Light acclimation interacts with thylakoid ion transport to govern the dynamics of photosynthesis in Arabidopsis.

Understanding photosynthesis in natural, dynamic light environments requires knowledge of long-term acclimation, short-term responses, and their mechanistic interactions. To approach the latter, we systematically determined and characterized light-environmental effects on thylakoid ion transport-mediated short-term responses during light fluctuations. For this, Arabidopsis thaliana wild-type and mutants of the Cl- channel VCCN1 and the K+ exchange antiporter KEA3 were grown under eight different light environments and characterized for photosynthesis-associated parameters and factors in steady state and during light fluctuations. For a detailed characterization of selected light conditions, we monitored ion flux dynamics at unprecedented high temporal resolution by a modified spectroscopy approach. Our analyses reveal that daily light intensity sculpts photosynthetic capacity as a main acclimatory driver with positive and negative effects on the function of KEA3 and VCCN1 during high-light phases, respectively. Fluctuations in light intensity boost the accumulation of the photoprotective pigment zeaxanthin (Zx). We show that KEA3 suppresses Zx accumulation during the day, which together with its direct proton transport activity accelerates photosynthetic transition to lower light intensities. In summary, both light-environment factors, intensity and variability, modulate the function of thylakoid ion transport in dynamic photosynthesis with distinct effects on lumen pH, Zx accumulation, photoprotection, and photosynthetic efficiency.

The time course of acclimation to the stress of triose phosphate use limitation.

Triose phosphate utilisation (TPU) limits the maximum rate at which plants can photosynthesise. However, TPU is almost never found to be limiting photosynthesis under ambient conditions for plants. This, along with previous results showing adaptability of TPU at low temperature, suggest that TPU capacity is regulated to be just above the photosynthetic rate achievable under the prevailing conditions. A set of experiments were performed to study the adaptability of TPU capacity when plants are acclimated to elevated CO2 concentrations. Plants held at 1500 ppm CO2 were initially TPU limited. After 30 h they no longer exhibited TPU limitations but they did not elevate their TPU capacity. Instead, the maximum rates of carboxylation and electron transport declined. A timecourse of regulatory responses was established. A step increase of CO2 first caused PSI to be oxidised but after 40 s both PSI and PSII had excess electrons as a result of acceptor-side limitations. Electron flow to PSI slowed and the proton motive force increased. Eventually, non-photochemical quenching reduced electron flow sufficiently to balance the TPU limitation. Over several minutes rubisco deactivated contributing to regulation of metabolism to overcome the TPU limitation.

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.

Photometric screens identified Arabidopsis peroxisome proteins that impact photosynthesis under dynamic light conditions.

Plant peroxisomes function collaboratively with other subcellular organelles, such as chloroplasts and mitochondria, in several metabolic processes. To comprehensively investigate the impact of peroxisomal function on photosynthesis, especially under conditions that are more relevant to natural environments, a systematic screen of over 150 Arabidopsis mutants of genes encoding peroxisomal proteins was conducted using the automated Dynamic Environment Photosynthesis Imager (DEPI). Dynamic and high-light (HL) conditions triggered significant photosynthetic defects in a subset of the mutants, including those of photorespiration (PR) and other peroxisomal processes, some of which may also be related to PR. Further analysis of the PR mutants revealed activation of cyclic electron flow (CEF) around photosystem I and higher accumulation of hydrogen peroxide (H2 O2 ) under HL conditions. We hypothesize that impaired PR disturbs the balance of ATP and NADPH, leading to the accumulation of H2 O2 that activates CEF to produce ATP to compensate for the imbalance of reducing equivalents. The identification of peroxisomal mutants involved in PR and other peroxisomal functions in the photometric screen will enable further investigation of regulatory links between photosynthesis and PR and interorganellar interaction at the mechanistic level.

PhenoCurve: capturing dynamic phenotype-environment relationships using phenomics data.

Motivation: Phenomics is essential for understanding the mechanisms that regulate or influence growth, fitness, and development. Techniques have been developed to conduct high-throughput large-scale phenotyping on animals, plants and humans, aiming to bridge the gap between genomics, gene functions and traits. Although new developments in phenotyping techniques are exciting, we are limited by the tools to analyze fully the massive phenotype data, especially the dynamic relationships between phenotypes and environments. Results: We present a new algorithm called PhenoCurve, a knowledge-based curve fitting algorithm, aiming to identify the complex relationships between phenotypes and environments, thus studying both values and trends of phenomics data. The results on both real and simulated data showed that PhenoCurve has the best performance among all the six tested methods. Its application to photosynthesis hysteresis pattern identification reveals new functions of core genes that control photosynthetic efficiency in response to varying environmental conditions, which are critical for understanding plant energy storage and improving crop productivity. Availability and Implementation: Software is available at phenomics.uky.edu/PhenoCurve. Contact: chen.jin@uky.edu or kramerd8@cns.msu.edu. Supplementary information: Supplementary data are available at Bioinformatics online.

Multi-level regulation of the chloroplast ATP synthase: the chloroplast NADPH thioredoxin reductase C (NTRC) is required for redox modulation specifically under low irradiance.

The chloroplast ATP synthase is known to be regulated by redox modulation of a disulfide bridge on the γ-subunit through the ferredoxin-thioredoxin regulatory system. We show that a second enzyme, the recently identified chloroplast NADPH thioredoxin reductase C (NTRC), plays a role specifically at low irradiance. Arabidopsis mutants lacking NTRC (ntrc) displayed a striking photosynthetic phenotype in which feedback regulation of the light reactions was strongly activated at low light, but returned to wild-type levels as irradiance was increased. This effect was caused by an altered redox state of the γ-subunit under low, but not high, light. The low light-specific decrease in ATP synthase activity in ntrc resulted in a buildup of the thylakoid proton motive force with subsequent activation of non-photochemical quenching and downregulation of linear electron flow. We conclude that NTRC provides redox modulation at low light using the relatively oxidizing substrate NADPH, whereas the canonical ferredoxin-thioredoxin system can take over at higher light, when reduced ferredoxin can accumulate. Based on these results, we reassess previous models for ATP synthase regulation and propose that NTRC is most likely regulated by light. We also find that ntrc is highly sensitive to rapidly changing light intensities that probably do not involve the chloroplast ATP synthase, implicating this system in multiple photosynthetic processes, particularly under fluctuating environmental conditions.

Inter-functional analysis of high-throughput phenotype data by non-parametric clustering and its application to photosynthesis.

MOTIVATION: Phenomics is the study of the properties and behaviors of organisms (i.e. their phenotypes) on a high-throughput scale. New computational tools are needed to analyze complex phenomics data, which consists of multiple traits/behaviors that interact with each other and are dependent on external factors, such as genotype and environmental conditions, in a way that has not been well studied. RESULTS: We deployed an efficient framework for partitioning complex and high dimensional phenotype data into distinct functional groups. To achieve this, we represented measured phenotype data from each genotype as a cloud-of-points, and developed a novel non-parametric clustering algorithm to cluster all the genotypes. When compared with conventional clustering approaches, the new method is advantageous in that it makes no assumption about the parametric form of the underlying data distribution and is thus particularly suitable for phenotype data analysis. We demonstrated the utility of the new clustering technique by distinguishing novel phenotypic patterns in both synthetic data and a high-throughput plant photosynthetic phenotype dataset. We biologically verified the clustering results using four Arabidopsis chloroplast mutant lines. AVAILABILITY AND IMPLEMENTATION: Software is available at www.msu.edu/~jinchen/NPM. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: jinchen@msu.edu, kramerd8@cns.msu.edu or rongjin@cse.msu.edu.

NPQ(T) : a chlorophyll fluorescence parameter for rapid estimation and imaging of non-photochemical quenching of excitons in photosystem-II-associated antenna complexes.

In photosynthesis, light energy is absorbed by light-harvesting complexes and used to drive photochemistry. However, a fraction of absorbed light is lost to non-photochemical quenching (NPQ) that reflects several important photosynthetic processes to dissipate excess energy. Currently, estimates of NPQ and its individual components (qE , qI , qZ and qT ) are measured from pulse-amplitude-modulation (PAM) measurements of chlorophyll fluorescence yield and require measurements of the maximal yield of fluorescence in fully dark-adapted material (Fm ), when NPQ is assumed to be negligible. Unfortunately, this approach requires extensive dark acclimation, often precluding widespread or high-throughput use, particularly under field conditions or in imaging applications, while introducing artefacts when Fm is measured in the presence of residual photodamaged centres. To address these limitations, we derived and characterized a new set of parameters, NPQ(T) , and its components that can be (1) measured in a few seconds, allowing for high-throughput and field applications; (2) does not require full relaxation of quenching processes and thus can be applied to photoinhibited materials; (3) can distinguish between NPQ and chloroplast movements; and (4) can be used to image NPQ in plants with large leaf movements. We discuss the applications benefits and caveats of both approaches.

Variations in chloroplast movement and chlorophyll fluorescence among chloroplast division mutants under light stress.

Chloroplasts divide to maintain consistent size, shape, and number in leaf mesophyll cells. Altered expression of chloroplast division proteins in Arabidopsis results in abnormal chloroplast morphology. To better understand the influence of chloroplast morphology on chloroplast movement and photosynthesis, we compared the chloroplast photorelocation and photosynthetic responses of a series of Arabidopsis chloroplast division mutants with a wide variety of chloroplast phenotypes. Chloroplast movement was monitored by red light reflectance imaging of whole plants under increasing intensities of white light. The accumulation and avoidance responses were differentially affected in different mutants and depended on both chloroplast number and morphological heterogeneity. Chlorophyll fluorescence measurements during 5 d light experiments demonstrated that mutants with large-chloroplast phenotypes generally exhibited greater PSII photodamage than those with intermediate phenotypes. No abnormalities in photorelocation efficiency or photosynthetic capacity were observed in plants with small-chloroplast phenotypes. Simultaneous measurement of chloroplast movement and chlorophyll fluorescence indicated that the energy-dependent (qE) and long-lived components of non-photochemical quenching that reflect photoinhibition are affected differentially in different division mutants exposed to high or fluctuating light intensities. We conclude that chloroplast division mutants with abnormal chloroplast morphologies differ markedly from the wild type in their light adaptation capabilities, which may decrease their relative fitness in nature.

Non-invasive, whole-plant imaging of chloroplast movement and chlorophyll fluorescence reveals photosynthetic phenotypes independent of chloroplast photorelocation defects in chloroplast division mutants.

Leaf chloroplast movement is thought to optimize light capture and to minimize photodamage. To better understand the impact of chloroplast movement on photosynthesis, we developed a technique based on the imaging of reflectance from leaf surfaces that enables continuous, high-sensitivity, non-invasive measurements of chloroplast movement in multiple intact plants under white actinic light. We validated the method by measuring photorelocation responses in Arabidopsis chloroplast division mutants with drastically enlarged chloroplasts, and in phototropin mutants with impaired photorelocation but normal chloroplast morphology, under different light regimes. Additionally, we expanded our platform to permit simultaneous image-based measurements of chlorophyll fluorescence and chloroplast movement. We show that chloroplast division mutants with enlarged, less-mobile chloroplasts exhibit greater photosystem II photodamage than is observed in the wild type, particularly under fluctuating high levels of light. Comparison between division mutants and the severe photorelocation mutant phot1-5 phot2-1 showed that these effects are not entirely attributable to diminished photorelocation responses, as previously hypothesized, implying that altered chloroplast morphology affects other photosynthetic processes. Our dual-imaging platform also allowed us to develop a straightforward approach to correct non-photochemical quenching (NPQ) calculations for interference from chloroplast movement. This correction method should be generally useful when fluorescence and reflectance are measured in the same experiments. The corrected data indicate that the energy-dependent (qE) and photoinhibitory (qI) components of NPQ contribute differentially to the NPQ phenotypes of the chloroplast division and photorelocation mutants. This imaging technology thus provides a platform for analyzing the contributions of chloroplast movement, chloroplast morphology and other phenotypic attributes to the overall photosynthetic performance of higher plants.

Plant photosynthesis phenomics data quality control.

MOTIVATION: Plant phenomics, the collection of large-scale plant phenotype data, is growing exponentially. The resources have become essential component of modern plant science. Such complex datasets are critical for understanding the mechanisms governing energy intake and storage in plants, and this is essential for improving crop productivity. However, a major issue facing these efforts is the determination of the quality of phenotypic data. Automated methods are needed to identify and characterize alterations caused by system errors, all of which are difficult to remove in the data collection step and distinguish them from more interesting cases of altered biological responses. RESULTS: As a step towards solving this problem, we have developed a coarse-to-refined model called dynamic filter to identify abnormalities in plant photosynthesis phenotype data by comparing light responses of photosynthesis using a simplified kinetic model of photosynthesis. Dynamic filter employs an expectation-maximization process to adjust the kinetic model in coarse and refined regions to identify both abnormalities and biological outliers. The experimental results show that our algorithm can effectively identify most of the abnormalities in both real and synthetic datasets. AVAILABILITY AND IMPLEMENTATION: Software available at www.msu.edu/%7Ejinchen/DynamicFilter .

Temporal Dynamics of Growth and Photosynthesis Suppression in Response to Jasmonate Signaling.

Biotic stress constrains plant productivity in natural and agricultural ecosystems. Repression of photosynthetic genes is a conserved plant response to biotic attack, but how this transcriptional reprogramming is linked to changes in photosynthesis and the transition from growth- to defense-oriented metabolism is poorly understood. Here, we used a combination of noninvasive chlorophyll fluorescence imaging technology and RNA sequencing to determine the effect of the defense hormone jasmonate (JA) on the growth, photosynthetic efficiency, and gene expression of Arabidopsis (Arabidopsis thaliana) rosette leaves. High temporal resolution was achieved through treatment with coronatine (COR), a high-affinity agonist of the JA receptor. We show that leaf growth is rapidly arrested after COR treatment and that this effect is tightly correlated with changes in the expression of genes involved in growth, photosynthesis, and defense. Rapid COR-induced expression of defense genes occurred concomitantly with the repression of photosynthetic genes but was not associated with a reduced quantum efficiency of photosystem II. These findings support the view that photosynthetic capacity is maintained during the period in which stress-induced JA signaling redirects metabolism from growth to defense. Chlorophyll fluorescence images captured in a multiscale time series, however, revealed a transient COR-induced decrease in quantum efficiency of photosystem II at dawn of the day after treatment. Physiological studies suggest that this response results from delayed stomatal opening at the night-day transition. These collective results establish a high-resolution temporal view of how a major stress response pathway modulates plant growth and photosynthesis and highlight the utility of chlorophyll fluorescence imaging for revealing transient stress-induced perturbations in photosynthetic performance.

A Review of 1228 In-Office Hand Surgery Procedures With Wide Awake Local Anesthesia No Tourniquet (WALANT) at a Single Private Practice.

BACKGROUND: This study examined the complication rate of Wide Awake Local Anesthesia No Tourniquet (WALANT) technique in the clinic setting with field sterility at a single private practice. We hypothesized that WALANT is safe and effective with a low complication rate. METHODS: This retrospective chart review included 1228 patients who underwent in-office WALANT hand procedures at a single private practice between 2015 and 2022. Patients were divided into groups based on type of procedure: carpal tunnel release, A1 pulley release, first dorsal compartment release, extensor tendon repair, mass excision, foreign body removal, and needle aponeurotomy. Patient demographics and complications were recorded; statistical comparisons of cohort demographics and risk factors for complications were completed, and P < .05 was considered significant for all statistical comparisons. RESULTS: The overall complication rate for all procedures was 2.77% for 1228 patients including A1 pulley release (n = 962, 2.7%), mass excision (n = 137, 3.7%), extensor tendon repair (n = 23, 4.3%), and first dorsal compartment release (n = 22, 8.3%). Carpal tunnel release, foreign body removal, and needle aponeurotomy groups experienced no complications. No adverse events (e.g. vasovagal reactions, digital ischemia, local anesthetic toxicity, inadequate vasoconstriction) were observed in any group. Patients with known autoimmune disorders and those who were currently smoking had a statistically significant higher complication rate. CONCLUSIONS: Office-based WALANT procedures with field sterility are safe and effective for treating common hand maladies and have a similar complication profile when compared to historical controls from the standard operating room in an ambulatory center or hospital.

An Arabidopsis mutant with high cyclic electron flow around photosystem I (hcef) involving the NADPH dehydrogenase complex.

Cyclic electron flow (CEFI) has been proposed to balance the chloroplast energy budget, but the pathway, mechanism, and physiological role remain unclear. We isolated a new class of mutant in Arabidopsis thaliana, hcef for high CEF1, which shows constitutively elevated CEF1. The first of these, hcef1, was mapped to chloroplast fructose-1,6-bisphosphatase. Crossing hcef1 with pgr5, which is deficient in the antimycin A-sensitive pathway for plastoquinone reduction, resulted in a double mutant that maintained the high CEF1 phenotype, implying that the PGR5-dependent pathway is not involved. By contrast, crossing hcef1 with crr2-2, deficient in thylakoid NADPH dehydrogenase (NDH) complex, results in a double mutant that is highly light sensitive and lacks elevated CEF1, suggesting that NDH plays a direct role in catalyzing or regulating CEF1. Additionally, the NdhI component of the NDH complex was highly expressed in hcef1, whereas other photosynthetic complexes, as well as PGR5, decreased. We propose that (1) NDH is specifically upregulated in hcef1, allowing for increased CEF1; (2) the hcef1 mutation imposes an elevated ATP demand that may trigger CEF1; and (3) alternative mechanisms for augmenting ATP cannot compensate for the loss of CEF1 through NDH.

Arthroscopic TFCC Ulnar Bone Tunnel Foveal Repair in Adult Patients.

Triangular fibrocartilage complex (TFCC) tears may cause persistent ulnar-sided wrist pain, loss of grip strength, and associated loss of function. Although the majority of TFCC tears can be treated nonoperatively, surgical repair is considered when conservative measures fail. TFCC tears with foveal disruption and instability of the distal radioulnar joint (DRUJ) require direct repair of the TFCC to the ulnar fovea. The traditional method of foveal TFCC repair involves an open surgical approach through the floor of the 5th dorsal compartment. However, this open approach causes disruption of structures such as the dorsal ulnocarpal capsule, the extensor retinaculum, and, potentially, the distal radioulnar ligament (DRUL). This article describes, in detail, the recently developed arthroscopic assisted ulnar foveal bone tunnel repair. This method spares dorsal structures that may be disrupted during an open surgical approach and creates a robust repair of the TFCC deep fibers with restoration of DRUJ stability.

The Arabidopsis T-DNA mutant SALK_008491 carries a 14-kb deletion on chromosome 3 that provides rare insights into the plant response to dynamic light stress.

In nature, plants experience rapid changes in light intensity and quality throughout the day. To maximize growth, they have established molecular mechanisms to optimize photosynthetic output while protecting components of the light-dependent reaction and CO2 fixation pathways. Plant phenotyping of mutant collections has become a powerful tool to unveil the genetic loci involved in environmental acclimation. Here, we describe the phenotyping of the transfer-DNA (T-DNA) insertion mutant line SALK_008491, previously known as nhd1-1. Growth in a fluctuating light regime caused a loss in growth rate accompanied by a spike in photosystem (PS) II damage and increased non-photochemical quenching (NPQ). Interestingly, an independent nhd1 null allele did not recapitulate the NPQ phenotype. Through bulk sequencing of a backcrossed segregating F2 pool, we identified an ~14-kb large deletion on chromosome 3 (Chr3) in SALK_008491 affecting five genes upstream of NHD1. Besides NHD1, which encodes for a putative plastid Na+/H+ antiporter, the stromal NAD-dependent D-3-phosphoglycerate dehydrogenase 3 (PGDH3) locus was eradicated. Although some changes in the SALK_008491 mutant's photosynthesis can be assigned to the loss of PGDH3, our follow-up studies employing respective single mutants and complementation with overlapping transformation-competent artificial chromosome (TAC) vectors reveal that the exacerbated fluctuating light sensitivity in SALK_008491 mutants result from the simultaneous loss of PGDH3 and NHD1. Altogether, the data obtained from this large deletion-carrying mutant provide new and unintuitive insights into the molecular mechanisms that function to protect the photosynthetic machinery. Moreover, our study renews calls for caution when setting up reverse genetic studies using T-DNA lines. Although second-site insertions, indels, and SNPs have been reported before, large deletion surrounding the insertion site causes yet another problem. Nevertheless, as shown through this research, such unpredictable genetic events following T-DNA mutagenesis can provide unintuitive insights that allow for understanding complex phenomena such as the plant acclimation to dynamic high light stress.

The effects of moderately high temperature on zeaxanthin accumulation and decay.

Moderately high temperature reduces photosynthetic capacities of leaves with large effects on thylakoid reactions of photosynthesis, including xanthophyll conversion in the lipid phase of the thylakoid membrane. In previous studies, we have found that leaf temperature of 40°C increased zeaxanthin accumulation in dark-adapted, intact tobacco leaves following a brief illumination, but did not change the amount of zeaxanthin in light-adatped leaves. To investigate heat effects on zeaxanthin accumulation and decay, zeaxanthin level was monitored optically in dark-adapted, intact tobacco and Arabidopsis thaliana leaves at either 23 or 40°C under 45-min illumination. Heated leaves had more zeaxanthin following 3-min light but had less or comparable amounts of zeaxanthin by the end of 45 min of illumination. Zeaxanthin accumulated faster at light initiation and decayed faster upon darkening in leaves at 40°C than leaves at 23°C, indicating that heat increased the activities of both violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE). In addition, our optical measurement demonstrated in vivo that weak light enhances zeaxanthin decay relative to darkness in intact leaves of tobacco and Arabidopsis, confirming previous observations in isolated spinach chloroplasts. However, the maximum rate of decay is similar for weak light and darkness, and we used the maximum rate of decay following darkness as a measure of the rate of ZE during steady-state light. A simulation indicated that high temperature should cause a large shift in the pH dependence of the amount of zeaxanthin in leaves because of differential effects on VDE and ZE. This allows for the reduction in ΔpH caused by heat to be offset by increased VDE activity relative to ZE.

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.

Metabolite profiling of Calvin cycle intermediates by HPLC-MS using mixed-mode stationary phases.

SUMMARY: A sensitive and robust mixed-mode high performance liquid chromatography-tandem mass spectrometry method was developed for the qualitative and quantitative determination of sugar phosphates, which are notoriously difficult to separate using reversed-phase materials. Sugar phosphates were separated on a Primesep SB column by gradient elution using aqueous ammonium formate and acetonitrile as mobile phases. Target analytes were identified by their precursor/product ions and retention times. Quantitative analysis was performed in negative ionization/multiple reaction monitoring mode with five different time segments. The method was validated by spiking authentic sugar phosphate standards into complex plant tissue extracts. Standard curves of neat authentic standards and spiked extracts were generated for concentrations in the low picomole to nanomole range, with correlation coefficients of R(2) > 0.991, and the degree of ion suppression in the presence of a plant matrix was calculated for each analyte. Analyte recoveries, which were determined by including known quantities of authentic standards in the sugar phosphate extraction protocol, ranged from 40.0% to 57.4%. The analytical reproducibility was assessed by determining the coefficient of variance based on repeated extractions/measurements (<20%). The utility of our method is demonstrated with two types of applications: profiling of Calvin cycle intermediates in (i) dark-adapted and light-treated tobacco leaves, and in (ii) antisense plants expressing reduced levels of the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase or ribulose-1,5-bisphosphate carboxylase/oxygenase (comparison with wild-type controls). The broader applicability of our method is illustrated by profiling sugar phosphates extracted from the leaves of five taxonomically diverse plants.

The long-term responses of the photosynthetic proton circuit to drought.

Proton motive force (pmf) across thylakoid membranes is not only for harnessing solar energy for photosynthetic CO(2) fixation, but also for triggering feedback regulation of photosystem II antenna. The mechanisms for balancing these two roles of the proton circuit under the long-term environmental stress, such as prolonged drought, have been poorly understood. In this study, we report on the response of wild watermelon thylakoid 'proton circuit' to drought stress using both in vivo spectroscopy and molecular analyses of the representative photosynthetic components. Although drought stress led to enhanced proton flux via a approximately 34% increase in cyclic electron flow around photosystem I (PS I), an observed approximately fivefold decrease in proton conductivity, g(H)(+), across thylakoid membranes suggested that decreased ATP synthase activity was the major factor for sustaining elevated q(E). Western blotting analyses revealed that ATP synthase content decreased significantly, suggesting that quantitative control of the complex plays a pivotal role in down-regulation of g(H)(+). The expression level of cytochrome b(6)f complex - another key control point in photosynthesis - also declined, probably to prevent excess-reduction of PS I electron acceptors. We conclude that plant acclimation to long-term environmental stress involves global changes in the photosynthetic proton circuit, in which ATP synthase represents the key control point for regulating the relationship between electron transfer and pmf.