experimental points. The L model (logarithmic model, dotted line) was commonly used (see Figure 6-05) until it has been shown that the H model (hyperbolic model, solid line) fitted better the experimental points (Perbal et al. 2002). The L model had the advantage to permit to determine the so-called presentation dose (or presentation time, tp), which corresponds to the intercept of the curve with the x-axis. In this particular example it is about 60 sec. This parameter was used to estimate gravisensitivity. This is not possible for the H model and gravisensitivity in this case can be measured as the slope of the curve at the origin.

These space experiments have led Perbal et al. (2002) to reconsider the way of determining the presentation time and presentation dose. The majority of the results concerning tp or dp were obtained by extrapolation assuming that the logarithmic model fitted the experimental points (see Figure 6-05B). Perbal et al. (2002) showed that very often the logarithmic model fitted correctly the data for stimulation time less than 10 min and greater than 1 min. They also demonstrated that the hyperbolic model corresponding to a ligand receptor system, (where response equals = ^ ^ d°se with a and b being constants) was better for almost all data published in the literature since the 1960's (Figure 6-09).

This model implies that there is a slight response even for a very short stimulation time. Thus, the only parameter directly related to the perception phase should be the perception time. According to these authors, the presentation dose should correspond to a dose of stimulation that induces a sufficient signal to provoke a visible curvature. Below this presentation dose, the quantity of stimulus should be too low to induce a curvature, or the curvature should be too slight to be measured.

Thus, space experiments have given the opportunity to reconsider and better estimate the parameters used for measuring graviperception and have led to new methods for estimating gravisensitivity.

* y * ■ ^ . A. Lentil seedlings were grown in f^ " ■'' -V microgravity for 27 h

\"> TCS "■* 3 V.oiiC1 and chemically fixed pjET'.1-^': " in space. They were

HK»«' - V. J '"« ' if.'- il^W^^JLjr. -1 treated on the ground

■■;-:. ' . ,■ f 'J sojLijiy jor routine micro-^^^ ^^^^^^^^^^^^ V^vm^av./.- ii,.,I //#.

' ** " were hydrated with a solution containing Cytochalasin B and grown on the ground for 27 h in the vertical position and chemically fixed as in A. N: nucleus; nu: nucleolus; mi: mitochondria; dw: distal wall; RE: endoplasmic reticulum; pw, proximal wall Courtesy ofDriss-Ecole, University Pierre et Marie Curie, Paris, France.

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