Shining Light into a "Black Box": Essential Rationale Underlying Multiphase Flash Methodology.

The world we live in is very fragile. Sustainable food production is increasingly under intense pressure due to changing environmental conditions on many levels. Understanding the complexities of how to optimize food production under increasingly deleterious environmental conditions is dependent upon accurate and detailed analyses of plant productivity from the molecular-to-the-remote scales. One method that can link many of these scales has been around for decades, namely, pulse amplitude modulation (PAM) chlorophyll a fluorescence. This technique is used to measure an assortment of important parameters based on chlorophyll a fluorescence. One of the parameters measured by this method is termed the steady state maximum fluorescence yield ( Φ Fm ' ). This parameter, while extremely informative when used to quantify an assortment of processes of intense scientific interest, is nonetheless subject to intrinsic underestimation. A clever approach has evolved over several decades to more accurately estimate Φ Fm ' . The underlying rationale of the methodology requires a thorough and nuanced explanation, which is lacking in the literature. Herein, we systematically develop the essential rationale for accurately measuring Φ Fm ' based on the latest evolution of this approach, called multiphase flash (MPF) methodology.

Short-term warming does not affect intrinsic thermotolerance but induces strong sustaining photoprotection in tropical evergreen citrus genotypes.

Consequences of warming and postwarming events on photosynthetic thermotolerance (PT ) and photoprotective responses in tropical evergreen species remain elusive. We chose Citrus to answer some of the emerging questions related to tropical evergreen species' PT behaviour including (i) how wide is the genotypic variation in PT ? (ii) how does PT respond to short-term warming and (iii) how do photosynthesis and photoprotective functions respond over short-term warming and postwarming events? A study on 21 genotypes revealed significant genotypic differences in PT , though these were not large. We selected five genotypes with divergent PT and simulated warming events: Tmax 26/20°C (day-time highest maximum/night-time lowest maximum) (Week 1) < Tmax 33/30°C (Week 2) < Tmax 36/32°C (Week 3) followed by Tmax 26/16°C (Week 4, recovery). The PT of all genotypes remained unaltered despite strong leaf megathermy (leaf temperature > air temperature) during warming events. Though moderate warming showed genotype-specific stimulation in photosynthesis, higher warming unequivocally led to severe loss in net photosynthesis and induced higher nonphotochemical quenching. Even after a week of postwarming, photoprotective mechanisms strongly persisted. Our study points towards a conservative PT in evergreen citrus genotypes and their need for sustaining higher photoprotection during warming as well as postwarming recovery conditions.

Fresh perspectives on an established technique: Pulsed amplitude modulation chlorophyll a fluorescence.

Pulsed amplitude modulation (PAM) chlorophyll a fluorescence provides information about photosynthetic energy transduction. When reliably measured, chlorophyll a fluorescence provides detailed information about critical in vivo photosynthetic processes. Such information has recently provided novel and critical insights into how the yield potential of crops can be improved and it is being used to understand remotely sensed fluorescence, which is termed solar-induced fluorescence and will be solely measured by a satellite scheduled to be launched this year. While PAM chlorophyll a fluorometers measure fluorescence intensity per se, herein we articulate the axiomatic criteria by which instrumentally detected intensities can be assumed to assess fluorescence yield, a phenomenon quite different than fluorescence intensity and one that provides critical insight about how solar energy is variably partitioned into the biosphere. An integrated mathematical, phenomenological, and practical discussion of many useful chlorophyll a fluorescence parameters is presented. We draw attention to, and provide examples of, potential uncertainties that can result from incorrect methodological practices and potentially problematic instrumental design features. Fundamentals of fluorescence measurements are discussed, including the major assumptions underlying the signals and the methodological caveats about taking measurements during both dark- and light-adapted conditions. Key fluorescence parameters are discussed in the context of recent applications under environmental stress. Nuanced information that can be gleaned from intra-comparisons of fluorescence-derived parameters and intercomparisons of fluorescence-derived parameters with those based on other techniques is elucidated.

Photosynthesis: a multiscopic view.

A recurring analogy for photosynthesis research is the fable of the blind men and the elephant. Photosynthesis has many complex working parts, which has driven the need to study each of them individually, with an inherent understanding that a more complete picture will require systematic integration of these views. However, unlike the blind men, who are limited to using their hands, researchers have developed over the past decades a repertoire of methods for studying these components, many of which capitalize on unique features intrinsic to each. More recent concerns about food security and clean, renewable energy have increased support for applied photosynthesis research, with the idea of either improving photosynthetic performance as a desired trait in select species or using photosynthetic measurements as a phenotyping tool in breeding efforts or for high precision crop management. In this review, we spotlight the migration of approaches for studying photosynthesis from the laboratory into field environments, highlight some recent advances and speculate on areas where further development would be fruitful, with an eye towards how applied photosynthesis research can have impacts at local and global scales.

Dehydration Stress Memory: Gene Networks Linked to Physiological Responses During Repeated Stresses of Zea mays.

Stress memory refers to the observation that an initial, sub-lethal stress alters plants' responses to subsequent stresses. Previous transcriptome analyses of maize seedlings exposed to a repeated dehydration stress has revealed the existence of transcriptional stress memory in Zea mays. Whether drought-related physiological responses also display memory and how transcriptional memory translates into physiological memory are fundamental questions that are still unanswered. Using a systems-biology approach we investigate whether/how transcription memory responses established in the genome-wide analysis of Z. mays correlate with 14 physiological parameters measured during a repeated exposure of maize seedlings to dehydration stress. Co-expression network analysis revealed ten gene modules correlating strongly with particular physiological processes, and one module displaying strong, yet divergent, correlations with several processes suggesting involvement of these genes in coordinated responses across networks. Two processes key to the drought response, stomatal conductance and non-photochemical quenching, displayed contrasting memory patterns that may reflect trade-offs related to metabolic costs versus benefits of cellular protection. The main contribution of this study is the demonstration of coordinated changes in transcription memory responses at the genome level and integrated physiological responses at the cellular level upon repetitive stress exposures. The results obtained by the network-based systems analysis challenge the commonly held view that short-term physiological responses to stress are primarily mediated biochemically.

Sub-saturating Multiphase Flash Irradiances to Estimate Maximum Fluorescence Yield.

Many intricacies of leaf-level photosynthesis can be probed by combining infrared gas analysis with pulse-amplitude-modulation chlorophyll a fluorometry. A key fluorescence yield (ΦF) parameter required for estimating many of the phenomena associated with the light reactions of photosynthesis is referred to as the maximum ΦF, which is termed Fm' when measured on a light-adapted leaf. While ubiquitously used to assess many aspects of photosynthesis, Fm' is problematic because it is prone to being underestimated. This error can be propagated to parameters and phenomena that are based on estimation of Fm'. Theoretical and experimental observations have shown that ΦF increases hyperbolically in response to increasing irradiance, asymptotically approaching the maximum ΦF, or Fm', at extreme irradiances. Importantly, depending upon the convexity of the hyperbolic response, ΦF exhibits a linear and inverse relationship with the reciprocal of irradiance, a relationship previously referred to as a reciprocal plot. Given the negative slope of the reciprocal plot, estimates of ΦF at infinite irradiance can be obtained, even over sub-saturating irradiances, by linear regression and extrapolation of the resultant reciprocal plot to the y-intercept. Here, we show how to obtain data from a dynamic multiphase flash of sub-saturating irradiance, occurring within the time span of ~1 s, to generate a reciprocal plot that subsequently provides an accurate estimate of ΦF at infinite irradiance, or Fm'.

Lutein accumulation in the absence of zeaxanthin restores nonphotochemical quenching in the Arabidopsis thaliana npq1 mutant.

Plants protect themselves from excess absorbed light energy through thermal dissipation, which is measured as nonphotochemical quenching of chlorophyll fluorescence (NPQ). The major component of NPQ, qE, is induced by high transthylakoid DeltapH in excess light and depends on the xanthophyll cycle, in which violaxanthin and antheraxanthin are deepoxidized to form zeaxanthin. To investigate the xanthophyll dependence of qE, we identified suppressor of zeaxanthin-less1 (szl1) as a suppressor of the Arabidopsis thaliana npq1 mutant, which lacks zeaxanthin. szl1 npq1 plants have a partially restored qE but lack zeaxanthin and have low levels of violaxanthin, antheraxanthin, and neoxanthin. However, they accumulate more lutein and alpha-carotene than the wild type. szl1 contains a point mutation in the lycopene beta-cyclase (LCYB) gene. Based on the pigment analysis, LCYB appears to be the major lycopene beta-cyclase and is not involved in neoxanthin synthesis. The Lhcb4 (CP29) and Lhcb5 (CP26) protein levels are reduced by 50% in szl1 npq1 relative to the wild type, whereas other Lhcb proteins are present at wild-type levels. Analysis of carotenoid radical cation formation and leaf absorbance changes strongly suggest that the higher amount of lutein substitutes for zeaxanthin in qE, implying a direct role in qE, as well as a mechanism that is weakly sensitive to carotenoid structural properties.

Lutein can act as a switchable charge transfer quencher in the CP26 light-harvesting complex.

Energy-dependent quenching of excitons in photosystem II of plants, or qE, has been positively correlated with the transient production of carotenoid radical cation species. Zeaxanthin was shown to be the donor species in the CP29 antenna complex. We report transient absorbance analyses of CP24 and CP26 complexes that bind lutein and zeaxanthin in the L1 and L2 domains, respectively. For CP24 complexes, the transient absorbance difference profiles give a reconstructed transient absorbance spectrum with a single peak centered at approximately 980 nm, consistent with zeaxanthin radical cation formation. In contrast, CP26 gives constants for the decay components probed at 940 and 980 nm of 144 and 194 ps, a transient absorbance spectrum that has a main peak at 980 nm, and a substantial shoulder at 940 nm. This suggests the presence of two charge transfer quenching sites in CP26 involving zeaxanthin radical cation and lutein radical cation species. We also show that lutein radical cation formation in CP26 is dependent on binding of zeaxanthin to the L2 domain, implying that zeaxanthin acts as an allosteric effector of charge transfer quenching involving lutein in the L1 domain.

Kinetic modeling of charge-transfer quenching in the CP29 minor complex.

We performed transient absorption (TA) measurements on CP29 minor light-harvesting complexes that were reconstituted in vitro with either violaxanthin (Vio) or zeaxanthin (Zea) and demonstrate that the Zea-bound CP29 complexes exhibit charge-transfer (CT) quenching that has been correlated with the energy-dependent quenching (qE) in higher plants. Simulations of the difference TA kinetics reveal two-phase kinetics for intracomplex energy transfer to the CT quenching site in CP29 complexes, with a fast <500 fs component and a approximately 6 ps component. Specific chlorophyll sites within CP29 are identified as likely locations for CT quenching. We also construct a kinetic model for CT quenching during qE in an intact system that incorporates CP29 as a CT trap and show that the model is consistent with previous in vivo measurements on spinach thylakoid membranes. Finally, we compare simulations of CT quenching in thylakoids with those of the individual CP29 complexes and propose that CP29 rather than LHCII is a site of CT quenching.

Architecture of a charge-transfer state regulating light harvesting in a plant antenna protein.

Energy-dependent quenching of excess absorbed light energy (qE) is a vital mechanism for regulating photosynthetic light harvesting in higher plants. All of the physiological characteristics of qE have been positively correlated with charge transfer between coupled chlorophyll and zeaxanthin molecules in the light-harvesting antenna of photosystem II (PSII). We found evidence for charge-transfer quenching in all three of the individual minor antenna complexes of PSII (CP29, CP26, and CP24), and we conclude that charge-transfer quenching in CP29 involves a delocalized state of an excitonically coupled chlorophyll dimer. We propose that reversible conformational changes in CP29 can "tune" the electronic coupling between the chlorophylls in this dimer, thereby modulating the energy of the chlorophyll-zeaxanthin charge-transfer state and switching on and off the charge-transfer quenching during qE.

Zeaxanthin radical cation formation in minor light-harvesting complexes of higher plant antenna.

Previous work on intact thylakoid membranes showed that transient formation of a zeaxanthin radical cation was correlated with regulation of photosynthetic light-harvesting via energy-dependent quenching. A molecular mechanism for such quenching was proposed to involve charge transfer within a chlorophyll-zeaxanthin heterodimer. Using near infrared (880-1100 nm) transient absorption spectroscopy, we demonstrate that carotenoid (mainly zeaxanthin) radical cation generation occurs solely in isolated minor light-harvesting complexes that bind zeaxanthin, consistent with the engagement of charge transfer quenching therein. We estimated that less than 0.5% of the isolated minor complexes undergo charge transfer quenching in vitro, whereas the fraction of minor complexes estimated to be engaged in charge transfer quenching in isolated thylakoids was more than 80 times higher. We conclude that minor complexes which bind zeaxanthin are sites of charge transfer quenching in vivo and that they can assume Non-quenching and Quenching conformations, the equilibrium LHC(N) <==> LHC(Q) of which is modulated by the transthylakoid pH gradient, the PsbS protein, and protein-protein interactions.

Regulating the proton budget of higher plant photosynthesis.

In higher plant chloroplasts, transthylakoid proton motive force serves both to drive the synthesis of ATP and to regulate light capture by the photosynthetic antenna to prevent photodamage. In vivo probes of the proton circuit in wild-type and a mutant strain of Arabidopsis thaliana show that regulation of light capture is modulated primarily by altering the resistance of proton efflux from the thylakoid lumen, whereas modulation of proton influx through cyclic electron flow around photosystem I is suggested to play a role in regulating the ATP/NADPH output ratio of the light reactions.

Plasticity in light reactions of photosynthesis for energy production and photoprotection.

Plant photosynthesis channels some of the most highly reactive intermediates in biology, in a way that captures a large fraction of their energy to power the plant. A viable photosynthetic apparatus must not only be efficient and robust machinery, but also well integrated into the plant's biochemical and physiological networks. This requires flexibility in its responses to the dramatically changing environmental conditions and biochemical demands. First, the output of the energy-storing light reactions must match the demands of plant metabolism. Second, regulation of the antenna must be flexible to allow responses to diverse challenges that could result in excess light capture and subsequent photoinhibition. Evidence is presented for the interplay of two types of mechanistic flexibility, one that modulates the relative sensitivity of antenna down-regulation to electron flow, and the other, which primarily modulates the output ratio of ATP/NADPH, but also contributes to down-regulation.

Dynamic flexibility in the light reactions of photosynthesis governed by both electron and proton transfer reactions.

Plant photosynthesis performs the remarkable feat of converting light energy into usable chemical forms, which involves taming highly reactive intermediates without harming plant cells. This requires an apparatus that is not only efficient and robust but also flexible in its responses to changing environmental conditions. It also requires that the output of the energy-storing reactions be matched with the demands of metabolism. This article addresses the mechanisms by which this flexibility is achieved for short-term environmental changes. We argue that chloroplasts need two types of flexible mechanisms: one for modulating the output ratio of ATP:NADPH, which involves cyclic electron flux around photosystem I; and another for changing the regulatory sensitivity of the light-harvesting antenna to electron (and proton) flow.

Modulation of energy-dependent quenching of excitons in antennae of higher plants.

Energy-dependent exciton quenching, or q(E), protects the higher plant photosynthetic apparatus from photodamage. Initiation of q(E) involves protonation of violaxanthin deepoxidase and PsbS, a component of the photosystem II antenna complex, as a result of lumen acidification driven by photosynthetic electron transfer. It has become clear that the response of q(E) to linear electron flow, termed "q(E) sensitivity," must be modulated in response to fluctuating environmental conditions. Previously, three mechanisms have been proposed to account for q(E) modulation: (i) the sensitivity of q(E) to the lumen pH is altered; (ii) elevated cyclic electron flow around photosystem I increases proton translocation into the lumen; and (iii) lowering the conductivity of the thylakoid ATP synthase to protons (g(H+)) allows formation of a larger steady-state proton motive force (pmf). Kinetic analysis of the electrochromic shift of intrinsic thylakoid pigments, a linear indicator of transthylakoid electric field component, suggests that, when CO(2) alone was lowered from 350 ppm to 50 ppm CO(2), modulation of q(E) sensitivity could be explained solely by changes in conductivity. Lowering both CO(2) (to 50 ppm) and O(2) (to 1%) resulted in an additional increase in q(E) sensitivity that could not be explained by changes in conductivity or cyclic electron flow associated with photosystem I. Evidence is presented for a fourth mechanism, in which changes in q(E) sensitivity result from variable partitioning of proton motive force into the electric field and pH gradient components. The implications of this mechanism for the storage of proton motive force and the regulation of the light reactions are discussed.