IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L.22 (3) 2009 Study o n The Effects of Boron on The Structure of Chloroplasts of Cauliflower , By Using Light Scattering Technique *H. M.A.Al-Qysi , R. M.M.AL Mola , N. K.Alani *Departme nt of Physics ,College of Science, Nahrain Unive rsity Departme nt of Biology ,College of Science, Baghdad unive rsity Abstract Effects of Boron on the structure of chlorop lasts membrane isolated from caulif lower are invest igated , using light scatt ering technique. Results obtained in this st udy suggest that Boron in the concentration range (0.1-5 µm) can fluidize the lip ids of the chlorop last membran e due to different extent. M echanisms by which Boron can change the lip id fluidity is discussed. Furt hermore, an exp erimental evidence is p resented to show that2µM Boron can mediate conformational chan ges in the membrane –bound p roteins of the cauliflower’s chlorop last . Introduction Although the essentiality of Boron for higher p lants is well established , the mechan isms of its action are far fro m bein g well underst ood. It has been indicated that Boron is involved in carbohy drate metabolism, translocation, p rotein sy nthesis , p henolic p roduction , and pollen germin ation. In an att empt to determine the p rimary role of Boron in p lants, Parr and loughman (1) reported that one asp ect which could underlie all typ es of resp onses to Boron is that of imp aired membrane function. One of the well known Boron deficiency sy mp toms is the reduction in cap acity for Ion transp ort. Robertson and Lough mann (2) showed that the reduced cap acity for p hosp hate absorp tion could be restored almost comp letely by supp ly ing 10µM H3BO3 for 0ne hour. Tanada(3) found out that induced a bioelectric f ield chan ge the hyp ocoty l tissues in mung beans. He att ribute these changes to modification of some membran e comp onents. Even though the control of membrane function by Boron is beyond doubt, its exact involvement in membr ane component is not y et understood .Thus in this st udy an attempt has been made to determine at a molecular level the direct effect of Boron up on the comp onents of chlorop last membrane as we ll as the concentration at which Boron maintainin g the structural integr ity of the membrane. Material and Methods Isolation of chlorop last : 10 g of mature clean cauliflower leaves cut into small p ieces ground in a p istil and mortar(kept in an ice bucket) using 50 ml of cold tris buffer (5 x10 M tris p H 7.8) . The homogenate were filtered through three layers of gauze cheese cloth. The filtrate were centrifuged at 200 xg for 10 min. the sup ernatant were again centrifuged at 1000xg for 15 min. The p ellet were collected and purified by resusp ended with 5 ml of tris IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L.22 (3) 2009 buffer, then 1 ml of this susp ension were added to sucrose gradient tube( 1.6,1.0,0.5,0.25M ), and st ored for 2 hours . Pure chlorop lasts were collected somewhere nearby region of 1.3 M concentration. Samp le p reparation: The st ock suspension of the isolated chlorop last was diluted by aqueous solution 3 ml sa mples of the diluted chlorop last susp ension were then p repared. Various con centrations of aqueous solutions of Boric acid were prepared. To p rep are the samp le for turbidity measurement, 1ml of certain solution was add ed to the chlorop last susp ension; the sample was allowed for 3minutes at room temp erature, turbidity was measured by Lovibond turbidometer. Light scatt ering measurement: Light scatt ering intensity at 90 (190) was measured by light scatt ering set up . The set up consists of He-Ne laser as light source , the samp le holder calibr ated with different angles , p hotomultip lier, digital voltmeter, and water bath for temp erature measurement. Results and Discussion It becomes fact that function of any biological sy stem can be greatly affected by variations in the structure of that system. Likewise, many functional asp ects of cell membran e have been shown to be impaired or mod ified by alteration in the structure of the membrane. Lip id fluidity p lay s a very imp ortant role in maintainin g the p rop er functions of the cell membran e. M any membrane – bound enzy mes require certain degree of lip id fluidity for their optimum activities. In addition, the rate of transp ort throughout the membrane is high ly dependent up on the amount of lip id fluid ity . Convincingly , Boron seems to be essential for p rop er functions of cell membrane in higher plants. T he activities of many membrane – bound enzy mes have been shown to be markedly affected by Boron deficiency (6,7). Transp ort of p otassium, phosp horus, and Rb appears t o be significantly altered by the action of Boron (1). Also, Boron has shown to facilitate the p ermeability of water throughout the liposomes (1). Yes, it is not clear whether the Boron – induced functional changes in the p lant cell membran es could be att ributed to a direct interaction of Boron with the membrane comp onents or might be mediated by some other actions of Boron. Finding p resented in this st udy p rovide exp erimental evidence required for the imp ortant conclusion that Boron may interact directly with chlorop last membrane comp onents. A decline in the turbidity of the chlorop last suspension , shown in fig1 , indicates that Boron in the concentration (0.1-4µM ) could be fluid ize the membran e lip ids. Boron in the concentration range (o.1-4µM ) seems to be incorp orated in the chlorop last membran e , p erturbing the lip id p acking and increasing the lip id fluidity . This comes in agr eement with a previous work done on different membrane(8). Therefore, Boron might be considered as regu lator of membrane fluid ity acting like cholest erol in the animal cell membran e (9). Furt hermore , results illustrated in fig 3 clear ly indicate that 2µM of Boron may act to induce thermal destabilization of membran e –bound p roteins of the chlorop last as result of st ructural changes. The observed structural changes in the membrane –bound p roteins of chlorop last that caused by 2µM boron might com as a result of a direct interaction of Boron with the membrane –bound p rotein or might be due to the Boron – induced lip id fluidity chan ges. IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L.22 (3) 2009 However, results shown in fig 3 rules out the first p ossibility that Boron could interact directly with membrane –bound p roteins since boron seems t o fluid ize the lecithin lip osomes in sp it of the absence of the membrane – bound proteins. Interest ingly, Boron with high concentration (4- 10µM ) seems not to be incorporated in the chlorop last membran e, since there are no effects could be observed on chlorop last membran e. Therefore, it is likely that Boron with high concentration could translocate through the chlorop last membrane , mediating the toxicity of the cell. Toxicity of high level of Boron applications to p lants has been well known. In order to analyze the temp erature –dependence of 190 accurately , differentiation of 190 with resp ect to temp erature (d190/dt) was accomp lished by comp uter p rogram. The temp erature – dep endent p lots of (d190/dt) with 2µm Boron and without Boron are depicted in fig 4. The p rofile without Boron consists of two bands that start at 50˚C and 73˚C characterist ic temp eratures.The p resence of 2µM Boron app ears to shift this p rofile toward the lower temp erature range by about 5˚C. Re ferences 1.Parr,A.J. and loughman, B.C., (1983) Boron and membrane function in p lants in (M etals and M icronutrients:uptake and utilization by p lants edi.by Robb,D.A.,and p ierp oint , W.S., Academic press. 2.Robertson,G.A., and Loughman,B.C., 1974 new p hytologist, 73: 821. 3.Tanada,T., 1974,p lant p hysiol., 53:775. 4.Chong,C. S.,and co lbow,R.A. 1976., Biochem.Biophys. Acta, 436:260. 5. Camp ell,I.D., and Dwek, R.A., 1984 Biological sp ectroscopy , Benjamin/cummings p ublishing comp any, Inc. 6.Pollard,A.S., parr,A.J., and lough man,B.C., 1977, j.exp .Bot.28:831. 7.Hirsch,A., and Torry ,T., 1980, can J.Bot. 58:856. 8.Wolfe,J.1978, p lant cell and environment 1:241. 9.Giese,A.C., 1979, cell p hy siology , fifth edition. 2009) 3( 22مجلة ابن الهیثم للعلوم الصرفة والتطبیقیة المجلد تأثیر عنصر البورون في تركیب غشاء البالسیدات الخضر لنبات القرنابیط باستعمال تقنیة تشتت الضوء ، رعد محسن مطر المولى، نبیل خلف العاني*هشام محمد علي القیسي قسم الفیزیاء، كلیة العلوم ،جامعة النهرین * قسم علوم الحیاة ، كلیة العلوم ،جامعة بغداد جامعة بغداد،قسم علوم الحیاة ، كلیة العلوم الخالصة تضمن البحث دراسة تأثیر عنصر البورون في بناء وعمل غشاء البالسیدات الخضراء المعزولة من نبات القرنابیط ء ) مایكرومول 5 – 0.1(اظهرت النتائج ان البورون بتراكیز . باستعمال تقنیة تشتت الضوء ادى الى سیولة دهون الغشا ادى الى تغیرات ) مایكومول 2(یبیة اوضحت ان تركیز البورون بدرجات متفاوتة فضال عن ذلك تم التوصل الى ادلة تجر .في هیئة بروتین الغشاء حفاظا على نفاذیة اغشیة البالسیدات Fig(1):Turbidity of cauliflower chl oroplast with different Boron concentration Fig2:Te mperature –dependent plot of light scattering intensity at angle of 90 degree (190) for caul iflower chl oroplast suspension (a)Without Boron (b) With 2µM Boron Fig( 3): Turbidity of lecithin liposomes containing different Boron concentration Fig 4: Te mperature –dependent plot of (d 190/d T) for caul iflower chl oroplast suspension (a) Without Boron (b) With 2µM Boron.