Effect of Heat Stress on Calcium Ultrastructural Distribution

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Environmental and Experimental Botany 48 (2002) 161168

Effect of heat stress on calcium ultrastructural distributionin pepper anther

Chun-Lan Yan, Jian-Bo Wang *, Rong-Qian LiKey Laboratory of MOE for Plant De elopmental Biology , College of Life Sciences , Wuhan Uni ersity ,

Wuhan 430072 , People s Republic of ChinaReceived 10 April 2001; received in revised form 12 March 2002; accepted 13 March 2002

Abstract

Potassium antimonate was used to locate loosely bound calcium in the pepper ( Capsicum annuum ) anther undernormal and heat stress environments. In the microspore mother cell, a few calcium precipitates were deposited on thesurface of the cell, a few in the cytoplasm and almost no precipitates were formed in the nucleus. After 12 h at 40 C,antimonate calcium deposits increased in the cytoplasm and the nucleus and many emerged on the inner surface of the vacuole membrane. After 24 h heat stress, some cells were partly deformed, numerous calcium precipitates

appeared in the cytoplasm and deposited on the surface of the vacuole membrane and in the vacuoles. Compared tothe pollen mother cell, there was a signicant increase in calcium deposit quantities on the surface and in thecytoplasm of the tetraspore. By heat stressing for 24 h, precipitates obviously increased in the cytoplasm and nucleusof the tetraspore in contrast to the control. In the microspore, many calcium precipitates were formed on the baculae,the inner surface of plasma membrane, vacuole membrane, but only a few in the cytoplasm and nucleus. After 12 hheat exposure, precipitates on plasma membrane became abundant, a few in the cytoplasm and the peripheralnucleus, while no precipitates were seen on the vacuole membrane. As for the anthers exposed to 24-h heat stress,precipitates increased on the inner surface of the plasma membrane, cytoplasm and nucleus. In mature pollen, therewas a layer of calcium-induced precipitates on the pollen wall, but few in the cytoplasm and plasma membrane. Noobvious calcium changes occurred on mature pollen after 12 or 24 h heat exposure. The relationship between heatstress and calcium distribution was discussed. 2002 Elsevier Science B.V. All rights reserved.

Keywords : Antimonate precipitation; Cytoplasm; Microspore; Pollen development; Vacuole

www.elsevier.com /locate /envexpbot

1. Introduction

Heat stress is a common environmental stress inthe development of plants and it may be themajor constraints to vegetable growth. Heat toler-

ance is becoming a desirable trait for vegetables inheat-stressed environments, so more and morestudies are focusing on the mechanism of heatstress and thermotolerance.

Physiological responses of plants to heat stress,such as the damage of structure and the disorderof physiological metabolism, have been well docu-mented (Vierling, 1991; Blum, 1996; Wang et al.,

* Corresponding author.E -mail address : [email protected] (J.-B. Wang).

S0098-8472 /02/$ - see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0098-8472(02)00021-7
mailto:[email protected]:[email protected]


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C .-L. Yan et al . / En ironmental and Experimental Botany 48 (2002) 161 168 162

1997). Although the damage and death of cells arecaused by very heavy heat stress, many plants cansurvive in otherwise lethal high-temperatures

treatments if they are rst subjected to a pretreat-ment at non-lethal high temperatures. Extensiveexperiments have shown that pretreatments whichlead to acquisition of thermotolerance are condi-tions under which heat-shock proteins (HSPs) aresynthesized (Vierling, 1991; Waters et al., 1996;Schof et al., 1998). However, the pathways bywhich heat shock signals are perceived and trans-ducted to activate the gene expression of HSPs inorder to induce thermotolerance are unclear(Schof et al., 1998).

As a secondary messenger in plant signaling,calcium plays a vital role in plant growth anddevelopment, in uences plant response and adap-tation to various environments. Intracellular cal-cium changed frequently with response to variousenvironmental stress signals, such as salinity(Lynch et al., 1989), oxidative stress (Price et al.,1994) and anoxia stress (Subbaiah et al., 1994).Klein and Ferguson (1987) found that the uptakeof calcium in pear cells was signi cantly enhancedunder heat stress and Biyaseheva et al. (1993)showed that heat shock resulted in increased in-

tracellular calcium in pea mesophyll protoplasts.Several authors have suggested that pretreatment,such as Ca 2 + , Ca 2 + chelator EGTA, plasmamembrane Ca 2 + channel blockers La 3 + or vera-pamil, could change the intrinsic heat tolerance of various plants (Gong et al., 1997; Zhang et al.,2000).

In the studies of heat stress, the responses of plant leaves to heat shock /stress have been widelyinvestigated (Gong et al., 1997; Zhang et al.,2000). In contrast, little is known about how heat

stresses affect the development of anthers. Somecytological data suggested that heat stress wouldlead to a decrease of pollen s viability (Han et al.,1996); some studies indicated that calcium wasinvolved in pollen germination and pollen tubeelongation (Tirlapur and Cresti, 1992; Tirlapurand Willemse, 1992; Gong and Cao, 1995), butthe interaction of heat stress and anther s develop-ment is not clear.

Presently, several approaches have been appliedto study the calcium localization and alteration in

plant cells. The cytochemical method of anti-monate precipitation is widely used for subcellularcalcium localization (Jian et al., 1999; Meng et al.,

2000). The present study was designed to locatethe loosely bound calcium in pepper anthers byusing antimonate precipitates. In this study, it washypothesized that: (i) there would be interactionnot only between calcium and the normal antherdevelopment, but also between calcium and theeffect of heat stress in the anther development. Assecondary messenger, calcium in the anther cellsmay increase under heat stress, while a prolongedexposure to increasing Ca 2 + may be toxic to thecells and be correlated with the decrease of pol-len s viability; (ii) the appropriate restoration of Ca 2 + homeostasis would be necessary to preventheat injury. To test the hypothesis, pepper antherswere treated under different levels of heat stressenvironments. The study was focused on the ef-fects of heat stress on calcium distribution inanther cells.

2. Materials and methods

Pepper ( Capsicum annuum L., cv. XiangYan

No. 1) was grown in a green house with thetemperature ranged from 25 to 28 C. When theplants were in the blossom period, they weretransferred to a 40 C growth chamber withoutlight. The anthers of different lengths were col-lected after being stressed for 0 h (control, beforeexposure), 12 and 24 h, respectively.

Subcellular calcium localization was analyzedaccording to the method of Slocum and Roux(1982) with minor modi cation. Anthers were im-mediately immersed in a xative containing 2.5%

gluteraldehyde and 2% potassium antimonate(K 2 H 2 Sb 2 O 7 4H 2 O) in 0.2 mol /l potassium phos-phate buffer (pH 7.8) for 4 h at 4 C. Then theanthers were washed four times, 30 min each, with0.2 mol / l potassium phosphate buffer (pH 7.8)containing 2% potassium antimonate. Then thesamples were xed in potassium phosphate buffer(pH 7.8) containing 1% osmium tetroxide (OsO 4 )and 2% potassium antimonate at 4 C overnight.After the second xation, the samples were bathedfour times in 0.1 mol / l phosphate buffer, 30 min


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each. Thereafter, the samples were dehydrated ina graded acetone series and embedded in Spurrresin. The embedded samples were sectioned with

a glass knife using a LKB8800 ultramicrotome.The sections were stained with uranyl acetate ornot, then observed and photographed with JEM-100 /CXII transmission electron microscopy(TEM) operated at 60 kV.

In order to con rm that the deposits containCa 2 + , chelation of calcium ions with EGTA (eth-ylene glycol-bis N ,N ,N N -tetraacetic acid) wasperformed. The grids mounted with tissue sectionsthat had been examined by TEM, were immersedin 100 mmol /l EGTA (pH 8.0) and incubated at37 C for 1.5 h. After incubation, the grids wererinsed brie y with distilled water, stained withuranyl acetate again and examined under TEM.

3. Results

Calcium distribution was determined from themicrospore mother cell stage to mature pollenstage in both normal and heat stressed anthers.We focused on four stages of development: (i)microspore mother cell; (ii) tetrad; (iii) mi-

crospore; and (iv) mature pollen.

3 .1. Calcium distribution differences betweennormal and treated microspore mother cell ( MMC )

MMCs in normal anthers were distinct fromthe tapetal tissue and the anther wall. They wereangular in outline and each cell had the featuresof a relatively undifferentiated cell bounded by asimple wall. In the MMC, a number of small

vacuoles were seen in the cytoplasm. A few cal-cium-induced precipitates occurred on the surfaceof the cell, few in the cytoplasm and nearly noprecipitates in the nucleus (Fig. 1A).

After 12 h heat stress at 40 C, calcium de-posits (40 59 ppt m 2 ), which can be observedon the surface of MMC, were more abundantthan those in the control (20 39 ppt m 2 ). Thenumber of deposits increased continuously inboth cytoplasm and the nuclei (Fig. 1B). At thesame time, many vacuoles formed in the MMC,

many calcium precipitates deposited on the innersurface of vacuole membrane and numerous cal-cium-induced precipitates occurred on the surface

of MMC opposite to the tapetum.As for the anthers treated with 24 h heat stress,numerous calcium precipitates emerged in the cy-toplasm and also occurred on the surface of vac-uole membrane and in the vacuoles (Fig. 1C).

3 .2 . Calcium distribution differences betweennormal and stressed tetrad

When anthers developed to the tetrad stage,tetrads of microspores, in a tetrahedral arrange-ment, were encased in callose each that was a light

Fig. 1. Electron micrographs of cells in MMC stage exposed tonormal environment (A: bar = 2 m), 12 h heat stress (B:bar = 3 m), 24 h heat stress (C: bar = 3 m) and tetrad stagein normal environment (D: bar = 3 m), 24 h heat stress (E:bar = 4 m). (F) The calcium-free control section incubated byEGTA (bar = 2 m). The difference in calcium precipitatesdistribution after different durations of heat treatment wasevident. Arrows indicate calcium precipitates. MMC, mi-crospore mother cell; N, nucleus; P, pollen; Sp, microspore; V,vacuole.


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layer of electron-lucent and nearly no precipitatesformed in it. Moreover, the number of calciumprecipitates on the surface of the tetraspore was

increasing and the volume became bigger. Cal-cium precipitates also increased in the cytoplasmand distributed regularly. Some calcium depositsformed on the surface of vacuole (Fig. 1D).

After exposure to 40 C for 24 h comparedwith the control, precipitates signi cantly in-creased and accumulated in the cytoplasm andnucleus of the tetraspores (Fig. 1E).

3 .3 . Calcium distribution differences betweennormal and stressed microspore

Released from the callose tetrad, the mi-crospores had irregular shape and dense cyto-plasm. Exine deposition on the wall of themicrospores, which just released from the tetrad,was well developed. At the early stage of mi-crospore development, baculae were irregularlyspaced between a spongy tectum and a dense footlayer that was complete around the microspore.Many big volume precipitates were on the baculaediscontinuously. There were many calcium precip-itates in the plasma membrane where the future

colporal regions were formed (data not shown).The later uninucleate microspores contained alarge central vacuole and a peripheral nucleus.Abundant calcium precipitates were deposited notonly on the surface of the baculae, but also on theinner surface of the plasma membrane and vac-uole membrane, with only a few precipitates inthe cytoplasm and nucleus (Fig. 2A).

In those microspores that endured 12 h heatstress, a few calcium deposits were found on thebaculae in the later uninucleate microspores, but

precipitates on the plasma membrane becameabundant. In addition, a few precipitates occurredin the cytoplasm and the peripheral nucleus.Compared with the control, no calcium depositsgranules were seen on the vacuole membrane (Fig.2B).

In microspores treated for 24 h, besides a layerof abundant calcium precipitates was present onthe baculae, precipitates also occurred on theinner surface of the plasma membrane and theprecipitates in the cytoplasm and nucleus were

Fig. 2. Electron micrographs of cells in microspore stageexposed to normal environment (A); 12 h heat stress (B); 24 hheat stress (C); mature pollen stage in normal environment(D); 12 h heat stress (E); and 24 h heat stress (F). Arrowsindicate calcium precipitates. L, lipid body; N, nucleus; P,

pollen; PM, cytoplasm membrane; S, starch grain; V, vacuole;(bar = 3 m).

abundant. In the nucleus, the volume of precipi-tates was bigger than in the cytoplasm (Fig. 2C).

3 .4 . Calcium distribution differences betweennormal and treated mature pollen

In mature pollen, cytoplasm became dense

again and storage materials, such as starch andlipids, accumulated inside the grains. The pollenwall was composed of a lightly sculptured tectumand a fully developed intine. On the pollen wall,there was a layer of calcium-induced precipitateson baculae, but few in the cytoplasm and plasmamembrane (Fig. 2D).

The quantities and the distribution of calcium-induced precipitates in mature pollen after 12 and24 h heat stress were similar to that in the control(Fig. 2E, F).


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Calcium accumulation trends are summarizedin Table 1.

4. Discussion

As an electron microscopic cytochemical tech-nique for in situ localization of exchangeable cel-lular Ca 2 + -calcium that may be readily convertedto free calcium upon environmental modi cation,potassium antimonate precipitation has beenwidely used in the relevant studies. Although themechanism of precipitate formation was not clear(Wick and Hepler, 1982), recent studies indicatethat the precipitates are calcium antimonate: (i)the speci c chelator EGTA was capable of chelat-ing the Ca 2 + precipitates (Meng et al., 2000); and(ii) energy-dispersive X-ray microanalysis (EDX)indicated that a ratio of Sb to Ca of nearly 2:1 isexpected (Slocum and Roux, 1982; Jian et al.,1999). Free cytoplasmic calcium is usually belowthe limit of detection (10 6 mol /l Ca 2 + ), whereasless soluble compound (e.g. calcium phosphatesor carbonates) did not appear to release calciumfor binding with antimonate. The localization of calcium antimonate seems useful in identifying

loosely bound calcium.

4 .1. Calcium distribution during normal antherde elopment

Calcium is known to in uence cell functionsthrough spatial and temporal changes induced bystimuli (Bush, 1995). Calcium levels in developingtissues of anther appear to be dynamic, pre-sumably allowing transfers of Ca 2 + between freecytoplasmic pools, loosely bound pools and

tightly bound pools. Antimonate preferentially la-bels the loosely bound pools.In microspore mother cell, calcium-induced pre-

cipitates were mainly found on plasma membrane.However in tetrad, calcium precipitates emergedequably in the cytoplasm. In microspore, calciumprecipitates accumulated on the inner surface of plasma membrane and the sites opposite to thefuture colporal region and some calcium depositsalso occurred in the nucleus, while few calciumprecipitates accumulated in the cytoplasm of ma-

ture pollen. The differentiation of calcium concen-tration and distribution in anther developmenthad also been reported in Casteria errucosa and

wheat (Tirlapur and Willemse, 1992; Meng et al.,2000).We also observed calcium-induced precipitates

gradually accumulated on the microspore surfaceof tetrad, evidently increased on the surface of themicrospore and deposited a frequent calciumlayer on the mature pollen exine. The same cal-cium deposits changes in the pollen wall forma-tion also appear in the rice and wheat (Tian et al.,1998; Meng et al., 2000). All these results indi-cated that strategically located concentrations of calcium were related to normal antherdevelopment.

4 .2 . The relationship between heat stress and Ca 2 + distribution

Calcium has been found to be involved in theregulation of responses of plants to environmentalstresses (Bush, 1995; Braam et al., 1996; Webb etal., 1996). Intracellular calcium levels in plant cellsoften signi cantly increase under various stresses,such as salinity (Lynch et al., 1989), touch, wind

stimulation and cold shock (Jian et al., 1999),anoxia (Subbaiah et al., 1994) and oxidative stress(Price et al., 1994) and there is increasing evidencethat the same affairs may happen in heat shock /stress (Biyaseheva et al., 1993; Gong et al., 1997;Torrecilla et al., 2000). In this study, we alsofound those calcium precipitates increased by heatstress.

Several studies have revealed that there aregenotypic differences in the sensitivity of maizepollen germination to high temperature (Herrero

and Johnson, 1980; Lyakh et al., 1991). Frova etal. (1989) demonstrated that mature maize pollenwas unable to mount a heat-shock response whenit was exposed to supraoptimal temperatures. Thesame phenomenon had not been demonstratedfrom cytochemical studies. In our study, we ob-served that calcium precipitates changed greatly instressed microspore mother cell and tetraspore,compared with the control, respectively, while noobvious calcium distribution changes occurred inmature pollen. Given the potential role of HSPs


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T a b l e 1

C o m p a r i s o n o f r e l a t i v e c o n c e n t r a t i o n o f c a l c i u m - i n

d u c e d p r e c i p i t a t e s i n a n t h e r s o f n o r m a l ( N ) a n d h e a t s t r e s s ( H ) p e p p e r p l a n t s

M a t u r e p o l l e n

M i c r o s p o r e

T e t r a d

M M C

H

N

H

N

H

H

N

N

1 2 h

2 4 h

1 2 h

2 4

h

1 2 h

2 4 h

2 4 h

+ +

+ +

+ + + +

S u r f a c e / p o l l e n w a l l

+ + + +

+ +

+ + + +

+ + + +

+ + +

+ +

+ + +

+ + +

+ +

+ +

+ + + +

+

+

+ + +

+

C y t o p l a s m

+ +

+ + +

+ +

+

+ + +

+ + + +

+ + + +

C y t o p l a s m

m e m b r a n e

+

+

+ +

V a c u o l e

+ +

+ +

+ +

V a c u o l e m e m b r a n e

+

+ + +

+ +

+ +

+ + + +

+ +

+

N u c l e u s

+ +

+ + +

R e l a t i v e a b u n d a n c e : , n

o p r e c i p i t a t e s ( p p t ) ; +

, u n c o m m o n ( 1

1 9 p p t m

2 ) ; + +

, c o m m o n ( 2 0 3 9 p p t m

2 ) ; + + +

, a b u n d a n t ( 4 0 5 9 p p t m

2 ) ; + + + +

, v e r y

a b u n d a n t ( 6 0 o r m o r e p p t m

2 ) .


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Klein, J.D., Ferguson, I.B., 1987. Effect of high temperatureon calcium uptake by suspension-cultured pear fruit cells.Plant Physiol. 84, 153 156.

Lyakh, V.A., Kravchenkv, A.N., Soroka, A.I., Dryuchina,

E.N., 1991. Effects of high temperatures on mature pollengrains in wild and cultivated maize accessions. Euphytica55, 203 207.

Lynch, J., Polito, V.S., La uchli, A., 1989. Salinity stress in-creases cytoplasmic Ca activity in maize toot protoplasts.Plant Physiol. 90, 1271 1274.

Magnard, J.L., Vergne, P., Dumas, C., 1996. Complexity andgenetic variability of heat-shock protein expression onisolated maize microspores. Plant Physiol. 111, 1085 1096.

Meng, X.H., Wang, J.B., Li, R.Q., 2000. Effect of photoperiodon calcium distribution in photoperiod-sensitive cytoplas-mic male-sterile wheat during anther development. ActaBot. Sin. 42, 15 22 in Chinese.

Minorsky, P.V., 1985. A heuritic hypothesis of chilling injuryin plants: a role for calcium as primary physiologicaltransducer of injury. Plant Cell Environ. 8, 75 94.

Price, A.H., Tayler, A., Ripley, S.J., Grif ths, A., Trewavas,A.J., Knight, M.R., 1994. Oxidative signals in tobaccoincrease cytosolic calcium. Plant Cell 6, 1301 1310.

Schof , F., Prandl, R., Reindl, A., 1998. Regulation of theheat-shock response. Plant Physiol. 117, 1135 1141.

Slocum, R.D., Roux, S.J., 1982. An improved method for thesubcellular localization of calcium using a modi cation of the antimonate precipitates technique. J. Histochem. Cy-tochem. 30, 617 629.

Subbaiah, C.C., Bush, D.S., Sachs, M.M., 1994. Elevation of cytosolic calcium precedes anoxic gene expression in maize

suspension-cultured cells. Plant Cell 6, 1747 1762.Thomson, L.J., Xing, T., Hall, J.L., Williams, L.E., 1993.Investigation of the calcium transporting ATPases at theendoplasmic reticulum and plasma membrane of red beet(Beta ulgaris ). Plant Physiol. 102, 553 564.

Tian, H.Q., Kuang, A.X., Musgrave, M.E., Russell, S.D.,1998. Calcium distribution in fertile and sterile anthers of aphotoperiod-sensitive genic male-sterile rice. Planta 204,183 192.

Tirlapur, U.K., Cresti, M., 1992. Computer-assisted videoimage analysis of spatial variations in membrane-associ-ated Ca 2 + and calmodulin during pollen hydration, germi-nation and tip growth in Nicotiana tabacum L. Ann. Bot.69, 503 508.

Tirlapur, U.K., Willemse, M.T.M., 1992. Changes in calciumand calmodulin levels during microsporogenesis, pollendevelopment and germination in Gasteria errucosa . Sex.Plant Reprod. 5, 214 223.

Torrecilla, I., Legane s, F., Bonilla, I., Francisca, F.-P., 2000.Use of recombinant aequorin to study calcium homeostasisand monitor calcium transients in response to heat andcold shock in cyanobacteria. Plant Physiol. 123, 161 175.

Vierling, E., 1991. The roles of heat shock proteins in plants.Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, 579 602.

Wang, G.Y., Liu, J.M., Zhang, Y., Yu, B.S., Shen, Z.Y., 1997.Studies on ultrastructure in common bean leaves duringheat acclimation and heat stress. J. Agric. Biotechnol. 7,151 156 in Chinese.

Waters, E.R., Lee, G.J., Vierling, E., 1996. Evolution, struc-ture and function of the small heat shock proteins inplants. J. Exp. Bot. 47, 325 338.

Webb, A.A.R., Mcainsh, M.R., Taylor, J.E., Hetherington,A.M., 1996. Calcium ions as intracellular second messen-ger in higher plants. Adv. Bot. Res. 22, 45 96.

Wick, S.M., Hepler, P.K., 1982. Selective localization of intra-cellular Ca 2 + with potassium antimonate. J. Histochem.

Cytochem. 30, 1190 1204.Zhang, Z.S., Li, R.Q., Wang, J.B., 2000. Effect of Ca 2 + ,La 3 + and EGTA treatment on the responses of pepperleaves to heat stress. J. Wuhan Univ. 46, 253 256 inChinese.

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Effect of Heat Stress on Calcium Ultrastructural Distribution