Hyperekplexia or startle disease is a serious neurological condition affecting newborn children and usually involves dysfunctional glycinergic neurotransmission.Glycine receptors (GlyRs) are major mediators of inhibition in the spinal cord and brainstem.A missense mutation, replacing asparagine (N) with lysine (K), at position 46 in the GlyR α1 subunit induced hyperekplexia following a reduction in the potency of the transmitter glycine; this resulted from a rapid deactivation of the agonist current at mutant GlyRs.These effects of N46K were rescued by mutating a juxtaposed residue, N61 on binding Loop D, suggesting these two asparagines may interact.Asparagine 46 is considered to be important for the structural stability of the subunit interface and glycine binding site, and its mutation represents a new mechanism by which GlyR dysfunction induces startle disease.Nmf11, which causes reduced body size, evoked tremor, seizures, muscle stiffness, and morbidity by postnatal day 21. Introducing the N46K mutation into recombinant GlyR α1 homomeric receptors, expressed in HEK cells, reduced the potencies of glycine, β‐alanine and taurine by 9‐, 6‐ and 3‐fold respectively, and that of the competitive antagonist strychnine by 15‐fold. Replacing N46 with hydrophobic, charged or polar residues revealed that the amide moiety of asparagine was crucial for GlyR activation. Co‐mutating N61, located on a neighbouring β loop to N46, rescued the wild‐type phenotype depending on the amino acid charge. Single‐channel recording identified that burst length for the N46K mutant was reduced and fast agonist application revealed faster glycine deactivation times for the N46K mutant compared with the WT receptor. Overall, these data are consistent with N46 ensuring correct alignment of the α1 subunit interface by interaction with juxtaposed residues to preserve the structural integrity of the glycine binding site. This represents a new mechanism by which GlyR dysfunction induces startle disease.Dysfunctional glycinergic inhibitory transmission underlies the debilitating neurological condition, hyperekplexia, which is characterised by exaggerated startle reflexes, muscle hypertonia and apnoea. Here we investigated the N46K missense mutation in the GlyR α1 subunit gene found in the ethylnitrosourea (ENU) murine mutant,  Hyperekplexia or startle disease is a serious neurological condition affecting newborn children and usually involves dysfunctional glycinergic neurotransmission.Glycine receptors (GlyRs) are major mediators of inhibition in the spinal cord and brainstem.A missense mutation, replacing asparagine (N) with lysine (K), at position 46 in the GlyR α1 subunit induced hyperekplexia following a reduction in the potency of the transmitter glycine; this resulted from a rapid deactivation of the agonist current at mutant GlyRs.These effects of N46K were rescued by mutating a juxtaposed residue, N61 on binding Loop D, suggesting these two asparagines may interact.Asparagine 46 is considered to be important for the structural stability of the subunit interface and glycine binding site, and its mutation represents a new mechanism by which GlyR dysfunction induces startle disease. DMEMDulbecco's modified Eagle mediumDRdose‐ratioENUethylnitrosoureaGFPgreen fluorescent proteinGLRA1GlyR α1 subunit geneGLRBGlyR β subunit geneGluCl˗ channelglutamate‐activated ClGlyRglycine receptorHEK‐293human embryonic kidney 293 cellsBKequilibrium dissociation constantpatchVtrans‐patch potentialWTwild typeet al. et al. GLRA1; Shiang et al. et al. GLRB; Rees et al. et al. Hyperekplexia or startle disease is a serious neurological condition affecting newborn children. It is characterised by exaggerated startle reflexes following tactile and acoustic stimuli, resulting in hypertonia and apnoea. Although considered as a rare orphan disease , this disorder can cause developmental delay and sudden infant death (Davies GLRA1), including: spasmodic, oscillator, cincinatti  in the extracellular domain of GlyR α1, caused a relatively mild phenotype, with homozygous mice appearing normal at rest but developing an exaggerated startle response to acoustic or tactile stimuli at around 2 weeks of age  in the extracellular domain of GlyR α1  is, however, puzzling, because neither α1 subunit protein levels nor the somatodendritic distribution of GlyRs are affected, discounting trafficking or clustering deficits  and transfected with recombinant murine GlyRs 1–3 h later using a modified calcium phosphate precipitation method. For homomeric GlyRα1, the cells were co‐transfected with 1.5 μg pcDNA3‐ wild‐type or mutant rGlyR α1 with 1.5 μg pRK5‐eGFP for identifying transfected cells (1:1 ratio). For heteromeric αβ GlyRs, cells were co‐transfected with pcDNA3‐rGlyR β subunit . Mutations were made using the Stratagene Quikchange method. To increase efficiency, the rGlyRα1 N61 mutation PCR products were subject to DpnI digestion , and methylation (NEB) prior to ligation. All mutated cDNAs were completely sequenced  to check the inclusion and position of the mutations.Human embryonic kidney (HEK293) cells were grown in DMEM supplemented with 10 % v/v fetal calf serum, 2 mm CaCl2 and 24 μl of double‐strength Hanks’ balanced salt solution  for 5–10 min prior to drop‐wise addition to the plated cells. After 16–48 h post transfection, cells were used for electrophysiological recording.DNA solutions were incubated with 20 μl of 340 mm): 120 KCl, 1 MgCl2, 1 CaCl2, 10 Hepes, 11 EGTA, 30 KOH, 2 ATP and 0.5 GTP, pH 7.11 with 1 m NaOH. The cells were continuously perfused with a physiological salt solution containing (mm): 140 NaCl, 4.7 KCl, 1.2 MgCl2, 2.5 CaCl2, 5 Hepes, and 11 d‐glucose, pH 7.4.Glycine‐activated currents were recorded from single transfected HEK293 cells using whole‐cell patch clamp recording with an Axopatch 200B amplifier. Patch electrodes (resistances of 3–4 MΩ) were filled an internal solution containing (m50) and Hill coefficients were determined from concentration–response data fitted using the Hill equation with non‐linear least squares routines (Origin 6.0) as previously described . The biphasic curve data that resulted from the modulation of GlyR function by Zn2+ was fitted using a modified Hill equation as previously described  sampling at 15 kHz and filtered at 5 kHz (6th order Bessel). The currents were normalised to the maximum response amplitude activated by a saturating glycine concentration applied to each cell. Maximal responses, half‐maximal concentrations (ECd) and presence (ds) of strychnine were measured. The dose‐ratios (DR = ds/d) were calculated and used in the Schild plot of log (DR − 1) against the log antagonist concentration ([B])  as expected for an antagonist that acts in a purely competitive manner. The data were then re‐fitted with the Schild equation using a constrained slope of 1:Glycine concentration–response curves were constructed in the absence and presence of three different concentrations of strychnine. From the parallel curve shifts, the glycine concentrations that produced 50% of the maximum response in the absence (KB) to be determined. The upper and lower 95% confidence limits of the regression line were also calculated.The intercept of the line when (DR − 1) = 1 enabled the equilibrium constant for strychnine (m): 102.7, NaCl; 20, sodium gluconate; 4.7, KCl; 2, CaCl2.2H2O; 1.2, MgCl2.6H2O; 10, Hepes; 14, glucose; 15, sucrose; 20, TEACl; pH 7.4; 320 mosmol l–1. For single‐channel experiments with WT GlyRs, the pipette solution incorporated an EC60 concentration of either glycine (0.05 mm) or taurine (0.4 mm). For the N46K experiments, the pipette solution was supplemented with equivalent EC60 concentrations of either glycine (0.5 mm) or taurine (0.9 mm).Single glycine‐activated channel currents were recorded from cell‐attached patches from transfected HEK293 cells expressing WT and N46K homomeric α1 subunit GlyRs. Patches were held at +100 mV and the recorded currents were filtered at 5 kHz and sampled at 30 kHz. Thick‐walled borosilicate patch electrodes (8–10 MΩ) were filled with external physiological salt solution containing . Open and shut durations were measured using a 50% threshold cursor applied to the main single‐channel current amplitude in each patch. As with all threshold cursor methods for detecting single‐channel state transitions, very brief shuttings can be missed, increasing the duration of adjacent open periods  contained the same internal solution used for whole‐cell recording. Cells were perfused with physiological salt solution. A theta glass electrode, pulled and cut with a diamond knife to a tip diameter of 50–100 μm, was used to apply adjacent solution streams for rapid exchange over the macropatch. Solution exchange was achieved by activating a piezoelectric transducer  that translated the solution interface for a specified time (2–200 ms). Open tip potentials were measured at the conclusion of each patch to confirm exchange rates . Application durations were programmed for multiple pulses, then averaged to reduce signal/noise ratios. Rise and deactivation times were calculated between 10 and 90 % of the peak current amplitude and reported as means ± SEM.C. elegans glutamate‐activated Cl˗ channel  was used to generate the simulated whole‐cell currents to glycine on WT and N46K GlyRα1. The binding/unbinding rate constants, and gating and preactviation constants, are generally in accord with values previously determined and published for GlyRs . Glycine‐activated currents for homomeric N46K GlyRα1 were comparable with those for WT receptors  with saturating concentrations of glycine  revealing only a small, but insignificant, decrease in maximal current: 3245 ± 570 pA  and 2209 ± 367 pA . However, the glycine concentration–response curves revealed differences in potency . For GlyRα1 the glycine EC50 was 41 ± 3 μm (n = 20); whereas for GlyRα1N46K, glycine was 9‐fold less sensitive with an EC50 of 372 ± 4 μm (n = 19).Here we compared the glycine sensitivities of both homomeric and heteromeric GlyRs incorporating the N46K mutation in the α1 subunit with wild‐type receptors and the phenotypically less severe A52S mutation. Asparagine 46 is located in the extracellular domain, within the β1 loop (Brejc n = 6) and 4871 ± 460 pA  with a smaller 5‐fold separation in receptor sensitivity to glycine; the respective EC50s being: 64 ± 6 μm  and 318 ± 79 μm . This suggested that the β subunit partly rescued the N46K hyperekplexia mutation effect on glycine current, which might be expected given that the β subunit is replacing mutant α subunits in the α1β heteromeric GlyR. Co‐assembly of α and β subunits was confirmed by a reduced inhibition of the EC50 glycine steady‐state response by picrotoxin (20 μm) . Thus, homomeric α1 GlyRs are the most sensitive to the N46K mutation.Heteromeric receptors GlyRα1β) for WT and N46K exhibited comparable maximal glycine currents: 4840 ± 1019 pA .As the phenotype for the A). For the less potent partial agonists taurine and GABA, the curve displacements were less apparent: only 3‐fold for taurine  and 1.5‐fold for GABA , again without any significant changes to the relative maximum responses . This supported the hypothesis that N46K reduced ligand binding rather than agonist efficacy.To examine if the predominant effect of N46K is centred on the agonist binding site, the partial agonists β‐alanine, taurine and GABA were studied. For a full agonist, a reduction in efficacy can manifest as just a rightward displacement of the concentration–response curve; whereas a changed efficacy is often more evident with a partial agonist, revealed by an additional lower maximum response. β‐Alanine was 6‐fold less potent at N46K receptors compared to WT – without any change in the relative maximum response Fig. A. For tet al. et al. WT and α1N46K GlyRs to inhibition by strychnine were assessed with glycine EC50 responses. Strychnine concentration–inhibition curves indicated that the potency of the antagonist was reduced 15‐fold by N46K , which is similar to the shift observed in glycine potency by N46K.To further probe the role of N46K on the orthosteric binding site, inhibition by the competitive antagonist strychnine was examined  from 27.2 ± 4.21 nm  to 122.5 ± 16 nm .To determine if the reduced strychnine inhibition was due to a reduction in the binding affinity, we performed a Schild analysis. Shifts in the glycine concentration–response curve following exposure to 0.1, 0.3 and 1 μ50 = 51 ± 4 μm; n = 5) was similar to that for wild‐type GlyRs . However, concentration–response curves were displaced to the right when the amide side‐chain was replaced with the negatively‐charged carboxyl groups in aspartate and glutamate, N46D  and N46E , and even more so with positively‐charged lysine .We analysed the importance of N46 by conservatively substituting it for glutamine (Q), which retains the amide side‐chain. The glycine concentration–response curve for N46Q  compared with N46K. Replacing N46 with phenylalanine , a large non‐polar group, reduced the sensitivity of the GlyR even more than for N46K. Further increasing side‐chain volume with tryptophan , or changing charge using the basic amino acid arginine  did not cause any greater displacement in the concentration–response curve compared with N46F . Smaller polar side‐chain residues, lacking an amide group, such as cysteine , serine  and threonine , again significantly reduced glycine potency compared with WT , and the atomic resolution structures for the human α3 . Furthermore, it seems unlikely that N46 fulfils an α‐helical capping action or stabilisation of a β loop, as the structural projections placed this residue in the middle of a β loop . Our receptor structures suggested that the side‐chains for loop A residues, N102 and E103, are directed away from N46 implying that direct interactions are unlikely , with N102 more likely to interact with its own backbone  or aspartate  resulting in 500‐ and 10‐fold shifts, respectively, in the glycine concentration–response curves compared to WT . Charge reversal at N46 (to K or D) and at N102 (to D or K) did not reverse the displacement of the glycine curves to that observed with WT , suggesting it was unlikely that N46 directly interacted with N102 . In addition, mutating N46 and N102 to the same charged residue resulted in non‐functional glycine receptors.Using structural homology modelling, based on the glutamate‐activated Clet al. A), was a prime candidate for interaction. Mutating N61 to lysine had no significant effect on the glycine concentration–response curve compared to WT. However, mutating N61 to aspartate displaced the curve towards lower glycine concentrations . The double mutant N46K‐N61K also rescued glycine potency back to WT levels, whilst N46D‐N61D significantly reduced glycine potency when compared with curves for WT and also N46K GlyRs , causing potentiation and inhibition at low and high micromolar concentrations, respectively . Co‐ or pre‐applying Zn2+ with a glycine EC20 revealed that N46K had no effect on Zn2+ potentiation of the glycine current , which was also unaffected by A52S (not shown). However, glycine‐activated currents were differentially inhibited by higher Zn2+ concentrations (100 μm), with homomeric WT receptors showing greater inhibition (80 ± 5% inhibition) compared to the N46K mutant . By contrast, A52S presented a similar Zn2+ inhibition profile to WT . Interestingly, another hyperekplexia mutation GlyRα1E103K located in loop A  and abolished 100 μm Zn2+ inhibition without affecting Zn2+potentiation (data not shown).GlyRs can be modulated by several allosteric ligands Lynch, . Notablyent Fig. A and B,et al. et al. et al. 80 response by pregnenolone sulphate  was unaffected by either the N46K or A52S mutations . However, potentiation by 10 μm tetrahydro‐deoxycorticosterone (THDOC) of glycine EC20  and glycine EC50  responses for GlyR α1 were significantly reduced by N46K . However, potentiation of the glycine EC20  and EC50  responses induced by 10 μm THDOC at the α1A52S mutant was similar to WT. By comparison, GlyRs containing the β subunit were relatively insensitive to THDOC .Neurosteroids are endogenous molecules in the brain that are capable of modulating GlyRs depending on the receptor subunit composition . Picrotoxin (20 μm) inhibited the EC50 peak glycine current for WT GlyRs by 61 ± 4 %, and for α1A52S mutant GlyR by 46 ± 3 % (n = 5). By comparison, it caused less inhibition of equivalent glycine currents at GlyR α1N46K . The inhibition by 20 μm PTX of steady‐state glycine‐activated currents was also greater for WT (81 ± 3 %) and A52S receptors  compared to N46K .Picrotoxin (PTX) inhibits homomeric GlyRs by blocking the ion channel, but heteromers, containing the β subunit, are relatively less sensitive . Using a patch potential of +100 mV, a comparison between wild‐type GlyRs and N46K mutant revealed a small but significant (P = 0.017) increase in channel current , which overall equates to a unitary conductance range of 65–80 pS. However, since we are not controlling the HEK cell membrane potential in cell‐attached recording mode, this small difference in single channel current could arise from small changes to the membrane potential and thus the driving force.To gain insight into how N46K affected the activation of GlyRs, single‐channel recording from cell‐attached patches was used. Single‐channel currents were activated by ECO1 and τO2 and areas AO1 and AO2  and C and Table P < 0.05).The channel open time distributions for WT and N46K α1GlyRs were best described by two exponentials with time constants τO2 Table . However60 concentrations of the partial agonist taurine were also investigated. Taurine‐activated channel current amplitudes were similar between WT and N46K GlyRs (7.63 ± 0.4 pA (WT) and 7.37 ± 0.5 pA (N46K); n = 5–6). Furthermore, the taurine open time distributions were very similar for WT and N46K GlyRs  compared with WT  with no change in the activation kinetics .The changes detected for burst durations of glycine‐activated channels were investigated further by using a fast application system to apply concentration jumps of glycine or taurine to outside‐out macropatches containing either WT or N46K GlyRs Fig . A 200 mics Fig. A and B.n = 6) compared with WT receptors  without affecting activation kinetics . Fast 200 ms application of EC60 taurine presented a different profile to glycine, with N46K exhibiting slower activation kinetics with no apparent change in deactivation/desensitisation kinetics. However, the 2 ms taurine pulse showed faster deactivation with N46K compared to WT .Similarly, 2 ms applications of equipotent glycine concentrations also revealed significantly faster current deactivation for N46K  in GlyRα1 , glycine fulfils an excitatory role as a consequence of high internal ClOverall, the correlation between phenotype severity and extent to which GlyR sensitivity to glycine is reduced, under conditions where receptor trafficking and maximal glycine currents are unaffected, may be an important criterion for future genotype‐phenotype studies of human hyperekplexia.N46K suggested that gating efficiency was possibly unaffected. However, if agonist efficacy (E) is very high, then a reduction in E would appear to displace the curve with minimal reduction in the relative maximum response. By using estimates of E for glycine activating the fully liganded GlyR α1  or charged  side‐chains. These all caused large displacements to the glycine curve that did not occur with N46Q, which retains the amide moiety. The location of N46 near the subunit interface and its apparent effect on glycine binding, suggested it might interact with residues either located on the adjacent α subunit or on β loops within the same α subunit that are important for ligand binding. GlyR structures suggest N46 points towards loop A, which is important for binding in nicotinic AChRs . The charge reversal experiments involving N46 and N61 demonstrated that these two asparagines could potentially interact, particularly given the likelihood of de‐protonated carboxyl groups in their side‐chains under physiological conditions. Possible interactions between the two carboxamide side‐chains could include: electronic delocalisation, dipole–dipole or charge–charge interactions. If these interactions are disrupted, as indicated by the charged residue substitutions, this could alter the structural integrity of the binding site located just above this plane, thus reducing glycine binding.A parsimonious explanation for the N46 phenotype may involve important ligand binding residues, on loop D, which are upstream of N61  constitute the Zn2+ inhibition site . Since N46 faces loop A, across the subunit interface, substitution with a positively charged lysine could disrupt loop A, perturbing Zn2+ coordination and compromising inhibition. This is supported by the results with A52S, which is located further along the β strand and thus away from loop A, and did not disrupt Zn2+ inhibition when compared with the WT. Interestingly, E103K (loop A), a mutation that causes hyperekplexia in humans . To describe the changes to the glycine current profiles and agonist concentration–response curves by N46K required several empirical changes to rates and constants. Primarily, the deactivation rates (and thus the agonist dissociation constants (K1–3)) between states AR, A2R and A3R were increased (∼5‐fold) together with a reduction in the pre‐activation constant, F3  from the pre‐activation state A3F to A2F by ∼5‐fold  and increase in K (∼3‐fold), which may reflect, given the relative proximity of N46 and A52 in terms of primary sequence, the impact this part of the extracellular domain has on GlyR function.To account for the effects caused by N46K, we constructed a three binding site model for the GlyR that incorporated pre‐activation states (Burzomato old Fig. ; Table 4In conclusion, the N46K missense mutation markedly reduced glycine sensitivity, resulting in a severe, lethal startle phenotype. N46 most likely interacts with N61 to stabilise binding loops D and E near the orthosteric site for glycine. By disrupting the structural integrity of the glycine site Fig.  N46K proThe authors have no competing interests to declare.The work for this study was carried out at University College London. M.E.W. designed and performed the experiments, acquired, analysed and interpreted the data, and helped write the manuscript; A.C. contributed data and performed data analysis; M.C.G. contributed towards the homology modelling and interpreted data; R.J.H. contributed to the conception of the work and to manuscript writing; T.G.S. conceived and designed the study, performed kinetic modelling and simulations, and helped to write the manuscript. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.This work was supported by the MRC, EU‐FP7 consortium, Neurocypres, and The Leverhulme Trust.


C and QD, in photosystem II (PSII). Here, we investigated the potential involvement of loosely bound plastosemiquinones in superoxide anion radical (O2•−) formation in spinach PSII membranes using electron paramagnetic resonance (EPR) spin-trapping spectroscopy. Illumination of PSII membranes in the presence of the spin trap EMPO (5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide) resulted in the formation of O2•−, which was monitored by the appearance of EMPO-OOH adduct EPR signal. Addition of exogenous short-chain plastoquinone to PSII membranes markedly enhanced the EMPO-OOH adduct EPR signal. Both in the unsupplemented and plastoquinone-supplemented PSII membranes, the EMPO-OOH adduct EPR signal was suppressed by 50% when the urea-type herbicide DCMU -1,1-dimethylurea) was bound at the QB site. However, the EMPO-OOH adduct EPR signal was enhanced by binding of the phenolic-type herbicide dinoseb  at the QD site. Both in the unsupplemented and plastoquinone-supplemented PSII membranes, DCMU and dinoseb inhibited photoreduction of the high-potential form of cytochrome b559 (cyt b559). Based on these results, we propose that O2•− is formed via the reduction of molecular oxygen by plastosemiquinones formed through one-electron reduction of plastoquinone at the QB site and one-electron oxidation of plastoquinol by cyt b559 at the QC site. On the contrary, the involvement of a plastosemiquinone formed via the one-electron oxidation of plastoquinol by cyt b559 at the QD site seems to be ambiguous. In spite of the fact that the existence of QC and QD sites is not generally accepted yet, the present study provided more spectroscopic data on the potential functional role of these new plastoquinone-binding sites.Recent evidence has indicated the presence of novel plastoquinone-binding sites, Q Thermosynechococcus elongatus and Thermosynechococcus vulcanus demonstrated that PSII consists of 20 protein subunits, 35 chlorophylls, 12 carotenoids and 25 lipids per monomer [A and QB sites, respectively. One-electron reduction of plastoquinone at the QB site forms plastosemiquinone (QB•−), which is subsequently stabilized by the protonation of proximal amino acid side chains (QBH•), whereas the sequential one-electron reduction and protonation of QBH• forms plastoquinol (QBH2).Photosystem (PSII) is a heterodimeric multiprotein-pigment complex embedded in the thylakoid membrane of photosynthetic organisms such as cyanobacteria, algae and higher plants. Recent X-ray crystallographic structural analyses of PSII from the cyanobacteria  monomer –5. In th monomer –9. It isA and QB sites [b559 (cyt b559) in the presence of exogenous plastoquinone, a third plastoquinone-binding site referred to as QC was proposed to be located closed to cyt b559 [b559 provided more biochemical data on the existence of QC site [C site [C site was not reported in the most recent PSII crystal structure at 1.9 Å resolution [C site is lower compared to the QB site [C site might be due to the different purification and crystallization procedures [D. The authors concluded that the QC site depicted in the PSII crystal structure is in a highly hydrophobic environment, while the QD site is located in a more polar environment. The urea-type herbicide DCMU -1,1-dimethylurea) blocks QB to QB•− reduction at the QB site, whereas the phenolic-type herbicide dinoseb  prevents the oxidation of plastoquinol (QDH2) to plastosemiquinone (QDH•) by cyt b559 at the QD site [Several biochemical studies have suggested that PSII contains two plastoquinone-binding sites in addition to the QQB sites –12. Baseyt b559 . Later,  QC site –12. Conssolution . Hasegaw QB site . In agreocedures . Recentlocedures  identifi QD site .2•−), which either spontaneously dismutates to hydrogen peroxide (H2O2) or forms bound peroxide through interactions with the non-heme [b559 [2O2 by free metals or bound peroxide by the non-heme iron forms hydroxyl radicals (HO•) [The limitations on electron transport both on the electron donor and acceptor sides of PSII are associated with the formation of reactive oxygen species (ROS) –19. Undenon-heme  or heme e [b559 . Subsequls (HO•) .2•−. These cofactors are highly reducing species with a midpoint redox potential lower than the standard redox potential of the O2/O2•− redox couple . Molecular oxygen may be reduced by pheophytin (Pheo•−) [A site (QA•−) [B site (QB•−) [•−) [b559 [Several studies have demonstrated that various cofactors on the electron acceptor side of PSII can reduce molecular oxygen, forming O(Pheo•−) , 22, the (QA•−) , the loo (QB•−) , free pl•−) [•−)  and the ) [b559 .Em (Pheo/Pheo•−) = – 505 to –610 mV, pH 6.5 to 7) [•− is likely. The favorable thermodynamic properties for reduction of molecular oxygen by Pheo•− are limited by kinetic restrictions. Forward electron transport from Pheo•− to QA•− is much more rapid than diffusion-limited reduction of molecular oxygen; thus, the reduction of molecular oxygen by Pheo•− is less likely. However, under certain circumstances, such as limitation of electron transport from Pheo•− to QA•−, the Pheo•− lifetime is prolonged, and the reduction of molecular oxygen is more likely.Due to a highly negative redox potential (.5 to 7) –28, the •−, reduction of molecular oxygen by QA•− and QB•− is less favorable from a thermodynamic perspective. In principle, the midpoint redox potentials of the QA/QA•−  [B/QB•−  [2/O2•− redox couple  [2 ~ hundreds μM) and product (O2•− ~ hundreds nM) differ, the operational redox potential of the O2/O2•− redox couple is shifted to 0 mV or even positive values based on the Nernst equation [A•− and QB•− seems to be more thermodynamically feasible. From a kinetic perspective, the lifetimes of QA•− and QB•− are sufficiently long for the diffusion-limited reduction of molecular oxygen. In addition to QA•− and QB•−, free PQ•− can reduce molecular oxygen  [b559 produces O2•− and may be thermodynamically feasible because the LP form of cyt b559 has a low midpoint redox potential  [In contrast to PheoV, pH 7) –30 and QV, pH 7)  redox coV, pH 7) . When thequation . Thus, tV, pH 7) ; howeverV, pH 7) . It has V, pH 7) , 26, 33.2•− formation in PSII membranes using an electron paramagnetic resonance (EPR) spin-trapping spectroscopy. We provide evidence that O2•− is produced via one-electron reduction of molecular oxygen by plastosemiquinones, which are formed through one-electron reduction of plastoquinone at the QB site (QB•−) and one-electron oxidation of plastoquinol by cyt b559 at the QC site (QCH•). By contrast, a role of plastosemiquinone formed at the QD site (QDH•) in O2•− formation is ambiguous.Herein, we studied whether loosely bound plastosemiquinones are involved in the light-induced Oet al. [2, 5 mM MgCl2 and 50 mM Mes-NaOH (pH 6.5) and stored at -80°C until further use. For PQ-supplemented PSII membranes, exogenous short-chain platoquinone containing one isoprenoid units in the side-chain (PQ-1) was added to the PSII membranes prior to illumination. 30 μM PQ-1 was added to the PSII membranes as an ethanol solution .PSII membranes were isolated from fresh spinach leaves using the method reported previously by Berthold et al.  with modet al. . The iso2•− was detected by EPR spin-trapping spectroscopy using EMPO (5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide; Alexis Biochemicals, Lausen, Switzerland) as the spin trap. PSII membranes (150 μg Chl ml-1) were illuminated with a continuous white light (1000 μmol photons m-2 s-1) in a glass capillary tube  with 25 mM EMPO, 100 μM Desferal and 40 mM MES buffer (pH 6.5). PSII membranes were illuminated using a halogen lamp with a light guide  at room temperature. The spectra were recorded using an EPR spectrometer Mini Scope MS400 . The following EPR conditions were used: microwave power, 10 mW; modulation amplitude, 1 G; modulation frequency, 100 kHz; sweep width, 100 G; and scan rate, 1.62 G s-1. For quantification, intensity of EPR signal was evaluated as the relative height of peak of the first derivative of the EPR absorption spectrum.Ob559 were studied using an Olis RSM 1000 spectrometer . The redox states of cyt b559 in PSII membranes (150 μg Chl ml-1) were determined based on the changes in the absorbance at 559 nm upon stepwise additions of 50 μM potassium ferricyanide, 8 mM hydroquinone, 5 mM sodium ascorbate and sodium dithionite in a cuvette at room temperature using the method in Tiwari and Pospíšil [b559 in the PSII membranes were determined by subtracting the control from the treatment spectra: for the HP form of cyt b559, the hydroquinone-reduced spectra were subtracted from the ferricyanide-oxidized cyt b559; for the IP form of cyt b559, the ascorbate-reduced spectra were subtracted from the hydroquinone-reduced cyt b559; and for the LP form of cyt b559, the dithionite-reduced spectra were subtracted from the ascorbate-reduced cyt b559. In photoreduction measurements, the photoreduced HP form of cyt b559 (PH) was calculated based on the difference between the absorbance spectra measured after illumination for 180 s and the dark-adapted ferricyanide oxidized spectrum and hydroquinone-reduced spectra were subtracted from photoreduced HP form of cyt b559 to get unreduced HP form of cyt b559. The PSII membranes were illuminated with continuous white light (1000 μmol photons m-2 s-1) in the cuvette, which was rotated by 90° at intervals of 15 s.The redox properties of cyt Pospíšil  with cer-1) was mixed with 3 ml of heptane and 30 μl of isobutanol, followed by vortexing for 1 h in the dark. The mixture was then centrifuged at 4000 x g for 10 min. The plastoquinone content in the upper organic layer was determined by HPLC based on the method of Kruk and Karpinski [The loosely bound plastoquinone was measured using the method in Wydrzynski and Inoue . A 1 ml arpinski .2•− formation in the unsupplemented PSII membranes was measured using EPR spin-trapping spectroscopy. For spin-trapping, we used the spin trap compound EMPO, which reacts with O2•− to form an EMPO-OOH adduct [• through the Fenton reaction [2•−.Light-induced OH adduct . No EMPOreaction , 39. Fig2•− formation, light-induced O2•− formation was measured in the presence of exogenous PQ-1. Because PQ-1 is smaller than the natural molecule PQ-9, PQ-1 can better penetrate the membrane and substitute for PQ-9 as an electron acceptor in PSII. The observation that the addition of PQ-1 to EMPO did not generate any EPMO-OOH adduct EPR spectrum indicates that PQ-1 does not directly interact with EMPO (data not shown). In the dark, the addition of PQ-1 to the PSII membranes in the presence of EMPO did not produce an EPR signal; however, exposure of PQ-supplemented PSII membranes to white light resulted in the formation of an EMPO-OOH adduct EPR signal  and dinoseb (bound at the QD site) on the EMPO-OOH adduct EPR signal were studied in the unsupplemented PSII membranes. When the unsupplemented PSII membranes were illuminated in the presence of DCMU, the EMPO-OOH adduct EPR signal was suppressed by 50%, whereas the remaining EPMO-OOH EPR signal (50%) was insensitive to DCMU  the DCMU-sensitive EMPO-OOH adduct EPR signal corresponds to O2•− formed at or after the QB site  and 2) the DCMU-insensitive EMPO-OOH adduct EPR signal corresponds to O2•−, which is formed before the QB site . When dinoseb was added to the unsupplemented PSII membranes prior to illumination, the EMPO-OOH adduct EPR signal was enhanced by 25%  O2•− is formed prior to the QB site by reduction of molecular oxygen by Pheo•− and QA•−. The intensity of the EMPO-OOH adduct EPR signal after the addition of DCMU was higher for the PQ-supplemented PSII membranes than for the unsupplemented PSII membranes  spectra, ascorbate-reduced minus hydroquinone-reduced (IP) spectra, and dithionite-reduced minus ascorbate-reduced (LP) spectra. In the unsupplemented PSII membranes, 40% of cyt b559 was in the hydroquinone-reducible HP form, 22% was in the sodium ascorbate-reducible IP form, and 38% was in the dithionite-reducible LP form , consistent with values in the literature  [A) and two plastoquinones are loosely bound (QB and QC or QD).To correlate the PQ-binding site and O Chl/RC) , 42. HPL2•− is formed through one-electron reduction of molecular oxygen on the electron acceptor side of PSII [2/O2•− redox couple is close to 0 mV or even positive due to the difference in concentration of molecular oxygen and O2•−, O2•− formation requires a suitable electron donor with a redox potential lower than the operational redox potential of O2/O2•− redox couple, and thus consequently, a high reducing power to reduce molecular oxygen. It was suggested that various cofactors on the electron acceptor side of PSII can fulfil such thermodynamic criteria and thus might serve as potential electron donors to molecular oxygen. Although light-induced O2•− formation in PSII has been examined by measuring oxygen consumption [2•− formation remains unclear. Here, we studied the role of loosely bound plastosemiquinone at the QB, QC and QD sites in light-induced O2•− formation in the PSII membranes supplemented with exogenous PQ-1. Addition of exogenous PQ-1 to the PSII membranes enhanced light-induced O2•− production, indicating the involvement of plastosemiquinones in O2•− production. Because the midpoint redox potentials for tightly bound plastosemiquinones at the QA site (Em (QA/QA•−) = -60 to -140 mV, pH 7) [B site (Em (QB/QB•−) = -45 mV, pH 7) [2/O2•− redox couple (close to 0 mV or even positive), the reduction of molecular oxygen by plastosemiquinones is feasible. Based on the presented data, we propose that O2•− is produced by one-electron reduction of molecular oxygen by plastosemiquinones formed by one-electron reduction of plastoquinone at the QB sites and one-electron oxidation of plastoquinol at the QC site but most likely not the QD site  –30 and lV, pH 7)  are lowe QD site .B site  and II (upper channel) [BH2 liberates from the QB site and diffuses through the bottom channel to the QC site located in the vicinity of the heme iron of cyt b559 at distance of 17 Å from the head group of plastoquinol. Plastoquinol binding at the QC site was proposed to favour electron donation to the ferric heme iron of cyt b559 [b559, demonstrating that QCH2 is oxidized by the ferric heme iron of cyt b559 to form QCH•. Here, we propose that QCH• reduces molecular oxygen to O2•−. Because addition of dinoseb to the PSII membranes partially enhanced O2•− formation . Consistent with this proposal, we assume that the reduction of molecular oxygen by QDH• is not feasible and thus O2•− formation at the QD site is ambiguous.The observation that the phenolic-type herbicide dinoseb, which binds at the Qoduction . QDH2 oxShuvalov  recentlyB and QC sites are involved in the formation of O2•− via one-electron reduction of molecular oxygen. Loosely bound plastosemiquinone QB•− is formed via one-electron reduction of plastoquinone at the QB site; however, one-electron oxidation of plastoquinol by cyt b559 at the QC site forms QCH•. By contrast, the results indicated that O2•− formation from plastosemiquinones at the QD site was ambiguous. In addition to loosely bound plastosemiquinone, previous studies have reported the formation of O2•- by free plastosemiquinone in the PQ pool [•−, which reduces molecular oxygen to form O2•−. Further studies are needed to elucidate a unifying mechanism for O2•- formation which involves PQ pool.The data presented in this study demonstrate that loosely bound plastosemiquinones at the Q PQ pool , 43–45.


B. taurus × B. grunniens) were investigated on five experimental pastures (Sites 1–5), following an up- and downward transhumance route . Hybrids were kept in replicated groups of two (low SD) and four animals per paddock (high SD). As a control, yaks  were included at low SD at Sites 2–4. Performance was described by body weight, milk yield and composition. Herbage samples as selected by the animals were analyzed. Activity patterns of the hybrids were assessed at Sites 2–4. At similar body weight change and milk composition, the individual hybrids on average produced 26% less milk at high than at low SD. However, at high SD on average still 49% more milk/m2 of pasture area was produced. Milk fat increased with time from 5.4 to 7.1%, milk protein decreased from 4.2 to 3.3%. At high SD, the hybrids spent less time standing and more time lying. The yaks gained weight at 4,500 m a.s.l. while the hybrids lost weight . In conclusion, high SD reduced individual milk yield and forced hybrids to spend more time for feeding. The yaks apparently had an advantage over the hybrids at very high altitude.Twelve lactating cattle × yak hybrids ( F1-hybrids of yaks and cattle are common in the yak-keeping high altitude areas of Asia  if increased stocking densities will result in losses of individual animal performance and changes in the activity pattern of the animals and (2) if, at high altitudes, cattle × yak hybrids do no longer perform better than yaks because of the hybrids’ incomplete inheritance of the yak’s specific high altitude adaptive capacity. In order to be able to test the hypotheses, a controlled field experiment was conducted along a transhumant route starting from 3,200 m a.s.l. up to 4,500 m a.s.l. and descending to 2,600 m a.s.l., including five pasture sites used for data collection. The performance of the hybrids was compared with that of purebred yaks at the three high altitude pasture sites. The pasture site effect included altitude, season and sward composition  as potential factors of influence.A traditional transhumant route used by herders of Olangchung Gola, Taplejung district, and five pasture sites  along this route, were selected for the experiment in 2010. The route is situated within the Kanchenjunga Conservation Area in the Eastern Himalayan Mountains of Nepal. In order to protect the areas from grazing, the pastures were fenced 2 weeks before the respective measurement periods started. All paddocks were relatively flat and had a North-East aspect. Sites 2 and 4 were established at the same pasture site; however, sites were not contiguous but separated by a small ridge. Sites 1–3  were grazed in spring and summer , while Sites 4 and 5 were used in late summer and autumn . A portable weather station was installed at each of the five pasture sites. Average temperatures measured at 01.30 p.m. and minimum temperatures, recorded at 06.30 a.m. (both in °C) for Site 1 were 16 ± 5 and 4 ± 3, for Site 2: 14 ± 1 and 2 ± 1, for Site 3: 12 ± 1 and 2 ± 2, for Site 4: 12 ± 2 and 1 ± 1 and for Site 5: 11 ± 1 and 2 ± 1, respectively. Average precipitation  recorded was 7 ± 11, 9 ± 7, 12 ± 8, 4 ± 6 and 4 ± 5 for Sites 1–5, respectively.Bos taurus × Bos grunniens (♂ × ♀) hybrids, locally called Dimjo chauries, which were shown to tolerate higher altitudes better than the Urang chauries (Bos grunniens × Bos indicus) . Only the green forage biomass was used for calculation of paddock size. It was separated into palatable biomass  and unpalatable biomass . A dry matter (DM) content of 200 g/kg was assumed. Feed intake was estimated based on the animals’ requirements using the equation stated in Wiener et al.  at Sites 1–5 was 861, 1,485, 1,125, 1,050, and 700 at low SD and 431, 743, 563, 525 and 350 at high SD, respectively. Both SD treatments were repeated by independent paddocks per site. Two additional paddocks, established at Sites 2–4, were stocked with two yaks per paddock (following the low SD scheme). The animals were moved into the paddocks after morning milking at about 09.00 a.m. on day 1 and were released at about 05.00 p.m. on day 9. Before starting the experimental periods, the animals had been kept for 5 days in the surroundings of the respective pasture site without entering the pre-fenced areas. In the time between the experimental periods, the animals were kept together with a larger herd of the respective genotype and were moved along the transhumance route according to the herder practice.The corresponding assessments for BW and MY were made one and two consecutive days before stocking the paddocks, respectively. Shortly before starting with the experimental periods, the final paddocks were established within the previously fenced areas. Additional space was allocated if part of the area was covered with unpalatable shrubs or stones. Estimates of trampling losses were considered. Based on these considerations, the amount of forage on offer was estimated to match the requirements of four adult hybrids at high SD during 9 days. Low SD was defined as stocking with only half of the animals for the same period of time in a paddock of similar size. At Sites 1–5, this resulted in paddock sizes (m) of 41 × 42, 55 × 54, 50 × 45, 50 × 42 and 40 × 35, respectively. The corresponding SD . The animals were accompanied and their selection behavior was mimicked for the whole duration of the respective observation period by randomly switching between animals following the procedure described by Berry et al. . The MY was measured with a digital balance . In the yaks, at first milk let-down had to be initiated by allowing the calves to suckle for 1–2 min for two times, then hand-milking was practiced and finally the calves were allowed to suckle again. By weighing them before and after suckling, the milk amount consumed by the calves was determined. Keeping yaks and their calves separately at night ensured that most of the milk was consumed in the morning. Still, the actual total MY of the yaks was probably higher as the calves may have suckled also during the day. Milk compositional analysis was done in duplicate after each milking in the 9-day experimental periods by a portable ultrasonic milk analyzer . For the hybrids, aliquots (weighted means) of morning and evening milk yields were used for calculation of the daily values. The contents measured were adjusted using regression equations obtained from 25 milk samples analyzed with the portable milk analyzer and the standard Milkoscan 4000   was assessed with a digital balance  covered by a wooden platform of a size of 200 × 50 × 5 cm. Calves were weighed before and after each milking. Adults were weighed after morning milking 1 day before the start of each experimental period and after evening milking on day 9. The daily BW change was calculated as /9 days.n = 8) by accelerometer-based sensors to record animal behavior . The sensors were fixed at the right hind leg in the morning of day 1 of the experimental periods during milking and detached when the animals were released at the evening of day 9. Thus, complete data sets were only available for 8 days. Data was evaluated by the corresponding software . Analysis was done per minute following Aharoni et al.  and n = 6 (Sites 2–4).The mixed procedure of SAS (version 9.3) was used to perform the analysis of variance. The botanical composition was analyzed with Model 1 treating pasture site (S) as single fixed effect and paddock as replicate For ECM yield, milk composition, BW and behavior data, a model was applied exclusively on data of the hybrids in order to determine site (S) and SD effects. This Model 2 reads:P < 0.05 as significant. The data on the chemical composition of the herbage selected by the experimental animals are arithmetic means. Means of variables subjected to analysis of variance and given in the tables are Least Square means.Model 3 compared the two genotypes for BW data, ECM yield and milk composition at Sites 2–4 with low SD only. It reads as follows:P < 0.05) at Site 2 (Table P < 0.05 compared to the other sites). Together, Poaceae and Cyperaceae contributed to >50% of forage biomass on Sites 2–5. Poaceae alone made up about half of the biomass at Site 5, whereas they accounted for <10% at Site 1. During the downward movement, the vegetation matured, was fully flowering at Site 4 and relatively dry with only some regrowth mostly by grasses underneath the senescent material at Site 5. Accordingly, the increasing maturity of the vegetation was associated with a decline in nutritional quality of the herbage selected. The CP content in the herbage selected was numerically highest at Site 1 and values were gradually lower when moving from Site 1 to Site 5. The relatively high CP content found at Site 1 could be related to the early sprouting stage in spring and in addition to the low prevalence of grass species. Cellulose and hemicellulose contents of the herbage selected increased numerically from Sites 1–5, but this pattern was not reflected in the lignin contents.The biomass available was largest ( 2 Table . In the P < 0.001) on initial BW, while BW remained unaffected (P > 0.1) by SD. There was daily BW gain in the hybrids at Sites 1–2 and at Site 4, whereas there was a BW loss at Sites 3 and 5. The ECM yield was affected by both, pasture site and SD (P < 0.001). At high compared to low SD, a significant reduction in ECM yield occurred at Sites 2 and 3. However, ECM yield/unit of pasture area available per animal was significantly higher with high SD on Sites 1–3, but not on Sites 4–5. Site, but not SD, had an effect on the contents of fat, protein and lactose in the milk of the hybrids. On average, the hybrids were heavier (P < 0.001) than the yaks. There was a significant interaction between genotype and pasture site in body weight change. While the hybrids lost 653 g BW/day/animal at the highest altitude at Site 3, which was different (P < 0.05) from the BW gain of 897 g/day/animal at Site 2, the yaks gained 836 g BW/day . Comparing the initial BW of yaks and hybrids measured at Sites 2 and 4, the hybrids gained 26 kg across this time, while the yaks increased their BW by 49 kg. Looking specifically at the high altitude Site 3, the hybrids numerically lost on average 2.3% of their BW across the 9 days of measurement period at low SD, whereas the yaks gained on average 3.6% of BW. An interaction of pasture site and genotype occurred with an initially higher (P < 0.05) ECM yield in the hybrids at Site 2  in hybrids and yaks at Site 2, respectively, data not shown in table). There was an interaction of genotype and pasture site in milk fat content and fat amount. No significant differences in milk fat content were found in the hybrids when comparing Sites 2 and 4 , but the yaks had a higher (P < 0.05) milk fat content at Site 4 compared to Site 2 . Yaks had higher (P < 0.05) milk protein and lactose contents than the hybrids.There were various effects of pasture site, SD and genotype in the traits describing performance of the experimental animals Table . In the P < 0.05) from Sites 2–3 (Table P < 0.05), whereas standing time did not change (P > 0.1). Overall, high SD resulted in more steps (P < 0.05) during the entire day and daytime. SD had overall an effect on lying and standing time (P < 0.05).During the time the hybrids stayed at altitudes between 4,000 and 4,500 m, the number of steps per day declined  than to the actual site conditions.Yaks are especially well adapted to high altitude environments  of the hybrids investigated was not superior to that of the yaks at the same low stocking density at very high altitude. It seems that the hybrids had surpassed their comfort zone at this altitude. However, long-term studies over several years are needed to be able to draw definitive conclusions.As hypothesized, overall a high stocking density limited the hybrid individuals’ milk yield even during a short period of 9 days. In the present study, SD had recurrent effects on milk yield as in the time between the experimental periods the animals were allowed to graze freely, and forced animals to perform overall more steps, likely to gather enough feed. However, the individual milk yield loss was compensated by a concomitantly about 50% higher overall milk yield per m


Varp (VPS9-ankyrin repeat protein) was originally identified as an activator of small GTPase Rab21 through its VPS9 domain, but it has subsequently been shown to function as a Rab32/38 effector through its first ANKR1 domain. Although these functions of Varp are important for melanogenesis, Varp contains a second ANKR2 domain, whose function remained completely unknown. Here we identified Rab40C, an atypical Rab containing a SOCS box that recruits a ubiquitin ligase complex, as a novel ANKR2-binding protein and investigated its involvement in melanogenic enzyme trafficking in melanocytes. The results showed that overexpression of Rab40C in melanocytes caused a dramatic reduction in melanogenic enzyme Tyrp1 signals by promoting proteasomal degradation of Varp in a SOCS-box-dependent manner and that knockdown of Rab40C in melanocytes caused an increase in the amount of Varp. Intriguingly, Rab40C knockdown also caused a dramatic reduction in Tyrp1 signals, the same as Varp overexpression did. These findings indicated that Rab40C is a previously unexpected regulator of Tyrp1 trafficking in melanocytes through controlling the proteasomal degradation of Varp. Melanosomes are specialized organelles that synthesize and store melanin pigments in melanocytes . Since tchocolate mice, and defects in BLOC-3 cause the hypopigmentation seen in type 1 and type 4 HPS patients.Cumulative recent evidence has indicated that several members of the Rab-type small GTPase family, a family of conserved membrane trafficking proteins in all eukaryotes , and theWe previously identified Varp (VPS9-ankyrin repeat protein) as a Rab32/38-specfic effector protein that specifically recognizes an active form of Rab32/38 and is involved in Tyrp1 trafficking to melanosomes together with Rab32/38 . Varp coIn this study, we investigated whether the ANKR2 domain was also involved in Tyrp1 trafficking in melanocytes. We identified Rab40C as a novel ANKR2-domain-binding protein in melanocytes and found that manipulation of Rab40C either by RNAi-mediated knockdown or by overexpression altered the amount of Varp protein in melanocytes. We also found that the effect of Rab40C on Varp expression is completely dependent on the presence of a SOCS  box in Rab40C that is associated with a ubiquitin ligase complex . Based oTo investigate the possible involvement of the ANKR2 domain of Varp in the trafficking of melanogenic enzymes in melanocytes, we overexpressed the ANKR2 domain of Varp with mStr (monomeric Strawberry)-tag in melanocytes and observed its effect on the signals of the melanogenic enzyme Tyrp1. If the ANKR2 domain was actually involved in the control of Tyrp1 trafficking, the same as the ANKR1 domain is, its overexpression in melanocytes would reduce Tyrp1 signals, the same as ANKR1 overexpression did  Fig. 1B. As antisupplementary material Fig. S1). Since the results of the reverse transcription (RT)-PCR analysis indicated that Rab40C, but not Rab40B, was expressed in mouse melan-a cells .We performed co-immunoprecipitation assays in COS-7 cells to determine whether the interaction between full-length Varp and Rab40C occurs in mammalian cells and, as shown in We next focused our attention on the Varp-binding site in Rab40C, because members of the Rab40 subfamily are unique in containing a SOCS box, which is associated with a ubiquitin ligase complex  Fig. 2EFig. 2E. supplementary material Fig. S3B). By contrast, strong mStr-Varp signals were easily detected, when EGFP alone was overexpressed in melanocytes . A similar tendency toward suppression of Varp expression was also observed in response to Rab40C-Q73L, a constitutive active Rab40C mutant, which preferentially bound Varp , whereas Rab40C-G28N, a constitutive negative mutant, which hardly bound Varp at all . Intriguingly, when the SOCS box of Rab40C was deleted, the Rab40C-ΔSOCS mutant, which still retained Varp binding ability . The SOCS-box-dependent and Varp-interaction-dependent suppression of Varp expression by Rab40C mutants was confirmed by immunoblotting .To identify the site of colocalization between Varp and Rab40C at the cellular level, mStr-tagged Varp and EGFP-tagged Rab40C were coexpressed in melanocytes. To our surprise, however, we were unable to detect a sufficient amount of mStr-Varp signals when the level of EGFP-Rab40C expression was relatively high  against Rab40C . Althouganocytes , it cleaanocytes , in sharanocytes . To our anocytes , the samanocytes . These oTo determine whether the increased amount of Varp protein in Rab40C-knockdown cells was a major cause of the reduction in Tyrp1 signals, we finally investigated the effect of overexpression of T7-tagged Varp on Tyrp1 signals in control melanocytes. As anticipated, expression of full-length Varp in melanocytes also dramatically reduced Tyrp1 signals , the samWe previously showed that Varp regulates not only Tyrp1 trafficking to melanosomes through interaction with Rab32/38 via its ANKR1 domain  but alsoXenopus Rab40 binds the Cullin5 E3 ubiquitin ligase complex . The findings in this study provide new insights into the regulatory mechanism of melanogenic enzyme trafficking to melanosomes in melanocytes.In conclusion, we have demonstrated a novel function of the ANKR2 domain of Varp, i.e., Rab40C-mediated ubiquitination of Varp in melanocytes, and that it contributes to Tyrp1 trafficking to melanosomes. This finding, together with our previous findings in regard to Varp, indicate that Varp is a pleiotropic regulator of three different types of Rabs in three different processes through three distinct domains, a regulator of Rab21 in dendrite formation through its VPS9 domain, a regulator of Rab32/38 in Tyrp1 trafficking through its ANKR1 domain, and a regulator of Rab40C in the control of its own degradation in melanocytes through its ANKR2 domain (summarized in S-transferase) essentially as described previously . All other reagents used in this study were analytical grade or the highest grade commercially available.Anti-Tyrp1 mouse monoclonal antibody (Ta99) and anti-β-actin mouse monoclonal antibody were obtained from Santa Cruz Biotechnology  and Applied Biological Materials , respectively. Anti-multi ubiquitin mouse monoclonal antibody (FK2) and HRP (horseradish peroxidase)-conjugated anti-GAPDH  mouse monoclonal antibody were from MBL . Anti-FLAG tag (M2) mouse monoclonal antibody and HRP-conjugated anti-FLAG tag (M2) mouse monoclonal antibody were from Sigma-Aldrich . HRP-conjugated anti-T7 tag mouse monoclonal antibody and HRP-conjugated anti-HA tag (3F10) rat monoclonal antibody were from Merck Millipore  and Roche Applied Science , respectively. Alexa 488/594-conjugated anti-mouse and/or anti-rabbit IgG goat antibodies were from Invitrogen . Anti-Tyrp1 rabbit polyclonal antibody was prepared as described previously . Anti-Vaeviously . The proBamHI linker (underlined) or a stop codon (bold): Met primer, 5′-GGATCCATGCAGATCTTCGTGAAGACCCT-3′ and stop primer, 5′-ACAGCTTTATTTGACCTTCTTCTTGG-3′. The cDNA of Ub was subcloned into the pEF-HA vector, which was modified from pEF-BOS (Rab40C (target site 1: 5′-AACTGCATGGCCTTCTTTGAA-3′ and target site 2: 5′-CCATCAAAAGCCACCTCAAG-3′) were chemically synthesized by Nippon Gene .The cDNAs of mouse Rab40C  and Varp were prepared as described previously . The cDN pEF-BOS . The del pEF-BOS . Unless supplementary material Fig. S1A) as described previously (Yeast two-hybrid assays were performed by using pGBD-C1-Rab(CA) (constitutive active) lacking the C-terminal geranylgeranylation site (ΔCys) and pAct2-Varp-ANKR2 see , pAct2-Veviously . The yeaeviously .Rab40C siRNA, control siRNA, or nothing was prepared with TRI-reagent (Sigma-Aldrich), and reverse transcription was performed by using ReverTra Ace®  according to the manufacturer's instructions. The pairs of oligonucleotides used for amplification were: for Rab40B, forward primer, 5′-GGATCCATGATGAGCTCCCTGGGCAG-3′ and reverse primer, 5′-CTAAGAAATTTTGCAGCTGT-3′; for Rab40C, forward primer 1, 5′-AGATCTATGGGCACCCAGGGCAGTCC-3′ and reverse primer 1, 5′-CTAGGAGATCTTGCAGTTGC-3′ . Data representative of the results of the independent experiments are also shown in t-test or Dunnett's test (for multiple comparisons), and p-values <0.05 were considered statistically significant .The black-mouse-derived immortal melanocyte cell line melan-a (generous gift of Dorothy C. Bennett) was cultured as described previously . PlasmidRab40C siRNA by using Lipofectamine 2000, and melan-a cells were transfected with control siRNA or Rab40C siRNA by using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. At 48 hours after transfection the B16-F1 cells (or melan-a cells) were washed with ice-cold PBS, scraped, and recovered by centrifugation at 800 g for 5 minutes at 4°C. The cells were lysed with the lysis buffer , and the lysates were subjected to 10% SDS-PAGE  sodium deoxycholate, 1% (w/v) SDS, and 10 mM N-ethylmaleimide supplemented with complete EDTA-free protease inhibitor cocktail by passing them through a 27-gauge needle. Inhibition of T7-Varp degradation was assessed by 10% SDS-PAGE followed by immunoblotting with HRP-conjugated anti-T7 tag antibody.B16-F1 cells were co-transfected with pEF-T7-Varp together with control siRNA or 5 cells/35-mm dish) were co-transfected with pEF-T7-Varp and pEF-FLAG-Rab40C  by using Lipofectamine LTX Plus (Invitrogen) according to the manufacturer's instructions. At 36 hours after transfection the cells were lysed with the lysis buffer . Co-immunoprecipitation assays with anti-FLAG tag antibody-conjugated agarose beads (Sigma-Aldrich) were performed as described previously (5 cells/60-mm dish) by using Lipofectamine LTX Plus according to the manufacturer's instructions. Proteasomes were inhibited by exposing the cells to the proteasome inhibitor MG132 (10 µM) for 3 hours before harvesting them. After lysing the cells with the lysis buffer containing 10 mM N-ethylmaleimide, T7-Varp was immunoprecipitated with anti-T7 tag antibody-conjugated agarose beads (Merck Millipore). Proteins bound to the beads were analyzed by 5% SDS-PAGE followed by immunoblotting with HRP-conjugated anti-T7 tag antibody and HRP-conjugated anti-HA tag antibody.COS-7 cells (2×10eviously . Ubiquit


SNPs in the GWD gene were associated with C6 phosphorylation, whereas polymorphisms in the SBEI and SBEII genes were associated with both C6 and C3 phosphorylation and the SNP in the SSIII gene was associated with C3 phosphorylation. These allelic variants have potential as genetic markers for starch phosphorylation in potato.Starch phosphorylation is an important aspect of plant metabolism due to its role in starch degradation. Moreover, the degree of phosphorylation of starch determines its physicochemical properties and is therefore relevant for industrial uses of starch. Currently, starch is chemically phosphorylated to increase viscosity and paste stability. Potato cultivars with elevated starch phosphorylation would make this process unnecessary, thereby bestowing economic and environmental benefits. Starch phosphorylation is a complex trait which has been previously shown by antisense gene repression to be influenced by a number of genes including those involved in starch synthesis and degradation. We have used an association mapping approach to discover genetic markers associated with the degree of starch phosphorylation. A diverse collection of 193 potato lines was grown in replicated field trials, and the levels of starch phosphorylation at the C6 and C3 positions of the glucosyl residues were determined by mass spectrometry of hydrolyzed starch from tubers. In addition, the potato lines were genotyped by amplicon sequencing and microsatellite analysis, focusing on candidate genes known to be involved in starch synthesis. As potato is an autotetraploid, genotyping included determination of allele dosage. Significant associations ( Starch is the major storage carbohydrate in plants and comprises most of the dry matter in potato tubers. Starch represents the largest source of carbohydrates in human food and has a range of industrial uses. Starch is made up of chains of α-D-glucose units which can be unbranched (amylose) or branched (amylopectin). The native starches of almost all plants are phosphorylated, the extent of which varies between plant species, and is greater in potato starch than in cereal starches. The degree of phosphorylation of starch determines its physicochemical properties and is therefore relevant for industrial uses of starch, which include the manufacturing of paper, adhesives, textiles, and foods Kraak, . PhosphoGWD (or R1) gene encodes the protein glucan/water dikinase (GWD) which catalyzes the addition of phosphate groups to the C6 position of the glycosyl residues   caused a small increase in C6 phosphorylation and an increase in amylopectin content  had no significant effect on starch phosphorylation  is a highly heterozygous, autotetraploid, outcrossing species which is propagated clonally. The breeding history of the cultivated potato has created a situation in which many current cultivars from around the world are related by a complex network of relationships. Genes from wild species  have been introduced during the introgression of disease resistance genes to try to overcome disease problems in commercial cultivars  was grown in field trials at Lincoln, Canterbury, New Zealand, during two consecutive seasons (2011 and 2012). Tubers were planted in October and harvested in March, and the plants were irrigated as required. Climate data for the growing seasons were obtained from the National Climate Database maintened by NIWA , New Zealand. The lines and their origins are listed in Supplementary Data A diverse collection of 193 tetraploid potato lines . Each plot consisted of six plants, planted in two adjacent rows. The 432 plots were planted in a 72 × 6 plot array, with a full replicate (including 24 controls) in each of two adjacent blocks. The position of test lines within the layout was derived from a resolvable block design with blocks of eight, created with CycDesign  were hydrolyzed with trifluoroacetic acid and prepared as described previously  method 2SO4, 0.01% Tween® 20, 0.2 mM each dNTP, 1 μM each primer, 0.02 μM tail primer, 0.75U Taq , approximately 10 ng genomic DNA and 2.5–4.0 mM MgCl2 as shown in Supplementary Data SSR markers for population structure were amplified by PCR using previously published primers  to identify alleles by product size, while allele copy number was estimated by peak area. When four alleles were present, these were scored as a single copy of each. When a single allele was present, this was scored as four copies of that allele. When two alleles were present, this was scored as 2:2, 3:1 or 2:1 depending on the relative peak areas . Where three alleles were present, the largest peak was scored as two copies and the other two as a single copy, except where the three peaks were all of a similar area in which case a null allele was assumed and the three alleles were scored as one copy each.SBEII, was amplified in two pieces as it was not possible to amplify the complete 20 kb gene. The products were normalized to ensure equal representation of all genes, then fragmented and used to construct four bar-coded libraries which were sequenced on a Genome Sequencer FLX instrument . The resulting sequence reads were aligned to reference sequences using gsMapper software , which identified a list of SNPs . The PCR was performed using the Expand long template PCR system  according to the manufacturers' instructions, and using the primers shown in Supplementary Data 4)2SO4, 0.01% Tween 20, 2.5 mM MgCl2, 0.2 mM each dNTP, 1μM each primer, 0.75U Taq , and approximately 10 ng genomic DNA. The PCR conditions were one cycle of 95°C for 2 min, 40 cycles of 95°C for 30 s, 58°C for 30s, 72°C for 1 min, then one cycle of 72°C for 7 min. Unincorporated primers were removed from the PCR products using exonuclease I and shrimp alkaline phosphatase  and used to group the heterozygotes into three genotypes.A region containing an insertion/deletion polymorphism (InDel) located 0.5 kb upstream of the ATG start codon of the The C6 and C3 phosphorylation (nmol/mg) for each of the two field trials were analyzed separately. For each type of phosphorylation, mean values were calculated which were adjusted to compensate for spatial variation in the field trial and any local trends in processing , using the “Red Rascal” control data. The data were analyzed using a mixed model approach, with the model fitted with restricted maximum likelihood  were combined for the q-value calculation.To address the problem of false discovery when multiple tests are performed simultaneously, Amino acid substitutions resulting from the DNA polymorphisms that were found to be significantly associated with starch phosphorylation were analyzed using PROVEAN software  Figure . HoweverThe four subpopulations indicated by STRUCTURE reflect the geographical origins of the samples  from within the candidate genes of 192 potato lines were sequenced in both directions. This included two amplicons for p < 0.001 as shown in Table GWD gene and were associated with C6 phosphorylation (p < 0.001) for the 2012 samples, and of these, three also gave association at p < 0.01 for the 2011 samples. Two SNPs in the SBEI gene were associated with both C6 and C3 phosphorylation. Sbei2ag was associated (p < 0.001) with C6 for both years and C3 2011. Sbei5tc was associated with C3 2011 at p < 0.001 and also with C3 2012 and C6 for both years at p < 0.01. Two SNPs in the SSIII gene were associated with C3 phosphorylation at p < 0.001. Both ssiii7tc and ssiii11gc were associated with C3 phosphorylation in 2012 (p < 0.001) while ssiii11gc was also associated with C3 2011 at a lower level of significance (p < 0.01). Only one of the SSR alleles (sbeii180) gave association results significant at p < 0.001. It was associated with both C6 and C3 phosphorylation in 2011(p < 0.001) and also with C3 in 2012 (p < 0.01). In total, 9 SNPs and one SSR allele gave association results significant at p < 0.001  is likely to be a false positive. Therefore, p < 0.001 makes a sensible threshold to use for confidence that the genetic markers discovered are truly associated with the phenotype, and that we have got the most important markers. Although this is likely to exclude some true positives, some of those excluded will involve SNPs that have been found to be associated at p < 0.001 with the alternative form of phosphorylation or for the alternative year, e.g., SNP gwd2gt was significant at p < 0.001 for C6 2012 and 0.001<p < 0.01 with C6 2011  had two alleles with fairly even frequency  was located within an intron, while two  were within synonymous codons, and would therefore be unlikely to affect protein function. The other six SNPs would result in amino acid substitutions in the protein encoded. These substitutions were analyzed using PROVEAN software  is consistent with published results, such as 8–25 nmol/mg  gave results that were significant at p < 0.001 for both field trials, several of the other results that were significant at p < 0.001 were backed up by results from the alternative year that were significant at p < 0.01  association was found between PWD SNPs and C3 phosphorylation, given the PWD encodes the enzyme which phosphorylates starch at the C3 position. However, this may simply reflect that we were looking at SNPs from a single fragment of 532 bp and these SNPs were not in LD with polymorphisms that may affect C3 phosphorylation. Although we have not characterized the extent of LD in this association panel, other studies have indicated that LD decays rapidly over relatively short distances in tetraploid potato  with the degree of both C6 and C3 phosphorylation. This is consistent with findings from antisense experiments that showed a decrease in expression of SBEI , encoded a serine/asparagine substitution at a position in the protein which tends to be conserved among homologous sequences, which have serine at this position. In potato the alleles encoding serine and asparagine are approximately equally common, and it is the asparagine variant which is associated with higher phosphorylation. It is possible that this SNP is a functional mutation which influences the degree of starch phosphorylation. Comparison with the crystal structure of the homologous protein in rice revealed that the equivalent serine residue is in the central catalytic α-amylase domain of the protein, but is not one of the seven residues known to be involved in enzymatic activity . This is a novel observation as other studies have not measured C3 phosphorylation in a large number of samples. However, in a QTL analysis, a CAPS marker in SSIII was found to be associated with total starch phosphorylation . The QTL analysis . The 140 bp InDel in the GBSS gene has been shown to have a major effect on GBSS activity and amylose content (van de Wal et al., Among the four starch synthase genes that we examined, only GWD, SBEI, and SBEII genes. As some of these markers appear to be closely linked, only a subset would be required for use as genetic markers. The gwd1ct, gwd2gt, sbei2ag SNPs and the sbeii180 SSR allele, either individually or in combination, would make useful markers for potato starch phosphorylation and its accompanying physicochemical characteristics. The ssiii7tc or ssiii11gc SNPs may also be useful if C3 phosphorylation is of interest. While the C6 and C3 phosphorylation of starch have different roles in starch structure and catabolism (Hansen et al., The markers that were associated with the greatest influence on C6 phosphorylation, and therefore on total phosphorylation, were in the MAC co-ordinated the project, contributed to genotyping and phenotyping of samples, analyzed data, and drafted the manuscript; NIJ did the mass spectrometry; RAG provided potato tubers and planted and harvested the field trials, RDC contributed to starch isolation and genotyping; SRM contributed to genotyping; ADN conducted the association analysis; RCB designed the field trials and analyzed phenotypic data; GMT conceived and initiated the project, secured funding, and contributed to the manuscript. All authors read and approved the final manuscript.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.


PlantAPA provides various interactive and dynamic graphics and seamlessly integrates a genome browser that can profile heterogeneous cleavage sites and quantify expression patterns of poly(A) sites across different conditions. Particularly, through PlantAPA, users can analyze poly(A) sites in extended 3′ UTR regions, intergenic regions, and ambiguous regions owing to alternative transcription or RNA processing. In addition, it also provides tools for analyzing poly(A) site selections, 3′ UTR lengthening or shortening, non-canonical APA site switching, and differential gene expression between conditions, making it more powerful for the study of APA-mediated gene expression regulation. More importantly, PlantAPA offers a bioinformatics pipeline that allows users to upload their own short reads or ESTs for poly(A) site extraction, enabling users to further explore poly(A) site selection using stored PlantAPA poly(A) sites together with their own poly(A) site datasets. To date, PlantAPA hosts the largest database of APA sites in plants, including Oryza sativa, Arabidopsis thaliana, Medicago truncatula, and Chlamydomonas reinhardtii. As a user-friendly web service, PlantAPA will be a valuable addition to the community of biologists studying APA mechanisms and gene expression regulation in plants.Alternative polyadenylation (APA) is an important layer of gene regulation that produces mRNAs that have different 3′ ends and/or encode diverse protein isoforms. Up to 70% of annotated genes in plants undergo APA. Increasing numbers of poly(A) sites collected in various plant species demand new methods and tools to access and mine these data. We have created an open-access web service called PlantAPA ( Chlamydomonas reinhardtii (Chlamy for short herein), APA affects transcripts from 33 to 68% of expressed genes  genes possess more than one poly(A) site  3′ end formation, including cleavage and polyadenylation, is a vital step in post-transcriptional processing in eukaryotic cells. Polyadenylation plays important roles in many mRNA functions, such as mRNA stability, exportation, and translation initiation  stores read alignments and poly(A) sites for human, chicken, and Arabidopsis using sequences from RNA-seq and direct sequencing, but it lacks datasets for other plant species and provides limited capability for data visualization and analysis. Recent studies have discovered an increasing number of APA genes in various plant species, including rice, Arabidopsis, Medicago, and Chlamy  to query, visualize, and analyze genome-wide poly(A) sites derived from various data sources, including RNA-seq, ESTs, 454, and poly(A) tag (PAT) sequencing  sites from previous studies  sites.A Perl script was written to extract the chromosome names, nucleotide contents, and chromosome lengths from a FASTA file of genome sequence and then insert the data into the database. For each species in the database, another Perl script parses the genome annotation in GFF format. Because a gene may contain more than one transcript, we proposed a method to determine unique gene models. If a region is not the same among different transcripts of the same gene, it is denoted as an AMB (AMBiguous) region Figure . The incA computational pipeline was implemented in Perl to detect poly(A) sites from next generation sequencing (NGS)  sites and gene expression levels between samples, including identification of 3′ UTR lengthening and shortening, identification of non-canonical APA site switching genes, detection of differentially expressed (DE) genes and significant changes in PAC levels Figure . First, s libraries and a gene with k poly(A) sites, read count and transcript length of the ith PAC in the jth library are Nij and Lj, respectively. Then, PACs in a gene are sorted by the 3′ UTR lengths, and the lengths are considered as scores. A table is constructed with each row denoting the indexes of samples Si and each column denoting the scores. Each element in the table is the read count Nij. Then a chi-squared test for trend in proportions is performed using the R function prop.trend.test, and the Pearson correlation r is calculated using the number of reads in the table as the values and the scores for rows and columns as coordinates. The correlation r falls between −1 and 1. The larger the correlation is in absolute value, the farther the data fall from independence in the linear dimension. Finally, p-values are adjusted using Benjamin method implemented by the R function p.adjust, and genes with adjusted p-values smaller than a given threshold are those with significant 3′ UTR shortening or lengthening.To discover significant 3′ UTR shortening or lengthening, genes with at least two PACs in 3′ UTRs are considered. The global lengthening or shortening of each gene is calculated by detecting a trend association for two-way tables with ordered levels Agresti, . Given sPA1 and PA2). Genes with both PA1 and PA2 located in 3′ UTRs are discarded. Then genes passing through the following filtering criteria are considered as potential APA switching instances  total read count for a gene more than N1; (3) PA1:PA2 read count ratio more than K (K > 1)-fold in one sample and PA2:PA1 also more than K (K > 1)-fold in another sample; (4) difference in read counts between PA1 and PA2 more than N2 within each library in which switching occurred; and (5) p-value of Fisher's exact test for PA1 and PA2 read counts between the two libraries less than a given significance level.In addition to the PACs located in 3′ UTRs, recent studies have uncovered an increasing number of PACs located in non-3′ UTRs, e.g., introns or CDS. To study such unconventional PACs, we proposed a method to detect non-canonical APA site switching genes. First, if a gene has more than two PACs, then the top two supported by the greatest number of PATs were used  is identified as a DE gene. We also designed a pipeline integrating DEXseq  and other JavaScript libraries to improve user experience. In order to make web interfaces more interactive and dynamic, integrated AJAX (Asynchronous Javascript and XML) technology was used to communicate with background pages and upgrade the front interface without reloading the website. Several open source plug-ins were also incorporated in our server, such as E-Charts (http://echarts.baidu.com/) and jTable (http://www.jtable.org), to display data dynamically. The poly(A) site extraction pipeline is composed of several Perl scripts and alignment tools, such as Bowtie  sites. We constructed the interactive web interfaces based on jQuery (PAC trap), query of platform-collected PACs (PAC search), browsing PACs through JBrowse (PAC browse), analysis of APA between conditions , and quantification and visualization of PACs (PAC viewer). In the PAC trap module, users can input their own short reads, ESTs, or known poly(A) sites to extract or upload their own PACs. Then users can visualize and analyze their uploaded PACs combined with stored PlantAPA PACs in various ways. In the PAC search module, users can query PACs by various kinds of keywords and choose to visualize detailed information, such as poly(A) signals, expression patterns, and sequences in the PAC viewer module. The PAC browse module integrates the Jbrowse genome browser to provide an interactive and graphical view of PACs on a genome-wide scale. In the PAC analysis module, users can compare general poly(A) site usage and changes in gene expression levels between libraries, combining user uploaded PACs, if any, and collected PlantAPA PACs. The PAC viewer module provides diverse representation modes to allow users to quantify and visualize PACs of a gene or intergenic region across different conditions.The goal of the portal is to provide biologists with a convenient and meaningful platform for studying plant APA. To this end, we implemented five modules in PlantAPA Figure , includioxt6 mutant for Arabidopsis; leaf, hairy root, and root tissues for Medicago; flower, leaf, root, grain, and seed tissues for rice; and reads from Sanger, 454, Illumina, and poly(A) tag sequencing for Chlamy   of PAC coordinates or file(s) of short reads or ESTs in FASTA or FASTQ format. A label, such as library, sample name, tissue, or cell type, can be assigned to each uploaded file to help distinguish each input. If the input is a file with a PAC list, then users can skip the PAC extraction pipeline and switch to other modules for PAC visualization and analysis, using both the user- uploaded PACs and stored PlantAPA PACs. Otherwise, the user needs to choose the sequence type by clicking a drop-down box that shows different file types, including unknown, reads with T stretches at the 5′ end (T), and reads with A stretches at the 3′ end (A). If “unknown” is chosen, a sequence type (A or T) will be automatically assigned by comparing the frequency of occurrence of stretch A and stretch T in the uploaded file. Predefined sets of parameters are provided in the PAC trap module for general users. Nevertheless, users can modify the options to meet their specific needs, such as discarding low-quality sequences or not, minimum read length, alignment tools, removing internal priming candidates or not, and distance for clustering adjacent cleavage sites. Upon completion of the PAC extraction process  for dynamic browsing of PACs, together with genomes, genes, transcripts, and annotations, on a genome-wide scale. Users can have quick access to the PAC browser by clicking the “PAC browse” tab in the main menu or the “Jbrowse” link in a PAC list. One or more datasets from each plant species can be quickly loaded and graphically browsed online by selecting the checkboxes of datasets in the “Available Tracks” panel. Users can conduct a search for a gene or chromosome fragment to zoom in on particular genomic regions. Data tracks of PACs from different cells, tissues, or conditions can be displayed in sync with tracks of PATs  is a cluster which spans 76 bp from Chr3:23311308 to Chr3:23311383 with the reference coordinate at Chr3:23311368. This PAC seems to be specific to root tissue in that it has 2389 PATs in WT root and 2743 PATs in oxt6 root, while only a few PATs (<3) were found in leaf or seed. Another PAC (PAC:4180@23309923) is located in 5′ UTR of the same gene, which spans 154 bp from Chr3:23309836 to Chr3:23309990 with the reference coordinate at Chr3:23309923 . Summary information is given on the top of the web page, which describes basic information about this gene, such as gene alias or potential function, and then tabulates all six PACS and the associated GO terms , one in intron (PAC:144@2693638), two in CDS (PAC:91@2691822 and PAC:12@2691769), and one in 3′ UTR (PAC:4814@2691118). Cleavage sites of a PAC are depicted in vertical lines with height representing the number of supporting PATs. The dominant cleavage site supported by the maximum number of PATs in a PAC is marked by a thick line. If the number of PATs of the dominant cleavage site exceeds the maximum scale value (default of 50) of the vertical axis, a small horizontal line will be shown on the top of the thick line. A text label is found under each dominant cleavage site to clearly indicate the expression level, i.e., total number of supporting PATs, of the respective PAC. Users can also view selected samples by choosing specific samples in the “Individual” drop-down list or group replicates within each individual sample by clicking the “Grouping” drop-down list. By clicking the “PAC usage” tab, an additional bar chart  with PACs in AMB regions. This gene has four annotated transcripts  is the most dominant PAC, while the one in blue (PAC:9@2884358) is supported by only nine PATs and specific in WT root. The bar chart summarizes all PATs of individual groups to show the usage of PACs more clearly  sites or raw sequences, which enables users to analyze APA site switching with samples provided by the user or PlantAPA. By following the “PAC analysis” tab in the main menu, users can choose to generate lists of differentially expressed genes, PACs with differential usage, genes with 3′ UTR lengthening or shortening, and APA site switching genes. Two groups of samples, each with one or more than one condition, need to be selected first. Then, the raw count or normalized count calculated by different methods, such as TPM, EdgeR, and DESeq, can be specified. Additional parameters can be set for each assay, such as minimum number of PATs for prefiltering of PACs, significance level, and p-value adjusted method. To make the result statistically significant, a p-value or adjusted p-value will be calculated and assigned to each output PAC or gene. Users can download the output list to their local computer, or continue to inspect the resulting PAC or gene by clicking the link on a particular item on the list using the PAC viewer or the PAC browse module.The PAC viewer module to view detailed information about a gene or PAC. For the example query of AT1G67680, the PAT figure  sites in various plant species demand new methods and platforms for data access and mining. PlantAPA is intended to leverage our enormous wealth of existing poly(A) site data and genome annotations in plants by providing users with easy-to-use web services for the study of APA-related mechanisms and functions in plants. Like other APA-related web services  site selections, 3′ UTR lengthening or shortening, differential uses of poly(A) sites, and differential gene expression between selected conditions, making PlantAPA convenient and powerful for the study of APA-directed gene regulations. Second, PlantAPA provides users with the capability to perform APA analysis with different parameter settings for prefiltering and custom analysis, which allows users to analyze PACs of interest and compare the results with different parameters for fine-tuning. Third, users may upload their own PACs from different samples and analyze them separately. Users can also analyze their PACs together with those collected in PlantAPA if they prefer to use a larger dataset or to compare their data with the existing data.APA has been shown to play an important role in the global control of gene expression in animals and plants. Tandem 3′ UTR switching can alter posttranscriptional regulation of gene expression in that it may lead to a gain or loss of miRNA binding sites. The APA site switching that occurs in CDS or introns may result in changes in translated proteins and their functions. Recent studies have investigated APA site switching, especially 3′ UTR lengthening or shortening, in various physiological states  sites from raw sequences without the problems involved with software installation and configuration are rare. To the best of our knowledge, PlantAPA is the first public web service that provides the biology community with a unique module for the extraction of poly(A) sites from uploaded reads or ESTs. Different options can be tuned to customize the poly(A) site identification process. For example, to investigate the impact of clustering distance for grouping heterogeneous cleavage sites, a user might want to compare poly(A) site detection using two different criteria, e.g., 24 nts vs. 50 nts. Using two separate network sessions, a user can upload the same dataset independently to do comparative processing. Particularly, if the user already has one or more than one list of poly(A) sites, he/she can upload the list(s) to PlantAPA for data visualization and analysis. More importantly, PlantAPA allows users to make a comprehensive analysis of their own poly(A) sites and associated information combined with platform-stored poly(A) sites. With robust functionalities in poly(A) site query, visualization, and analysis, a user can easily appreciate the differences and relationships between their own poly(A) sites and the shared poly(A) sites in PlantAPA. Thus, PlantAPA provides users with an easy-to-use tool that can be customized and fully explored in terms of individual research demands.We believe that PlantAPA will be a valuable addition to researchers engaged in the investigation of the polyadenylation and 3′ UTR, as well as those studying transcriptome and gene expression. Data updates will occur regularly upon the availability of new poly(A) site data. Continuous improvement will involve the retrieval of more sequencing data to obtain poly(A) sites, importation of data from additional organisms, and integration of new functionalities and tools to the web interface. Efforts are also underway to augment existing, related resources to provide the user with additional evidence for the usage and regulatory implications of poly(A) sites.QQL and XW conceived the study. XW developed the back-end pipelines contained in PlantAPA. YZ and XW implemented the web server. XW and QQL wrote the manuscript. All authors read and approved the final manuscript.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.