CC BY
96
УДК 581.132
I. S. Zulfugarov1, A. Pashayeva2, Zh. M. Okhlopkova3, Choon-Hwan Lee1
Practical Guide to Measure Chlorophyll Fluorescence in Plants and Calculate Main Chlorophyll Fluorescence Parameters
'Pusan National University, Korea 2Azerbaijan National Academy of Sciences, Azerbaijan 3M.K. Ammosov North-Eastern Federal University, Yakutsk, Russia
Abstract. Chlorophyll fluorescence is one of the most widely used and powerful techniques in many scientific research areas, because it is a non-destructive intrinsic probe of several aspects of oxygenic photosynthesis. In leaf-absorbed light energy can undergo one of three outcomes: 1) it can be used in photosynthesis; 2) it can be dissipated in heat (nonphotochemical quenching - NPQ); and 3) it can be released as fluorescence. Because all these processes occur in competition, by measuring chlorophyll fluorescence yield, we can get information about efficiency of photosynthesis and NPQ. In this research we provide introduction to the principle, methodology and application of chlorophyll fluorescence analysis. Although the measurement of chlorophyll fluorescence is simple, the underlying theory and the data interpretation remain complex and sometimes controversial. We will introduce several parameters derived from the saturation pulse-induced chlorophyll fluorescence analysis that can provide information on various processes on photosynthesis. To accurately estimate electron transport rate and NPQ, as well as several other important parameters the maximum fluorescence yield, called Fm, must be known. Estimation of the Fm was achieved using short and high intensities flashes of light termed 'saturation flashes'. High intensity flashes are required because in response to progressively increasing light intensity the yield of fluorescence increases hyperbolically. Therefore, the estimation of the maximum fluorescence yield when measured in an illuminated leaf (Fm), requires the use of extreme light intensities never encountered by plants in nature. The experimental conditions should be selected very carefully.
ZULFUGAROV Ismayil Sokhbatovich - PhD, Researcher Professor, Department of Molecular Biology, Pusan National University, Busan 46241, Korea; Department of Biology, M.K. Ammosov North-Eastern Federal University.
E-mail: Iszulfugarov@pusan.ac.kr
ЗУЛЬФУГАРОВ Исмаил Сохбатович - профессор-исследователь кафедры молекулярной биологии Национального университета Пусана, Пусан 46241, Корея; кафедра биологии ИЕН СВФУ им. М.К. Аммосова.
PASHAYEVA Aynura - PhD student, Institute of Molecular Biology and Biotechnology, Azerbaijan National Academy of Sciences, Azerbaijan .
ПАШАЕВА Айнура - аспирант Института молекулярной биологии и биотехнологии Национальной академии наук Азербайджана, Азербайджан.
OKHLOPKOVA Zhanna Mikhailovna - Candidate of Biological Sciences, Associate Professor, Head and Lead Researcher of the "Molecular-genetic and cell technologies" Laboratory of the Department of Biology, Institute of Natural Sciences, M.K. Ammosov North-Eastern Federal University.
E-mail: zhm.okhlopkova@s-vfu.ru
ОХЛОПКОВА Жанна Михайловна - к. б. н., доцент, научный руководитель и ведущий научный сотрудник лаборатории «Молекулярно-генетические и клеточные технологии» ИЕН СВФУ им. М.К. Аммосова.
CHOON-HWAN Lee - PhD, Professor, Head of the Laboratory of Molecular Biology, Department of Molecular Biology, Pusan National University, Busan 46241, Korea.
E-mail: chlee@pusan.ac.kr
ЧУН-ХВАН Ли - PhD, профессор, научный руководитель лаборатории молекулярной биологии факультета молекулярной биологии Национального университета Пусана, Пусан 46241, Корея.
Keywords: Chlorophyll fluorescence parameters, Photosynthetically active radiation, Photosynthesis, Photochemical quenching, Nonphotochemical quenching, Electron transport rate, Maximum quantum yield, Quantum yield of PSII, Variable fluorescence, Coefficient of photochemical quenching assuming interconnected.
Acknowledgments
This research was partially supported by a grant from the Next-Generation Biogreen 21 Program (SSAC, Grant No. PJ013155012018), Rural Development Administration, Republic of Korea and by the Science Development Foundation under the President of the Republic of Azerbaijan - Grant No. EIF-KETPL-2-2015-1(25) -56/35/3.
DOI 10.25587/SVFU.2018.64.12129
Исмаил С. Зульфугаров', Айнура Пашаева2, Жанна М. Охлопкова3, Чун-Хван Ли'
Практическое руководство по измерению уровня флуоресценции хлорофила в растениях и расчету основных параметров флуороесценции хлорофила
'Национальный университет Пусана, Корея 2Национальная академия наук Азербайджана, Азербайджан 3СВФУ им. М.К. Аммосова, Якутск, Россия
Аннотация. Флуоресценция хлорофилла - один из наиболее широко применяемых и эффективных методов, используемых во многих областях научных исследований, т. к. она представляет собой неразрушающий объективный способ забора проб для изучения некоторых аспектов кислородного фотосинтеза. Поглощаемая листьями световая энергия может подвергнуться одному из трех процессов: 1) она может быть использована при фотосинтезе; 2) она может рассеяться вместе с теплом (нехимическое охлаждение - НХО); 3) она может высвободиться в виде флуоресценции. Так как все эти процессы носят конкурентный характер, измерение выхода флуоресценции хлорофилла позволит получить информацию об эффективности фотосинтеза и НхО. В рамках данного исследования мы знакомим читателей с принципом, методологией и способами применения анализа флуоресценции хлорофилла. Процедура измерения флуоресценции хлорофилла несложна, однако в ее основе теория, а также метод обработки данных по-прежнему носят сложный, а зачастую противоречивый характер. Мы представим несколько параметров, полученных при помощи анализа насыщения импульсной флуоресценции хлорофилла, которые могут дать дополнительную информацию относительно различных процессов фотосинтеза. Для точного расчета скорости переноса электронов и НХО, а также нескольких других важных параметров необходимо знать уровень максимального выхода флуоресценции, обозначенного Фм. Подсчет Фм был выполнен путем применения коротких интенсивных вспышек света, обозначенных как «вспышки насыщения». Вспышки должны быть высокой интенсивности, т. к. реакцией на последовательное усиление интенсивности света является гиперболическое увеличение выхода флуоресценции. Таким образом, подсчет максимального выхода флуоресценции при замере на освещенном листе (Фм) требует применения экстремальной световой интенсивности, с которой в естественных условиях растения не сталкиваются. Поэтому необходимо тщательнейшим образом соблюдать условия проведения эксперимента.
Ключевые слова: параметры флуоресценции хлорофилла, фотосинтетически активная радиация, фотосинтез, фотохимическое охлаждение, нефотохимическое охлаждение, скорость переноса электронов, максимальный объем выхода, объем выхода PSII, переменная флуоресценция, коэффициент фотохимического охлаждения при условии взаимосвязи.
Introduction
When photosynthetic tissues/organisms illuminated by the light of approximately 400-700 nm photosynthetically active radiation (PAR), chlorophylls are emitting red to far-red light known as fluorescence (McCree 1972).
Chlorophyll fluorescence parameters and techniques for measuring chlorophyll fluorescence are used extensively in many areas of fundamental and applied research. Chlorophyll fluorescence is used to study the mechanism of electron transport between and elsewhere within the two photosystems of photosynthetic organisms and light harvesting in fundamental and applied investigations in order to analyze the effects of biotic and abiotic stresses on the photosynthetic efficiency (Papageorgiou and Govindjee, 2004). Chlorophyll fluorescence is an advantageous tool for experimental research in many scientific areas including agriculture, forestry, marine science, material science, remote sensing, and others. Table 1 shows top 50 scientific research areas where chlorophyll fluorescence is applied.
Table 1
Distribution of the published articles about chlorophyll fluorescence in research areas
Research Areas Published articles % of 20.255
PLANT SCIENCES 8.829 43,56
ENVIRONMENTAL SCIENCES ECOLOGY 2.654 13,10
BIOCHEMISTRY MOLECULAR BIOLOGY 2.622 12,95
AGRICULTURE 1.979 9,77
MARINE FRESHWATER BIOLOGY 1.920 9,48
BIOPHYSICS 1.597 7,88
CHEMISTRY 1.160 5,73
OCEANOGRAPHY 1.135 5,60
FORESTRY 673 3,32
SCIENCE TECHNOLOGY OTHER TOPICS 607 3,00
CELL BIOLOGY 547 2,70
BIOTECHNOLOGY APPLIED MICROBIOLOGY 541 2,67
REMOTE SENSING 391 1,93
LIFE SCIENCES BIOMEDICINE OTHER TOPICS 330 1,63
TOXICOLOGY 330 1,63
PHYSICS 314 1,55
IMAGING SCIENCE PHOTOGRAPHIC TECHNOLOGY 313 1,55
FOOD SCIENCE TECHNOLOGY 295 1,46
MICROBIOLOGY 286 1,41
ENGINEERING 282 1,39
GEOLOGY 242 1,20
OPTICS 215 1,06
WATER RESOURCES 179 0,88
SPECTROSCOPY 146 0,72
BIODIVERSITY CONSERVATION 145 0,72
METEOROLOGY ATMOSPHERIC SCIENCES 144 0,71
PHARMACOLOGY PHARMACY 128 0,63
MATERIALS SCIENCE 118 0,58
INSTRUMENTS INSTRUMENTATION 110 0,54
ENTOMOLOGY 109 0,54
PHYSIOLOGY 89 0,44
ELECTROCHEMISTRY 79 0,39
FISHERIES 66 0,33
GENETICS HEREDITY 66 0,33
ENERGY FUELS 60 0,30
GEOCHEMISTRY GEOPHYSICS 49 0,24
PHYSICAL GEOGRAPHY 49 0,24
MYCOLOGY 41 0,20
COMPUTER SCIENCE 39 0,19
MATHEMATICAL COMPUTATIONAL BIOLOGY 36 0,18
ASTRONOMY ASTROPHYSICS 30 0,15
ENDOCRINOLOGY METABOLISM 30 0,15
EVOLUTIONARY BIOLOGY 30 0,15
ZOOLOGY 28 0,14
NUTRITION DIETETICS 23 0,11
RADIOLOGY NUCLEAR MEDICINE MEDICAL IMAGING 22 0,11
URBAN STUDIES 21 0,10
PUBLIC ENVIRONMENTAL OCCUPATIONAL HEALTH 20 0,10
EXPERIMENTAL MEDICINE 19 0,09
DEVELOPMENTAL BIOLOGY 16 0,08
Web of Science (WoS) search (https://apps.webofknowledge.com/WOS_GeneralSearch_input. do?product=WOS&search_mode=GeneralSearch&SID=C5GwWDWIk7ZNXr5bo3r&preferenc esSaved=) resulted in 20.255 published articles. The analysis of the published articles per year starting from 1971 shows that during the last two decades chlorophyll fluorescence was used in scientific research quite extensively (Figure 1). Over the last decades chlorophyll fluorescence parameters and techniques were used very extensively in the scientific literature, but only few as measuring protocols. For this article we prepared a protocol for measuring chlorophyll fluorescence parameters to assess and quantify the photosynthesis in crop plants.
Principle
In leaf-absorbed light energy can undergo one of three outcomes: 1) it can be used for photosynthesis; 2) it can be dissipated as heat (nonphotochemical quenching - NPQ); and 3) it can be released as fluorescence. Because all these processes occur in competition, by measuring chlorophyll fluorescence yield, we can get information about efficiency of photosynthesis and NPQ. The most chlorophyll fluorescence comes from photosystem II (PSII). The plastoquinone bound to QA binding site of D2 protein of PSII, called QA, is the first electron acceptor of PSII. In dark-adapted leaves, all QAs are in oxidized state or all the PSII reaction centers are in open state, and the thermal dissipation of the light energy absorbed by chlorophyll
Fig. 1. The extensive growth of the numbers of published articles which used chlorophyll fluorescence
is minimal. Application of weak light that does not significantly reduce QA allows finding the minimal fluorescence of the system Fo. This low fluorescence is indicative of the energy inevitably lost by the system by the competitive pathway of energy dissipation.
Upon application of a saturating flash on dark-adapted leaves, fluorescence rises from basal level (Fo) to maximum value (Fm). Under this condition, all QAs are fully reduced and then photochemistry is blocked, or all the PSII reaction centers come to a closed state. Upon application of a saturating flash on illuminated leaves, fluorescence rises from a basal level (Fo') to a maximum value (Fm'). Upon the onset of photochemical and non-photochemical process, the fluorescence yield is quenched, and the varying fluorescence yield - the steady state yield of fluorescence in the light is called Ft.
These parameters are measurable parameters, and we show some calculated chlorophyll fluorescence parameters in Table 2. Figure 2 shows typical chlorophyll fluorescence transient measured using a rice leaf.
Table 2
Basic fluorescence parameters
Name Description Equation Note (Reference)
Fv Variable fluorescence Fv = Fm — Fo Maxwell and Johnson, 2000
Fv/Fm Maximum quantum yield or maximum photosynthetic efficiency of PSII Fm - Fo Fm Oxborough and Baker, 1997
°PSn or Y(II) Quantum yield of PSII Fm ' - Ft Fm Genty et al., 1989
qP Photochemical quenching or proportion of open PSII Fm' — Ft Fm — Fo' Busch et al, 2009
qL Coefficient of photochemical quenching assuming interconnected PSII antenna qP x- F ' 1 or Fm' - Ft Fo' -x- Fm'- Fo' F' Kramer et al., 2004
qN Non-photochemical quenching Fm — Fm Fm — Fo Maxwell and Johnson, 2000
NPQ Non-photochemical quenching f Fm \ 1 V Fm J Maxwell and Johnson, 2000
ETR Electron transport rate (AF ) V Fm j A x PAR x 0.5 x 0.84 F = Fm- Ft Zulfugarov et al., 2014
Y(NO) Quantum yield of non-light induced non-photochemical quenching Y(NO) = [NPQ +1+ qL x(Fm/ Fo-1)] or Kramer et al., 2004
Y(NPQ) Quantum yield of light-induced (D Y(NPQ) =1 - Y(II) - Y(NO) Kramer et al., 2004
The parameters, Fo, Fo', Ft, Fm and Fm', are measurable and they are introduced in the text. Photosynthetically active radiation (PAR) is - the amount of light available for
photosynthesis.
0 5 10 15 20 25
Time {min)
Fig. 2. A typical chlorophyll fluorescence transient showing basic principle of chlorophyll fluorescence measurement
Fig. 3. A photograph of the PAM-2000 (Walz Co., Germany)
Materials and Reagents
One-month-old wild-type and PsbS-KO mutant seedlings of rice (Oryza sativa L.) were grown in rice soil (pH 4.5-5.5; Samhwa Greentech, Seoul, Republic of Korea) in a greenhouse under natural sunlight. Growth conditions included a 16-h photoperiod and temperatures of 28/22 ± 2 °C (day/night). PsbS-KO mutant leaves are lacking the qE component of the NPQ. We usually use 2 cm long middle part of the 2nd leaves.
Equipment
Modulated chlorophyll fluorimeters (for example, PAM2000 shown in Figure 3; PAM101/102/103; PAM-100; Imaging PAM, Walz Co., Germany).
Actinic and saturation pulse light sources (such as Schott KL1500, Mainz, Germany). Imaging PAM is using a blue light source, and all other instruments use white light sources. But, colors of the light sources can be white, red or blue, because they do not interfere with measurements.
Procedure
A. Measurement of the chlorophyll fluorescence parameters
1. Turn on the instrument.
2. Put dark-adapted (Note 1) leaf segments (Note 2) or whole plants in a leaf (plant) chamber.
3. Cover the chamber with a black curtain.
4. Turn on measurement (modulated) light to detect Fo level.
5. Wait 10 s and then apply saturation pulse (0.8 sec, 5.000 ^mol photons m-2 s-1) (Note 3) to detect Fm level.
6. Wait 10 to 60 s and then apply actinic light (100~1.000 ^mol photons m-2 s-1) (Note 4) to detect Ft level.
7. Apply a saturation pulse (0.8 sec, 5.000 ^mol photons m-2 s-1) every 10 to 30 sec to detect Fm' levels.
8. After 5 to 10 min illumination, turn off the actinic light, and apply saturation pulses to monitor dark recovery processes of NPQ.
9. Use the detected (measured) chlorophyll fluorescence units to calculate specific parameters such as Fv/Fm, ®PSII, qP, NPQ using equations shown in Table 1.
10. Repeat the same experiment with other leaf segment or whole plant at least 4-5 times.
11. Calculate standard deviation or standard error.
12. Draw the graph for Fv/Fm, ®PSII, qP, NPQ or prepare a table.
Fig. 4. Light response curves for electron transport rates
B. Recording light curve of chlorophyll fluorescence to measure the electron transport rate
1. Turn on the instrument;
2. Put dark adapted leaf segment or whole plant in a leaf (plant) chamber;
3. Cover chamber with black curtain;
4. Turn on measurement (modulated) light to detect Fo level;
5. Wait 10 s and then apply saturation pulse (0.8 s, 5.000 ^mol photons m-2 s-1) to detect Fm level;
6. Apply actinic light (Note 5) for 30 sec (25-50* ^mol photons m-2 s-1) after than apply saturation pulse (0.8 s, 5.000 ^mol photons m-2 s-1) to detect Fm' levels;
7. Turn off actinic light and keep in the dark (under measurement light) for 30 s;
8. Again apply actinic light for 30 s (50-100* ^mol photons m-2 s-1) after than apply saturation pulse (0.8 s, 5.000 ^mol photons m-2 s-1);
9. Turn off actinic light and keep in the dark (under measurement light) for 30 s;
10. Repeat the steps 7 and 8 with higher step-by-step increasing light intensities (up to 2.500 ^mol photons m-2 s-1);
11. Use the detected (measured) chlorophyll fluorescence units to calculate electron transport rate as well as to calculate light curves for qP and NPQ.
12. Use the subsequence equations from Table 1.
13. Repeat the same experiment with other leaf segment or whole plant at least 4-5 times.
14. Calculate standard deviation or standard error.
15. Draw the graph for ETR, NPQ and qP.
Representative data
The examples of the electron transport rate measurements result are shown in Figure 4. Figure 5 shows light curve (Note 6) for development of non-photochemical quenching.
Leaves of 1-month-old seedlings of wild-type and NPQ-less PsbS-Knockout plant leaves grown in greenhouse were dark-adapted for 10 min before measurements. Photosynthetically active radiation of 0, 70, 110, 180, 250, 400, 550, 900, 1.400 or 2.200 ^mol photons m-2 s-1 was then applied for 10 min. Each point represents mean of at least 4 experiments (SD indicated by bar).
Leaves of 1-month-old seedlings of wild-type and NPQ-less PsbS-Knockout plant grown in greenhouse were dark-adapted for 10 min before measurements. Photosynthetically active radiation of 0, 70, 110, 180, 250, 400, 550, 900, 1.400 or 2.200 ^mol photons m-2 s-1 was then applied for 10 min. Each point represents mean of at least 4 experiments (SD indicated by bar).
Fig. 5. Light response curves for NPQ
Notes
Dark adaptation period could range from minimum 10 min up to several hours depending on experiment purpose.
Please be sure that for fluorescence detection you are using upper surface of the rice leaf.
Intensity of saturation pulse may be less if the pulse gives the same Fm.
Actinic light intensity can be varied depending on plant species, particular growth conditions and physiological status of the sample as well as depending on the experiment purpose.
Please note that almost all chlorophyll fluorimeters used automatic step-by-step increasing light intensities.
Depending on plant species, its particular growth conditions and physiological state the same light intensity may be more or less excessive. Additional information on the photosynthetic performance of a leaf can be obtained with the help of light curves. While induction curves normally are measured with dark-adapted samples, light curves preferentially are measured with pre-illuminated samples, e.g. after an induction curve recording. Light response curves provide valuable information on the efficiency with which photosynthetically active radiation is used by plants. It provides detailed information on electron transport capacity and limitations of the two photosystems.
R e f e r e n c e s
1. Busch F., Hunter N. P. A. and Ensminger I. (2009). Biochemical constrains limit the potential of the photochemical reflectance index as a predictor of effective quantum efficiency of photosynthesis during the winter spring transition in Jack pine seedlings. Funct Plant Biol 36: 1016-1026.
2. Genty B., Briantais J. M. and Baker N. R. (1989). The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990: 87-92.
3. Kramer D. M., Johnson G., Kiirats O., Edwards G. E. (2004). New fluorescence parameters for the determination of QA redox state and excitation energy fluxes. Photosynth Res 79: 209-218.
4. McCree K. J. (1972). The action spectrum, absorptance and quantum yield of photosynthesis in crop plants. Agric Meteorol 9: 191-216.
5. Maxwell K. and Johnson G. N. (2000). Chlorophyll Fluorescence - a practical guide. J Expt Bot 51: 659-668.
6. Oxborough K. and Baker N. R. (1997). Resolving chlorophyll a fluorescence images of photosynthetic
efficiency into photochemical and non-photochemical components - calculation of qP and Fv'/Fm' without measuring Fo'. Photosynth Res 54: 135-142.
7. Papageorgiou G. C., Govindjee (eds), 2004. Chl a Fluorescence: a signature of photosynthesis, advances in photosynthesis and respiration, vol 19. Springer, Dordrecht.
8. Walz company instruction manual for Junior PAM-100. http://www.walz.com/downloads/manuals/ junior-pam/jpm_071206.pdf.
9. Web of Science (https://www.webofknowledge.com/)
10. Zulfugarov I. S., Tovuu A. Eu. Y. J., Dogsom B., Poudyal R. S., Nath K., Hall M., Banerjee M., Yoon U. C., Moon Y. H., An G., Jansson S. and Lee C.-H. (2014). Production of superoxide from Photosystem II in a rice (Oryza sativa L.) mutant lacking PsbS. BMC Plant Biol 14: 242.
Л и т е р а т у р а
1. Буш Ф., Хантер Н. П. А. и Энсмингер И. (2009) Биохимические рамки ограничивают потенциал индекса фотохимического отражения в качестве предиктора эффективности фотосинтеза во время зимне-весеннего перехода у саженцев черной сосны. Funct Plant Biol 36: 1016-1026.
2. Генти Б., Брианте Ж. М. и Бейкер Н. Р. (1989). Связь между объемом выхода фотосинтетического переноса электронов и охлаждением флуоресценции хлорофилла. Biochim Biophys Acta 990: 87-92.
3. Крамер Д. М., Джонсон Г., Кииратс О., Эдвардс Г. Е. (2004). Новые параметры флуоресценции для определения состояния окислительно-восстановительного потенциала и изменений возбужденной энергии. Photosynth Res 79: 209-218.
4. МакКри К. Дж. (1972). Спектр действия, уровень поглощения и объем выхода фотосинтеза у сельскохозяйственных культур. Agric Meteorol 9: 191-216.
5. Максвелл К. и Джонсон Г. Н. (2000). Флуоресценция хлорофилла - практическое руководство. J Expt Bot 51: 659-668.
6. Оксборо К. и Бейкер Н. Р. (1997). Анализ фотосинтетической эффективности примеров флуоресценции хлорофилла в фотохимических и не-фотохимических компонентах - расчет qP и Fv'/ Fm' без измерения Фо. Photosynth Res 54: 135-142.
7. Папагеоргиу Г. С., Говинджи (ред.), 2004. Флуоресценция хлорофилла: сигнатура фотосинтеза, развитие фотосинтеза и дыхания, том 19. Шпрингер, Дордрехт.
8. Руководство компании Waltz для Junior PAM-100. http://www.walz.com/downloads/manuals/junior-pam/jpm_071206.pdf.
10. Web of Science (https://www.webofknowledge.com/)
11. Зульфугаров И. С., Товуу А., Ю. й. Дж. Догсом Б., Пудял Р. С., Нат К., Холл М., Банерджи М., Юн У. С., Мун й. Х., Ан Г., Янссон С., и Ли С.-Г. (2014). Производство супероксида из фотосистемы II в рисе (Oryza sativa L.) с нехваткой PsbS. BMC Plant Biol 14: 242.
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