Baba, Sango

Miki-Hirosige, Hisako

Shinke, Namio

English

Kyoto University Research Information Repository

RIGHT:URL:CITATION:AUTHOR(S):ISSUE DATE:TITLE:Isoelectric Zones of Vegetative andGenerative Nuclei in Pollen Grainsof TradescantiaBaba, Sango; Shinke, Namio; Miki-Hirosige, HisakoBaba, Sango ...[et al]. Isoelectric Zones of Vegetative and Generative Nuclei in PollenGrains of Tradescantia. Memoirs of the College of Science, University of Kyoto. Series B1961, 28(3): 359-3631961-12-20http://hdl.handle.net/2433/258217MEMOIRSOF THE COLLEGE    Vol. XXVIII,OF SCIENCE, UNIVERS!TY OF KYOI[•O,Ne. 3, Article 7 (Biology), 1961SERIES B,Isoelectric Zones ofill PollenVegetativeGrains ofand GenerativeTradescantiaNi}cleiBySango BABA,      BotanicalNamioInstitute,SHINKECollege efand Hisake MIKI-HIROSIGEScience, University ef Kyoto(Received June 15, 1961)   It is well known that vegetative and generative nuclei in pollen grainsdiffer in their reaction to dyes, e.g. a combination as cyanin (basic dye) andeerythrosin (acid dye) staining the vegetative nucleus red and the generativenucieus blue (cf. CouLTER and CHAMBERLAiN, 1909).   While, from the cytophotometric studie$ of Feulgen staining in the pollengrains of Tradescantia, it has been found that DNA synthesis in the generativenucleus occurs during the first third of interphase while the DNA content ofthe vegetative nucleus remained unchanged (SwiFT, 1950; WooDARD, 1956).   From the facts mentioned above, it may be assumed that the differencein the reaction to the dyes is due to the different amounts of DNA betweenthe vegetative and gelterative nuclei in the pollen grains. The purpose ofthe pyesent study is to see whether difference of isoe}ectric po!nts betweenthese two nuclei exists or not, and to determine whether the difference ofthe isoelectric point of these nuclei has an important relation to the amountof DNA in these nuclei or not.Material and   Young pollen grains of Tradescantiagrew up inte crescent-shaped ones, were   Methods of the study are describedscriptive parts of the following pages.IY{ethods refiexa, before the generative nuclei used in the present investigation.in the 'appropyiate places in the de-Results   I) Isoelectric zones cletermined by the tu?'bidily method. Isoelectric zones ofthe vegetative and generative nuclei in the fresh pollen grains were determinedby the turbidity method with a' dark fie}d microepcope (cf. SHiNKE, 1939).   This method is based on the fact that the maximum flocculation of proteinsaRd other ampholytes takes place at the isoelectric point of the substances'.360 Sango BABA, Namio SmNi<E and Hisako MIKI-HIRoslGEIn this study, the living pollen grains were observed in the rnedia of differeRtpH and the brightRess of the nuclei was measured by a dene.itometric method.The pH of the media was controlled by acetic acid solutions of differentconcentrations and also by MciLvAiNE's buffer solutions. '   The results obtained by the above rnetbod on tke vegetative and generaSivenuc}ei in the pollen grains are shown in Plate I and Table 1.    Table 1. Turbidity of nuciei and cyteplasm in polleR grains caused by acetiÅë acid       in different concentrations.Composition of media50/o sucrose solution20 ml20 ml20 ml20 ml20 ml2e m}20 ml20 mlG}acial acetic   acid46.6 ml 3.0 ml 2.0 ml l.e ml 0.7 ml 0.4 ml 0.2 ml 0.1 mlpH1.002.352.402.552.622.752.952.98Turbidity            iGenerat. nucleus i Vegetat. nucleus-{-F-x-eÅÄÅÄ" +ÅÄ++-im++or - l-FCytoplasmiÅ}"i''L-m.+"   In Plate I, the generative nuclei in Figs. B (pH, 2.35), C (pff, 2.40) andD (pH, 2.55) are illuminated brightly, and are easily distingui.ehable from thevegetative nuclei and the cytoplasm. In Fig. .E (pH, 2.95), the generativenuclei decrease somewhat in brightness and are of the same brightness as thoseof the vegetative ones.. In Fig. F (pH, 2.98), the vegetatlve nuclei and thecytoplasm increase in brightness. These results are summarized in Table 1.   From the Table 1, it ls seen that the isoelectric peint or zone of tkevegetative Bucleus is larger than pH 2.98 and that of the generatlve one isfottnd between pH 2.35 and 2.62.   II) Isoelectvic zones determined by acid a?td basic dyes. The isoelectric zonesof the nuclei and cytoplasm in the pollen grains were determined by a modi-fied NAyLoR's method (1926). The fixative used in this experiment was 109o!neutral formalin. The mixtures of 5ml of M/10 sodium acetate solution aRddiffereltt amount of M/10 or IM acetic acid were used for buffer solutions(cf. SHiNKE, 1939).   Tke limits of retention of eosin and toluidine blue by the cytoplasm aRdboth the vegetative and generative nuclei in the pollen grains are shown inTable 2. As shown in this table, the isoelectric zones of the cytoplasm aRdboth tke vegetative and generative nuclei are pHi 4.05-4.62, 3.90-4.35 and 3.40-3.90 respectively.          Isoelectfic Zones of Nuc]ei in Pollen Gralns of Tradescantia 361Table 2. Limits of retention of eesin and toluidine blue by nuclei and cytoplasm of   pollen grains.renynwunyp-mdmul       lparts l       ,DyesGenerativenucleusVegetativenucleusCytoplasml Eosinil Toluidine bluei' Eosinl Toluidine bluejEosin 'i' Toluidine bluei3.10 3.40       pH values3.70 3.90 4.05 4.35 4.62 5.00-i{"-i-i-{"-i-yÅ}-i'lu-l++ --Y er ww}- +or ----F + -Y      + {- or+-y + +          Å} or -i-+or -"- {+  Å}  {" -ff  +Å}  -e t""mi'i-IYF    III) Effect of DNAase treatment. The pollen grains were fixed with 10fOo/neutral formalin, washed with dist. water and were treated wkh the enzymesolution such as 0.5mg of crystaline DNAasei) per 1m! of dist. water contain-ing O.OO125MMgS04 for 20 ltours at 35"C (PEARsE, 1960). '    In the polien grains treated with the DNAase solution, the isoelectriczones of botk the vegetative and generative nuclei determined by the seainingmethod are shifted to alkaline side by the treatment and the zone is pHi 4.05-4.62, which is tke sarae zone as that of the cytoplasm. The$e results areshown in Table 3.   Table 3. Limits of retention of the dyes by nuclei and cytoplasm of po}len grains       treated vvith DNAase.Parts DyesI  3.10 3.40          EosinGenerativenucleus          Toluidine          EosinVegetativenucleus          Toluidine          EosinCytoPlaSrn l Toluidineblueblueblue3.70 pH values3.90 4.05 4.35 4.62 5.00-l-l•-my-{'l"---•H-{'l+++l ElrÅ} L  ++ or -i-' "-   lÅÄ Qr -l-  +Å} Or -f--- F++ÅÄ-l-+Å}-l-l•-Å}"ÅÄÅ}-l-F-l-•I-{+"   IV) Eff ect of proteinase treatment. The pollen grains fixed with 10.0o!neutral formalin, after being washed with dist. water, were treated with asolution containing 1mg of proteinase2) per 1ml of dist. watey foi' 20 hoursat 3sOC.    The isoelectric zones of the cytoplasm and both the vegetative andgenerative nuclei determined by the staining method are pHi 4.05-4.62, 3.90-   1) The DNAase used was the products of Sigma Chernical Co.   2) The proteinase used was Pronase A S, the preducts of l<aken Chemical Co.362 Sango BABA, Namio SHINi<E and Hisako MI--HrRoslGE4.35 and 3.40-3.90 respectively, and these zones do not skow any changes bythe enzyrne treatment as compared with those of the untreated pollen grains.                        Discussioll and ConelusioR    The isoelectric zone of the generative nucleus in tke fresh pollen grainsdetermined by the turbidity method is found to be smalier value than pHi 2.62,while that of the vegetative one is larger thaR pHi 2.98. 0n the other hand,the isoelectric zones of the cytoplasm and both the vegetative and generativenuclei in the fixed pollen grains as determined by the dye staining arepHi 4.05-4.62, 3.90-4.35 and 3.40rr3.90 respectlvely. In both cases, the iso-electriczones of the generative nuclei are in more acid-side than those of thevegetative ones, and those of the vegetative nuclei are in rnore acid side thanthose of the cytop}asm. These results are harmonious with the yeports ofPiscHiNGER (1926), YAMAHA (1932) and others that the isoelectric point of thenucieus is'in more acid siqe than that of the cytoplasm. Tkese results arealso consistent with the report that the isoelectric point of nucleoprotein is nearpHi 4.0 (HALL, 1941).    In the pollen grains treated with the DNAase, the isoe}ectric zones ofboth of the vegetative and generative nuclei are skifted to more alkaline sideby the treatment, and the difference in isoelectric zones among the cytoplasmand the vegetative and generative nuclei does not exist. Whereas, in tkepollen grains treated with the proteinase, the isoelectric zoBes of the cytoplasmand the vegetative and generative nuclei do not change by the treatmeRt, andthe zones aye the same as those of ulttreated polleR grains.    From these facts mentioned above, it may be concluded that the differencein the reactions to the dyes between tke vegetative and generative nuclei isbased upon the difference of the DNA content in these nuclei. This is inagreement with the results of SwiFT (1950) and WooDARD (1956).                                Summary    1) Isoelectric zones of the vegetative and generative nuclei in the freshpollen grains of Tradescantia refiexa were determined by the turbidity method.These zones of the generative nucleus are between pHi 2.35 and 2.62, and tkoseof the vegetative one are of largei' value than pHi 2.98.    2) Isoelectric zones of the vegetative and generative nuclei in the fixedpollen grains of Tradescantia were also determined by acid (eosin) and basic(toluidine blue) dyes. The isoelectric zones of the generative and vegetativenuclei and the cytoplasm were pHl 3.40-3.90, 3.90-4.35 and 4.05-4.62 respectively.    3) Isoelectrlc zones of the generative and vegetative nuclei and the cyto-plasm in the pollen grains treated with DNAase or proteinase were also deter-mined by the dyes. After the treatment of tke DNAase, the !$oeiectric zonesIsoelectric Zones of Nuclei in Pollen Grains of Tradescantia 363of both the generaÅíive and vegetative nuc}ei were skifted to more alkalineside by the treatment and tke difference in isoelectric zones amoRg the genera-tive and vegetative nuclei and tlte cytoplasm did Rot exist. The treatment ofpollen grains with proteinase did not give any changes in the isoelectric zones.    4) Tke difference in the reactions to the dyes between the generative andvegetative nuclei in the pollen grains may be based upon the different amountsof DNA contained in these nuclei.                             niterature citedCovLTER, J. M., & C. J. CHAMBERLAIN, 1909. Morphology of Angiosperms.HALL, I. L., i941. Jour. Amer. Chem. Soc., 63:794.NAyLeR, E. E., 1926. Amer. Jour. Bot., 13:265.PEARsE, A. G. E., l960. Histochemistry. London.PISCI{INGER, A., 1926. Zeitschr. f. Zellforschg. u. mikr. Anat., 3: 169.SmNi<E, N., 1939. Mem. Coll. Sci. Univ. Kyoto, (B), 15; 1.Swlit'T, H., 1950. Proc. Nat. Acad. Sci., 36:463.WooDARD, J., 2956. 3our. Biophysic. and Biochem. Cyto!., 2: 93.YAMAHA, G., 1932. Proc. Imp. Acad. Tokyo, 8: 315.NewYork.                    Explanation of Plate I   Figs. tw-E , All these figures are photomicrographs taken by a dark fieldillumination showing turbidity of nuclei and cytoplasm in pollen grains ofTradescantia reflexa.   Fig. A shows the pollen grains observed in the medium at pK 1.00; Fig. Bat pH 2.35; Fig. C at pH 2.40; Fig. I) at pH 2.55; Fig. E at pH 2.95; Fig. F atpH 2.98.Mem.Coll. Sci., Univ. Kyoto, Ser. B, Vol. XXVIII, No. 3 Pl.

Isoelectric Zones of Vegetative and Generative Nuclei in Pollen Grains of Tradescantia

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Isoelectric Zones of Vegetative and Generative Nuclei in Pollen Grains of Tradescantia

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