Senescence of Rice Leaves XXIV. Involvement of Calcium and Calmodulin in the Regulation of Senescence

JSPP © 1990

Senescence of Rice Leaves XXIV. Involvement of Calcium and

Calmodulin in the Regulation of Senescence

Yuanman Huang, Chien Teh Chen and Ching Huei Kao

Department of Agronomy, National Taiwan University, Taipei, Taiwan, Republic of China

Effects of compounds that influenced calcium uptake and calmodulin inhibitors on the

senes-cence of detached rice leaves were examined. Chelators, ethyleneglycol-bis-03-aminoethyl

ether)-N,N,N",W-tetraacetic acid (EGTA) and l,2-bis-(o-aminophenoxy)-ethane-/v",N,7V,A''-tetraacetic

acid (BAPTA), significantly promoted senescence of detached rice leaves in the dark and light.

The effect of EGTA can be reversed by treating detached rice leaves with calcium. Verapamil, a

calcium channel blocker, and lanthanum chloride, a calcium antagonist, promoted dark-induced,

and suppressed BA- and light-retarded senescence of detached rice leaves. Calcium ionophore

A23187 and ruthenium red, believed to raise cytosolic level of Ca

, were quite effective in

retard-ing dark-induced and ABA-promoted senescence of detached rice leaves. Calmodulin inhibitors,

W-7, compound 48/80, chlorpromazine and trifluoperazine, significantly promoted

dark-in-duced, and suppressed BA- and light-retarded senescence of detached rice leaves. It is concluded

that cytosolic level of Ca

may regulate senescence of detached rice leaves through a

calmodulin-dependent mechanism.

Key words: Calcium — Calmodulin — Leaf senescence — Oryza sativa.

Calcium is known to retard ripening process of fruit.

Specifically, maintenance of relatively high calcium

concen-trations in fruit tissue results in a slower rate of ripening, as

seen in lower respiration, reduced ethylene production, and

slower softening of fruit flesh (Ferguson 1984). Leaf

senes-cence has also been shown to be retarded by the application

of calcium. Chi loss and protein degradation were both

re-duced in corn and Rumex leaf discs by calcium (Poovaiah

and Leopold 1973). In whole, excised cucumber

coty-ledons, calcium treatment reduced the rate of Chi

degrada-tion and peroxide accumuladegrada-tion (Ferguson et al. 1983).

They related the calcium effect to maintain cellular

mem-branes (Ferguson et al. 1983, Poovaiah and Leopold

1973). It appears that the senescence-effect of calcium is

an effect of external calcium. Using pea leaf system,

Leshem et al. (1982, 1984, 1986) were able to show that

se-nescence was promoted by increasing intracellular calcium.

They concluded that elevated intracellular calcium

pro-moted senescence through a calmodulin-mediated effect.

Abbreviations: BA, benzyladenine; BAPTA, l,2-bis-(o-aminophenoxy)-ethane-;V,MAf,yV-tetraacetic acid; CPZ, chlor-promazine; EGTA, ethyleneglycol-bis-(/?-aminoethyl ether)-yV,yV,W,JV-tetraacetic acid; TFPZ, trifluoperazine.

The present investigation was undertaken to examine

the effects of calcium chloride, calcium chelators (EGTA

and BAPTA), a calcium antagonist (lanthanum chloride),

a calcium channel blocker (verapamil), a calcium

ion-ophore (A23187), and anti-calmodulin drugs (W-7,

com-pound 48/80, CPZ and TFPZ) on the senescence of

detach-ed rice leaves. Results indicate that elevatdetach-ed cytosolic

calcium retards senescence of detached rice leaves through

a calmodulin-dependent mechanism.

Materials and Methods

Rice {Oryza sativa cv. Taichung Native 1) seedlings

were cultured as previously described (Kao 1980). The

apical 3-cm segments excised from the third leaves of

12-day-old seedlings were used. A group of 10 segments were

floated in a Petri dish containing 10 ml of test solutions.

All chemicals used in this investigation were purchased

from Sigma. Incubation was carried out at 27°C in

darkness or the light (16.7 W m ~

) provided by fluorescent

lamps.

Chi was extracted and quantitated as described

previ-ously (Kao 1980). All experiments were repeated at least

twice. Data are presented as the results of a single

ment typical of the trends seen in the repeated experiments.

Results

The effectiveness of calcium in retarding Chi degrada-tion of detached rice leaves was tested as shown in Figure 1. Calcium was effective in retarding the decrease of Chi under both light and dark conditions. The effect of calci-um in the dark was more effective than that in the light. The effect of EGTA on Chi content of detached rice leaves in the dark is presented in Figure 2. The amount of Chi was decreased by EGTA treatment. This effect was revers-ed by treating detachrevers-ed rice leaves with calcium (data not shown). Ca2+ chelator BAPTA was found to be more effective than EGTA in decreasing Chi content (Fig. 2).

Experiments were carried out with putative calcium channel blocker to further characterize the role of calcium in the regulation of senescence of detached rice leaves in the dark. Verapamil, a calcium channel blocker, or lan-thanum chloride, a calcium antagonist, was applied to de-tached rice leaves in the dark. The results in Figure 3 in-dicate that verapamil was very effective in causing the decrease of Chi content. Lanthanum chloride also decreas-ed Chi content in the dark (Fig. 3). These experiments sug-gest that Chi loss of detached rice leaves in the dark is regulated by a block of transport of calcium ions into the cytosol. Further evidence of this role for calcium was ob-tained using the calcium ionophore A23187 (Campbell 1983). Treatment with the ionophore at 0.01 DIM resulted in higher Chi content than the control (Table 1). Ruthenium red is known to block calcium ion transport (Hinds et al. 1981, Watson et al. 1971) and is believed to

o con t o lorophy i 1 I U 100 80 60 40 20 0 • - • • EGTA T O — O ' BAPTA I AT~~~~~~~-| V I \ \

1 \

Fig. 2 Effects of EGTA and BAPTA on the Chi content of de-tached rice leaves in the dark. Chi was determined after 4 days in the dark. Values are averages with standard errors (n=4).

raise the intracellular calcium level (Bednarska 1989, Moll and Jones 1982, Vashington et al. 1972). We investigated the effect of this compound on the Chi content of detached rice leaves in the dark. Ruthenium red was found very effective in retarding Chi loss (Table 1).

We also investigated the Chi content of detached rice leaves in the dark in the presence of calmodulin an-tagonists. Results shown in Table 1 and Figure 4 indicate that calmodulin antagonists, W-7, compound 48/80, CPZ and TFPZ, significantly accelerated the decrease of Chi

con-I

4

Fig. 1 Effect of calcium chloride on the Chi content of detached rice leaves. Chi was determined after 4 days in the dark or light. Values are averages with standard errors (n=4).

— 100 c o

f

6

Fig. 3 Effects of verapamil and lanthanum chloride on the Chi content of detached rice leaves in the dark. Values are averages with standard errors (n=4).

Table 1 Effect of A23187, ruthenium red, W-7 and

com-pound 48/80 on the Chi content of detached rice leaves in

the dark

Table 2 Effects of BA alone and BA in the presence

lan-thanum chloride, verapamil, CPZ, TFPZ, W-7 and

com-pound 48/80 on the Chi content of detached rice leaves in

the dark

Treatment

Chi (A^/10 segments)

Treatment

Chi (A^/10 segments)

A23187 (mM)

0

0.01

Ruthenium red (mM)

0

0.5

1.0

2.0

W-7 (mM)

0

0.1

0.5

Compound 48/80 0*g/ml)

0

100

0.44±0.01

0.67 ±0.02

0.40 ±0.02

0.49±0.04

0.61+0.02

0.71 ±0.03

0.72±0.03

0.46±0.02

O.34±0.03

0.55 ±0.03

0.43 ±0.01

Control

BA

BA + LaCl,

BA + Verapamil

BA + CPZ

BA + TFPZ

Control

BA

BA + W-7

Control

BA

BA +Compound 48/80

The concentrations for BA, lanthanum CPZ are 1 mM, whereas those for TFPZ

— ^ OA r* vn A C m i r A 1 m w n « A 1 AA , . A / m l car

0.23 ±0.02

O.78±O.O3

0.55±0.02

0.29±0.03

0.45 ±0.02

O.39±O.O2

0.72 + 0.03

0.95 ±0.02

0.84±0.03

O.55±O.O3

0.96±0.01

0.82±0.01

chloride, verapamil, and , W-7 and compound 48/ Chi was determined after 4 days in the dark. Values are averages

with standard errors (n=4). mined after 4 days in the dark. Values are averages with standard errors (n=4).

tent.

BA, a synthetic cytokinin, is known to retard Chi

deg-radation of detached rice leaves in the dark (Kao 1980).

To see whether there is a requirement for calcium channels

when BA retards Chi degradation, agents known to block

such channels were incorporated in the incubation media.

Lanthanum chloride and verapamil significantly reduced

the retardation effect of Chi loss by BA (Table 2). We also

0.5 0.4 0.3 0.2 0.1 !

1

i

Fig. 4 Effects of TFPZ and CPZ on the Chi content of detached rice leaves in the dark. Chi was determined after 4 days in the dark. Values are averages with standard errors (n=4).

investigated the ability of BA to retard Chi loss in the

ence of calmodulin antagonists in the dark. In the

pres-ence of W-7, compound 48/80, CPZ and TFPZ, the effect

of BA was significantly suppressed (Table 2), indicating

there is a requirement of calmodulin for BA to retard Chi

loss of detached rice leaves in the dark.

c_a. o o O 100 so 60 40 20 o • — » : £ G T A O— O:BAPTA \ \ 0 1 2 5 10 Concentration C mM 5

Fig. 5 Effects of EGTA and BAPTA on the Chi content of de-tached rice leaves in the light. Chi was determined after 4 days in the light. Values are averages with standard errors (n=4).

Table 3 Effects of lanthanum chloride, verapamil, CPZ,

TFPZ, W-7 and compound 48/80 on the Chi content of

de-tached rice leaves in the light

Treatment

Chi (A^/10 segments)

Control

LaCl3

Verapamil

CPZ

TFPZ

Control

W-7

Control

Compound 48/80

0.56±0.02

0.34±0.02

0.21+0.12

0.46±0.01

0.45±0.01

0.80±0.01

0.59±0.01

0.79±0.01

0.64 ±0.02

The concentrations for lanthanum chloride, verapamil and CPZ are 1 mM, wherease those for TFPZ, W-7 and compound 48/80 are 0.5 mM, 0.1 mM and 100/ig/ml, respectively. Chi was deter-mined after 4 days in the light. Values are averages with standard errors (n=4).

Light has been shown to retard Chi loss of detached

rice leaves (Hurng et al. 1986). Similar to the effect of BA

in the dark, calcium and calmodulin appear to be required

for the effect of light. This conclusion is supported by the

observations that calcium chelators, calcium channel

blockers and calmodulin antagonists significantly reduced

the effect of light (Fig. 5 and Table 3).

ABA significantly promoted Chi loss of detached rice

leaves in the light (Table 4). ABA effect was totally

abolished by ruthenium red and significantly reduced by

A23187 (Table 4). These experiments seem to suggest that

ABA promoted Chi loss may mediate through blocking

cal-cium ions into the cytosol.

Table 4 Effects of ABA alone and ABA in the presence

of ruthenium red or A23187 on the Chi content of detached

rice leaves in the light

Treatment

Chi (.4 6*5/10 segments)

Control

ABA

ABA+Ruthenium red

Control

ABA

ABA + A23187

0.88±0.01

0.26±0.02

0.87 ±0.03

0.58 ±0.03

0.36±0.01

0.43 ±0.02

The concentrations of ABA, ruthenium red and A23187 are 0.1, 2.0 and 0.01 mM, respectively. Chi was determined after 4 days in the light. Values are averages with standard errors (n = 4).

Discussion

Several compounds expected to increase or decrease

cytosolic levels of calcium were used to examine the

re-quirement of calcium in regulating the senescence of

detach-ed rice leaves, measurdetach-ed as Chi loss. We also investigatdetach-ed

effects of four inhibitors known to inhibit action of

Ca-calmodulin in animals.

EGTA is a specific Ca

chelator (Campbell 1983)

used in many systems to affect endogenous Ca

concentra-tions (Daye 1984, Karege et al. 1982, Lehtonen 1984) by

causing a depletion of cytosolic Ca

(Campbell 1983,

Gilroy et al. 1986, Poovaiah and Reedy 1987). The

chelator BAPTA is much more selective for Ca

than

EGTA (Tsien 1980). In the present study, we found that

both EGTA and BAPTA were effective in promoting Chi

loss. The effect of EGTA could be reversed by the

addi-tion of Ca.

Of particular interest is the finding that ruthenium red,

a hexavalent dye, significantly retarded dark-induced and

ABA-promoted senescence of detached rice leaves.

Ruthenium red has been shown to be a potent inhibitor of

Ca

uptake and efflux by animal cells and mitochondria

(Hinds et al. 1981, Watson et al. 1971). This compound

also intereferes with Ca

efflux from aleurone layers of

barley (Moll and Jones 1983). Recently, Bednarska (1989)

reported that ruthenium red raised the amount of

Ca

taken up by germinating pollen grains relative to the

con-trol. Based on the known mechanism of ruthenium red

ac-tion in animal and plant cells, the most likely explanaac-tion

of its effect on Chi loss involves its role in the regulation of

cytosolic Ca

content.

The importance of the cytosolic Ca

in the regulation

of rice leaf senescence is further supported by the

observa-tions that (a) lanthanum chloride, a calcium antagonist,

and verapamil, a calcium channel blocker, promoted

dark-induced, and reduced light- and BA-retarded senescence

of detached rice leaves, and (b) calcium ionophore A23187,

a compound that induces an increases in the cytosolic Ca

,

retarded dark-induced and ABA-promoted senescence of

detached rice leaves.

Our data thus strongly suggest that an elevated level of

cytosolic level of Ca

is required for retarding senescence

of detached rice leaves. However, our results are

inconsis-tent with those reported by Leshem et al. (1982, 1984,

1986), who found that senescence of pea leaves was

pro-moted by increasing cytosolic Ca

. The use of different

plant species may have led this discrepancy. Recently,

Leshem (1987) proposed that elevated cytosolic Ca

might

promote senescence through a mechanism that triggered

the catabolic process by binding to calmodulin and

activat-ing phospholipase A2.

The use of calmodulin inhibitors on intact cells can

lead to complex responses, and it cannot be assumed that

the primary target reaction or system is the only one affected. It was for this reason that we used four different calmodulin inhibitors, and it is important that we obtained essentially similar results with all four. The concentra-tions of calmodulin inhibitors used in this investigation are comparable with those used by others who concluded that Ca-calmodulin is probably essential for physiological pro-cesses (Elliott 1983, Elliott et al. 1983, Friedman et al. 1989, Muto and Hirosawa 1987). Furthermore, no toxi-city (rolling or loss of turgor) was visually observable at the inhibitory concentrations.

Since calmodulin inhibitors were found to promote dark-induced and reduce BA- and light-retarded senes-cence, the cytosolic Ca2+ seems to regulate senescence through a calmodulin-mediated effect. Calcium- and calmodulin-stimulated phosphorylation of membrane pro-teins has been found to decrease during senescence of ap-ples (Paliyath and Poovaiah 1984, 1985). In a recent study, we demonstrated that proton secretion activity of de-tached rice leaves, which was found to be originated from ATP-driven H+ pump located in the plasma membrane, played an important role in regulating senescence of detach-ed rice leaves (Chen et al. 1990). Proton secretion activity decreased during senescence of detached rice leaves. Phos-phorylation of the plasma-membrane H+-ATPase has been shown to be regulated by calcium-stimulated protein kinase (Bidwai and Takemoto 1987, Schaller and Sussman 1988). Hanson and Trewavas (1982) suggested that calci-um and calmodulin might regulate the activity of H+ -ATP-ase of the plasma membrane. It seems that the promotion of Ca2+ and calmodulin-dependent protein kinase or phos-phorylation by Ca2+ and calmodulin provides a mechan-ism by which intracellular Ca2+ _{can regulate senescence of}

detached rice leaves. The possible changes of protein ki-nase activity or protein phosphorylation during senescence of detached rice leaves are now the subject of further research.

This research was supported financially by the National Science Council, Republic of China under Grant NSC 79-0409-B002-04.

References

Bednarska, E. (1989) The effect of exogenous Ca ions on pollen grain germination and pollen growth. Investigations with

45

Ca2+ together with verapamil, La3+, and ruthenium red. Sex-ual Plant Reproduction 2: 53-58.

Bidwai, A. P. and Takemoto, J. Y. (1987) Bacterial phytotoxin, syringomycin, stimulates a protein kinase mediated phosphory-lation of red beet plasma membrane polypeptides (Abstr.). J. Cell Biochem. (Suppl.) 11B: 93.

Campbell, A. K. (1983) Intracellular Calcium: Its Role as

Regulator, p. 537, Wiley, New York.

Chen, C.T., Chou, I.T. and Kao, C. H. (1990) Senescence of rice leaves XX. Changes of proton secretion during senescence. Plant Sci. 66: 29-34.

Daye, S., Bico, R. L. and Roux, S. J. (1984) Inhibition of gravitropism in oat coleoptiles by the calcium chelator, ethyleneglycol-bis-0?-aminoethylether)-7V, /V-tetraacetic acid. Physiol. Plant. 61: 449^154.

Elliott, D. C. (1983) Inhibition of cytokinin-regulated responses by calmodulin-binding compounds. Plant Physiol. 72: 215— 218.

Elliott, D. C , Batchelor, S. M., Cassar, R . A . and Marinos, N. G. (1983) Calmodulin-binding drugs affect responses to cyto-kinin, auxin, and gibberellic acid. Plant Physiol. 72: 219-224. Ferguson, I. B. (1984) Calcium in plant senescence and fruit

ripening. Plant Cell Environ. 7: 477-489.

Ferguson, I. B., Watkins, C. B. and Harman, J. E. (1983) Inhibi-tion by calcium of senescence of detached cucumber coty-ledons: effect on ethylene and hydroperoxide production. Plant Physiol. 71: 182-186.

Friedman, H., Goldschmidt, E. E. and Halevy, A. H. (1989) In-volvement of calcium in the photoperiodic flower induction process of Pharbitis nil. Plant Physiol. 89: 530-534.

Gilory, S., Hughes, W. A. and Trewavas, A. J. (1986) The meas-urement of intracellular calcium level in protoplasts from higher plant cell. FEBS Lett. 199: 217-223.

Hanson, J. B. and Trewavas, A. J. (1982) Regulation of plant cell growth: The changing perspective. New Phytol. 90: 1-18. Hinds, T. R., Raess, B. U. and Vincenzi, F. F. (1981) Plasma membrane Ca2+ transport: antagonism by several potential in-hibitors. J. Membr. Biol. 58: 57-65.

Hurng, W. P., Su, L. Y. and Kao, C. H. (1986) Senescence of rice leaves. XVI. Regulation by light. Bot. Bull. Academia Sinica 27: 163-174.

Kao, C. H. (1980) Senescence of rice leaves. IV. Influence of ben-zyladenine on chlorophyll degradation. Plant Cell Physiol. 21: 1255-1262.

Karage, F., Pencel, C. and Greppin, H. (1982) Rapid correlation between the leaves of spinach and the photocontrol of perox-idase activity. Plant Physiol. 69: 437-441.

Lehtonen, J. (1984) The significance of Ca2+ in the mor-phogenesis of Micrasterias studied with EGTA, verapamil, LaCl3 and calcium ionophore A23187. Plant Sci. Lett. 33: 5 3

-60.

Leshem, Y. Y. (1987) Membrane phospholipid catabolism and Ca2+ activity in control of senescence. Physiol. Plant. 69: 551— 559.

Leshem, Y. Y., Wurzburger, Y., Frimer, A. A., Bar ness, G. and Ferguson, I. B. (1982) Calcium and calmodulin metabolism in senescence: interaction of lipoxygenase and superoxide dismutase with ethylene and cytokinin. In Plant Growth Substances 1982. Edited by Wareing, P. F. pp. 569-578. Academic Press, London.

Leshem, Y. Y., Sridhara, S. and Thompson, J. E. (1984) Involve-ment of calcium and calmodulin in membrane deterioation

dur-ing senescence of pea foliage. Plant Physiol. 75: 329-335. Leshem, Y. Y., Frend-Silverberg, M., Warzburger, J., Bar-Nes,

G., Malik, Z. and Langsam, Y. (1986) Ca2 +: calmodulin phytohormone-linked plant senescence control. In Plant Growth Substances 1985. Edited by Bopp, M. pp. 159-168. Springer-Verlag, Berlin.

Moll, B. A. and Jones, R. L. (1982) a-Amylase secretion by single barley aleurone layer. Plant Physiol. 70: 1149-1155.

Muto, S. and Hirosawa, T. (1987) Inhibition of adventitious root growth in Tradescantia by calmodulin antagonists and cal-cium inhibitors. Plant Cell Physiol. 28: 1569-1574.

Paliyath, G. and Poovaiah, B. W. (1984) Calmodulin-inhibitor in senescing apples and its physiological and pharmacological significances. Proc. Natl. Acad. Sci. USA 81: 2065-2069. Paliyath, G. and Poovaiah, B. W. (1985) Calcium- and

calmodulin-promoted protein phosphorylation of membrane proteins during senescence in apples. Plant Cell Physiol. 26: 977-986.

Poovaiah, B. W. and Leopold, A. C. (1973) Deferral of leaf se-nescence with calcium. Plant Physiol. 52: 235-239.

Poovaiah, B. W. and Reedy, R. W. (1987) Calcium messenger systems in plants. CRC Crit. Rev. Plant Sci. 6: 47-103. Schaller, G. E. and Susman, M. R. (1988) Phosphorylation of

the plasma-membrane H+-ATPase of oat roots by calcium-stim-ulated protein kinase. Planta 173: 509-519.

Tsien, R. Y. (1980) New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry. 19: 2396-2404.

Vasington, F. D., Gazzotti, P. and Cavafoli, E. (1972) The effect of rutehium red on Ca transport and respiration in rat liver mitochondria. Biochim. Biophys. Ada 256: 43-54.

Watson, E. L., Vincenzi, F. F. and Davis, P. W. (1971) Ca2+ -ac-tivated ATPase: selective inhibition by ruthenium red. Bio-chim. Biophys. Ada 249: 606-610.