Proc. Natl. Acad. Sci. USA Vol. 89, pp. 3591-3595, April 1992 Plant Biology
Gibberellic acid and abscisic acid coordinately regulate cytoplasmic calcium and secretory activity in barley aleurone protoplasts (confocal sanning optical microscopy/plant hormones/ratio imaging/secretion)
SIMON GILROY AND RUSSELL L. JONES* Department of Plant Biology, University of California, Berkeley, CA 94720
Communicated by Winslow R. Briggs, December 16, 1991
made after 36 h of GA3 treatment, whereas a 5- to 50-fold increase in a-amylase synthesis and secretion occurs after only 8-12 h (reviewed in ref. 1). In this paper we show that GA3 treatment increases [Ca2+]i 3-fold and that this increase precedes the effects of the hormone on a-amylase synthesis and secretion. We also show that the increased (Ca2+]i is localized to the peripheral cytoplasm of the cell and that the stimulatory effects of GA3 on [Ca2+]i are reversed by treatment with ABA.
ABSTRACT The effects of gibberellic acid (GA3) and abscisic acid (ABA) on the temporal and spatial dynamics of cytosolic calcium concentration ([Ca2+]o) in aleurone protoplasts of barley (Hordeum vulgare L. cv. Himalaya) were measured by using fluorescence ratio analysis and confocal microscopy. After 4-6 h of treatment, GA3 induced a sustained increase in [Ca2+]1 from 50 to 150 nM in aleurone protoplasts. The increase in [Ca2+]1 preceded the GA3-induced increase in a-amylase synthesis and secretion by 4 h. The elevation of [Ca2+]1 was highest in the peripheral cytoplasm and may play a role in coordinating the secretory events there. Reducing Ca2+ levels in the incubation medium to below 500 FM inhibited the increase in [Ca2+]1 and the GA3-induced stimulation of a-amylase synthesis and secretion. These data suggest that GA3 may increase [Ca2+]1 by increasing the influx of Ca2+ at the plasma membrane. ABA reversed the effect of GA3 on [Ca21]J within 3 h, 2 h before its effect on a-amylase production could be detected. Thus changes in [Ca2+] may play a role in mediating the effects of GA3 and ABA on the synthesis and secretion of a-amylase in the aleurone cell.
MATERIALS AND METHODS Fluorescent Dyes. Fluorescent dyes were obtained from Molecular Probes. Calcium green linked to a dextran of Mr 10,000 has Ca2+-dependent fluorescence characteristics similar to fluo-3 but is approximately 5 times brighter. Isolation and Incubation of Aleurone Protoplasts. Protoplasts were prepared from aleurone layers isolated from deembryonated grain of Hordeum vulgare L. cv. Himalaya (1985 harvest) and purified on a discontinuous Nycodenz density gradient (10). Protoplasts were incubated in modified Gambourg's B-5 medium (10) supplemented with 10 mM CaCl2 and, where specified, 5 ,uM GA3 or 5 ,uM GA3 followed by 5 uM ABA. a-Amylase was assayed according to Jones and Varner (12). Radiolabeling. Protein synthesis was monitored by radiolabeling with [35S]methionine (13). Protoplasts (5 X 10' per ml) were incubated with 100 uCi (1 Ci = 37 GBq) of Tran-35S-label reagent (ICN) for 1 h, then homogenized in buffer supplemented with 10% (wt/vol) trichloroacetic acid and 10 mM methionine. [35S]Methionine incorporated into trichloroacetic acid-precipitable material was measured by liquid scintillation counting (13). Acid Loading of Protoplasts with Indo-1 and Fluo-3. Protoplasts were loaded with indo-1 and fluo-3 essentially as described by Bush and Jones (10). Carry-over of extracellular dye contributed %
40
u
0
,2-
dextran. The large size of dextran-linked dyes is thought to prevent their uptake by organelles. Confocal imaging of protoplasts microinjected with calcium green-dextran revealed a peripheral ring of increased Ca2+ concentration in GA3-treated protoplasts (Fig. lc). Imaging of the distribution of rhodamine-dextran that had been microinjected simultaneously with the calcium green-dextran showed no evidence of localization of dextran-linked dyes within the cell (Fig. lc). The fluorescence from rhodamine-dextran was not affected by Ca2+ (data not shown). These results suggest that the increased Ca2+ observed in the cortical cytoplasm of GA3treated protoplasts was due to an increase in [Ca2+]j. After 1- to 3-h treatment with 5 ,uM ABA, the [Ca2 ]i in GA3-treated protoplasts was reduced (Figs. lb and 6A). This reduction in [Ca2W]i preceded the first measurable effects of ABA on a-amylase synthesis and secretion by about 2 h (Fig. 6B). The GA3-Induced Increase in [Ca2+] Depends upon Extracellular Ca2+. The localized increase in (Ca2+]i induced by GA3 could have arisen from increased Ca2` influx at the plasma membrane and/or by release of Ca2" from intracellular stores. Influx at the plasma membrane can be reduced by lowering the extracellular Ca2` concentration ([Ca2+]k). Addition of 5 mM EGTA to the protoplasts to chelate extracellular Ca2` led to a rapid loss in protoplast viability (data not shown) and relocalization of indo-1 or fluo-3 to the vacuoles within 5 min (Fig. 2b). Lowering [Ca2W+ to 10-4 M
.
250 200
c
4.0 2.0 3.0 1.0 0.0 Time after ABA treatment (h)
t
4
150
u
t
E 100
0
n
x
3
5
E 0~
c 0
10
2
1-01
0
f._
1
(A
uj
Gibberellic acid and abscisic acid coordinately regulate cytoplasmic calcium and secretory activity in barley aleurone protoplasts (confocal sanning optical microscopy/plant hormones/ratio imaging/secretion)
SIMON GILROY AND RUSSELL L. JONES* Department of Plant Biology, University of California, Berkeley, CA 94720
Communicated by Winslow R. Briggs, December 16, 1991
made after 36 h of GA3 treatment, whereas a 5- to 50-fold increase in a-amylase synthesis and secretion occurs after only 8-12 h (reviewed in ref. 1). In this paper we show that GA3 treatment increases [Ca2+]i 3-fold and that this increase precedes the effects of the hormone on a-amylase synthesis and secretion. We also show that the increased (Ca2+]i is localized to the peripheral cytoplasm of the cell and that the stimulatory effects of GA3 on [Ca2+]i are reversed by treatment with ABA.
ABSTRACT The effects of gibberellic acid (GA3) and abscisic acid (ABA) on the temporal and spatial dynamics of cytosolic calcium concentration ([Ca2+]o) in aleurone protoplasts of barley (Hordeum vulgare L. cv. Himalaya) were measured by using fluorescence ratio analysis and confocal microscopy. After 4-6 h of treatment, GA3 induced a sustained increase in [Ca2+]1 from 50 to 150 nM in aleurone protoplasts. The increase in [Ca2+]1 preceded the GA3-induced increase in a-amylase synthesis and secretion by 4 h. The elevation of [Ca2+]1 was highest in the peripheral cytoplasm and may play a role in coordinating the secretory events there. Reducing Ca2+ levels in the incubation medium to below 500 FM inhibited the increase in [Ca2+]1 and the GA3-induced stimulation of a-amylase synthesis and secretion. These data suggest that GA3 may increase [Ca2+]1 by increasing the influx of Ca2+ at the plasma membrane. ABA reversed the effect of GA3 on [Ca21]J within 3 h, 2 h before its effect on a-amylase production could be detected. Thus changes in [Ca2+] may play a role in mediating the effects of GA3 and ABA on the synthesis and secretion of a-amylase in the aleurone cell.
MATERIALS AND METHODS Fluorescent Dyes. Fluorescent dyes were obtained from Molecular Probes. Calcium green linked to a dextran of Mr 10,000 has Ca2+-dependent fluorescence characteristics similar to fluo-3 but is approximately 5 times brighter. Isolation and Incubation of Aleurone Protoplasts. Protoplasts were prepared from aleurone layers isolated from deembryonated grain of Hordeum vulgare L. cv. Himalaya (1985 harvest) and purified on a discontinuous Nycodenz density gradient (10). Protoplasts were incubated in modified Gambourg's B-5 medium (10) supplemented with 10 mM CaCl2 and, where specified, 5 ,uM GA3 or 5 ,uM GA3 followed by 5 uM ABA. a-Amylase was assayed according to Jones and Varner (12). Radiolabeling. Protein synthesis was monitored by radiolabeling with [35S]methionine (13). Protoplasts (5 X 10' per ml) were incubated with 100 uCi (1 Ci = 37 GBq) of Tran-35S-label reagent (ICN) for 1 h, then homogenized in buffer supplemented with 10% (wt/vol) trichloroacetic acid and 10 mM methionine. [35S]Methionine incorporated into trichloroacetic acid-precipitable material was measured by liquid scintillation counting (13). Acid Loading of Protoplasts with Indo-1 and Fluo-3. Protoplasts were loaded with indo-1 and fluo-3 essentially as described by Bush and Jones (10). Carry-over of extracellular dye contributed %
40
u
0
,2-
dextran. The large size of dextran-linked dyes is thought to prevent their uptake by organelles. Confocal imaging of protoplasts microinjected with calcium green-dextran revealed a peripheral ring of increased Ca2+ concentration in GA3-treated protoplasts (Fig. lc). Imaging of the distribution of rhodamine-dextran that had been microinjected simultaneously with the calcium green-dextran showed no evidence of localization of dextran-linked dyes within the cell (Fig. lc). The fluorescence from rhodamine-dextran was not affected by Ca2+ (data not shown). These results suggest that the increased Ca2+ observed in the cortical cytoplasm of GA3treated protoplasts was due to an increase in [Ca2+]j. After 1- to 3-h treatment with 5 ,uM ABA, the [Ca2 ]i in GA3-treated protoplasts was reduced (Figs. lb and 6A). This reduction in [Ca2W]i preceded the first measurable effects of ABA on a-amylase synthesis and secretion by about 2 h (Fig. 6B). The GA3-Induced Increase in [Ca2+] Depends upon Extracellular Ca2+. The localized increase in (Ca2+]i induced by GA3 could have arisen from increased Ca2` influx at the plasma membrane and/or by release of Ca2" from intracellular stores. Influx at the plasma membrane can be reduced by lowering the extracellular Ca2` concentration ([Ca2+]k). Addition of 5 mM EGTA to the protoplasts to chelate extracellular Ca2` led to a rapid loss in protoplast viability (data not shown) and relocalization of indo-1 or fluo-3 to the vacuoles within 5 min (Fig. 2b). Lowering [Ca2W+ to 10-4 M
.
250 200
c
4.0 2.0 3.0 1.0 0.0 Time after ABA treatment (h)
t
4
150
u
t
E 100
0
n
x
3
5
E 0~
c 0
10
2
1-01
0
f._
1
(A
uj
