To address this question, we measured photosynthetic parameters of gas exchange and chlorophyll fluorescence in tobacco leaves at 2-min intervals while irradiance levels alternated between 100 and 1200 μmol photons m−2 s−1. Compared with leaves exposed to a constant light of 1200 μmol photons m−2 s−1, both stomatal and mesophyll conductances were significantly restricted in leaves treated with fluctuating light condition. Meanwhile, CO2 assimilation rate and electron flow devoted to RuBP carboxylation at 1200 μmol photons m−2 s−1 under fluctuating light were limited by the low chloroplast CO2 concentration. Analysis based on the C3 photosynthesis model indicated that, at 1200 μmol photons m−2 s−1 under fluctuating light, the CO2 assimilation rate was limited by RuBP carboxylation. Electron flow devoted to RuBP oxygenation at 1200 μmol photons m−2 s−1 under fluctuating light remained at nearly the maximum level throughout the experimental period. We conclude that fluctuating light restricts CO2 assimilation by decreasing both stomatal and mesophyll conductances. Under such conditions, photorespiration plays an important role in the regulation of photosynthetic electron flow.Plants usually experience dynamic fluctuations of light intensities under natural conditions. However, the responses of mesophyll conductance, CO Under constant high light, the rate of CO2 assimilation (An) is maintained at an elevated level due to high gs and mesophyll conductance (gm)  plants grown with adequate water and optimum temperature, gm is mainly dependent upon the growth light intensity . In tobacco, a model C3 plant, the rate of CO2 assimilation under high light is influenced by leaf nitrogen (N) content. CO2 assimilation rate under high light tends to be limited by RuBP regeneration for plants grown at high N concentration  Is gm restricted by fluctuating light? (2) What is the limiting step of An under fluctuating light? and (3) Does the photorespiratory pathway play an important role in regulating photosynthetic electron flow under fluctuating light?In this study, we measured the photosynthetic parameters of gas exchange and chlorophyll fluorescence to investigate the responses of Following seed germination, seedlings of tobacco cv. y87 were cultivated in phytotron for 7 weeks. Afterwards, they were grown in plastic pots in an open field at Kunming Institute of Botany, Yunnan, China . During our experiment period (10 May to 24 June 2013), none of the plants experienced any water or nutrient stresses. The average temperature at Kunming was 20.9°C in May and 20.6°C in June. Fully expanded mature leaves on 13-week-old plants were used for photosynthetic measurements.2 and water vapor analyzers  and a 2-cm2 measuring head . Measurements were made in a phytotron where relative air humidity (60%) and air temperature (25°C) were controlled. The atmospheric CO2 concentration was maintained at 400 μmol mol−1 by the Li-6400XT. To generate a light response curve, we initially exposed the mature leaves to strong irradiance (2000 μmol photons m−2 s−1) for 20 min to obtain steady, high levels of gs and CO2 assimilation. Afterward, photosynthetic parameters were evaluated at 2-min intervals at photosynthetic photon flux densities (PPFDs) of 2000, 1600, 1200, 800, 500, 300, 200, 100, 50, 20, or 0 μmol photons m−2 s−1. To investigate the responses of gs, gm, CO2 assimilation rate, and photosynthetic electron flow to fluctuating light, we also evaluated those photosynthetic parameters under light levels that alternated every 2 min between 100 and 1200 μmol photons m−2 s−1 after dark-adaptation for 30 min. Photosynthetic induction curves were also developed at 1200 μmol photons m−2 s−1 after 30 min of darkness. Values for those parameters were recorded automatically by the Li-6400XT at 2-min intervals.Photosynthetic parameters for gas exchange and chlorophyll fluorescence were monitored with an open gas exchange system that incorporated infrared CO2 assimilation rate vs. chloroplast CO2 concentration (Cc) was examined at 1200 μmol photons m−2 s−1  and RuBP carboxylation (Vcmax) according to the method of Long and Bernacchi  was calculated as (Fm′–Fo′)/Fm′. Coefficient of PSII photochemical quenching (qP) was calculated as (Fm′–Fs)/(Fm′–Fo′). Effective quantum yield of PSII (ΦPSII) was calculated as (Fm′–Fs)/Fm′  and oxygenation (JO). Values for JC and JO were estimated according to the method of Valentini et al.  at 1200 μmol photons m−2 s−1 after plants were exposed to either fluctuating or constant light for 60 min. For our comparisons, gm was also estimated at 1200 μmol photons m−2 s−1 in light response curves. Values for gm were estimated through a combination analysis of gas exchange and chlorophyll fluorescence, and according to the following equation  and Ko (mmol mol−1) were the Michaelis constants for CO2 and O2, respectively  to examine differences among treatments involving fluctuating vs. constant light. Those differences were considered significant at gs was maintained at high levels (>0.3 mol m−2 s−1) when plants were exposed to light intensities above 100 μmol photons m−2 s−1  at 1200 μmol photons m−2 s−1 was significantly higher under constant illumination than under fluctuating light, i.e., 225 vs. 185 μmol electrons m−2 s−1, respectively  at 1200 μmol photons m−2 s−1 changed only slightly between constant- and fluctuating-light treatments  at 1200 μmol photons m−2 s−1 was higher under constant light (151 μmol electrons m−2 s−1) than under fluctuating light (115 μmol electrons m−2 s−1)  Figure . ConsequC Figure . These r−2 s−1 under fluctuating light, we determined that gs was linearly and positively correlated with An, JT, and JC  to that of RuBP carboxylation (Vcmax) was 0.92, and the chloroplast CO2 concentration at which the transition from RuBP carboxylation to RuBP regeneration occurred (Ctrans) was 135 μmol mol−1  or under constant light (0.28 mol m−2 s−1) , the rate of CO2 assimilation at 1200 μmol photons m−2 s−1 under fluctuating light was limited by RuBP carboxylation.To analyze the limiting step of CO1 Figure . After e) Figure . For theAn is mainly determined by the relative values of Ctrans and Cc. For tobacco plants supplied with high concentrations of nitrogen, photosynthesis tends to be limited by RuBP regeneration because Cc is higher than Ctrans  in fluctuating light. We found that, under fluctuating light, gm was regulated by both high and low light levels; i.e., although the former induced an increase in gm, this effect could be partially reversed when plants were then exposed to reduced irradiance.Carriquí et al.  have dem1 Figure . We beli2 assimilation in C3 plants is constrained by RuBP carboxylation and/or RuBP regeneration. Therefore, based on that model, the limiting step can be altered in two ways: (1) adjustments in the balance between the maximum rates of RuBP regeneration and RuBP carboxylation, or (2) changes in the chloroplast CO2 concentration  depends upon the leaf-N content and is mainly determined by Jmax/Vcmax. Furthermore, at high leaf-N content, A380 is limited by RuBP regeneration due to the low ratio of Jmax/Vcmax  and O2-dependent alternative electron sinks in regulating photosynthetic electron flow under fluctuating light  to dissipate excess light energy harmlessly as heat  via cytochrome b6/f (Tikkanen and Aro, A. thaliana (Kono et al., Suppression of COCc will accelerate RuBP oxygenation and, ultimately, the production of those intermediates. If the photorespiratory pathway is maintained at a low level under such conditions, the accumulation of those intermediates inhibits CO2 assimilation as well as photosynthetic electron flow, causing acceleration of photodamage (Chastain and Ogren, 2 assimilation, rapid acceleration of photorespiratory pathway results in low glycine and glycerate contents (Timm et al., 2 assimilation and photosynthetic electron flow. In addition, the operation of this pathway is necessary for the regeneration of RuBP (Takahashi et al., 2 fixation, the rates of RuBP oxygenation and RuBP regeneration through photorespiratory pathway must be balanced. Therefore, under fluctuating light conditions, strong activation of the photorespiratory pathway accelerates RuBP regeneration, preventing a decrease in the RuBP pool and favoring the Calvin cycle.Although photorespiratory intermediates such as glycine and glycerate inhibit the Calvin cycle (Chastain and Ogren, 2 concentration. Consequently, the rate of CO2 assimilation is limited by RuBP carboxylation under such conditions. Meanwhile, the photorespiratory pathway is highly activated to regulate photosynthetic electron flow and benefit photosynthetic CO2 fixation. Thus, strong activation of this pathway is an important strategy by which sun-grown plants adapt to fluctuating light.In summary, our results provide evidence that, for sun-grown tobacco leaves, fluctuating light conditions significantly decrease both stomatal and mesophyll conductances, as well as chloroplast COThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study investigates differential neural activation patterns in response to reward-related feedback depending on various reward contingencies. Three types of reward contingencies were compared: a “gain” contingency ; a “lose” contingency ; and a “combined” contingency . Sixteen undergraduate students were exposed to the three reward contingencies while performing a series of perceptual judgment tasks. The fMRI results revealed that only the “gain” contingency recruited the ventral striatum, a region associated with positive affect and motivation, during overall feedback processing. Specifically, the ventral striatum was more activated under the “gain” contingency than under the other two contingencies when participants received positive feedback. In contrast, when participants received negative feedback, the ventral striatum was less deactivated under the “gain” and “lose” contingencies than under the “combined” contingency. Meanwhile, the negative feedback elicited significantly stronger activity in the dorsal amygdala, a region tracking the intensity and motivational salience of stimuli, under the “gain” and “lose” contingencies. These findings suggest the important role of contextual factor, such as reward contingency, in feedback processing. Based on the current findings, we recommend implementing the “gain” contingency to maintain individuals’ optimal motivation. Motivation, a major determinant of behavior, helps individuals to engage in goal-directed behaviors , and the amygdala  or prevention-focused (lose context), resulting in differential emotional and motivational responses. For example, in a behavioral study comparing the effects of three reward contingencies on performance satisfaction of middle school students, Bong and Kim  showed tIn the present study, we aimed to investigate how reward contexts with different focuses  would influence the neural responses during feedback processing. We manipulated three different types of reward contingencies  and compared the brain activities in various regions of interest (ROI)  during feedback processing. We expected different brain activation patterns in response to feedback depending upon the type of reward contingency.SD = 2.28) volunteered to participate in this study via online postings. All participants reported no prior experience with neurological experiments and neurological or psychiatric disorders, and signed a written consent form according to the protocols of Korea University’s Institutional Review Board. All participants received a monetary reward (Korean Won (KRW) 30,000, equivalent to $27) after completing the experiment.The study was approved by Korea University’s Institutional Review Board for human participants. A total of sixteen healthy, right-handed undergraduate students from several private universities in Seoul  during scanning. Each contingency consisted of 40 trials, yielding 120 trials in total. Each trial lasted 11 s, with each contingency lasting 7 min and 20 s. The entire scan took 22 min. For each trial, the task stimulus was presented for 2 s, followed by a random fixation. The durations of the fixation were determined by a sequence of random numbers generated by Excel, ranging from 1 to 4 s with an interval of 0.5 s. The average duration of fixation was 2 s. After the random fixation, the participants received facial feedback for 1 s, notifying them of whether they had succeeded or failed. After another random fixation, participants received monetary feedback that lasted 2 s. This was followed by a random inter-trial interval also ranging from 1 to 4 s  or the right button (if they believed that the number did not meet the criteria) on a keyboard connected to the fMRI machine. To prevent the practice effect as well as fatigue effect from repetitive performance of the same tasks, we incorporated slightly different tasks into each contingency. Specifically, during the “combined” contingency, participants looked at a computer screen filled with 14 to 16 figures for 2 s. The task was to determine whether the number of figures on the screen was 15 or not. Similarly, participants were asked to detect whether or not the number of letters was 16 during the “gain” contingency and to determine whether or not the number of digits was 16 during the “lose” contingency. The pilot test showed that there was no difference among these tasks in terms of task difficulty and task interest.Three patterns of feedback were used in accordance with the three types of reward contingencies. Under the “combined” contingency, participants started with KRW 20,000. For a correct answer, an image of a smiley face was accompanied and followed by monetary feedback indicating that the participant received KRW 1000. For an incorrect answer, on the other hand, an image of a sad face was accompanied and followed by monetary feedback indicating that the participant lost KRW 1000. Under the “gain” contingency, participants started with KRW 0. For a correct answer, a smiley face was presented and followed by monetary feedback indicating that the participant received KRW 1000. For an incorrect answer, a sad face was presented and followed by monetary feedback indicating no monetary change. Under the “lose” contingency, participants started with KRW 40,000. For a correct answer, a smiley face was presented and followed by monetary feedback indicating no monetary change. For an incorrect answer, a sad face was presented and followed by monetary feedback indicating that the participant lost KRW 1000. Figure Unbeknownst to the participants, the feedback was predetermined regardless of their actual performance and all of the participants received identical feedback throughout the whole experiment. We used this method to prevent potential large differences in performance among the participants so that they could end up with the same amount of rewards within each contingency. The success rate was set as 50% for all three contingency conditions, and the sequence of positive and negative feedback was randomized within each contingency. Because the task stimulus was presented for a relatively short period of time (2 s), the high level of task difficulty made the bogus performance feedback credible to the participants. In their post-scanning interviews, all participants reported that they believed the feedback was based on their actual performance. After they had finished the whole experiment, participants were fully debriefed.For the run sequence, the “combined” contingency was always presented first because it creates a reward context with no specific focus (neither promotion nor prevention). Therefore, it was used as a baseline condition. For the purpose of counterbalancing the order effect of the “gain” and “lose” contingencies, half of the participants were scanned in a “combined-gain-lose” sequence and the other half were scanned in a “combined-lose-gain” sequence.The experiment was conducted at Ewha Womans University Mokdong Hospital. Images were acquired using a Philips Intera Achieva 3T MRI scanner . Functional data were obtained using a single-shot gradient echo-planar imaging (EPI) sequence  = 240 mm, ascending, 36 3-mm-thick slices, with no gap). After the first run, high-resolution T1-weighted three-dimensional volumes were acquired for anatomical localization .Imaging data were preprocessed and analyzed by Statistical Parametric Mapping  in the Matlab  environment. During preprocessing, functional images were first realigned to the first volume to compensate for subtle head motions. All participants’ head motions were less than 3 mm in any translation within each run. Data were then corrected for differences in timing of slice acquisition, normalized to EPI templates implemented in the SPM, and spatially smoothed using an 8 mm full width at half maximum isotropic (FWHM) Gaussian kernel.After the preprocessing, statistical analyses were performed on each participant’s data using a general linear model (GLM) in SPM. The analyses were performed by modeling facial feedback (success and failure events for each contingency), monetary feedback (success and failure events for each contingency), and task stimuli as regressors. Participants’ response times during task phases and realignment parameters were also included as regressors in the statistical model. Changes in the Blood-Oxygen-Level-Dependent (BOLD) signal were assessed by linear combinations of the estimated GLM parameters . Because participants were informed of the contingency condition before they started each run, the present study particularly focused on comparing the facial feedback phase. In other words, once the participants received facial feedback, they were automatically aware that they would receive reward, lose money, or obtain no monetary reward/penalty.p < 0.05 false discovery rate (FDR) corrected for multiple comparisons at the voxel level, with an extent threshold of 10 contiguous voxels. Activations in a priori ROI but failed to survive in the whole-brain correction were then subjected to a small-volume correction (SVC). ROI masks for SVC were created based on apriori anatomical structures rather than observed activation from present result. Specifically, the masks for the bilateral ventral striatum were created as 8-mm spheres centered on the coordinates  coordinates: x, y, z = −10, 12, −6; 16, 12, −12) indentified in a quantitative meta-analysis which tested the role of the ventral striatum in reward processing  reported in a previous empirical study which specifically investigated the role of the dorsal amygdala subregion in modulating attentional resources during emotional processing  and contingency  as factors to test the main effects of the feedback and the contingency as well as the potential interaction of the two factors on brain activation. We also conducted two separate one-way ANOVAs with each feedback valence as factors to explicitly test how positive and negative feedback may recruit distinct patterns of brain activation under various contingencies. The statistical criterion was set at post hoc analyses using the least significant difference method with adjusted alpha levels of 0.05 to quantify the mean beta value and activation patterns of the significantly activated brain regions. Functional ROIs were defined by the full cluster of activated regions and were analyzed with the Marsbar toolbox  in the “combined” contingency, 1399.64 ms (SD = 220.05) in the “gain” contingency, and 1392.81 ms (SD = 224.44) in the “lose” contingency. A one-way ANOVA revealed no significant difference in response times among the three reward contingencies  = 0.089, p = 0.92). Likewise, the average missing frequencies (a failure to respond within 2 s of task stimulus time) were 2.67 times (SD = 4.38) in the “combined” contingency, 2.67 times (SD = 3.75) in the “gain” contingency, and 2.08 times (SD = 4.12) in the “lose” contingency. There was also no significant difference among the three reward contingencies  = 0.081, p = 0.92).Participants’ average response times were 1326.88 ms . Functional ROI result indicated that the positive feedback elicited significantly higher activation in the ventral striatum than the negative feedback . Functional ROI analyses revealed that the ventral striatum showed positive activation only under the “gain” contingency during the overall feedback processing  = 5.06, p < 0.01; right: t(15) = 5.97, p < 0.01) and was less deactivated under the “lose” contingency (left: t(15) = 4.07, p < 0.01; right: t(15) = 4.56, p < 0.01) than under the “combined” contingency  = 4.30, p < 0.01; right: t(15) = 4.21, p < 0.01) and the “lose” (left: t(15) = 4.87, p < 0.01; right: t(15) = 2.65, p = 0.02) contingencies than under the “combined” contingency during the overall feedback processing , a key brain region involved in perceiving and evaluating emotional stimuli. Specifically, the sgACC showed significantly higher activation under the “lose” contingency than under the “gain” (t(15) = 3.32, p = 0.01) and the “combined” (t(15) = 5.16, p < 0.01) contingencies during the overall feedback processing  under the “gain” contingency than under the “lose” (t(15) = 2.23, p = 0.04) and the “combined” (t(15) = 5.15, p < 0.01) contingencies. Meanwhile, when participants received negative feedback, the ventral striatum  was significantly less deactivated under the “gain” (t(15) = 5.50, p < 0.01) and “lose” (t(15) = 3.94, p < 0.01) contingencies than under the “combined” contingency  under the “gain” (t(15) = 4.23, p < 0.01) and the “lose” (t(15) = 4.16, p < 0.01) contingencies than under the “combined” contingency when participants received negative feedback  only in the one-way ANOVA with negative feedback. Moreover, the activation pattern of the sgACC in the one-way ANOVA with negative feedback was exactly the same as the pattern observed in the factorial ANOVA. The sgACC is a well-documented brain region that engages in negative mood, particularly sadness , we found significant activation in the corresponding sgACC region (MNI coordinates: There was also a significant main effect of feedback on the lateral OFC in the 2 × 3 ANOVA analysis. Specifically, positive feedback has produced significantly stronger activation in the lateral OFC than negative feedback. The OFC is the critical brain region for value judgment (Grabenhorst and Rolls, Taken together, the findings of this study have practical implications for designing reward contexts that could beget positive affect and enhance individuals’ motivation. Depending upon the reward contingency, individuals could perceive an identical feedback differently and in turn experience different emotions and motivations. Among the three types of contingencies, we recommend implementing the “gain” contingency, in which a reward is given for success and no punishment is given for failure, because it shows the most adaptive pattern of emotional and motivational responses to both positive and negative feedback. Our interview data support this argument as most of the participants felt more satisfied with the “gain” contingency. Among the sixteen participants, nine rated the “gain” contingency, four rated the “combined” contingency, and only three rated the “lose” contingency as most satisfactory.Several limitations of the present study as well as suggestions for future research need to be addressed. First, we used bogus feedback regardless of participants’ actual performance. Although all the participants believed that the feedback was based on their actual performance, it would be ideal for future research to use real feedback based on participants’ actual performance. Second, it would be worthwhile to further investigate if verbal feedback without a monetary reward would have a similar effect because verbal praise and punishment are more frequently used in educational settings.The present study investigated individuals’ emotional and motivational responses to three different types of reward contingencies during feedback processing. It contributes to the existing literature by demonstrating that contextual effect of reward could elicit distinct neural activities during feedback processing. In particular, the results indicate that the “gain” contingency is more likely to produce positive affect and maintain individuals’ motivation. Therefore, we suggest implementing reward/punishment systems based on the “gain” contingency to maintain individuals’ motivation during task performances.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arabidopsis thaliana to characterise the importance of Rubisco activase (Rca), stomatal conductance (gs), non-photochemical quenching of chlorophyll fluorescence (NPQ) and sucrose phosphate synthase (SPS) on photosynthesis in fluctuating irradiance. Leaf gas exchange and chlorophyll fluorescence were measured in leaves exposed to stepwise increases and decreases in irradiance. rwt43, which has a constitutively active Rubisco enzyme in different irradiance intensities (except in darkness), showed faster increases than the wildtype, Colombia-0, in photosynthesis rates after step increases in irradiance. rca-2, having decreased Rca concentration, showed lower rates of increase. In aba2-1, high gs increased the rate of change after stepwise irradiance increases, while in C24, low gs tended to decrease it. Differences in rates of change between Colombia-0 and plants with low levels of NPQ  or SPS (spsa1) were negligible. In Colombia-0, the regulation of Rubisco activation and of gs were therefore limiting for photosynthesis in fluctuating irradiance, while levels of NPQ or SPS were not. This suggests Rca and gs as targets for improvement of photosynthesis of plants in fluctuating irradiance.A better understanding of the metabolic and diffusional limitations of photosynthesis in fluctuating irradiance can help identify targets for improving crop yields. We used different genotypes of  However, plants grow in a variable environment, with changes occurring in the time range of seconds or lessArabidopsis thaliana possesses a wide, well documented genotypic diversity, which has been extended by selecting for mutations and by transgenic modifications.Our understanding of the metabolic constraints of photosynthesis in fluctuating irradiance (hereafter: ‘dynamic photosynthesis’) have mainly come from biochemical studies7132 assimilation and its activation limits An after irradiance increases13Arabidopsis thaliana, there are two isoforms of Rca, the larger α-isoform and the smaller β-isoform18Rubisco catalyses CO2 assimilation by Rubisco, a fraction of the triose phosphates leaves the chloroplast in exchange for orthophosphate (Pi) from the cytosol. In the cytosol, triose phosphate is converted to sucrose, and sucrose phosphate synthase (SPS) plays a central role in this pathway2, irradiance-dependent activation of SPS can be slower than that of Calvin cycle enzymes, making the Calvin cycle transiently Pi-limitednn after irradiance increases, and a smaller transient dip in An after irradiance decreases.After COPSII). Model calculations indicate that slow relaxation of NPQ could decrease canopy photosynthesis by ~13–24%n in genotypes with faster NPQ buildup after irradiance increasesn following a decrease in irradiance.Leaves protect themselves from absorbed irradiance that is in excess of the capacity of photochemistry using non-photochemical quenching (NPQ). This protection, however, may come at a price. Sustained high levels of NPQ after irradiance decreases may result in transient limitations of the quantum efficiency of photosystem II for electron transport  and diffusional (gs) limitations affect dynamic photosynthesis. In addition to measuring their steady-state photosynthetic irradiance and CO2 responses, we exposed these genotypes to stepwise increases and decreases in irradiance, while measuring gas exchange and chlorophyll fluorescence. To investigate the effects of Rca regulatory properties or concentrations, we used the transformant rwt43  synthesis, leading to constitutively high gsss limit dynamic photosynthesis in A. thaliana, while wildtype levels of SPS and NPQ do not.We used several genotypes, i.e. plants containing point mutations, transformants, T-DNA insertion lines . Rates of An in Col-0 were comparable to studies using plants grown under similar conditions343536rca-2, irradiance-saturated An was lower than for Col-0, and saturation occurred around 600 μmol m−2 s−1 , maximum rate of electron transport  and maximum rate of triose phosphate utilisation  compared to Col-0 , An did not differ from Col-0 in its irradiance response (i (max (−14%) and TPU (−23%). The ABA-deficient mutant, aba2-1, showed larger irradiance- and CO2-saturated photosynthesis rates compared to Col-0, while the accession C24 showed the opposite , while they were smaller in C24 . The supply liness between C24, Col-0 and aba2-1: the steeper the slope, the smaller the difference between external CO2 concentration (Ca) and Ci, and the larger gs. Irradiance and Ci responses of photosynthesis of low-NPQ mutants  were similar to Col-0 (max in npq4-1 (−7%). The response of quantum yield of photosystem II (ϕPSII) to Ci largely paralleled that of An, with the exception that ϕPSII decreased at high Ci in many genotypes . Rates of photosynthetic induction were initially similar between all genotypes (except rwt43) until ~60% induction was reached (rwt43 reached 50% of photosynthetic induction (tA50) significantly faster than Col-0  by ~10 minutes compared to Col-0. spsa1 showed slightly slower induction rates  to stepwise increases in irradiance, namely 70 → 800 and 130 → 600 μmol mansition . rwt43 ean Col-0 . This rean Col-0 . C24 tenan Col-0 , leading−2 s−1 transition. Changes in ϕPSII largely paralleled those of An  in Col-0. In rca-2, C24 and npq4-1, Fv/Fm was marginally, but significantly, smaller, possibly due to photoinhibition that was not completely removed by dark adaptation. In spsa1, it was slightly but significantly higher than in Col-0 , decreased with increasing background irradiance  and between background irradiances, with a τR of ~22 minutes in dark-adapted leaves decreasing to ~4 minutes in leaves adapted to an irradiance of 130 μmol m−2 s−1  was not significantly different to that of Col-0 , but was significantly (P < 0.001) smaller at 70 and 130 μmol m−2s−1 background irradiance  after different low-to-high irradiance transitions  and found that the threshold between limiting and non-limiting initial gs was between 0.09 and 0.17 mol m−2 s−1, with no discernible trend between time points or background irradiance levels.Before and after stepwise increases in irradiance, g and C24 . In dark in tA90 . This shs on the rate of An increase, we also analysed the effects of gs increase after stepwise increases in irradiance (s after the 0 → 1000 μmol m−2 s−1 increase (until 60 minutes after the start of illumination) and tA90 correlated positively .Apart from the effect of initial gradiance . In C24 sitively . Because−2 s−1), relative changes in An were similar for all genotypes  or regulation (rwt43) had strong effects on dynamic photosynthesis. The observed effects were likely caused by different kinetics of Rubisco activation, as the initial increase in assimilation after dark-light transitions .Changes affecting Rca concentration  during transients. With respect to gm, contrasting responses to irradiance have been reported  did not show such large differences in stomatal opening , and faster responses of An to an increasing irradiance could be reached at the expense of lower intrinsic water use efficiency. Rapid screening for high gs could be achieved by thermal imagingThe mutant with high initial gnpq1-2 . Importantly, the decrease in SPS hardly affected photosynthetic responses to fluctuating irradiance. The only significant difference was a longer time to reach 90% of full induction after dark-light transitions  limited by Rubisco, ϕPSII increases with Ci; when An is (b) limited by RuBP regeneration, ϕPSII is constant with increases in Ci and when An is (c) limited by TPU, ϕPSII decreases with increasing Ci54PSII that would signify a phase of RuBP regeneration limitation, with spsa1 showing an extreme form of that behaviour  different limitations occur simultaneously within different layers of the leaf, (c) changes in the rate of cyclic electron transport around photosystem I and/or strength of alternative electron sinks or (d) with increasing Ci during the phase of limitation by RuBP regeneration photosynthetic electron transport is sometimes restricted, and ϕPSII is reduced, due to the increased inhibition of starch synthesis following the inhibition of phosphoglucoisomerase by phosphoglycerateThe relationship between ϕehaviour . This suA. thaliana, the presence of the redox-regulated α-isoform of Rca in the wildtype, and wildtype levels of gs, are limiting for dynamic photosynthesis. Furthermore, reductions in Rca strongly decrease (dynamic) photosynthesis. We also show that wildtype levels of NPQ and SPS are not limiting in A. thaliana. This suggests Rca and gs as targets for improvement of photosynthesis in fluctuating irradiance.In conclusion, in npq4-1, spsa1 (SALK_148643C) and rca-2 (SALK_003204C) were obtained from NASC , npq1-2 was obtained from Dr. Shizue Matsubara  and rwt43 was obtained from Dr. Elizabete Carmo-Silva .Seeds of −2 s−1 as supplied by LED lights  were sprayed with an aqueous solution containing 10 μmol mol−1 ABA  when plants were 2, 4 and 6 weeks old. This increases rosette growth compared to untreated aba2-1 plants (data not shown). There was a period of 15 days between the last application of ABA and the first measurements on aba2-1 plants.Plants were grown in 0.37 L pots using soil with a 4:1 peat:perlite mixture. Pots were placed on irrigation mats, and mats were saturated daily to full capacity. Plants were fertilized weekly using a nutrient solution especially developed for ArabidopsisSingle genotypes were grown sequentially (approx. one batch per week). Five plants per batch were used for measurements. To monitor the quality of the growth system over time, Col-0 was grown in three batches, each batch separated by several weeks. The number of replicates was therefore 15 for Col-0, and 5 for all other genotypes. The growth system produced very reproducible photosynthetic phenotypes of Col-0 .2, diameter: 1.6 cm) were used. Conditions in the cuvette were as follows: 23 °C air temperature, 70% relative humidity, 90/10% red/blue light mixture and 500 μmol s−1 air flow rate. The choice of flow rate was a compromise between getting a fast time response of the measuring system (necessary in dynamic gas exchange studies), and the difference in CO2 concentration between sample and reference air stream. Except for the CO2-response curves, the external CO2 mole fraction was kept at 400 ppm. The oxygen mole fraction was always 21%.Measurements were performed using the LI-6400 portable photosynthesis system  equipped with the leaf chamber fluorometer (Part No. 6400-40) on single leaves of plants that were 6–8 weeks old. Leaves large enough to cover the leaf chamber gasket (area: 2 cm−2 s−1) for 30–60 minutes (until An and gs had visibly reached a steady state), and then exposed to single-step increases in irradiance, namely 0 → 1000, 70 → 800 and 130 → 600 μmol m−2 s−1. These intensities were chosen, after preliminary irradiance-response curves on Col-0 had shown that all but the highest (1000 μmol m−2 s−1) intensity were in the sub-saturating range , or 60 minutes after switching to 1000 μmol m−2 s−1. Before and after the 0 → 1000 μmol m−2 s−1 increase, ϕPSII and NPQ were measured, using a measuring beam intensity of ~1 μmol m−2 s−1 and a saturating pulse of ~7600 μmol m−2 s−1 intensity and 1 s duration. In preliminary measurements on Col-0, the saturating pulse was sufficient to saturate Fm’ in leaves adapted to 1000 μmol m−2 s−1 . The Fo and Fm relative fluorescence yields were measured in dark-adapted leaves. After the increase in irradiance, the Fm’ relative fluorescence yield was measured every minute for the first ten minutes, and every two minutes thereafter. The regular application of saturating flashes transiently increased the leaf temperature by 0.4–0.7 °C across genotypes  indicate that the regular application of saturating flashes of similar intensity and frequency in tomato (Lycopersicon esculentum) had no effects on leaf gas exchange during photosynthetic induction. The steady-state relative fluorescence yield, Fs, was measured continuously. Dark-adapted Fv/Fm, ϕPSII and NPQ were calculated as Fv/Fm = (Fm − Fo)/Fm, ϕPSII = (Fm’ − Fs)/Fm’ and NPQ = (Fm − Fm’)/Fm’, respectively.Leaves were adapted to several background irradiances  were assumed to be constant because, to our knowledge, changes in gm and Rd have never been assessed for irradiance transients. Rd in the light was considered similar to genotype-specific steady-state respiration in the dark; this assumption is supported by measurements on several speciesm, a value of 0.2 mol m−2 s−1 was assumed for all genotypes, which is an average of three values determined on Col-0 of comparable photosynthetic capacity35During transients, g50 and t90) of steady-state An was calculated for each irradiance increase. To increase robustness of these indices to experimental noise and outliers, time series were smoothed using a local polynomial regressionThe time to reach 50 and 90% (i.e. tn was corrected for transient changes in chloroplast CO2 concentration (Cc). For diffusional limitation, An was multiplied by the relative rate by which An would increase if Cc during induction was equal to ambient CO2 concentration, Ca  is the steady-state value of An at Ca (i.e. at 400 ppm), and f(Cc) is the steady-state value of An at Cc. The relative effects of Cc on An were taken from steady-state An/Cc response curves by fitting local polynomial regressions (LOESS) in the range 50–500 ppm . For biochemical limitation and τR, An was multiplied , following Woodrow and MottWhere Acf) is the solution for An at Ccf. Biochemical limitation was calculated after Allen and PearcyWhere f was calculated after Woodrow and MottThroughout induction, biochemical limitation decreases from 100 to 0%, and therefore indicates the additional limitation imposed on Atime) for calculating τR differed between background irradiances  and 40 (for rwt43) or 60  in the case of 70 → 800 and 130 → 600 μmol m−2 s−1. These ranges were selected by visual inspection. The average root mean squared error of the linear fits was 1.2 μmol m−2 s−1 (range: 1.0–3.0 μmol m−2 s−1).The range of timepoints .Each genotype was compared to Col-0 using a Student’s How to cite this article: Kaiser, E. et al. Metabolic and diffusional limitations of photosynthesis in fluctuating irradiance in Arabidopsis thaliana. Sci. Rep. 6, 31252; doi: 10.1038/srep31252 (2016).


The results showed that biological mass, total chlorophyll, chlorophyll a, and carotenoids content, photosynthetic rate, stomatal conductance, PS II maximum quantum yield, PS II effective quantum yield, PS II electron transport rate, photochemical quenching, maximal P700 change, photochemical quantum yield of PS I, and PS I electron transport rate decreased with increasing herbicide concentrations, whereas initial fluorescence, quantum yield of non-regulated energy dissipation in PS II and quantum yield of non-photochemical energy dissipation due to acceptor side limitation in PS I increased. It suggests that nicosulfuron ≥1 mg L−1 causes the damage of chloroplast, PS II and PS I structure. Electron transport limitations in PS I receptor side, and blocked dark reaction process may be the main cause of the significantly inhibited growth and decreased photosynthetic rate of Radix Isatidis seedlings.Radix Isatidis (Isatis indigotica Fort.) is one of the most important traditional Chinese medicine plants. However, there is no suitable herbicide used for weed control in Radix Isatidis field during postemergence stage. To explore the safety of sulfonylurea herbicide nicosulfuron on Radix Isatidis (Isatis indigotica Fort.) seedlings and the photosynthetic physiological response of the plant to the herbicide, biological mass, leaf area, photosynthetic pigment content, photosynthetic rate, chlorophyll fluorescence characteristics, and P Isatis indigotica Fort.), one kind of cruciferous plants, is extensively cultivated in China. However, weeds seriously limit its yield and quality. Compared with conventional manual control of weeds, chemical control is more effective, yet there is no suitable herbicide used for weed control in Radix Isatidis field during postemergence stage As one of the most important traditional Chinese medicine plants, Radix Isatidis  cornfield, goosegrass (Eleusine indica), spiny amaranth (Amaranthus spinosus L), amaranthus blitum (Amaranthus ascendens Loisel.), speargrass (Imperata cylindrica), and purslane (Portulaca oleracea) in maize field It is reported that nicosulfuron can effectively control large crabgrass  seeds were supplied by Anguo Lixin Medicinal Materials Co., Ltd., Hebei province, China. Nicosulfuron  was provided by Xianda Chemical Co., Ltd., Shandong province, China.Radix Isatidis .2 concentration in the cuvette. Photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs) and intercellular CO2 concentrations (Ci) were measured simultaneously with the light intensity at (800±0.4 µmol·m−2·s−1) and CO2 concentration (379±0.4 µmol·mol−1). Air flow rate was set at 750 µmol s−1 and air temperature (20.9±0.4°C) was also recorded automatically by the instrument. Stomatal limitation value (Ls) = 1–Ci/Ca (Ca is the atmospheric CO2 concentration). Non-stomatal limitation value was calculated by Ci/GsPhotosynthetic gas exchange was analyzed with a GFS-3000 optical instrument (Germany WALZ company) which can control photosynthesis by means of light intensity, leaf temperature, air flow rate and COP700 parameters were measured simultaneously by Dual-PAM-100 measurement system (Germany WALZ company), using the automated ‘‘Induction Curve’’ routine provided by the Dual PAM software P700 mode’’. Then, the kinetics of chlorophyll fluorescence induction and P700 oxidation were recorded simultaneously by the instrument. Firstly, the initial fluorescence (Fo) was established and subsequently the maximum fluorescence (Fm) was determined by the ‘‘Saturation Pulse’’ method. Secondly, the maximal P700 change (Pm) was determined by application of a saturation pulse (SP) after far-red pre-illumination. Thirdly, actinic illumination was started and SP was given every 20 s, with the same pulses serving for fluorescence and P700 analysis.Chlorophyll fluorescence and Fv/Fm) was evaluated as Fv/Fm = (Fm–Fo)/Fm. Other PS II energy dissipation parameters were estimated by the Dual PAM software. qP = (Fm’–F)/(Fm’–Fo’) was used as indicator to reflect a ratio of light energy absorbed in PS II being used to photochemical electron transport. Apparent electron transfer efficiency in PS II in light was calculated according to ETR(II) = PAR×0.84×0.5×Y(II), and was used to measure electron transfer of carbon fixation resulted from photochemical reactions. Three complementary quantum yields of energy conversion in PS II were calculated: PS II effective quantum yield (Y(II)) was evaluated as (Y(II)) = (Fm’–F)/Fm’, the yield of non-photochemical losses via non-regulated pathways of PS II as Y(NO) = 1/(NPQ+1+qL·(Fm/Fo–1))), NPQ = Fm/Fm’–1, qL = qP·Fo’/F, where quantum yield of regulated energy dissipation in PS II as Y(NPQ) = 1–Y(II)–Y(NO) PS II maximum quantum yield (P700 oxidation was monitored by absorbance changes in the near-infrared (830–875 nm) P700 signal observed upon full oxidation was denoted by Pm. Y(NA), the quantum yield of non-photochemical energy dissipation due to acceptor-side limitation, was calculated according to: Y(NA) = (Pm’–Pm)/Pm’. Photochemical quantum yield of PSI as Y(I) was estimated according to Y(I) = (Pm’–P)/Pm; Quantum yield of non-photochemical energy dissipation due to donor side limitation in PS I as Y(ND) was calculated by Y(ND) = (P–Po)/Pm. The total value of three quantum yields was one: Y(I)+Y(ND)+Y(NA) = 1. The electron transfer efficiency of PS I as ETR (I) was provided by the Dual PAM software.Microsoft Office Excel 2003 and Statistics Analysis System 8.0 were used in statistical analysis of the data. Mean values were compared by a one-way analysis of variance (ANOVA), and Duncan’s test was used to determine the significant differences among the treatments. We used P = 0.05 as the statistical significance threshold.−1, whereas this was not affected by other treatment. The whole plant fresh weight and shoot fresh weight showed the similar trend, and differences between the treatment of nicosulfuron at 0.5 mg L−1 and the control were significant. Shoot fresh weight declined by 41.64%, 52.90%, 53.58% and 59.04%, respectively, from 0.5 to 4 mg L−1 of nicosulfuron. The reduction in root fresh weight was not significant by nicosulfuron, suggesting that the suppression of nicosulfuron on the aboveground parts of Radix Isatidis was greater than root.The effects of nicosulfuron on agronomic characteristics of Radix Isatidis seedlings are shown in −1 of nicosulfuron inhibited Chl by 33.57%, Chl a by 37.04% and Car by 47.37, and the differences between the treatment and the control were significant. However, Chl b was not significantly decreased until the herbicide concentrations reached up to 2 mg L−1, and it was inhibited by 37.14%. Although nicosulfuron declined chlorophyll a/b, there was no significant effect.As shown in Pn and Gs in leaves of Radix Isatidis seedling significantly. As concentration of nicosulfuron increases, inhibition of Pn changed by 58.21%, 70.02%, 70.69%, and 78.89%, respectively  from 1 to 4 mg L−1 of nicosulfuron. Ci increased first then declined with increasing of nicosulfuron concentrations, peaked at 0.5 mg L−1, and was the lowest at 4 mg L−1. Compared to Ci, the change trend of Ls was just the opposite. Ls was the least at 4 mg L−1, and the largest at 1 mg L−1. For Ci/Gs, the value for treated plants was higher than the control in different degrees.Nicosulfuron decreased ectively . Gs inh4 mg L−1 . HoweverFo increased with the increase in nicosulfuron concentration. Fo was a bit higher than the control for 2 mg L−1 treatment, but significantly higher for 4 mg L−1 treatment. Fv/Fm was slightly higher than the control for 0.5 mg L−1 concentration, and then decreased with the increase concentrations of nicosulfuron.As shown in qP were consistent, and declined in a concentration-dependent manner. Compared with the control, Y(II) was reduced by 7.01%, 36.07%, 70.57% and 81.95%, ETR declined by 7.10%, 36.14%, 70.44% and 81.91%, and qP decreased by 8.64%, 29.54%, 70.19% and 82.51%, respectively (Changes of Y(II), ETR and ectively -B, 1-D.−1 and 1 mg L−1 concentration, and significantly (49.70%) higher for 2 mg L−1. Compared to Y(NO), the trend of Y(NPQ) was the opposite (Y(NO) showed a fluctuant increase as nicosulfuron concentration increases, slightly higher than the control for 0.5 mg Lopposite -C. It shPm, ETR (I), and PS I. Nicosulfuron treatment at 1 mg L−1 had dramatically influence on Pm, while the differences between 1 mg L−1 and 2 mg L−1 were not significant  was not safe to Radix Isatidis seedlings, which was reflected by reduced biomass. It was coincident with Yuan et al. Pn.The safety of herbicides on plants may be represented through agronomic traits and physiological indexes Pn resulted by stomatal factors or non-stomatal factors? In our current research, Pn and Gs at 0.5 mg L−1 were significant lower than the control, however, Ci and Ci/Gs were a little higher than the control. It may suggest that the decline in Pn is mainly caused by stomatal limitation. Pn, Gs and Ci treated by nicosulfuron ≥2 mg L−1 were significantly lower than the control, and Ls and Ci/Gs were significant higher than the control, showing that both stomatal and non-stomatal factors may limit the photosynthesis. Nicosulfuron at 0.5 mg L−1 decreased Chl a and Car content significantly. It may suggest that nicosulfuron destructs the chloroplast structure of Radix Isatidis leaf, reduces the thylakoid stacking level Is the decline in Fo−1 significantly reduced Fv/Fm, Y(II) and qP in leaves of Lemna minor, but enhanced NPQ markedly Fv/Fm, Y(II) and qP significantly, and lead to increase in initial fluorescence, quantum yield of non-regulated energy dissipation in PS II in Radix Isatidis seedlings. Similar to the previous, Fo and Y(NO) increased, whereas Fv/Fm, Y(II), ETR(II), qP and Y(NPQ) decreased in leaves treated by nicosulfuron in this study. It may suggest that nicosulfuron causes excess excitation energy accumulation in PS II reaction center, the higher reduction state of QA, net loss of D1 protein, reversible deactivation or destruction in PS II reaction centers Chloroplasts of photosynthetic apparatus, PS II and PS I in thylakoid membranes are the most sensitive parts to environmental changes. Reversible inactivation or damage of PS II reaction center can cause increase in Pm, Y(I) and ETR (I) declined with the increase of nicosulfuron usage, and which at 1 mg L−1 were significantly lower than the control. It may suggest that herbicide suppressed the activity of PS I in Radix Isatidis leaves, and electron transfer was blocked at its receptor side. Y(ND) reflects the state of electron donor in PS I, and it is affected by the transmembrane proton gradient and PS II damage degree. Y(NA) reflects the state of electron acceptor in PS I, and it is affected by dark adaptation and CO2 fixation damage level. Y(NA) >0.5 mg L−1 increased significantly, showing that nicosulfuron aggravated the injury of PS II in Radix Isatidis leaves, declined transmembrane proton gradient, blocked dark reaction process, and reduced the fixed amount of CO2.Typical feature of PS I photoinhibition is the decline in maximum oxidation-reduction ability of PS I In conclusion, recommended usage of nicosulfuron for maize is not safe to Radix Isatidis seedlings. It causes the damage of chloroplast, PS II and PS I structure. Electron transport limitations in PS I receptor side, blocked dark reaction process may be the main cause of the significantly inhibited growth and decreased photosynthetic rate of Radix Isatidis seedlings. Effect of nicosulfuron on the activities of key enzymes in the Calvin cycle of Radix Isatidis seedlings will be researched in the later experiment.


Here, we extend this investigation by evaluating the influence of phosphatidylethanolamine (PE) lipids. Using experimental bilayer form factors derivedfrom X-ray diffuse scattering data as a guide, our simulations indicate that Tat peptides localize close to the carbonyl-glycerol group in the headgroup region of bilayers composed of either DOPC or DOPC:DOPE (1:1) lipid. Our results also suggest that Tat peptides may more frequently insert into the hydrophobic core of bilayers composed of PC:PE (1:1) lipids than into bilayers composed entirely of PC lipids. PE lipids may facilitate peptide translocation across a lipid bilayer by stabilizing intermediate states in which hydrated peptides span the bilayer.The interactions of the basic, cell-penetrating region (Y Many ceembranes ,4. It isembranes ,7,8,9,10hin 30 s . In anot with PC . Therefo47–57-induced perturbation of simplified membrane mimetics with varying compositions of PC, PE, PS, phosphatidylinositol (PI) lipids, and cholesterol [47–57 interacting with PC bilayers, which were scored based on their goodness of fit to experimental values [L) and Tat47–57 immersion depth were restrained to a series of prescribed values. The advantages of this approach are that it does not require brute-force equilibration in these two slowly-relaxing degrees of freedom (AL and peptide immersion depth), and that it does not rely entirely upon the force field parameters to generate accurate models [47–57 most likely resides in the PC bilayer’s headgroup region, ~18 Å from the bilayer center [Previously, X-ray scattering was used to characterize Tatlesterol . Concurrl values . Since sl values ,15, we ee models ,17. Thesr center .47–57-bilayer interactions. Interestingly, while the experimental X-ray scattering form factors of Tat47–57 in PC:PE (1:1) bilayers are most consistent with simulation models in which the center of mass of Tat47–57 resides in the bilayer's headgroup region, we also obtain good fits to experiment for some simulations in which Tat47–57 is much more deeply inserted, ~8 Å from the bilayer center.Here, the simulation approach employed in the previous work is extended to include mixed PC:PE (1:1) bilayers. We identify simulation conditions whose resulting form factors are maximally consistent with experimental X-ray scattering form factor data and then use these simulations to obtain plausible atomistic depictions of Tat47–57 in several lipid mixtures. This bimodal result was the motivation to compare the form factors from experiment to those from MD simulation in which Tat47–57 was restrained in various regions of the bilayer. In the previous work, only DOPC bilayers were compared to MD simulations, with the result that good agreement between X-ray experiment and MD simulation was found only when Tat47–57 was located in the headgroup region, and not in the hydrocarbon region, of the bilayers [Previously, X-ray diffuse scattering were collected for DOPC, DOPC:DOPE (3:1), DOPC:DOPE (1:1), DOPC:DOPS (3:1) and a nuclear membrane mimic . The Scabilayers . In the L, and can provide relatively accurate predictions of lipid order parameters and X-ray form factors [L in an attempt to circumvent systematic sampling errors related to slow conformational relaxation in atomistic simulations. Therefore, our initial evaluation of a neat lipid bilayer is also conducted with systematic variation of fixed AL values.In the absence of an applied surface tension or fixed dimensions of the unit cell, simulations of neat bilayers can reproduce experimental values of bilayer thickness and A factors ,20,21. HL values of 66 and 70 Å2 yield the best fit to experiment for DOPC:DOPE (1:1) and DOPC bilayers, respectively | quite well, capturing lobes L1–L4 and minima M1–M3 and having an overall χ2 measure of the goodness of fit (see Methods) between |F(qz)| from the simulation and experiment of 2.3 and 2.1 for DOPC:DOPE (1:1) and DOPC bilayers, respectively (z)| do not capture the skew of lobe 2 towards smaller values of qz which is likely an experimental artifact. More importantly, the simulations slightly over-estimate the height of lobe 2 relative to lobes 1 and 3 , equal to 0.0087, 0.016, 0.034, and 0.059 [47–57 is included again here as 47–57, x = 0.0087  mixture with mole fractions of Tatnd 0.059 . Form fathinning .47–57 interaction with a DOPC:DOPE (1:1) bilayer, we conducted simulations in which we systematically varied peptide distance from the bilayer center along its normal, z, and bilayer AL using peptide mole fractions of either 0.015 or 0.030 . These are the same two concentrations previously investigated with MD simulations and pure DOPC bilayers [2 values reporting the goodness of fit between simulation and experiment are shown in To generate atomistic models of the Tatbilayers . Form fa47–57 mole fraction of 0.015 indicates that these peptides reside either in the DOPC:DOPE (1:1) bilayer’s headgroup region  or undergo much deeper insertion   (z)| (left) and corresponding electron density profile (right) calculated from those parameters that yielded the smallest χ2 values are shown in L = 66 Å2, while 47–57 mole fraction of 0.015 (A) z = 18 Å, AL = 68 Å2 (B) z = 8 Å, AL = 72 Å2, and 47–57 mole fraction of 0.030, z = 18 Å, AL = 68 Å2. In Comparison to experiment for simulations with a Tatvie SM1) A,B. Conv ± 0.16) C,D. The 47–57 up to x = 0.034 thin the bilayer only slightly as determined from DHH . Greater thinning was observed in the hydrocarbon thickness, 2DC. The greatest thinning, 2.5 Å, was determined in the 2DC value for DOPC/DOPE(1:1)/2 Tats (x = 0.015) when AL = 72 Å2 with Tat47–57 located at 8 Å from the bilayer center. For this sample only, there is also a large increase in area (6 Å2)  .L with a Tat47–57 mole fraction of 0.015. Specifically, we reevaluated peptide insertion into the bilayer headgroup region at z = 18 Å and AL = 68 Å2, and deep peptide insertion at z = 0 or 8 Å and AL = 72 Å2, using different initial conformations in each repeat simulation. Values of χ2 are reproducibly low when Tat47–57 is inserted into the bilayer’s headgroup region  simulations at z = 8 Å, AL = 72 Å2, x = 0.015 in which χ2 < 3.4 . Representative snapshots from two such simulations are shown in − traversed the bilayer by moving through the column of water surrounding two interacting Tat47–57 peptides , released the restraints, and simulated for another 100 ns. In this simulation, both peptides retain their interactions with lipid headgroups from both leaflets (not shown), the average values of |z| for the two peptides over the last 50 ns are 6.9 and 9.7 Å, and the form factor computed from the final 50 ns has a χ2 value of 3.5 when compared to experiment . It is therefore possible that at x = 0.030 some subset of the Tat47–57 peptide molecules migrate toward the bilayer center. It is also possible that Tat47–57 aggregates at x = 0.030, which could affect the peptide’s ability to translocate across a bilayer. However, intermolecular peptide contacts are absent and rare at x = 0.015 and x = 0.030, respectively, in our simulations at z = 18 Å2, AL = 68 Å2 (47–57 aggregation (Movie SM2), and further studies are necessary.At the higher concentration of x = 0.030 (four peptides/128 lipids), our results suggest that Tat = 68 Å2 . NeverthMany simulations have attempted to assess the cost of placing either an arginine or an arginine-rich peptide into the membrane hydrocarbon core, which must overcome the energetic barrier of Born self-energy of a charged particle . Table 3et al. found a larger free energy for insertion of arginine into the center of a POPE bilayer , compared to a POPC bilayer  [47–57 may partition closer to the DOPC:DOPE (1:1) bilayer center than in the pure DOPC bilayer. Inclusion of PE may therefore even overcome the effect of an increased thickness. So in spite of the high cost of placing a protonated arginine, or even a Tat peptide at the center of the bilayer, our present result that an interior location of Tat47–57 at low concentration in DOPC:DOPE (1:1) membranes may be nearly as good as in the headgroup region, suggests that the curvature-inducing PE headgroup may play an important role.In contrast to the high cost of placing an arginine in the center of the bilayer, it has been suggested that the free energy cost for charged amino acids entering the headgroup region is similar to that for partitioning into octanol, about an order of magnitude smaller free energy cost than partitioning into cyclohexane . Similarcal/mol) . This cocal/mol) . Yet, ascal/mol) ,12. As mcal/mol) , yet our47–57 is restrained at 8 Å from the DOPC:DOPE (1:1) bilayer center, water immediately fills the space adjacent to the peptide  bilayer center (L = VolumeLipid (VL)/DC [L with Tat47–57 and water within the membrane is nearly identical to the neat lipid bilayer, thus leading to a 6 Å2 increase in AL.Also shown in r center . Since A (VL)/DC , this in47–57 is located close to the carbonyl-glycerol group in both DOPC:DOPE (1:1) and DOPC bilayers [47–57 in DOPC:DOPS (80:20) bilayers [48–60 in DMPC:DMPG (8:7) bilayers [At the higher concentration, Tatbilayers . This rebilayers , and NMRbilayers . A posit47–57 in DOPC bilayers [47–57 does not partition near the center of DOPC bilayers.In the present work, we show that relatively long conformational autocorrelations can exist at values of z near zero, where final conformations and form factors from 100-ns simulations depend strongly on initial conformations . Becausebilayers  did not et al., which showed that arginine nonamers permeabilize DOPC and DOPC:DOPG (3:1) bilayers to ions [47–57-dependent ionic conduction does not require large toroidal pore structures. This is consistent with experiments in GUVs that showed AlexaFluor647 dye molecules were completely released from PC:PE:cholesterol (50:30:20) vesicles, but that 3 and 70 kDa fluorescent dextrans were not released [The peptide-induced ion transport shown in  to ions . In addireleased .47–57 peptides along the bilayer normal. However, we have not computed PMFs for two reasons. First, simulations of 100-ns per umbrella are very likely to be subject to systematic sampling errors [Multiple approaches exist to synergistically combine experimental diffraction data with molecular simulation. In one approach, the experimental data is used as a restraint to guide conformational sampling in simulations . In the g errors . Such sa47GRKKRRQRRR57) was purchased from the Peptide Synthesis Facility ; mass spectroscopy revealed >95% purity. Tat47–57 was dissolved in HPLC trifluoroethanol (TFE)  and added to the lipid mixture in mole fractions, x = (Tat47–57/lipid + Tat47–57), of 0.0087, 0.016, 0.034 and 0.059. The mass of Tat in these mole fractions was corrected for protein content (the remainder being 8 trifluoroacetate counter-ions from the peptide synthesis). Solvents were removed by evaporation in the fume hood.Lipids were purchased from Avanti Polar Lipids  and used without further purification. Dioleoylphosphatidylcholine:dioleolphosphatidylethanolamine (DOPC:DOPE) (1:1) membrane mimics were prepared by first dissolving lyophilized DOPC or DOPE in chloroform and then mixing these stock solutions in a 1:1 mole ratio. Tat peptide (Y3) via the rock and roll method [Four mg dried lipid/peptide mixture was dissolved in 200 μL HPLC chloroform/TFE (2:1 v:v) and plated onto silicon wafers (15 × 30 × 1 mml method  to produLAXS. Low-angle X-ray scattering data from oriented fluid phase lipid mixtures at 37 °C were obtained at the Cornell High Energy Synchrotron Source (CHESS) using previously described methods [z)| were obtained as previously described [ methods ,41. Back methods . Absolutescribed . FurtherNpAT ensemble is achieved as follows (with deviations from this protocol during system setup and equilibration outlined where appropriate). Temperature is controlled using velocity Langevin dynamics [−5 and 0 bar−1 in the Cartesian z dimension and xy plane, respectively, thus prohibiting changes in the dimensions of the bilayer's transverse plane while allowing fluctuations of the box dimension along the global bilayer normal (the Cartesian z axis).Molecular dynamics simulations are conducted with a single-precision compilation of version 4.6.5 of the GROMACS simulation package . Proteindynamics  at 310 Kdynamics ,54 at 1 47–57 peptides in each leaflet . There are a total of 128 sets of simulation conditions for the pairwise combination of AL values of 62, 64, 66, 68, 70, 72, 74, or 76 Å2, 2 or 4 peptides, and displacements between the peptide and bilayer centers of mass along the bilayer normal, z, of 0, 5, 8, 10, 12, 14, 16, or 18 Å, where 0 Å indicates the bilayer center . Two simulations are conducted for each of the aforementioned conditions using different initial conformations. Each simulation system is constructed independently using bilayer and peptide conformations drawn from the bilayer- and peptide-only simulations outlined in the SM text. To construct a unique bilayer environment for each simulation, we randomly select a snapshot from the last 10 ns of our 20-ns simulation of a neat bilayer at the desired AL, translate it by a random distance in the bilayer plane (resetting the periodic unit cell), and randomly decide whether to rotate the bilayer by 180° to exchange upper and lower leaflets. For each peptide that is to be inserted, we randomly select a snapshot from the last 30 ns of our 40-ns simulation of Tat47–57 in water and rotate it with randomly selected magnitudes about each of the three Cartesian axes. Peptides are arranged in the bilayer plane so as to maximize the separation of their centers of mass. Specifically, for simulation systems with two Tat47–57 molecules, one peptide is placed at the desired value of z in the +x, +y quadrant of the upper bilayer leaflet and the other peptide is placed in the −x, −y quadrant of the lower leaflet  simulation systems consist of a hydrated 128-lipid bilayer with 32 DOPC and 32 DOPE lipids and either one or two Tat leaflet A. Analog leaflet B.L, or because they are trapped in the wrong metastable state.The actual embedding of the peptide in the bilayer is conducted with 40 cycles of the InflateGRO2 routine  and doubAL values of 66, 68, 70, 72, 74, or 76 Å2; here, each condition is simulated only once.Simulations with pure DOPC bilayers are set up and conducted analogously for z axis are constructed from the last 50 ns of each simulation trajectory using a modified version of the g density tool from GROMACS 5.0.4 (this custom modification ensures that the bilayer is properly centered in each trajectory frame). These density profiles are converted through the Fourier transform to form factors, |F(qz)|, using the SIMtoEXP software [Electron density profiles along the Cartesian software .z)| evaluated from simulation are well defined and are expressed in units of e/Å2. However, the experimental values of |F(qz)| include an unknown scaling factor. Therefore, when evaluating the goodness of fit between simulation and experimental data, one may (and should) scale either form factor to minimize the inter-form factor deviation. To facilitate comparison of measures of the goodness of fit between experimental data sets, we initially scale all experimental form factors such that the maximum value in the second lobe is equal to one | from simulation such that qz(i) from simulation and experiment are equal for all data points.The simulation scaling factor, a2, as:Next, we quantify the goodness of fit between experiment and simulation, χz)| from simulation to match those from experiment in the determination of χ2 (Equations (1) and (2)). All plots of |F(qz)| comparing results from simulation and experiment use unscaled ordinate values (expressed in units of e/Å2) from the simulation having the lowest χ2 value; experimental values of |F(qz)|  are multiplied by 1/asim(best), where asim(best) is the value of asim obtained for the simulation having the lowest χ2 value; and all other simulation values of |F(qz)| are multiplied by asim/asim(best), where asim differs for each simulation.As outlined above, we scale the values of |F(q2 are taken straight from Equations (1) and (2) and are reported without further modification. Additional discussion of fitting procedures is included in the SM text.All reported values of χ47–57 peptide from HIV-1 that are maximally consistent with experimental DOPC:DOPE (1:1) bilayer form factors derived from X-ray diffuse scattering data. Whereas Tat47–57 appears to reside exclusively at the surface of DOPC bilayers [47–57 bilayer penetration. Nevertheless, our results suggest that Tat47–57 translocation across PE-rich bilayers may involve a metastable intermediate caused by the tendency for PE to adopt a negative curvature, in which monomeric and/or dimeric peptides adopt an extended conformation and interact with the headgroups of lipids from both bilayer leaflets, drawing both water and chloride ions into the bilayer interior.We use atomistic molecular dynamics simulations to identify bilayer insertion depths of the Tatbilayers , we show


AbstractArachnothera longirostra Temminck, 1826  in India. Pedanodectes angustilobussp. n. differs from all known Pedanodectes species by having opisthosomal lobes short, at base wider than long, roughly rounded apically in males, and strongly elongated and narrowed lobar region with wide terminal appendages in females. A key to species of the genus Pedanodectes is presented.The article describes a new species of the feather mite subfamily Pterodectinae from the Little Spiderhunter  Arachnothera longirostra (including Pedanodectes species), but unfortunately this material has never been described.Feather mites are commensals or ectoparasites permanently living on birds. In India, the diversity of feather mites is poorly investigated, and only 26 species have been mentioned so far in various taxonomic papers . Atyeo  reportedPedanodectes found on Arachnothera longirostra in India and we present a key for all species of this genus.In this paper, we describe a new species of The material used in the present paper was collected in Meghalaya (India), in January 2014. The birds were trapped by means of ornithological mist nets, identified and visually checked for the presence of mites and, after mites were collected, released back to the wild. Mite specimens were placed in tubes with 95% ethanol. Later, in the laboratory, the mite specimens were cleared in lactic acid and mounted on microscope slides in Hoyer’s medium. Drawings were made using an Olympus CX21 microscope, using a camera lucida drawing device.The bird specimens were identified according to Passeriformes  in Africa ; INDIA: Meghalaya, Jaintia Hills, Shnongrim village, ; 1151 m alt; 24.01.2014, collector Costică Adam.Male holotype (ANA256), 3 male  and 4 female  paratypes from the Little Spiderhunter PageBreakPageBreakPageBreakse and si, antero-lateral extensions short and rounded, posterior margin slightly convex in median part, total length of shield 102 (102–106), greatest width 90 (84–90), surface without ornamentation (se separated by 38 (36–40). Humeral shields absent, setae cp situated ventrally, setae c2 situated dorsally, in anterior angles of hysteronotal shield. Subhumeral setae c3 lanceolate, 20 (19–20) × 6 (5–6). Length of hysteronotal shield from anterior margin to bases of setae h2 195 (199–203), greatest width in anterior part 88 (80–86), anterior margin concave, anterior angles acute, surface without ornamentation with strongly sclerotized transverse fold between h1 setae. Opisthosomal lobes short, at base wider than long, roughly rounded apically, lobar apices bearing setae h2. Terminal cleft almost semicircular, with narrow membranous margin in anterior part, length of terminal cleft 10 (12–13). Supranal concavity absent. Hysteronotal setae c1, d1, e1 absent; setae h3 narrowly lanceolate, length 24 (24–25), greatest width 6 (4–6); setae h2 represented by macrosetae, length 130 (132–140), greatest width 5 (4–5); setae ps2 slightly thickened, 18 (12–16) long; setae ps1 filiform, minute, length 6 (5–6), situated slightly anterior to bases of setae h3 and h2, approximately equidistant from inner and outer margins of opisthosomal lobe. Dorsal measurements: se-c2 72 (66–70), c2-d2 92 (90–104), d2-e2 68 (60–66), e2-h3 32 (32–34), h1-h3 10 (10–12), h2-h2 38 (38–42), h3-h3 26 (26–28), ps2-ps2 50 (46–49). Epimerites I fused into a Y, sternum about ½ of the total length of epimerites, posterior end of sternum with pair of postero-lateral extensions not connected to epimerites II. Epimerites II short, not extending to level of sejugal furrow. Coxal fields I–III open, without wide sclerotized areas. Epimerites IVa long, their anterior ends extending to midlevel of epimerites IV, their basal parts connected by semicircular transverse sclerite  and almost completely encircling genital apparatus , 4b-4a 50 (44–50), 4a-g 24 (24–28), g-ps3 32 (28–31), ps3-ps3 40 (32–38), ps3-h3 24 (23–24).MALE  long, with apical claw and with small apico-ventral extensions bearing seta r and w; setae d, e button-like, seta d bigger than e, situated in basal and apical parts of segment, respectively (ω1I 15 (14–15), ω1II 12 (13–16), φI 42 (34–46), φII 29 (32–34), φIII 22 (22–24), φIV 32 (28–32).Legs I slightly longer and thicker than legs II, femora I and II with ventro-basal crests . Seta mGectively . Length PageBreakPageBreakPageBreakse and si, antero-lateral extensions short and angular, posterior margin concave, total length of shield 118–122, greatest width 104–112, surface without ornamentation  from Deleornis fraseri cameroonensis  (Passeriformes: Nectariniidae). Males of both species have setae ps1 situated antero-lateral to the adanal suckers, epimerites I fused into Y, similar shape of opisthoventral shields and epimerites IVa with narrow anterior projections. Males of Pedanodectes angustilobus sp. n. are easily to distinguish from those of Pedanodectes mesocaulus by the following features: epimerites IVa have free the anterior projections and are connected posterior to the genital arch by a transversal sclerite, the tip of aedeagus does not extend beyond the posterior margin of idiosoma. In males of Pedanodectes mesocaulus, the anterior projections of epimerites IVa are fused forming a pregenital sclerite while the transverse sclerite connecting the bases of these epimerites is absent, the tip of aedeagus extends beyond the posterior margin of idiosoma. Females of the new species are clearly different from those of the other species of the genus by the following unique combination of characters: the lobar region has the same width in anterior and posterior part; the terminal cleft is parallel-sided, with the margins almost touching, except for the anterior one third; and the terminal appendages are thick, their basal half is approximately as wide as opisthosomal lobes. In females of the other Pedanodectes species, the lobar region in the anterior part is wider than in the posterior part and the terminal appendages are narrower than lobes. The terminal cleft in females of the other species has the following shape: with the lateral margins parallel and almost touching in Pedanodectes andrei and Pedanodectes mesocaulus, with lateral margins sinuous and almost touching in certain parts in Pedanodectes marginatus and Pedanodectes latior; as a narrow inverted V in Pedanodectes blaszaki, and as a narrow inverted U in Pedanodectes hologaster.PageBreakPedanodectes, some authors considered that males of this genus practically have no opisthosomal lobes and setae ps3 are usually situated lateral or postero-lateral to the adanal suckers (ps3 antero-lateral to the adanal suckers can be found in some species. The new species found on Arachnothera longirostra in India and described in this paper demonstrates the example of a Pedanodectes species having distinct opisthosomal lobes and setae ps3 situated antero-lateral to the anal suckers in males.According to the diagnosis of the genus  suckers . However


We examined spatial δ15N gradients in the California Upwelling Ecosystem (CUE), determining bulk δ15N values of mussel tissue from 28 sites between Port Orford, Oregon and La Jolla, California, and applying CSI-AA at selected sites to decouple trophic effects from isotopic values at the base of the food web. Bulk δ15N values showed a strong linear trend with latitude, increasing from North to South . In contrast, CSI-AA trophic position estimates showed no correlation with latitude. The δ15N trend is therefore most consistent with a baseline δ15N gradient, likely due to the mixing of two source waters: low δ15N nitrate from the southward flowing surface California Current, and the northward transport of the California Undercurrent (CUC), with15N-enriched nitrate. This interpretation is strongly supported by a similar linear gradient in δ15N values of phenylalanine (δ15NPhe), the best AA proxy for baseline δ15N values. We hypothesize δ15NPhe values in intertidal mussels can approximate annual integrated δ15N values of coastal phytoplankton primary production. We therefore used δ15NPhe values to generate the first compound-specific nitrogen isoscape for the coastal Northeast Pacific, which indicates a remarkably linear gradient in coastal primary production δ15N values. We propose that δ15NPhe isoscapes derived from filter feeders can directly characterize baseline δ15N values across major biochemical provinces, with potential applications for understanding migratory and feeding patterns of top predators, monitoring effects of climate change, and study of paleo- archives.We explored δ Isoscapes, are maps of systematic isotope variation and provide important biogeochemical information. Isoscapes are becoming increasingly important tools to characterize major biogeochemical zones and gradients in the ocean, and have been also used in ecological studies to help constrain animal migration and fish stock patterns  can be particularly informative, because such measurements have the potential to identify major ocean transitions between eutrophic/mesotrophic and oligotrophic regions, the balance of fundamental N cycle processes , and also basic ecological and food web relationships across major habitat zones. For example, water-column denitrification has a very large isotope effect (ε) of 25–30‰ 15N value of all organisms in areas where this process is important. However, this could be rapidly changing in many ocean regions, linked to oceanographic climate events associated with a shifting climate  of ∼3.4‰ is often assumed 15N value that is of primary interest . Because the bulk isotope value in a consumer is the combined signal of the baseline value and subsequent trophic effects, it is extremely difficult to isolate either factor with confidence.However, the information potential inherent in δ15N values, a seminal study by McClelland & Montoya (2002) demonstrated strong differential 15N enrichment of different groups of amino acids (AA) with trophic transfer. One group of AA has strongly elevated δ15N values with each trophic transfer (∼4–8‰), and are now termed the “Trophic AAs.” A second group of AA, now termed the “Source AAs,” in contrast has relatively constant δ15N values with trophic transfer, and so largely preserves δ15N values from the base of the food web. This pattern of AA differential enrichment has now been verified across a wide range of photoautotrophs and primary consumers Compound-specific isotope analysis of amino acids (CSI-AA) is a rapidly evolving technique that can address many inherent issues with bulk isotope data. For δ15N CSI-AA studies to date have focused on nitrogen isotopic values of two main AAs: phenylalanine (Phe), as the best indicator of baseline δ15N value, and glutamic acid (Glu), as the best indicator for relative trophic transfer. The relative predictability of 15N offsets between Glu and Phe with trophic transfer has also led to an explicit equation now used widely to calculate CSI-AA based trophic position (see methods). Based on these findings, CSI-AA patterns (δ 15NAA) have now been used to not only estimate trophic position (TP) 15NAA is applied in appropriate heterotrophic organisms, the source AA should be able to indicate baseline δ15N isoscapes, decoupled from influence of trophic transfer.Most δMytilus californianus) is a sessile resident of intertidal zones, which continuously filters particulate organic matter (POM). As such, mussels correspond closely to an ideal “baseline indicator” organism , as well as to attempt reconstruction of ocean water composition and conditions The California mussel  across 10 degrees of latitude in the coastal zone of the California Upwelling Ecosystem (CUE). The CUE is part of the greater California Current System (CCS), and is a highly dynamic region where we would anticipate not only large potential variation in baseline δ15N values, but also the potential for rapid future change linked to a warming climate 15N values of source AA, and in particular Phe (δ15NPhe), may can serve as a new, direct proxy for constructing isoscapes of integrated δ15N values of primary production within highly dynamic coastal regions. We compared bulk δ15N and δ15N AA patterns in mussels to first test dependence of bulk isotopic variability on baseline δ15N values, using CSI-AA to constrain variations in TP. We also compared mussel δ15N values with literature values in more offshore sample types (zooplankton and POM), to examine if our results may also apply to the larger CCS. Our results indicate that source AA values in mussels are likely represent a direct record of variation in baseline δ15N values, and suggest that in the CA coast region isoscapes based on δ15NPhe closely follow variations in nitrate δ15N values.Here we examined δMytilus californianus) analyzed for this study were collected from 28 different sites between Coos Bay, Oregon and San Diego, California , 1±0.05 mg of lyophilized tissue was weighed and combined for each individual mussel (n = 5). Further CSI-AA preparation proceeded as described below.Mussels were collected in the winter (Dec – Feb) of 2009–2010. Sites were chosen to be approximately evenly distributed along the CA coastline, with ∼80 km geographic separation between each sampling site. Our main goal here was to sample mussels from a wide geographic range across the CCS, although for observing finer scale local or regional variations, a finer-scale sampling strategy would like be required. Typically 5 individual mussels were collected from each site, all between 30–40 mm maximum shell length, which were immediately placed on dry ice until further preparation. The adductor muscle of each individual was dissected for analysis. This tissue was selected because isotopic values in muscle tissue have shown relatively long turnover times; based on past growth data, mussels of this size would be expected to integrate approximately annual variability in suspended food source isotopic values for each location sampled 15N =  [(Rsample/Rstandard) − 1] ×1,000, where R is the ratio of heavy to light isotope, Rsample is from the sample, and the Rstandard is atmospheric N2 (air) for carbon, as provided by pulses of calibrated CO2 reference gas. For details on correction calculations and normalization to international standards refer to UCSC-SIL website (http://es.ucsc.edu/~silab/index.php).Stable nitrogen isotope analyses were conducted using standard protocols in the Stable Isotope Lab at the University of California, Santa Cruz (UCSC-SIL). Briefly, homogenized muscle tissue of each individual was weighed into tin capsules and combusted. Isotope values determined on a Carlo Erba 1108 elemental analyzer  coupled to a Thermo Finnigan Delta Plus XP isotope ratio mass spectrometer  (EA-IRMS). Analytical error associated with this measurement was typically <±0.15‰ based on sample replicates. Stable isotopes are reported using standard delta (δ) notation in parts per thousand (‰):δ15N values were measured as Trifluoroacetyl isopropyl ester (TFA-IP) AA derivatives, following protocols described in detail elsewhere  under nitrogen, and TFA derivatives subsequently prepared from free AA using a modified version of the protocol described by Silfer : isopropyl esters were made with a 1∶5 mixture of Acetyl Chloride (AcCl):2-propanol , and then acylated using a 1∶3 mixture of Dichloromethane:Trifluroacetyl acetate (DCM:TFAA) . Derivatized AAs were dissolved in DCM to a final ratio of approximately 4 mg of original tissue to 250 µl DCM.Amino acid δ15N values could be reproducibly measured for alanine (Ala), aspartic acid + asparagine (Asp), glutamic acid + glutamine (Glu), leucine (Leu), isoleucine (Ile), proline (Pro), valine , glycine (Gly), lysine (Lys), serine (Ser), phenylalanine (Phe), threonine (Thr), and tyrosine (Tyr) (n = 4 injections), and the average mean deviations for individual AA δ15N measurements across all tissue sample replicates was 0.5‰.After derivatization, samples were analyzed by a Varian gas chromatograph coupled to a Finnegan Delta-Plus isotope ratio mass spectrometer (GC-IRMS). AAs were separated using a 50 m, 0.32 ID Hewlett Packard Ultra-1 column with 1 µm film thickness. Under our analytical conditions, δne (Tyr) . Most AAafter Popp et al. 2007 after Germain et al. 2013 15N with trophic transfer) were Glu, Asp, Ala, Ile, Leu, Pro and Val. The measured Source AA (with expected little to no change in δ15N at higher trophic levels) were Phe, Gly, Ser, Lys and Tyr. The δ15N values of Thr exhibit an apparent inverses isotopic fractionation with trophic transfer, however are also highly variable with organism type In all results and discussion, measured AA are grouped into one of three categories: “Trophic” vs. “Source” , and +7.6 is the assumed 15N enrichment in Glu relative to Phe with each trophic transfer from food source to consumer, also called the Δ value For explicit TP calculations, we used the “canonical” AA's (Glu and Phe) to calculate TP of mussels in the CUE, after Chikraishi et al. 2009:15NPhe values vs. latitude . Our second model  were conducted using the JMP statistical software package . We used Arc-GIS Spatial Analyst (version 10.1) to produce visual isoscapes of the CUE. Our first model is based on the line for δnd model  is based15N values in the adductor muscle of Mytilus californianus ranged from 7.4‰ to 11.5‰  collected from each site to gauge intra-site variability in individuals. Standard deviations on average δ15N values for individual mussels from the same sites ranged from 0.1 to 0.5‰ . We note that all sampling sites were located in exposed waters, and variable habitat type  was 5.1 to 10.3‰  and the averaged Trophic AA values are 10.3 to 15.9‰ . While precision for individual AA δ15N measurements varied (n = 160).Of the 28 sites measured for bulk analysis, 13 were chosen for CSI-AA . Samples5N trend . Measureto 16.0‰ . In all N values ; Fig. S2s varied , it was 15N values as the best proxies for Trophic and Source AA groups, respectively, as has been indicated by a number of recent papers 15N values both correlated significantly with average values for Trophic and Source AA groups respectively , confirming the validity of this approach . In contrast, there was more variability in the Glu data. The relationship of Glu vs. bulk δ15N was not significant at 95% confidence (P>0.05), however a Fit Model run of Analysis of Covariance shows that δ15N values of bulk, Phe and Glu all share a common slope ; in other words, the slope of δ15N change with latitude for Glu and Phe are not significantly different from the slope of bulk δ15N change with latitude.We focused on Glu and Phe δsee also . Both Phlatitude . The δ15P = 0.706), indicating that despite local variability, mussels' suspended POM food sources had similar average TP in all CA coastal regions. Across all mussel samples analyzed with CSI-AA, the average δ15NGlu – δ15NPhe offset was 6.5‰. Prior work indicates that these mussels feed primarily on microalgae 15NGlu – δ15NPhe offsets in phytoplankton and marine macroalgae Glu-Phe for Mytilus californianus of 3.1‰ , continental US (30–48°N) and British Columbia and Alaska (48–60°N) 15N change, wind forcing alone cannot not explain the clear linear decrease in δ15N values with increasing latitude.A complimentary forcing for δ15N baseline values. Water column denitrification in the CCS has been documented in borderland basins , the specific tissue analyzed  and mussel size class were specifically selected to isotopically integrate over an approximate yearly time frame. This assumption is supported by preliminary data of samples collected in both summer and winter season of the same year for selected sampling sites, for which no significant effect on the observed latitudinal trend was observed Local denitrification is another process that could also contribute to regional δ15N trend recorded in mussel tissues seems most consistent with the endmember mixing outlined above, consistent with both modeling and prior discrete sampling. For example, δ15N values of sediment traps and sediment cores contrasted between central CA vs. the Southern CA bight have indicated δ15N values are generally more enriched in the Southern CA bight vs. Northern CA, consistent with our measurements 15N variation 15N values, driven by the ETNP denitrification endmember. Our study therefore represents perhaps the strongest confirmation to date of both model predictions, and also prior discrete-location sampling results. However, no prior sample set has ever tested CCS latitudinal δ15N variation at such high resolution, based on an archive coupling unambiguous source location with approximately annual signal integration. In particular, the striking linear trend in our data is a novel, and also perhaps a surprising finding. This indicates that the diminution in 15N-enriched nitrate supply via the CUC (or relative mixing with southerly CCS) is remarkably regular in the CUE: across the 10 degrees of latitude that we sampled, δ15N change was remarkably consistent (0.41‰±0.04 per degree). We suggest that the ability to capture this regional trend at such high precision is linked to the integrative property of filter feeding consumers, as well as the longer-turnover tissue that we sampled. Overall, we propose that 15N values in coastal CUE waters with latitude. If correct, this also suggests that surveys of costal mussel δ15N values might constitute a powerful new tool for constraining physical mixing and circulation models, since they would show the effective mixing of two source waters in great detail.Overall, the strong latitudinal δ15N values in the CUE, and potentially also a change in the slope of the clear latitudinal gradient we observe. At the same time, increasing stratification in CCS waters is also proposed as one main consequence of warming, and this has already been documented 15N values. However, the potential effects of natural climatic perturbations  are currently very difficult to decouple from longer term trends. Given that our current understanding of physical and biological responses of the CUE to a changing climate remains poor. Repeated sampling of mussels could provide a time and geographically integrated record of baseline isotopic change in this system, revealing longer term trends in the regional δ15N gradient, due to either natural fluctuations or climate change, at near annual resolution.While satellite data has documented changing global ocean surface chlorophyll concentrations, leading to predictions of declining primary production in the world's oceans due to increased stratification associated with warming 15N data derived from mussels may reflect isotopic values of broader coastal waters, as opposed to only littoral sources and process. This is likely to be a function of relative time scale: the time frame over which mussels integrate δ15N of primary production, vs. the mixing time scale for littoral water with more seaward coastal water masses. If water mixing is relatively rapid vs. sampled tissue isotopic turnover, then it is possible mussels would reflect isotopic values within the broader CUE, and possibly into shoreward extent of the CCS. In contrast, if upwelling and nutrient utilization in the littoral zones are rapid and strongly localized, then littoral mussel δ15N values could be mostly decoupled from values in more offshore coastal waters. To definitively address this question, an extensive sampling program would likely be required, comparing offshore/onshore POM isotope values with those recorded in mussel tissues.An important question is to what degree δ15N values in both organisms and detrital OM. We therefore compiled δ15N values for a range of sample types from the Monterey Bay region, and compared these with δ15N values for mussels at our sampling sites in or near the bay , requiring no correction. For carnivorous zooplankton (secondary consumers) we assumed one additional trophic transfer (TP = 3), and therefore adjusted reported δ15N values by 3.4‰ . For sediment trap and surface sediment samples, we used recent results from Monterey Bay long-term sediment trap records, which have indicated an average trophic position (determined by CSI-AA) of 1.6 . To fully explore the potential of littoral mussels as integrators of coastal isoscapes, we suggest a synoptic sampling program comparing offshore/onshore mussel isotopic values will be required.While we acknowledge that this approach can provide only a very general initial comparison, the results are nevertheless quite encouraging . For mos15N values at the base of the food web. Based on differential enrichment behavior of the Trophic vs. Source AAs introduced above 15N value trends. Our calculations of mussel TP for an extended population along the entire CA coast represents, to our knowledge, the first wide-ranging CSI-AA survey of any filter feeding mollusk population in nature. The lack of any trend in TP with latitude . The similarity of TP from all locations therefore supports the conclusion that δ13C variation is primarily driven by changes in baseline δ13C values. Since TP does not change with latitude also strongly supports our basic hypothesis that the overall δ15N trend with latitude , also are lower than would be expected. Mussels predominately feed on POM derived from primary production see Text S1). However, δ13CAA source fingerprinting applied to these same mussels has verified a dominantly marine microalgal diet 13CAA patterns do account for the offset, it is more likely due to variations among different microalgal groups. Given the relatively limited current δ13CAA data on different marine algal lineages, this is possible. An alternate possibly, is that the change in Glu δ15N values with trophic transfer in mussels may be smaller than the TEF factor now most commonly applied . Accumulating evidence now suggests that CSI-AA trophic enrichment factors may be specific for different groups of organisms However, the exact TP values , when compared with previous literature data 15N offsets, nor the actual TEF values for mussels. In contrast, our proposed CSI-AA approach uses δ15NPhe values as a direct proxy baseline δ15N. The fact that the slope of δ15NPhe vs. latitude is identical within error to bulk δ15N (see results) strongly supports the idea of a constant offset from primary production. The δ15NPhe regression-derived baseline values are universally heavier than the bulk- δ15N derived data discussed above, and are a closer match for reported NO3− values in northern vs. southern CA waters.A direct comparison between bulk δhis idea . If we aN values  should a15NPhe is ultimately more accurate representation of baseline δ15N, because it requires no assumptions about TEF values in any specific organism. However, in order to derive precise baseline δ15N predictions based on δ15NPhe, it will also be necessary to have robust calibrations for the offset between δ15NPhe and average algal δ15N values. The close match we observe between the δ15NPhe regression values and expected NO3− δ15N values of this region , and therefore might be used to examine temporal change in isoscapes in many offshore instrumented locations. Finally, our results also suggest potential for paleoceanographic reconstructions. Mussel shells are often the major species found in archeological middens widely distributed from Baja California to Alaska along the US west coast. If source AA δ15N patterns were also well preserved in archeological shell, this could potentially extend the reconstruction of coastal baseline isoscapes back through much of the Holocene.Overall, a CSI-AA approach for constructing baseline isoscapes could have broad importance in both modern and paleoceanographic studies. While CSI-AA based isoscapes could also be generated from other consumers, we suggest that for coastal zones mussels may be a particularly useful bioarchive. The combination of ubiquitous occurrence in many coastal regions, relatively long-term integration of microalgal isotopic signatures, and unambiguous source locations together would provide strong confidence in geographic patterns. We suggest that the degree to which mussel-derived isoscapes also reflect more offshore coastal waters will be an important topic for future work. If our mussel data indicates broad similarity to near-shore coastal isotopic data as a general result , then saFigure S115N Isoscape approach.Alternate δ Alternate δ15N Isocape of the California Upwelling Ecosystem, showing δ15N gradients between sampling stations. As in text 15N values. However, this isoscape interpolates between δ15N at each specific site. While CSI-AA data coverage was not large in this study, preliminary re-sampling has indicated offsets are reproducible. While clearly additional sampling would be required to verify such variation, this approach directly suggests the potential for high resolution isoscapes that capture finer scale regional patterns. Given the ubiquity of mussels along the CA coast, as well as relative ease of sampling, such high resolution coastal isoscapes of baseline δ15N might be readily constructed.(TIF)Click here for additional data file.Figure S215NAA patterns in the California Mussel . δ15N amino acid signatures of Mytilus californianus from 13 sampling sites selected for CSI-AA. Absolute δ15N values normalized to the δ15NPhe, so that patterns can be compared. Measured amino acids are categorized into Trophic, Source, and Metabolic (M), based on relative changes with trophic transfer (see main text). Site and amino acid abbreviations are as defined in main text. Overall δ15NAA patterns conform closely to those expected from other heterotrophic organisms, with Trophic AA enriched in 15N vs. Source AA, and Thr strongly depleted in 15N.(TIFF)Click here for additional data file.Figure S3Low CSI-AA based Mussel Trophic Position Results. Relationships between measured δ15NGlu-Phe values vs. expectations for standard CSI-AA TP equations. Measured δ15NGlu-Phe of mussels are plotted vs. latitude (filled diamonds). Shaded bar on average value represents ± 1SD for entire data set. Assumed β values for primary producers are indicated by lower dotted line, . Commonly assumed TEFGlu-Phe for a single trophic transfer for a primary consumer (7.6 per mil ) is represented by upper dashed line. Arrow represents ΔGlu-Phe the theoretical isotopic enrichment from a TP1 to a TP2.(TIFF)Click here for additional data file.Figure S4Representative chromatogram of a GC-IRMS analysis of amino acids. Mussel amino acid gas chromatogram. A representative gas chromatogram of derivatized individual amino acids from Mytilus californianus. Abbreviations: Ala, alanine; Gly, Glycine; Thr, threonine; Ser, serine; Val, valine; Leu, leucine; Ile, isoleucine; Nor, Norleucine ; Pro, proline; Asp, aspartic acid, Met, Methionine; Glu, glutamic acid; Phe, phenylalanine; Lys, Lysine.(TIFF)Click here for additional data file.Text S1Mussel trophic position discussion.(DOCX)Click here for additional data file.