To determine if starch statoliths do, in fact, act as gravisensors in cereal grass shoots, starch was removed from the starch statoliths by placing 45-day-old intact barley plants (Hordeum vulgare cv 'Larker') in the dark at 25°C for 5 days. Evidence from staining with I2-KI, scanning electron microscopy, and transmission electron microscopy indicated that starch grains were no longer present in plastids in the pulvini of plants placed in the dark for 5 days. Furthermore, gravitropic curvature response in these pulvini was reduced to zero, even though pulvini from vertically oriented plants were still capable of elongating in response to applied auxin plus gibberellic acid. However, when 0.1 molar sucrose was fed to the dark pretreated, starch statolith-free pulvini during gravistimulation in the dark, they not only reformed starch grains in the starch-depleted plastids in the pulvini, but they also showed an upward bending response. Starch grain reformation appeared to precede reappearance of the graviresponse in these sucrose-fed pulvini. These results strongly support the view that starch statoliths do indeed serve as the gravisensors in cereal grass shoots.Recent studies in our laboratory (19-21) have been concerned with the cereal grass leaf-sheath pulvinus as a graviresponsive organ system and whether or not the starch-containing plastids in pulvinus cells of this system are indeed the gravisensors (statoliths). The young, undifferentiated grass shoot pulvinus does not possess any starch-containing statoliths, and it shows no upward bending curvature response when oriented horizontally (9,21,29). However, when the pulvinus is mature, starch statoliths are present in statenchyma cells along the inside of each of the longitudinally oriented vascular bundles, as seen in transverse sections of the pulvinus (Fig. 1). The structure of starch statoliths in grass shoots as examined by means of LM,2 SEM, and TEM (9, 19-21, 23, 26), as well as their purported function as gravisensor organelles (1,14,17,22,25,27), have been studied extensively. Furthermore, it has been shown that these organelles descend within the presentation time (1-2 min) for initiation of an upward bending graviresponse in the grass shoot pulvinus (21). Even so, and in spite of all previous work, we still have no definitive proof that starch statoliths are the gravisensors that trigger an upward bending response in prostrated cereal grass shoots. In other graviresponsive systems, such as in rootcaps of starch-free Arabidopsis mutants, they are not required (7,25); that is, the roots respond to gravistimulation to the same extent as do nonmutant plants, with starch statoliths present in the root ' Supported by National Aeronautics and Space Administration grant NAGW-34.2 Abbreviations: LM, light-microscopy; SEM, scanning electron microscopy; TEM, transmission electron microscopy.caps.In light of the above, we set out to determine whether or not gravitropic curvature could occur in barley shoots in the absence of starch in the pulvinus pla...

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Pulvini of excised stem segments from barley (Hordeum vulgare cv 'Larker') were pretreated with 1 millimolar coumarin before gravistimulation to reduce longitudinal cell expansion and exaggerate radial cell enlargement. The cellular localization and pattern of graviresponse across individual pulvini were then evaluated by cuffing the organ in cross-section, photographing the cross-section, and then measuring pulvinus thickness and the radial width of cortical and epidermal cells in enlargements of the photomicrographs. With respect to orientation during gravistimulation, we designated the uppermost point of the crosssection 0°and the lowermost point 1800. A gravity-induced increase in pulvinus thickness was observable within 400 of the vertical in coumann-treated pulvini. In upper halves of coumarintreated gravistimulated pulvini, cells in the inner cortex and inner epidermis had increased radial widths, relative to untreated gravistimulated pulvini. In lower halves of coumarin-treated pulvini, cells in the central and outer cortex and in the outer epidermis showed the greatest increase in radial width. Cells comprising the vascular bundles also increased in radial width, with this pattem following that of the central cortex. These results indicate (a) that all cell types are capable of showing a graviresponse, (b) that the graviresponse occurs in both the top and the bottom of the responding organ, and (c) that the magnitude of the response increases approximately linearly from the uppermost point to the lowermost. These results are also consistent with models of gravitropism that link the pattern and magnitude of the graviresponse to graviperception via statolith sedimentation.

Ultrastructural analyses of the cell walls from top and bottom halves of gravistimulated pulvini from oat leaves show a decrease in the density of material within the cell walls from the lower halves of pulvini after 24 h of gravistimulation. Assays of cellulose synthesis with a 14C-sucrose pulse-chase experiment indicate no difference in the amount of new cellulose synthesized in top compared with bottom halves of gravistimulated pulvini. The highest rate of cellulose synthesis occurs with 12-24 h of gravistimulation. Treatment of graviresponding pulvini with 2,6-dichlorobenzonitrile (DCBN) had only a minor effect on segment gravitropic curvature. We also found that there is no difference in the activities of either glucan synthase I or glucan synthase II in top halves as compared with bottom halves of gravistimulated pulvini. We conclude that the graviresponse in oat stems is not driven by new cell wall synthesis but, rather, by changes in cell wall plasticity and osmotic potential.

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