Genetically-determined variations in photosynthesis indicate roles for specific fatty acid species in chilling responses.

Using a population of recombinant inbred lines (RILs) cowpea (Vigna unguiculata. L. Walp), we tested for co-linkages between lipid contents and chilling responses of photosynthesis. Under low-temperature conditions (19°C/13°C, day/night), we observed co-linkages between quantitative trait loci intervals for photosynthetic light reactions and specific fatty acids, most strikingly, the thylakoid-specific fatty acid 16:1Δ3trans found exclusively in phosphatidylglycerol (PG 16:1t). By contrast, we did not observe co-associations with bulk polyunsaturated fatty acids or high-melting-point-PG (sum of PG 16:0, PG 18:0 and PG 16:1t) previously thought to be involved in chilling sensitivity. These results suggest that in cowpea, chilling sensitivity is modulated by specific lipid interactions rather than bulk properties. We were able to recapitulate the predicted impact of PG 16:1t levels on photosynthetic responses at low temperature using mutants and transgenic Arabidopsis lines. Because PG 16:1t synthesis requires the activity of peroxiredoxin-Q, which is activated by H2 O2 and known to be involved in redox signalling, we hypothesise that the accumulation of PG 16:1t occurs as a result of upstream effects on photosynthesis that alter redox status and production of reactive oxygen species.

Light potentials of photosynthetic energy storage in the field: what limits the ability to use or dissipate rapidly increased light energy?

The responses of plant photosynthesis to rapid fluctuations in environmental conditions are critical for efficient conversion of light energy. These responses are not well-seen laboratory conditions and are difficult to probe in field environments. We demonstrate an open science approach to this problem that combines multifaceted measurements of photosynthesis and environmental conditions, and an unsupervised statistical clustering approach. In a selected set of data on mint (Mentha sp.), we show that 'light potentials' for linear electron flow and non-photochemical quenching (NPQ) upon rapid light increases are strongly suppressed in leaves previously exposed to low ambient photosynthetically active radiation (PAR) or low leaf temperatures, factors that can act both independently and cooperatively. Further analyses allowed us to test specific mechanisms. With decreasing leaf temperature or PAR, limitations to photosynthesis during high light fluctuations shifted from rapidly induced NPQ to photosynthetic control of electron flow at the cytochrome b6f complex. At low temperatures, high light induced lumen acidification, but did not induce NPQ, leading to accumulation of reduced electron transfer intermediates, probably inducing photodamage, revealing a potential target for improving the efficiency and robustness of photosynthesis. We discuss the implications of the approach for open science efforts to understand and improve crop productivity.

Charting the native architecture of Chlamydomonas thylakoid membranes with single-molecule precision.

Thylakoid membranes scaffold an assortment of large protein complexes that work together to harness the energy of light. It has been a longstanding challenge to visualize how the intricate thylakoid network organizes these protein complexes to finely tune the photosynthetic reactions. Previously, we used in situ cryo-electron tomography to reveal the native architecture of thylakoid membranes (Engel et al., 2015). Here, we leverage technical advances to resolve the individual protein complexes within these membranes. Combined with a new method to visualize membrane surface topology, we map the molecular landscapes of thylakoid membranes inside green algae cells. Our tomograms provide insights into the molecular forces that drive thylakoid stacking and reveal that photosystems I and II are strictly segregated at the borders between appressed and non-appressed membrane domains. This new approach to charting thylakoid topology lays the foundation for dissecting photosynthetic regulation at the level of single protein complexes within the cell.

Chloroplast ATP Synthase Modulation of the Thylakoid Proton Motive Force: Implications for Photosystem I and Photosystem II Photoprotection.

In wild type plants, decreasing CO2 lowers the activity of the chloroplast ATP synthase, slowing proton efflux from the thylakoid lumen resulting in buildup of thylakoid proton motive force (pmf). The resulting acidification of the lumen regulates both light harvesting, via the qE mechanism, and photosynthetic electron transfer through the cytochrome b6f complex. Here, we show that the cfq mutant of Arabidopsis, harboring single point mutation in its γ-subunit of the chloroplast ATP synthase, increases the specific activity of the ATP synthase and disables its down-regulation under low CO2. The increased thylakoid proton conductivity (gH+) in cfq results in decreased pmf and lumen acidification, preventing full activation of qE and more rapid electron transfer through the b6f complex, particularly under low CO2 and fluctuating light. These conditions favor the accumulation of electrons on the acceptor side of PSI, and result in severe loss of PSI activity. Comparing the current results with previous work on the pgr5 mutant suggests a general mechanism where increased PSI photodamage in both mutants is caused by loss of pmf, rather than inhibition of CEF per se. Overall, our results support a critical role for ATP synthase regulation in maintaining photosynthetic control of electron transfer to prevent photodamage.

Limitations to photosynthesis by proton motive force-induced photosystem II photodamage.

The thylakoid proton motive force (pmf) generated during photosynthesis is the essential driving force for ATP production; it is also a central regulator of light capture and electron transfer. We investigated the effects of elevated pmf on photosynthesis in a library of Arabidopsis thaliana mutants with altered rates of thylakoid lumen proton efflux, leading to a range of steady-state pmf extents. We observed the expected pmf-dependent alterations in photosynthetic regulation, but also strong effects on the rate of photosystem II (PSII) photodamage. Detailed analyses indicate this effect is related to an elevated electric field (Δψ) component of the pmf, rather than lumen acidification, which in vivo increased PSII charge recombination rates, producing singlet oxygen and subsequent photodamage. The effects are seen even in wild type plants, especially under fluctuating illumination, suggesting that Δψ-induced photodamage represents a previously unrecognized limiting factor for plant productivity under dynamic environmental conditions seen in the field.

Varicella-zoster virus-specific cell-mediated immunity in Ramsay Hunt syndrome.

OBJECTIVES/HYPOTHESIS: The etiology of Ramsay Hunt syndrome (Hunt syndrome) is reactivation of latent varicella-zoster virus (VZV) in the geniculate ganglion of the facial nerve, leading to neuritis. Although the mechanism of the VZV reactivation is unclear, one possibility is that the reactivation involves a low level of VZV-specific cell-mediated immunity (CMI). The aim of this study was to clarify the characteristics of the VZV-specific CMI in Hunt syndrome compared to that in Bell's palsy, and to obtain clues to its role in the development of Hunt syndrome. STUDY DESIGN: Prospective study. METHODS: We determined the median spot numbers and examined VZV-specific CMI in patients with Hunt syndrome and with Bell's palsy using interferon-γ enzyme-linked immunospot (ELISPOT) assays. We analyzed the relationship between the value of VZV-specific CMI and days from disease onset. RESULTS: The median spot number in Hunt syndrome (87.3 spot-forming cells [SFCs]/4 × 10(5) peripheral blood mononuclear cells [PBMCs]) was higher than that in Bell's palsy (62.3 SFCs/4 × 10(5) PBMCs). Hunt syndrome showed a strong relationship between the ELISPOT count and days from onset (r = 0.65). Within the first 5 days from onset, no ELISPOT counts higher than 80 SFCs/4 × 10(5) PBMCs were observed. On the other hand, no correlation was observed between the ELISPOT count and days from onset in patients with Bell's palsy (r = -0.19). CONCLUSIONS: These results suggest that VZV-specific CMI in Hunt syndrome is low at disease onset and increases rapidly thereafter. Consequently, reduced VZV-specific CMI may play an important role in the reactivation of VZV in the facial nerve, leading to Hunt syndrome.

The site of regulation of light capture in Symbiodinium: does the peridinin-chlorophyll a-protein detach to regulate light capture?

Dinoflagellates from the genus Symbiodinium form symbiotic associations with cnidarians including corals and anemones. The photosynthetic apparatuses of these dinoflagellates possess a unique photosynthetic antenna system incorporating the peridinin-chlorophyll a-protein (PCP). It has been proposed that the appearance of a PCP-specific 77K fluorescence emission band around 672-675 nm indicates that high light treatment results in PCP dissociation from intrinsic membrane antenna complexes, blocking excitation transfer to the intrinsic membrane-bound antenna complexes, chlorophyll a-chlorophyll c2-peridinin-protein-complex (acpPC) and associated photosystems (Reynolds et al., 2008 Proc Natl Acad Sci USA 105:13674-13678).We have tested this model using time-resolved fluorescence decay kinetics in conjunction with global fitting to compare the time-evolution of the PCP spectral bands before and after high light exposure. Our results show that no long-lived PCP fluorescence emission components appear either before or after high light treatment, indicating that the efficiency of excitation transfer from PCP to membrane antenna systems remains efficient and rapid even after exposure to high light. The apparent increased relative emission at around 675nm was, instead, caused by strong preferential exciton quenching of the membrane antenna complexes associated with acpPC and reaction centers. This strong non-photochemical quenching (NPQ) is consistent with the activation of xanthophyll-associated quenching mechanisms and the generally-observed avoidance in nature of long-lived photoexcited states that can lead to oxidative damage. The acpPC component appears to be the most strongly quenched under high light exposure suggesting that it houses the photoprotective exciton quencher.

Continuous 6-year follow-up study of sensitization to Japanese cedar pollen and onset in schoolchildren.

BACKGROUND: Intra individual longitudinal data has been lacking for IgE-mediated seasonal allergic rhinitis (SAR), especially in young children. Little is known about the development and natural course of SAR in terms of prevalence and incidence rates in schoolchildren. METHODS: In May or June each year from 1994 to 2007, schoolchildren were assessed for serum Japanese cedar pollen (JCP)-IgE and house dust mite (HDM)-IgE levels, and surveyed regarding nasal symptoms. RESULTS: Among the 220 children initially assessed in the first grade, 69 (31.4%) were already sensitized to JCP at first grade and 119 (54.1%) did not develop JCP sensitivity during the 6-year study at all. In the first grade children who were HDM-sensitized but JCP non-sensitized, JCP-IgE level was significantly elevated compared to the JCP and HDM non-sensitized group. This seems to indicate that HDM sensitization was very strongly associated with JCP sensitization. CONCLUSIONS: Elevated serum IgE is a consequence of specific sensitization to HDM and HDM sensitization appeared to develop prior to the start of primary school which distinguishes HDM sensitization from JCP sensitization.

The midline electroneurography method for facial palsy reflects total nerve degeneration.

CONCLUSION: The midline electroneurography (ENoG) method might reflect total facial nerve degeneration. OBJECTIVE: We compared ENoG values in patients with facial palsy using two different methods, the midline method and five electroneurogram recordings, to reveal whether the ENoG value obtained with the midline method reflects total facial nerve degeneration. METHODS: Forty patients with facial palsy were enrolled. Compound muscle action potentials (CMAPs) were recorded using the midline method, in which the anode was placed on the mental protuberance and the cathode was placed on the philtrum. Additionally, five electroneurogram recordings were obtained by placing the anode on the skin of the parietal region and five cathodes on the skin over five facial muscles (frontalis, orbicularis oculi, nasalis, orbicularis oris, and depressor anguli oris muscles). ENoG values recorded using the two methods were compared. RESULTS: The ENoG values of the five facial muscles did not differ from those obtained using the midline method. The total ENoG value calculated by summing five CMAPs from five facial muscles, which is considered to reflect total facial nerve degeneration, was not significantly different from that using midline methods; moreover, a strong positive correlation coefficient (r = 0.87) was found between them.

Essential role of macrophages in the initiation of allergic rhinitis in mice sensitized intranasally once with cedar pollen: regulation of class switching of immunoglobulin in B cells by controlling interleukin-4 production in T cells of submandibular lymph nodes.

The production of allergen-specific IgE antibodies (Abs) in allergen-sensitized patients or animals has a mutual relationship with the immunologic response leading to allergic rhinitis. We recently reported that, after an intranasal injection of cedar pollen into mice, an interleukin-4 (IL-4)-dependent increase in serum nonspecific IgE Abs was a prerequisite for the production of serum allergen-specific IgE Abs. Here, we explored which lymphoid organs were responsive to the intranasally injected allergen and how IL-4 and IgE Abs were produced in the lymphocytes. Time-dependent changes in the total cell numbers and in in vitro IgE Ab production in various lymphoid organs revealed that the submandibular lymph nodes were the main responsible organ. After treatment with allergen (for IgE production) or allergen and complete Freund's adjuvant (for IgG production), we separated submandibular lymph node cells into macrophage-, lymphocyte-, and granulocyte-rich populations by discontinuous Percoll density-gradient centrifugation. Unexpectedly, bulk cells, but not the lymphocyte- or macrophage-rich populations, produced significant amounts of IL-4, IgE, and IgG; whereas production was restored by addition of Mac-1(+) cells from the macrophage-rich to the lymphocyte-rich fraction. Furthermore, a combination of the lymphocyte-rich population (for IgG [or IgE]) production) and the macrophage-rich population (for IgE [or IgG]) production) produced a large amount of IgE (or IgG). These results indicate that, in the initiation of allergic rhinitis, macrophages in the submandibular lymph nodes are essential not only for IL-4 or immunoglobulin production, but also for class switching of immunoglobulin in lymphocytes.

Role of cytokines in lavage or drainage fluid after hemithyroidectomy in wound healing: involvement of histamine in the acceleration and delay of wound healing.

Wound healing is a sophisticated biologic process. In the case of hemithyroidectomy, the operation time is relatively short with small tissue damage and without skin excision, and bacterial contamination before, during, and after the operation is uncommon. Here, we explored which cytokine(s) affected the rates of healing of skin wounds after hemithyroidectomy of 29 patients. We assessed the amounts of cytokines (e.g., interleukin-6, platelet-derived growth factor, basic fibroblast growth factor, vascular endothelial growth factor, and tumor necrosis factor-α) in either the preoperative or postoperative lavage fluids, or in the drainage fluids on postoperative days (PODs) 1-8. All of these cytokines showed a similar pattern; after reaching a peak on POD1, the production fell sharply on POD2-8, revealing that wound healing commenced on POD1. The rates of wound healing were inversely related to the levels of histamine in six patients (i.e., those with the three largest and those with the three smallest total volumes of drainage fluid on POD1): high (or low) levels of histamine in the postoperative lavage fluids with low (or high) levels in the drainage fluids on POD1 caused earlier (or the delay of) wound healing, suggesting involvement of histamine in the acceleration and delay of wound healing.

Thioredoxin-insensitive plastid ATP synthase that performs moonlighting functions.

The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and acts as a key feedback regulatory component of photosynthesis. Arabidopsis possesses two homologues of the regulatory γ subunit of the ATP synthase, encoded by the ATPC1 and ATPC2 genes. Using a series of mutants, we show that both these subunits can support photosynthetic ATP synthesis in vivo with similar specific activities, but that in wild-type plants, only γ(1) is involved in ATP synthesis in photosynthesis. The γ(1)-containing ATP synthase shows classical light-induced redox regulation, whereas the mutant expressing only γ(2)-ATP synthase (gamma exchange-revised ATP synthase, gamera) shows equally high ATP synthase activity in the light and dark. In situ redox titrations demonstrate that the regulatory thiol groups on γ(2)-ATP synthase remain reduced under physiological conditions but can be oxidized by the strong oxidant diamide, implying that the redox potential for the thiol/disulphide transition in γ(2) is substantially higher than that for γ(1). This regulatory difference may be attributed to alterations in the residues near the redox-active thiols. We propose that γ(2)-ATP synthase functions to catalyze ATP hydrolysis-driven proton translocation in nonphotosynthetic plastids, maintaining a sufficient transthylakoid proton gradient to drive protein translocation or other processes. Consistent with this interpretation, ATPC2 is predominantly expressed in the root, whereas modifying its expression results in alteration of root hair development. Phylogenetic analysis suggests that γ(2) originated from ancient gene duplication, resulting in divergent evolution of functionally distinct ATP synthase complexes in dicots and mosses.

Regulation of cyclic electron flow in C3 plants: differential effects of limiting photosynthesis at ribulose-1,5-bisphosphate carboxylase/oxygenase and glyceraldehyde-3-phosphate dehydrogenase.

Cyclic electron flow around photosystem I (CEF1) is thought to augment chloroplast ATP production to meet metabolic needs. Very little is known about the induction and regulation of CEF1. We investigated the effects on CEF1 of antisense suppression of the Calvin-Benson enzymes glyceraldehyde-3-phosphate dehydrogenase (gapR), and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (SSU), in tobacco (Nicotiana tabacum cv. Wisconsin 38). The gapR, but not ssuR, mutants showed substantial increases in CEF1, demonstrating that specific intermediates, rather than slowing of assimilation, induce CEF1. Both types of mutant showed increases in steady-state transthylakoid proton motive force (pmf) and subsequent activation of the photoprotective q(E) response. With gapR, the increased pmf was caused both by up-regulation of CEF1 and down-regulation of the ATP synthase. In ssuR, the increased pmf was attributed entirely to a decrease in ATP synthase activity, as previously seen in wild-type plants when CO2 levels were decreased. Comparison of major stromal metabolites in gapR, ssuR and hcef1, a mutant with decreased fructose 1,6-bisphosphatase activity, showed that neither the ATP/ADP ratio, nor major Calvin-Benson cycle intermediates can directly account for the activation of CEF1, suggesting that chloroplast redox status or reactive oxygen species regulate CEF1.

A novel electroneurography method in facial palsy.

CONCLUSION: The novel midline electroneurography (ENoG) method may have advantages over the standard method in terms of ease of electrode setting, and the ENoG value may be a useful prognostic factor. OBJECTIVE: We compared ENoG performed in patients with facial palsy using two different methods--the new midline method and standard method--in terms of the amplitudes of the compound muscle action potentials(CMAPs) and relationship between the ENoG value and clinical course. METHODS: A total of 64 patients with facial palsy were enrolled. CMAPs were recorded using the midline method, in which the recording electrodes were placed on the mental protuberance and philtrum over the orbicularis oris muscle, and the standard method, in which the recording electrodes were set close to the nasolabial fold. Percutaneous electrical stimulation was applied to the main trunk of the facial nerve.The amplitudes of the CMAPs and the relationship between the ENoG value and the period to full recovery from the facial palsy were compared. RESULTS: The midline method had larger CMAP amplitudes on both sides and a stronger negative correlation in the relationship between the ENoG value and period to full recovery from palsy than the standard method statistically.

Endoscopy-assisted surgery with topical mitomycin for a cholesterol granuloma in the petrous apex.

OBJECTIVES/HYPOTHESIS: We present the case of a 21-year-old woman who underwent endoscopy-assisted surgery with topical mitomycin for a large cholesterol granuloma in the petrous apex that involved the eustachian tube. STUDY DESIGN: Case report. METHODS: Minimally invasive surgery was performed on the cholesterol granuloma with endoscopic assistance, and topical mitomycin (0.4 mg/mL) was administered to prevent scarring of the newly created drainage/ventilation route. With the endoscopic approach, the drainage/ventilation route was secured not only from the tympanic cavity, but also using a guidewire inserted from the pharyngeal orifice of the eustachian tube. RESULTS: The drainage/ventilation route between the nasopharynx and middle ear cavity involving the cholesterol granuloma has been preserved more than for 9 months postoperatively. Her hearing has recovered. CONCLUSIONS: This less invasive technique is a surgical option for patients with a large cholesterol granuloma in the petrous apex involving the eustachian tube.

Postoperative aeration in the middle ear and hearing outcome after canal wall down tympanoplasty with soft-wall reconstruction for cholesteatoma.

OBJECTIVE: Canal wall down (CWD) tympanoplasty with soft-wall reconstruction (SWR) is a unique technique for cholesteatoma surgery. The external auditory canal shape after surgery-retracted like a radical mastoid cavity or preserved intact-depends on postoperative aeration in the mastoid cavity. However, the relationship between postoperative middle ear aeration and hearing outcome with this procedure is unknown. We characterized this relationship and propose an ideal state of middle ear aeration to obtain satisfactory postoperative hearing after CWD tympanoplasty with SWR. STUDY DESIGN: Retrospective case series. PATIENTS: Seventy-eight patients (78 ears) with fresh cholesteatomas treated surgically at our hospital by planned 2-stage CWD tympanoplasty and SWR were included. MAIN OUTCOME MEASURES: Postoperative middle ear aeration was scored 1 year after second-stage surgery by computed tomography. The patients were divided into 4 bins according to postoperative audiometric air-bone (A-B) gaps: 0-10, 11-20, 21-30, and greater than 30 dB. RESULTS: Postoperative middle ear aeration was significantly greater in the smaller gap bins (0-10 and 11-20 dB) compared with the larger A-B gap bins (21-30 and >30 dB). In contrast to the larger A-B gap bins, those with smaller A-B gaps showed reaeration of the antrum and mastoid cavity. No significant differences were observed in postoperative middle ear aeration or hearing outcome between the 2 cholesteatoma types. CONCLUSION: Promoting postoperative aeration of the entire middle ear is necessary to achieve better hearing outcome in patients undergoing CWD tympanoplasty and SWR for cholesteatoma.

A drug-selected Plasmodium falciparum lacking the need for conventional electron transport.

Mitochondrial electron transport is essential for survival in Plasmodium falciparum, making the cytochrome (cyt) bc(1) complex an attractive target for antimalarial drug development. Here we report that P. falciparum cultivated in the presence of a novel cyt bc(1) inhibitor underwent a fundamental transformation in biochemistry to a phenotype lacking a requirement for electron transport through the cyt bc(1) complex. Growth of the drug-selected parasite clone (SB1-A6) is robust in the presence of diverse cyt bc(1) inhibitors, although electron transport is fully inhibited by these same agents. This transformation defies expected molecular-based concepts of drug resistance, has important implications for the study of cyt bc(1) as an antimalarial drug target, and may offer a glimpse into the evolutionary future of Plasmodium.

Depletion of stromal P(i) induces high 'energy-dependent' antenna exciton quenching (q(E)) by decreasing proton conductivity at CF(O)-CF(1) ATP synthase.

This work tests two models to account for the effects of depletion of stromal inorganic phosphate (P(i)), which results in down-regulation of light capture via the exciton quenching (q(E)) mechanism and has been proposed to act in feedback regulation of the light reactions. In both models, antenna down-regulation is activated by acidification of the lumen, despite the fact that linear electron flow (LEF) (and associated proton flux) is decreased upon P(i) depletion. In one model, an imbalance of ATP or NADPH activates cyclic electron transfer around photosystem I (CEF1), increasing proton influx to the lumen. In the second, the effective conductivity of the CF(O)-CF(1) ATP synthase to protons (g(H)(+)) is decreased, retarding proton efflux from the lumen. Sequestering of P(i) by mannose infiltration increased sensitivities of q(E) and pmf to LEF. The effects were attributable to decreases in g(H)(+), but not to CEF1 and were largely reversed by subsequent P(i) feeding. Rapid recovery of g(H)(+) in the dark suggested that dark-labile metabolic pools are responsible for regulation of the ATP synthase. Overall, these results support models where accumulation of Benson-Calvin cycle intermediates or lowering of stromal P(i) below its K(M)at the ATP synthase, retards proton efflux from the lumen, leading to build-up of pmf and subsequent down-regulation of photosynthetic light capture.

Prognosis for Bell's palsy: a comparison of diabetic and nondiabetic patients.

CONCLUSION: The present study indicates that recovery from Bell's palsy in a diabetic group (DG) is delayed, and the facial movement score remains low in comparison with a nondiabetic group (NDG). More aggressive treatments, such as higher-dose corticosteroid administration and/or facial nerve decompression surgery, might be considered in diabetic patients with severe Bell's palsy. OBJECTIVES: The purpose of this study was to reveal prognostic differences for Bell's palsy in the DG and NDG. PATIENTS AND METHODS: The grades of facial palsy in 19 diabetic and 57 nondiabetic patients with Bell's palsy were assessed using the House-Brackmann grading system (HB system). Recovery was defined as grade I. The average of HB system grades and recovery rates were compared in the DG and NDG at the start of the treatment, and 1 month, 3 months, and 6 months after onset. RESULTS: There were no differences in the HB system between the DG and NDG at the start of treatment and at 1 month after onset. However, facial movement in the DG was poorer than that in the NDG at 3 months and 6 months after onset. In terms of the recovery rate, the rate in the DG (52.6%) was much lower than that in the NDG (82.5%) at 6 months after onset.

The thylakoid proton motive force in vivo. Quantitative, non-invasive probes, energetics, and regulatory consequences of light-induced pmf.

Endogenous probes of light-induced transthylakoid proton motive force (pmf), membrane potential (Deltapsi) and DeltapH were used in vivo to assess in Arabidopsis the lumen pH responses of regulatory components of photosynthesis. The accumulation of zeaxanthin and protonation of PsbS were found to have similar pK(a) values, but quite distinct Hill coefficients, a feature allowing high antenna efficiency at low pmf and fine adjustment at higher pmf. The onset of "energy-dependent' exciton quenching (q(E)) occurred at higher lumen pH than slowing of plastoquinol oxidation at the cytochrome b(6)f complex, presumably to prevent buildup of reduced electron carriers that can lead to photodamage. Quantitative comparison of intrinsic probes with the electrochromic shift signal in situ allowed quantitative estimates of pmf and lumen pH. Within a degree of uncertainly of approximately 0.5 pH units, the lumen pH was estimated to range from approximately 7.5 (under weak light at ambient CO(2)) to approximately 5.7 (under 50 ppm CO(2) and saturating light), consistent with a 'moderate pH' model, allowing antenna regulation but preventing acid-induced photodamage. The apparent pK(a) values for accumulation of zeaxanthin and PsbS protonation were found to be approximately 6.8, with Hill coefficients of about 4 and 1 respectively. The apparent shift between in vitro violaxanthin deepoxidase protonation and zeaxanthin accumulation in vivo is explained by steady-state competition between zeaxanthin formation and its subsequent epoxidation by zeaxanthin epoxidase. In contrast to tobacco, Arabidopsis showed substantial variations in the fraction of pmf (0.1-0.7) stored as Deltapsi, allowing a more sensitive qE response, possible as an adaptation to life at lower light levels.