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 Biology, Ohio State University, Columbus, Ohio 43210-1293

ABSTRACT sive than WT3 (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 . These results curvature was only slightly inhibited in NS 458, the mutation are consistent with a based statolith hypothesis for probably affects gravity perception rather than differential shoots as well as roots. growth. Our data suggest that starch deficiency reduces gravi- tropic 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 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 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 apices and the gravity vector was measured from photo- graphic 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 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). Orientation was further quantified by calculating the mean seedlings angle ± SD from the vertical (Table I). The mean angle from the vertical of dark-grown mutant Figure 1. Diagram of the infrared (IR) video system that was used hypocotyls was 47.0°, whereas it was 10.6° for the WT, and to measure curvature and growth of Nicotiana hypocotyls. Details the ratio of the standard deviations of the mutant:WT hypo- are in "Materials and Methods." cotyls was 5.3 (Table I). Despite this severe disorientation, REDUCED GRAVITROPISM IN STARCH-DEFICIENT NICOTIANA 1 869

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Figure 2. Whole mounts of dark-grown Nicotiana hypocotyls stained for starch with IKI. Direction of gravity is indicated by arrows. a, apical of the shoot; C, cortical cells; S, stele (x140). a, WT. Amyloplasts are sedimented (arrowheads) in the endodermis including cells in the petiole (asterisk). b, Starch-deficient mutant NS 458. jI; mutant hypocotyls were somewhat oriented to gravity since of mutant hypocotyls to curve upward regardless of starting the mean angle was 470 rather than 900 (agravitropic). orientation. Mutant roots were less oriented (in terms of both mean While curvature of mutant hypocotyls did not increase angle and SD) relative to the vertical than WT roots, especially beyond approximately 120 after 72 h, WT hypocotyls contin- when grown in the dark (Table I). Both WT and mutant roots ued to curve upward after 72 h. Thus, WT hypocotyls (n = were less oriented (mean angle and SD) when grown in the 14) curved 75.90 ± 5.5° (±SE) and 85.9° ± 5.00 at 7 and 14 d, dark than in the light. While WT hypocotyls and WT roots respectively, while mutant hypocotyls (n = 13) curved 11.9° (both dark-grown) had comparable mean angles from the ± 5.8° and 12.30 ± 5.80 at the respective times. vertical, dark-grown mutant hypocotyls were much more disoriented (mean angle and SD) than dark-grown mutant Growth Rates roots. Furthermore, in all cases studied, the mutant organs were less oriented around the gravity vector compared to Growth rates of dark-grown vertical hypocotyls were meas- the WT. ured using seedlings of age and size comparable to those used The orientation of light-grown hypocotyls was not com- in the time course of curvature studies. The mean values (in pared because of the interaction between gravitropism and um-h-') were 28.5 ± 1.3 (n = 83) for the WT and 25.8 ± 1.6 light effects including phototropism (5, 22), whereas the ori- (n = 73) for the mutant. A t test indicated that these growth entation of light-grown roots could be measured since they rates were not significantly different (P > 0. 1). are not phototropic (10). Phototropism Time Course of Gravitropic Curvature In response to exposure to unilateral blue light, dark-grown mutant hypocotyls curved 49, 65, and 79% as much as When the time course of gravitropic curvature was meas- dark-grown WT hypocotyls at 4, 8, and 24 h, respectively ured with an infrared video system, curvature was greatly (Table II). reduced in dark-grown mutant hypocotyls compared to the WT, but mutant hypocotyls did curve upward slightly (Fig. DISCUSSION 6). Since mutant hypocotyls were severely disoriented (Fig. 4b), they were not all gravistimulated in a horizontal orien- The NS 458 mutant of Nicotiana sylvestris has altered tation by reorienting the dishes 900. However, qualitative plastid phosphoglucomutase activity and consequently is observations indicated there was no difference in the ability starch deficient. Small starch grains were detected in mutant 1870 KISS AND SACK Plant Physiol. Vol. 94, 1990

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Figure 3. Light (LM) and electron micrographs (EM) of dark-grown Nicotiana hypocotyls. Arrow indicates the direction of gravity; N, nucleus; p, prolamellar body; Pa, parenchyma cell of cortex; s, starch grain; St, stele. a, EM. The WT has typical amyloplasts filled with starch (x7,200). b, EM. Mutant plastids have much less starch than those of the WT (x20,000). c, LM of WT. Proximal region of dark-grown hypocotyl. The amyloplasts are sedimented (arrowheads) in endodermal cells (E) (x290). d, EM of mutant. Proximal region of dark-grown hypocotyl. Plastids (arrowheads) are not sedimented in endodermal cell (E). Electron dense granules of unknown composition (asterisks) are present in the cytoplasm (x3,000). REDUCED GRAVITROPISM IN STARCH-DEFICIENT NICOTIANA 1871

are less sensitive to gravity than WT hypocotyls. These differ- ences are not likely to be due to hidden background muta- tions, since the NS 458 mutant was backcrossed. These data reinforce previous data for roots (9, 10) that a full complement of starch is necessary for full gravitropic sensitivity and that amyloplasts are likely to function as statoliths in hypocotyls as well as in roots. Although preliminary observations suggested that the dark- grown mutant Nicotiana hypocotyls were agravitropic, quan- tification permitted us to conclude that these mutant hypo- cotyls were weakly gravitropic because (a) mutant hypocotyls curved upward 12° (versus 68° for the WT) after 72 h of gravistimulation, and (b) vertically grown hypocotyls of the mutant had a mean angle of 470; presumably, if they were agravitropic, this angle would be 900. It would be valuable to determine whether other mutants described as agravitropic (12) are actually weakly gravitropic. While dark-grown Nico- tiana hypocotyls are weakly gravitropic, it also would be important to determine whether light affects the relative grav-

Figure 4. Dark-grown Nicotiana seedlings. Dishes were maintained cn in the same orientation throughout the growth period, and the gravity 4-) vector is toward the bottom of the figure (x1.5). a, WT hypocotyls 0 are upright and oriented around the gravity vector; b, most mutant 0 (M) hypocotyls are disoriented relative to gravity. 0

0~ plastids of the root (10) and hypocotyl (Fig. 3b). In contrast, Arabidopsis mutant TC7, which also is deficient in plastid 6 phosphoglucomutase activity, is starchless (3, 9, 17). z Our results demonstrate that dark-grown NS 458 hypoco- tyls are less responsive to gravity than dark-grown WT hypo- cotyls since: (a) WT hypocotyls were oriented while those of U,. the mutant were severely disoriented during vertical growth, 0 and (b) curvature of mutant hypocotyls was greatly reduced 0 compared to WT hypocotyls in time course studies. In pre- 4.- vious work with roots of Arabidopsis and Nicotiana, gravi- 0 tropic sensitivity was measured by calculating the presentation and perception times, which estimate thresholds for gravi- 6 (9, 10). Because of the difficulty of using a clinostat in conjunction with the infrared recording system used U, for hypocotyl measurements, these threshold values were 4- not determined. 0 Since threshold values were not measured, the question 0 arises as to whether reduced curvature in mutant hypocotyls results from lowered sensitivity (perception) or whether im- pairment of later stages of gravitropism contributes as well. C:i- We suggest that the primary effect of the mutation is upon gravity perception rather than the later stages. First, el ongation was not impaired by the mutation since hypocotyl growth rates were equal in both genotypes. Second, mutant hypocotyls were capable of differential growth that produced phototropic curvature. Although the phototropic curvature of Degrees from Vertical mutant hypocotyls 24 h after unilateral irradiation was some- Figure 5. Histograms of orientation with respect to the gravity vector what reduced compared to the WT (79% of WT curvature), of populations of Nicotiana hypocotyls and roots that were vertically gravitropic curvature of the mutant was only 13% of the WT grown. Each bar represents a 100 interval, and 00 represents the after 24 h ofgravitropic stimulation. Thus, it is likely that the vertical (i.e. up for hypocotyls and down for roots; 900 is the horizon- NS 458 hypocotyls are impaired more in gravitropic sensing tal). Means, sample sizes, and standard deviations are given in than in differential growth and that these mutant hypocotyls Table I. 1 872 KISS AND SACK Plant Physiol. Vol. 94, 1990

Table I. Orientation of Vertically Grown Organs of Nicotiana Table II. Phototropic Curvature of Dark-Grown Hypocotyls Toward 00 = vertical (i.e. up for hypocotyls and down for roots). Unilateral Blue Light Orientation Treatment Curvature Treatment M SD/WT SD Duration Wild-type Mutant Mutant Wild-type degrees h degrees Hypocotyls in dark 47.0 ± 41.9a 10.6 ± 7.9 5.3 4 29.9±5.1a 14.6±4.2 (1 53)b (155) (41)b (26) Roots in dark 23.5 ± 23.1 12.2 ± 14.4 1.6 8 41.2 ± 4.7 26.6 ± 4.4 (133) (157) (46) (43) Roots in light 8.9 ± 9.4 8.1 ± 7.3 1.3 24 78.6 ± 3.8 62.1 ± 6.0 (157) (158) (49) (41) b a Mean ± SD. b Sample size. a Mean ± SE. Sample size. itropic performance ofmutant and WT hypocotyls, since light contributes to sensing and functions in addition to amylo- is known to enhance gravitropism in stems (2). plasts. If the mutant plastids were statoliths in these hypoco- Because there are extensive correlations between the capa- tyls, then plastid sedimentation is not required for sensing, bility for gravitropic curvature and the presence ofamyloplast although sedimentation (which occurs with a full complement sedimentation in the stem endodermis, or starch sheath, (7, ofstarch) may be necessary for full sensitivity. This conclusion 13), we investigated plastid sedimentation in these cells. Amy- is consistent with data for starch-deficient mutant roots loplasts were sedimented in endodermal cells in the proximal (9, 10). regions of WT Nicotiana hypocotyls. However, the starch- Previous data showed that starch-deficient Nicotiana roots deficient plastids were not sedimented in comparable regions were more closely oriented around the vertical than starchless of mutant hypocotyls. Arabidopsis roots (9, 10). We suggested that this improved As mentioned, the severely reduced gravitropic perform- orientation resulted from the presumed relative increase in ance ofthe dark-grown mutant Nicotiana hypocotyls suggests plastid buoyant mass (Nicotiana mutant plastids > Arabidop- that gravitropic sensing is affected by the starch deficiency. It sis mutant plastids). However, we report here that starch- is likely that starch deficiency reduces gravitropic sensitivity deficient Nicotiana hypocotyls are less oriented about the by reducing plastid buoyant mass, although it cannot be ruled vertical than starchless Arabidopsis hypocotyls (Fig. 8 in ref. out that other effects of changes in carbohydrate metabolism 3). These observations suggest that comparisons between dif- might inhibit sensing instead or as well. Clearly, the mutant ferent species may not be appropriate. plastids have less mass than WT amyloplasts since the mutant Nevertheless, it is useful to compare the gravitropic per- plastids are not sedimented. formance of organs within a species (i.e. roots versus shoots) The residual gravitropic sensing present in the mutant can and between genotypes (i.e. WT versus mutant). Both Nico- be attributed to either (a) starch-deficient plastids functioning tiana and Arabidopsis mutant hypocotyls were more disori- as statoliths, or (b) an unknown, second component that ented than mutant roots of their respective species (3, 10, this paper). This suggests that starch deficiency reduces gravitropic sensitivity more severely in shoots than in roots, and that co 75- gravity perception in shoots may require a greater plastid WT o buoyant mass than in roots. In all cases studied, the starch-deficient mutants showed U60 0 reduced gravitropism (3) and reduced gravitropic sensitivity 0 (9, 10, this paper) compared to their respective WT organs. 45- These data are consistent with a plastid-based statolith hy- Lli pothesis for gravity perception in both roots and shoots. 0 30 0 ACKNOWLEDGEMENTS "' 0o/ mM 1 5- We thank Roger Hangarter, Helen Kiss, and Jeff Young for valu- ah able discussions. Thanks also to Jeff Osborn for help with graphics u O and Jeff Young for helping to develop the infrared video recording k0 1 2 24 36 48 60 72 system. TIME (Hours) LITERATURE CITED Figure 6. Time course of curvature of dark-grown Nicotiana (n = 1. Bjorkman T ( 1988) Perception of gravity by plants. Adv Bot Res 113) and mutant (n = 111) hypocotyls. Upward curvature following 15: 1-41 placement in a horizontal orientation was measured with an infrared 2. Britz SJ, Galston AW (1982) Light-enhanced perception of video system (Fig. 1). All SE bars are shorter than the diameter of the gravity in stems of intact pea seedlings. Planta 154: 189-192 symbols. 3. Caspar T, Pickard BG (1989) Gravitropism in a starchless mutant REDUCED GRAVITROPISM IN STARCH-DEFICIENT NICOTIANA 1 873

of Arabidopsis: implications for the starch-statolith theory of 13. Perbal C, Riviere S (1980) Perception et reaction geotropiques gravity sensing. Planta 177: 185-197 de l'epicotyle d'Asparagus officinalis L. Physiol Plant 48: 51- 4. Hanson KR, McHale NA (1988) A starchless mutant ofNicotiana 58 sylvestris containing a modified plastid phosphoglucomutase. 14. Roberts JA (1984) Tropic responses of hypocotyls from normal Plant Physiol 88: 838-844 tomato plants and the gravitropic mutant Lazy-l. Plant Cell 5. Hart JW, MacDonald IR (1981) Phototropism and geotropism Environ 7: 515-520 in hypocotyls of cress (Lepidium sativum L.). Plant Cell En- 15. Sack FD (1987) The structure ofthe stem endodermis in etiolated viron 4: 197-201 pea seedlings. Can J Bot 65: 1514-1519 6. Haughn GW, Somerville C (1986) Sulfonylurea-resistant mutants 16. Sack FD (1991) Plant gravity sensing. Int Rev Cytol (in press) ofArabidopsis thaliana. Mol Gen Genet 204: 430-434 17. Sack FD, Kiss JZ (1989) Rootcap structure in wild-type and in 7. Hawker LE (1932) A quantitative study of the geotropism of a starchless mutant ofArabidopsis. Am J Bot 76: 454-464 seedlings with special reference to the nature and development 18. Sack FD, Kiss JZ (1989) Plastids and gravity perception. In CD of their statolith apparatus. Ann Bot 46: 12 1-157 Boyer, JC Shannon, RC Hardison, 8. Hertel R, de la Fuente RK, Leopold AC (1969) Geotropism and eds, Physiology, Biochem- the lateral transport of in the corn mutant amylomaize. istry, and Genetics of Nongreen Plastids. American Society of Planta 88: 204-214 Plant Physiologists, Rockville, MD, pp 171-181 9. Kiss JZ, Hertel R, Sack FD (1989) Amyloplasts are necessary 19. SAS (1982) Statistical Analysis System user's guide: statistics. for full gravitropic sensitivity in roots ofArabidopsis thaliana. SAS Institute, Cary, NC Planta 177: 198-206 20. Song I, Lu CR, Brock TG, Kaufman PB (1988) Do starch 10. Kiss JZ, Sack FD (1989) Reduced gravitropic sensitivity in roots statoliths act as the gravisensors in cereal grass pulvini? Plant ofa starch-deficient mutant ofNicotiana sylvestris. Planta 180: Physiol 86: 1155-1162 123-130 21. Volkmann D, Sievers A (1979) Graviperception in multicellular 11. O'Brien TP, McCully ME (1981) The study of plant structure: organs. In W Haupt, ME Feinleib, eds, Encyclopedia of Plant principles and selected methods. Termarcarphi Pty Ltd, Mel- Physiology (New Series), Vol 7, Physiology of Movements. bourne, Australia Springer-Verlag, Berlin, pp 573-600 12. Olsen GM, Iversen T-H (1980) Growth and curvature in seed- 22. Woitzik F, Mohr H (1988) Control ofhypocotyl gravitropism by lings of Pisum sativum and an ageotropic mutant. Physiol in a dicotyledonous seedling (Sesamum indicum Plant 50: 269-274 L.). Plant Cell Environ 11: 663-668