Journal of Experimental Botany, Vol. 52, No. 358, pp. 1029-1040,
May 1, 2001
© 2001 Oxford University Press
Original Papers |
Changes in starch content in oat (Avena sativa) shoot pulvini during the gravitropic response
1 Molecular, Cellular and Developmental Biology Group, Department of Biology, University of Michigan, Ann Arbor, Michigan 48109-1048, USA
2 Department of Biology, Yonsei University, Seoul 120 749, Korea
Received 4 July 2000; Accepted 29 November 2000
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Abstract |
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In order to determine if components of the signal transduction pathway are involved in starch metabolism during the gravitropic response, the effects of inhibitors of phosphoprotein phosphatases and protein kinases (OA), and calcium channel blockers (LaCl3), on gravitropic bending and starch levels in gravisensitive node/pulvini of oat shoots were examined. Among the compounds tested, okadaic acid (OA) and lanthanum chloride (LaCl3) showed the strongest inhibitory effects on the negative gravitropic curvature response in oat shoot node/pulvini. At the same time, they caused a rapid loss of starch in graviresponding pulvini based on a quantitative analysis of starch levels in the bending tissues over 48 h periods. These two compounds act initially to block the net increase in starch content that occurs during the early stages (0–9 h) in graviresponding oat shoot pulvini. As a result, starch levels drop precipitously in shoots treated with OA and LaCl3, starting at time zero of gravistimulation by reorientation. These findings suggest that protein dephosphorylation and calcium play a role in starch metabolism in oat shoot pulvini in response to a gravistimulation signal. They also indicate that the amount of starch present in the chloroplast gravisensors in oat shoot pulvini may determine the rate of upward bending in graviresponding pulvini.
Key words: Calcium, gravitropism, protein dephosphorylation, oat, pulvini, starch metabolism.
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Introduction |
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Biologists have long been interested in discovering how the gravitropic response mechanism works in both shoots and roots of plants. So far, it has been learned that all three components of this mechanism, graviperception, gravity signal transduction and asymmetric growth, are much more complex than originally thought. The starch-statolith hypothesis indicates that the gravisensors in roots are starch-filled amyloplasts, and in shoots, starch-filled chloroplasts (Perbal and Riviere, 1980
; Bjorkman, 1988; Song et al., 1988; Kiss et al., 1989; Sack, 1991, 1997). Among these works (Song et al., 1988), it has been shown that the size of starch grains in the chloroplast gravisensors was increased in pulvini when sucrose was added to barley stems; this resulted in an increase in the gravitropic curvature response. Among studies using starch-deficient mutants, it was reported that the degree of graviresponsiveness is proportional to the total mass of plastids (amyloplasts) per cell in Arabidopsis hypocotyls (Kiss and Sack, 1990; McCleary and Kiss, 1999; Kiss et al., 1997). However, to present knowledge, there has been no quantitative evidence reported on the relationship between the rate of gravitropic curvature and starch levels in the gravisensors of shoots.
It has been proposed that once gravity is perceived, the earliest events may involve the opening of voltage-gated channels in cell membranes, rapid asymmetric distribution of cytosolic calcium and of inositol 1,4,5-triphospahte (IP3), and changes in protein phosphorylation/dephosphorylation (Belavskaya, 1996; Friedmann et al., 1998; Kaufman et al., 1995; Perera et al., 1999; Trewavas and Malho, 1997; Chang and Kaufman, 2000). Taken together, these studies suggest that calcium and other components of the signal transduction pathway may be involved in the gravitropic response. Thus, there are at least two signal transduction pathways in the gravitropic response. One is the sensing pathway, which includes variations in cytosolic calcium and protein phosphorylation, and the other is the hormonal (auxin) signal transduction pathway, which can also involve calcium fluxes and protein phosphorylation.
In the present study, the graviresponsive cereal grass leaf sheath pulvinus is used as a model experimental system in order to gain a better understanding of how gravity is perceived and transduced in plants. Its response to gravistimulation by reorientation involves the basipetal movement of the gravisensors (starch-filled plastids located in cortical cells lying just inside each of the vascular bundles which pass longitudinally through the pulvinus) and differential cell elongation (Kaufman et al., 1995). The hypothesis that is to be tested here is as follows: during the gravitropic response, a certain level of starch may be required for graviresponding pulvini to maintain gravisensing capability, and correspondingly, that the amount of starch present at any given time is directly correlated with rate of upward bending. A corollary to this hypothesis is that protein phosphorylation and calcium, both components of the signal transduction pathway, may be involved in changes in starch metabolism that occur in response to gravistimulation treatment. The results indicate that starch level is changed upon gravistimulation by reorientation of plants in a correlative fashion with rate of gravitropic curvature and that protein dephosphorylation and calcium may mediate starch metabolism in response to a gravistimulation signal.
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Materials and methods |
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Plant materials and growth conditions
Seeds of oats (Avena sativa cv. ‘Victory’) were obtained from the Swedish Seed Association, Svalof International AB, Svalof, Sweden. Oat plants were grown at the University of Michigan Matthaei Botanical Gardens research greenhouse under continuous illumination provided by high pressure sodium vapour lamps (PL-Light Systems, Grimsby, Ontario L3M 4G3, Canada) at a light intensity of 1000 µmol m-2 s-1 1 m above the plants. The greenhouse temperatures were 27 °C for 16 h and 23 °C for 8 h in 24 h cycles. Plants are harvested 42–45 d after sowing seeds at a stage when next-to-last formed internodes were in log phase growth. From these shoots, stem segments are excised from the next-to-last nodes (p-1 node/pulvinus). They include 3 cm below and 6 cm above the p-1 node/pulvinus. As they are cut, stem segments are kept in a vertical position with their bases immersed in water. The segments are kept at 4 °C until use in experiments. Under these conditions, the gravitropic responsiveness is not significantly reduced within a week.
Pretreatments with inhibitors
The inhibitors employed in this investigation were selected on the basis of their being specific inhibitors of signal transduction processes in plants, animals, and microorganisms. They include: (l) okadaic acid (OA) (Christopher et al., 1997) and calyculin A (Gopalakrishna et al., 1992), potent inhibitors of protein phosphatases types 1 and 2A; NaF, an inhibitor of protein phosphatases; (2) staurosporine (STA) (Couldwell et al., 1994), K-252a (Hashimoto, 1988), and H-89 (Chijiwa et al., 1990), all inhibitors of protein kinase; (3) LaCl3 (Friedmann et al., 1998) verapamil (Lonsberry et al., 1994), and ruthenium red (Phillippe and Basa, 1996), all calcium channel blockers. Each of these inhibitors was obtained from Calbiochem (San Diego, CA) except NaF, staurosporine, and LaCl3 which were obtained from Sigma Chemical Co. (St Louis, MO). The inhibitors that were chosen reduced gravitropic curvature with no, or only small, effects on stem elongation.
Inhibitor pretreatments were made as follows: stem segments were gently abraded by rotating them three to four times with thumb and forefinger in a silicic acid paste. This is done in order to remove cuticular wax from the pulvinus, and thus, to improve inhibitor uptake by the pulvini. The respective inhibitors were made up in solutions containing 0.1 M sucrose and 50 mM Hepes-NaOH buffer (pH 7.5). Bases of stem segments were positioned vertically in the respective inhibitor/buffer solution, the heights of which were 4 cm or more, and thus, sufficient to soak the p-1 pulvini. Then, the stem segments were subjected to mild vacuum for 2 min, then placed in the dark at 25+1 °C for 2 h.
Gravistimulation treatments
Following pretreatments with the inhibitors, 120–150 oat stem segments were gravistimulated by positioning the segments horizontally between four paper towels (two above and two below) saturated with a solution containing 0.1 M sucrose and 50 mM Hepes-NaOH buffer (pH 7.5). Two 5 mm thick glass plates are placed above and below the paper towels so as to maintain the stem segments in a horizontal position during gravistimulation treatment and to prevent the segments from rotating during upward bending. While positioning the stem segments horizontally in this ‘sandwich’, it is critical to have the swollen p-1 node/pulvinus of each segment located just outside the edge of the two glass plates so that upward bending of the upper 6 cm portion is unimpaired. Once segments are positioned horizontally, the glass plate ‘sandwich’ is placed horizontally atop a stainless steel rack that sits inside a PlexiglasTM box (50x40x14 cm) containing 1 cm of water and covered with perforated SaranwrapTM (to provide 100% relative humidity). This box was then placed inside a controlled environmental chamber (Precision Scientific Co., Dual Program Illuminated Incubator 818) programmed for continuous darkness at 25±1 °C for 24 or 48 h. After each gravistimulation treatment, pulvini were cut and stored at -80 °C until used for starch analysis.
Gravitropic curvature and stem elongation measurements
Angles of curvature of p-1 node/pulvini were measured with a protractor after 24 h of gravistimulation for the inhibitor dose–response experiments and at 1, 3, 8, 24, and 48 h of gravistimulation for the inhibitor time-course of response experiments. At the same time, amount of linear extension growth of the internodes above the non-growing sheath portions of the stem segments was measured with a mm rule. The latter stem elongation measurements were made in order to determine the specificity of the inhibitor treatments to the gravitropic curvature response.
I2KI staining for observation of starch in chloroplasts
Next-to-last (p-1) node/pulvini from both vertical and gravistimulated shoots were hand-sectioned with a fresh and very sharp single-edge razor blade from top and bottom portions of each pulvinus. The best sections for observing the starch-filled chloroplasts were located in parenchyma tissue that lies just inside each vascular bundle (Fig. 1). Starch is not stored in any other tissues of the pulvinus than in aggregates of cortical cells referred to above.
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The freshly prepared sections were immediately stained with an aqueous solution of I2KI (1 g KI plus 0.3 g I2 in 100 ml distilled water). This stain is specific for starch (Juliano, 1980
). A blue-black colour is obtained in the chloroplasts if starch is present. The stained sections were observed with phase contrast optics at a magnification of x100 with a Nikon Orthophot-2 research microscope. Kodak T-Max 100 film was used to take photomicrographs.
Transmission electron microscopy (TEM)
The methods employed for TEM are basically the same as those employed earlier (Song et al., 1988). Small pieces of pulvinus tissue were fixed in 3% glutaraldehyde in 50 mM cacodylate buffer (pH 7.0) for 2 h at room temperature, then washed in three changes (10 min each) of cacodylate buffer. They were post-fixed with 1.5% osmium tetroxide for 2 h, then dehydrated in a graded series of ethanol. They were placed in Spurr's resin at 30% (1 h), 50% (1 h), and 100% (overnight), then embedded in 100% Spurr's resin and allowed to polymerize at 60 °C overnight. Specimens were sectioned with a diamond knife, stained with uranyl acetate followed by lead citrate, and observed in a Zeiss EM 10-CA transmission electron microscope.
Analysis of starch levels in oat shoot pulvini
The methods employed for starch analysis are essentially those of Hubbard et al. and are as follows: (1) 0.2–0.4 g of fresh pulvini tissues were ground in a mortar in 2.8–3.5 ml of 80% ethanol; (2) this was followed by heating the ground sample in a hot (80 °C) water bath for 5 min, then centrifuging it at 3000 rpm for 10 min; (3) the tissue was extracted three times as above and the supernantants were discarded; (4) 0.2 M KOH was added to the remaining pellets, followed by immersion of the tubes in a boiling water bath for 30 min; (5) after the pHs were adjusted to 7.0 with 1.0 M acetic acid, each sample was treated with 3 ml of dialysed amyloglucosidase (240 units per ml) with stirring, then placed in a water bath at 55 °C for 1 h with occasional stirring; (6) the samples were next placed in a boiling water bath for 5 min, cooled, and centrifuged at 15 000 rpm for 10 min; (7) the supernatants were decanted, filtered if necessary, made to a known volume, and analysed for D-glucose content using the hexokinase-glucose-6-phosphate dehydrogenase method (Hubbard et al., 1990). This method is based on the conversion of the glucose-6-phosphate to 6-phosphogluconolactone in the presence of NADP and glucose-6-phosphate dehydrogenase and thence to 6-phosphogluconate in the presence of phosphoglucose isomerase. Absorbance was read at 340 nm against distilled water after 30 min at room temperature. All reagents used in this starch analysis were purchased from Sigma Chemical Co. (St Louis, MO).
It should be noted that starch in pulvinus tissue occurs primarily in the plastid gravisensors. These are located in groups of cells lying just inside each longitudinal vascular bundle of the pulvinus and not in other cells that make up the pulvinus tissues (Kaufman et al., l995; Song et al., 1988). So the starch, as analysed by the above protocol, is essentially that which occurs in the plastid gravisensors.
Replication of experiments and statistical analysis of data
All experiments were repeated at least three times. The number of oat stem segments used in each experiment was 18–30 per treatment. The significance of the means was verified by the use of Student's t-test.
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Results |
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In order to study the behaviour of the starch-filled gravisensors (statoliths) during the gravitropic curvature response, the I2KI staining method was employed (see Materials and methods). As shown in Fig. 1
, the clearest image that could be obtained shows that the starch statoliths move downward in response to gravistimulation treatment. Subsequent experiments showed evidence that, following gravistimulation treatment, the downward movement of the starch statoliths is altered in response to treatment with several inhibitors of gravitropic bending. However, it was not possible to determine the kinetics of the observed changes with the I2KI staining method, nor could any quantitative data on changes in starch levels in the statoliths with this approach be obtained. Therefore, a kinetic analysis of starch levels was performed in order to gain an insight on statolith movement as expressed in terms of changes in starch levels following gravistimulation and inhibitor treatments (see Materials and methods).
The kinetics for gravitropic curvature response and changes in starch content were compared to see the possible relationship between the two phenomena. The gravitropic curvature response proceeded at a high rate between 0 and 8 h, then progressed at a slightly lower rate from 8–24 h following gravistimulation treatment (Fig. 2). During the period 24–48 h, the rate of upward bending was decelerated still further. The starch content increased from 0–8 h, then decreased steadily between 8 h and 48 h (Fig. 2). From these results, it appears that there is a correlation between the two changes, although any exact proportional relationship was not found. This kind of correlation is also described for the results described below.
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In order to determine if components of the signal transduction pathway are involved in gravity-induced changes in starch content of the gravisensors, the effects of several inhibitors of protein phosphorylation and dephosphorylation on gravitropic curvature response and changes in starch content of graviresponding pulvini were examined. Staurosporine (STA) and okadaic acid (OA) showed the strongest inhibitory effects among inhibitors of protein kinases and phosphoprotein phosphatases, respectively (Table 1
). Calyculin A at 0.5 µM and NaF at 1 mM caused a 40% and 85% decrease, respectively, in the gravitropic curvature response and had no significant effect on stem elongation. Another inhibitor of protein kinases, H-89 at 10 µM decreased gravitropic curvature by 30% as compared with control pulvini and had no effect on stem elongation, while 1 µM K252a did not show any significant effect on gravitropic curvature. Taken together, these results imply that protein phosphatases are more likely to be involved in the gravitropic response than are protein kinases.
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The effect of different concentrations (10-10-10-6 M) of okadaic acid (OA) on the gravitropic response after 24 h of gravistimulation is shown in Fig. 3A
. At concentrations of 10-7 and 10-6 M OA, gravitropic curvature was inhibited about 15% and 85%, respectively. In this same figure, it is clear that stem elongation above the p-1 node/pulvinus was not inhibited by OA at any of the concentrations employed. Figure 3B shows a time-course plot of the effect of 1 µM OA on gravitropic curvature. The kinetics indicate that inhibition of upward bending in the segments began within 1 h after initiation of the gravistimulation by reorientation of shoots. By 48 h, the inhibition of curvature elicited by OA treatment is nearly 85%. It is clear from these results that 10-6 M OA effectively ‘shuts down’ the gravitropic response and that its inhibitory effect on upward bending occurs very rapidly once stem segments are gravistimulated. OA has IC50 values of 10 nM for protein phosphatase 1 and 0.1 nM for protein phosphatase 2A (Christopher et al., 1997). Therefore, 10-6 M OA is thought not to be too high a concentration in order to manifest its in vivo physiological effects. It is also possible that cuticle layers are not completely removed by gentle abrasion with silicic acid, and this could limit the uptake of OA.
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Based on the results that OA exerted an inhibitory effect on gravitropic bending, its effect was determined on starch levels in the p-1 node/pulvini.
Figures 3C and 3D depict time-course changes in starch levels in pulvini of segments held vertically versus those that were gravistimulated (held horizontally), respectively, following + and -10-6 M OA pretreatment. For vertically-held segments, starch levels in control pulvini increase slightly during the first 8 h, while OA-treated ones show a rapid decline after 3 h of gravistimulation by positioning the shoots vertically over this same time period. After 8 h, both + and -OA treatments show similar rates of decline in starch levels. By 48 h, the decline in amount of starch from the initial level is 20% for -OA treatment, while that for +OA treatment is 40%. For the gravistimulated segments, the effect of OA in causing a decline in starch levels in the pulvini is even more profound. In contrast with control pulvini, where there was even greater starch synthesis during the first 8 h of gravistimulation, as compared with vertically held pulvini, -OA treatment caused an immediate decline in starch levels that reached a level by 48 h of about 14% of initial levels shown at the start of gravistimulation treatment. In contrast, control (-OA) pulvini starch levels at 48 h have lost only 25% of their initial level. OA pretreatment of oat stem segments completely abolished the net increase in starch level seen in control pulvini during the first 8 h of gravistimulation. Instead, it caused a continuous and rapid decline in starch levels in the pulvini over the entire gravistimulation period.
The next question asked is what would be the effect of OA on the gravitropic bending response and starch levels once shoots had already started to respond to gravistimulation? For this purpose, a 3 min pulse treatment with 3x10-7 M OA was given after 3 h of gravistimulation of oat stem segments by reorientation. Here, even with such a short pulse treatment, OA caused an immediate and continuous decrease in the rate of upward bending as compared with that of the control (Fig. 4A). By 24 h after the OA pulse treatment, the curvature response was only half that of control segments (mean amount of bending is 18° for +OA pulse treatment and 36° for the control). Kinetic changes in starch levels (Fig. 4B) clearly show that a 3 min pulse treatment with OA given at 3 h after initiation of the gravistimulation treatment abolished the obvious rise in starch levels seen in control pulvini 3–11 h after the initiation of gravistimulation treatment. Between 11 h and 27 h, the level of starch in OA-treated segments stabilized, whereas it dropped to the +OA level by 27 h in the control. The decrease in starch level is irreversible during the 27 h of gravistimulation, even though OA was administered for just 3 min. This is contrary to the case of LaCl3, which shows little difference from the control over a long-term gravistimulation period (Fig. 7). The results of Figs 3 and 4 indicate that administration of OA causes a drastic reduction in rate of gravitropic curvature and a very significant diminution in starch content in the pulvini during the early stages of gravitropic bending.
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Staurosporine (STA) suppressed gravitropic bending by 30% at 10-6 M and 65% at 10-5 M, but it had no adverse effect on on stem elongation (Fig. 5A
). So, the inhibitory effect is specific to the gravitropic bending response. A kinetic analysis of the effect of 3x10-6 M STA on the upward bending response is shown in Fig. 5B. This analysis shows that STA starts to suppress the upward curvature response within 3 h. This inhibitory effect increased with time, such that by 48 h, a 60% inhibition of upward curvature elicited by STA as compared with that in control, -STA, segments is observed. Figure 5C shows the effect of STA on starch content. Again, the control shows the early increase in starch level between 0 and 3 h after the initiation of gravistimulation treatment. In contrast with OA, 3x10-6 M STA treatment did not cause any difference in starch content from that of the control to occur over the 48 h gravistimulation period. The inhibitory effect of STA on the gravitropic response, like that caused by OA, started early. In contrast with OA, STA caused no adverse effect on the movement of the starch statoliths as checked with I2KI staining of fresh sections (data not shown). The starch-filled plastids fall normally to basal positions in graviresponding cells, as they did in control cells. This is corroborated by the data shown in Fig. 5C.
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The effects of three known calcium channel blockers, namely, verapamil, ruthenium red and LaCl3, on gravitropic curvature and stem elongation were examined next. The results shown in Table 2
indicate that 50 µM verapamil and 0.l mM ruthenium red caused 18% and 28% inhibition of gravitropic curvature compared with that for control pulvini. LaCl3 at 10 mM, a suboptimum concentration for inhibition, caused 59% inhibition of gravitropic curvature. However, because oat shoot pulvini have cuticularized epidermal outer cell walls that would form a barrier to rapid uptake of many large molecules such as these inhibitors, the concentrations of verapamil and ruthenium red were increased to 1 mM. It can be seen that the inhibition levels of 49% and 95% are obtained for verapamil and ruthenium red, respectively. These results show that calcium channel blockers are effective in the inhibition of the oat stem gravitropic curvature response. They also indicate that calcium channels are involved in the regulation of cytosolic calcium levels in the gravitropic response mechanism.
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LaCl3, over a concentration range of 0.3–30 mM shows a marked inhibition of gravitropic bending without causing any significant effect on stem elongation (Fig. 6A
). A concentration of 10 mM LaCl3 was chosen to examine the kinetics of the inhibitory response (Fig. 6B). It is clear that LaCl3 acts rapidly (within 3 h). By 48 h, it inhibited upward bending to the extent of almost 60%. Based on the result that it acted so quickly, to repress gravitropic curvature, its effect was analysed on starch levels in vertical and horizontal graviresponding pulvini (Fig. 6C, D, respectively). Comparison of Fig. 6B with Fig. 6C and D indicates that during the time when LaCl3 was causing the greatest amount of deceleration in upward bending rate (3–20 h after the start of the gravistimulation treatment), it simultaneously caused the greatest decrease in starch levels in the pulvini. Like OA, LaCl3 abolished the net increase in starch level that occurred between 0 and 3 h after the start of the gravistimulation treatment (compare Figs 3C and D with 6C and D).
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Figure 7 shows the effect of a 3 min pulse treatment with LaCl3 on oat stem segment gravitropic curvature response (Fig. 7A
) and on starch levels in the gravisensors in graviresponding node/pulvini (Fig. 7B). Even with such a short-time pulse treatment, upward bending rate decreases, such that by 27 h after the initiation of the gravistimulation treatment, LaCl3 pulse treatment had caused a 40% decrease in the amount of curvature compared with that of control stem segments (20° and 35° curvature, respectively). However, the amount of decrease of starch in the chloroplast gravisensors is only a transient one that is expressed for c. 3 h after the administration of the pulse treatment. During these 3 h, there is apparently a sufficient decrease in the amount of starch in the gravisensors to account for the 40% decrease in upward bending response evident 24 h following the LaCl3 pulse treatment.
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Discussion |
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The gravisensing mechanism in the shoots of green plants involves the sedimentation of starch-filled plastids. Based on studies with cereal grass shoots, when these gravisensors are depleted of starch in the dark, gravitropic curvature is absent, but when they are reconstituted by feeding shoots with sucrose, starch is resynthesized and gravitropic curvature occurs (Song et al., 1988
; Kaufman et al., 1995). In addition, starchless or low starch mutants of Arabidopsis thaliana and tobacco show no or very slow gravitropic curvature responses (Bjorkman, 1988; Caspar, 1994; Kiss et al., 1989; Kiss and Sack, 1990; McCleary and Kiss, 1999; Sack, 1991, 1997; Vitha et al., 1998). Taken together, these two lines of evidence lend credence to the idea that starch-filled plastids in green plants are indeed the gravisensors when plants are gravistimulated. What is still not known is how they function as gravisensors in the shoots of plants.
The present study may provide some insights into the nature of how gravisensing occurs in cereal grass shoots. At first, it was found that the downward movement of starch statoliths was altered in response to treatment with several inhibitors. This would explain why the gravitropic response is slower. Next, it was found that after the onset of gravitropic curvature, there is a net increase in the amount of starch in the gravisensors during the first 8 h of upward bending. This is followed by a continuous decline in the level of starch during the remainder of the time that the shoot is bending upward. OA and LaCl3 treatments abolish this early net starch increase and cause starch levels to decrease rapidly (Figs 3D, 6D). These effects are also shown when a 3 min pulse of the two chemicals, used separately, are applied at an early stage of the gravitropic response. Wright has suggested that when a sufficient number of starch-filled statoliths becomes evident in developing wild oat (Avena fatua) shoot pulvini, gravitropic competence is established (Wright, 1986). The analyses of starch levels in graviresponding cultivated oat shoot pulvini reported here corroborate Wright's assertion. When these starch-filled statoliths remain sedimented due to their mass over extended periods of time (up to 48 h in the case of oat shoots), gravitropic curvature will continue to occur. The new finding reported here is that the rate of curvature appears to be directly correlated with the amount of starch present in the gravisensors (compare Figs 3B and 6B with 3D and 6D). As described in the results, the net increase in starch content that occurs in the gravisensors during the early stages of gravitropic bending (first 8 h) may be critical for the gravisensing process. It would result in enhanced mass per plastid and in the population of plastids in each of the gravisensor cells (statocytes). Such an effect would favour rapid rates of upward bending, which, in fact, do occur during this period. The net decrease in amount of starch in the gravisensors which occurs from c. 8 h until the time when bending ceases would be expected to result in a loss in mass per plastid in each of the gravisensor cells. This would be expected to cause a decrease in the rates of downstream gravity signal transduction processes that could explain why a deceleration was seen in the rate of upward bending during this period. In summary, it is necessary to emphasize that the gravity signal should be transduced continuously via the statoliths in order to sustain the gravitropic response. In other words, gravisensing is maintained when a certain level of starch is present in the gravisensors. One possible explanation is that the function of gravity signal transduction is to maintain the starch level in the gravisensors so as to maintain continuous gravity perception required to sustain continuous gravitropic bending.
In roots, it has been shown previously (Aarrouf and Perbal, 1996; Salisbury, 1993) that there is no relationship between starch content in the root cap and the responsiveness of the roots to a gravistimulus, except when the amount of starch is small. In roots with starch-depleted plastids, graviresponsiveness is maintained, but the curvature response is slower. In contrast, in cereal grass shoots such as oats, a correlation was found between the level of starch present in plastids of graviresponsive pulvini and the rate of gravitropic curvature in these pulvini (the present studies). Further, when the plastids in graviresponsive shoot pulvini of barley shoots are depleted of starch by placing shoots in the dark for 5 d, graviresponsiveness in the pulvini is lost; but when starch synthesis is induced in the pulvinus plastids by feeding the stem segments with 0.1 M sucrose, graviresponsiveness is restored (Song et al., 1988).
That the phosphoprotein phosphatase inhibitor, okadaic acid, not only strongly inhibits gravitropic curvature, but also, causes a very rapid loss in starch in the gravisensors, suggests that phosphoprotein dephosphorylation may be one of the essential steps that is involved in sucrose metabolism. In this connection, it has been shown that phosphorylation of sucrose synthase (SuSy), which like invertase, functions in sucrose hydrolysis, is an important component of the gravitropic response mechanism in maize shoot pulvini (Winter et al., 1997). These authors postulate that the phosphorylation of SuSy is involved in the release of the membrane bound enzyme, partly as a result of decreased surface hydrophobicity. If sucrose synthesis or sucrose hydrolysis are negatively regulated by okadaic acid's inhibitory effect on phosphoprotein dephosphorylation, this could have a downstream repressing effect on net increase in starch level in the starch statoliths.
That staurosporine and H-89 (inhibitors of protein kinases) and okadaic acid, calyculin A and NaF (inhibitors of phosphoprotein dephosphorylation), strongly and selectively inhibit gravitropic bending in oat shoot pulvini suggest that protein phosphorylation and dephosphorylation reactions are key steps in gravity signal transduction pathways. This is corroborated by recent studies on protein phosphorylation in oat shoot pulvini, where differential phosphorylation of 38 and 50 kDa soluble proteins was reported within 30 min after the onset of gravistimulation treatment (Chang and Kaufman, 2000). In addition, the fact that these inhibitors did not change movement of the gravisensors indicates that they may affect other transduction pathways than the gravisensing pathway.
Calcium is a well-known second messenger in the gravitropic response of maize roots (Lee et al., 1983) and oat coleoptiles (Daye et al., 1984).
Other studies show it to play an essential role in many diverse physiological processes (Subbaiah et al., 1994; Knight et al., 1997; Wymer et al., 1997; Legue et al., 1997; Shimazaki et al., 1997; Roelfsema and Prins, 1997; Tahtiharju et al., 1997; Chandra et al., 1997; Sedbrook et al., 1996). This study's results with LaCl3, ruthenium red and verapamil indicate that cytosolic calcium is likely to exert an important role in starch metabolism in response to gravistimulation treatment. These results also indicate that the sources of cytosolic calcium may be both the cell wall and cellular organelles, as is true in the case of Arabidopsis seedlings in response to anoxia (Sedbrook et al., 1996). However, it should be mentioned that these results on the inhibitors’ effects do not exclude the possibility that both sensing and hormonal signal transduction pathways can be perturbed by the inhibitors because the two transduction pathways occur in the pulvini of oat shoots.
How can one account for the increase, then decrease, in the level of starch in the starch statoliths during the course of upward bending? One possible explanation is the observed increase in amount of soluble B-fructofuranosidase (invertase) mRNA and invertase activity starting within 1 h and 3 h, respectively, after the initiation of gravistimulation (Wu et al., l993). After 9 h of gravistimulation, invertase activity decreases rapidly, so that by 24 h, it is slightly above that seen at time zero of gravistimulation by reorientation. The consequent increase, then decrease in level of hexoses (D-glucose and D-fructose) resulting from invertase-catalysed hydrolysis of sucrose might be expected to cause a corresponding increase, then decrease in the level of starch in the pulvinus tissue. The kinetics of these changes in invertase activity (Fig. 3 in Wu et al., 1993) and starch levels in the pulvinus tissue following gravistimulation of oat shoots (Fig. 2D) are remarkably similar. Even though this is merely a correlation, its validity can be checked experimentally by means of pulse-chase treatments with 14C-labelled sucrose in order to answer this question.
In conclusion, changes in the levels of starch in gravisensors (the statoliths) are correlated with the rate of gravitropic curvature response, and that gravity signal transduction may be involved in starch metabolism through the mediation of calcium and protein dephosphorylation so as to sustain the gravitropic bending process.
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Acknowledgments |
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This research was supported by Research Grant No. IS-2434-94 from BARD, The United States–Israel Binational Agricultural Research and Development Fund, and in part, by a KOSEF grant (1998G0203) to Bin Goo Kang, Hormone Research Center, Chonnam National University. We thank David M Pharr, North Carolina State University, for providing us with methods for quantitative analysis of starch and Sonia Philosoph-Hadas of the Volcani Agricultural Research Center, Bet-Dagan, Israel for helpful discussions.
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3 To whom correspondence should be addressed. Fax: +734 647 0884. E-mail: pbk{at}umich.edu
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