BULLETIN Actin and myosin during pollen germination

BULLETIN Actin and myosin during pollen germination REN Dongtao, HAN Shengcheng and YAN Longfei (YEN Lungfei) College of Biological Sciences, Chinese Agricultural University, Beijing 100094, China

Abstract Actin and myosin from pollen tubes of Lilium davidii were studied by using imrnunoblotting, Dot-Blot and myosin ca2+-ATPaseanalysis. On imrnunoblotting of the total soluble pollen tube proteins, anti-a-actin antibody labelled a polypeptide approximately 43 ku, which is considered to be the actin of lily. The mRNA encoding actin in ungerminated pollen and germinated pollen were both undetectable in our experiments. A myosin exhibited Ca2+-ATPaseactivity, with a native molecular weight of 460 ku has been identified by using immunoblotting. A polypeptide of about 205 ku and a polypeptide of about 20 ku were the heavy chain and a set of light chain of the myosin, which can crossreact with anti-skeletal muscle myasin heavy chain monoclonal antibody and anti-skeletal muscle myosin light chain (20 ku) monoclonal antibody, respectively. The Ca2+-ATPase activities of myosin in crude extracts of germinated pollen were pitively related to the growth rates of pollen tubes. Keywords: pollen of lily, actin, myosin.

THEcontraction of striated muscle involves the interaction of two types of filaments, thick filaments consisting mainly of myosin and thin filaments consisting primarily of actin. Myosin is a well-known mechanochemical enzyme capable of producing force and sliding along the actin filament, which is coupled with hydrolysis of ATP. It had been established that actomyosin system is universally distributed in higher plants[11. Since the discovery of actomyosin system in higher plants, it is considered to be involved in the cytoplasmic streaming, intracellular transport of organelles, cell motility and mitosis[21. Although the concentrations of actin and mysin in plant are lower than they are in animal tissues, many authors have identified and purified actin in various plants, and proved that its biochemical characteristics are similar to that of muscle actin13]. However, the studies on purification of myosin from plant are limited, that postponed to understand the molecular mechanism which was related to the actomyosin of plants[3'41. We found previously that pollen is a rich source of actin and myosin for analysing cell motility in plant. In this

Chinese Science Bulletin

Vol . 4 3 No. 8

April 1998

BULLETIN note, actin and myosin from pollen tubes are studied by using immunoblotting, Dot-Blot and Ca2+-ATPase analysis. The results suggest that, there are a 205 ku heavy chain and at least one 20 ku light chain in pollen tubes myosin. There is neither a storage mRNA e n d i n g actin in ungerminated pollen, nor actin synthesis during its germination. 1

Materials and methods

(1) Plant materials. Pollen was obtained from Lillium davidii Duch flowers growing in Lanzhou, Gansu Province, and was dried at room temperature. The dried pollens were kept at - 80C until use. Pollen was allowed to germinate in culture medium containing 15% sucrose, 1. 27 rnrnol/L Ca( NO3) 2 , 0.162 mmol/L boric acid, 0.99 mmol/L KN03, 1-66 mmol/L MgS04, pH 5.61, then the pollen tubes were collected on filter paper every 2 h and observed under an Olympus microscope. ( 2 ) Methods ( i ) Extract of actin from pollen tubes. The crude extract of actin from pollen tubes was performed according to Ma and ( ii ) Dot-Blot analysis of RNA. Pollen or pollen tubes were ground in liquid nitrogen for 3-5 min. For extract of total RNA, the SSC buffer (25 mmol/L sodium citrate, pH 7.0, 4 mol/L guanidinium isothiocyanate, 1 . 5 % ( w/v) sodium laurylsarcosine and 100 mmol/L P-mercaptoethanol) was added and stirred for 30 s, and standing on ice for 10 min. After double extraction with phenol/chloroform, the upper phase was extracted with chloroform. Then the upper phase was precipitates with 2-fold volumes of absolute ethanol, at - 20C for 30 min. The precipitates were rinsed thoroughly with 70 % ethanol, and dissolved in sterilized water. The phenol/chloroform and chloroform extractions were repeated twice. The upper phase was precipitated with one-tenth volume of 4 mol/L of LiCl and 2-fold volumes of absolute ethanol. The precipitates were rinsed with 70 % ethanol, and dissolved in sterilized water and diluted with RNA buffer (DEPC-H20:20 x SSC: Formamide, 5 :3 :2 ) , Dot-Blot upon nylon membrane with each 10 pg RNA, the Pea actin gene was used as standard. The RNA was fixed on membranes by UV-crosslinking for 2 min. Pea actin gene inserted in pBlue script plasmid was digested by Not I and separated from the vector sequence by electrophoresis. The purified 1 . 7 kb pea actin gene was labeled by the standard random primed DNA labeling reaction and the blotted membranes were detected by DIG Detection Kit. ( iii ) Extraction of myosin from pollen tubes. Extraction of myosin was performed according to Yokata and himm men'" . Ungerminated pollen or pollen tubes were suspended in EMP buffer (10 mmol/ L EGTA, 6 mmol/L MgC12, 0 . 5 mmol/L PMSF, 50 pg/mL TPCK, 50 pg/mL pepstating, 2 mmol/L DTT, 30 mmol/L PIPES-KOH, pH 7 . 0 ) and ground in a pestle and motor. The homogenate was centrifuged at 10000 g, at 4°C for 15 min. The supernant was used as crude extract of myosin. ( iV ) Native-polyacrylamide gel electrophoresis (PAGE) and SDSPAGE. Native-PAGE was carried out according to the method of ~,aemmili[~~ without SDS, using a 3 . 5 % stacking gel and 5 % -10 % gradient running gel. Cd+-ATPase activity of myosin was identified in a buffer containing 100 mmoVL Tris-HC1, pH 8.0, 10 mmoVL EDTA, 5 mmol/L ATP, 50 mmol/L CaC12, at 35C for 30 min. The bands containing myosin would produce a white band of precipitate of calium phosphate. Myosin was subjected to S D S SDSPAGE was performed according to the method of ~aemmili'~]. PAGE using 7.5 % -15 % gradient running gel, actin using 10 % running gel, and 4.5 % stacking gel. The gel was stained with Commasie blue RzSoor with a silver staining. ( V ) Immunoblotting. After native-PAGE and SDSPAGE, proteins were electrotransferred to a nitrocellulose membrane (0.45 pm) according to Towbin et 4 1 . ['I The membrane was incubated with anti-muscle a-actin monoclonal antibody (Sigma product, with 1 : 4000 dilution), anti-skeletal muscle myosin polyclonal antibody (Sigma product, with 1 :2 500 dilution), anti-skeletal muscle myosin heavy chain monoclonal antibody (Sigma product, with 1 : 2 000 dilution), anti-skeletal light chain (20 ku) monoclonal antibody (Sigma product, with 1: 1 000 dilution), respectively. Excess antibodies were removed by washing the membranes. The membranes were then incubated with anti-mouse IgG (Sigma product, with 1: 20 000 dilution) or anti-rabbit IgG (Sigma product, with 1 : 4000 dilution) conjucted with alkaline phosphatase . Color development was performed with an alkaline phosphatase development kit (Promega product) .


Vol .43 No. 8

Apri 1 1998

69 1

BULLETIN 2

Results

2.1

SDS-PAGE and immunobloting of pollen tubes actin The crude extract of actin from lily pollen tubes was electrophoresized on a SDSPAGE gel and was transferred to a nitrocellulose membrane. The proteins separated were visualized by Commassie blue staining ( fig. 1 ( a ) ) and by immunoblotting (fig. 1( b ) ) . The results showed that, the pollen tubes contained an actin with a molecular weight of 43 ku and the actin was immunochemically identical to the muscle a-actin. 2.2

Dot-blot of mRNA encoding pollen tubes actin RNA extracted from pollen or pollen tubes (per 2 h after germination) were dot-blotted. The membrane was hybridized with pea actin gene. The ungerminated pollen and germinated pollen tubes gave negative hybridization signals, whereas the pea actin gene showed positive spot. Therefore, the ungerminated pollen and pollen tubes all lacked detectable mRNA encoding actin. 2 . 3 Native-PAGE of crude extract and immunoblotting of myosin After native-PAGE of crude extract, the myosin was identified by c a Z f-ATPase activity on polyacrylamide gels. The white band on gel, with a native molecular weight of 460 ku could crossreact with the anti-rabbit

Fig. 1 . SDS-PAGE of crude extracts ( a ) and immunoblotting of actin ( b ) from lily pollen and pollen tubes. Lane 1, Molecular weight marker; lane 2, rabbit muscle actin; lane 3, crude extracts from lily pollen and

skeletal muscle myosin polyclonal antibody, was the lily pollen tubes, myosin. A weak band on the immunoblotting membrane with a molecular weight less than lily myosin was suggested to be a proteolytic fragment of myosin (figure 2 ) . 2.4

SDS-PAGE of crude extract and immunoblotting of myosin Immunoblotting of the crude extract from pollen tubes showed that, a polypeptide of about 205 ku was labeled by antimyosin heavy chain and a polypeptide of about 20 ku was labeled by anti-myosin 20 ku light chain. Silver stained molecular weight markers were shown on the SDS-polyacrylamide gel (fig. 3 ( a ) , lane I ) , as well as the same gel of heavily loaded pollen tube extract (fig. 3 ( a ) , lane 2 ) . These results demonstrated that the bands in pollen tube extracts bound the anti-myosin heavy chain antibody (fig. 3 ( b ) , lane 3 ) and the anti-myosin light chain antibody (fig. 3 ( c) , lane 3 ), and this corresponded to a band of about 205 and 20 ku in the silver stained gel respectively, which were the heavy chain and a light chain of the lily myosin.

-

2 . 5 Changes of mvosin Ca2+-ATPase activities of crude exFig. 2 . CaZt -ATPase activity staining of crude tracts during pollen germination extracts from lily pollen tubes separated on NaDuring pollen germination, we sampled the pollen tube for tive-PAGE( a ) and immunoblotting of myosin every 2 h and extracted the protein. The crude extracts were ( b ) , Lane 1 , Mo~ecu~ar weight marker; lane electrophoresized on a Native-polyacrylamide gel and stained by 2, crude extracts of lily pollen tubes. its ca2+-ATPase activity. The results revealed that, there was no myosin CaZ+-ATPase activity band in the extract of ungerminated pollen, however, the c a 2 +-ATPase activities of myosin in the crude extracts of germinated pollen were positively related to the growth rate of

692


Vol .43 No. 8

April 1998

pollen tubes. The extract with a higher activity of myosin ca2+ - A T P ~ S ~was found 2 h after germination, with stable activities from 4 to 10 h, and then declined gradually from 12 to 20 h (figure

4). 3

Discussion

Evidence shows that, the known myosins can be grouped into nine distinct and evolutionary ancient families[81. Recently, the crystal structure of myosin II head has been solved[91, the biophysical and biochemical properties of myosin are being characterized in exquisite find and detail[3951,myosins I , Ill, V were analyzed by mutation[10'"1. These gave an Fig. 3 . SDS-PAGE of crude extract of pollen tubes (a) and immunoblotting abundance of molecular information to of heavy chain (b) and 20 ku light chain of myosin (c) . Lane 1, Molecular weight marker; lane 2, rabbit muscle myosin; lane 3, crude extract of lily

myosin molecular structure. But, there is pollen tubes, dearth of functional information of myosin['01. Because of the lower content of myosin in higher plants, our knowledge of plant myosin is rather poor. The limited results revealed that there were a 200 ku myosin heavy chaih in Nittela flexis and Allium cepa[lZ1, a 165 ku in Pisum sativvum tendril^'^], a 175 ku in Nicotiana tabacum pollen tube"31, and a 100 ku ~ . rein Lycopersion e s c ~ l e n t u m [ ' ~Our sults prove that there exists a myosin in Lilium davidii with a native molecular weight of about 460 ku, which exhibited immunological similarity to skeletal muscle myosin ( fig. 2 ) . And a 205 ku polypeptide was the heavy chain of the myosin, it can also crossreact with the anti-skeletal muscle Fig. 4. Relationships among the ca2+-ATPaseactivity of myosin in crude exmyosin monoclonal antibody. yokata tracts from pollen and pollen tubes of lily and growth ratio of pollen tubes. 1, and shimmen reported that there was a Ca2'-ATPase activity staining of myosin in crude extracts from pollen and pollen tubes; 2, curve of pollen tubes growth; 3, ratio of pollen tubes growth.

myosin with a 175 ku heavy chain in Lilium longiflorum, which did not crossreact with anti-pan myosin antibody[']. These results disagreed with ours. It may be caused by the difference between the myosins in Lilium davidii and Lilium Longiflorum , or by the extracted methods (see Materials and methods). The evidence shows that, phosphorylation of the regulated light chain of myosin played an important role in regulating the ATPase activities of myosin and its onf formation[''^. Up to now, there is still lack of knowledge about the light chain of myosin in higher plants. Ma and Yen have found two sets of light chain of Pisum sativvum tendrils of about 17 and 15 ku"]. We report here a set of light chain of about 20 ku in Lilium davidii pollen tubes using immunoblotting for the first time (fig. 3 ( c ) , lane 3 ) . The functions of the light chain are in progress. The electron-microscopic evidence indicated that the ungerminated pollen grain of Endymion non-


Vol. 43 No. 8

April 1998

693

BULLETIN scriptus contained numerous fusiform bodies, the bodies were dispersed during the activation of the pollens, being replaced by aggregates of slender F-actin, which converge towards the germination apertures and were present in emerging tube[16]. The same results have also been described by Pierson in pollen tubes of Lilium longifl~rum["~.Whether the changes of actin from fusiform to net form were caused by synthesis of actin or the convergence from storage form is still unknown. Our result proved that the actin from Lilium davidii with a molecular weight of 43 ku can crossreact with anti-muscle a-actin monoantibody. Many reports have shown mRNAs synthesis during pollen maturation and stored in matured pollen[181.The mRNA encoding actin in ungerminated pollen and germinated pollen was undetectable in our experiment. The result suggested that the changes of actin form are caused by convergence of storage form of actin. Myosin is a molecular motor responsible for movements associated with actin containing microfilaments. Can the changes of actin form affect the myosin? Study on ca'' -ATPase activity staining of myosin has found that the band of myosin &+ -ATPase activities is undetectable in ungerminated pollen, meanwhile the myosin Ca2+-ATPase activities are positively related to the growth ratio of pollen tubes (fig. 3 ) , suggesting the change of myosin. Since myosin plays an important role in growth of pollen tubes, study on the changes of myosin form is needed. Acknowledgement This work was supported by the National Natural Science Foundation of China (Grant No. 39600073).

References 1

2 3 4 5

6

Yen, L. F., Shih. T. C . , The presence of a contractile protein in higher plants, Acta Biochimica et Biophysica Sinica (in Chinese), 1963, 3: 490. Williamson, R. E., Organelle movements, Annu. Rev. Plant Physiol. Plant Mol Biol., 1993, 44: 181. Ma, Y. Z.. Yen, L. F., Actin and myosin in Pea tendrils, Plant Physiol.. 1989, 89: 586. Yokata, E . , McDonald, A. R . , Liu, B. et a1 . , Localization of a 170 KDa myosin heavy chain in plant cells. Pmtoplasma, 1995, 185: 178. Yokata, E.. Shimmen. T . , Isolation and characterization of plant myosin from pollen tubes of Lily, Pmtoplasma, 1994, 17: 153. I.aemmli, U. K. , Cleavages of structural proteim during the assembly of the head of bacteriophage T,, Nature, 1970, 227: 680. 'Towbin, J . , Sachelin, T. , Cordon. J . Electrophoretic transfer of proteins from poly-acrylamide gels to nitrocellulose sheets: Procedure and some applications, Proc . Natl . Acad . Sci . USA, 1979, 76: 4350. Cheney, R. E . , Riley, M. A.. Mooseker, M. S., Phylogenetic analysis of the myosin superfamily, Cell Motil Cytoskl, 1993, 24: 215. Rayment, I.. Rypniewski. W . K., Schmidt-he, K. et a1 . , Three-dimensional structure of myosin sub-fragment I: A molecular motor, Science, 1993, 261 : 50. Bement, W . M., Mooseker, M. S.. Molecular motors keeping out the rain, Nature, 1993, 365: 785. Kato, T. , Tonomura, Y. , ldentification of myosin in Nitella flexilis, J . Biochem . , 1977, 88: 777. Parke, J . Miller, C. , Anderton, B. H. Higher plant myosin heavy-chain identified using a monoclonal antibody, Eur . J . G l l . Biol., 1986, 41: 9 . Tang, X. J . , Hepler, P. K . , Scordilis, S. P . , Immunochemical and immunocytochemical identification of a myosin heavy chain peptide in Nicotiaiiu pollen tubes, 1. Cell Sci . , 1989. 92: 569. Vahey. M., Titus, M.. Trautwein. R. et a l . . Tomato actin and myosin: Contractile proteins from a higher land plant, G11 M o t i l . , 1982, 2: 131. Trybus, K . M . , Filamentous smooth muscle myosin is regulated by phosphorylation, J . Cell Biol., 1989, 109: 2887. HeslopHarrison, J . , Hcslop-Harrison, Y . , Cresti, M. et a1 . Actin during pollen germination. J . Cell Sci . 1986, 86: 1. Pierson, E. S. , Rhodamine-phalloidin staining of F-actin in pollen after dimethyl-sulphoxide permeabilization, Scr Plant Re1988, 1: 83. p&., Frankis, R., Mascarenhas, J . P. Messenger RNA in the ungerminated pollen grain: A direct demonstration of its presence, Ann. B o t . . 1980, 45: 595.

.

.

.

.

.

.


Vol .43 No. 8

April 1998