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Leaf chlorophyll fluorescence: background and fundamentals for plant biologists.

Introduction

Chlorophyll a fluorometry bas come a long way in the past few decades to become the single most common spectroscopic technique used in photosynthetic studies, particularly in assessing the functionality of PSII. Measurements of Chl fluorescence, both at physiological and low temperatures, have provided critical information on almost every aspect of the light energy absorption and conversion process that occurs in photosynthesis, with special relevance to our understanding of excitation energy migration within the antennae and to the cognate reaction centers, the energetic connectivity between antennas and reaction centers, the primary photochemistry and the secondary electron transport associated with the primary reactions.

In the past few years, the development of portable, user-friendly fluorometers extended the accessibility of the technique to leaf measurements in the field, where it round prompt and useful applications in many physiological and eco-physiological studies. As a result of the increasing utilization of chlorophyll fluorescence technique, a number of comprehensive reviews have discussed the subject matter to varying depths (Krause & Weis, 1991; Govindjee, 1995; Lazar, 1999; Maxwell & Johson, 2000; Strasser et al., 2000; Rohacek, 2002; Baker & Rosenqvist, 2004; Oxborough, 2004; Baker, 2008). However, most are biased towards its physics aspects and dwell on elaborate theoretical considerations not readily accessible to the more field-oriented plant researcher. This review aims primarily at providing a basic understanding of the origins of fluorescence and the functional meaning of those fluorescence parameters most routinely measured in the field, based on present knowledge of the light energy conversion process in photosynthesis.

While this review was in preparation, an authoritative work on the same subject was published by Baker (2008), in which some aspects that are dealt with here are treated in greater depth and whose reading is recommended to those interested in a more specialized knowledge of the subject.

Abbreviations: Car, carotenoid; Chl, chlorophyll; DCMU, 3(3,4-dichlorophenyl)-1,1-dimethylurea; [F.sub.0], Fm, Fv, minimum, maximum and variable fluorescence levels; [J.sub.F], photosynthetic electron transport rate; LHCII, light-harvesting complex of photosystem II; [P.sub.680], photochemically active Chl of PSII; PFD, photon flux density; Pheo, pheophytin a; PQ, plastoquinone; PSI, PSII, photosystems I and II, respectively; [Q.sub.A], primary quinone acceptor of PSII; [Q.sub.B], secondary quinone acceptor of PSII; qE, energy-dependent component of qN; qN, non-photochemical quenching of variable Chl fluorescence; qP, photochemical quenching of variable Chl fluorescence; RCII, reaction center of photosystem II: [Y.sub.Z], secondary electron donor of PSII.

Overview of the Photosynthetic Energy Conversion Process

In the photosynthetic process light is principally utilized to drive an energetically uphill transport of electrons from water to [NADP.sup.+]. Coupled with this process, a proton translocation across the thylakoid membrane builds-up a difference in electrochemical potential that is harnessed for ATP synthesis. The NADPH and ATP thus formed are then utilized in a number of different biochemical reactions, of which the quantitatively most important is the carbon dioxide assimilation. In oxygenic photosynthesis, the electron transport from water to [NADP.sup.+] is mediated by two photosystems, operating in series through a chain of redox transporters that comprises plastoquinone, the cytochrome [b.sub.6],f complex and plastocyanin. The electrons extracted from the water flow through PSII, the intersystems transport chain and PSI, in this order, to reduce [NADP.sup.+] and generate the transthylakoid difference in proton electrochemical potential that drives ATP formation. The [DELTA]pH component of this difference constitutes an absolute requirement for the induction of a down-regulatory mechanism of excitation energy in the PSII antenna that accounts for part of the Chl fluorescence quenching observed in the light, as will be discussed.

In the reaction centers of the two photosystems excitation energy generated in the antenna as a result of light absorption is converted into electrochemical potential, through the transfer of the high energy electron located in excited specialized chlorophyll molecules (the primary electron donors) to neighbouring acceptor molecules (the primary electron acceptors). This charge separation process (primary photochemistry) can be schematically represented as follows:

P R [right arrow] [P.sup.*] R [right arrow] [P.sup..+] [R.sup..-],

where P represents the photochemically active "pigment" of the reaction center, [P.sup.*] is its excited state, and R is the primary acceptor. For PSII, one can write the charge separation reaction more specifically as

[P.sub.680][Q.sub.A] [right arrow] [P.sub.680.sup.*][Q.sub.A] [right arrow] [P.sup..+.sub.680][Q.sup..-.sub.A],

where the cationic radical [P.sub.680.sup..+] is a very strong oxidant potentially capable of oxidizing the water, and the anionic radical [Q.sub.A.sup..-] is a moderately reductant plastoquinone unable to reduce the [NADP.sup.+]. The formation of NADPH thus requires an additional energetic promotion of the electron extracted from water by a new light quantum, which is now mediated by PSI. In this process, a very strong reductant is formed, which ultimately leads to the reduction of [NADP.sup.+]. The electron transport from water to [NADP.sup.+] is graphically represented by the usual Z scheme, shown in Fig. 1.

Primary Photochemistry and Secondary Electron Transport at PSII

Given the importance of understanding the physical basis of Chl variable fluorescence, the why, when and where of its origin, we will dwell a little longer on the charge separation process that occurs at the PSII as well as on the secondary electron transport that neutralizes the cationic and anionic radical species there formed. First, it should be noted that [Q.sub.A] is not really the immediate acceptor of the electron transferred from [P.sub.680.sup.*]; rather, the primary charge separation in PSII occurs between [P.sub.680.sup.*] and a pheophytin a molecule (Pheo, often termed the intermediate acceptor, I), but the electron is rapidly transferred to the [Q.sub.A] molecule to stabilize the charge separated state (for a recent review of this subject sec, for example, Nelson & Yocum, 2006). Indeed, the radical pair [P.sub.680.sup..+][I.sup..-] is readily reversible and rapid transfer of the separated electron to [Q.sub.A] is required to prevent it from going back to [P.sub.680.sup..+]. After being reduced, [Q.sub.A.sup..-] passes the electron to a second plastoquinone molecule ([Q.sub.B], the secondary electron acceptor), and the semiquinone [Q.sub.B.sup..-] attaches to the binding site B of the reaction center subunit [D.sub.1], near the stromal side of the thylakoid membrane. After a second turnover of the reaction center, that is, upon a new excitation of [P.sub.680], [Q.sub.B.sup..-] receives a second electron from [Q.sub.A.sup..-], takes up two protons from the stromal side of the thylakoid membrane and forms the neutral plastoquinol ([Q.sub.B][H.sub.2]=PQ[H.sub.2]). The double reduced, doubly protonated [Q.sub.B] molecule frees itself from the B site, and diffuses laterally in the thylakoid membrane to dock at the [Q.sub.0] site of the cytochrome [b.sub.6],f complex where it is sequentially reoxidized by the "high" (redox) and "low" (redox) potential chains of the complex (Fig. 1). Meanwhile, [P.sub.680.sup..+] has been reduced by an electron from a redox-active tyrosine residue of the [D.sub.1] protein ([Y.sub.Z], the secondary electron donor) which, in mm, is neutralised by an electron extracted from water bound at the [Mn.sub.4]Ca center. This linear electron transport from water to the secondary acceptor [Q.sub.B] is, thus, rather long and complex and can be represented as follows:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII],

where [S.sub.1] represents the prevalent oxidation state of the [Mn.sub.4]Ca center in the dark. As already mentioned, in a second photoact, [Q.sub.B.sup..-] is reduced to [Q.sub.B.sup.2-], protonated and released into the membrane to be reoxidized by the cyt [b.sub.6],f complex. These electron transfer steps with their corresponding timescales are depicted in Fig. 2.

[FIGURE 1 OMITTED]

The primary charge separation and stabilization processes occur in the range of picoseconds (ps) and the subsequent electron transfer from [Q.sub.A.sup..-] to [Q.sub.B] takes a couple hundredths of a microsecond ([micro]s), so that the transfer of the excited electron from [P.sub.680.sup.*] to the PQ pool occurs in less than 1 millisecond (ms). The reoxidation of the PQ[H.sub.2], however, is much slower, requiring a few ms to be completed. Thus, in most current field fluorometers, in which the light flashes utilized induce multiple turnovers of PSII, one can consider the PQ pool as the terminal sink for the electrons extracted from water at PSII. With very fast light pulses that cause a single PSII turnover and equally fast recording systems, it is possible to catch the reaction centers before any forward electron transfer from [Q.sub.A.sup..-] has had time to occur and these two different situations can be used to measure the sizes of the whole set of quinones associated with PSII ([Q.sub.A], [Q.sub.B] and PQ pool) or just that of [Q.sub.A], respectively. We will return to this aspect later on.

"Open" and "Closed" PSII Reaction Centers

When both [P.sub.680] and [Q.sub.A] are in the (electrically) neutral state, the PSII reaction center is in a state [P.sub.680].[Q.sub.A]] said to be open, meaning that is ready to carry out the charge separation process upon excitation of [P.sub.680]. As a result of charge separation, the reaction center is then in the new state [P.sub.680.sup..+][Q.sub.A.sup..-]] and is now said to be closed, indicating that it cannot use excitation energy to repeat the charge separation until the photoproducts, oxidized [P.sub.680] and reduced [Q.sub.A], are neutralized by secondary electron transport reactions. (Actually, it is still possible for charge separation to occur at a very low rate when [Q.sub.A] is reduced, but this is followed by a rapid charge recombination that may lead to the generation of harmful singlet oxygen species; see accompanying Box on singlet oxygen production in PSII.) While the reaction centers remain close& the excitation energy generated in the PSII antenna cannot be used for driving photochemistry and has to be rapidly dissipated by other mechanisms in order not to cause damage to the photosynthetic machinery, in particular to components of the PSII reaction center. During the lifetime of an exciton, two major alternative pathways compete with photochemistry to dissipate the energy of Chl excited states, namely fluorescence and heat loss (Butler, 1978). When the reaction centers are open, photochemistry generally overrides (that is, is much faster than) these two other mechanisms in deactivating Chl excited states, and consequently their yields are low. However, when some impairment slows photochemistry down or the irradiance is high, these other mechanisms take over and their yields rise. Thus, changes in the yields of fluorescence and heat dissipation can be used to monitor changes in the yield of PSII photochemistry (1) or, said in a different way, any increase in fluorescence or heat dissipation rates means a decrease in PSII photochemistry rate and, accordingly, a decrease in the photosynthetic efficiency of light energy use. When viewed in this way, the two alternatives to photochemistry could be construed as wasteful processes. However, it should be considered that whenever light energy is absorbed in excess, that is, at a rate higher than the reaction centers are able to process it (overexcitation of reaction centers), it is extremely important to dispose of the excess excitation in an efficient and harmless manner, and those "wasteful" processes provide effective mechanisms to achieve that. Under most natural conditions light intensity is highly variable, both dally and seasonally, and, therefore, there is a permanent need for regulating the rate of excitation energy transfer from the antennas to the reaction centers of PSII. Generally, most absorbed light not used for photochemistry is converted into heat, with only a small percentage being reemitted as fluorescence. In vivo, the fluorescence yield varies between a maximum of between 3% and 5% and a minimum of ca. 0.5% of the absorbed light energy (Walker, 1987; Krause & Weis, 1991), values that correspond to situations in which the reaction centers are fully closed or maximally open, respectively. This lowering (quenching) of fluorescence yield results from both the utilization of excitation energy to drive photochemistry at the PSII reaction center--photochemical quenching (of Chl fluorescence)--and from its dissipation as heat at the PSII antenna--non-photochemical quenching (of Chl fluorescence). The relative contributions of these two types of (fluorescence) quenching to total (fluorescence) quenching depend mostly on the redox state of [Q.sub.A]: when [Q.sub.A] remains mainly oxidized (open reaction centers), the photochemical quenching predominates, but as a larger fraction of [Q.sub.A] remains reduced (closed reactions centers), the non-photochemical quenching becomes progressively more important.

[FIGURE 2 OMITTED]

The relationship between the yield of photochemistry and those of fluorescence and heat loss would be simpler to interpret if one could create conditions under which only one of the two alternatives to photochemistry would be available, in which case variations in the yield of this process would inversely reflect changes in the yield of photochemistry. We will see below that it is possible under some conditions to eliminate thermal dissipation as an alternative to photochemistry for the use of excitation energy. However, before we discuss such conditions, we have briefly to consider the relative contributions of PSII and PSI to the total fluorescence yield of photosynthetic samples under physiological conditions.

PSII and PSI Fluorescence Characteristics

It is now well established that, at room temperature, Chl fluorescence originates mostly at the PSII, and, consequently, fluorescence measurements provide functional information principally about this photosystem (Krause & Weis, 1991; Govindjee, 1995). At physiological temperatures, PSI Chl emits very little fluorescence and this shows no component that reflects the photochemistry going on in this photosystem. We do not fully understand the reasons behind these differences between the two photosystems, but they seem to be related to differences in the manner the antennas are coupled to the reaction centers, and also to characteristics and organization of the electron transport chain downhill of the charge separation at PSI. Apparently, both PSII and PSI are shallow traps for the excitonic energy migrating from the antennas, but the probability of excitation energy escaping from the reaction center and returning to the antenna is much lower in PSI. Indeed, the rates for the early steps of forward electron transport beyond [P.sub.700] are extremely fast, and the electron separated at the PSI reaction center moves away from the primary acceptor very rapidly. Thus, any fluorescence that may occur no longer reports to the redox state of the primary electron acceptor, as is the case in PSII. Further, and very importantly, the closed reaction center is left in the state [[P.sub.700.sup..+][A.sub.1]] and [P.sub.700.sup..+] is an effective quencher of fluorescence (Butler, 1978). [P.sub.680.sup..+] is also a fluorescence quencher, but it is such a strong oxidant that it doesn't normally exist under physiological conditions. There is, then, no fluorescence component reflecting PSI primary photochemistry, that is, there is no variable fluorescence associated with PSI. Regardless, some minimal fluorescence from PSI also contributes to the total fluorescence measured in our instruments and this may somewhat jeopardize the validity of fluorescence measurements as a probe for PSII functioning. This may constitute a significant problem when fluorescence measurements are carried out at long wavelengths, beyond 700 nm, where light emission from PSI could assume a relatively larger contribution. In such cases, there is evidence that the contribution of PSI fluorescence for the non-variable component of total fluorescence can be as high as 30% in [C.sub.3] plants or even 50% in C4 plants (Genty et al., 1990b; Pfundel, 1998). Undoubtedly, an accurate interpretation of fluorescence measurements in relation to PSII photochemistry requires both an accurate separation of PSI and PSII emission spectra or the quantification of PSI-related emission (Gilmore et al., 2000; Blankenship, 2002; Franck et al., 2002; Oxborough, 2004; Baker, 2008)

Under most circumstances, Chl fluorescence arises mainly from the PSII antenna. When the reaction centers are closed or working at slow pace, rapid exciton equilibration between the antenna and the reaction centers favours the return of the exciton to the antenna, from where it is reemitted (Butler, 1978). Some authors argue that under photoinhibitory conditions, the fluorescence is emitted principally from the damaged reaction centers, but this is questionable; anyway, the fast excitation equilibration renders the fluorescence emission site a question of mainly theoretical interest.

Fluorescence of Dark-Adapted Leaves

Let us first discuss what happens when one illuminates a dark-adapted leaf, i.e., a leaf that has been in the dark for a period of time long enough so that all the components of the photosynthetic electron transport chain are in the oxidized state (2). The PSII reaction center is then open and ready to carry out photochemistry, as shown before. We have also seen that, in general, the Chl excited states that are not used for photochemistry can be deactivated by heat loss or fluorescence emission. However, for leaves that have been in the dark for some time, the heat dissipation mechanism is not operational, as it requires that the photosynthetic electron transport had been working for some time to build up a transthylakoidal pH difference, and this is not the case. Fluorescence is then the only alternative to photochemistry for deactivation of the excitation energy generated in the PSII antenna (3) and thus becomes immediately apparent that changes in fluorescence yield inversely reflect changes in the yield of photochemistry.

This is a particular situation that illustrates well how fluorescence measurements can be used to monitor PSII photochemistry, but which is obviously not representative of field conditions where leaves are continuously exposed to light during the day. However, we can analyse this situation a little further to derive some useful fluorescence parameters and introduce their functional meaning.

Suppose that the illumination of the dark-adapted leaf is done with a light flash intense enough to close all the reaction centers (a saturating flash) through multiple turnovers of the PSII. The closure of the reaction centers though rapid, as we have seen, is not an instantaneous process; it requires several photoacts that result in the transfer of successive electrons to the PQ pool, via [Q.sub.A] and [Q.sub.B]. While the PQ pool is not fully reduced, [Q.sub.A.sup..-] is rapidly re-oxidized, the reaction centers open again and the fluorescence yield stays transiently low--the excitons are being preferentially utilized for carrying out photochemistry. However, as the PQ pool becomes gradually more reduced, the [Q.sub.A.sup..-] re-oxidation becomes progressively more difficult and the fluorescence yield increases. Finally, when the PQ pool is fully reduced and [Q.sub.A.sup..-] can no longer pass on its electron, the reaction centers remain closed, the use of excitation energy for photochemistry nullifies, and fluorescence reaches its maximum level (Fm, maximum fluorescence). On average, Fm is reached in about 200 ms. After reaching its peak, the fluorescence decays slowly as the reaction centers reopen because of electron transfer from the reduced pool of quinones to oxidized downhill transporters (4).

Figure 3 shows a typical response of fluorescence intensity to a saturating flash of a dark-adapted leaf, plotted on different timescales. Figure 3A shows the fluorescence emission changes plotted on a linear timescale, and the progressive closure of the reaction centers is represented as a vertical straight line, the maximum value of which (P) is observed when [Q.sub.A], [Q.sub.B] and PQ pool are fully reduced. The decrease in fluorescence level observed afterwards reflects mostly the progressive reopening of the reaction centers as electrons are transferred from the PQ pool to oxidized electron acceptors located downhill, as discussed above. Now, if one resorts to a recording system capable of a much higher time resolution (Fig. 3B; note the logarithmic timescale in this figure), it can be seen that the upwards time course of fluorescence represented by the vertical line in Fig. 3A is indeed polyphasic, with two major inflections at J and I, from its origin (O) to its peak (P; P is identical to Fm when the light pulse is fully saturating) (Neubauer & Schreiber, 1987 (5); Schreiber & Neubauer, 1987; Govindjee, 1995; Strasser et al., 1995; Lazar, 2001). The O-J-I-P curve reflects the progressive filling up of PSII electron acceptors pools--[Q.sub.A], [Q.sub.B] and PQ; the O-J fluorescence rise reflects the closure of PSII reaction centers due to the transient reduction of [Q.sub.A]; however, the reduced [Q.sub.A.sup..-] is rapidly re-oxidized by transfer of its electron to the neighbour [Q.sub.B], reopening the centers and causing the fluorescence curve to level off. The subsequent fluorescence rise to I characterizes the new closure of the reopened centers upon reduction of oxidized [Q.sub.A] after a new turnover of PSII. Subsequently, the newly reduced [Q.sub.A] transfers its electron to [Q.sub.B.sup..-] sites and the centers reopen again, causing the flattening of the fluorescence curve seen at inflection point 1. Finally, the fluorescence rise after this last "plateau" up to P corresponds to the full closure of PSII reaction centers as [Q.sub.A.sup.-][Q.sub.B.sup.2-]/[Q.sub.A.sup.-][Q.sub.B][H.sub.2] states accumulate (Strasser et al., 1995). This is a correct though somewhat simplified explanation of the fluorescence curve kinetics. Indeed, one must note that there is some overlapping in electron transfer from [Q.sub.A.sup.-] after point J and a mixed population of [Q.sub.A].[Q.sub.B] redox states can be found at any instant during the rise of the curve to its maximum (see legend of Fig. 3). Note that the fluorescence rise from [F.sub.0] to Fm, the variable fluorescence component (Kitajima & Butler, 1975), is related to, but it is not directly proportional to the redox state of [Q.sub.A]. Also, if the applied light pulse is truly saturating and brief enough, the inflection point J reflects the maximum amount of [Q.sub.A]. This basic pattern for fluorescence kinetics holds for all plants. The actual pattern, however, depends on a number of PSII structural and functional features, such as connectivity between PSII units, state of the water oxidation complex, redox state of the intersystem electron transport chain and other factors beyond the scope of this review.

Note that the fluorescence level at the start of the illumination (Fig. 3), when all reaction centers are still open, has a value different from zero, although small ([F.sub.0], initial, basal or minimum fluorescence). This value is characteristic of the system and reflects excitation energy that is lost for photochemistry under the most favourable conditions, i.e., when all reaction centers are open for energy conversion. In fluorometers equipped with only one light source (type Plant Efficiency Analyzer, PEA, of Hansatech), [F.sub.0] is estimated by extrapolating the fluorescence level to the very start of the illumination with a saturating pulse. In fluorometers that use modulated light to measure fluorescence (type PAM fluorometers), [F.sub.0] is measured directly by illuminating the sample with a modulated beam of very low intensity unable to trigger photochemistry to any appreciable extent, therefore keeping the reaction centers open. Estimation of [F.sub.0] with high precision is very important to reduce bias in calculating several fluorescence parameters in which [F.sub.0] is included, as will be shown shortly.

The fluorescence emitted when a dark-adapted leaf is illuminated by a saturating flash varies, then, from a minimum value ([F.sub.0]), measured in the absence of any significant photochemistry, to a maximum value ([F.sub.m]), observed when all possible photochemistry has occurred. The difference between these two fluorescence values, [F.sub.v] = [F.sub.m] - [F.sub.0], constitutes the variable fluorescence and its level at each instant reflects the photochemistry going on at PSII, indicated by the redox state of [Q.sub.A]. The fraction of the total absorbed light that is used to carry out photochemistry can then be estimated from Fv/Fm=(Fm-[F.sub.0])/Fm=1 - [F.sub.0]/Fm = [[PHI].sub.PSII] and measures the quantum yield of PSII photochemistry (6) (Kitajima & Butler, 1975). In dark-adapted leaves, where all reaction centers are initially open and the excitation energy not used for photochemistry is all re-emitted as fluorescence (no thermal energy dissipation can occur, as we have seen; see also footnote 3), Fro reaches its absolute maximum and [[PHI].sub.PSII] measures the maximum, intrinsic or potential quantum yield of PSII photochemistry. In the great majority of healthy leaves, under optimal conditions, [[PHI].sub.PSII] has a theoretical maximum value of about 0.832 [+ or -] 0.004 (corresponding to a typical Fm/[F.sub.0] ratio of 6) and this was experimentally verified for a large number of higher plants of diverse origins (Bjorkman & Demmig, 1987; Johnson et al., 1993). [[PHI].sub.PSII] values down to 0.8 are indicative of highly active PSII units, but decreases below 0.8 indicate that the number of PSII reaction centers capable of performing photochemistry has been decreased, revealing the presence of damaged RCIIs. These damaged centers can result from the effects of either (a) a number of stresses that lower the photochemical ability of PSII making excessive the light intensity to which the plant is subjected, or (b) direct exposure to sustained high light intensity and both situations originate the so-called PSII photoinhibition. Photoinhibition is, thus, a reduction in photosynthetic activity due to light-induced inactivation of PSII reaction centers that results primarily, but not exclusively, from oxidative damage of the D1 subunit of RCII; D1 is then proteolytically degraded to be replaced by a newly synthesized protein, with consequent functional recovery of the damaged centers (see, for example, Aro et al., 2004). When the rate of DI damage exceeds its repair rate, there is a transient loss of competent RCIIs and a consequent decrease in the maximum quantum yield of PSII photochemistry. Some authors consider that the photoinhibitory state exists only when permanently damaged PSII units accumulate in the thylakoid membrane, before that, though, there may be damaged PSII units that may recover during the dark period following excess irradiance. Lowering of the Fv/Fm ratio means a potentially decreased PSII performance under illuminated conditions. However, under many light conditions, as those often encountered by plants in the field, photochemistry is not occurring at its maximum possible rate and, therefore, there are a number of RCIIs that are not operating at all. In such conditions, a decreased Fv/Fm ratio does not necessarily imply a decreased photochemical efficiency of PSII in the light, and it is important to consider this when analysing the effects of stresses on the plant's photosynthetic performance.

[FIGURE 3 OMITTED]

The true Fm level is only reached when the pQ pool is fully reduced, as we have discussed before. This Fro level is then proportional to the number of electrons required to reduce the whole set of active quinones ([Q.sub.A], [Q.sub.B] and PQ pool) associated with PSII, and reflects its size rather than that of QA only--as is often taken to represent. The [Q.sub.A] concentration--and, thus, the "concentration" of active PSII units, as each PSII unit possesses only one [Q.sub.A] molecule--is sometimes estimated from measurements of Fm in the presence of DCMU, which blocks the electron transport beyond [Q.sup.A.sup..-]. However, because the herbicide does not penetrate the leaf in an uniform manner, this technique is prone to significant errors; also, because the PQ pool remains oxidized in DCMU-treated leaves and causes static fluorescence quenching, the technique suitability for measuring [Q.sub.A] absolute concentration in leaves is further compromised, although it may still be of interest for some comparative purposes. As mentioned above, when available, an alternative way to estimate the number of PSII active units is to use single turnover flashes and recording systems with a time resolution that are capable of catching [Q.sub.A.sup..-] before forward electron transfer to [Q.sub.B] has occurred, but this is so far not possible for most currently used field fluorometers.

Fluorescence of Light-Adapted Leaves

The change in Chl fluorescence kinetics associated with a dark-light transition has been widely used to investigate the physiological status of PSII, but it yields essentially no information on this photosystem's performance under continuous light conditions. To measure Chl fluorescence kinetics of leaves under field conditions, the development of fluorometers using the pulse amplitude modulation (PAM) technique (Schreiber, 1986; Schreiber & Neubauer, 1987) was fundamental. In this technique, one uses an actinic light of variable intensity to drive photosynthesis and a weak modulated light to excite fluorescence; the detector is only sensitive to the modulated radiation, whose low signal is recovered by selective amplification. Any reflected or refracted actinic light is filtered out and, thus, fluorescence yield can be measured in the presence of actinic light, including that of full sunlight in the field (Maxwell & Johson, 2000). Modulated fluorometers are also relatively insensitive to disturbances induced by brief saturating flashes. These are applied in order to maximally close PSII reaction centers and eliminate the photochemical quenching, thus revealing the non-photochemical quenching (7). Discrimination of the photochemical and non-photochemical components of Chl fluorescence quenching is critical to a full understanding of PSII functioning under continuous light, as we will see below.

Let us start by illuminating a dark-adapted sample with a weak beam of modulated light (less than 1 [micro]mol quanta [m.sup.-2] [s.sup.-1], often 0.1 [micro]mol quanta [m.sup.-2] [s.sup.-1]) that doesn't cause closure of PSII reaction centers, to determine [F.sub.0] (Fig. 4). In this manner, [F.sub.0] is measured with high accuracy and this minimizes bias error in the determination of other fluorescence parameters that include [F.sub.0]. Next, apply a saturating light pulse (SP) capable of closing all RCIIs, which causes a sudden rise in fluorescence up to its maximum value Fm. The fluorescence intensity decreases slowly afterwards, as we have discussed above, until it eventually returns to the [F.sub.0] level. This return to [F.sub.0] can be speeded up if one applies a light of a wavelength preferentially absorbed by PSI (far-red, [lambda] [greater than or equal to] 700 nm) for a few seconds, which causes the rapid oxidation of the PSII-associated pool of quinones and the reopening of the reaction centers. After reaching [F.sub.0], if an actinic light is turned on, there occurs a fluorescence rise reflecting the closure of RCIIs, the extent of which depends on the intensity of the actinic light applied to the sample. As the photosynthetic process is turned on and electrons start flowing from PSII acceptors to downstream components of the electron transport chain the RCIIs reopen. This in turn determines a decline in fluorescence intensity over a timescale of a few minutes to successively lower levels (Ft') until it eventually reaches a minimum, steady state level (Fs'), corresponding to an equilibrium between electron transport processes and coupled "dark" biochemical reactions (Rohacek, 2002). At any point in time during this decline, one can apply to the sample a series of brief saturating light pulses (8) (SP; Fig. 4) that close all RCIIs that were still open, leading to a fluorescence rise to its maximum value, termed Fm' (it is usual that fluorescence parameters referring to light-adapted leaves are denoted with a prime to contrast to the same parameters obtained from dark-adapted leaves). Note that Fm' is lower than Fro obtained from dark-adapted leaves (Fig. 4), even though both fluorescence levels correspond to a state of full reduction of [Q.sub.A] and the level of this quinone has not changed meanwhile. Such decrease results from the fact that part of the absorbed radiation is being dissipated as heat and this difference in the maximum fluorescence level is the non-photochemical quenching (qN), as we have seen earlier, qN is formally measured as [(Fm-[F.sub.0])-(Fm'-[F'.sub.0])]/[(Fm-[F.sub.0])] [approximately equal to] [(Fm-Fm')/(Fm-[F.sub.0])] and represents the decrease in variable fluorescence levels observed in the light in relation to that measured in dark, in which there is no qN (9). From this, it follows that in order to validly compare the qN of different samples, it is necessary that they show the same quenching characteristics in the dark-adapted state, and a good indicator of this are identical Fv/Fm ratios (Baker & Rosenqvist, 2004). More precisely, meaningful comparisons can only be made when Fv/Fm values in the dark-adapted leaves are close to 0.83, indicating that the photochemical quenching is at its maximum and that no non-photochemical quenching is occurring.

[FIGURE 4 OMITTED]

When the non-photochemical quenching is calculated as (Fm-Fm')/(Fm-[F.sub.0]), its values vary between 0 (Fm'-Fm, hence no qN) and 1 (Fm'-[F.sub.0], hence maximum qN) and, thus, its sensitivity to variations in qN when this reaches elevated values is rather poor. This led some authors (Bilger & Bjorkman, 1990) to propose another parameter, NPQ (Non-Photochemical Quenching)=(Fm-Fm')/Fm'-Fm/Fm'-1 that is linearly related to heat dissipation in the light but which can now range from 0 to [infinity] (see common values for NPQ in Rohacek, 2002, for example); additionally, the calculation of NPQ eliminates the need for the determination of [F.sub.0]', which is always problematic. For these reasons, the parameter NPQ gained wide acceptance and has found widespread use. The non-photochemical quenching occurs under continuous illumination and causes loss of part of the excitation energy collected at the antenna as heat, thus reducing the energy transferred to RCIIs to drive photochemistry. Consequently, [[PHI].sub.PSII] = Fv'/Fm'=(Fm'-[F.sub.0]')/Fm', the maximum PSII photochemical efficiency in the light, is generally lower than [[PHI].sub.PSII] of dark-adapted leaves. [F.sub.0]' values are close to those of [F.sub.0] but are not necessarily the same, namely because the non-photochemical quenching not only decreases the maximum fluorescence level but also somewhat quenches [F.sub.0].

The fluorescence quenching that occurs under actinic light due to photochemistry at PSII is represented by Fq' (=Fm'-Fs') in Fig. 4 and its proportion of the maximum variable fluorescence is (Fm'-Fs')/(Fm'-[F.sub.0]')=qP (short notation for photochemical quenching) (Bradbury & Baker, 1984; Quick & Horton, 1984). qP is, of course, dependent on the fraction of RCIIs that is actually carrying out photochemistry, but it should not be used as a measure of the open RCIIs as the two are not linearly related. Indeed, because of the connectivity between different PSII units, it is possible that an exciton that finds a closed reaction center moves on to another one that is open, thus driving further photochemistry without a change in the number of open RCIIs (10). The photochemical and non-photochemical quenching jointly account for practically (11) all fluorescence quenching found in light-adapted leaves. Each conveys important information about the manner in which light is being used by the photosynthetic apparatus: qP gives the proportion of absorbed light energy that is used to drive photochemistry at the RCIIs and qN the proportion that is dissipated as heat at the antenna. However, one should keep in mind that the parameters qP and qN are not directly related: the first refers only to the illuminated state of the sample, whereas the latter reports to both the light- and dark-adapted states (Baker & Rosenqvist, 2004).

The effective, actual or operational quantum efficiency of PSII photochemistry in the light is given by the ratio Fq'/Fm'=(Fm'-Fs')/Fm' (Genty et al., 1989) and is the most used indicator of the functioning condition of PSII in studies of plants under both normal and stress conditions. It measures the fraction of the absorbed light energy that is actually being used to drive photochemistry at PSII and is linearly related to the quantum yields of linear electron transport (Genty el al., 1989), [O.sub.2] evolution (Genty et al., 1992) and C[O.sub.2] assimilation (Genty et al.. 1990a: Harbinson et al., 1990; Krall & Edwards, 1990; Cornic & Ghashghaie, 1991; Krall et al., 1991; Edwards & Baker, 1993; Johnson et al., 1993) under many ambient conditions (we will come back to this point below). There are a number of possible causes of error in the determination of Fq'/Fm' (Oxborough, 2004; Baker 2008), some of which have been discussed earlier. An important one respects to the contribution of PSI fluorescence to the [F.sub.0] value when fluorescence is measured at wavelengths above 700 nm. This error could theoretically be minimized by carrying out the measurements at [lambda] [less than or equal to] 700 nm. However at these wavelengths the measured signal would reflect mainly the fluorescence emitted by chloroplasts located in the upper layers of the leaf rather than in the whole sampled region, as shorter wavelengths are scattered within the leaf and readily reabsorbed by chloroplasts located more to the interior (Schindler & Lichtenthaler, 1996; Nobel, 1999). Also, when the PQ/PQ[H.sub.2] ratio is particularly high immediately before application of the saturating light pulse, as occurs under low ambient light intensity, there could be an overestimation of Fm' if the saturating pulse induces multiple RCII turnovers, as happens in most commercial fluorometers (Baker, 2008). Then, part of the PQ pool would be reduced and no longer quench chlorophyll fluorescence, causing higher levels of Fm' to be reached. A third and important cause of error in estimating Fq'/Fm' arises from using light pulses that are not truly saturating, that is, that do not cause complete closure of RCIIs. This is more probable to occur under intense actinic light, as we discussed before, and would cause an underestimation of Fm'. In general it is round that these various potential sources of rotor have only limited effects on Fq'/Fm' values and require particular attention only under special circumstances.

Analysis of PSII Operational Efficiency

The operational efficiency (Fq'/Fm') of PSII can be viewed as the product of two other fluorescence parameters: Fv'/Fm', the maximum quantum efficiency of PSII photochemistry in the light, and Fq'/Fv', a parameter often referred to as the PSII efficiency factor because it reflects the photosynthetic system's capacity to keep RCIIs open in the light [Fq'/Fm'=(Fv'/Fm')x(Fq'/Fv')] (Baker, 2008). Indeed, the maximum PSII quantum efficiency (Fv'/Fm') is attained only when the maximum number of reaction centers are open and, thus, the operational efficiency can be obtained from the maximum efficiency by taking into account the system's ability to maintain RCIIs open in the light. Therefore, changes in the operational efficiency of PSII can best be understood by looking at causes that change either one or both of its components. Fv'/Fm' is inversely related to the extent of the non-photochemical quenching, as we have seen, and the latter tends to increase with increasing PFDs up to a certain point, leading to a minimum, practically constant value of Fv'/Fm' (Fig. 5). From this point on, further decreases of the operational efficiency of PSII caused by higher irradiances result mostly from reductions in Fq'/Fv', reflecting the system's inability to keep RCIIs open in the light. When the non-photochemical quenching reaches its maximum, any excitation energy still available in the antenna is transferred to the RCIIs; if this transferred energy finds some open RCIIs, it can still be used photochemically, but if it finds all RCIIs close& it will cause oxidative damage to PSII (photoinhibition), as we have discussed before.

The ability of the system to keep RCIIs open in the light, as measured by the parameter Fq'/Fv', may turn out to be critical under a number of environmental conditions, namely under high light intensities (Baker, 2008). The rate of reopening of RCIIs in high light may result from either an enhanced rate of the linear electron transport that leads to the formation of ATP and NADPH, or from enhanced rates of alternative electron transport pathways (pseudo-cyclic or cyclic) that are associated with sink processes such as photorespiration, [O.sub.2] reduction (Mehler reaction), protein synthesis, and nitrogen assimilation, among others. At the same time, as electrons are increasingly diverted to reactions other than the production of ATP and NADPH utilized for C[O.sub.2] assimilation, the linear proportionality between Fq'/Fm' and photosynthetic C[O.sub.2] fixation is obviously lost. The linear proportionality between the yield of C[O.sub.2] assimilation and the operational efficiency of PSII is only verified in the absence (or at minimal rates) of electron sink processes alternative to C[O.sub.2] assimilation. Notice that, complementarily, whenever the rate at which NADPH and ATP are being consumed decreases, for example because of C[O.sub.2] limitation, the rate of electron transport is proportionally slowed down in the absence of alternative electron sinks. Then, the RCIIs remain closed longer and Fq'/Fv' is consequently lowered, bringing about a reduction of Fq'/Fm'. Undoubtedly, under many situations, particularly at high PFD values, changes in Fq'/Fv' determine the changes in Fq'/Fm' and thus a correct analysis of the changes of each of its two components (Fv'/Fm' and Fq'/Fv') can be revealing of what is going on in the chloroplast. Under many stressful conditions, measurements of Fq'/Fv' may convey important information on how plants tolerate adverse environmental conditions. In his recent review on chlorophyll fluorescence as a probe of photosynthesis, Baker (2008) discusses how changes in Fv'/Fm' and Fq'/Fv' in plants subjected to certain important abiotic stresses can be used to help understand the mechanisms underlying the injuries brought about by such stresses. Maxwell and Johson (2000) also discuss the use of fluorescence parameters to study specific and interesting cases of plant adaptation to its environment, as well as in plant screening for acclimation to different microenvironments.

[FIGURE 5 OMITTED]

We have stated above that [F.sub.0]' values are close to those of [F.sub.0] but are not necessarily the same, although this is expediently assumed many times. Experimentally, [F.sub.0]' is often measured by applying a pulse of far-red light to the sample after turning off the actinic light, in order to preferentially stimulate the PSI activity and reoxidize reduced electron transporters located uphill. However, this procedure is rather controversial as it has been rightly argued that enhancement of PSI activity may not only cause relaxation of the non-photochemical quenching but also open some PSII reaction centers that were closed m the light (Baker & Rosenqvist, 2004), leading to an underestimation of the [F.sub.0]' value. The measurement of [F.sub.0]' is also sometimes problematic in the field because it is necessary to completely shield the leaf from any sunlight and this may turn out to be impracticable. To overcome these limitations, Oxborough and Baker (1997) proposed that [F.sub.0]' be instead estimated from the following equation: [F'.sub.0] = [F.sub.0]/[(Fv/Fm)+([F.sub.0]/Fm')]. However, it has been argued (Maxwell & Johson, 2000) that while this estimate has been shown to work well for the fluorescence imaging technique it was devised for, it does not necessarily perform equally well under other conditions, namely in stressed plants where extensive photoinhibition may be present. This was contested by Lawson et al. (2002), and [F.sub.0]' values seem to be conveniently calculated according to the above equation.

Scaling Up From Fluorescence Data to Whole Leaf Photosynthesis

The operational efficiency of PSII measures the quantum yield for electron flux through PSII to downhill acceptors, such as cyt [b.sub.6],f and PSI, and can be used to estimate the actual rate of the photosynthetic electron transport ([J.sub.F]). One can write, then, [J.sub.F]=([F.sub.q]'/Fm') x absorbed PFD x [a.sub.II], where [a.sub.II] is the relative absorption cross section of PSII. Accurate measurements of absorbed PFD and [a.sub.II] raise some difficulties and generally the former is calculated from the incident PFD assuming a leaf absorptance of 0.84 and the latter is taken as 0.5. This practice introduces some errors in the calculation of [J.sub.F], as leaf absorptance depends on various leaf characteristics and may vary significantly from the given value, and it is also doubtful that absorbed quanta are partitioned equally between the two photosystems (Nobel, 1999). Yet, it is found that [J.sub.F] values calculated in this manner often correlate well with rates of C[O.sub.2] assimilation and major differences are detected only at high PFDs (Maxwell & Johson, 2000). These differences seem to arise mostly from difficulties in accurately determining Fm' at high irradiances, as was discussed before. Recently, a method was devised (Earl & Ennahl, 2004) for measuring Fm' using multiple light pulses that seems to overcome this problem and restore the direct proportionality between PSII operating efficiency and the yield of photosynthetic electron transport at high irradiances.

We have characterized and discussed the physiological meaning of a number of fluorescence parameters commonly used in both laboratory and field studies. From these parameters it is possible, by their rearrangement and/or combination, to derive additional ones that provide further or more direct insight into specific aspects of the photosynthetic process (see, for example, Rohacek, 2002). The reader should be cautioned to pay close attention to the symbols and expressions used by different authors for the various fluorescence parameters as, regretfully, no agreement on terminology has been achieved so far. Chl fluorometry is undoubtedly a valuable technique to analyse aspects not only related to PSII functioning but also to whole leaf photosynthesis when a number of specific conditions are met. The technique is very often used to investigate the manner in which certain stress conditions affect the photosynthetic process, but there are a number of situations in which it cannot be used at all or does not allow to make valid comparisons between treated samples and controls. Two of such situations are rather common and worth recalling: first, when the stress causes the leaves to display a non-uniform colour or even changes its colour relative to the controls, as sometimes happens with mineral deficiency/ toxicity situations; second, when the stress causes important changes in the leaves' internal structure or hydration status, as for example under severe water deficiency conditions. Also, in most situations, one has to resort to information provided by several fluorescence parameters to correctly interpret the measured results. Extrapolations from the PSII operational efficiency to electron transport and C[O.sub.2] assimilation rates, particularly under stressful conditions, also require assumptions that are often not met and may lead to erroneous interpretations. Changes in photorespiration or cyclic/pseudo-cyclic electron transport rates, for example, relative to controls, make the relationships between PSII operational efficiency and whole leaf photosynthetic features not valid.

Appendix

BOX: PSII and singlet oxygen production

When the PSII reaction center is in its state [P.sub.680].Pheo.[Q.sub.A.sup.-] (closed), charge separation between [P.sub.680] and Pheo is strongly restrained by the repulsive electrostatic effect of [Q.sub.A.sup.-], but it still can occur. Subsequent stabilization of the separated charges, however, is prevented because [Q.sub.A] is already reduced. The high energy electron in [Pheo.sup..-] can then return to [P.sub.680.sup..+], where it may undergo spin reversion to generate the lower energy triplet state of the excited primary electron donor of PSII, [sup.3][P.sub.680.sup.*]. This long-lived triplet state is unable to initiate any productive photochemistry, but it can react rather readily with ground state molecular oxygen, [sup.3][O.sub.2], exciting it to the higher energy singlet state, [sup.1][O.sub.2.sup.*]. Remember that the ground state of oxygen is a triplet state with two unpaired electrons of parallel spins, which makes its effectiveness as an oxidant very low; in the new excited singlet state, the spin of one of the unpaired electrons is reversed and the oxidizing ability of oxygen towards organic molecules is greatly enhanced. Singlet oxygen is thus a highly reactive form of oxygen that can cause extensive modifications to lipids, proteins and nucleic acids, and the chloroplasts have developed a number of defence mechanisms against its potential damaging effects. Among the molecules that protect the thylakoid membranes against singlet oxygen are carotenoids and tocopherols and it is proposed that a [beta]-carotene molecule located at the PSII reaction center serves to quench singlet oxygen that may there be produced.

[sup.3][P.sub.680.sup.*] + [sup.3][O.sub.2] [right arrow] [sup.1][P.sub.680] + [sup.1][O.sub.2.sup.*]

[sup.1][O.sub.2.sup.*] + [sup.1][beta]-Car [right arrow] [sup.3][O.sub.2] + [sup.3][beta]-[Car.sup.*]

[sup.3][beta]-[Car.sup.*] [right arrow] [sup.1][beta]-Car + heat

In the PSII triplet chlorophyll molecules can also be formed directly in the antenna by intersystem crossing of singlet excited chlorophylls. This occurs particularly under conditions where there is an accumulation of excitation energy which lengthens the lifetime of singlet excited chlorophylls and increases the likelihood of spin inversion. Carotenoid molecules appropriately located throughout the antenna complex can directly quench these chlorophyll triplets and subsequently loose their excess energy as heat, thus preventing the formation of the singlet oxygen and, therefore of oxidative stress.

[sup.3][Chl.sup.*] + [sup.1]Car [right arrow] [sup.1]Chl + [sup.3][Car.sup.*]

[sup.3][Car.sup.*] [right arrow] [sup.1]Car + heat

Because of its short lifetime in vivo and its high reactivity, it is likely that singlet oxygen damages principally thylakoid components close to the site of its production. The subunit DI of PSII reaction center is a preferential target of [sup.1][O.sub.2.sup.*], and this protein was shown to display an extremely high turnover rate, particularly under excess light conditions in which chlorophyll triplets are mainly formed. This selective destruction of D1, followed by its repair, has thus been regarded as a safety valve that serves to prevent further damage to PSII.

Acknowledgements The author would like to thank his colleague Victor Conceicao Martins for drawing the figures included in this review.

Published online: 17 June 2009

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(1) Strictly, this relationship is only valid when a number of assumptions are made, which do not hold under most conditions. In particular, the rate constants for fluorescence and thermal energy dissipation should not change during photochemistry, but it is known that the rate constant for the latter processes increases significantly as the number of closed reaction centers increases. This relationship also implies that all Chl excited states generated in the antenna are equivalent, which is certainly not true given the heterogeneity of PSII organization in the thylakoid membrane.

(2) It should be noted that there is experimental evidence for a light-independent reduction of PQ, possibly with electrons from different origins. Lately, a dark electron transport chain associated with chlororespiration is being characterized that is a probable source of electrons for PQ reduction in the dark, although it is quantitatively a minor process, at least in higher plant chloroplasts. Because of equilibration of PQ with [Q.sub.A] it is possible that some PSII reaction centers may remain closed even in the dark. To assure maximal opening of reaction centers, sometimes the sample is initially illuminated with a far-red light that stimulates preferentially PSI activity and reoxidizes any reduced plastoquinone molecules.

(3) There is some simplification in this statement. Indeed, there is always part of excited states that are deactivate non-radiatively, for example, by inter-crossing system. Also, the higher energy of excited states created by absorption of blue photons is partly deactivated as heat loss.

(4) These changes in fluorescence yield following illumination with actinic light of a dark-adapted leaf, termed fluorescence induction, are also known as Kautsky effect, after his discoverer. Indeed, Kautsky and Hirsch (1931) were the first to report such changes and Kautsky postulated that the rise phase of this transient reflected the primary photochemistry of photosynthesis, whereas the declining phase was correlated to the onset of C[O.sub.2] assimilation. This interpretation was advanced ca. 30 years before Duysens and Sweers (1963) provided the basic understanding of variable fluorescence in PSII and has proven essentially correct, being now considered a cornerstone of photosynthesis research.

(5) Schreiber and Neubauer (1987) used a different notation for the fluorescence rise (O-[I.sub.1]-[I.sub.2]-M) that is currently less used.

(6) The reciprocal of the quantum yield is called quantum requirement and it is often used in efficiency analysis of photosynthetic energy conversion.

(7) The use of saturating light pulses on a background of actinic light is called the "light-doubling" technique and was first introduced by Bradbury and Baker, in 1981; it allows the transient closure of all PSII reaction centers and, thus, momentarily turns off the photochemical quenching, revealing the maximum fluorescence level in the light required to calculate the non-photochemical quenching.

(8) To achieve full closure of PSII reaction centers in the light, a pulse of very high light intensity, of the order of several thousand [micro]mol photons [m.sup.-2] [s.sup.-1] as to be used. In the field, when the plant is under high PFD and excitation energy is being effectively dissipated by non-photochemical quenching, maximum fluorescence level is often not attained and this leads to errors in the estimation of several fluorescence parameters.

(9) Note that in the light it is not possible to fully turn off the non-photochemical quenching in order no obtain a fluorescence level that could be used as a reference (zero level) for calculation of the extent of that process; in dark-adapted leaves, however, there is no qN, and thus measurements of non-photochemical quenching are made using as reference the Fm level measured in the dark-adapted state.

(10) It becomes clear, then, that the quantity 1-qP=(Fs'-[F.sub.0]')/(Fm'-[F.sub.0']) doesn't exactly indicate the proportion of closed RCII. This quantity is sometimes referred to as "excitation pressure".

(11) Formation of triplet states and state 2 transitions, that occur preferentially under high and low irradiance, respectively, also account for some fluorescence quenching, but generally for a very minor fraction of this.

Fernando S. Henriques [1,2,3]

[1] Plant Biology Unit, Department of Life Sciences, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal

[2] Grupo de Disciplinas de Biologia Vegetal, Departamento de Ciencias da Vida, Faculdade de Ciencias e Tecnologia da UNL, Quinta da Torre, 2829-516 Caparica, Portugal

[3] Author for Correspondence; e-mail: ffh@fct.unl.pt
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