Chloramphenicol effects on chlorophyll degradation and photosystem I assembly in the chlorina CD3 wheat mutant

We previously reported that applications of chloramphenicol to the chlorina wheat mutant, CD3, decreased the leaf Chl a/b ratio and enhanced accumulations of LHC proteins and LHC complexes during greening (Duysen et al. 1985). We have now examined Chl degradation and the change in Chl a/b ratios in wheat leaves kept in the dark as a measure of LHC destruction. Chl b was stable in chloroplasts of the CD3 wheat kept in darkness up to 5 days. Chloramphenicol significantly increased Chl b accumulations and impaired Chl a degradation in both CD3 mutant and normal wheat relative to untreated plants. Our Chl data suggest that the chloramphenicol induced accumulation of the LHC complex in the mutant wheat results from enhanced processing of LHC into the membrane rather than impairment of LHC degradation. The photosystem I (PSI) fraction of the CD3 wheat mutant was examined relative to that of normal wheat after 3 days greening. PSI was deficient in 25, 26, 26.5 kD LHCI protein in the mutant but both wheats accumulated low quantities of the 27–29 kD LHCII protein as detected by Western blot analysis. Chloramphenicol enhanced accumulations of several LHCI proteins primarily near 25 kD in the mutant and the 27–29 kD LHCII protein in normal wheat. The fluorescence emission and absorbance spectra suggest that chloramphenicol enhances accumulations of dissociated LHC in the PSI preparation of normal and CD3 mutant wheat.A contribution of North Dakota Agricultural Experiment Station. Published with approval of the Director as Journal Paper Number 1563.     

Light-dependent, chilling effects on phosphorylation of thylakoid proteins and consequences for associated photochemical activities in maize

When maize ( Zea mays L. cv. LG11) leaves are exposed to low temperatures and high light modifications to both photosystem 2 (PS2) and the light-harvesting chlorophyll a/b protein complex associated with photosystem 2 (LHC2) occur. This study examines the consequences of these modifications for phosphorylation of LHC2 and PS2 polypeptides and the associated changes in electron transport. Maize leaves were chilled at 5°C for 6 h under photon flux densities of 1 500 and 250 μmol m^-2 s^-1. Thylakoids were then isolated from the leaves and their abilities to phosphorylate LHC2 and PS2 polypeptides and modify electron transport activities were determined. Measurements of chlorophyll fluorescence induction in the thylakoids were also made. Thylakoids isolated from leaves chilled under high light and from leaves kept in the ambient growth environment had similar phosphoprotein profiles. However, polypeptide phosphorylation in thylakoids from the chilled leaves did not produce a decrease in PS2 electron transport. Chilling leaves under low light produced a decrease in the ability of isolated thylakoids to phosphorylate PS2, but not LHC2, polypeptides, which was not associated with any change in the phosphorylation-induced decrease in PS2 electron transport. Chilling under high, but not low, light appears to produce changes in membrane organisation that do not affect the ability of the thylakoids to phosphorylate PS2 and LHC2 polypeptides, but which do prevent the phosphorylation-induced decrease in excitation energy transfer from LHC2 to PS2.     

Influence of thylakoid protein phosphorylation on photosynthetic electron transport and photophosphorylation

Data are reported which show that thylakoid protein phosphorylation decreases photosystem II fluorescence yield and enhances the photosystem I dependent photophosphorylation catalyzed by phenazinemethosulphate in the presence of DCMU. The stimulation is larger at low light intensity, but is still observed at high intensity. These observations are interpreted to demonstrate that thylakoid protein phosphorylation causes a transfer of excitation energy from PS II to PS I, but may also have an independent stimulatory effect on PS I dependent photophosphorylation.     

The effect of the dark interval in intermittent light on thylakoid development: photosynthetic unit formation and light harvesting protein accumulation

Etiolated bean plants were grown in intermittent light with dark intervals of shorter or longer duration, to modulate the rate of chlorophyll accumulation, relative to that of the other thylakoid components formed. We thus produced conditions under which chlorophyll becomes more or less a limiting factor. We then tested whether LHC complexes can be incorporated in the thylakoid. It was found that an equal amount of chlorophyll, formed under the same total irradiation received, may be used for the stabilization of few and large-in-size PS units containing LHC components (short dark-interval intermittent light), or for the stabilization of many and small-in-size PS units with no LHC components (long dark-interval intermittent light). The size of the PS units diminishes as the dark-interval duration is increased, with no further change after 98 minutes. The PSII/cytf ratio remains constant throughout development in intermittent light and equal to that of mature chloroplasts (PSII/cytf = 1) except in the case of very long dark-interval regimes, where about half PSII units per cytf are present. The PSII/PSI ratio was found to be correlated with the PSII unit size (the larger the size, the lower the ratio). The number of PSI units operating on the same electron transfer chain varied depending on the size of the PSII unit (the larger the PSII unit size, the more the PSI units per chain). The results suggest that it is not the chlorophyll content per se which regulates the stabilization of LHC in developing thylakoids and consequently the size of the PS units, but rather the rate by which it is accumulated, relative to that of the other thylakoid components.Abbreviations Chl Chlorophyll - CL Continuous light - CPa the reaction center complex of PSII - CPI the reaction center complex of PSI - CPIa Chlorophyll protein complex containing the CPI and the light harvesting complex of PSI - fr w fresh weight - LDC Light dark cycles - LHC-I Light-harvesting complex of PSI - LHC-II Light harvesting complex of PSII - PS photosystem - PSI photosystem I - PSII photosystem II     

Correlation between persistent forms of zeaxanthin-dependent energy dissipation and thylakoid protein phosphorylation

High light stress induced not only a sustained form of xanthophyll cycle-dependent energy dissipation but also sustained thylakoid protein phosphorylation. The effect of protein phosphatase inhibitors (fluoride and molybdate ions) on recovery from a 1-h exposure to a high PFD was examined in leaf discs of Parthenocissus quinquefolia (Virginia creeper). Inhibition of protein dephosphorylation induced zeaxanthin retention and sustained energy dissipation (NPQ) upon return to low PFD for recovery, but had no significant effects on pigment and Chl fluorescence characteristics under high light exposure. In addition, whole plants of Monstera deliciosa and spinach grown at low to moderate PFDs were transferred to high PFDs, and thylakoid protein phosphorylation pattern (assessed with anti-phosphothreonine antibody) as well as pigment and Chl fluorescence characteristics were examined over several days. A correlation was obtained between dark-sustained D1/D2 phosphorylation and dark-sustained zeaxanthin retention and maintenance of PS II in a state primed for energy dissipation in both species. The degree of these dark-sustained phenomena was more pronounced in M. deliciosa compared with spinach. Moreover, M. deliciosa but not spinach plants showed unusual phosphorylation patterns of Lhcb proteins with pronounced dark-sustained Lhcb phosphorylation even under low PFD growth conditions. Subsequent to the transfer to a high PFD, dark-sustained Lhcb protein phosphorylation was further enhanced. Thus, phosphorylation patterns of D1/D2 and Lhcb proteins differed from each other as well as among plant species. The results presented here suggest an association between dark-sustained D1/D2 phosphorylation and sustained retention of zeaxanthin and energy dissipation (NPQ) in light-stressed, and particularly photoinhibited, leaves. Functional implications of these observations are discussed.This revised version was published online in October 2005 with corrections to the Cover Date.     

Excitation energy trapping in photosystem I complexes depleted in Lhca1 and Lhca4

We report a time-resolved fluorescence spectroscopy characterization of photosystem I (PSI) particles prepared from Arabidopsis lines with knock-out mutations against the peripheral antenna proteins of Lhca1 or Lhca4. The first mutant retains Lhca2 and Lhca3 while the second retains one other light-harvesting protein of photosystem I (Lhca) protein, probably Lhca5. The results indicate that Lhca2/3 and Lhca1/4 each provides about equally effective energy transfer routes to the PSI core complex, and that Lhca5 provides a less effective energy transfer route. We suggest that the specific location of each Lhca protein within the PSI-LHCI supercomplex is more important than the presence of so-called red chlorophylls in the Lhca proteins.     

An investigation of the mechanistic aspects of excitation energy redistribution following thylakoid membrane protein phosphorylation

Accompanying thylakoid membrane protein phosphorylation is a redistribution of energy between PS II and PS I; mechanistic aspects of this redistribution have been investigated in both a mature and a developing chloroplast system. Data are presented which suggest that the mechanism of these changes is dependent upon the developmental status and/or morphological characteristics of the chloroplast.     

Radiation inactivation analysis of thylakoid protein kinase systems in light and in darkness

Chloroplast thylakoid contains several membrane-bound protein kinases that phosphorylate thylakoid polypeptides for the regulation of photosynthesis. Thylakoid protein phosphorylation is activated when the plastoquinone pool is reduced either by light-dependent electron flow through photosystem 2 (PS2) or by adding exogenous reductants such as durohydroquinone in the dark. The major phosphorylated proteins on thylakoid are components of light-harvesting complex 2 (LHC2) and a PS2 associated 9 kDa phosphoprotein. Radiation inactivation technique was employed to determine the functional masses of various kinases for protein phosphorylation in thylakoids. Under the photosynthetically active radiation (PAR), the apparent functional masses of thylakoid protein kinase systems (TPKXs) for catalyzing phosphorylation of LHC2 27 and 25 kDa polypeptides were 540±50 and 454±35 kDa as well as it was 448±23 kDa for PS2 9 kDa protein phosphorylation. Furthermore, the functional sizes of dark-regulated TPKXs for 25 and 9 kDa proteins were 318±25 and 160±8 kDa. The 9 kDa protein phosphorylation was independent of LHC2 polypeptides phosphorylation with regard to its TPKX functional mass. Target size analysis of protein phosphorylation mentioned above indicates that thylakoid contains a group of distinct protein kinase systems. A working model is accordingly proposed to interpret the interaction between these protein kinase systems.     

Estimation of the light distribution between photosystems I and II in intact wheat leaves by fluorescence and photoacoustic measurements

Usisng intact leaves, the extent of the decrease in chlorophyll a fluorescenece caused by the addition of continuous 710 nm light superimposed on modulated (20 Hz) 550 nm light was used to determine the distribution of this absorbed light between photosystems I () and II (). The F_o and F_m levels, which defined the total variable fluorescenece, were taken as equal to those obtained with excess 710 nm light and with saturating blue-green light, respectively.An analogous procedure was used with a photoacoustic detector, saturating white light defining a base line for oxygen yield, the levels with an without 710 nm light being used to define and respectively.The two methods gave similar values for the distribution of light between the two photosystems for the experimental conditions used, averaging 0.55 for a range of Triticum genotypes and Brachypodium sylvaticum grown in high or low light.     

Chloroplast biogenesis at cold-hardening temperatures. Development of photosystem I and photosystem II activities in relation to pigment accumulation

Chloroplast biogenesis during continuous illumination at either low, cold-hardening temperatures (5°C) or non-hardening temperatures (20°C) was examined by monitoring the etioplast-chloroplast transformation with respect to pigment accumulation and the development of PSI- and PSII-associated electron transport activities in winter rye (Secale cereale L. cv Puma). Generally, chlorophyll and carotenoid accumulation during greening at 20°C were characterized by rapid initial rates in contrast to pronounced, initial lag times during biogenesis at 5°C. Although greening temperature had no effect on the sequential appearance of PSI relative to PSII, greening temperature significantly altered the pattern of appearance of PSI relative to chlorophyll accumulation. Thylakoid biogenesis under continuous illumination at 20°C imposed a pattern whereby the development of PSI activity was antiparallel to chlorophyll accumulation. In contrast, the development of PSI activity under continuous illumination at 5°C was paralllel to chlorophylll accumulation. These developmental patterns were independent of the temperature experienced during etiolation. However, rye seedlings etiolated at 20°C and subsequently subjected to continuous illumination at 5°C exhibited a 70% reduction in the maximum PSII activity (100 mol DCPIP reduced.mg Chl^-1.h^-1) attained relative to that observed for similar etiolated seedlings greened at 20°C (300 mol DCPIP reduced.mg Chl^-1.h^-1). This low temperature-induced inhibition could be alleviated by an initial 2 h exposure to continuous light at 20°C prior to greening to 5°C. Rye seedlings etiolated at 5°C attained similar maximal PSII activities (300 mol DCPIP reduced.mg Chl^-1.h^-1) regardless of the greening temperature. We suggest that the altered kinetics for pigment accumulation, the low temperature-induced change in the pattern for the appearance of PSI activity relative to chlorophyll accumulation and the differential sensitivity of 20° and 5° etiolated seedlings to greening temperature reflect an alteration in membrane organization incurred as a consequence of thylakoid assembly at low temperature.Abbreviations RH cold-hardened rye - RNH non-hardened rye - MV methylviologen - ASC ascorbate - Chl chlorophyll - DCPIP dichlorophenol indophenol     

Thylakoid protein phosphorylation,state 1-state 2 transitions,and photosystem stoichiometry adjustment: redox control at multiple levels of gene expression

In photosynthesis in chloroplasts, control of thylakoid protein phosphorylation by redox state of inter-photosystem electron carriers makes distribution of absorbed excitation energy between the two photosystems self-regulating. During operation of this regulatory mechanism, reduction of plastoquinone activates a thylakoid protein kinase which phosphorylates the light-harvesting complex LHC II, causing a change in protein recognition that results in redistribution of energy to photosystem I at the expense of photosystem II, thus tending to oxidise the reduced plastoquinone pool. These events correspond to the transition from light-state 1 to light-state 2. The reverse transition (to light-state 1) is initiated by transient oxidation of plastoquinone, inactivation of the LHC II kinase, and return of dephosphorylated LHC II from photosystem I to photosystem II, supplying excitation energy to photosystem II and thereby reducing plastoquinone. State 1-state 2 transitions therefore operate by means of redox control of reversible, post-translational modification of pre-existing proteins. A balance in the rates of light utilization by photosystem I and photosystem II can also be achieved, on longer time-scales and between wider limits, by adjustment of the relative quantities, or stoichiometry, of photosystem I and photosystem II. Recent evidence suggests that adjustment of photosystem stoichiometry is also a response to perturbation of the redox state of inter-photosystem electron carriers, and involves specific redox control of de novo protein synthesis, assembly, and breakdown. It is therefore suggested that the same redox sensor initiates these different adaptations by control of gene expression at different levels, according to the time-scale and amplitude of the response. This integrated feedback control may serve to maintain redox homeostasis, and, as a result, quantum yield. Evidence for the components required by such systems is discussed.     

低温对水稻类囊体膜蛋白磷酸化及光合机构光能分配的影响

研究了低温胁迫对水稻类囊体膜蛋白磷酸化和光能分配的影响。类囊体膜蛋白组分的SDS-PAGE和免疫印迹分析结果显示,低温弱光条件下光系统Ⅱ(PSⅡ)功能蛋白的稳态水平均有所降低。低温(77K)荧光分析表明,低温处理后类囊体膜光能吸收明显下降,而且FPSⅡ/FPSⅠ的比值均较对照组下降,表明低温弱光条件下有更多的激发能被分配到PSⅠ。低温处理同时还改变了类囊体膜蛋白磷酸化水平,捕光天线LHCⅡ蛋白中lhcb1的磷酸化水平明显降低,lhcb2的磷酸化水平增加,进一步证实lhcb2向PSⅠ移动,改变光能分配。PSⅡ反应中心D1、D2蛋白和核心天线CP43的磷酸化水平增高,有利于PSⅡ二聚体的稳定。     

Spinach-thylakoid phosphorylation: Studies on the kinetics of changes in photosystem antenna size, spill-over and phosphorylation of light-harvesting chlorophyll a/b protein

The kinetics of LHCP phosphorylation and associated changes in photosystem cross-section and energy ‘spill-over’ from PS II to PS I have been examined in isolated spinach chloroplasts. During an initial phosphorylation period of 3–6 min, in the presence of saturating concentrations of Mg²⁺, the increase in PS I and decrease in PS II cross-section are largely completed, as judged by both measurements of the steady-state redox state of Q and fluorescence yield changes. This corresponds to a period of rapid ³²P incorporation into the low-molecular weight LHCP polypeptide. Subsequent to this initial 3–6-min period there is substantial further phosphorylation of both LHCP polypeptides, which is not accompanied by significant changes in photosystem cross-section, even after the chloroplasts had been unstacked with extensive mixing of PS I and PS II by Mg-removal. It is suggested that there exists a specific ‘mobile’ population of LHCP molecules which is rapidly phosphorylated and which may be enriched in the low-molecular-weight polypeptide. In addition, measurements of the kinetics of the ‘spill-over’ changes upon either Mg²⁺ addition or removal indicate that the continued phosphorylation of LHCP is able to increase the ‘spill-over’ process under favourable ionic conditions.     

Spectroelectrochemistry of P700 in native photosystem I particles and diethyl ether-treated thylakoid membranes from spinach and Thermosynechococcus elongatus

The redox potentials (E(composite function')) of P700 in intact and diethyl ether-treated thylakoid membranes as well as native photosystem (PS) I particles from spinach and Thermosynechococcus elongatus have been measured by a spectroelectrochemistry with an error range of +/-2-3 mV. Stepwise removal of antenna pigments by ether treatment caused distinct shifts of the E( composite function') value with increasing degree of water saturation in ether; negatively from +471 to +428 mV for spinach, but positively from +423 to +436 mV for T. elongatus. Such a contrasting behavior is discussed by invoking the mode of action of ether on the microenvironments around P700.     

Two forms of the Photosystem II D1 protein alter energy dissipation and state transitions in the cyanobacterium Synechococcus sp. PCC 7942

Synechococcus sp. PCC 7942 (Anacystis nidulans R2) contains two forms of the Photosystem II reaction centre protein D1, which differ in 25 of 360 amino acids. D1: 1 predominates under low light but is transiently replaced by D1:2 upon shifts to higher light. Mutant cells containing only D1:1 have lower photochemical energy capture efficiency and decreased resistance to photoinhibition, compared to cells containing D1:2. We show that when dark-adapted or under low to moderate light, cells with D1:1 have higher non-photochemical quenching of PS II fluorescence (higher q_N) than do cells with D1:2. This is reflected in the 77 K chlorophyll emission spectra, with lower Photosystem II fluorescence at 697–698 nm in cells containing D1:1 than in cells with D1:2. This difference in quenching of Photosystem II fluorescence occurs upon excitation of both chlorophyll at 435 nm and phycobilisomes at 570 nm. Measurement of time-resolved room temperature fluorescence shows that Photosystem II fluorescence related to charge stabilization is quenched more rapidly in cells containing D1:1 than in those with D1:2. Cells containing D1:1 appear generally shifted towards State II, with PS II down-regulated, while cells with D1:2 tend towards State I. In these cyanobacteria electron transport away from PS II remains non-saturated even under photoinhibitory levels of light. Therefore, the higher activity of D1:2 Photosystem II centres may allow more rapid photochemical dissipation of excess energy into the electron transport chain. D1:1 confers capacity for extreme State II which may be of benefit under low and variable light.Abbreviations D1 the atrazine-binding 32 kDa protein of the PS II reaction centre core - D1:1 the D1 protein constitutively expressed during acclimated growth in Synechococcus sp. PCC 7942 - D1:2 an alternate form of the D1 protein induced under excess excitation in Synechococcus sp. PCC 7942 - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - Fo minimal fluorescence in the dark-adapted state - Fo minimal fluorescence in a light-adapted state - F_M maximum fluorescence with all quenching mechanisms at a minimum, measured in presence of DCMU - F_M maximal fluorescence in a light-adapted state, measured with a saturating flash - F_Mdark maximal fluorescence in the dark-adapted state - F_V variable fluorescence in a light-adapted state (F_M-Fo) - PAM pulse amplitude modulated fluorometer - q_N non-photochemical quenching of PS II fluorescence - q_N (dark) q_N in the dark adapted state - q_P photochemical quenching of fluorescence     

Kinetics of pigment-acceptor interaction induced by continuous illumination in Synechocystis spaeroides photosystem I preparations cooled to 160 K in the dark and light

The kinetics of dark reduction of chlorophyll P700 oxidized by continuous light in preparations of photosystem I reaction centers from cyanobacterium Synechosystis spharoides cooled in the dark to 160 K is essentially nonexponential. The characteristic times of the components range from fractions of a second to minutes or more. During the cooling of reaction center preparations under illumination with actinic light, most of the chlorophyll P700 molecules are fixed in the oxidized state at 160 K. The kinetics of dark reduction of P700⁺ in the fraction of reaction centers that retain photochemical activity under these conditions is somewhat faster compared to the samples cooled in the dark. A theoretical analysis of substantial deceleration of P700⁺ dark recovery kinetics was done for preparations of photosystem I reaction centers oxidized by continuous light at 160 K in comparison to the experiments where reaction centers were oxidized by short single light flashes. This slowing down of the kinetics in samples excited by continuous illumination can be explained by microconformational relaxation processes related to proton shifts in the reaction center.     

Evidence for the operation of alternative electron transport routes through photosystem I in intact barley leaves under weak and moderate white light

The functioning of alternative routes of photosynthetic electron transport was analyzed from the kinetics of dark reduction of P700⁺ , an oxidized primary donor of PSI, in barley (Hordeum vulgare L.) leaves irradiated by white light of various intensities. Redox changes of P700 were monitored as absorbance changes at 830 nm using PAM 101 specialized device. Irradiation of dark-adapted leaves caused a gradual P700⁺ accumulation, and the steady-state level of oxidized P700 increased with intensity of actinic light. The kinetics of P700⁺ dark reduction after a pulse of strong actinic light, assayed from the absorbance changes at 830 nm, was fitted by a single exponential term with a halftime of 10–12 ms. Two slower components were observed in the kinetics of P700⁺ dark reduction after leaf irradiation by attenuated actinic light. The contribution of slow components to P700⁺ reduction increased with the decrease in actinic light intensity. Two slow components characterized by halftimes similar to those observed after leaf irradiation by weak white light were found in the kinetics of dark reduction of P700⁺ oxidized in leaves with far-red light specifically absorbed by PSI. The treatment of leaves with methyl viologen, an artificial PSI electron acceptor, significantly accelerated the accumulation of P700⁺ under light. At the same time, the presence of methyl viologen, which inhibits ferredoxin-dependent electron transport around PSI, did not affect three components of the kinetics of P700⁺ dark reduction obtained after irradiations with various actinic light intensities. It was concluded that some part of PSI reaction centers was not reduced by electron transfer from PSII under weak or moderate intensities of actinic light. In this population of PSI centers, P700⁺ was reduced via alternative electron transport routes. Insensitivity of the kinetics of P700⁺ dark reduction to methyl viologen evidences that the input of electrons to PSI from the reductants (NADPH or NADH) localized in the chloroplast stroma was effective under those light conditions.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 5–11.Original Russian Text Copyright © 2005 by Bukhov, Egorova.     

Plastoquinone redox control of chloroplast thylakoid protein phosphorylation and distribution of excitation energy between photosystems: discovery,background, implications

Chloroplast thylakoid protein phosphorylation was discovered, and the most conspicuous phosphoproteins identified, by John Bennett at Warwick University. His initial findings were published in 1977. The phosphoproteins included apoproteins of chloroplast light harvesting complex II. Thylakoid protein phosphorylation was shown to influence distribution of excitation energy between Photosystems I and II in 1979, during a visit by Bennett to the laboratory of Charles J. Arntzen at the University of Illinois at Urbana-Champaign. That work was published by Bennett, Katherine E. Steinback and Arntzen in 1980. Control of both protein phosphorylation and excitation energy distribution by the redox state of the plastoquinone pool was first established in 1980 during the author's visit to Arntzen's laboratory. The experiments were prompted by the realization that coupling between redox state of an inter-photosystem electron carrier and excitation energy distribution provides a concrete mechanism for adaptations known as state transitions. This work was published by Allen, Bennett, Steinback, and Arntzen in 1981. This discovery and its background are discussed, together with some implications for photosynthesis and for research generally. This minireview is a personal account of the Urbana-Warwick and related collaborations in 1979–83: it includes impressions, conjectures, and acknowledgements for which the author is solely responsible. This revised version was published online in June 2006 with corrections to the Cover Date.     

Determining the limitations and regulation of photosynthetic energy transduction in leaves

The light-dependent production of ATP and reductants by the photosynthetic apparatus in vivo involves a series of electron and proton transfers. Consideration is given as to how electron fluxes through photosystem I (PSI), using absorption spectroscopy, and through photosystem II (PSII), using chlorophyll fluorescence analyses, can be estimated in vivo. Measurements of light-induced electrochromic shifts using absorption spectroscopy provide a means of analyzing the proton fluxes across the thylakoid membranes in vivo. Regulation of these electron and proton fluxes is required for the thylakoids to meet the fluctuating metabolic demands of the cell. Chloroplasts exhibit a wide and flexible range of mechanisms to regulate electron and proton fluxes that enable chloroplasts to match light use for ATP and reductant production with the prevailing metabolic requirements. Non-invasive probing of electron fluxes through PSI and PSII, and proton fluxes across the thylakoid membranes can provide insights into the operation of such regulatory processes in vivo.