Proximity to Photosystem II is necessary for activation of Plastid Terminal Oxidase (PTOX) for photoprotection.

The Plastid Terminal Oxidase (PTOX) is a chloroplast localized plastoquinone oxygen oxidoreductase suggested to have the potential to act as a photoprotective safety valve for photosynthesis. However, PTOX overexpression in plants has been unsuccessful at inducing photoprotection, and the factors that control its activity remain elusive. Here, we show that significant PTOX activity is induced in response to high light in the model species Eutrema salsugineum and Arabidopsis thaliana. This activation correlates with structural reorganization of the thylakoid membrane. Over-expression of PTOX in mutants of Arabidopsis thaliana perturbed in thylakoid stacking also results in such activity, in contrast to wild type plants with normal granal structure. Further, PTOX activation protects against photoinhibition of Photosystem II and reduces reactive oxygen production under stress conditions. We conclude that structural re-arrangements of the thylakoid membranes, bringing Photosystem II and PTOX into proximity, are both required and sufficient for PTOX to act as a Photosystem II sink and play a role in photoprotection.

The electron transport chain of Shewanella oneidensis MR-1 can operate bidirectionally to enable microbial electrosynthesis.

Extracellular electron transfer is a process by which bacterial cells can exchange electrons with a redox-active material located outside of the cell. In Shewanella oneidensis, this process is natively used to facilitate respiration using extracellular electron acceptors such as Fe(III) or an anode. Previously, it was demonstrated that this process can be used to drive the microbial electrosynthesis (MES) of 2,3-butanediol (2,3-BDO) in S. oneidensis exogenously expressing butanediol dehydrogenase (BDH). Electrons taken into the cell from a cathode are used to generate NADH, which in turn is used to reduce acetoin to 2,3-BDO via BDH. However, generating NADH via electron uptake from a cathode is energetically unfavorable, so NADH dehydrogenases couple the reaction to proton motive force. We therefore need to maintain the proton gradient across the membrane to sustain NADH production. This work explores accomplishing this task by bidirectional electron transfer, where electrons provided by the cathode go to both NADH formation and oxygen (O2) reduction by oxidases. We show that oxidases use trace dissolved oxygen in a microaerobic bioelectrical chemical system (BES), and the translocation of protons across the membrane during O2 reduction supports 2,3-BDO generation. Interestingly, this process is inhibited by high levels of dissolved oxygen in this system. In an aerated BES, O2 molecules react with the strong reductant (cathode) to form reactive oxygen species, resulting in cell death.IMPORTANCEMicrobial electrosynthesis (MES) is increasingly employed for the generation of specialty chemicals, such as biofuels, bioplastics, and cancer therapeutics. For these systems to be viable for industrial scale-up, it is important to understand the energetic requirements of the bacteria to mitigate unnecessary costs. This work demonstrates sustained production of an industrially relevant chemical driven by a cathode. Additionally, it optimizes a previously published system by removing any requirement for phototrophic energy, thereby removing the additional cost of providing a light source. We also demonstrate the severe impact of oxygen intrusion into bioelectrochemical systems, offering insight to future researchers aiming to work in an anaerobic environment. These studies provide insight into both the thermodynamics of electrosynthesis and the importance of the bioelectrochemical systems' design.

Structural rather than catalytic role for mitochondrial respiratory chain supercomplexes.

Mammalian mitochondrial respiratory chain (MRC) complexes are able to associate into quaternary structures named supercomplexes (SCs), which normally coexist with non-bound individual complexes. The functional significance of SCs has not been fully clarified and the debate has been centered on whether or not they confer catalytic advantages compared with the non-bound individual complexes. Mitochondrial respiratory chain organization does not seem to be conserved in all organisms. In fact, and differently from mammalian species, mitochondria from Drosophila melanogaster tissues are characterized by low amounts of SCs, despite the high metabolic demands and MRC activity shown by these mitochondria. Here, we show that attenuating the biogenesis of individual respiratory chain complexes was accompanied by increased formation of stable SCs, which are missing in Drosophila melanogaster in physiological conditions. This phenomenon was not accompanied by an increase in mitochondrial respiratory activity. Therefore, we conclude that SC formation is necessary to stabilize the complexes in suboptimal biogenesis conditions, but not for the enhancement of respiratory chain catalysis.

Optimizing the electron transport chain to sustainably improve photosynthesis.

Genetically improving photosynthesis is a key strategy to boosting crop production to meet the rising demand for food and fuel by a rapidly growing global population in a warming climate. Many components of the photosynthetic apparatus have been targeted for genetic modification for improving photosynthesis. Successful translation of these modifications into increased plant productivity in fluctuating environments will depend on whether the electron transport chain (ETC) can support the increased electron transport rate without risking overreduction and photodamage. At present atmospheric conditions, the ETC appears suboptimal and will likely need to be modified to support proposed photosynthetic improvements and to maintain energy balance. Here, I derive photochemical equations to quantify the transport capacity and the corresponding reduction level based on the kinetics of redox reactions along the ETC. Using these theoretical equations and measurements from diverse C3/C4 species across environments, I identify several strategies that can simultaneously increase the transport capacity and decrease the reduction level of the ETC. These strategies include increasing the abundances of reaction centers, cytochrome b6f complexes, and mobile electron carriers, improving their redox kinetics, and decreasing the fraction of secondary quinone-nonreducing photosystem II reaction centers. I also shed light on several previously unexplained experimental findings regarding the physiological impacts of the abundances of the cytochrome b6f complex and plastoquinone. The model developed, and the insights generated from it facilitate the development of sustainable photosynthetic systems for greater crop yields.

Interface engineering of cellobiose dehydrogenase improves interdomain electron transfer.

Cellobiose dehydrogenase (CDH) is a bioelectrocatalyst that enables direct electron transfer (DET) in biosensors and biofuel cells. The application of this bidomain hemoflavoenzyme for physiological glucose measurements is limited by its acidic pH optimum and slow interdomain electron transfer (IET) at pH 7.5. The reason for this rate-limiting electron transfer step is electrostatic repulsion at the interface between the catalytic dehydrogenase domain and the electron mediating cytochrome domain (CYT). We applied rational interface engineering to accelerate the IET for the pH prevailing in blood or interstitial fluid. Phylogenetic and structural analyses guided the design of 17 variants in which acidic amino acids were mutated at the CYT domain. Five mutations (G71K, D160K, Q174K, D177K, M180K) increased the pH optimum and IET rate. Structure-based analysis of the variants suggested two mechanisms explaining the improvements: electrostatic steering and stabilization of the closed state by hydrogen bonding. Combining the mutations into six combinatorial variants with up to five mutations shifted the pH optimum from 4.5 to 7.0 and increased the IET at pH 7.5 over 12-fold from 0.1 to 1.24 s-1 . While the mutants sustained a high enzymatic activity and even surpassed the IET of the wild-type enzyme, the accumulated positive charges on the CYT domain decreased DET, highlighting the importance of CYT for IET and DET. This study shows that interface engineering is an effective strategy to shift the pH optimum and improve the IET of CDH, but future work needs to maintain the DET of the CYT domain for bioelectronic applications.

Insulin-Degrading Enzyme Interacts with Mitochondrial Ribosomes and Respiratory Chain Proteins.

Insulin-degrading enzyme (IDE) is a highly conserved metalloprotease that is mainly localized in the cytosol. Although IDE can degrade insulin and some other low molecular weight substrates efficiently, its ubiquitous expression suggests additional functions supported by experimental findings, such as a role in stress responses and cellular protein homeostasis. The translation of a long full-length IDE transcript has been reported to result in targeting to mitochondria, but the role of IDE in this compartment is unknown. To obtain initial leads on the function of IDE in mitochondria, we used a proximity biotinylation approach to identify proteins interacting with wild-type and protease-dead IDE targeted to the mitochondrial matrix. We find that IDE interacts with multiple mitochondrial ribosomal proteins as well as with proteins involved in the synthesis and assembly of mitochondrial complex I and IV. The mitochondrial interactomes of wild type and mutant IDE are highly similar and do not reveal any likely proteolytic IDE substrates. We speculate that IDE could adopt similar additional non-proteolytic functions in mitochondria as in the cytosol, acting as a chaperone and contributing to protein homeostasis and stress responses.

Cyclophilin 37 maintains electron transport via the cytochrome b6/f complex under high light in Arabidopsis.

Plants have evolved multiple mechanisms to cope with diverse types of light stress, particularly the regulation of the electron transport chain (ETC). Under high light (HL) conditions, the balance of electron flux in the ETC is disturbed, which leads to the overaccumulation of reactive oxygen species (ROS) and results in photodamage and photoinhibition. The cytochrome (Cyt) b6/f complex, which coordinates electron transfer between photosystems I and II (PSI and PSII), plays an essential role in regulating the ETC and initiating photoprotection. However, how the Cyt b6/f complex is maintained under HL conditions remains unclear. Here, we report that the activity of the Cyt b6/f complex is sustained by thylakoid-localized cyclophilin 37 (CYP37) in Arabidopsis (Arabidopsis thaliana). Compared with wild-type plants, cyp37 mutants displayed an imbalance in electron transport from Cyt b6/f to PSI under HL stress, which led to increased ROS accumulation, decreased anthocyanin biosynthesis, and increased chlorophyll degradation. Surprisingly, CYP37's role in regulating ETC balance was independent of photosynthesis control, which was indicated by a higher Y (ND), an indicator of P700 oxidation in PSI. Furthermore, the interaction between CYP37 and photosynthetic electron transfer A (PetA), a subunit of the Cyt b6/f complex, suggests that the central function of CYP37 is to maintain Cyt b6/f complex activity rather than to serve as an assembly factor. Our study provides insights into how plants balance electron flow between PSII and PSI via Cyt b6/f complex under HL.

Rubisco deactivation and chloroplast electron transport rates co-limit photosynthesis above optimal leaf temperature in terrestrial plants.

Net photosynthetic CO2 assimilation rate (An) decreases at leaf temperatures above a relatively mild optimum (Topt) in most higher plants. This decline is often attributed to reduced CO2 conductance, increased CO2 loss from photorespiration and respiration, reduced chloroplast electron transport rate (J), or deactivation of Ribulose-1,5-bisphosphate Carboxylase Oxygenase (Rubisco). However, it is unclear which of these factors can best predict species independent declines in An at high temperature. We show that independent of species, and on a global scale, the observed decline in An with rising temperatures can be effectively accounted for by Rubisco deactivation and declines in J. Our finding that An declines with Rubisco deactivation and J supports a coordinated down-regulation of Rubisco and chloroplast electron transport rates to heat stress. We provide a model that, in the absence of CO2 supply limitations, can predict the response of photosynthesis to short-term increases in leaf temperature.

An n-Type Conjugated Oligoelectrolyte Mimics Transmembrane Electron Transport Proteins for Enhanced Microbial Electrosynthesis.

Interfacing bacteria as biocatalysts with an electrode provides the basis for emerging bioelectrochemical systems that enable sustainable energy interconversion between electrical and chemical energy. Electron transfer rates at the abiotic-biotic interface are, however, often limited by poor electrical contacts and the intrinsically insulating cell membranes. Herein, we report the first example of an n-type redox-active conjugated oligoelectrolyte, namely COE-NDI, which spontaneously intercalates into cell membranes and mimics the function of endogenous transmembrane electron transport proteins. The incorporation of COE-NDI into Shewanella oneidensis MR-1 cells amplifies current uptake from the electrode by 4-fold, resulting in the enhanced bio-electroreduction of fumarate to succinate. Moreover, COE-NDI can serve as a "protein prosthetic" to rescue current uptake in non-electrogenic knockout mutants.

Tissue-preferential recruitment of electron transfer chains for cytochrome P450-catalyzed phenolic biosynthesis.

Cytochrome P450 system consists of P450 monooxygenase and redox pattern(s). While the importance of monooxygenases in plant metabolism is well documented, the metabolic roles of the related redox components have been largely overlooked. Here, we show that distinct electron transfer chains are recruited in phenylpropanoid-monolignol P450 systems to support the synthesis and distribution of different classes of phenolics in different plant tissues. While Arabidopsis cinnamate 4-hydroxylase adopts conventional NADPH-cytochrome P450 oxidoreductase (CPR) electron transfer chain for its para-hydroxylation reaction, ferulate 5-hydroxylase uses both NADPH-CPR-cytochrome b5 (CB5) and NADH-cytochrome b5 reductase-CB5 chains to support benzene ring 5-hydroxylation, in which the former route is primarily recruited in the stem for syringyl lignin synthesis, while the latter dominates in the syntheses of 5-hydroxylated phenolics in seeds and seed coat suberin. Our study unveils an additional layer of complexity and versatility of P450 system that the plants evolved for diversifying phenolic repertoires.

Reversible down-regulation of photosystems I and II leads to fast photosynthesis recovery after long-term drought in Jatropha curcas.

Jatropha curcas is a drought-tolerant plant that maintains its photosynthetic pigments under prolonged drought, and quickly regains its photosynthetic capacity when water is available. It has been reported that drought stress leads to increased thermal dissipation in PSII, but that of PSI has been barely investigated, perhaps due to technical limitations in measuring the PSI absolute quantum yield. In this study, we combined biochemical analysis and spectroscopic measurements using an integrating sphere, and verified that the quantum yields of both photosystems are temporarily down-regulated under drought. We found that the decrease in the quantum yield of PSII was accompanied by a decrease in the core complexes of PSII while light-harvesting complexes are maintained under drought. In addition, in drought-treated plants, we observed a decrease in the absolute quantum yield of PSI as compared with the well-watered control, while the amount of PSI did not change, indicating that non-photochemical quenching occurs in PSI. The down-regulation of both photosystems was quickly lifted in a few days upon re-watering. Our results indicate, that in J. curcas under drought, the down-regulation of both PSII and PSI quantum yield protects the photosynthetic machinery from uncontrolled photodamage.

Algal PETC-Pro171-Leu suppresses electron transfer in cytochrome b6f under acidic lumenal conditions.

Linear photosynthetic electron flow (LEF) produces NADPH and generates a proton electrochemical potential gradient across the thylakoid membrane to synthesize ATP, both of which are required for CO2 fixation. As cellular demand for ATP and NADPH varies, cyclic electron flow (CEF) between Photosystem I and the cytochrome b6f complex (b6f) produces extra ATP. b6f regulates LEF and CEF via photosynthetic control, which is a pH-dependent b6f slowdown of plastoquinol oxidation at the lumenal site. This protection mechanism is triggered at more alkaline lumen pH in the pgr1 (proton gradient regulation 1) mutant of the vascular plant Arabidopsis (Arabidopsis thaliana), which contains a Pro194Leu substitution in the b6f Rieske Iron-sulfur protein Photosynthetic Electron Transfer C (PETC) subunit. In this work, we introduced the equivalent pgr1 mutation in the green alga Chlamydomonas reinhardtii to generate PETC-P171L. Consistent with the pgr1 phenotype, PETC-P171L displayed impaired NPQ induction along with slower photoautotrophic growth under high light conditions. Our data provide evidence that the ΔpH component in PETC-P171L depends on oxygen availability. Only under low oxygen conditions was the ΔpH component sufficient to trigger a phenotype in algal PETC-P171L where the mutant b6f was more restricted to oxidize the plastoquinol pool and showed diminished electron flow through the b6f complex. These results demonstrate that photosynthetic control of different stringency are established in C. reinhardtii depending on the cellular metabolism, and the lumen pH-sensitive PETC-P171L was generated to read out various associated effects.

Physiological Benefits of Oxygen-Terminating Extracellular Electron Transfer.

Extracellular electron transfer (EET) is a process via which certain microorganisms, such as bacteria, exchange electrons with extracellular materials by creating an electrical link across their membranes. EET has been studied for the reactions on solid materials such as minerals and electrodes with implication in geobiology and biotechnology. EET-capable bacteria exhibit broad phylogenetic diversity, and some are found in environments with various types of electron acceptors/donors not limited to electrodes or minerals. Oxygen has also been shown to serve as the terminal electron acceptor for EET of Pseudomonas aeruginosa and Faecalibacterium prausnitzii. However, the physiological significance of such oxygen-terminating EETs, as well as the mechanisms underlying them, remain unclear. In order to understand the physiological advantage of oxygen-terminating EET and its link with energy metabolism, in this review, we compared oxygen-terminating EET with aerobic respiration, fermentation, and electrode-terminating EET. We also summarized benefits and limitations of oxygen-terminating EET in a biofilm setting, which indicate that EET capability enables bacteria to create a niche in the anoxic zone of aerobic biofilms, thereby remodeling bacterial metabolic activities in biofilms.

Rieske FeS overexpression in tobacco provides increased abundance and activity of cytochrome b6 f.

Photosynthesis is fundamental for plant growth and yield. The cytochrome b6 f complex catalyses a rate-limiting step in thylakoid electron transport and therefore represents an important point of regulation of photosynthesis. Here we show that overexpression of a single core subunit of cytochrome b6 f, the Rieske FeS protein, led to up to a 40% increase in the abundance of the complex in Nicotiana tabacum (tobacco) and was accompanied by an enhanced in vitro cytochrome f activity, indicating a full functionality of the complex. Analysis of transgenic plants overexpressing Rieske FeS by the light-induced fluorescence transients technique revealed a more oxidised primary quinone acceptor of photosystem II (QA ) and plastoquinone pool and faster electron transport from the plastoquinone pool to photosystem I upon changes in irradiance, compared to control plants. A faster establishment of qE , the energy-dependent component of nonphotochemical quenching, in transgenic plants suggests a more rapid buildup of the transmembrane proton gradient, also supporting the increased in vivo cytochrome b6 f activity. However, there was no consistent increase in steady-state rates of electron transport or CO2 assimilation in plants overexpressing Rieske FeS grown in either laboratory conditions or field trials, suggesting that the in vivo activity of the complex was only transiently increased upon changes in irradiance. Our results show that overexpression of Rieske FeS in tobacco enhances the abundance of functional cytochrome b6 f and may have the potential to increase plant productivity if combined with other traits.

Prying into the green black-box.

Life-long efforts of the Tartu photosynthesis research group have been summarized. The measurements were facilitated by self-designed instruments, distinct in multifunctionality and fastresponse time. The black-box type kinetical analysis on intact leaves has revealed several physiologically significant features of leaf photosynthesis. Rubisco studies reflected competition for the active site between the substrates and products, linearizing in vivo kinetics compared with the low-Km in vitro responses. Rubisco Activase usually activates only a small part of the Rubisco, making the rest of it a storage protein. Precisely quantifying absorbed photons and the responding transmittance changes, electron flow rates through cytochrome b6f, plastocyanin and photosystem I were measured, revealing competition between the proton-uncoupled cyclic electron flow from PSI to Cyt b6f to P700+ and the proton-coupled linear flow from PSII to Cyt b6f to P700+. Analyzing responses of O2 evolution and Chl fluorescence to ms-length light pulses we concluded that explanation of the sigmoidal fluorescence induction by excitonic connectivity between PSII units is a misconception. Each PSII processes excitation from its own antenna, but the sigmoidicity is caused by rise of the fluorescence yield of the QA-reduced PSII units after their QB site becomes occupied by reduced plastoquinone (or diuron). Unlike respiration, photosynthetic electrons must prepare their acceptor by coupled synthesis of 3ATP/4e-. Feedback regulation of this ratio leads to oscillations under saturating light and CO2, when the rate is Pi-limited. The slow oscillations (period 60s) indicate that the magnitudes of the deflections in the 3ATP/4e- ratio, corrected by regulating cyclic and alternative electron flow (including the Mehler type O2 reduction), are only a fraction of a per cent. The Pi limitation causes slip in the ATP synthase, slightly increasing the basic 12H+/3ATP requirement.

The circadian night depression of photosynthesis analyzed in a herb, Pulmonaria vallarsae. Day/night quantitative relationships.

Although many photosynthesis related processes are known to be controlled by the circadian system, consequent changes in photosynthetic activities are poorly understood. Photosynthesis was investigated during the daily cycle by chlorophyll fluorescence using a PAM fluorometer in Pulmonaria vallarsae subsp. apennina, an understory herb. A standard test consists of a light induction pretreatment followed by light response curve (LRC). Comparison of the major diagnostic parameters collected during day and night showed a nocturnal drop of photosynthetic responses, more evident in water-limited plants and consisting of: (i) strong reduction of flash-induced fluorescence peaks (FIP), maximum linear electron transport rate (Jmax, ETREM) and effective PSII quantum yield (ΦPSII); (ii) strong enhancement of nonphotochemical quenching (NPQ) and (iii) little or no change in photochemical quenching qP, maximum quantum yield of linear electron transport (Φ), and shape of LRC (θ). A remarkable feature of day/night LRCs at moderate to high irradiance was their linear-parallel course in double-reciprocal plots. Photosynthesis was also monitored in plants subjected to 2-3 days of continuous darkness ("long night"). In such conditions, plants exhibited high but declining peaks of photosynthetic activity during subjective days and a low, constant value with elevated NPQ during subjective night tests. The photosynthetic parameters recorded in subjective days in artificial darkness resembled those under natural day conditions. On the basis of the evidence, we suggest a circadian component and a biochemical feedback inhibition to explain the night depression of photosynthesis in P. vallarsae.

Integration of physiologically relevant photosynthetic energy flows into whole genome models of light-driven metabolism.

Characterizing photosynthetic productivity is necessary to understand the ecological contributions and biotechnology potential of plants, algae, and cyanobacteria. Light capture efficiency and photophysiology have long been characterized by measurements of chlorophyll fluorescence dynamics. However, these investigations typically do not consider the metabolic network downstream of light harvesting. By contrast, genome-scale metabolic models capture species-specific metabolic capabilities but have yet to incorporate the rapid regulation of the light harvesting apparatus. Here, we combine chlorophyll fluorescence parameters defining photosynthetic and non-photosynthetic yield of absorbed light energy with a metabolic model of the pennate diatom Phaeodactylum tricornutum. This integration increases the model predictive accuracy regarding growth rate, intracellular oxygen production and consumption, and metabolic pathway usage. Through the quantification of excess electron transport, we uncover the sequential activation of non-radiative energy dissipation processes, cross-compartment electron shuttling, and non-photochemical quenching as the rapid photoacclimation strategy in P. tricornutum. Interestingly, the photon absorption thresholds that trigger the transition between these mechanisms were consistent at low and high incident photon fluxes. We use this understanding to explore engineering strategies for rerouting cellular resources and excess light energy towards bioproducts in silico. Overall, we present a methodology for incorporating a common, informative data type into computational models of light-driven metabolism and show its utilization within the design-build-test-learn cycle for engineering of photosynthetic organisms.

Hysteresis light curves: a protocol for characterizing the time dependence of the light response of photosynthesis.

Photosynthesis vs. light curves (LCs) have played a central role in photosynthesis research for decades. They are the commonest form of describing how photosynthesis responds to changes in light, being frequently used for characterizing photoacclimation. However, LCs are often interpreted exclusively regarding the response to light intensity, the effects of time of exposure not being explicitly considered. This study proposes the use of 'hysteresis light curves' (HLC), an experimental protocol focused on the cumulative effects of light exposure to obtain information on the time dependence of photosynthetic light responses. HLC are generated by exposing samples to a symmetrical sequence of increasing and decreasing light levels. The comparison of the light-increasing and the light-decreasing phases allows the quantification of the hysteresis caused by high-light exposure, the magnitude and direction of which inform on the activation, and subsequent relaxation of high-light-induced photosynthetic processes. HLCs of the chlorophyll fluorescence indices rETR (relative electron transport rate of photosystem II) and Y(NPQ) (index of non-photochemical quenching) were measured on cyanobacteria, algae, and plants, with the aim of identifying main patterns of hysteresis and their diversity. A non-parametric index is proposed to quantify the magnitude and direction of hysteresis in HLCs of rETR and Y(NPQ). The results of this study show that HLCs can provide additional relevant information on the time dependence of the light response of photosynthetic samples, not obtainable from conventional LCs, useful for phenotyping photosynthetic traits, including photoacclimation state and kinetics of light activation and relaxation of electron flow and energy dissipation processes.

Role of guard cell- or mesophyll cell-localized phytochromes in stomatal responses to blue, red, and far-red light.

MAIN CONCLUSION: Guard cell- or mesophyll cell-localized phytochromes do not have a predominant direct light sensory role in red- or blue-light-mediated stomatal opening or far-red-light-mediated stomatal closure of Arabidopsis. The role of phytochromes in blue- and red-light-mediated stomatal opening, and far-red-light- mediated decrease in opening, is still under debate. It is not clear whether reduced stomatal opening in a phytochrome B (phyB) mutant line, is due to phytochrome acting as a direct photosensor or an indirect growth effect. The exact tissue localization of the phytochrome photoreceptor important for stomatal opening is also not known. We studied differences in stomatal opening in an Arabidopsis phyB mutant, and lines showing mesophyll cell-specific or guard cell-specific inactivation of phytochromes. Stomatal conductance (gs) of intact leaves was measured under red, blue, and blue + far-red light. Lines exhibiting guard cell-specific inactivation of phytochrome did not show a change in gs under blue or red light compared to Col-0. phyB consistently exhibited a reduction in gs under both blue and red light. Addition of far-red light did not have a significant impact on the blue- or red-light-mediated stomatal response. Treatment of leaves with DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), a photosynthetic electron transport (PET) inhibitor, eliminated the response to red light in all lines, indicating that stomatal opening under red light is controlled by PET, and not directly by phytochrome. Similar to previous studies, leaves of the phyB mutant line had fewer stomata. Overall, phytochrome does not appear have a predominant direct sensory role in stomatal opening under red or blue light. However, phytochromes likely have an indirect effect on the degree of stomatal opening under light through effects on leaf growth and stomatal development.