Plant Physiol. (1994) 105: 937-940

The Response to Gravity 1s Correlated with the Number of Statoliths in Chara Rhizoids’

John Z. Kiss*

Department of Botany, Miami University, Oxford, Ohio 45056

In contrast to higher plants, Chara rhizoids have single mem- brane-bound compartments that appear to function as statoliths. Rhizoids were generated by germinating zygotes of Chara in either soil water (SW) medium or artificial pond water (APW) medium. Differential-interference-contrast microscopy demonstrated that rhizoids from SW-grown plants typically contain 50 to 60 statoliths per cell, whereas rhizoids from APW-grown plants contain 5 to 10 statoliths per cell. Rhizoids from SW are more responsive to gravity than rhizoids from APW because (a) SW rhizoids were oriented to gravity during vertical growth, whereas APW rhizoids were rela- tively disoriented, and (b) curvature of SW rhizoids was 3 to 4 times greater throughout the time course of curvature. l h e growth rate of APW rhizoids was significantly greater than that of SW- grown rhizoids. This latter result suggests that APW rhizoids are not limited in their ability for gravitropic curvature by growth and that these rhizoids are impaired in the early stages of gravitropism (i.e. gravity perception). Plants grown in APW appeared to be healthy because of their growth rate and the vigorous cytoplasmic streaming observed in the rhizoids. This study is comparable to earlier studies of gravitropism in starch-deficient mutants of higher plants and provides support for the role of statoliths in gravity perception.

Gravitropism in plants has been divided into the following stages: perception, transduction, and response, i.e. differen- tia1 growth leading to curvature (Evans et al., 1986). The perception of gravity is generally thought to involve stato- liths, which are dense, movable intracellular particles (Salis- bury, 1993).

In higher-plant roots and shoots, a great deal of evidence supports the hypothesis that amyloplasts serve as statoliths. This evidence includes the observation that starch-deficient plants have a weaker graviresponse compared to plants with the normal leve1 of starch (Hertel et al., 1969; Iversen, 1969; Sack and Kiss, 1989; Sack 1991). In these studies, starch deficiency was induced by treatment (e.g. applied chemicals, darkness) or mutation. Two particularly valuable mutants in plant gravitropism studies have been a starchless mutant of Arabidopsis and a low-starch mutant of Nicotiana that are deficient in the activity of the enzyme phosphoglucomutase (Kiss et al., 1989; Kiss and Sack, 1989, 1990). Both of these mutants are healthy plants that have identical growth rates

This research was supported by grants from the National Aero- nautics and Space Administration (NAGW-3783) and the Committee for Faculty Research (Miami University).

* Fax 1-513-529-4243.

relative to their respective wild types, yet they have greatly reduced gravitropic sensitivity.

In contrast to those in higher plants, the presumed stato- liths in rhizoids of the alga Chara are barium- and sulfur- containing vesicles that are located near the apex of the rhizoid and that settle to the lower cell wall within minutes after horizontal reorientation (Sievers and Volkmann, 1979; Sievers and Schnepf, 1981). When these vesicles are centri- fuged from the apex of the rhizoid, growth continues but the rhizoids do not respond to gravity (Buder, 1961). Following regeneration of these vesicles at the apex, the rhizoid again becomes graviresponsive. These and other observations (eg. Friedrich and Hertel, 1973) suggest that the vesicles function as statoliths in Chara rhizoids.

In this paper I report that the number of statoliths in Chara rhizoids can change depending on the type of growth me- dium used to culture the plants. Rhizoids with a greater number of statoliths are more oriented relative to the gravity vector and have a stronger graviresponse compared to rhi- zoids with fewer statoliths.

MATERIALS AND METHODS

Plant Material and Culture Conditions

Zygotes (fertilized oospores) of Chara contraria were col- lected from both Coot Lake in Boulder County, CO, and from the Scharbauer Ranch in Andrews County, TX, and were stored at 20°C. Zygotes were surface sterilized in 7% (v/v) commercial bleach and 0.002% (v/v) Triton X-100 for 30 min. After severa1 rinses in sterile, distilled water, zygotes were sown in Petri dishes (100 X 15 mm) either on SW extract in 1.2% (w/v) agar or on APW in 1.2% (w/v) agarose. SW extract was prepared by filtering 1 L of distilled water that was incubated for 24 h with 100 g of soil (collected from Lubbock, TX) and 1 g of agricultura1 limestone. APW con- sisted of 1 mM Hepes (pH 7.0), 1 mM NaCI, 0.05 mM KzSO4, 0.05 mM Caso4, 7 mM MgClZ, and 5 m~ CaC12.

Zygotes were sown onto the medium in Petri dishes that were sealed with Parafilm and placed on edge in a rack so that the surface of the agar or agarose was vertical. A line was drawn on each Petri dish to indicate the gravity vector. Approximately 7 to 14 d after incubation at 20°C with continuous illumination of 90 to 100 pmol m-’s-’ (from 40- W ”warm light” fluorescent bulbs), zygotes genninated to yield a rhizoid and a protonema. Rhizoids used in curvature

Abbreviations: APW, artificial pond water; SW, soil water. 93 7

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938 Kiss Plant Physiol. Vol. 105, 1994

Figure 1. Living Chara rhizoids viewed with differential-interfer-ence-contrast optics. Direction of gravity vector is indicated by thearrow in A. Scale bars = 20 fim. A, Rhizoid (grown in SW) has adistinctive structural polarity with statoliths (S) located near theapex and the nucleus (N) near the large vacuole (V) region. B, ThisSW-grown rhizoid contains approximately 50 statoliths (arrow-heads); however, most statoliths are not in the plane of focus. C,This APW-grown rhizoid has 5 statoliths (arrowheads), but onlythree are in the plane of focus.

and growth studies were from plants generated from zygotes(30-60 d after sowing).

Curvature and Growth Studies

The time course of curvature was determined by rotating(90°) dishes containing vertically grown plants and thenphotographing the rhizoids with bright-field optics througha compound microscope at various times after reorientation.Vertical growth rates were determined in a similar mannerand were calculated from increases in length over a 24-hperiod. Rhizoids that deviated more than 10° from the gravityvector before gravistimulation were excluded from all exper-iments. Curvature and growth measurements were madefrom photographic prints and are reported as mean values ±SE. A t test was used to determine if the differences in growthrates were significant (P < 0.05), and all experiments wererepeated at least three times.

Microscopy

Living rhizoids were examined with an Olympus BH-2compound microscope equipped with differential-interfer-ence-contrast optics. To determine the orientation of therhizoids during vertical growth, low-magnification observa-tions of plants were made with a Wild (Leica Inc., Deerfield,

IL) stereomicroscope. Photomicroscopic observations wererecorded with either Kodak Technical Pan film at ASA 50 orKodak T-Max 100 film.

RESULTS

Number of Statoliths in Rhizoids from SW versus APW

Chara rhizoids are relatively large cells that are approxi-mately 6 to 10 mm in length; a vacuole occupies most of thevolume of these cells (Fig. 1A). Single membrane-boundvesicles that appear to function as statoliths are located at theapex of the rhizoid. Living rhizoids were observed withdifferential-interference-contrast microscopy. Representativemicrographs of rhizoids from plants cultured in SW mediumand APW medium are shown in Figure 1, B and C, respec-tively. Rhizoids from SW-grown plants typically contain 50to 60 statoliths per cell (Fig. IB), and rhizoids from APW-grown plants typically contain 5 to 10 statoliths per cell (Fig.1C). There is greater variability in APW rhizoids, since somerhizoids observed had approximately 20 statoliths and otherswere observed with no statoliths. Rhizoids from both SW-and APW-grown plants exhibited vigorous cytoplasmicstreaming that is typical of characean cells (data not shown).

Orientation of Rhizoids

During vertical growth, rhizoids from plants in SW (Fig.2A) are more uniformly oriented with respect to gravity, arestraighter than rhizoids from APW plants (Fig. 2B), and arepositively gravitropic. Although most rhizoids from APW-grown plants are positively gravitropic, some of these rhizoids

Figure 2. Chara plants observed with a stereomicroscope. Directionof gravity vector is indicated by the arrow in B. R, Rhizoids; t,photosynthetic thallus; z, zygote. Scale bars = 1 mm. A, Rhizoidsfrom SW plant are oriented to the gravity vector. B, Rhizoids fromAPW plant are less oriented. Rhizoids that deviate from the gravityvector are indicated by arrowheads, and those that are out of theplane of focus are indicated by asterisks. www.plantphysiol.orgon June 16, 2020 - Published by Downloaded from

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Response to Gravity Is Correlated with Statolith Number 939

deviate from the gravity vector and are considerably undulate(Fig. 2B). In a few instances, rhizoids from APW plants werenegatively gravitropic (Fig. 3A). These negatively gravitropicrhizoids had only one to few statoliths per cell (Fig. 3B).

Time Course of Curvature

The time course of curvature after gravistimulation isshown in Figure 4. Rhizoids from SW plants curved morerapidly than rhizoids from APW plants throughout the courseof curvature. At all time points throughout the experiments,the curvature of SW rhizoids was three to four times that ofAPW rhizoids.

Growth Rates

Growth rates of vertical rhizoids were measured usingplants of age and size comparable to those used in the timecourse of curvature studies. The mean values in /urn h"1 are43.3 ± 2.8 (n = 15) for APW-grown plants and 26.6 ± 6.2 (n= 15) for SW-grown plants. A t test indicates that growth inAPW is significantly greater (P < 0.05; t = 1.761) than growthin SW. Zygotes sown in SW medium have a higher germi-nation rate than those sown in APW medium (data notshown).

Figure 3. Rhizoid from an APW-grown plant that was photographedwith bright-field optics through agaroseand a Petri dish. N, Nucleus.Direction of gravity vector is indicated by the arrow in A. B is ahigher-magnification view of the apex of the rhizoid in A. Thisrhizoid has two statoliths (arrowheads) and is negatively gravitropic.Scale bars = 200 ^m (A) and 50 ^m (B).

80

70

60

50

40

30

20

10

0

Soil Water

8 12 16

Time (hours)20 24

Figure 4. Time course of curvature of Chara rhizoids from plantsgrown in either SW or APW medium. The downward curvature ofrhizoids was measured after placement in a horizontal orientation.Data points are mean curvature ± SE (15 < n < 24).

DISCUSSION

These results demonstrate that rhizoids from Chara grownin SW medium are more responsive to gravity than rhizoidsfrom plants grown in APW medium because (a) SW rhizoidsare oriented to gravity during vertical growth, whereas APWrhizoids are relatively disoriented, and (b) curvature of SWrhizoids is three to four times greater throughout the timecourse of curvature. Furthermore, the data suggest that APWrhizoids are altered in their ability to perceive gravity ratherthan in the later stages of gravirropism, since APW rhizoidswere not limited in their growth rate. In fact, APW rhizoidshad a significantly greater growth rate than SW rhizoids.Plants grown in APW medium appeared to be healthy be-cause of their growth rate and the vigorous cytoplasmicstreaming observed in the rhizoids.

Rhizoids that deviated more than 10° from the verticalprior to gravistimulation were excluded from curvature andgrowth experiments. For rhizoids grown in both types ofmedium, the sampling was biased toward rhizoids that weremore graviresponsive, but more APW rhizoids than SWrhizoids were rejected in these experiments. If this criterionwere not applied, the differences between the APW and SWrhizoids probably would have been greater, and the APWrhizoids would have been considered even less responsive togravity.

The number of statoliths (50-60 per cell) observed in SWrhizoids is similar to values previously reported in the litera-ture for mature Chara rhizoids (generated from thallus cut-tings) grown in either Foresburg medium (which is a moreenriched medium than APW) or agar without any nutrients(Sievers, 1971; Sievers and Volkmann, 1979; Hejnowicz andSievers, 1981; Bartnik and Sievers, 1988; Volkmann et al.;1991; Braun and Sievers, 1993). However, several studieshave demonstrated that immature, developing Chara rhizoidscontain fewer statoliths than do mature rhizoids (Schroter etal., 1973; Kiss, 1992). Others have also used SW medium toculture Chara, but these workers focused on cytoplasmicstreaming in the thallus rather than gravirropism in therhizoids (Staves et al., 1992). Although a variation in thenumber of statoliths has been observed, no previous study www.plantphysiol.orgon June 16, 2020 - Published by Downloaded from

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940 Kiss Plant Physiol. Vol. 105, 1994

has reported so few statoliths (typically 5-10 per cell) as I found in APW rhizoids, except for a report by Gerber et al. (1993).

In this and a previous study (Kiss and Staehelin, 1993) rhizoids were generated by germinating zygotes, in contrast to most other investigations in which rhizoids were produced from cuttings of the thallus. The zygote method served our purposes better because (a) axenic cultures can be maintained since zygotes were surface sterilized, and (b) the number of statoliths per cell can be better controlled in this method than by using thallus cuttings. The reason for the second advan- tage is that since the statolith compartment in Chara rhizoids is rich in barium and sulfur (Sievers and Schmitz, 1982), the major source of statolith components in rhizoids germinated from zygotes is from the growth medium because of a limited pool of precursors in the zygote. In contrast, rhizoids gener- ated from thallus cuttings should have a larger pool of statolith precursors because of the presumably larger pool available in the thallus.

Previous studies have demonstrated a correlation between statolith number and graviresponsiveness in Chara rhizoids. Buder (1961) found that when statoliths were centrifuged away from the tip, the centrifuged rhizoids grew at the same rate as the control but did not respond to gravity. Hours after centrifugation, when the rhizoid began to regenerate stato- liths, graviresponsiveness was restored. In addition, Sievers and Schnepf (1981) reported that mature rhizoids with fewer statoliths have a weaker graviresponse.

In the present study growth conditions were manipulated so that statolith number was altered in Chara rhizoids, and rhizoids with a greater number of statoliths were 3 to 4 times more responsive to gravity compared to rhizoids with fewer statoliths. This system is in many ways comparable to the studies with higher-plant starch-deficient mutants (Kiss et al., 1989; Kiss and Sack, 1989, 1990), which provide strong support for the importance of statoliths in plant gravity perception.

ACKNOWLEDCMENTS

Thanks are due to Dr. Vemon Proctor (Texas Technical University, Lubbock, TX) for providing Chara zygotes.

Received January 21, 1994; accepted March 24, 1994. Copyright Clearance Center: 0032-0889/94/105/0937/04.

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