CHLOROPHYLL CONTENT QUANTIFICATION IN ACCLIMATED IN VITRO PLUM PLANTS (PRUNUS DOMESTICA, L.) Zs. Jakab-Ilyefalvi, D. Pamfil

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1 Annals of RSCB Vol. XVI, Issue CHLOROPHYLL CONTENT QUANTIFICATION IN ACCLIMATED IN VITRO PLUM PLANTS (PRUNUS DOMESTICA, L.) Zs. Jakab-Ilyefalvi, D. Pamfil UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE, DEPARTMENT OF BIOTECHNOLOGY, CLUJ-NAPOCA Summary The apparition of the symptoms caused by the plum pox virus through formation of mosaic surfaces and necrotic zones at the stone fruits are known as reduction factors of the chlorophyll content and influences negatively the physiological factors inclusively the affection of the metabolic processes in the accumulating of sugar content. Spectroscopy results revealed the fact that the chlorophyll a content is higher in the case of healthy plants, this varying from 7. mg/l to the quantity.93 mg/l in comparison with the infected plants at which the chlorophyll a content was at a lower level. The content in chlorophyll a of the infected leaves varied between. mg/l respectively.38 mg/l, concluding the fact that the chlorophyll content was significantly reduced regarding the analyzed in vitro plant leaves. The analyzed leaves presented chlorothic bands, circles, and the reduction of the chlorophyll content cannot due because of the physiological stadium of leaves, the in vitro plantlets have presented a high juvenile stadium being regenerated by meristematic tissue culture technique. The low chlorophyll content was probable due to alterations in the biosynthesis of chlorophylls. Similar phenomena were observed in the case of the chlorophyll b content analysis at the healthy and infected plum leaves. According the analysis the chlorophyll b content varied mg/l at the healthy in vitro plants. In the case of the infected in vitro plants the chlorophyll b content varied between,5 3. mg/l. Results show that the total chlorophyll content in the plum leaves had lower values in the case of infected samples, comparative with the healthy ones. Key words: chlorophyll, plum, leaves, plum pox virus zsolt.jakab@yahoo.com Introduction The apparition of the symptoms caused by the plum pox virus through formation of mosaic surfaces and necrotic zones at the stone fruits are known as reduction factors of the chlorophyll content and influences negatively the physiological factors inclusively the affection of the metabolic processes in the accumulating of sugar content (Bulgaru and Isac, 988). Many studies showed the fact that the whole photosynthetic and metabolic system can be affected in the case of severe 55 infection with PPV, influencing thus the main vital processes of the plants ( Zhang, et al,, Wang, et al., 7). The complex photosynthetic process also in greenhouse conditions are open field conditions are influenced by the degradation of chlorophylls, thus the quantification of the exact chlorophyll content of the two types of chlorophyll a and b can be used in the characterization of the photosynthetic capacity of the plants. The photo-inhibition in additional way is associated but not necessary accompanied by the degradation of the chlorophylls

2 Annals of RSCB Vol. XVI, Issue (Critchley, 998). The study effectuated by Baumgartnerova and Slovakova, 998, regarding the chlorophyll containment and the viral concentration of PPV particles have concluded the fact that in the symptomatic leaves of apricot the assimilatory pigment containment was affected by the plum pox viral infection. The affected leaves presented a lower level of chlorophylls and the carotene content was higher than those used for control. Material and methods In order to elucidate the a and b chlorophyll amount from the in vitro plum plants at the cultivars Ivan, Iulia, Geta, Jubileu 5, it has been proceeded to the extraction and determination of chlorophylls after the method described by Arnon, (98), and Baumgartnerova, (998). Micropropagated in vitro plantlets were labeled and numbered starting with the acclimation process. There have been effectuated serological and molecular tests in order to confirm the presence of the PPV virus. Healthy and PPV infected leaves were sampled according to the effectuated tests. Leaves were put in zip bags and introduced in a refrigerating box and transported to the Biochemistry Department of USAMV Cluj-Napoca where took place the extraction and determination of chlorophyll content. Samples were weighted,. g for every individual sample. Refrigerated 8 % cc. acetone was used in order to rive the protein-chlorophyll complexes and to extract the chlorophylls. The vegetal material was introduced in a grinding mortar and 8 % cc. acetone was used for extraction. The chlorophyll extract were put inb Falcon tubes and introduced in an ultrasonic cleaner (Tellsonic, TPC-5) for 5 minutes. Fig. - Preparing of vegetal samples Fig.. Weighing of the leaf samples Fig. 3 Extraction of chlorophylls a+b in 8 % acetone Fig. Centrifugation of the plant extract at 7 rpm 5

3 Annals of RSCB Vol. XVI, Issue Fig.5 Chlorophyll extracts ready for UV-VIS spectroscopy The homogenate was centrifuged at 7 rpm, decanted, and procedure was repeated once again. The final volume was measured for every sample in order to effectuate the final chlorophyll concentration. The quantification of the chlorophyll content was effectuated based on the absorbance registered by UV VIS spectroscopy tehnique. Based by the formula published by Arnon, (98) subsequently there were effectuated the exact chlorophyll content calculations. Total chlorophyll (µg/ml) =. (A 5 ) + 8. (A 3 ) Chlorophyll a (µg/ml) =.7 (A 3 ) -.9 (A 5 ) Chlorophyll b (µg/ml) =.9 (A 5 ) -.8 (A 3 ) Sample number acclimated plant TABLE I. Determination of the optical density by spectroscopy of the chlorophyll extracts Sample Phytoviral No. Status A 3 A 5 A 33 A 5 8 Healthy,89,3,9398, Healthy,98,7,75, Healthy,98,397,395, Healthy,87,87,7, Healthy,8,97,938,355 7 Infected,535,98,5937,87 Infected,3857,598,97, Infected,3,9,85,898 5 Infected,3355,38,,773 3 Infected,555,93,793,58 3 Infected,337,8,8, Healthy,977,87,83, Infected,,5,778, 89 8 Healthy,75,93,5558,85 8 Healthy,3333,95,3987, Healthy,9,7,35,58 57

4 Annals of RSCB Vol. XVI, Issue Results and discussion There have been analyzed the individual content of a chlorophylls respectively b type chlorophylls at both of the plant categories also at the healthy ones and those infected with plum pox virus. The major importance in the light assimilation process is due to chlorophyll a due to the fact that these are the main responsible for the satisfactory functioning of the PS II photo-system in natural light conditions. The b type chlorophylls the main components of the proteins which collect LHCP light and the content of this pigment in the leaves is important for the capacity of the leave to accommodate in shadow conditions (conditions typical in greenhouse). Comparative analyses of chlorophyll "a" in plum leaves (Prunus domestica,l.) 9 8 mg/ L Healthy leaves Infected leaves Fig. Comparative analysis of the chlorophyll a content in the analyzed samples Comparative analyses of chlorophyll "b" in plum leaves (Prunus domestica,l.) mg/ L Healthy leaves Infected leaves Fig. 7. Comparative analysis of the chlorophyll b content in the analyzed samples Analyzing the fig. it is revealed the fact that the chlorophyll a content is higher in the case of healthy plants, this varying from 7. mg/l to the quantity.93 mg/l in comparison with the infected plants at which the chlorophyll a content was at a lower level. The content in chlorophyll a of the infected leaves varied 58 between. mg/l respectively.38 mg/l, concluding the fact that the chlorophyll content was significantly reduced regarding the analyzed in vitro plant leaves. The analyzed leaves presented chlorothic bands, mosaicated circles, and the reduction of the chlorophyll content cannot due because of the leaves sencescence, the in vitro plantlets

5 Annals of RSCB Vol. XVI, Issue have presented a high juvenile stadium being regenerated by meristematic tissue culture technique. The low chlorophyll content was probable due to alterations in the biosynthesis of chlorophylls. Similar phenomena was observed in the case of the chlorophyll b content analysis at the healthy and infected plum leaves ( Fig. 7). According the analysis the chlorophyll b content varied mg/l at the healthy in vitro plants. In the case of the infected in vitro plants the chlorophyll b content varied between,5 3. mg/l. A lower value of chlorophylls can be influenced by an excess of irradiation of these pigments at the plants grown in open field condition (Jason et al., ). The analysis of the absorbance spectra. The chlorophyll molecules are components in different photo-systems integrated in the interior of the tylacoid membranes of the chloroplasts. The chlorophylls are absorbing the most intense light from the blue region of the electromagnetic spectra of light, followed by the red region, but they absorb very weak in the green region, from this derives the fact that the vegetal tissues containing the chlorophylls are becoming the green color after this pigment. The absorption of the chlorophylls are between the -7 nm, and this region is named the photosynthetically active region (PAR). sursa: Fig. 8 Spectral absorbance of chlorophylls in the visible light Chlorophyll a molecules extracted in acetone in our experiences have absorbed at optical densities of A=3nm respectively A=3 nm, and the chlorophyll b at optical densities of A=5 nm and A=5 nm for the two extremities of the spectra. Proba nr. - Nediluata 5 7 Fig. 9 of the chlorophyll extract, plum cultivar Ivan, healthy plant 59

6 Annals of RSCB Vol. XVI, Issue In fig.9 it is shown the absorption spectra of the sample no., the specific absorption at the level of chlorophylls having a value of.89 OD at the maximum value of A=3 nm respectively.57 OD at the maximum value of A=33. This sample has been diluted to an appropriate reading at the maximum value of A=33 nm obtaining optimal values Proba nr. - Nediluata which could be interpreted properly obtaining the value,3333 at the maximum value of A=33 nm respectively,38 at the maximum value of A=33 nm. The specific absorbance at the chlorophyll b at the absorbance maximum of A=5 nm has presented the values,3 at the absorbance of A=5 nm the optical density was,9398. at the maxima of A=33 nm presented the value of,598 DO and the A=33 nm the optical density was,97. As shown above in our experiences, the total chlorophyll content in the plum leaves had lower values in the case of infected samples, comparative with the healthy ones. 5 7 Comparison between the absorbance of chlorophyll extracts of healthy / infected plum leaves Fig. of the chlorophyll extract, plum cultivar Iulia, infected plant In the case of sample no. the registred specific absorbance had the value,3857 OD at the A=33 nm point respectively,7397 OD at the maxima of A=33 nm at the chlorophyll a level. The specific absorbance at the chlorophyll b The comparative analysis of the spectra of the two plant categories reveals the fact that there are notable differences between the absorbance of the chlorophyll content of healthy and infected plants, in the frame of the same cultivar Geta there being differences between the infected plants and healthy plants. Proba nr. - Nediluat Proba nr. 8 - Nediluat Proba nr. 5 - Nediluata Proba nr. 9 - Nediluata Fig. Comparations between the absorbance of healthy and infected samples at cultivar Geta The spectral curve of the healthy plants where noted with red color and the spectra of the infected leaves were noted with black color, being revealed the existing differences. The symptoms of healthy leaves without the mosaic shaped

7 Annals of RSCB Vol. XVI, Issue surfaces on the leaves have revealed higher levels of chlorophyll content. According to the effectuated studies, we consider that among other factors which contributes to the decrease of the chlorophyll content, the incidence of the plum pox virus influences the biosynthesis and metabolic system of chlorophylls, experiences confirmed by other researches (Zhang, at al., ). References Arnon, D.I., Copper enzymes in isolated chloroplast, polyphenoloxidase in beta vulgaris, Plant physiology, Vol., Number, -, 99. Baumgartnerova, H., Slovakova, L., Petrusova, N., Relationship between concentration of plum pox virus and content of pigments in virusinfected symptomatic apricot leaves, Acta Virol., ():-8, 998. Critchley, C., Photoinhibition in photosynthesis, a comprehensive treatise, Cambridge University Press, Cambridge, Jason, J.G., Thomas and D.M. Pharr, Photosynthesis, chlorophyll florescence, and carbohydrate content of illicium taxa grown under varied irradiance, J. Am. Soc. Hort. Sci., 9:-53,. Hong, W., Falin Wang, Gang Wang, Khalid Majourhat, The responses of photosynthetic capacity, chlorophyll fluorescence and chlorophyll content of nectarine ( Prunus persica var Nectarina Maxim) to greenhouse and field grown conditions, Scientia Horticulturae, :-7, 7. Bulgaru, L., Isac, M. Some biochemical changes induced by plumn pox in plum, Acta Horticulturae,Vol.35,5-9, 988. Zhang, Y., Simeone, A. M., Cappelini, P., Physiological modifications caused by plum pox virus in the leaves of peach and apricot tree, Agris, ISMEA (Italy), 39(3), p. 7-5,.