Severely Reduced Gravitropism in Dark-Grown Hypocotyls of a Starch-Deficient Mutant of Nicotiana Sylvestris'

Plant Physiol. (1990) 94, 1867-1873 Received for publication July 5, 1990 0032-0889/90/94/1867/07/$01 .00/0 Accepted September 1, 1990 Severely Reduced Gravitropism in Dark-Grown Hypocotyls of a Starch-Deficient Mutant of Nicotiana sylvestris' John Z. Kiss*2 and Fred D. Sack Department of Plant Biology, Ohio State University, Columbus, Ohio 43210-1293 ABSTRACT sive than WT3 maize coleoptiles (8). Light-grown hypocotyls Gravitropism in dark-grown hypocotyls of the wild type was of a starchless mutant ofArabidopsis exhibited 70 to 80% of compared with a starch-deficient Nicotiana sylvestris mutant (NS the gravitropic curvature of the WT (3). In general, there are 458) to test the effects of starch deficiency on gravity sensing. In relatively few studies on structural/functional aspects ofgrav- a time course of curvature measured using infrared video, the itropic sensing in stems (14, 15). response of the mutant was greatly reduced compared to the In the present study, we evaluate gravitropism in dark- wild type; 72 hours after reorientation, curvature was about 100 grown hypocotyls of a starch-deficient Nicotiana mutant. We for NS 458 and about 700 for wild type. In dishes maintained in a report here that the gravitropic performance of mutant hy- vertical orientation, wild-type hypocotyls were predominantly ver- pocotyls is less than that ofthe and that tical, whereas NS 458 hypocotyls were severely disoriented with WT, starch deficiency about 5 times more orientational variability than wild type. Since reduces gravitropic sensitivity more in dark-grown Nicotiana the growth rates were equal for both genotypes and phototropic hypocotyls than in dark- or light-grown roots. These results curvature was only slightly inhibited in NS 458, the mutation are consistent with a plastid based statolith hypothesis for probably affects gravity perception rather than differential shoots as well as roots. growth. Our data suggest that starch deficiency reduces gravitropic sensitivity more in dark-grown hypocotyls than in dark- or light-grown roots in this mutant and support the hypothesis that MATERIALS AND METHODS amyloplasts function as statoliths in shoots as well as roots. Plant Material and Culture Conditions Seeds of the WT of Nicotiana sylvestris Speg. et Comes, According to the starch statolith hypothesis, amyloplasts and of the starch-deficient mutant NS 458 (F3 generation are involved in gravity perception in both roots and shoots from the first backcross) were obtained from Dr. Kenneth R. (reviewed in refs. 1, 16, 21). To test this hypothesis, gravitrop- Hanson (Connecticut Agricultural Experimental Station, New ism has been examined in starch-deficient plants obtained by Haven, CT). NS 458 has a recessive mutation in a single treatment (e.g applied chemicals, darkness) or mutation (re- nuclear gene which makes it deficient in the activity ofplastid viewed in ref. 18). phosphoglucomutase (4). Because roots of a starchless Arabidopsis mutant are grav- For gravitropic curvature, growth, and structural studies, itropic, starch, amyloplasts, and amyloplast sedimentation are seedlings were grown at 24°C under sterile conditions in not necessary for gravitropic sensing in these roots (3, 9). square polystyrene Petri dishes (100 x 15 mm, VWR Scien- However, because the roots ofthe mutant Arabidopsis and of tific) on 1% (w/v) agar containing the nutrients described in a starch-deficient Nicotiana mutant have decreased gravi- Haughn and Somerville (6) with 1% (w/v) sucrose added. tropic sensitivity, it is likely that amyloplasts and a full com- Seeds were surface sterilized (6) and sown on the agar medium plement of starch are necessary for full gravitropic sensitivity in rows, and the Petri dishes were sealed with Parafilm. The (9, 10). dishes were placed on edge in a rack so that the surface ofthe Few data are available about the effects of starch deficiency agar was vertical. To promote germination, the seeds were on shoot gravitropism. In Hordeum pulvini, the degree of incubated on the medium for 3 d under continuous illumi- gravitropic curvature was proportional to the amount of nation (90-100 E * m-2 s-' PAR from 40-W General Electric starch present, and pulvini that were completely destarched 'warm white' fluorescent lamps), and then the dishes were (by treatment) did not curve upward at all (20). Coleoptiles placed in complete darkness for an additional 6 to 7 d. of the low starch amylomaize mutant were less gravirespon- Seedlings were used when hypocotyls were 16 to 20 mm long, i.e. 9 to 10 d after sowing. All experiments were repeated two ' This research was supported by grant NAGW-780 from the National Aeronautics and Space Administration. to five times. 2 Present address: Department of Molecular, Cellular, and Devel- In one set of experiments, the orientation of roots was opmental Biology, University ofColorado, Boulder, CO 80309-0347. measured from seedlings grown in the light; details of these 'Abbreviations: WT, wild-type; IKI, iodine potassium iodide. culture conditions were described previously (1O). 1867 1 868 KISS AND SACK Plant Physiol. Vol. 94, 1990 Microscopy illumination. Seedlings were illuminated (with infrared) only for a few seconds for recording at each point in the time To fix dark-grown seedlings in vertically oriented Petri course, a lighting regime which did not increase the temper- dishes, cotton gauze was gently placed over the seedlings in ature inside the box. Images of the seedlings were obtained dim green light (15-W incandescent Westinghouse bulb fil- using a Panasonic WV-1410 Vidicon videocamera. Curvature tered through two layers of Roscolux Dark Yellow Green was measured as increments over a starting value from the plastic; Rosco Labs, Port Chester, NY). Fixative (1% [v/v] video monitor. glutaraldehyde, 1% [w/vJ paraformaldehyde, 5 mm calcium Growth rates were calculated from increases in length of chloride buffered with 50 mm sodium cacodylate buffer [pH hypocotyls over a 3 d period (using the infrared video system). 7.2] at 24°C) was then introduced into each dish, and dishes All growth and curvature data are mean values ± SE (except were maintained in their original orientation for 2.5 h in the for Table I which lists SD). A t test of means was performed dark. The seedlings were transferred to fresh fixative for 2 h, according to the procedures of S.A.S. (19). aspirated at 0.5 atm, rinsed in buffer, and postfixed in buffered 2% (w/v) osmium tetroxide for 15 h at 4°C. The tissue was Phototropism dehydrated through an acetone series, infiltrated for 48 h, and embedded in Spurr's resin. For light microscopy, 1.5 to 2.0 For phototropism studies, culture conditions were as de- ,um sections were stained with toluidine blue. For electron scribed above except that the seedlings were grown in trans- microscopy, silver-to-gold sections were stained with uranyl parent polycarbonate Magenta GA-7-3 vessels (7.6 cm3, acetate and lead citrate and viewed at 80 kV in a Zeiss 10 Sigma). Vertically grown seedlings were continuously illumi- transmission electron microscope. In both cases, approxi- nated from one side using a 460 nm blue filter (No. 2045, mately median longitudinal sections were examined. In ad- Rohm and Haas, Philadelphia, PA) with an intensity (at the dition, whole mounts of seedlings fixed in place were stained seedlings) of 3.0 to 5.5 usE m-2 s'. Twice as many mutant with IKI (11) and examined using light microscopy. as WT seeds were sown to obtain a sufficient yield of usable (vertical ± 200) mutant hypocotyls. To record the response, Measurement of Vertical Orientation, Curvature, and seedlings were placed under fluorescent light for immediate Growth photography. To determine hypocotyl curvature, angles were measured from photographic prints (9). To quantify the extent of deviation from the gravity vector ofvertically grown plants, the angle between the hypocotyl or RESULTS root apices and the gravity vector was measured from photographic prints as described (9), except that a scale of0 to 180° Starch Content and Plastid Sedimentation was used in the present study. The vertical was defined as 00 (i.e. up for hypocotyls and down for roots). Staining with IKI indicated that dark-grown WT hypocotyls To avoid possible interactions between visible light and (Fig. 2a) contained much more starch than mutant NS 458 gravitropism or growth, the time course ofcurvature following hypocotyls (Fig. 2b). However, by careful observation, small gravistimulation and growth rates ofhypocotyls was measured starch grains could be detected in IKI stained mutant hypo- using an infrared video system (Fig. 1). Dark-grown seedlings cotyls. Most of the starch in the WT and mutant was found were placed in a light-tight box and illuminated with a Syl- in the endodermis or starch sheath (Figs. 2, 3). This distri- vania infrared lamp filtered through a long pass infrared filter bution and the relative amounts of starch were confirmed RG830-850 (Schott Glass, Duryea, PA). The inside of the by transmission electron microscopy (Fig. 3a, b). Prolamel- box was lined with aluminum foil to reflect the infrared lar bodies were observed in plastids of both genotypes (Fig. 3a, b). Amyloplast sedimentation was observed in endodermal cells in vertical upright parts of WT hypocotyls (Figs. 2a, 3c). light-tight box Light and transmission electron microscopy revealed that zjf-> IR source plastids of the mutant were not sedimented in endodermal cells (Fig. 3d). videocamera < 0 XI~~~~~~~~~~Rfilter Orientation of Vertically Grown Seedlings Dark-grown WT hypocotyls were closely oriented around the gravity vector (Fig. 4a) whereas mutant hypocotyls were severely disoriented (Fig. 4b). The distribution around and rotatable extent of deviation from the gravity vector of hypocotyls in arm vertically oriented Petri dishes are shown in histograms (Fig. IR filter Petri dish with 5).

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