Chlorophyll Fluorescence and Photosynthesis Under Abiotic Stress
287
14 Chlorophyll Fluorescence and Photosynthesis Under Abiotic Stress SHIVAM YADAV1, ALKA SHANKAR1, SHWETA RAI1, RUCHI RAI1, SHILPI SINGH1 AND L.C. RAI1*
ABSTRACT
Chlorophyll fluorescence, a non-invasive technique commonly used to analyze photosynthetic performance of plants, is now being widely employed in studying stress responses of plants by plant physiologists and eco-physiologists. Environmental stresses commonly target photosynthetic apparatus thereby affecting the PSII functioning and linked de-excitation pathways, subsequently chlorophyll fluorescence. Chlorophyll fluorescence analysis provides information about efficiency of both photochemical and non-photochemical processes. Present chapter, provides an overview of the principles of fluorescence analysis followed by usefulness of this technique in detecting the responses of photosynthetic organism to various abiotic stresses such as UV-B, salinity, drought, extremes of temperature, heavy metals and herbicides. Moreover, we also report few constraints of this important technique. Key words: Chlorophyll fluorescence, Photosynthesis, Heavy metals, Drought, Herbicides, Salinity, UV-B radiation INTRODUCTION
Nowadays, chlorophyll a fluorescence measurement has emerged as a powerful tool to study photosynthetic performance for agricultural, environmental and ecological studies in algae and plant system and enough evidence are present that witnesses above statement (Krause, 1988; Maxwell and Johnson, 2000; Papageorgiou and Govindjee, 2004; Baker and Rosenquist, 2004; Henriques, 2009; Calatayud et al., 2004; Guidi et al., 2007; Guidi and Degl’Innocenti, 2008; Massacci et al., 2008). Environmental stresses mostly cause an imbalance in cellular redox state thus causing oxidative damage of cell structure and inhibition of cellular activities. All photosynthetic organisms are challenged by abiotic stresses in their natural habitats which result in disruption of photosynthetic apparatus, thus an ultimate decline in productivity and yield is observed. Since photosynthesis is considered as highly sensitive phenomenon towards environmental fluctuations, photosynthetic measurements are important parameters to be studied under stress (Chen and Cheng, 2007; Singh et al., 2012, 2014, 2015, 2016). Specifically, chloroplast/thylakoid 1
Molecular Biology Section, Centre of Advanced Study in Botany, Institute of Science, Banaras Hindu University, Varanasi - 221005, India *Corresponding author: E-mail:
[email protected]
288
Environment and Photosynthesis a Future Prospect
is considered as a major stress sensor in photosynthetic organisms because it represents a site where light drives redox reactions and simultaneously O2 is evolved from water, thus marks the availability of O2 near the electron transport chain which may lead to the formation of (ROS) reactive oxygen species, ii) chloroplast can produce PSII associated strong oxidants responsible for splitting of H2O molecules, however can also oxidise components of thylakoid membrane such as pigments, lipids and proteins (Biswal and Biswal, 1999). However, the earlier methods did not provide required information and were time consuming. Contrary to them, Chl fluorescence measurements are comparatively rapid and simple method that provide a holistic view of overall photosynthetic function (Govindjee, 1995). Chlorophyll fluorescence imaging, an advanced form of chl fluorescence analysis is also used since many years. It has also been applied widely in response to drought (West et al., 2005), high light (Zuluaga et al., 2008) and ozone pollution (Guidi et al., 2007; Guidi and Degl’Innocenti, 2008). Using Chl fluorescence imaging stress mediated alterations can be analysed at very early stages of stress. Therefore the close relationship between the actual photochemical efficiency of photosystem II and CO2 assimilation rate in leaves determines that chlorophyll fluorescence can be used to identify alterations in the response towards changing environmental conditions and subsequently to screen for tolerance to abiotic stresses. In view of above, present chapter is an attempt to summarize chlorophyll fluorescence responses of photosynthetic organisms under various abiotic stresses along with a basic idea of chlorophyll fluorescence and its limitations. CHLOROPHYLL FLUORESCENCE AND PHOTOSYNTHESIS: AN ELEMENTARY IDEA
Total light energy arriving on a leaf surface have three fates i) absorbed by the leaf, ii) dissipated as heat energy and iii) re-emitted as light termed as chlorophyll fluorescence (Fig. 1). All these three processes operate in competition with each other. The key events involved in alterations in chlorophyll fluorescence are largely dependent on redox states (oxidation-reduction) of key electron carriers.
Fig. 1: A model displaying the possible fate of light energy absorbed by photosystem II (PSII)
A transient rise in the chlorophyll fluorescence occurs when adequate amount of light enough to drive photosynthesis strikes the leaf after a period of darkness. This rise is due to the reduction of electron carriers in the thylakoid membrane. Following the transient rise P680, special chlorophyll in photosystem II ejects one electron originated from water splitting to
Chlorophyll Fluorescence and Photosynthesis Under Abiotic Stress
289
electron acceptor QA via phaeophytin. Further acceptance of electron by QA occurs only when it has passed its electron to the next carrier QB. This state of reaction centers is termed as ‘closed’ which depends on various external conditions. Closed reaction centers in avoidably cause decrement in photosystem II quantum efficiency. Kautsky and coworkers observed changes in the yield of chlorophyll fluorescence for the first time (Kautsky et al., 1960). They found that transfer of photosynthetic material from dark into the light results in an increase in the yield of chlorophyll fluorescence over a time period of around one second, termed as Kautsky effect which is a consequence of reduction of electron acceptors in the photosynthetic pathway. The initial rise in fluorescence is followed by a decline in fluorescence signal over a period of minutes termed as quenching (Krause and Weis, 1991). Conversion of light energy to chemical energy that is used to drive photosynthesis is termed as ‘photochemical quenching’. Various processes of photosynthesis require light activation for example to achieve complete activity various key enzymes of Calvin cycle require light activation (Buchanan and Balmer, 2005). Furthermore, light energy required to drive photosynthesis is often small, the excess energy is dissipated as heat and called as ‘non-photochemical quenching’ (NPQ). Non-photochemical quenching represents a photoprotective process that prevents the formation of damaging free radicals and removes excess excitation energy within chlorophyllcontaining complexes. The key components involved in NPQ are regulatory protein PsbS and xanthophylls (Murchie and Niyogi, 2011; Demmig-Adams, 1990). NPQ is induced as consequence of conformational changes in PSII antenna due to prolongation of PsbS and formation of zeaxanthin (Ruban et al., 2012). Moreover generation of zeaxanthin from violaxanthin occur in light in the presence of violaxanthin de-epoxidase and light and activated by acidification of the thylakoid lumen. A clear discrimination between photochemical quenching and non-photochemical quenching is required in order to gain useful information about the photosynthetic performance of a plant utilizing chlorophyll fluorescence data. The usual approach is to block any one of the contributor which earlier was done by using a herbicide diuron, that inhibits PSII. Nowadays a method has been developed, termed as ‘light doubling technique’ (Bradbury and Baker, 1981; Quick and Horton, 1984). This technique uses a high intensity short duration flash of light which immediately closes all the PSII reaction centers. As the duration of flash is very short it would not cause increase in non-photochemical quenching (or a negligible increase). The flash causes the fluorescence to reach a value equivalent to that could be attained only in the absence of photochemical quenching, termed as the maximum fluorescence (Fm), fluorescence yield in the absence of an actinic (photosynthetic) light is termed as F0 and steady state fluorescence yield in light is termed as Ft. A comparative study of Fm, F0 and Ft provides information about the efficiency of photochemical quenching and ultimately PSII performance. The photochemical quenching co-efficient qp is measured as qP = (Fm’ – Ft)/(Fm’ – F0’) where Fm’ is the maximum Chl fluorescence yield in light conditions, Ft is the steady-state Chl fluorescence immediately prior to the flash. For determination of F0’ in the light state, the leaf has to be transiently darkened and it has to be assured that QA is quickly and fully oxidized, before there is a substantial relaxation of non-photochemical quenching. Efficiency of PSII photochemistry is calculated by following formula PSII = (Fm’ – Ft)/Fm’ Maximum efficiency of PSII, Fv/Fm is determined as Fv/Fm = (Fm – F0)/Fm
290
Environment and Photosynthesis a Future Prospect
It is strictly related with both qP and PSII and has an optimal value of 0.83 in leaves of healthy plants (Bjorkman and Demmig, 1987). Decreases in Fv/Fm are frequently observed when plants are exposed to abiotic and biotic stresses. There are a large number of other coefficients that quantify photochemical and non-photochemical quenching and often the same parameter is used in different ways. Moreover, different terminology for the same parameter can be also found in literature. CHL FLUORESCENCE AND PHOTOSYNTHESIS UNDER ABIOTIC STRESS
Stress is very challenging to define because of the complex interaction between organism and environment. However, in common terms, it can be defined as a biotic or abiotic factor that prevents organism from usual functioning and thus causes reduction in their growth and reproduction (Osmond et al., 1987). As earlier described, photosynthesis being a sensitive process towards environmental stresses, becomes essential parameter to be studied under diverse stresses. This section of chapter reviews photosynthetic response with special reference to chl fluorescence following various abiotic stresses.
UV-B Radiation Although UV-B (320–280 nm) accounts for
