Analysis of the chlorophyll a fluorescence transient of Spirodela polyrhiza by JIP-test Hongtao Li1, Yu Zhao2, Ke Zhu1, Jinju Wang1,2, Qingdai Liu2, Hongjie An2,3 1Dept. of plant biology and ecology, Nankai University, Tianjin, China 300071 2Key Laboratory of Food Nutrition and Safety (Tianjin University of Science & Technology), Ministry of Education, Tianjin, China 300457 3Bio-X Center for Life Science and Technology, Shanghai Jiao Tong University, Shanghai, China 200030 Correspondence author: hjanloghotmail.com, htli2004126.com AbstractChlorophyll a fluorescence has been used as non-invasive probe for stress physiology and environmental investigation. The changes of photosynthetic apparatus in Spirodela polyrrhiza was studied by using chlorophyll a fluorescence technique and JIP-test. During L-serine-induced senescence, photosynthetic performance index (PI), reaction center (RC) decreased gradually and reached zero on the 8th day of cultivation. However, the oxygen evolving complex (OEC) remained nearly 60% and the capacity of Hill reaction remained about 43%. JIP-test also offers a platform to analyze the energy flux in fronds. Keywords- chlorophyll a fluorescence, JIP-test, Spirodela polyrhiza I. INTRODUCTION (HEADING 1) Chlorophyll a fluorescence techniques have been used for many years to investigate the effect of environmental factors on plants and become very useful in ecotoxicological studies 1,2. Recently, a method of measuring the fluorescence kinetics by means of direct, time-resolved fluorescence measurements has provided detailed information at even 10 s on fast fluorescence rise 3. The fluorescence transient is a biophysical signal that is extremely rich in terms of information and reflects the time course of photosynthesis. Although chlorophyll a fluorescence imaging offers the visualization, it is very important to make the links between the fluorescence signal and the behavior of the photosynthetic apparatus. The JIP-test based on the theory of energy fluxes in biomembranes has been used to analyze the chlorophyll a fluorescence kinetics transient widely helping us to have a deeper insight into the primary photochemical reaction in photosynthetic apparatus 4,5. When leaves are exposed to saturating actinic light the Chl a fluorescence curves start from the initial FO intensity (the origin, O) and increased to the highest intensity (P or FM). When the curve is plotted on logarithmic timescale, two intermediate steps in the fluorescence induction transient are observed, which are designated as the J step at approximately 2 ms and the I step at approximately 30 ms. The analysis of these intermediate data points forms the basis of JIP-test. Spirodela polyrhiza could serve as a water purifer since it accumulates three times the medium concentration into its cells 6. Spirodela polyrhiza is also often used as biosensors to test many pollutants in water 2,7. In previous work Spirodela polyrrhiza have been shown to be one of the suitable systems to study leaf senescence and metal pollution, when aseptically cultivated like microorganisms within laboratory under controlled conditions 8,9. By using this system we have investigated the effect of L-serine in medium on leaf senescence 10. In this study, we analyze the effect of L-serine on the photosynthetic apparatus by JIP-test. II. MATERIALS AND METHODS A. Experimenal Materials Spirodela polyrrhiza strain P143 plants were grown aseptically on a culture medium in flasks 9. All cultures of intact plants were kept under long-day conditions (16h light and 8h dark) with a light intensity of c.a. 45 molm-2s-1. Cultures were kept at 20-25oC during the light phase and 18-22oC during the dark phase. 1mM L-serine was added into the medium at the time as described in each experiment. Plants were collected at the time according to experimental design. B. Experimenal Methods Chl a fluorescence measurements Chlorophyll a fluorescence was measured by using the Handy Plant Efficiency Analyzer (Handy PEA built by Hansatech, Kings Lynn, Norfolk, England). Fronds were dark-acclimated for 12 min before measurements, as instructed by the manufacturer. Samples were exposed to a saturation light pulse of 1s at 100% actinic light (3000 molm-2s-1) to obtain the OJIP Chl a fluorescence rise of dark-adapted sample. All the data were digitized and stored for further analysis. At least 20 samples were measured for each treatment at a given time. JIP-test The fluorescence value at five defined times are extracted 978-1-4244-4713-8/10/$25.00 2010 IEEE50010001500200025003000350040000.010.11101001000Times(ms)Fluorescence Intensity (a.u.) from the trace data (T1= 50 s, T2= 100s, T3= (K step) 300 s, T4 = (J step) 2ms and T5 = (I step) 30ms). The maximum quantum yield of primary photochemistry (Po) and the efficiency with which a trapped exciton can move an electron into the electron transport chain further than QA (0) was calculated as follows11: TR0/ABS = Po = 1 (FO/FM) ET0/ABS =0 = (1 VJ), where VJ is variable fluorescence at 2ms. The estimation about ABS/CS, OEC, RC/CS (CS=cross section) and PI are calculated as follows: ABS/CS FM. ABS refers to the photon flux absorbed by the antenna. Fraction of Oxygen Evolving Complexes (%OEC) = 1-(VK/VJ) sample/1-(VK/VJ)Contral, where VK is variable fluorescence at 300s. RC/CS = FMPoVJ/M0, where Po = 1 (FO/FM) and M0 = 4 (F300s FO)/(FM FO) RC/CS is the active reaction center (RC) per leaf cross-section. PI = (RC/ABS) (Po/(1 Po) (0/(1 0), where 0 = (1 VJ). Data were analyzed by analysis of variance with the software Statistical Package for the Social Science (SPSS) version 13.0. Hill Reaction Chloroplasts were isolated and hill reaction activity was measured in terms of photoreduction of 2,6-dichlorophenol indophenol (DCPIP) as described by Holt and French 12 . The reaction mixture contained 0.003 % DCPIP and chloroplast suspension. The absorbance change of 600 nm was recorded after 5 min of exposure to light with a intensity of about 335mol m-2s-1. III. RESULTS AND DISCUSSION A. OJIP Transient The freshly fronds of S. polyrrhiza were used to measure the Chl a fluorescence and the results are shown in Fig. 1 as a typical OJIP transient. The OJIP transient of S. polyrrhiza changed remarkably during cultivation of the leaves on a medium with L-serine under long-day conditions. After 2 days of cultivation, the fluorescence intensity of the explants was lowered at all the J I P steps and P-step decreased to 76% of fresh fronds. The J step remained almost unchanged within 6 days of cultivation. After 5 days of cultivation, the J step began to decrease significantly. On the 6th day of cultivation, both the I and P steps disappeared, only a clear J band at about 2ms occurred. Figure 1. Chl a fluorescence transient (OJIP) of fresh leaves (C), leaves cultivated for 2 (S2), 4 (S4), 5 (S5) and 6 (S6) days on a medium with L-serine under long-day conditions, respectively. B. Electron Flow Energy fluxes for absorption (ABS), trapping (TR), and electron transport were the functional parameters of photosynthetic apparatus and the flux ratios were calculated by JIP-test. TR/ABS decreased slightly within 4 days of cultivation with L-serine, but a sharp decrease was observed thereafter (from 85% down to 57%) (fig. 2). However, the decrease of ET/TR was more dramatic than TR/ABS and down to 70% after one day of cultivation. The probability that an absorbed photon will move an electron into the electron transport chain, ET/TR was 11% on 8th day. The energy fluxes were affected by the L-serine induced senescence, and a correlation can be made with the partial destruction of the light-harvesting antennas organization and the consequent difficulty of funneling the excitation energy from the incoming photon to the reaction center (RC). The decrease in ET/TR confirmed that the trapped energy did not go to the RC and was dissipated. Damages within any part of photosynthetic apparatus will block the transfer of electrons to PSI and affect the photosynthesis of the leaves. 020406080100120012345678Times(days)Percentage(%)TR/ABSET/TR050100012345678ABSRCOECPI 02040608010012002468Times(days)Relative Activity oHill Reaction(%) Figure 2. TR/ABS and ET/TR of the energy flux during cultivation of leaves on a medium with L-serine. C. Photosynthetic Apparatus In order to understand more precisely the part of photosynthetic apparatus, the photosynthetic performance index (PI), the oxygen evolving complexes (OEC), reaction center (RC) and ABS of PSII were analyzed by JIP test (fig. 3). Photosynthetic performance index (PI) decreased gradually and reached zero on the 8th day of cultivation. The changing patterns of RC and ABS were basically similar to that of PI. However, OEC was kept at a high level (50%). Figure 3. Photosynthetic performance index (PI), OEC, RC and ABS of PSII during cultivation of leaves on a medium with L-serine by radar plot. During cultivation of fronds on the medium with L-serine the capacity of Hill reaction in chloroplasts decreased slightly. The capacity of Hill reaction remained about 43 % of the original (fig. 4). Figure 4. Changes in Hill reaction. K band at 300m was not found in fig1, which correlated with the damage of OEC 13. These data presented here suggest that the activity of OEC is less affected than the reaction center and ABS. Impressive advances in molecular biology have made access to the genome and structure of the photosynthetic organisms feasible, the JIP-test can help access the function and, thus, promote an understanding of the Structure-function relation. In practical terms, the JIP-test can be a handy tool in molecular breeding and transgenic strategies for crop improvement for stress tolerance. On the other hand, JIP-test may be used in environmental stress assessment. With JIP-test, it will be possible to trace the damage of photosynthetic apparatus and provide the information needed to develop strategies that would help to decrease the impact of environmental stress in agriculture 15. ACKNOWLEDGMENT This study was supported by 2006 NECT grant, the National Natural Science Foundation (No. 30800255, 30900339), and TUST Funds (20060424, 20060432, 20080216). REFERENCES 1 Clark AJ, Landolt W, Bucher JB, Strasser RJ. Beech (Fagus sylvatica L.) response to ozone exposure assessed with a chlorophyll a fluorescence performance index. Environ Pollut 109:501507 (2000). 2 Appenroth KJ, Stckel J, Srivastava A, Strasser RJ. Multiple effects of chromate on the photosynthetic apparatus of Spirodela polyrhiza as probed by OJIP chlorophyll a fluorescence measurements. Environ. Pollut 115:4964 (2001). 3 Strasser RJ, Srivastava A, Tsimilli-Michael. MScreening the Vitality and photosynthetic activity of plants by fluorescence transient. In: Behl RK, Punia MS, Lather BPS (eds) Crop Improvement for Food Security. SSARM, Hisar, India, pp 72115(1999). 4 Liu QD, Zhu YR, Tao HL, Wang NN, Wang Y. Damage of PS II during senescence of Spirodela polyrrhiza explants under long-day conditions and its prevention by 6-benzyladenine. J. Plant. Res. 119, 145-152(2006). 5 Bueno M, Fillat MF, Strasser RJ, Maldonado-Rodriguez R, Marina N, Smienk H, Gmez-Moreno C, Barja F. Effects of lindane on the photosynthetic apparatus of the cyanobacterium Anabaena. Environ Sci Pollut Res, 11:98-106 (2004). 6 Staves RP, Knaus RM. Chromium removal from water by three species of duckweeds. Aquat Bot 23: 261273(1985). 7 Kaszycki P, Gabrys H, Appenroth KJ, Jaglarz A, Sedziwy S, Walczak T, Koloczek H. Exogenously applied sulphate as a tool to investigate transport and reduction of chromate in the duckweed Spirodela polyrhiza. Plant Cell Environ, 28(2): 260 268(2005). 8 Susplugas S, Srivastava A, Strasser RJ. Changes in the photosynthetic activities during several stages of vegetative growth of Spirodela polyrhiza: effect of chromate. J Plant Physiol 157:503512(2000). 9 Li WM, Liu QD, Xiong Y, Wang SF, Wang NN, Wang Y. Significant role of cytokines in maintaining the life of fronds in Spirodela polyrhiza. J Plant Physiol Mol Biol 29 (3):215220(2003). 10 Zhu YR, Tao HL, Lv XY, Wang SF, Wang NN, Wang Y. High level of endogenous L-serine initiates senescence in Spirodela polyrrhiza. Plant Sci 166:11591166(2004). 11 Srivastava A, Guisse B, Greppin H, Strasser RJ. Regulation of antenna structure and electron transport in PS II of Pisum sativum under elevated temperature probed by the fast polyphasic chlorophyll a fluorescence transient: OKJIP. Biochim Biophy Acta 1320:95106(1997). 12 Holt AS, French CS. Oxygen production by illuminated chloroplasts suspended in solution of oxidants. Arch Biochem, 19:368-378(1948). 13 Srivastava A, Guisse B, Greppin H, Strasser RJ. Regulation of antenna structure and electron transport in PS II of Pisum sativum under elevated temperature probed by the fast polyphasic chlorophyll a fluorescence transient: OKJIP. Biochim Biophy Acta 1320:95106 (1997). 14 Maldonado-Rodriguez R, Pavlov S, Gonzalez A, Oukarroum A, Strasser RJ. Can machines recognise stress in plants? Environ Chem Lett, 1: 201-205 (2003).