Carbohydrate metabolism is a key feature of vascular plant architecture, and is of particular importance in large woody species, where lignocellulosic biomass is responsible for bearing the bulk of the stem and crown. Since Carbohydrate Active enZymes (CAZymes) in plants are responsible for the synthesis, modification and degradation of carbohydrate biopolymers, the differences in gene copy number and regulation between woody and herbaceous species have been highlighted previously. There are still many unanswered questions about the role of CAZymes in land plant evolution and the formation of wood, a strong carbohydrate sink.Eucalyptus grandis and Populus trichocarpa, highlighting the major CAZyme classes involved in xylogenesis and lignocellulosic biomass production.Here, twenty-two publically available plant genomes were used to characterize the frequency, diversity and complexity of CAZymes in plants. We find that a conserved suite of CAZymes is a feature of land plant evolution, with similar diversity and complexity regardless of growth habit and form. In addition, we compared the diversity and levels of CAZyme gene expression during wood formation in trees using mRNA-seq data from two distantly related angiosperm tree species Eucalyptus and Populus are indicative of gene balance preservation.CAZyme domain ratio across embryophytes is maintained, and the diversity of CAZyme domains is similar in all land plants, regardless of woody habit. The stoichiometric conservation of gene expression in woody and non-woody tissues of The online version of this article (doi:10.1186/s12864-015-1571-8) contains supplementary material, which is available to authorized users. Carbohydrate metabolism in plants is responsible for a diverse array of developmental processes, including energy metabolism, signaling, defense, cell wall (CW) structure , and carCellulose synthase A (CESA) gene superfamily) [GTs catalyze glycosyl bonds between a donor sugar substrate and another molecule, typically another sugar . Along wrfamily)  and hemirfamily) -21. GH drfamily) . They plrfamily) . PLs arerfamily) ,24. CEs rfamily) ,26. CBMsrfamily) ,28. Due rfamily) .P. trichocarpa than that of A. thaliana [P. trichocarpa, specifically those involved in cellulose and hemicellulose biosynthesis, are more abundant and diverse than those in non-wood forming tissue such as young leaves [Previous studies have found higher frequency and diversity of CAZyme genes in the genome of thaliana , which ithaliana . Furtherg leaves . Based oProtein domains, as the functional and evolutionary building blocks of plant proteins, are informative of the functional capacity of the genome ,32. A reE. grandis, along with mRNAseq data for E. grandis and P. trichocarpa [P. trichocarpa and E. grandis woody and non-woody tissues. In this way, we could identify the common expressed CAZyme repertoires involved in carbohydrate metabolism in wood-forming tissues of two evolutionary divergent tree genera. Specifically, we asked: Does the frequency and diversity of CAZyme domains between plants reflect their evolution and developmental complexity? Is the expression of CAZyme domains related to wood formation in E. grandis and P. trichocarpa? We hypothesized that the genomes of woody trees would exhibit gains or expansions of CAZyme domain containing genes that contribute to carbohydrate biopolymer formation and deposition in wood formation. Further hypotheses were that the expression investment of CAZyme domain-containing genes abundant in the developing xylem would be higher than in non-xylogenic tissue as a reflection of focused carbohydrate metabolism in this sink tissue. This study is the first systematic analysis of genome-wide and expressed CAZyme domains across a diversity of plant species, with a focus on CAZyme utilization for the production of lignocellulosic biomass.Fundamental questions about the evolution of woody plant secondary growth still exist , and witchocarpa , some ofCarica papaya- 1,341 CAZyme domains) have more CAZyme containing genes and CAZyme domains than non-seed plants such as the bryophyte Physcomitrella patens  and the lycophyte Selaginella moellendorffii , and almost double that of the green algal species Volvox carteri and Chlamydomonas reinhardtii   are remarkably similar among species Figure  a, b. A 2 = 0.68), either present in all species, ubiquitous to all land plants considered, or present only in one or two species .Next, we asked what the diversity of CAZyme families within each class is between plant species. Here the objectives were to analyze the CAZyme domain families within each broader functional class present in each genome to determine whether the presence of unique domain families contributed to the organismal complexity of vascular and/or seed producing plants. All twenty-two species from the previous analysis were analyzed to determine the diversity of the domain families present in each species  followed the power law of gene complexity and gene number  are expressed in at least one tissue  contains seven GT41 repeats and had the highest expression of all GT41 containing proteins at >6 million FPKM in the xylem Figure . GT41 haE. grandis . GH9 domain families were also preferentially expressed in the immature xylem compared to the other tissues, the overall higher expression investment in the immature xylem being due to fewer genes (18) being expressed at higher levels than in the other tissues, similar to the GT41 domain containing genes  gene, which is known to be involved in cellulose synthesis [GH domain family expression investment across six tissues in r genes 1 being exynthesis .E. grandis. The expression investment of PL domain families across six tissues in E. grandis showed that all four PL domain families are expressed at diverse levels in all tissues  across all species, including the four expressed in residues .E. grandis and P. trichocarpa, showed the highest expression investment of all the CBM domains expressed in the mature leaf, immature xylem and the shoot tips, while CBM43 has the highest expression investment in the flowers and phloem. CBM43 and CBM57 together contributed 51% of the total average expression investment out of 17 CBM domain families in all tissues. CBM57 was first described in malectin [E. grandis, all of the proteins containing these domains are kinases, including mainly LRR protein kinases. Some of the ones highly expressed in wood include HERK1 and THESEUS (brassinosteroids responsive and required for cell elongation during vegetative growth) [Most CBM domain families did not show preferential expression investment, and were expressed at the same (relative) level in three or more tissues  ,53.E. grandis and P. trichocarpa xylem and leaf tissue  of expression investment in the immature xylem compared to the other five tissues in E. grandis  homolog, Potri.010G141600 , and was more highly expressed in P. trichocarpa xylem than in the leaf, while E. grandis showed the opposite trend  and SCW metabolism employs a standard set of CAZyme domains in different tissue types of different land plant species. Within woody species, the diversity of CAZyme domain families may contribute to wood formation via unique combinations and regulatory mechanisms of ancestral domains within the genomic and transcriptomic context. We have found that combinations of CAZyme domains do not differentiate woody plants from non-woody plants, as the majority of the types of domain combinations in complex proteins are common between lineages, with low promiscuity of domains  [FRAGILE FIBER8) [PARVUS, GALACTURONOSYLTRANSFERASE, and GALACTURONOSYLTRANSFERASE-LIKE (GAUT/GATLI) genes [At4g21060 in A. thaliana has been shown to encode a galactosyltransferase that is responsible for arabinogalactan protein galactosylation during backbone formation [A. thaliana Golgi apparatus, including GT14, GT8 and GT31 domain containing proteins [GT43 family members d IRX14)  and GT47 FIBER8)  are know FIBER8) . GT8 domI) genes ,59. The ormation . GT and proteins , lendingEucalyptus and Populus. This study highlights the importance of transcriptional regulation in the evolution of wood development as opposed to genomic innovations in the enzymatic domains responsible for carbohydrate metabolism.A key finding of this study is that the CAZyme containing genes in plant genomes have a conserved ratio between species, regardless of their carbohydrate metabolic strategy or the tandem, segmental or documented whole genome duplication events in their evolutionary history. Although we find evidence for lineage specific diversity of CAZyme families in plant genomes, the domain family diversity of CAZymes cannot be used to discriminate the eudicot and monocot lineages, or woody and herbaceous species. The expression pattern of the CAZyme domains responsible for cellulose and xylan biosynthesis appears to be stoichiometrically conserved between C. reinhardtii and V. carteri), Embryophyta, encompassing P. patens onwards, Tracheophyta, encompassing S. moellendorffii onwards and monocot and dicot representatives of the Magniliophyta.All CAZyme domains for twenty-two plant species Table  in Phytowithin annotated genes. Covariance analysis to determine within and between species CAZyme domain class relative frequency variation was done using SAS v9.3 .Analysis was performed using custom Python scripts  into their families based on dbCAN annotations. Complexity analysis was performed on a subset of ten species representing major lineages of land plant evolution  . FPKM pa


Fasciola hepatica is a helminth parasite of global importance in livestock, with major economic impact. However information on F. hepatica infections in Irish pasture-based dairy herds is limited. Therefore this study was conducted in order to determine the prevalence, seasonality and management factors associated with F. hepatica. A total of 319 Irish dairy herds were selected for this study. Bulk tank milk (BTM) samples were collected from 290 dairy farms on a quarter year basis, while from a further 29 dairy farms BTM samples were collected on a monthly basis to provide a more detailed pattern of F. hepatica exposure in Irish herds. BTM samples were analysed using a commercially available F. hepatica antibody detection ELISA. Furthermore, within-herd prevalence of F. hepatica was assessed in a subset of these 29 herds (n = 17); both individual serum samples and bulk tank milk samples were collected.F. hepatica shows elevated antibodies as the grazing season progressed, reaching a peak in January. A significant association was found between F. hepatica and age at first calving.A within-herd prevalence of ≤ 50 % was found for herds with negative bulk tank milk samples. The mean prevalence of the 290 study herds was 75.4 % (Range 52 %–75.1 %), with the highest prevalence being observed in November (75.1 %). The seasonal pattern of F. hepatica is present in a large proportion of Irish dairy herds and provides a basis on which control practices, particularly in adult dairy cows, can be reviewed.This study demonstrates that  Fasciola hepatica (liver fluke) is an important helminth parasite in livestock worldwide. The lifecycle of F. hepatica, similar to other trematodes, involves both a final  and an intermediate snail  host. F. hepatica outbreaks have a seasonal pattern with two waves of infection in summer and winter  and specificity [Sp] of 98 % . All tests were carried out “in-house” according to kit manufacturer’s instructions and previously described in detail by Selemetas et al. [F. hepatica cathepsin L1 antigen (CL1) [Pichia pastoris. Plates are coated with 0.01 mg/ml antigen in carbonate coating buffer in alternate columns on a 96-well ELISA plate  leaving each alternate strip uncoated. Samples were done in duplicate. Positive and negative bovine serum controls were supplied with the kit and used for S/P determination. The optical density (OD) reading of the uncoated well was subtracted from the reading of the coated well to yield a corrected OD. The ratio of the sample OD to the positive control OD was subsequently calculated to yield the S/P ratio. Likewise antibodies in serum were analysed using the same F. hepatica ELISA kit. A 1:20 ratio of bovine sera to sample buffer was used, 190 μl of sample buffer was mixed with 10 μl of control serum in each well.s et al. . The assen (CL1) , which iA positive cut-off of 15 and 20 S/P was used for BTM and serum samples respectively.A questionnaire was used to collect management data from ‘HerdAhead’ farms and has previously been described by Bloemhoff et al., . BrieflyStudy farms were allocated to three regions, described as Region-1 , Region-2  and Region-3  and outlined in Fig. T-Test using Excel (Version MS Office 2003). Univariable and multivariable generalized estimating equation (GEE) analysis was completed using PROC GENMOD .Descriptive analysis was performed using PROC FREQ and PROC UNIVARATE . Graphical representations were generated and Student F. hepatica  for each herd was calculated using Rogan Gladen Epitools [T-test using a one tailed distribution with two sample unequal variance, was used to compare the proportion of F. hepatica positive ‘HerdAhead’ herds in each Region  at each sampling date  of ‘HerdAhead’ herds was calculated for each sampling date for ca Table . The oveEpitools . The sean -1,-2,- at each F. hepatica bulk tank milk result and independent variables, with herd included as a repeated measure. A binomial distribution was assumed and a logit link function used. An exchangeable correlation was applied for the analysis. Independent variables significant at a p < 0.10 in the univariable analysis were included in the multivariable model. A manual backward regression with a forward step was performed to build the final model. Two-way interactions deemed biologically significant were included in the analysis and retained in the final model at a significance level of p < 0.05.As a first step a univariable analysis was performed using binomial dependent variable The herds are a national representative sample spread across three regions  Fig. . Due to All of the study herds had full access to pasture throughout most of the year . The mean herd size of study herds was 97.5 cows (range 28 to 400 cows). February  was the month when the majority of farmers turned out their adult cows, while housing was mostly performed in November . Close to 50 % of the study herds had a livestock enterprise of dairy only. An average of 24.5 first lactation heifers, were added to the adult herds (range 3–150) with an age of approximately 24.9 months (range 22–36 months).F. hepatica of the ‘Dairy17’ herds was calculated as the number of individual cows positive at the >20 S/P value as a percentage of all cows serum sampled. Both Figs. F. hepatica. The three herds that had a negative BTM result had less than half of the cows’ serum samples positive to F. hepatica. In addition these negative BTM herds had considerably lower mean S/P values (S/P = 21), compared to positive BTM herds (S/P = 73). There was a good correlation between BTM S/P results and the individual blood S/P results in the subset of BTM positive herds  and June (OR = 1.46).Results on prevalence from the ‘HerdAhead herds n = 277) are shown in Table 77 are shF. hepatica for each farm the percentage of herds recording BTM values positive (P= > 15 S/P) and negative (n = <15 S/P) were tabulated. For example, a farm recording a negative result in March and June and positive results in August and November, this would be described as ‘nnPP’. The positive/negative time course for ‘HerdAhead’ herds is shown in Table F. hepatica. In general study herds were positive to F. hepatica towards the end of the grazing season in November. Approximately 8 % of study farms had three positive results out of four sampling dates, of which all had a positive result in November. Approximately 8 % of the herds had two positive results, of which 97 % were positive in November. A total of 34 farms had one positive result in November, whereas 19 farms had two positive results namely in August and November.To investigate the seasonality of ve P= > 1 S/P and F. hepatica infections in ‘DairyMIS’ herds was also calculated. A reduced number of herds were sampled in December, January and February, as cows were in the dry period. The majority of ‘DairyMIS’ herds were spring-calving herds , and dry-off their dairy cows in November or December. The monthly seasonality of F. hepatica infection is shown in Fig. Monthly bulk milk samples from 2009 of the ‘DairyMIS’ herds (n = 29) were used to evaluate the seasonality. Monthly bulk milk sample S/P values were plotted for each herd, with the mean S/P value of all 29 farms also calculated and plotted Fig. . The seaF. hepatica compared to herds with heifers calving <24 months (p = 0.0269). No statistical regional differences were found in the multivariable analysis, however region was found to be significant in the univariable analysis. Table F. hepatica in Region–1 is higher than Regions–2 and–3. However the Student T-test did not find a significant difference between Regions–1 vs.-2 (p = 0.075) and–2 vs.-3 (p = 0.393), although there was a significant difference between Region 1 vs. 3 (p = 0.0296).The results of the multivariable analyses are shown in Table F. hepatica in Irish dairy herds using a BTM F. hepatica ELISA. This study revealed that a large proportion (75 %) of the ‘HerdAhead’ study herds were positive to F. hepatica across the 2009 lactation. This is slightly higher than previously reported in an Irish abattoir study, which found that 65 % of the livers of culled dairy and beef cows were infected, but is consistent with a recent study carried out by Selemetas et al. [F. hepatica prevalence is considerably lower [F. hepatica was found in Belgium, while in Germany a prevalence of 23.6 % was found in adult cows [F. hepatica (76 %) based on BTM samples was similar to that found in this study [F. hepatica and its intermediate host Galba truncatula [F. hepatica as pasture infectivity is the highest during the autumn [F. hepatica prevalence [F. hepatica [F. hepatica as a problem in their area.This study was used to determine the prevalence of s et al. , which uly lower , 22. In ly lower , 22. Morult cows . Howeveris study , which cuncatula . Moreoveuncatula . These mF. hepatica infection with an anthelmintic can be achieved [F. hepatica and therefore a subsequent treatment is required. To adhere to withdrawal periods the main time for flukicide treatments is during the dry period for spring-calving herds [F. hepatica infections. Therefore treatment with flukicides in these herds might not be optimal. This study highlights the importance of implementing adequate F. hepatica control strategies across the island of Ireland. In addition, approximately 50 % of the herds had four positive results to F. hepatica, suggesting that significant production losses due to F. hepatica in about half of Irish dairy herds are likely, further emphasizing the need to implement adequate control-strategies in Irish dairy herds. Losses due to F. hepatica have been estimated to be around €299 per infected cow, due to reduced milk yield, compromised reproductive performance, liver condemnations and reduced meat production [F. hepatica infections can reduce the reproductive performance and life-time milk production of heifers. In-calf heifers calving around 2 years of age have a considerable advantage over heifers that calve later, as they are more likely to produce more milk and calves over their productive lifetime [F. hepatica, compared to heifers that calved at 24 months and younger. Although reproductive performance in high-input high-output systems is a very important factor, it is of more importance in seasonal calving systems. This is to maintain a compact calving pattern. Ideally breeding of the majority of the cows in such systems should be achieved within a 6 week breeding period in May and June [F. hepatica infections during the breeding period. In this study, over half of the dairy herds were positive to F. hepatica during the crucial breeding month of June [F. hepatica infections in heifers and dairy cows.The seasonal pattern found in this study shows a rise in September towards the end of the grazing season, which has previously been described in literature , 28. Theachieved . Howeverachieved . The limachieved . In adding herds , which ioduction , 10, 29.lifetime . In thisand June . While m of June . TherefoF. hepatica antibodies. In addition there was a good correlation between the negative BTM results and the within-herd prevalence of F. hepatica positive animals in these herds. Therefore, the F. hepatica ELISA is a useful and easy to use tool for farmers and veterinarians.The ELISA clearly showed seasonal changes in bulk-milk F. hepatica in Irish dairy herds at the end of the grazing season, but also a considerable amount during the grazing season. There is a need for appropriate control measures in adult dairy cows, especially during the lactation. Additionally the Ildana ELISA showed clearly the seasonal changes of F. hepatica and is therefore a useful and easy to use diagnostic tool. This study shows a high prevalence of


Basement membrane (BM) extracellular matrices are crucial for the coordination of different tissue layers. A matrix adhesion receptor that is important for BM function and stability in many mammalian tissues is the dystroglycan (DG) complex. This comprises the non-covalently-associated extracellular α-DG, that interacts with laminin in the BM, and the transmembrane β-DG, that interacts principally with dystrophin to connect to the actin cytoskeleton. Mutations in dystrophin, DG, or several enzymes that glycosylate α-DG underlie severe forms of human muscular dystrophy. Nonwithstanding the pathophysiological importance of the DG complex and its fundamental interest as a non-integrin system of cell-ECM adhesion, the evolution of DG and its interacting proteins is not understood. We analysed the phylogenetic distribution of DG, its proximal binding partners and key processing enzymes in extant metazoan and relevant outgroups. We identify that DG originated after the divergence of ctenophores from porifera and eumetazoa. The C-terminal half of the DG core protein is highly-conserved, yet the N-terminal region, that includes the laminin-binding region, has undergone major lineage-specific divergences. Phylogenetic analysis based on the C-terminal IG2_MAT_NU region identified three distinct clades corresponding to deuterostomes, arthropods, and mollusks/early-diverging metazoans. Whereas the glycosyltransferases that modify α-DG are also present in choanoflagellates, the DG-binding proteins dystrophin and laminin originated at the base of the metazoa, and DG-associated sarcoglycan is restricted to cnidarians and bilaterians. These findings implicate extensive functional diversification of DG within invertebrate lineages and identify the laminin-DG-dystrophin axis as a conserved adhesion system that evolved subsequent to integrin-ECM adhesion, likely to enhance the functional complexity of cell-BM interactions in early metazoans. Basement membranes are a specialised and structurally distinct form of metazoan extracellular matrix (ECM). They are composed of a plethora of interacting glycoproteins, predominantly laminin, collagen IV, perlecan and nidogen, that are secreted from surrounding cellular layers and assembled into dense, thin, proteinaceous sheets by cell-ECM and extracellular interactions . Basemen(LARGE), which adds a repeating disaccharide unit [-α3-GlcA-β3-Xyl-] to a mucin-like, central region of α-DG. These carbohydrate decorations of α-DG are important for the calcium-dependent, high-affinity binding of α-DG to the LG4 and LG5 domains of laminin α-subunits ; S6 domain   A (Table stomata) . It is wstomata) . We previced out . In the aponicum .Table 1Cionaintestinalis and Ciona savignyi are surprisingly weakly related to human DG  DGs both IG domains as well as the S6 domain are conserved and the domain organization is equivalent to that of vertebrate DGs. The DG of the hemichordate Saccoglossuskowalevskii is well conserved with vertebrate DGs, yet includes clear repetition of the IG2_MAT_NU module . These data imply that duplication of the IG2_MAT_NU module must have taken place very early in the arthropod lineage. Apart from the IG2_MAT_NU module, the most highly conserved region in the arthropod DGs is the cytoplasmic domain. A striking distinction of the DGs of diptera is the absence of the IG1 domain . Interestingly, the characterized alternatively-spliced isoforms of D. melanogaster DG also include an isoform that lacks a central mucin-like region .In many cases, the DGs of Arthropoda are longer (comprising 1000 to 1200 residues) than the DGs of vertebrates (∼900 residues) . This isaster DG . We idene region . AlthougC. elegans and Caenorhabditis remanei, lack a central mucin-like region and thus these are the shortest (<600 aa) DG sequences identified . Phylogenetic analysis revealed that the sixth, most C-terminal module is distinct in being the most closely related to human IG2_MAT_NU, whereas each of the other modules are more closely related to each other than to the sixth repeated region .DG-like proteins were identified in multiple classes of spongescarmela . The mosd 500 aa . By use mplicata . AlthougAs introduced above, the domain architecture of αDG includes the N-terminal IG1 and S6 domains, followed by the central mucin-like region and the IG2 domain; the latter anticipates the α/β maturation interface A. A numbC. elegans α-DG, the topological equivalent of Thr192, as well as the entire first β-strand of the S6 domain are missing (C. savignyi (Chordata), Amblyomma triste  or T. adhaerens (Placozoa) α-DGs is occupied by Ala, Val or Asn, respectively. These residues that are less bulky than Met and so possibly are more compatible with the structure of the domain.In human α-DG, Thr192 lies within the first β-strand of the S6 domain which has a basket-like structure . This β- missing B. IntereStrongylocentrotus purpuratus and some mollucs (H. magnipapillata the consensus is absent (N. vectensis (Acroporadigitifera (not shown) contain the consensus motif. The furin site is not present in the DGs of C. elegans (Nematoda), T. adhaerens (Placozoa) or the insect Camponotusfloridanus . The following Thr residues that correspond to Thr317 and Thr319 in human DG (asterisks in Lottiagigantea (Molluscs), A. triste (Arachnida), C. elegans (Nematoda), Capitellateleta (Annelida), S. purpuratus (Echinodermata), C. floridanus (Insecta), T. adhaerens (Placozoa), H. magnipapillata (Cnidaria), C. savignyi (Urochordata) or O. carmela (Porifera), , C, indicaO. carmela DG-like protein  of the IG2 domain  are show protein J the DAF protein D. The “s protein A,D.β-DG is liberated upon proteolysis at the α/β cleavage site. It represents the transmembrane subunit of the DG complex and includes the natively unfolded region  in its eO. carmela DG-like protein , urochordates, several classes of arthropods, and N. vectensis β-DG contains a GP motif . The GS motif is commonly preceded by a basic residue and followed by a triplet of hydrophobic residues; both are likely to be important for the cleavage event . The latter are now considered to be the earliest-diverging lineage of metazoans .The glycosyltransferases POMK, B4GAT1 and LARGE1 are present in choanoflagellates, etazoans . The intA. queenslandica, the placazoan T. adhaerens and cnidarians . As previously reported, no secreted laminin proteins were identified in the choanoflagellates or C. owczarzaki . These domains are present in DGs from all phyla in which DGs were identified. Since these domains include the β-DG binding site, the α/β processing site and the transmembrane domain, the region is likely under strong, uniform selection for correct retention and presentation of DG at the plasma membrane. Direct functional evidence for the importance of the cytoplasmic domain of DG in non-muscle tissues include the establishment of oocyte polarity in nogaster  and visuc domain . Of the c domain  and 4. Ic domain . Becausec domain , clearlyc domain B, it is In contrast, the N-terminal region, comprising the IG1 domain, S6 domain, and mucin-like region shows much greater variability, including extreme sequence divergence of the IG1 or S6 domains, great variability in the length of the mucin-like region, or complete loss of the S6 or mucin-like regions . This vaBrancaccio et al., 1995The variability of the mucin-like region is of particular interest in view of the central role of this region in laminin-binding and also with regard to the general role of mucins that can sterically affect integrin clustering . WhetherS. kowalewskii, all Arthropoda and T. adhaerens (Placozoa), and a six-fold repetition in N. vectensis DG. We speculate that these enlargements of the DG molecule may have conferred evolutionary advantages by imposing a more rigid structure of the DG extracellular region and the presentation of the N-terminal domain and laminin-binding region at a greater distance from the cell-surface. A curious related observation is the extreme sequence divergence of the Ciona DGs from all other DGs, even within the IG2-MAT-NU region. Uniquely, in the DGs of urochordates, a repeated IG-MAT-NU region is present as an insertion within the mucin-like region. Because the LARGE modification site is missing (Ciona laminin subunits and dystrophin have around 40% sequence identity to the human orthologues (BLASTP search results). Thus, the extreme sequence divergence of urochordate DGs is not typical for other proteins within the DGC. Neither sarcospan nor sarcoglycan are encoded in the currently available urochodate genomes. It is possible that DG in modern urochordates may have evolved a distinct function that does not depend on the DGC.We identified that duplication events involving the IG2_MAT_NU module have occurred in multiple phyla. Tandem duplication of the IG2_MAT_NU module was observed in  missing C it is uPhylogenetic trees constructed on the basis of the highly-conserved IG2_MAT-NU region returned a consistent tree topology, with division of the DGs of arthropods, deuterostomes, and other invertebrates into three separate clades. Nevertheless, within the deuterostome clade, it is notable that the DGs of invertebrate deuterostomes branch separately and are clearly distinct from the vertebrate DGs. We speculate that DGs may have functional distinctions in these lineages. The arthropod clade was strongly supported, with the caveat that the taxon representation of non-arthropod protostomes and early-diverging metazoans is more patchy than for the arthropods. The clade comprising molluscs and early-diverging metazoans was the least stable under different methods of phylogenetic analysis and contained relatively divergent sequences. In view of the moderate or low level of overall sequence identity between these DGs and others e.g.  and the In view of the many examples of divergence of DG domain organisation that we identified in invertebrate DGs, the consistency of domain architecture amongst vertebrates becomes a point of note. We suggest that there have been several contributory factors: first, in vertebrates, the major function of DG as a component of the DGC may have resulted in a strong selection pressure to conserve the domain architecture and post-translational modifications. Indeed, relatively few disease-causing mutations of DG have been identified and these are all point mutations rather than domain deletions. Secondly, vertebrate DG genes have an unusual gene organization in which all of the protein except for the N-terminus is encoded in a single very large exon . This macis-acting, transmembrane partners of DG and subsequent lineage-specific divergence of DGs in certain phyla  (http://www.metazome.net); the Mnemiopsis Genome Project Portal (http://research.nhgri.nih.gov/mnemiopsis/) (Pleurobrachia Genome (http://neurobase.rc.ufl.edu/pleurobrachia) (http://compagen.zoologie.uni-kiel.de/index.html)  and the stony coral Acropora digitifera (Cnidaria); the JGI Trichoplax Genome Page (http://genome.jgi-psf.org/Triad1/Triad1.home.html) (Ciona savignyi CSAV 2.0 resources at ENSEMBL (www.ensembl.org/Ciona_savignyi/Info/Index), and the Broad Institute Initiative on Multicellularity  by TBLASTX searches of dbest or the transcriptomic resources at NCBI or the above-mentioned genome databases. From the many DG sequences identified, a curated dataset of 58 sequences from species representative of the major phyla and classes was used for further phylogenetic analyses. DG sequences from parasitic animals: Schistosoma mansoni (Platyhelminthes), Trichinella spiralis and Loa loa (Nematodes), showed extreme sequence divergence and were excluded from this set. The curated dataset of DG sequences is listed in The human DG protein sequence (GI:229462879) was used in systematic BLASTP or TBLASTX searches of NCBI GenBank according to entrez-specified terms for each of the major phyla of metazoans, or species of choanoflagellates and filasterians. Databases of specific genome projects were also searched, including the Japanese Lamprey Genome Project (edu.sg/) ; the datiopsis/) ; the Plebrachia) ; the plaex.html)  for Oscame.html) ; Ciona slularity . SequencC. owczarzaki. The dataset is listed in supplementary material Table S1.A similar approach, with the respective human protein sequences as the search queries, was taken to identify dystrophin (P11532), laminin-α2 (P24043), laminin-β1 (AAI13456), laminin-γ1 (NP_002284), sarcospan (Q14714), sarcoglycan-alpha (Q16586) and relevant glycosyltransferases  homologues in early-diverging metazoan species, choanoflagellates and the filasterean, http://www.ebi.ac.uk/Tools/pfa/iprscan5/ . Illustrations of multiple sequence alignments are presented in BoxShade 3.21 (http://www.ch.embnet.org/software/BOX_form.html). Phylogenetic trees were prepared from PRANK alignments of the IG2_MAT-NU region (245 positions) from DGs from 46 species representative of all phyla except urochordates and annelids. Trees were prepared in PhyML 3.0 (http://itol.embl.de/)  .


Questionnaire data on management were collected, and faecal samples from 252 kids were analysed by the McMaster technique. From all herds with a mean faecal egg count (FEC) above 300 eggs per g of faeces, pooled samples were stained with peanut agglutinin (PNA) for specific detection of Haemonchus contortus. Strongyle eggs were detected with an individual prevalence of 69%, including Nematodirus battus (3.6%) and other Nematodirus species (15.0%). Eimeria spp. were observed in 99.6% of the kids. H. contortus was found in 11 of 12 (92%) tested herds. Anthelmintics were used in 89% of the herds with mean treatment frequencies of 0.96 and 0.89 treatments per year for kids and adults, respectively. In 2011, new animals were introduced into 44% of the herds of which 25% practised quarantine anthelmintic treatments. In 10 herds the presence of AR was analysed by egg hatch assay and FEC reduction tests using ivermectin (0.3 mg/kg) or fenbendazole (10.0 mg/kg). AR against both fenbendazole and ivermectin was detected in seven herds; AR against fenbendazole in one herd, and AR against ivermectin in another herd. In conclusion, resistance to the most commonly used anthelmintics is widespread in larger goat herds throughout Denmark.The prevalence of gastrointestinal parasites in Danish goats and the presence of anthelmintic resistance (AR) in 10 selected herds were investigated during April–September 2012. All Danish herds ( Helminth infections, in particular gastrointestinal nematodes, are found worldwide and are among the most economically important diseases in goats , 35, 37.Haemonchus contortus is a species that has repeatedly been associated with AR [AR is believed to develop faster in goats than in sheep , 47, 51, with AR , 40, 41. with AR , 33, 43  with AR .H. contortus, and to assess the occurrence of AR in selected Danish goat herds.The objectives of the present study were to determine the prevalence of gastrointestinal parasites in Danish goats with a special focus on n = 137 herds). In addition, the invitation was posted on the Danish Goat Association’s website (www.goat-dgu.dk). Twenty-seven (19.7%) herds met the inclusion criterion of more than 10 young kids never treated with anthelmintics. Questionnaire data on herd management and practices were collected from all owners (n = 27), who also submitted rectally obtained faecal samples from at least 10 kids for initial screening. In a subset of herds (n = 10) with a minimum of seven kids and a mean faecal egg count (FEC) > 150 eggs per g faeces (epg), the presence of AR against ivermectin (IVM) and fenbendazole (FBZ) was analysed using a faecal egg count reduction test (FECRT) and egg hatch assay (EHA). A single herd (#18) was examined earlier in 2012 [In April 2012 an invitation to participate in the prevalence study was submitted to all Danish goat herds registered with 10 or more adult goats, according to the Central Animal Husbandry Register , vacuum-packed and stored in the dark at room temperature until analysis within one-three days. Helminth eggs and oocysts were quantified using a modified McMaster method  with a sH. contortus eggs [Peanut Agglutinin Staining (PNA) was performed to detect tus eggs . From alA FECRT was performed according to the World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines . On eachAn EHA for detection of BZ resistance was performed as described by Coles et al.  with modAll 27 participating farmers were asked to fill in a questionnaire concerning farm details (e.g. size and composition of goat flock), management practices , drug use  and knowledge about AR. The questionnaire was either answered by e-mail or the farmers were interviewed by telephone or face-to-face.x + 1)-transformed counts. The FEC reduction percentage (FECR) was calculated using three different methods:1 and T2 are the arithmetic means of FECs of the treatment group before and after treatment, and C1 and C2 are the arithmetic means of FECs of the control group at the same time points. According to this method resistance is indicated when FECR < 90% [Data from the prevalence study, FECRT and questionnaire were summarised by descriptive statistics. FECs of different groups were compared by means of analysis of variance on log(CR < 90% .(2)FECR=2 and C2 are the arithmetic means of FECs of the treatment group and the control group after treatment. When using this method of calculation, AR is present if the FECR < 95% and the lower confidence limit is <90%. If only one of these criteria is present, AR is suspected [1 and T2 are the arithmetic mean FECs of the treatment group before and after treatment, respectively. This method uses a threshold value of FECR < 95% to detect AR [A 95 % confidence interval was calculated:etect AR .In the present study, AR was considered present if the FECR was below the threshold value in one or more of the above-mentioned methods.50), the 95% confidence interval, and the coefficient of determination (R2). According to Coles et al. [50 value is ≥0.1 μg/mL. Resistance in only a minor nematode species may be declared when EC50 < 0.1 μg TBZ/mL and concurrent hatching of larvae is seen in 0.3 μg TBZ/mL [In the EHA, the hatching percentage was calculated for each well (larvae/(eggs + larvae) × 100). The data was analysed in GraphPad Prism Version 5.1  to determine the concentration required to inhibit hatching of 50% of strongyle eggs  before June. There were incidental findings of lungworm larvae (species not identified). A large variation was seen in the FECs, mainly due to the 10% samples in late season , agreement as regards IVM resistance was observed between the results of the different methods. Resistance against FBZ was present in all three farms tested , by one or more methods. In farm 2, resistance was only present according to Coles et al. [p < 0.05).In six herds no controls were included in the FECRT due to low numbers of kids, and thus only the McKenna method  for FECRs et al. , but the50 ranged from 0.069 to 9.803 × 106 μg TBZ/mL, and the coefficients of determination (R2) were all above 0.85. The hatching percentage in the negative controls was above 80% in all herds, except herd #18, in which the hatching percentage ranged between 41 and 51%. In herds #3 and #25, EC50 was below 0.1 μg TBZ/mL, but hatching of larvae was seen at a concentration of 0.3 μg/mL. When summarising the results from the FECRT and the EHA, seven of the ten herds had AR against both IVM and BZ in their nematode populations.By this method resistance to BZ was detected in all herds except #3 and #25 . The thrQuestionnaire data were obtained from 27 of the 137 herds which were invited to participate in the study. Herd size ranged from 4 to 500 adult goats, with a mean of 55 goats and a median of 17 goats. Of the 27 herds included in the prevalence study of gastrointestinal nematodes and AR, 15 were hobby herds, of which two were registered as organic and four were zoological gardens. Among the 12 professional herds, 7 were organic, producing either milk, meat or both, and 5 were conventional, producing meat, milk or mohair fibres. Fifty-five percent of the herds had Boer goats, being the dominant breed, followed by “mixed breed” (44%).The three largest participating herds had a combination of milk-producing breeds . A total of 1129 kids were born in the kidding period from January to July with most kiddings during March–April in 19 of the herds (70%).The average size of the professional herds in the study was 95 adult goats, whereas the hobby farms had an average size of 23 adult goats. No major differences regarding management were found between hobby and professional goat herds. However, all of the professional herds used pasture rotation, whereas this was only the case in 41% of the hobby farms. In addition, the professional herds treated more intensively with anthelmintics: 82% of the professional herds used whole-flock treatments compared with 50% of the hobby farms; and while hobby farmers treated 0.8 times per year in kids as well as adults, the professional farmers treated 1.2 and 1.0 times per year in kids and adults, respectively.The mean number of treatments for kids and adult goats, respectively, was 0.96 and 0.89 treatments per year. Treatment frequencies are summarised in Of the 24 herds that utilised anthelmintics, 15 (63%) used macrocyclic lactones at their last treatment, 4 (17%) used benzimidazoles, 2 (8%) used other drugs and 3 (13%) did not know which drug they had used. In addition, 15 (63%) herds had used the same drug repeatedly. As regards anthelmintic efficacy, only one farmer (4%) had noticed insufficient anthelmintic efficacy, 6 (25%) farmers did not know if the anthelmintic had any effect, and 17 (71%) farmers believed the anthelmintic treatment had the desired effect. All except one of the 27 herds in the study claimed to have a good understanding of AR.Data concerning deworming of new animals prior to introduction into the flocks were obtained from 24 herds. Of these 12 (50% corresponding to 44% of the entire study population) introduced new animals in 2011; and 3 herds (25%) treated with anthelmintics before the introduction. Among the 12 herds which did not introduce new animals in 2011, 7 stated that they would use quarantine anthelmintic treatment prior to introduction of new goats.As regards grazing practices, the goats were grazing in all herds except three zoological gardens where the animals only had access to dirt paddocks. Kids and adult goats were grazing together in 22 herds, while they grazed separately in one herd and the kids were kept indoors in the remaining herd. Of the 23 herds with grazing kids, pasture rotation was used in 16 (70%), and 12 (52%) had a specific grazing strategy including: rotating untreated goats to clean pasture (30%), “dose and move” (13%), co-grazing with other animal species (9%), strip grazing (4%) or “other strategies” (17%).n = 252), and practically all kids  were infected with Eimeria spp.The prevalences of gastrointestinal parasites found in the present study are similar to previous studies from Norway and Poland , 20, witn = 12). Had the samples been collected later the FECs would probably have been substantially higher, since egg excretion generally rises during the grazing season [H. contortus was found in 11 of the 12 tested herds (92%). Since only samples with high FECs were stained, the PNA results are subject to selection bias, and the detected herd prevalences may be falsely high. However, recent studies [H. contortus primarily over-winters as arrested larvae within the host [Due to generally low FECs PNA was performed in less than half of the participating herds  most likely is a better estimate of the actual prevalence in the study population. Regardless of this, it is concluded that H. contortus is a widespread and well-established nematode species in Danish goats.Considering the prepatent period of around 2.5–3 weeks , it is asampling , and wouN. battus was only identified in 4 herds and 3.6% of all individual samples and diarrhoea was not reported by the owners, suggesting that N. battus overall is not a major pathogen in Danish goats. Since the majority of samples were collected during the peak period for N. battus egg excretion in Northern Europe [n Europe , this prin vitro assays [Resistance against one or more anthelmintics was detected in 9 out of 10 herds (90%). In the FECRT, resistance against BZ was present in all of the three herds tested, and against IVM in eight herds (80%). Based on the EHA, resistance against BZ was found in 8 of the 10 herds (80%), and thus, dual resistance against both BZ and IVM was present in 7 herds (70%). A similar occurrence of AR was seen in Danish goat farms in 1996, where the efficacy of BZ, IVM and levamisol was tested using the FECRT and o assays . At thatNo anthelmintics are approved for use in goats in Denmark. Thus, IVM and FBZ were applied at 1½ and 2 times the dose levels recommended for sheep, which is common practice , 19, 29.2 was above 0.85 in all tests, indicating a good test performance [In the present study the various methods used for calculation of FECR generally showed good agreement, and as expected, the largest differences were recorded in farms where there was a large increase or decrease in the FEC in the control group. A statistically significant difference in the FEC was recorded in control groups from herds #2 and #22. Most importantly, the methods agreed on the discrimination of resistance and susceptibility in all cases but one (#2). Regarding the EHA performed in this study, the hatching percentage in negative controls was high (>80%), and Rformance . BZ effiformance , 13, 32.H. contortus) to substantially lower in others, resulting in a higher or lower reduction percentage depending on species composition and resistance status. Pre and posttreatment larval cultures enable calculation of the specific FECR for each nematode species, thereby clarifying each species’ susceptibility level [Mixed infections with both susceptible and resistant species may complicate interpretation of FECRT results, especially in cases with the FECR around the threshold level , 39. Egg50. This may explain the wide confidence intervals seen in the present study. Furthermore, the two herds  with EC50 below 0.1 μg TBZ/mL in the EHA had larval hatching at the discriminating dose (0.3 μg TBZ/mL). This indicates that resistant nematode strains were present in all of the ten samples, despite the calculated EC50 suggesting otherwise [50 to 0.05 μg TBZ/mL if H. contortus is present in the sample [H. contortus eggs were detected in samples from 9 of 10 herds tested in the EHA a decreased EC50 threshold may have been more appropriate, and would have supported the detection of BZ-resistant strains.Similarly, mixed infections can complicate interpretation of EHA results since varying susceptibility between species could result in a range of different ECtherwise . It has e sample . Thus, aGoats are mainly held as large animal pets in Denmark, and 89% of Danish goat herds have less than 10 animals . In the Anthelmintic drugs were used in 89% of the herds, and 75% of these had a predetermined treatment plan. Examples of such plans were treatments at turn-out, kidding or housing. Treatment at signs of disease was the most common practice, and was used in 29% of the herds, not necessarily as a deliberate strategy but in several cases as an emergency solution. If selected individual animals are treated, e.g. at signs of disease, this is defined as a targeted selective treatment (TST) . TST is The mean number of treatments per year was 0.96 and 0.89 for kids and adult goats, respectively. In only 26% of the herds, kids and adults were treated with anthelmintics ≥ 2 times per year, and the maximum frequency was 3 treatments per year. This is markedly lower than observed in 1996 by Maingi et al. , who fouNew animals were introduced into approximately 50% of the participating herds in 2011; and of these only 25% treated new animals with anthelmintics prior to introduction into the flocks. The lack of anthelmintic treatment and quarantine measures for goats that are moved between herds or imported from other countries as breeding animals facilitates the spread of nematode species, and is an important factor for dissemination of AR , 50, 54.H. contortus in a large proportion of the tested goat herds; and widespread AR against the most commonly used anthelmintics at the dose levels recommended for goats, and management practices that are known to increase the risk of AR development. Efficiency of other anthelmintic classes, which were not tested in the present study, may still be sufficient against gastrointestinal nematodes in Danish goats. Nevertheless, there are several reasons for concern, and efforts to monitor and control further development of AR are warranted.In conclusion, the present study demonstrated


The Golgi complex is the central sorting compartment of eukaryotic cells. Arf guanine nucleotide exchange factors (Arf-GEFs) regulate virtually all traffic through the Golgi by activating Arf GTPase trafficking pathways. The Golgi Arf-GEFs contain multiple autoregulatory domains, but the precise mechanisms underlying their function remain largely undefined. We report a crystal structure revealing that the N-terminal DCB and HUS regulatory domains of the Arf-GEF Sec7 form a single structural unit. We demonstrate that the established role of the N-terminal region in dimerization is not conserved; instead, a C-terminal autoinhibitory domain is responsible for dimerization of Sec7. We find that the DCB/HUS domain amplifies the ability of Sec7 to activate Arf1 on the membrane surface by facilitating membrane insertion of the Arf1 amphipathic helix. This enhancing function of the Sec7 N-terminal domains is consistent with the high rate of Arf1-dependent trafficking to the plasma membrane necessary for maximal cell growth.DOI:http://dx.doi.org/10.7554/eLife.12411.001 The cells of plants, animals and other eukaryotes are subdivided into different membrane-bound compartments. One of these compartments – called the Golgi complex – has been likened to the 'Grand Central Station' of the eukaryotic cell, because it serves as the cell’s centralized sorting compartment. Small, spherical structures called vesicles arrive at the Golgi complex from other cellular compartments, and the material within these vesicles is then sorted, packaged into new vesicles, and sent out to different destinations.Regulatory proteins are responsible for making decisions about when to turn on different incoming and outgoing pathways to or from the Golgi complex. In particular, one regulatory protein named Sec7 controls many of the outgoing vesicles that leave the Golgi complex. Sec7 is a fairly large protein and has different parts, or domains, that regulate how the protein works. It was known that two of these regulatory domains (named 'DCB' and 'HUS') were required for Sec7 to work, but it was not known what these domains actually did.Richardson et al. have now used a technique called X-ray crystallography to reveal the three-dimensional structure of the regulatory DCB and HUS domains of Sec7 from a species of yeast. The Sec7 protein has been conserved throughout evolution with few changes, and so the structure of this yeast protein is expected to be the same as that of the corresponding protein in humans. Unexpectedly, Richardson et al. discovered that DCB and HUS are not structurally separate domains and actually form a single 'DCB/HUS' domain.Biochemical experiments then showed that the DCB/HUS domain helps Sec7 work on the surface of membranes. One of the jobs of Sec7 is to insert another regulatory protein  into the membranes of the Golgi complex, and these new findings reveal that the DCB/HUS domain helps Sec7 overcome the challenges associated with this task.Researchers have now uncovered structural information for approximately half of the Sec7 protein, and so an important future challenge will be to reveal the structure of the remaining regulatory domains of Sec7. This achievement will help researchers to figure out how all of the parts of Sec7 work together to control how this protein makes decisions.DOI:http://dx.doi.org/10.7554/eLife.12411.002 Interestingly, the mutation E209K in human BIG2 associated with neuronal disease (The DCB and HUS domains together form a single continuous armadillo repeat joined by a conserved ~1400 Å domain. . We ther disease  maps to  disease .10.7554/T. terrestris Sec7 construct comprising the entire DCB/HUS domain (residues 1–492), for analysis by SAXS. While slight divergence is seen, the intact T. terrestris DCB/HUS domain SAXS data closely fits that predicted from a monomer  to identify the oligomeric state of a series of truncation constructs. We found that full-length T. terrestris Sec7 and the corresponding S. cerevisiae Sec7f (residues 203–2009) construct were both dimeric  (Sec7ΔC) suggests that the GEF domain is flexibly connected to the DCB/HUS domain rather than continuing the armadillo repeat  and monomeric (T. terrestris) constructs, we infer that DCB/HUS domain stimulation of GEF activity occurs independently of dimerization. Two potential mechanisms of action thereby present themselves: either the DCB/HUS domain binds to the membrane surface to recruit the GEF domain to its site of action, or the DCB/HUS domain assists in membrane-insertion of the N-terminal amphipathic helix of Arf1.Similar results were also observed using equivalent rescence . As the To distinguish between these two possibilities, we changed the lipids in the reaction mixture from physiologically relevant TGN-like liposome membranes to non-physiological DMPC/cholate micelles known to support nucleotide exchange of myristoylated Arf1 . In the Our interpretation of these observations is that the DCB/HUS domain assists in overcoming a kinetic activation barrier associated with insertion of the myristoylated Arf1 N-terminal helix into membranes. An alternative possibility, that the DCB/HUS domain has indiscriminate affinity for lipid surfaces and therefore increases the rate of membrane-localized encounters between the GEF domain and its substrate, is addressed further below.S. cerevisiae, selecting conserved residues found at the surface of the T. terrestris DCB/HUS domain structure (ARF1 gene (sec7-1 temperature sensitive mutation (S402L in S. cerevisiae)   maps to evisiae) . The leued patch .10.7554/arf1Δ strain. All of the new mutants that we examined expressed well and exhibited largely punctate localization  reduced both cell viability and membrane-dependent GEF activity of Sec7ΔC, whereas mutations in the positively charged patch  had little effect in vitro despite modestly affecting growth. None of the tested mutations reduced GEF activity in the absence of membranes  in batch, followed by MonoQ ion exchange and Superdex 200 gel filtration , with a final buffer composition of 20 mM HEPES pH 7.5, 150 mM NaCl, and 2 mM DTT.Sec7 and Arf1 constructs were purified as previously described , with no2 added to all buffers. Full-length Arf1 was coexpressed with NMT1 to myristoylate, then following lysis was purified via batch incubation with DEAE-sephacel, batch incubation with ToyoPearl phenyl resin, and Superdex 200 gel filtration. Arf1 L8K purification followed an almost identical protocol, but without NMT1 coexpression. Myristoylated Arf1 was assumed to be fully GDP-bound; Arf1 L8K and ΔN17Arf1 were treated with EDTA in the presence of excess GDP to convert them to their GDP-bound form prior to activity assays.ΔN17Arf1 constructs were purified as per Sec7, with additional 2 mM MgClT. terrestris His6-Sec7(1–458) at 16 mg/ml was crystallized via the hanging drop method, mixing 1:1 with 6% Jeffamine ED-2001, 150 mM MES pH 6, 3% DMSO, and 10 mM DTT at 4°C. Crystals were cryoprotected in a three-stage shift to synthetic well solution plus 30% DMSO. The native construct was crystallized and cryoprotected similarly, using a well solution of 5% Jeffamine ED-2001, 150 mM MES pH 6, and 6% DMSO.Selenomethionine substituted Diffraction data were collected locally at CHESS beamline A1 on an ADSC Quantum-210 CCD detector and processed using HKL-2000 ; to imprSmall-angle X-ray scattering data were collected at CHESS beamline F2 at room temperature on samples purified to homogeneity as described. Multiple serial exposures of each construct at 4 mg/ml and 3–4 successive twofold dilutions were collected against exactly matched blanks and processed using BioXTAS RAW  to confiProteins purified to homogeneity were exchanged into fresh buffer by serial concentration and redilution to a final concentration of 5 mg/ml, and run through a Wyatt WTC-050S5 gel filtration column coupled to DAWN HELEOS-II light scattering and Optilab T-rEX refractive index detectors (Wyatt Technology) at room temperature. Data were analyzed via ASTRA 6 software to obtain the molecular weight of the sample, and compared to that predicted from the sequence to determine oligomeric state.2), 200 μM lipids (lipsomes or micelles), 670 nM Sec7 construct, 670 nM Arf1 construct, and 200 μM GMPPNP were sequentially added while monitoring fluorescence, waiting 1–3 min between steps for the fluorescence to stabilize. After addition of GMPPNP, the fluorescence was monitored for an additional 40 min and fit to a single exponential curve to determine the rate of exchange. In the absence of Sec7, fluorescence traces were observed to remain stable, indicating that the observed changes in fluorescence were solely due to the activity of Sec7, and were not a lipid-mediated effect. The calculated rate of exchange was then normalized to the native fluorescence of the input Sec7 construct to obtain kreact. Note that the per-molecule exchange rates calculated from these reactions are lower than reported previously, in part due to the higher concentrations of GEF constructs used here and variations in behavior between liposome batches. Triplicates of each condition were collected for statistics; error bars represent 95% confidence as determined by t test or ANOVA with Tukey’s or Dunnett’s test in postprocessing, as appropriate.Arf1 nucleotide bound state was measured in real-time as described previously , with thLEU2 marked), and transformed into yeast containing genomic sec7 and arf1 disruptions and a URA3 plasmid harboring wild-type SEC7 (CFY863). Following overnight growth in –Leu synthetic media, serial half-log dilutions were spotted on 5-FOA and synthetic complete or dropout media and grown for three days at 37°C.Sec7 alleles to be assayed were cloned with an N-terminal GFP tag into pRS415 (600 ~ 0.5). For the temperature shift experiment, cells were grown shaking in a 37°C water bath for 30 min before imaging. Live cells were imaged on a DeltaVision RT wide-field deconvolution microscope (Applied Precision). Images were deconvolved using SoftWoRx 3.5.0 software (Applied Precision). Images were further processed in ImageJ, adjusting only min/max light levels for clarity, and using equivalent processing for all images within an experiment.Cells were grown in synthetic dropout media and imaged in log phase .The reviewers agreed that the major conclusion of the paper, that the DCB/HUS domain facilitates lipid insertion of the Arf1 amphipathic helix, is not adequately supported by the data. While they consider the authors' interpretation of their data to be reasonable, it is not directly supported with experimental data; all that is shown is Trp fluorescence measurements as a proxy for nucleotide exchange. This is necessary to address, particularly since early work on Arf1 (see below) raises an alternative interpretation than the authors' whereby the DCB/HUS domain increase the specific activity of the GEF domain without directly promoting membrane binding by the N-terminal helix. Related, the reviewers are also concerned that the Trp fluorescence assay used to infer nucleotide exchange may be misleading when assayed in the presence of lipids due to the potential that these agents influence the local environment of the Trp residue that reports the occupancy state of the nucleotide binding site. In order to be further considered by intrinsic rate of hydrolysis on Arf1 (Khan and Gilman 1986). This underscores a concern with the authors' models. Does the DCB-HUS domain promote Arf1 membrane insertion or, alternatively, does DCB-HUS increase the specific activity of the GEF domain when presented when the N-domain of Arf1 is – through whatever mechanism – already pried away from its pocket in Arf1:GDP. Indeed, that specific conformational change (characterized by NMR) has been suggested to be a prerequisite for GTP binding:Background: DPMC micelles stimulate the "The [yeast Myr-Arf1:GDP] structure clearly places the myristoyl chain in a hydrophobic groove between a C-terminal helix and a loop connecting b strands 3 and 4. The positioning suggests a clash with this loop would occur as it exists in the GTP-bound form, a fact that may require membrane association to accommodate an expelled myristoyl chain before GDP exchange can occur." . A possibility consistent with the authors' membrane-first model is that the DCB-HUS domain might actually allow Arf1-GDP to stably associate with the membrane (in the absence or presence of the GEF domain). That might be a pretty straightforward floatation or pelleting experiment. We thank the editor and reviewers for their supportive and constructive comments, which we have addressed through additional experiments and changes to the text. In our view the primary claim of this study, namely that the DCB/HUS domain of Sec7 forms a single structural unit that facilitates activation of Arf1 at the surface of a membrane, is now firmly supported by the data.We have performed additional experiments to address the core issue raised by the reviewers, namely to provide direct evidence (aside from Trp fluorescence) that the DCB/HUS domain facilitates Arf1 membrane association (and not just nucleotide exchange). We now show that this is indeed the case, using a membrane-binding (flotation) experiment. We also explain how the fluorescence experiments are controlled to avoid potential confounding artifacts arising from lipids.The reviewers agreed that the major conclusion of the paper, that the DCB/HUS domain facilitates lipid insertion of the Arf1 amphipathic helix, is not adequately supported by the data. While they consider the authors' interpretation of their data to be reasonable, it is not directly supported with experimental data; all that is shown is Trp fluorescence measurements as a proxy for nucleotide exchange. This is necessary to address, particularly since early work on Arf1 (see below) raises an alternative interpretation than the authors' whereby the DCB/HUS domain increase the specific activity of the GEF domain without directly promoting membrane binding by the N-terminal helix. Related, the reviewers are also concerned that the Trp fluorescence assay used to infer nucleotide exchange may be misleading when assayed in the presence of lipids due to the potential that these agents influence the local environment of the Trp residue that reports the occupancy state of the nucleotide binding site. In order to be further considered by eLife, the paper will need to provide direct evidence supporting the model that the DCB-HUS domain acts directly on the N-terminal helix of Arf to promote its insertion into the membrane. With regard to the effects of lipids on Arf Trp fluorescence, the reviewers noted that this latter concern might be adequately addressed by a more thorough description of the assay methods, including the strategy used to account for changes in fluorescence not due to nucleotide binding .Background: DPMC micelles stimulate the intrinsic rate of hydrolysis on Arf1 (Khan and Gilman 1986). This underscores a concern with the authors' models. Does the DCB-HUS domain promote Arf1 membrane insertion or, alternatively, does DCB-HUS increase the specific activity of the GEF domain when presented when the N-domain of Arf1 is – through whatever mechanism – already pried away from its pocket in Arf1:GDP. Indeed, that specific conformational change (characterized by NMR) has been suggested to be a prerequisite for GTP binding:"The [yeast Myr-Arf1:GDP] structure clearly places the myristoyl chain in a hydrophobic groove between a C-terminal helix and a loop connecting b strands 3 and 4. The positioning suggests a clash with this loop would occur as it exists in the GTP-bound form, a fact that may require membrane association to accommodate an expelled myristoyl chain before GDP exchange can occur." . A possibility consistent with the authors' membrane-first model is that the DCB-HUS domain might actually allow Arf1-GDP to stably associate with the membrane (in the absence or presence of the GEF domain). That might be a pretty straightforward floatation or pelleting experiment.Regarding possible artifacts arising from any affects of lipids on Arf Trp fluorescence, we can rule these out with certainty based on control experiments. To convince the reader of this, we now include additional descriptions in the Results and Methods sections. The most important thing to note is that we do directly monitor how the addition of each component changes the fluorescence of the reaction. Notably, exchange rates are extracted from time-course changes in kinetics that only occur subsequent to the addition of GTP, and we include a baseline control  in each set of experiments to confirm that no change in fluorescence is seen over time in the absence of exchange . In the new We note that our experiments do directly address the possibility that the DCB/HUS domain might simply increase the specific activity of the GEF domain: The fact that the DCB/HUS domain does not stimulate the activity of the GEF domain towards the deltaN17-Arf1 or L8K-Arf1 substrates  demonstrAdditionally, the competition experiments presented in We agree that it is very likely that membrane association of Arf1- GDP occurs prior to the exchange reaction. However, this interaction is known to be very transient . If the DCB/HUS domain stabilizes this transient intermediate state (which we think it does), this state is still likely to be quite transient compared to the time-scale of a pelleting or flotation experiment. We previously performed an experiment similar to that suggested above – as reported in our 2012 paper , Arf1-GDWe note that we propose two alternatives for how the DCB/HUS domain might facilitate Arf1 membrane-proximal activation and insertion of the amphipathic helix: either through direct transient interaction with the Arf1 N-terminus or by positioning Arf1 optimally for membrane insertion . We think that both of these possibilities are interesting, as either involves function of the DCB/HUS domain to overcome the activation barrier associated with membrane insertion.We expect that further determination of exactly how the DCB/HUS domain facilitates membrane insertion of the Arf1 N-terminus will await structures of longer constructs of Sec7 or its homologs “caught in the act” of activating Arf1.


Citrus shoot tips abscise at an anatomically distinct abscission zone (AZ) that separates the top part of the shoots into basal and apical portions (citrus self-pruning). Cell separation occurs only at the AZ, which suggests its cells have distinctive molecular regulation. Although several studies have looked into the morphological aspects of self-pruning process, the underlying molecular mechanisms remain unknown.Arabidopsis and tomato shoot apical meristem (SAM) activity genes were investigated in sweet orange by real-time PCR and in situ hybridization, and the results indicated that these genes were preferentially expressed in SAM as well as axillary meristem.In this study, the hallmarks of programmed cell death (PCD) were identified by TUNEL experiments, transmission electron microscopy (TEM) and histochemical staining for reactive oxygen species (ROS) during self-pruning of the spring shoots in sweet orange. Our results indicated that PCD occurred systematically and progressively and may play an important role in the control of self-pruning of citrus. Microarray analysis was used to examine transcriptome changes at three stages of self-pruning, and 1,378 differentially expressed genes were identified. Some genes were related to PCD, while others were associated with cell wall biosynthesis or metabolism. These results strongly suggest that abscission layers activate both catabolic and anabolic wall modification pathways during the self-pruning process. In addition, a strong correlation was observed between self-pruning and the expression of hormone-related genes. Self-pruning plays an important role in citrus floral bud initiation. Therefore, several key flowering homologs of Based on these findings, a model for sweet orange spring shoot self-pruning is proposed, which will enable us to better understand the mechanism of self-pruning and abscission.The online version of this article (doi:10.1186/1471-2164-15-892) contains supplementary material, which is available to authorized users. Most perennial plants undergo a rhythmic periodicity for shoot growth, in which phytomers are initiated but do not fully expand, and flowering and fruiting occur only after a dormancy period . Apical Poncirus trifoliata L. Raf.), abortion of the shoot tips (0.5-1 cm) takes place in spring or early summer and is rapidly followed by a decline in the growth of the distal portion of the extending shoot. The entire shoot tip soon turns yellow and abscises at the base of the shoot apex; this physiological phenomenon is called “self-pruning” in citrus. Self-pruning is a necessary but not sufficient condition for citrus flowering. Previous cytological studies revealed that the floral buds of spring shoots in an early-flowering mutant of trifoliate orange (precocious trifoliate orange) initiated differentiation immediately after self-pruning [In citrus, there are three important types of shoots produced during the growing season. The main type grows in late winter or early spring (spring shoots), and two additional types grow at the end of June (summer shoots) and late in September (autumn shoots) . The spr-pruning . In swee-pruning .Arabidopsis protein phosphatase type 2C, ABI1, a negative regulator of abscisic acid signaling, was also involved in the regulation of ethylene biosynthesis under oxidative stress conditions. Meanwhile, ABI1 interacted with ACS6 and dephosphorylates its C-terminal fragment, a target of the stress-responsive mitogen-activated protein kinase, MPK6 [During self-pruning of citrus, shoot tip separates from the top part of the shoots at a predetermined position  and transmission electron microscopy (TEM) analyses suggest that PCD occurs systematically at shoot tips during the self-pruning process, and ROS-induced PCD may be involved in the process of self-pruning. In addition, we carried out a high-throughput microarray analysis of the specific gene expression occurring during self-pruning. Our results notably increase the current catalogue of genes related to the abscission process and provide new candidate genes for future biotechnological applications in citrus. To our knowledge, this is the first comprehensive report of a direct link between citrus self-pruning and PCD.Although sweet orange is an evergreen tree, there were no apparent developmental changes in the size or form of buds when it entered dormancy at the end of the growing season. Bud enlarge somewhat and fold back due to differential enlargement, and presumably more growth occured on the adaxial than on the abaxial surfaces in the spring. Following the period of bud opening in the current study, the shoot underwent a period of rapid elongation during which the successive leaves and the stem continued to enlarge and mature Figure A. Early The paraffin sections and TEM analysis of shoot tip cells showed no visible evidence of cellular breakdown or death before self-pruning Figures  and 3. Oin situ, the TUNEL procedure was used to assess and confirm the degradation of nuclear DNA in shoot tips  and diaminobenzidine (DAB) was performed to check the levels of HP ≤ 0.001 and four fold changes. Among these DEGs, 30 genes were up-regulated and 124 were down-regulated  than down-regulated  from stage 1 to stage 2. However, more genes were down-regulated  than up-regulated  from stage 1 to stage 3. A total of 154 DEGs were identified from stage 1 to stage 2 based on ,764 thanIn this study, we classified the 1,378 DEGs of the shoot tips into four clusters based on the similarity of the kinetic expression patterns. Cluster 1 genes 571) were induced immediately at stage 2 and most maintained high expression levels at stage 3 , a homology search was conducted using the NCBI database  and three genes related to vegetative growth  were also investigated  and three FT homologues from citrus were isolated in previous studies [CiFT homologues showed high identities in open reading frame. Thus, total CiFT and CiSOC1/2 were investigated in this study. Overall, real-time PCR revealed the same expression trend as the microarray data for 32 of the genes except Cit.14181.1 and Cit.1497.1 , possibly because of low abundance of the transcript. In addition, recent studies of Arabidopsis also demonstrated that protein of FT is produced in the vascular tissues of leaves and moves from the leaves to the SAM as a mobile flowering signal [To assess the physiological functions of n Figure .Primer sm Figure A, B. Howg Figure A and teng Figure B. It is g Figure D as compg Figure C. CiFLC n citrus . CiFLC wg Figure E and itsg Figure  F. In thg Figure H compareg Figure G. The leg Figure I and teng Figure  J. CiSP d Figure  L compard Figure  K. HowevZ Figure  M–O. Cong signal .Figure 6LATERAL ORGAN BOUNDARIES DOMAIN PROTEIN 1, WUS, KNAT6, BELL-like protein 1, and JOINTLESS as well as axillary meristem genes BLIND and LATERAL SUPPRESSOR in tomato [WUS and KNAT6. It is possible that some different genes, perhaps a member of a different subfamily, perform similar functions during citrus development. In this study, homologs of KNAT6 displayed expression profiles similar to those previously described in SAM [P ≤ 0.001 and 4-fold change. One possible reason for this observation is that the regulatory mechanism of self-pruning differs between model plants and woody plants.Self-pruning is a demarcation point for SAM to initiate leaf bud or floral bud development. However, the phenological and morphological plasticity of self-pruning in citrus have not been examined by experimental manipulation. Abscission typically occurs at spring shoots when the citrus shoot separates from the top part of the shoots. This process has been widely characterized at AZ level , 28, 29,n tomato –34. Althn tomato , no cleaMany hormones regulate the process of plant organ abscission, such as auxin, ABA, GA, jasmonic acid (JA), and ethylene, among which auxin and ethylene play important roles , 14. EthArabidopsis[Arabidopsis[Arabidopsis[2O2 and O2- levels in shoot tips during the self-pruning process. Although we could not decipher the mechanism of action regarding H2O2 and O2-, our work demonstrates that ROS accumulate at higher level probably because they may play a role in stimulating the expression of abscission-related genes during the induction of shoot tip abscission. On the other hand, many genes involved in ROS detoxification were also identified in this study  detesis AZ s , 48. Thesis AZ s . Previousis AZ s , 50, 51.In this study, according to function clustering of the 1,378 DEGs by GO analysis, about 30% of the genes related to macromolecule and protein metabolism were expressed preferentially in the shoot tips during the self-pruning period  and regulator of self-pruning process of sweet orange spring shoots, were down-regulated in shoot tips before self-pruning, and up-regulated in lateral buds after self-pruning. Based on these results, we conclude that a high expression level of three flowering time genes might help to maintain the terminal buds in a dormant state before self-pruning, whereas the down-regulation of these genes after self-pruning induction might be closely related to the shift of cell activity or the change in flowering competence of spring shoot lateral buds.In addition, self-pruning in citrus affects the SAM development program and therefore the architecture of the plant as well as the production of fruits. The floral bud induction could be paralleled with the mechanism occurring in the other fruit plants promoting the return of the bloom or the onset of alternative bearing, this latter being an unwanted trait that negatively affects fruit production. Several classes of TFs exhibited significant changes in expression, including ERF/AP2 TFs, bZIP proteins, MADS-box and MYB domain proteins  were grown under natural environmental conditions in experimental fields of the National Citrus Breeding Center of Huazhong Agricultural University, Wuhan, China . The shoot tips of spring shoots were collected at three distinct phases . Tissue permeabilization was performed with 20 μg/mL proteinase K  in 10 mM Tris (pH 7.5) and 5 mM EDTA (pH 8) at 37°C for 30 min. After washing the tissue twice with phosphate-buffered saline (PBS), lysing enzyme (4 mg/mL) in 5 mM EDTA (pH 8) was added and incubated for 20 min at 37°C. TUNEL reaction was performed on slides using the DeadEnd Fluorometric TUNEL System  according to the manufacturer’s instructions.2- and H2O2 was detected by a histochemical staining method by using NBT and DAB, respectively [Accumulation of Oectively .4 in PBS (pH 7.2) for 3 h. The tissues were then rinsed twice in PBS and stained with uranyl acetate. The samples were dehydrated by passing them through an ethanol series and acetone, and they were then embedded in Agar100 epoxy resin (Agar Scientific). Thin sections were cut, treated with uranyl acetate/lead citrate, and examined with a Tecnai G2 Spirit transmission electron microscope . Representative photographs are presented.The shoot tips of spring shoot were washed with PBS (pH 7.2) at room temperature and post-fixed in 2% (w/v) OsOGenomic DNA from the shoot tips of spring shoot was isolated using cethyltrimethylammonium bromide (CTAB) method . DNA quaCitrus species and hybrids. Also included are sequences from Poncirus species and Poncirus × Citrus hybrids. Hybridization signals were normalized using the Affymetrix Microarray Suite program (version 5.0) and visualized using the software tool of The Institute for Genomic Research (TIGR) MeV [P ≤ 0.001 compared with the control.Three total RNA samples  from shoot tips including AZ were independently isolated from each sample, according to a previous protocol . HybridiIGR) MeV . AffymetIGR) MeV . In ordeIGR) MeV  was usedAnnotations of putative functions for DEGs were performed using the program Blast2GO , which wThe transcriptional profiles of 32 genes were analyzed by real-time PCR using the SYBR Green PCR master mix , as described previously . Primer in vitro transcription according to the manufacturer’s instructions. Prehybridization, hybridization, washing, and detection were performed as described in the Cold Spring Harbor Arabidopsis Molecular Genetics Course (http://www.Arabidopsis.org/cshl-course/5-in_situ.html).Digoxigenin-labeled RNA probes were prepared using a DIG Northern Starter Kit . T7 and SP6 RNA polymerase were used to generate the sense and antisense RNA probes by The microarray data have been submitted to Gene Expression Omnibus (GEO) under accession no. GSE53579.Additional file 1: Figure S1: Spring shoot of sweet orange regions used in this study: (A) shoot tip regions; (B) microarray analysis regions; (C) AZ; (D) apical portions; and (E) basal portions. (DOC 1 MB)Additional file 2: Figure S2: Histochemical staining assay of ROS accumulation with nitro blue tetrazolium (NBT) and diaminobenzidine (DAB) in shoot tips during self-pruning process. (A–F) control: shoot tips subjected to dehydration by alcohol. (G–L) NBT staining of shoot tips during self-pruning process. (M–R) DAB staining of shoot tips during self-pruning process.  10 days before self-pruning;  beginning of self-pruning of spring shoots;  3 days after self-pruning;  7 days after self-pruning;  before formation of protective layer of AZ;  after protective layer of AZ forms. (DOC 268 KB)Additional file 3: Table S1: Annotation of the 1,378 DEGs during self-pruning process. (XLS 370 KB)Citrus sinensis), (A) molecular function; (B) biological process; (C) cellular component. (DOC 1 MB)Additional file 4: Figure S3: Characterization of 1,378 differentially expressed genes by gene ontology categories in sweet orange Additional file 6: Figure S4: Cluster analysis of expression profiles of hormones related DEGs at three stages. Each column represents a sample, and each row represents a single citrus transcript sequence. The bar represented the scale of relative expression levels of DEGs, and colors indicate relative signal intensities. a: these genes involved in multiple hormones metabolism and signaling; b: SA-related genes; c: Cytokinin riboside 5-monophosphate phosphoribohydrolase. TF, transcription factor. (DOC 3 MB)Additional file 7: Figure S5: Cluster analysis of expression profiles of cell wall related DEGs at three stages. Each column represents a sample, and each row represents a single citrus transcript sequence. The bar represented the scale of relative expression levels of DEGs, and colors indicate relative signal intensities. XEH indicated Xyloglucan endotransglucosylase hydrolase. (DOC 1 MB)β-actin control expression. The primers used for the analyses are given in Additional file Additional file 8: Figure S6: Cluster analysis of expression profiles of DEGs at three stages of sweet orange spring shoots by real-time PCR (qPCR) and microarray analysis. Each column represents a sample, and each row represents a single citrus transcript. The bar represented the scale of relative expression levels of DEGs, and colors indicate relative signal intensities. For qPCR analysis, data points represent mean ± SE of at least four replicates for the relative expression, which were normalized by the amount of the in situ hybridization. (XLS 31 KB)Additional file 9: Table S3: Specific primers for real-time PCR and Citrus sinensis Osbeck) spring shoot during the second self-pruning process. Red arrows represent AZ. (DOC 12 MB)Additional file 10: Figure S7: Phenotypic characteristics of ‘Cara Cara’ navel orange (