The presence of single, non-motile “primary” cilia on the surface of epithelial cells has been well described since the 1960s. However, for decades these organelles were believed to be vestigial, with no remaining function, having lost their motility. It wasn't until 2003, with the discovery that proteins responsible for transport along the primary cilium are essential for hedgehog signaling in mice, that the fundamental importance of primary cilia in signal transduction was realized. Little more than a decade later, it is now clear that the vast majority of signaling pathways in vertebrates function through the primary cilium. This has led to the adoption of the term “the cells's antenna” as a description for the primary cilium. Primary cilia are particularly important during development, playing fundamental roles in embryonic patterning and organogenesis, with a suite of inherited developmental disorders known as the “ciliopathies” resulting from mutations in genes encoding cilia proteins. This review summarizes our current understanding of the role of these fascinating organelles in a wide range of signaling pathways. The primary cilium is a long, thin organelle protruding from the apical surface of almost all cell types, most commonly from epithelial cells. This structure is formed when the cell is in G0 or G1 phase, and often during S/G2 phase  particles, which carry protein and other cargos into the ciliary compartment. IFT is the process by which all proteins are transported into and along the ciliary compartment, as protein translation cannot occur here. Anterograde IFT transports proteins along the cilium from base to tip, catalyzed by cytoplasmic dynein 2/1b motor whereas retrograde IFT transports proteins from the tip to the base of the cilium, catalyzed by kinesin-2 motor protein  from the cilium. Gli transcription factors are sequestered and suppressed by Suppressor of Fused (SuFu) at the tip of the primary cilium in the unstimulated state  and gain of function Hh phenotypes in the limb   screen revealed a number of Hh pathway genes which, when knocked down, led to cilia disassembly, including a protein called Stk11, also known as Lkb1, which also regulates the Wnt signaling pathway  show a loss of basal canonical Wnt signaling activity leading to cystic kidney disease  non-canonical Wnt signaling pathway , leading to reduced cell proliferation  asymmetry of the embryo. Glycosylation of Notch1 activates the signaling pathway which increases specification of non-motile primary cilia at the node. Loss of this process leads to situs defects as a result of disturbed fluid flow at the node , epidermal growth factor receptor (EGFR) and insulin growth factor receptor (IGFR) signaling. A comprehensive review of these can be found in Christensen et al. .All of these RTK signaling cascades contribute directly or indirectly to regulation of mTor signaling, and conversely, mTOR signaling inhibits PDGFR alpha levels.PKD1, mutated in the common ciliopathy autosomal dominant polycystic kidney disease (ADPKD)  pathway acts through mTOR complex 1 and 2  and integrates information from a number of upstream pathways, including the tuberous sclerosis complex proteins such as tuberin. Polycystin-1 (PC1), the protein product of ) Figure , Table 1Ofd1 mutant mice showed hyperactivation of mTOR in the kidney epithelia, which was successfully lowered with rapamycin treatment to significantly reduce cystogenesis   which forms after the neuronal cells have completed their migration and their mother centrioles dock to the cell membranes to form the basal body. The pro-cilium matures into a cilium postnatally over a period of 8–12 weeks. During mouse brain development migratory progenitor neurons from the ventricular zone differentiate into neural cells which are mostly directed to the upper layers of the neocortex. In the later stages of development, the presence of a pro-cilium coincides with the non-migrating neuronal population of the developing cortical plate  . When GPCR retrieval fails, for example in Arl6 knock-out cells, SSTR3 mutant cells or NPY2R mutants lacking motifs for recognition by BBSome and β-arrestin, the GPCRs concentrate at the ciliary tip and are subsequently ectocytosed alongside significant amounts of β-arrestin2 and the BBSome. Thus, neuronal cells with defects in cilia can have significant defects in GPCR signaling . Recent whole genome sequencing studies, proteomics studies and reverse genomics studies are leading toward better understanding of the primary cilium and its function in human health and disease (Wheway et al., Over the coming years, whole genome sequencing is likely to provide significant insight into the genetics underlying cilia function and dysfunction, as genomic testing becomes integrated into standard clinical healthcare in the UK National Health Service through the 100,000 Genomes Project. Around 50% of ciliopathy patients still do not receive a genetic diagnosis, even after genetic screening of all known ciliopathy genes, suggesting that much remains to be discovered about the genes and proteins important for cilium function and dysfunction. Population-scale whole genome sequencing studies, such as the 100,000 Genomes Project are enormously exciting opportunities both for patients to receive diagnoses, and researchers to gain insights into previously unknown pathways of disease. Widespread whole genome sequencing of ciliopathy patients will uncover mutations in novel ciliopathy genes, many of them likely to be private mutations, providing new understanding of the role of cilia in diverse cell signaling pathways, and potentially new targets for therapies. Perhaps the biggest challenge for researchers in this field is to develop techniques to identify which genetic variants from an estimated 50 million per individual are the variants likely to be significantly contributing to disease and warranting further investigation.GW was the main author and wrote the draft, while LN was a contributor, being a researcher in GW's laboratory. JH edited the paper and suggested some references to be cited.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.


Wnt and transforming growth factor-β (TGF-β) signaling pathways are known to be involved in the pathogenesis of androgenetic alopecia (AGA). However, the way that Wnt and TGF-β signaling is altered in patients with AGA and whether there exists a crosstalk between them in pathogenetic process of AGA remain unclear.To investigate the expression of Wnt and TGF-β signaling and the crosstalk between these 2 signaling pathways in AGA.Fifteen male patients with AGA were recruited for our research. Fifteen scalp specimens of the balding were collected from frontal areas, and 9 nonbalding were collected from occipital areas. We analyzed the expression and activation of downstream Wnt and TGF-β signaling molecules in both balding and nonbalding hair follicles isolated from scalp specimens. Furthermore, we evaluated the activation of Wnt and TGF-β signaling after either of them was blocked with the inhibitor in balding and nonbalding dermal papilla (DP) cells.Wnt10a and LEF1 was decreased. But TβRI and TβRII, and the protein expression of TGF-β1 was elevated in balding hair follicles. To investigate the crosstalk between Wnt and TGF-β signaling, we used SB431542 to inhibit the TGF-β signaling in balding DP cells and found that SB431542 significantly attenuated the phosphorylation of Smad2 and Akt. However, the mRNA level of Wnt10a, LEF1, and the nuclear translocation of β-catenin was increased. On the other hand, we suppressed the Wnt signaling by XAV939 in nonbalding DP cells, which displayed that the level of β-catenin and LEF1 was significantly inhibited; however, the level of active TGF-β1 and the phosphorylation of Smad2 and Akt were up-regulated.Compared with the nonbalding counterparts, the mRNA level of These data indicate that crosstalk between Wnt/β-catenin and TGF-β signaling pathways may exist as one of the important mechanisms contributing to AGA. Despite the fact that specific molecules regulated by androgen in the balding scalp are not fully clear, several cytokines, hormones, growth factors, enzymes, and neuropeptides are contributors to hair cycle control and may be involved in the pathogenesis of AGA.,5 Recent studies have revealed that Wnt/β-catenin signaling was required to trigger epidermal stem cell in the process of hair follicle formation, and it induced the hair follicle dermal papilla (DP) formation. The results from transgenic mice showed that stabilization of β-catenin could promote the process of placode maturity and development, while the genetic or pharmacologic ablation of β-catenin could cause the failure of hair follicles formation. Furthermore, crosstalk between androgen and Wnt/β-catenin signaling is thought to play an important role in AGA pathogenesis. It is also well demonstrated, in the DP cells and keratinocytes co-culture system, that the secretion of TGF-β1 induced by androgen is essential in DP cells from balding frontal scalp via influencing the epithelial cell growth. In addition, TGF-β1 modulates androgen receptor transactivation and androgen sensitivity in DP cells, which contributes to the pathology of AGA.The cell fate choice of stem cell of complex organs and tissues is regulated by microenvironment during the embryonic stage. the development of prostate cancer, wound healing, and tissue fibrosis in skeletal muscle and kidney. However, whether there is a crosstalk between these 2 important signaling pathways during AGA pathological procession is still unknown.Several studies have shown that cooperation between TGF-β and Wnt signaling pathways plays an important role in controlling certain developmental events. Crosstalk between the TGF-β and Wnt signaling pathways participates in the formation of tooth eruption passage,Wnt10a, LEF1, β-catenin) and TGF-β  signaling in balding versus nonbalding hair follicles isolated from 15 scalp specimens and DP cells. Furthermore, the study examined the activation of the Wnt/β-catenin signaling after the TGF-β signaling pathway was inhibited in balding DP cells, and also evaluated the activation of the TGF-β signaling after the Wnt/β-catenin signaling pathway was blocked in nonbalding DP cells. These data confirm that both regulations of Wnt/β-catenin and TGF-β signaling pathways are involved in pathologic process of AGA and support the hypothesis that crosstalk between Wnt/β-catenin and TGF-β signaling pathway may exist in the pathogenesis of AGA.This study, therefore, explored the differences in the levels of Wnt , and exclusion of traumatic and inflammatory treatment. A total of 15 men, aging between 22 and 47 years (average 30 years), were involved in this study. Scalp specimens of the balding  areas were collected from all 15 patients with AGA, and specimens of the nonbalding  were collected from 9 of them.2.2 In brief, we obtained skin specimens during plastic surgery and thoroughly washed them with D-Hanks buffer (containing 5% [v/v] penicillin and streptomycin). Then, we carefully removed the subcutaneous fat and connective tissue surrounding the collagen capsules by microscissors and digested the separated tissues with 0.1% collagenase type I  at 37°C for 1 h. Afterward, the papillae were dissociated and cultured in DMEM/F-12 medium  with 10 ng/mL basic fibroblast growth factor and 10% fetal bovine serum. Finally, we used 0.25% trypsin  with 0.02% EDTA to digest DP cells and planted them into a new culture dish at 1 × 104 cells/mL and maintained at 37°C under a humidified atmosphere of 95% O2 and 5% CO2 for 24 h prior to complement with serum-free DMEM/F-12 medium. The DP cells used in this research were in the second and third passages of subculture.The isolation and cultivation of DP cells from balding and nonbalding scalps were carried out as previously described.2.3 Total RNA of hair follicles and DP cells was isolated by using TRIzol Reagent . RNA was reverse-transcribed using the PrimeScriptTMRT reagent Kit . Real time quantitative PCR (RT-qPCR) was performed with the corresponding primers  to measure the concentration of total (latent and active) and active TGF-β1, according to the manufacturer's instructions.2.5DP cells (the second passages of subculture) grown on glass coverslips were fixed in 4% paraformaldehyde, incubated with phosphate-buffered saline (PBS) containing 100 mmol/L glycine, and washed with PBS. Cells were permeabilized in PBS containing 0.1% Triton X-100, blocked with 5% albumin for 1 h, and incubated with mouse monoclonal antibody against β-catenin  overnight. After several rounds of washing, goat anti-mouse-fluorescein or rabbit anti-mouse-fluorescein  was added for 1 h, and the coverslips were washed and mounted using fluorescence microscopy.2.6Protein samples from hair follicles or cultured DP cells were extracted using RIPA buffer . Equal amounts of protein were loaded in each lane, resolved on 10% sodium dodecyl sulfate poly-acrylamide gradient gels and transferred onto a 0.45-μm NC membrane (Milipore). The membrane was blocked with 5% bovine serum albumin and incubated with anti-p-Smd2, anti-Smd2, anti-p-Akt, anti-Akt, anti-β-catenin  or Laminin B1  overnight at 4°C. Followed, then by anti-rabbit or anti-mouse IgG peroxidase conjugate. Immunoreactive bands were visualized by the Immobilon Western hemiluminescent HRP Substrate (Milipore).2.7P < 0.05.A statistical analysis was performed using SPSS software version 17.0. These data were presented as the mean ± standard error of the mean from at least 3 independent experiments. Multiple group comparisons were made using 1-way analysis of variance. Differences between groups were considered statistically significant at 33.1Wnt10a and LEF1  to inhibit the TGF-β signaling, and evaluated the activation of Wnt signaling subsequently by the methods of western blotting and RT-qPCR. The significant down-regulation of phosphorylation level of Smad2 and Akt confirmed that SB431542 was sufficient to inhibit the TGF-β signaling in balding DP cells  to inhibit the Wnt/β-catenin signaling, and assessed activation of TGF-β signaling afterward. As the data showed, the expression of β-catenin and the mRNA level of 4,17 For instance, dihydrotestosterone (DHT)-treated DP cells from AGA inhibits the Lymphoid enhancer factor/T-cell factor induced transcriptional activity and hair follicle stem cell differentiation,,19 while Wnt signaling reactivation will restore the DHT induced hair follicle stem cell differentiation.The regulation activity of androgen on hair growth and cycling varies on different body sites in puberty males, which promotes beard growth but inhibits hair growth in frontal and vertical scalp, known as “androgen paradox.” More and more evidence proves that androgen induced hair growth suppression is tightly related to Wnt/β-catenin signaling, which promotes perspective crosstalk occurring between androgen and Wnt/β-catenin signaling in AGA pathogenesis. but also TGF-β signaling promotes the AGA development,21 and TGF-β1 deficiency delays catagen progression and perifollicular fibrosis in AGA. Furthermore, we also demonstrated that the TGF-β signaling was remarkably up-regulated during the AGA pathogenesis  to block the Wnt/β-catenin signaling in cultured nonbalding DP cells, which were isolated from occipital scalp, then evaluated the activation of TGF-β signaling (Fig. LEF1 expression, while the TGF-β signaling was up-regulated with the increased concentration of active TGF-β1 and phosphorylation of Smad2 and Akt. It indicated that the Wnt/β-catenin might also contribute to TGF-β signaling regulation during AGA development.In this study, we confirmed that the activation of Wnt/β-catenin signaling was indeed down-regulated in the AGA pathogenetic process Fig. . On the sis Fig. . The resing Fig. . The datWnt/β-catenin and TGF-β/Smad signaling pathways are involved in the pathogenesis of AGA. Our clinical data are consistent with this view. In addition, the results of the inhibition of Wnt/β-catenin signaling resulting in up-regulation of TGF-β signaling and inhibition of TGF-β signaling resulting in up-regulation of Wnt/β-catenin indicate that there may exist a crosstalk between these 2 important signaling pathways, which would add more knowledge to understand the whole picture of mechanism that regulates AGA development. However, which cross points are involved between these 2 signaling pathways and how they interact with each other, needs to be explored more thoroughly in the future.TβRII inhibitor might be effective to prevent male pattern baldness.The available treatment methods of AGA mainly include drug therapy and hair transplantation. Due to some unacceptable side effects of the oral drugs and the limited hair follicle units of donor, it is difficult to carry out these therapies extensively and effectively. An understanding of the Wnt/β-catenin and TGF-β/Smad signaling pathways and the crosstalk between them involved in AGA may facilitate discovery of pharmacophores that modulate these pathways. Our data might have therapeutic implications for the development of anabolic drugs, for example, the combination of a Smad2 inhibitor, a TGF-β1 antagonist, or We want to thank our many colleagues who have helped to achieve the clinical specimens and those lab researchers who have helped make the experiment complete.


The binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein 1b-IX (GP1b-IX) leads to activation of platelets. GP1b was shown to signal via the FcRγ-ITAM (Fc Receptor γ-Immunoreceptor tyrosine-based activation motif) pathway, activating spleen tyrosine kinase (Syk) and other tyrosine kinases. However, there have been conflicting reports regarding the role of Syk in GP1b signaling. In this study, we sought to resolve these conflicting reports and clarify the role of Syk in VWF-induced platelet activation. The inhibition of Syk with the selective Syk inhibitors, OXSI-2 and PRT-060318, did not inhibit VWF-induced platelet adhesion, agglutination, aggregation, or secretion. In contrast, platelets stimulated with the Glycoprotein VI (GPVI) agonist, collagen-related peptide (CRP), failed to cause any aggregation or secretion in presence of the Syk inhibitors. Furthermore, GP1b-induced platelet signaling was unaffected in the presence of Syk inhibitors, but GPVI-induced signaling was abolished under similar conditions. Thus, we conclude that Syk kinase activity does not play any functional role downstream of GP1b-mediated platelet activation. IIbβ3 activation and consequent stable platelet adhesion, spreading, and aggregation -2-oxo-1H-indole-5-sulfonamide). Both the inhibitors are adenosine triphosphate (ATP)-competitive inhibitors and inhibit the kinase-activity of Syk [In this study, we evaluated the role of Syk in VWF signaling in human platelets by using two different small molecule pharmacological inhibitors of Syk, PRT 060318 (or PRT-318)  in the presence or absence of the Syk inhibitors. Syk−/− murine platelets [Additionally, to identify the role of Syk kinase activity in the initial adhesion of platelets to the immobilized VWF, we performed a flow over VWF assay. As shown in latelets , furtherSyk-independent pathways in platelet activation downstream of GP1b have also been suggested in previous reports. Yin et al.  showed tIn conclusion, our results demonstrate that Syk, although phosphorylated upon GP1b stimulation, does not play any kinase-dependent functional role in ristocetin/VWF-induced platelet adhesion, aggregation, or secretion, or in GP1b signaling. Thus, not only is GP1b a central player in hemostasis, but it may also be able to signal by an essentially different mechanism than other ITAM-pathways in platelets. The results presented in this study extend our knowledge of the VWF-GP1b signaling pathways in platelets and support the evidence that Syk kinase activity is not required for GP1b signaling. Understanding the versatility of GP1b signaling may therefore prove helpful in developing a rationale for the design of clinically useful antithrombotic agents targeted to the GP1b function.


Northern Ireland has been and continues to be deeply divided on the basis of religion. This paper examines and compares contemporary fertility in the two communities given the sharp declines that have occurred in recent decades. The data are drawn from the Northern Ireland Longitudinal Study and cover 108,000 women aged 16–44 in the period 1997–2007. A logit analysis of births in the period is undertaken based on individual demographic data and also the characteristics of the locality in which the woman is resident. The effect of religion is measured by its individual marginal effect averaged over the total sample, over time and by the age of the woman. The estimated average marginal effect is 4 % of the probability of a woman having a birth over the entire period. The effect peaks when the woman is between 29 and 30 years and is stable over time. When the fertility behaviour of each religious group is compared separately with those women that came from the same background but had lost their religion, it is found to differ substantially. Thus, community background is rejected as a possible explanation of the difference in fertility between declared Catholics and Protestants. Although the fertility rates of former Catholics and Protestants appear to be converging, the size of these groups is relatively small. There is no evidence to suggest that the small but distinct difference in the fertility rates of the two religious communities is likely to change in the immediate future.The online version of this article (doi:10.1007/s10680-016-9399-8) contains supplementary material, which is available to authorized users. The state of Northern Ireland was formed in 1921 and comprised six of the thirty-two counties of Ireland. The division of the island was an attempt to accommodate violently contradictory unionist and nationalist aspirations. In the South of Ireland, the abandonment of the six northern counties precipitated a civil war in which the Republican forces were defeated. In the North, a Protestant state was formed excluding a substantial Catholic minority. In 1926, when the first Census of Northern Ireland was undertaken, one-third of the population of one and a quarter million was Catholic and overwhelmingly nationalist. Politically, they were committed to a single state on the island of Ireland  was undertaken and its findings published in a book by Compton and Coward . NortherThis paper seeks to determine the contribution of religion to fertility in contemporary Northern Ireland, based on self-designated religion in the 2001 Census. The different fertility regimes associated with self-defined Protestants and Catholics are of central interest. In the 2001 Census, 40.3 % of the population stated their religion to be Catholic versus 45.6 % Protestant and 13.9 % either had no religion or declined to state it. In this last group, 43.8 % have a Catholic background (referred to as former Catholics further) and 53.1 % are former Protestants NISRA . While tIn the three decades since the NIFS was undertaken, union and family formation have changed. For example, the role of marriage considerably lost importance. In 2010, 40 % of births in Northern Ireland occurred outside marriage compared to 9 % in 1983. Over a similar period, the total period fertility rate (TPFR) decreased from 2.79 in 1980 to 1.75 in 2000 though in 2008 it rebounded to 2.05  of Catholics (Protestants) classed themselves as either ‘extremely’ or ‘very religious’ according to the Life and Times Survey (NILTS). Equivalent figures exist for 2008 (12 vs. 17 %). Those who considered themselves ‘somewhat religious’ grew from 56 % (42 %) to 60 % (44 %) in the same period  effect of religion to be established; comparing those who have lost the Protestant or Catholic religion of their upbringing gives an important measure of the extent of secularization in Northern Ireland. Across Western Europe this has been associated with the decline of religion  is used. In this study, 2001 and 1991 Census returns and birth registrations can be linked at the individual level. This results in annual fertility data of a panel of over 108,000 women covering the years 1997–2007. The construction of these data, referred to hereafter as the fertility panel, is central for addressing the research questions outlined above.The Northern Ireland Longitudinal Survey provides the individual data that are used in the construction of the fertility panel. The NILS does not conduct any population surveys itself but rather anonymizes the individual returns used in the construction of the official, aggregate data. The official data on birth registrations and the Censuses are reviewed in the first two subsections which are then followed by a subsection that outlines how these data are combined by the NILS. The final two subsections are devoted to the statistical nature of the study.Birth registrations are a legal obligation and fall under the responsibility of the General Register Office (GRO). The data derived from the registrations (and mid-year population projections based on the Census) are aggregated and summarized in tables that accompany the Annual Report of the Registrar General .The enumeration form of the Census asks whether the individual belongs to any particular religion. For those respondents answering ‘yes’, religious affiliation can be aggregated into Catholic, Protestant (and other Christian) and Other. In the statistical analysis, Catholics and Protestants are represented by dummy variables. If respondents answered ‘no’, then and only then, the respondent is asked what religion he or she was brought up in (which can be aggregated as mentioned before). This information permits former religion also to be included as dummy variables.It is important in this work that the Census information on religion is unbiased. In 1981, 18.5 % of respondents declined to state their religion. In 2001, ‘no religion or not stated’ amounted to 13.5 % of the enumerated population. Unfortunately, the two groups were combined during processing of the Census. In an analysis of the raw data, approximately 10 % stated ‘no religion’ and 4 % left the question unanswered  of which there were 890 in 2001. The rotated principal components (RPC) of eleven relevant socio-economic variables from the 2001 Census provided four components, named RPC1–4, that explain 91 % of the variation in the data. Details can be found  in the online Appendix 1*.Published Census data relating to the locality that an individual resided in was employed as an indicator of determinants relevant to individual fertility decisions (such as income) but which are not available at an individual level. The proportion of persons from a Catholic community background, RPC1 which is the only rotated variable where the Census education variables load substantially. It may thus be considered as inversely related to potential economic well-being. It accounts for a third of the variance in the locality variables. RPC2 is the only other rotated component that is significant in the regression analysis   model is frequently employed  the explanatory variables and so the estimated coefficients for the variables The key concept in attempting to quantify the effect of religion is the marginal effect .The variables employed in the statistical model were  defined in Table CATHOLIC dummy variable. The results are presented on the left-hand side of Table PERIOD and LOCALITY sets and predominantly relate to the Catholic subset (period and cohort variables were only dropped if the entire set was insignificant). The discussion below centres first on the variables that were dropped and then interprets the remaining ones.Equation  was estiPERIOD variables is an important result as it demonstrates that the two communities responded in similar ways to shocks suffered by the Northern Ireland economy. It thus suggests that the forces that stimulated the revival of fertility early in the twenty-first century affected religious groups in a similar fashion. The proportion of A8 migrants in a locality did affect domestic fertility but in a similar fashion in both communities.The insignificance of specifically the Catholic set of Secondly, the Northern Ireland economy changed appreciably in the period 1997–2007, but this development had a similar effect on the two communities given that the period effects did not differ. The correlation between the estimated period effects and the female activity rate is positive and strong (the correlation coefficient between the two series is 0.82).PP, the proportion of Protestants in the locality if the woman herself is a Protestant. Its effect on the latent index is negative which suggests that the more numerous Protestants are in a locality, the lower is their fertility. CATHOLIC, the religion dummy variable, on its own was insignificant and thus dropped. This implies that the constant in the parsimonious model is the same for Catholics and Protestants. Thus, there is no general increase in Catholic fertility, but rather it is restricted to those variables that are significant in the final column of Table It is possible that a woman’s religiosity would be influenced by the proportion constituted by her co-religionists in her particular locality. The only variable that is significant is In contrast to the above variables where the Catholic dummy was insignificant, the cohort variables were found to be highly significant and indicate that differential decline in Catholic cohort effects increased for women born between 1958 and 1967 and then plateaued before increasing again for those born 1983–1992. That the demographic profile differs between the two communities is clearly demonstrated by the highly significant differentials for Catholics across all the variables. The effect of this can be best viewed through considering the marginal effects.MECatholic, is reported in Table MECatholic is 0.0051 which occurs when the woman is 29. Except for a slight decline at ages 20 and 21, the marginal effect steadily increases to 29 and then declines to near zero by age 44. Thus, the fertility differences between the two communities follow their demographic profiles with the Catholic one being slightly above the Protestant one.The individual marginal effect is defined as the difference in the predicted probabilities of a woman with a particular set of characteristics having a child in a particular year when her religion is changed from Protestant to Catholic. The average marginal effect of being a Catholic, Further evidence on the extent of convergence in fertility between the two religious groups is provided in Figs. MECatholic is averaged by year and graphed, showing little variation. Catholic fertility is slightly but distinctly higher than that of Protestants, and this differential is stable in the new millennium.Again, the graph suggests that there was very limited convergence in fertility rates between 1997 and 2007. In Fig. The methodology employed in the previous two sections has also been applied to test differences in fertility between current and former Catholics and Protestants. First, those who declare themselves as Catholics are compared to former Catholics. The equality of coefficients between the two logits is firmly rejected by the Likelihood Ratio Test, and it is possible to reduce the full model from 69 to 36 variables (full results can be found  in online Appendix 2*).The mean marginal effect for the panel is −0.0033, which is greater in absolute value than that between self-declared Protestants and Catholics. Figure The differences between Protestants and former Protestants are more marked compared to the Catholic case. The Likelihood Ratio Test rejects the equality of coefficients, although the reduction process leads to only 13 variables being dropped. The mean marginal effect is −0.0017, though the median is 0.0010. Figure The final part of the analyses compares the fertility of former Protestants and Catholics. Secularization is considered an important element of the second demographic transformation (van de Kaa L01) and another which combines the higher groups 4 and 5 (L45). This is reproduced at the individual level by the dummy variables DL01 and DL45.Table 2 is almost six times greater, the logit explaining former Catholics is considerably better than that explaining former Protestants. According to the 2001 Census, 14 % of Protestants by background declared they had no current religious affiliation compared to 8 % of Catholics. The second result of note is that the greater the proportion of the population of the neighbourhood made up of people with the same community background, the less likely a woman loses her religious affiliation. The final point relates to the role of education. For both Protestants and Catholics, the signs of the locality effects are negative and thus the larger the proportion of poorly or highly qualified women in it makes it less likely that the woman concerned will lose her religion. On the other hand, if a particular woman herself is lower or higher educated, then she is more likely to lose her religion. The marginal effect sheds light on this apparent contradiction.There are three striking results of these analyses Table . FirstlyThe definition of the marginal effect of education on the probability of losing a religious affiliation has to take into account that there are three educational categories. The average change of a Protestant woman as she moves from the lowest educational attainment to the middle one (groups 0 and 1 to groups 2 and 3) is −0.0274 while moving to the highest groups, 4 and 5, from the middle has an average change of 0.0801. Thus, those with middling education are likely to be the most religious, while those at the extremes, with little or higher education, are more likely to lose their religious affiliation. For Catholics also, those with middling education are least likely to lose their religious affiliation with the marginal effects being −0.0466 moving up to middling education and 0.0604 moving beyond it. Over time, the increase in higher education is likely to have discouraged religious affiliation.The central issue tackled in this paper is the contribution of religion to fertility in contemporary Northern Ireland. To address this, the Northern Ireland Longitudinal Study has been employed which in this case matches the records of women from a sample of the Census and the Birth Registration Data. This results in the construction of a panel consisting of over one hundred thousand women in the period 1997–2007. Using these data has two major benefits. Firstly, the difference between groups in the population can be expressed quantitatively by the marginal effect estimated from regression analysis where the latter controls for differences in demographic and socio-economic structure between groups. The marginal effect thus expresses the differential fertility of subgroups of the population as a statistic. This can be disaggregated over time so that the extent of convergence between groups in the period considered can be assessed. Secondly, the NILS allows the definition of groups to be developed. This allows the effect of religion on fertility to be compared not just between Protestants and Catholics but also, to highlight contribution of religion by itself, by comparing Protestants with former Protestants and Catholics with former Catholics. Finally, comparing the fertility of former Protestants and Catholics provides insight into the extent of secularization in Northern Ireland.The results show that the marginal effect of a being a Catholic woman is a 4 % higher fertility compared to a Protestant. This difference is small but significant though it is likely to fall in the future because there has been a marked decline in the fertility across older Catholic cohorts and these will drop out with the passage of time. Among younger cohorts, the difference between Catholic and Protestant fertility is less marked. Against this, there is no evidence of convergence for the panel period. No significant difference between the period effects of Catholics and Protestants is found. This suggests that economic and social change during the time examined impacted both communities in a similar way. Changes in the trade-off between childbearing and labour market participation have been put forward as an explanation of the recent fertility revival Kalwij . The picMarginal effects are particularly informative when comparing those with religion to those without it. The magnitude of the marginal effect comparing Catholics with former Catholics is larger than that between Catholics and Protestants. This suggests that the pure fertility impact of religion in this case is considerable since both groups would have a similar background. When such an analysis is carried out on Protestants, the results are similar but less robust. Of particular interest then is the fertility behaviour of former Protestants and former Catholics. The marginal effect in this case is the smallest of the four comparisons though it is still just significant at the 5 % level. Over time the marginal effect declines and gives a firm indication of convergence.The overall picture of fertility in contemporary Northern Ireland by these statistical results can be presented in terms of the groups concerned. Protestants and Catholics form the religious groups, and these are distinctive in a small but definite fashion. It is arguable when considering former Protestants and Catholics that they together comprise one group. While the marginal effect is just significant between the two sets, its magnitude is the smallest of the four sets considered. Moreover, over the period considered, the two birth rates show definite convergence. If a single group is designated, then it can be considered as secular. Secularism though is not a central element within the future of fertility in Northern Ireland. The group is small as it comprises only 13 % of the population in 2001. Moreover, the Northern Ireland Life and Times Survey gives no impression of a fall in religious commitment in the period considered. Given the higher birth rate of the religious groups, if anything secularism is under demographic pressure. In this it reflects a general European trend Below is the link to the electronic supplementary material.


With this study, the authors hope to alert clinicians regarding the presence ofhuman T-cell lymphotropic virus type 1 and 2 (HTLV-1/-2) infections in patientswith viral hepatitis B and C in Brazil. HTLV-1/-2 were detected in 1.3% ofhepatitis B virus (HBV)- and 5.3% of hepatitis C virus (HCV)-infected bloodsamples sent for laboratory viral load measurements. A partial association ofhuman immunodeficiency virus (HIV)-1 and HTLV-1/-2 infection was detected inpatients with HCV , whereas this association was almost 100% inHBV-infected patients . The high prevalence of HTLV-1/-2infection among patients with hepatitis C was of concern, as HTLV-1/-2 couldchange the natural course of subsequent liver disease. The authors suggestincluding HTLV-1/-2 serology in the battery of tests used when followingpatients with viral hepatitis in Brazil, regardless of the HIV status. Human retroviruses, particularly human immunodeficiency virus (HIV)-1 and the humanT-cell lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2), are endemic in Brazil. NotablyThe Epidemiological Bulletins of HIV/AIDS and Viral Hepatitis from the Brazilian MHdisclose the number of infected individuals according to geographic region, state, sex,and age , b. HoweIn Brazil, HTLV-1 is endemic among individuals of African origin , whereasAlthough HIV-1, HTLV-1/-2, HBV, and HCV share some common routes of infection, the risksof acquiring these infectious agents vary considerably in Brazil, and are dependent onthe genetic backgrounds of individuals, geographic region, socioeconomic conditions, andrisk behaviours .Dual and/or triple co-infections involving HIV-1, HTLV-1, HTLV-2, HBV, or HCV can occurand may contribute to changes in the natural course of each subsequent viral disease.For instance, HIV/HTLV-1 co-infection has been associated with a rapid progression toAIDS and a shorter survival time . FurtherThe mechanism by which viral co-infections might affect the outcomes of subsequentdiseases is not completely understood, although some hypotheses have been devisedregarding the higher odds of HCV clearance in HTLV-1 and/or HIV/HTLV-1 co-infectedpatients. Changes in the immunological environment, particularly the balance between theTh2 immunological response induced by HCV and the Th1 response induced by HTLV-1, couldbe an explanation. Specifically, an increase in the spontaneous production of IL-1,IL-2, and IFN-g, reduction in the production of IL-4 by mononuclear cells, and elevatedserum levels of pro-inflammatory cytokines might explain this finding .However, as the benefit of HTLV-1 co-infection had not been confirmed in patients withhepatitis C from other parts of the world, except in Brazil , furtherStudies describing the clinical outcomes of HBV and HCV single-infected individualsversus those co-infected with HTLV-1/-2 in Brazil are scarce, and no studies havediscussed the prevalence of such co-infections. Therefore, we conducted a preliminarystudy on the prevalence of HTLV-1/-2 among HBV- and HCV-infected individuals.Plasma samples from 1,244 individuals with viral hepatitis were sent to Instituto AdolfoLutz, a public health laboratory in São Paulo, for HCV and HBV viral load analysis. Thesamples were screened for HTLV-1/-2 infection using an enzyme immunoassay , and were confirmedby a line immunoassay . HIV-1infection was investigated using an immunochromatographic assay . All assays were performed accordingto the respective manufacturers’ instructions. Plasma samples were collected from 622HCV-infected individuals , and 622 HBV-infectedindividuals . The study was approved by the EthicsCommittee for Research of Instituto Adolfo Lutz (CTC #21I-2016), under the MH protocolnumber CAAE - 55837316.0.0000.0059.During the HTLV-1/-2 screening of 1,244 plasma samples, 44 reacted positively; 25confirmed HTLV-1 infection , 16 samples confirmed HTLV-2 infection. Two samples were indeterminate (both G1), and one was negative (G2).The HTLV-1/-2 serology results by group (G1 and G2) and the HIV results are presented inthe The results obtained from the present study indicate that 5.3% of HCV-infectedindividuals from the city of São Paulo were co-infected with HTLV-1/-2. This percentageis concerning, as it is higher than that reported in populations considered at “highrisk” for HTLV infection, such as men who have sex with men (MSM) and HIV/AIDS patients.In fact, in one study on MSM from Campinas, a city 100 km distant from São Paulo, theHTLV-1/2 infection rate was 1.5% . In receAnother important point that emerged from the present study was the finding that only27.3% of HCV/HTLV-1-2 co-infected patients were also HIV-1 seropositive. As theBrazilian MH has recommended a HIV-1 serologic analysis during viral hepatitis follow-upand as HTLV-1/2 serology has been suggested during the follow-up of HIV-1-infectedpatients, we might speculate that HIV-1 seropositive patients could indirectly knowtheir HTLV status. However, HIV-1 seronegative patients with HCV infection (72.7%)likely would not know their HTLV status.In addition, although HCV/HTLV-1 co-infection has been associated with better outcomesamong patients in Brazil , co-infeRegarding hepatitis B, only 1.3% of patients were co-infected with HTLV-1/-2, and thesedata corroborate our previous finding of a minor OR for HBV/HTLV relative to HCV/HTLVco-infection among HIV/AIDS patients . Among HRegarding patient sex, although HTLV-1/-2 acquisition tends to occur preferentially amongfemale subjects , in the Of note, the Global Virus Network Task Force on HTLV-1 recommends that transplantsocieties should make extensive use of screening assays and should instruct donors andrecipients about HTLV-1/2 infection, transmission, and disease prevention, as high ratesof ATLL and HAM have been detected in HTLV-1-infected patients after organtransplantation .Taken together, our data lead us to suggest the inclusion of HTLV serology in the batteryof tests used to follow up hepatitis virus-infected patients, regardless of their HIVstatus, particularly in HTLV-1/-2-endemic regions of Brazil. Surveillance of the currentstatuses of these viral infections could assist physicians with performing accuratepatient follow-ups .


However, direct translation of these models and settings to animal studies might be sub-optimal. Therefore, this study evaluates pharmacokinetic models for the quantification of [11C]flumazenil binding in the rat brain.Preferred models for the pharmacokinetic analysis of [11C]flumazenil brain PET scans were performed in two groups of male Wistar rats (tracer dose (TD), n = 10 and pre-saturated (PS), n = 2). Time-activity curves from five regions were analyzed, including the pons (pseudo-reference region). Distribution volume (VT) was calculated using one- and two-tissue compartment models (1TCM and 2TCM) and spectral analysis (SA). Binding potential (BPND) was determined from full and simplified reference tissue models with one or two compartments for the reference tissue . Model preference was determined by Akaike information criterion (AIC), while parameter agreement was assessed by linear regression, repeated measurements ANOVA and Bland-Altman plots.Dynamic (60 min) flumazenil in the rat brain based on (1) higher fit quality, (2) lower AIC values, and (3) ability to provide reliable fits for all regions. Reference models resulted in negatively biased BPND and were affected by specific binding in the pons of the rat.Although the 1TCM was sufficient for the quantification of high specific-binding regions, the 2TCM was found to be the most adequate for the quantification of [ For plasma input methods, the one tissue compartment model (1TCM) was determined to be the preferred model [11C]flumazenil quantification, the simplified reference tissue model (SRTM) was considered valid and robust flumazenil binding have only been assessed in human studies and, therefore, are related to the behavior of this radiotracer in the human brain. Surprisingly, there has not yet been a careful assessment of the performance of the different pharmacokinetic models for rat studies. Nonetheless, pharmacokinetic modeling can be of particular relevance in the pre-clinical setting, since animal studies are often applied in drug development, longitudinal disease monitoring, or treatment response assessment. There, assessing the full tracer kinetic profile in tissue is preferable above semi-quantitative measures such as standard uptake values (SUVs) or SUV ratios (SUVR) [11C]flumazenil brain kinetics can be questioned. Therefore, the purpose of this study was to investigate which pharmacokinetic model is most appropriate for the quantification of [11C]flumazenil binding in pre-clinical rat studies. In order to do so, we retrospectively analyzed PET rat brain data, as well as simulated data, by a number of different methods, including compartmental (plasma input and reference tissue based) and non-compartmental methods.However, the superiority, validity, and accuracy of the 1TCM and the SRTM for the quantification of flumazenil over 60s. Dynamic PET data were acquired in list mode for 60 min, starting from the moment the tracer entered the body. For the PS group, unlabeled flumazenil  was injected intravenously 5 min before tracer administration flumazenil and the cold flumazenil were obtained from ABX  in order to ensure high chemical purity (>95%). The labeled [11C]flumazenil was then synthesized in our laboratory at the UMCG as described elsewhere [The precursor of [lsewhere . MoreoveList-mode data was reconstructed into 21 frames  including all necessary corrections to obtain quantitative emission images. Sinograms were Fourier rebinned and reconstructed by a 2D-OSEM algorithm  with 4 i11C]flumazenil template flumazenil in human studies [11C]flumazenil in animal studies. Therefore, the present study evaluated different pharmacokinetic models for the quantification of [11C]flumazenil binding specifically for the rat brain.The 1TCM and the SRTM were previously established as methods of choice for the quantification of flumazenil are polar compounds and, as such, are expected not to enter the brain flumazenil binding in rat studies should not be performed with the 1TCM, as it is the case in human studies. In fact, the 2TCM is required for an accurate VT estimation in low-density regions such as the pons, and it provides VT values equivalent to 1TCM for the high-density regions. In the case of reference-based modeling, the use of the pons as a reference region requires careful consideration. Since the rat pons demonstrated a considerable level of specific binding, any direct estimation of BPND via reference models will suffer from underestimation of tracer binding. However, the pons might be considered a pseudo-reference region flumazenil kinetics is different between rats and humans, and indicate that kinetic models used in clinical studies are not necessarily suited in the pre-clinical setting. For high-density regions, both 1TCM and 2TCM estimated comparable VT values and resulted in similar quality of fits (AIC). However, the application of the 2TCM in low-density regions resulted in higher quality of fits, both from visual inspection and AIC values. Moreover, the clinically applied 1TCM was not able to satisfactorily fit the data  of those regions, nor in the case of pre-saturation with cold flumazenil. Therefore, the 2TCM is the preferred plasma input model for the quantification of [11C]flumazenil in the rat brain. Among the various reference tissue models tested, SRTM was preferred providing BPND estimates in good agreement with plasma input 2TCM based DVR–1 values.This study showed that [


Iron is an essential micronutrient for plants, and plants have evolved adaptive mechanisms to improve iron acquisition from soils. Grafting on iron deficiency-tolerant rootstock is an effective strategy to prevent iron deficiency-chlorosis in fruit-tree crops. To determine the mechanisms underlying iron uptake in iron deficiency, two iron deficiency-tolerant citrus rootstocks, Zhique (ZQ) and Xiangcheng (XC), as well as iron deficiency-sensitive rootstock trifoliate orange (TO) seedlings were studied. Plants were grown in hydroponics system for 100 days, having 50 μM iron (control) and 0 μM iron (iron deficiency) nutrient solution. Under iron deficiency, more obvious visual symptoms of iron chlorosis were observed in the leaves of TO, whereas slight symptoms were observed in ZQ and XC. This was further supported by the lower chlorophyll concentration in the leaves of TO than in leaves of ZQ and XC. Ferrous iron showed no differences among the three citrus rootstock roots, whereas ferrous iron was significantly higher in leaves of ZQ and XC than TO. The specific iron absorption rate and leaf iron proportion were significantly higher in ZQ and XC than in TO, suggesting the iron deficiency tolerance can be explained by increased iron uptake in roots of ZQ and XC, allowed by subsequent translocation to shoots. In transcriptome analysis, 29, 298, and 500 differentially expressed genes (DEGs) in response to iron deficiency were identified in ZQ, XC, and TO, respectively (Fold change ≥ 2 and Probability ≥ 0.8 were used as thresholds to identify DEGs). A Gene Ontology analysis suggested that several genotype-specific biological processes are involved in response to iron deficiency. Genes associated with cell wall biosynthesis, ethylene and abscisic acid signal transduction pathways were involved in iron deficiency responses in citrus rootstocks. The results of this study provide a basis for future analyses of the physiological and molecular mechanisms of the tolerance of different citrus rootstocks to iron deficiency. Iron is an essential nutrient for plant growth since it takes part in most important primary metabolic processes, including photosynthesis, respiratory electron transport, nitrogen assimilation, and so on. Although the total iron content in soil is relatively high, its acquisition by crop plants is often limited owing to its low bioavailability in well-oxygenated and alkaline soil conditions . Chloros+-ATPases in roots, which lowers the rhizosphere soil pH  Raf  Raf . A set o(L.) Raf .In our previous study, we found that Zhique (ZQ), a novel citrus rootstock native to China, shows strong tolerance to iron deficiency in calcareous soil . Here, tCitrus wilsonii Tanaka) were obtained from Chenggu Fruit Industry Technical Guidance Station, seeds of Xiangcheng (C. junos Sieb. ex Tanaka) were collected and provided by the Institute of Horticulture Research, Sichuan Provincial Academy of Agricultural Science, China, and seeds of TO [P. trifoliate (L.) Raf.] were obtained from the National Citrus Breeding Center in Huazhong Agricultural University, China. As described in our previous work  was determined using a leaf area meter . The root samples were scanned using an Epson digital scanner  and images were analyzed using WinRhizo Pro (S) version 2009c . The root traits examined included total root length (cm), root surface area (cm2), root volume (cm3) and root number. The fresh materials were placed into a forced air oven at 105°C for 15 min, and then at 75°C until constant weights were reached to determine dry weights (g). All dried samples were ground into a fine powder to determine iron concentration in various tissues. Seedling height (cm) and taproot length (cm) were measured using a measuring scale.At the end of the experiment (100 days treatment), nine plants per treatment were harvested randomly and were rinsed with deionized water. Then the samples were divided into leaf, stem, and root samples. Leaf area  = [(lnR2 - lnR1)/(T2 - T1)] × [(M2 - M1)/(R2 - R1)], where R1 and R2 are the initial and final root dry weights, respectively; T2 and T1are the treatment durations; M1 and M2 are the initial (T1) and final (T2) iron contents per plant.Specific uptake rate  or 0 μM Fe-EDTA (-Fe) treatments for 24 h, and were immediately frozen in liquid nitrogen and stored at -80°C for further analysis.TM 2000 . The raw reads in fasta format were deposited in the NCBI Sequence Read Archive (SRA) database1 and the corresponding run accession number (SRR5272210) was offered.Roots from nine plants were pooled as an independent biological replicate and three independent biological replicates were used in transcriptomic analysis. RNAs were extracted from ZQ, XC and TO roots and used to construct cDNA libraries, with three replicates for each treatment . RNA library construction and sequencing experiments were conducted at Huada Genomics Co. Ltd. . During the QC step, the Agilent 2100 Bioanalyzer  and ABI StepOnePlus Real-Time PCR System were used for qualitative and quantitative characterization of the sample library. The library products were sequenced using the IlluminaHiSeq2 to obtain high quality reads as the clean reads (clean data). The procedure included following steps: (1) Remove reads with adaptor sequences; (2) Remove reads in which the percentage of unknown bases (N) is greater than 10%; (3) Remove low quality reads. If the percentage of the low quality base  is greater than 50% in a read, we define this read as low quality. Subsequently, the proportion of clean reads in raw reads of the libraries was classified. The clean reads were mapped to the sweet orange genome3 . RSEM is a quantification tool that computed Maximum likelihood abundance estimates using the Expectation Maximization (EM) algorithm for its statistical model, including the modeling of paired-end (PE) and variable-length reads, fragment length distributions, and quality scores, to determine which transcripts are isoforms of the same gene . Here FPKM (A) is the expression level of gene A, C is number of reads that uniquely aligned to gene A, N is total number of reads that uniquely aligned to all genes, and L is number of bases of gene A. The FPKM method is able to eliminate the influence of different gene length and sequencing discrepancy on the calculation of gene expression level. Therefore, the FPKM values can be directly used for comparing the difference of gene expression among samples.FPKM method is used in calculating expression level, the formula is following: FPKM = 105. Main functional categories for differentially expressed genes were analyzed according to MapMan functional plant categories  annotation was performed by using Blast2GO software (GO association done by a BLASTX against the NCBI nr database). For the pathway enrichment analysis, all DEGs were mapped to pathway terms in the KEGG databasetegories .260/OD280 ratio) and concentration were measured. Based on the estimated RNA concentration, one microgram of total RNA was used per cDNA synthesis reaction to avoid potential bias in the transcript evaluation. The actin gene was used for normalization. The primer sequences are listed in Supplementary Table The detailed protocol for the quantitative real-time PCR analysis procedure was described previously . BrieflyAll experiments data were tested for differences using analysis of variance, and mean separation within samples by Duncan’s multiple range test .Figure 1A). Consistent with the observed phenotypes, the reduction in chlorophyll a and b content was greater in TO (41 and 41%) than in ZQ (1 and 16%) and XC (31 and 32%) under iron deficiency conditions . The symptoms of iron deficiency and the reductions in chlorophyll a and b content were also obviously greater in XC than in ZQ. However, no differences were detected in leaf dry weight and leaf area among the three rootstocks under iron deficiency .Three citrus rootstock seedlings, ZQ , XC  and TO , were grown in hydroponic culture and subjected to iron deficiency. After 100 days of iron deficiency, the young leaves of TO showed obvious interveinal chlorosis under iron deficiency conditions. By contrast, the ZQ and XC leaves showed only slight chlorosis under iron deficiency treatments .As shown in Figures 3A,D, the total and ferrous iron in ZQ and XC showed no differences in leaves from those of control plant leaves under iron deficiency treatments. However, in leaves of TO both total and ferrous iron were significantly lower than those of control plant leaves. In stems under iron deficiency treatments total and ferrous iron were significantly decreased in ZQ and XC . In roots, total and ferrous iron level in all the three rootstocks were significantly decreased under iron deficiency conditions .Total and ferrous iron concentrations in different tissues were determined to explore iron uptake and allocation in citrus seedlings. As shown in Figure 4). It is worth noting that the specific iron absorption rate was higher in ZQ and XC than in TO under iron deficiency (though the difference between ZQ and TO was not rich a significant), but no difference among the three rootstocks were detected under control conditions (control). With respect to the iron distribution , iron deficiency treatment induced an increase in the leaf iron proportion. Notably, the leaf iron proportions in ZQ and XC were significantly higher than those in TO, regardless of iron treatments. The opposite trend was observed for relative root iron proportion.Iron content per plant and specific iron absorption rate in all the three genotypes were significantly lower under iron deficiency conditions than in control seedlings . Among these DEGs, only two (Cs4g18450 and Orange1.1t00580) were common to the three rootstocks. Five DEGs were common to both ZQ and XC, 7 DEGs were common to both ZQ and TO, and 52 DEGs were common to both XC and TO. Functional categories of the DEGs are summarized in Figure 7. The major categories were “transport” (13.79%),“protein” (6.90%), and “stress” (6.90%) in ZQ, “RNA” (12.65%), “signaling” (8.24%), and “protein” (7.65%) in XC, and “protein” (11.19%), “stress” (10.07%), and “RNA” (9.17%) in TO. In addition, genes involved in “hormone metabolism,” “cell,” and “secondary metabolism” were also highly represented in the three rootstocks. To validate the RNA-seq results, expression levels of 20 DEGs in ZQ, XC, and TO were determined by real-time PCR. As expected, 17 out of 20 genes in ZQ, 19 out of 20 in XC, and 16 out of 20 in TO showed the same expression as detected by RNA-seq , suggesting that the transcriptomic data is reliable.In ZQ, only 29 genes were differentially expressed in response to iron deficiency, including 10 up-regulated and 19 down-regulated genes. In XC, 298 genes were differentially expressed, including 232 up-regulated and 66 down-regulated genes. In TO, 400 genes were differentially expressed, including 234 up-regulated and 166 down-regulated genes . The genes were associated with hormone synthesis and signaling, transcription, transportation, cell wall modification, and degradation, indicating that these genes or biological processes might be conserved in iron homeostasis in both woody and herbaceous plants.The conservation of the mechanisms underlying the response to iron deficiency among citrus and other plant species were examined. The DEGs identified in ZQ, XC, and TO were compared with iron deficiency-related transcriptome data obtained for other species, including thaliana . The DEGFRO gene coding the plasma membrane ferric chelate reductase enzymes, and IRT gene coding for the iron transporter , were analyzed by quantitative real-time PCR at 0, 24h, 48, 72, and 96 h of iron deficiency treatments.Iron related genes were identified by a homology search with genes from sweet orange genome . Iron reFRO gene did not show any significant changes with regard to 24, 48, and 72 h iron deficiency in roots of ZQ and XC whereas FRO expression in both rootstocks increased significantly at 96 h iron deficiency. The FRO expression in TO was same to ZQ and XC at 24 and 48 h iron deficiency, whereas it was up-regulated at both 72 and 96 h iron deficiency in TO.The expression of the Iron-regulated transporter expression in ZQ and XC showed significantly up-regulated at 48, 72, and 96 h iron deficiency. However, the expression of IRT in TO was not significantly changed during the iron deficiency treatments.Zhique and XC are more tolerant to iron deficiency than TO , consistAs predicted, under iron deficiency treatment, the total iron and ferrous iron concentrations in leaves were significantly decreased in TO, but not in ZQ and XC. It is interesting that, under iron deficiency, though the total iron concentrations did not differ among ZQ, XC, and TO, the ferrous iron concentration iron in ZQ and XC were much higher than that in TO. These results supported a previous hypothesis that the ferrous concentration is an important indicator of iron deficiency-tolerance , which cA decrease in the ferrous iron concentration in leaves might be a consequence of lower iron uptake by roots and/or impaired iron translocation from roots to shoots. To clarify this, the iron content per plant, specific iron absorption rate, and iron distribution in different tissues of three rootstocks were investigated. Both the iron content per plant and specific iron absorption rate in iron deficiency-tolerant ZQ and XC were higher than those in iron deficiency sensitive TO under iron deficiency. Consistent with our results, A comparative transcriptome analysis was performed to investigate the molecular mechanism underlying iron uptake in the roots of ZQ, XC, and TO under iron deficiency. More DEGs were detected in iron deficiency-sensitive TO than in ZQ and XC . These results imply that the degree of molecular response could be arranged as TO > XC > ZQ, contrary to the performance of three rootstocks in filed conditions, in which ZQ is the most tolerant to iron deficiency while TO is the most sensitive.FRO and IRT genes, were not detected in our transcriptome analysis. One DEG was annotated as iron transport protein 3 (Cs4g18450), but it was down-regulated by iron deficiency treatment in all three rootstocks. Iron transporters, such as IRT1 are up-regulated in other species, in transcriptome experiments regardless of growth conditions and duration of iron deficiency  for iron deficiency treatment is not long enough, which was further confirmed by our quantitative PCR results. It was suggested that, different from herbaceous plants, the iron may be stored in apoplast which could be available for normal growth in woody plants  genes in XC and TO were up-regulated under iron deficiency. XTH plays roles in cell wall modification by regulating cell wall strength and extensibility, and is involved in diverse environmental stress responses . NotablyMalus xiaojinensis, suggesting that the ethylene signal pathway plays an important role in iron-tolerant species under iron deficiency conditions. Another ethylene transcription factor family including ETHYLENE INSENSITIVE3 (EIN3) and ETHYLENE INSENSITIVE3-LIKE1 (EIL1), physically interact with FIT and activate iron acquisition-related genes under iron deficiency conditions (One gene in ZQ (down-regulated), 16 genes in XC (15 up-regulated and 1 down-regulated), and 10 genes in TO (6 up-regulated and 4 down-regulated) involved in ethylene metabolism were detected in our study. A total of 13 genes involved in ethylene metabolism increased more than twofold in XC under iron deficiency, and only one gene in TO show an increase of twofold. In total, 13 up-regulated genes in XC encoded ethylene-responsive transcription factors (ERF) involved in the ethylene signal transduction pathway. Consistent with our results, several ERF genes are also activated in response to early iron deficiency in iron deficiency-tolerant nditions . Moreovenditions . These scis epoxycarotenoid dioxygenase, NCED), a key enzyme in the biosynthesis of ABA (It is notable that eight genes involved in the abscisic acid (ABA) pathway, an early response to iron deficiency, were detected in TO, including 2 up-regulated and 6 down-regulated genes, but genes in this pathway were not detected in ZQ and XC. Cs6g19380 (ABA 8′-hydroxylase) involved in ABA catabolism , was up-s of ABA , was dows of ABA  was alsoOur study revealed two new findings: (i) iron deficiency-tolerance in citrus rootstocks could be due to a higher uptake rate of iron and increased iron translocation from roots to shoots, and (ii) the cell wall modification, ethylene, and ABA signaling pathways seemed are involved in the acclimation to iron deficiency in citrus rootstocks. This study investigated the physiological and molecular changes underlying tolerance to iron deficiency, and it will contribute to further understand the tolerance response in citrus plants.LF, ZP, and SP conceived the design of this research. LF and QZ performed the bioinformatics data analysis. LF wrote the paper. ZP contributed to the statistical analyses and in the revising of the manuscript. QZ, YS, and WD contributed to the revising of the 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.


Halimium halimifolium and Cistus ladanifer, under controlled and natural conditions, respectively. For the first time, diurnal emission patterns of the diterpene kaurene were detected in real-time by Proton-Transfer-Reaction-Time-of-Flight-Mass-Spectrometer. Kaurene emissions were strongly variable among H. halimifolium plants, ranging from 0.01 ± 0.003 to 0.06 ± 0.01 nmol m−2 s−1 in low and high emitting individuals, respectively. They were in the same order of magnitude as monoterpene (0.01 ± 0.01 to 0.11 ± 0.04 nmol m−2 s−1) and sesquiterpene (0.01 ± 0.01 to 0.52 nmol m−2 s−1) emission rates. Comparable range and variability was found for C. ladanifer under natural conditions. Labelling with 13C-pyruvate suggested that emitted kaurene was not derived from de novo biosynthesis. The high kaurene content in leaves, the weak relationship with ecophysiological parameters and the tendency of higher emissions with increasing temperatures in the field indicate an emission from storage pools. This study highlights significant emissions of kaurene from two Mediterranean shrub species, indicating that the release of diterpenes into the atmosphere should probably deserve more attention in the future.Mediterranean vegetation emits a wide range of biogenic volatile organic compounds (BVOCs) among which isoprenoids present quantitatively the most important compound class. Here, we investigated the isoprenoid emission from two Mediterranean Cistaceae shrubs,  Quantitatively, the most important group of BVOCs are isoprenoids, which fulfil many essential functions in plants. At the (sub-) cellular level, they can protect against oxidative damage4 while at plant and ecosystem level, isoprenoids can serve as signalling compounds within plant-plant and plant-insect interactions as well as protection against biotic and abiotic stress6. Isoprenoids can be synthesized either via the plastidic non-mevalonate  pathway or the cytosolic mevalonate (MVA) pathway7. Emission of volatile isoprenoids can result from de novo biosynthesis, which is directly linked to photosynthetic CO2 assimilation, or from the release from specialized structures serving as storage pools of isoprenoids. Henceforth, when isoprenoid emissions are controlled by de novo biosynthesis, the driving abiotic factors are solar radiation as a factor controlling net CO2 assimilation, and temperature, since it influences (i) enzymatic processes and consequently de novo isoprenoid biosynthesis, and (ii) the volatility of stored isoprenoids. Importantly, de novo biosynthesis ceases when temperatures are higher than the optimum temperature of enzymes involved in isoprenoid biosynthesis, whereas emissions from storage pools may further increase9. In addition, biotic stresses such as herbivory or pathogen attack10, and other abiotic factors such as ambient ozone mixing ratios11, water stress, or soil fertility may indirectly influence isoprenoid emissions13 by affecting plant physiology and thereby the enzymatic production of isoprenoids.Vegetation is the largest global emission source of biogenic volatile organic compounds (BVOCs) with an estimated 1000 Tg of carbon yr5), monoterpenes (C10), and sesquiterpenes (C15), plants also synthesize larger isoprenoids such as diterpenes (C20), sesterterpenes (C25), triterpenes (C30) and even more complex compounds, which are overall assumed to be non-volatile17. High molecular weight isoprenoids carry a plethora of other essential functions as they are involved in crucial processes such as photosynthesis , membrane integrity (e.g. tocopherol), as well as plant growth and development 21. In particular, diterpenes (C20 isoprenoids) have been reported to play an important role in ecological interactions between plants and other organisms, such as signalling and defence against herbivores25; in addition, they provide an essential building block for chlorophyll, which is a diterpene conjugate, thus, diterpenes are also involved in light-dependent reactions of photosynthesis. Moreover, diterpenes can serve as growth regulators controlling crucial processes, namely germination, cell elongation and division, or flower and fruit development24. So far diterpenes were generally considered as non-volatile compounds, even though first records of emissions in three plant species were reported27. However, these measurements were sampled with low time resolution based on pre-concentration techniques, and thus very little is known on the dynamics of diterpene emissions or their role in the atmosphere.In addition to highly volatile isoprenoids, such as isoprene , we classified the plants into the groups of high and low emitters. Plants exceeding the mean emission rates for a specific group of isoprenoids were classified as high emitters, plants showing lower than mean emission rates were defined as low emitters. Mean emission rates were 0.008 ± 0.005 nmol m−2 s−1, 0.032 ± 0.028 nmol m−2 s−1, 0.062 ± 0.104 nmol m−2 s−1 and 0.022 ± 0.016 nmol m−2 s−1 for isoprene, monoterpenes, sesquiterpenes and kaurene, respectively.Substantial emission of monoterpenes, sesquiterpenes and even the diterpene kaurene were found in 2 assimilation rate and stomatal conductance  during the light period than low emitters   Fig. . Interesly) Fig. , the patH. halimifolium, generally highest emission rates occurred before noon  to 0.023 ± 0.02 nmol m−2 s−1 (high emitters), whereas emission rates of monoterpenes and sesquiterpenes of the strongly emitting plants were significantly higher . Although all plants were kept under similar environmental conditions and no visible pest or damaged was observed, biotic factors could have caused such high sesquiterpene emissions.For the other isoprenoids released by oon Fig. . Particuoon Fig. : isopren2 assimilation rates or stomatal conductance  and stomatal conductance (r2 = 0.70), while correlations for the low emitters were weak -β-ocimene and γ-terpinene -β-farnesene, were well correlated with their high emission rates. Interestingly, the very high abundance of eugenol did not drive any emission of this oxygenated sesquiterpenoid. Importantly, kaurene was found in high amounts in the leaves of H. halimifolium in accordance with the high emission rates of this diterpene, and suggesting that emissions were derived from storage pools.To further characterize individual isoprenoids released by ene Fig. . Interesene Fig.  was in l13C-labelling experiments which resulted in a detectable isotopic signal in isoprenoids are shown in Table 2 assimilation rates of 2–3 µmol m−2 s−1 and stomatal conductance of 20–40 mmol m−2 s−1 during the light period. The theoretical natural abundance of the first isotopologue (i.e. abundance of the molecule containing one 13C-atom relative to the light molecule exclusively containing 12C) is: 5.2% for isoprene, 9.7% for monoterpenes, 13.8% for sesquiterpenes and 17.5% for diterpenes . Our measured natural abundance of the isoprenoid isotopologues matched well the theoretical expected percentage, supporting the accuracy and precision of our measurements. Both, monoterpenes and sesquiterpenes quickly incorporated 13C after 30 minutes of labelling with 13C-pyruvate fed to the branches, amounting to 21.2 and 24.6% for monoterpenes and sesquiterpenes, respectively, even though labelling of sesquiterpenes showed a high variability . Nevertheless, it is important to note that these emission rates might be underestimated, since kaurene losses due to ozonolysis, i.e., reactions with OH-radicals, as well as adsorption of kaurene onto tubing surfaces might have occurred in the field.Measurements of kaurene emission from leaves of ons Fig. . Similar2 assimilation rates, which declined with the diurnal rise in temperature .A tendency of higher kaurene emissions along with the diurnal increase in air temperature, vapour pressure deficit (VPD) and to a lower extent photosynthetically active radiation became evident for each measured shrubs Fig. . Clear dH. halimifolium and C. ladanifer, emit the diterpene kaurene into the atmosphere. The observed emission patterns of kaurene were comparable to those of monoterpenes and sesquiterpenes. To the best of our knowledge, only three studies have reported kaurene emissions from vegetation so far, i.e. from Cryptomeria japonica and Chamaecyparis obstusa, the dominant conifer trees in Japan26, and from the moss Physcomitrella patens27. Therefore, BVOC studies of different species or ecosystems have rarely addressed diterpene emissions eventually because of former technical difficulties to detect diterpenes due to the low volatility and high molecular weight17. On the other hand, the Mediterranean plant species studied so far may either not emit this diterpene, or the compound was just overlooked. We speculate that diterpene emission may be restricted to plant species with a highly active secondary metabolism as shown for Cryptomeria japonica30, C. ladanifer31, and H. halimifolium33.Here, we demonstrate that two characteristic Mediterranean shrub species of the Cistaceae family, H. halimifolium were on average 0.034 nmol m−2 s−1  matching those reported earlier for C. japonica and C. obstusa26. Furthermore, monoterpene emission rates of H. halimifolium were comparable with those of other Mediterranean shrubs, such as Cistus albidus or Cistus monspeliensis (0.3–1.03 µg gdw−1 h−1)31 and matched those reported earlier for H. halimifolium34, whereas the sesquiterpene emission rates were slightly higher than those found for other Cistaceae species, but still in the same order of magnitude (0.6–0.63 µg gdw−1 h−1)31. In agreement with earlier studies on species of the Cistaceae31 family and similar to other Mediterranean species, which are rather characterised by high monoterpene and sesquiterpene emissions35, isoprene emission rates were very low (ca. 0.013 nmol m−2 s−1 or 0.05 µg gdw−1 h−1), thus, classifying H. halimifolium as a low isoprene emitter.The observed kaurene emission rates of de novo biosynthesis, i.e. direct emissions after production without long-term storage in particular structures. Our results indicate that kaurene emissions in H. halimifolium and C. ladanifer are not driven by de novo biosynthesis but mainly by the release from storage pools, which contrasts the emission pattern of monoterpenes and sesquiterpenes. Therefore, we postulate that temperature is a major factor determining kaurene emissions, as this is the main driver for release from storage pools36.Isoprenoid emission might be driven by a temperature dependent release of leaf internally stored compounds and/or from H. halimifolium coinciding with observed emission pattern. These included monoterpenes and monoterpenoids  and sesquiterpenes -β-farnesene). In contrast, some monoterpenes ((E)-β-ocimene, γ-terpinene) were less abundant in leaves but were strongly emitted, suggesting de novo biosynthesis and direct emission after production. Moreover, kaurene emission from leaves of H. halimifolium clearly occurred during night, which strongly suggests a release from storage pools. In addition, kaurene emissions were not directly linked to CO2 assimilation rates, differing from monoterpenes and sesquiterpenes that were better correlated with ecophysiological parameters.The assumption that kaurene emissions and some other isoprenoids derived from storage pools is based on the high abundance of kaurene and other isoprenoids in leaves of H. halimifolium, we performed 13C-labelling studies by applying position labelled 13C-pyruvate, which has been proven to rapidly be incorporated into freshly synthesized BVOCs34. The observed incorporation of 13C derived from pyruvate into the emitted monoterpenes and sesquiterpenes suggests that these compounds are at least partially derived from de novo biosynthesis. This is supported by the clear discrepancy between considerable emissions of (E)-β-ocimene, γ-terpinene and β-pinene, and the low abundance of these compounds in the leaves. It is generally thought that the biosynthesis of monoterpenes occurs via the MEP pathway within the plant chloroplasts, which directly depends on the plastidic availability of pyruvate and glyceraldehyde-3-phosphate. Therefore, it must be assumed that the pyruvate fed through the transpiration stream to the leaves, was taken up by mesophyll cells and subsequently channelled into chloroplasts37. On the other hand, sesquiterpenes are mainly synthesized through the cytosolic MVA pathway41 and the incorporated 13C of pyruvate most likely entered this pathway after conversion of pyruvate into acetyl-CoA7. In contrast to monoterpenes and sesquiterpenes, no sign of 13C-incorporation was obtained for kaurene. This strongly indicates that kaurene emission was mainly mediated by the release from storage pools as also suggested by the high kaurene abundance in leaves.To further disentangle the origin of the isoprenoids emitted by C. ladanifer under natural conditions. This tendency further indicates the important role of temperature for isoprenoid emissions from storage pools, in accordance with earlier work16 on temperature effects for kaurene emission rates. Furthermore, in the field, kaurene emission rates were highest at high temperatures when rates of net CO2 assimilation and stomatal conductance were low. During the Mediterranean summer, high CO2 assimilation often occurs only in the early morning hours. Thereafter, temperature and VPD increases inducing stomatal closure to protect plants against excessive water loss on very hot and dry summer days. Therefore, this is another hint of evidence that kaurene emissions were mainly driven by the diurnal increase in temperature and to a lesser extent by plant physiological processes and delivers further support to the premise that diterpene emissions may be mainly derived from storage pools and not by de novo biosynthesis.The important role of temperature for the release of isoprenoid from storage pools is further supported by the field measurements that showed a clear tendency of higher kaurene emission rates at higher temperatures for H. halimifolium and C. ladanifer, not only for kaurene but also for other isoprenoids, which is in good agreement with similar studies for other plant species43. Given that in the laboratory experiments all plants were kept under equal conditions, there might be a strong genetic plasticity among H. halimifolium individuals explaining the observed high variability. A high ecological45, phenological46 and genetic diversity47 has already been reported for the Cistaceae family. Therefore, as expected, C. ladanifer individuals differed also considerably in the magnitude of emissions, although under field conditions, other factors beyond genetic or phenotypic plasticity might have played a role. For instance the observed strong variability of kaurene emission rates by different individuals of the same species under field conditions could also be related to defence responses against pathogens and herbivores, as diterpenes have been found to act as phytoalexins, as constituents of oleoresins, or as activators of the systemic acquired resistance24. Furthermore, in the field, heat stress usually co-occurs with high radiation levels. Such excessive radiation levels constitute stress to plants under summer drought41; biosynthesis of kaurene could be involved in the prevention of photooxidation or light damage, by dissipating excessive energy from the electron transport chain into organic compounds. This is in line with the fact that H. halimifolium and C. ladanifer possess highly protective mechanisms against excessive irradiance and temperature48, as well as water stress48, similar to other Cistaceae species50. This study demonstrates that the kaurene released into the atmosphere by H. halimifolium and C. ladanifer seems to originate mainly from storage pools, and that plant internal kaurene contents together with prevailing temperature are most likely decisive drivers of emission rates. Nevertheless, the genetic or phenotypic plasticity of the Cistaceae family and their impact on modulating isoprenoid emissions together with the physiological and ecological function of kaurene in Cistaceae and other plant families deserve further attention.The results of this work point towards a vast intra-specific variability of emission rates for both, H. halimifolium is a species able to successfully colonize dry and mesic dune habitats51, while C. ladanifer has a high spatial abundance52 and in the last decades, most probably due to environmental and land use changes, has rapidly expanded in drier regions53. Although we cannot state the ubiquitous character of the kaurene emissions in Mediterranean ecosystems, our results show that kaurene is emitted at least by two Cistaceae species, and therefore the atmospheric relevance of their diterpene emission should be investigated. Up to date, there is no information about possible atmospheric implications of diterpenes, as they were considered non-volatile and, thus, have not been studied in an atmospheric chemistry context. Despite the assumed low reaction rate constant of kaurene towards ozone and hydroxyl radicals of 1.2 × 10−17 cm3 molec−1 s−1 and 7.2 × 10−13 cm3 molec−1 s−1, respectively , new studies should investigate the role of diterpenes in atmospheric chemistry and their potential to form secondary organic aerosols. Importantly, neglecting this type of compounds for atmospheric chemistry could also add to the so-called missing reactivity observed at some locations in the world56.13C-labelling indicated that emission of kaurene is most likely not derived from recently synthesized metabolites, whereas monoterpenes and sesquiterpenes revealed a partial de novo biosynthesis of labelled metabolites. Furthermore, concurrence of high leaf content and emissions of kaurene support the assumption that storage pools are the source of emission. This study demonstrates emissions of diterpenes by vegetation and indicates that this compound class may deserves further attention, similar as other, extensively studied, isoprenoids.Here, we demonstrated emissions of the diterpene kaurene under natural and laboratory conditions from two Cistaceae species and quantified emission rates by PTR-TOF-MS and TD-GC-MS. Both species revealed a strong individual variability in the magnitude of kaurene emissions, which were comparable to that of other emitted isoprenoids. Pyruvate position specific Halimium halimifolium L., a typical Mediterranean dune shrub species, which is a main component of stable sand vegetation and is a generalist able to successfully colonize dry and mesic dune habitats51. All H. halimifolium individuals used for the diurnal courses of isoprenoid emissions (n = 7), labelling experiments of isoprenoid emissions (n = 10) and plant material extraction (n = 5) were grown from seeds in plastic pots (size: 15 × 15 × 11 cm) on a mixture of sand (1/3) and soil substrate (2/3) of medium structure in a greenhouse for 1.5 and 5 years. Plants were supplied with 100 ml of a modified Hoagland solution46 once a week33. Two months prior to the experiments, plants were transferred to a well-ventilated walk-in growth chamber, with fully controlled environmental conditions . Light conditions were set to 500 µmol m−2 s−1 for 12 hours (9:00 to 21:00 h). Temperature and humidity were constant at 25 °C and 60%, respectively for all the experiments.We measured a semi-malacophyll shrub from the Cistaceae family, 2 assimilation and BVOC emission measurements was used. This system provides 1 L of purified air, produced by a custom build zero air generator, to inert borosilicate glass cuvettes (volume 0.6 L). This air is free of oxidants and volatile organic compounds (VOC), and it is supplied at a known and constant humidity of 30% and CO2 mixing ratio of 400 ppm. Besides the inertness of the materials, all tubing lines  were made of perfluoroalkoxy (PFA) and were heated and isolated (50 °C) to avoid condensation and compound losses to tube walls. The sharp changes between plant and empty control cuvette in isoprenoid emissions (4 seconds) confirm minimum memory effect in the sampling lines. Determination of leaf isoprenoid emissions of H. halimifolium with this system took place in July and August 2016. At the cuvette outlet, air was drawn over to the PTR-TOF-MS, which was further connected to the differential infrared gas analyser  for determination of leaf CO2 assimilation (A) and stomatal conductance (gs). Furthermore, air from the cuvette outlet was drawn  over adsorbent tubes filled with 20 mg of Tenax TA 60/80 and 30 mg of Carbotrap B 20/40 for subsequent isoprenoid analysis by Thermodesorption-Gas chromatograph-Mass Spectrometer (TD-GC-MS). For this purpose, air samples were taken for 60 min at a flow of 200 cm3 min−1 (STP), leading to a collection of 12 L of cuvette air.Measuring system of isoprenoid emissions and ecophysiological parameters: A newly designed measuring system for plant COH. halimifolium were analysed under standard diurnal conditions  along with net CO2 assimilation and stomatal conductance in seven H. halimifolium individals. Diurnal courses were measured from 07:00 h to 23:00 h, where the light period was from 09:00 h to 21:00 h.Diurnal courses of isoprenoid emissions by et al.57. For this purpose, leaf samples were homogenized under liquid N2 and aliquots of 50 mg were added to 2000 µl methanol. Following 30 min of stirring at 30 °C and a subsequent centrifugation, five PDMS tube pieces (5 mm length) were put into 400 µl supernatant, which was diluted with 1600 µl H2Odemin. Stirring occurred at 1,400 rpm for 60 min at 30 °C. After quantitative adsorption of isoprenoids onto PDMS-tubes, they were shortly dried with a lint free paper tissue, placed into glass tubes and analysed without further storage. Analysis occurred as described for air samples after TD-GC-MS.Plant isoprenoid extraction: Isoprenoids in leaf storage pools were determined by a modified method of Kallenbach 13C labelled pyruvate, a central metabolite of plant carbon metabolism with fast turn over times and a precursor for many BVOCs. 13C labelled pyruvate was fed via plant transpiration stream in order to evaluate if the emissions of isoprenoids were derived from de novo plant biosynthesis (i.e. emission of carbon that has been recently photosynthesized) or from other sources, such as storage pools. First, intact branches (put inside cuvette the day prior to measurement) of H. halimifolium were measured, then in the morning the branches were cut at the petiole. The branches were immediately recut under water and the stem was placed in deionized water. Two hours later the water solution was replaced by a 13C-labelled pyruvate solution (10 mM). Pyruvate was 99% labelled with 13C at the C2 carbon position 33. Measurements were performed during three days in August 2016 and December 2017, in which two H. halimifolium individuals per day were measured sequentially. These experiments were also analysed under standard diurnal conditions  along with net CO2 assimilation and stomatal conductance measurements.Labelling scheme: Labelling studies were performed with position-specific Cistus ladanifer L. shrubs growing in a cork oak (Quercus suber L.) woodland in Vila Viçosa , Portugal, in June, July and August 2017 at different times of the day (from 08:00 h to 14:00 h). On each sampling day, three to five different twigs of the same individual were chosen for isoprenoid emission measurements. A total of four different C. ladanifer individuals were screened each month. Ecosystem variables such as air temperature, photosynthetically active radiation (PAR), solar radiation, air humidity, soil water content, VPD, precipitation, as well as eco-physiological parameters of the monitored plants were measured. Mean annual precipitation in the study site is 585.3 mm mostly distributed between October and May and mean temperatures reach 31.1 °C in July and 5.8 °C in January  . More details of the site can be found elsewhere53.Field measurements of kaurene fluxes were conducted in C. ladanifer individuals were enclosed in custom-made cuvettes (~460 ml) made of Nalophan foil and PFA, and connected via PFA tubing to air sampling pumps . These materials were chosen to minimize compound adsorption to tube surfaces due to their inertness. Care was taken that leaves were not damaged and did not touch the cuvette walls when enclosed. To acclimatize branches to the new conditions, cuvettes containing twigs with ca. 8 to 20 leaves were flushed for five minutes at a flow of 200 cm3 min−1 (STP); after that time CO2 assimilation rates and presumably also kaurene emission rates were stable. In addition, one empty cuvette was installed approx. 2 m above ground (at the same level as the plant cuvettes), and measurements from this cuvette were used for background correction for the plant cuvettes. The air supplied to the plant and empty control cuvettes was ambient air with no oxidant removal due to field measurement restrictions. We therefore denote that a possible underestimation of our fluxes in the field could be present due to ozonolysis and OH reactivity sink. For determination of kaurene emissions, air leaving the cuvettes were drawn over adsorbent tubes filled with polydimethylsiloxane- (PDMS) foam . Air samples were taken for 90 minutes in June and for 60 minutes in July and August at an air flow of 200 cm3 min−1 (STD).Selected twigs from the four chosen During sampling, since the monitored branches were normally shaded by the upper canopy, a neutral-density shading mesh was placed above the cuvettes so shading conditions would prevail as constant as possible during measurements. In addition, adsorbent tubes were covered with aluminium foil during the sampling period. Immediately after sampling, twigs were cut and the leaves stored in a cooler for determination of leaf area and dry weight. Adsorbent tubes were stored in a fridge before taking them to the laboratory in Freiburg for TD-GC-MS analysis.In addition, a portable photosynthesis system  was used for A and gs measurements, made sequentially after isoprenoid sampling on leaves that were previously in the cuvettes. The recording frequency of the eco-physiological parameters was 1 second, and for better graphic representation, hourly averages were calculated for each measured plant.m/Δm)58. This newest 4000ultraPTR-TOF-MS model has some improvements such as a built-in internal mass calibration standard , which allows for the calibration of higher m/z scales. In addition, this version has an ion funnel at the end of the drift tube. The PTR-TOF-MS was operated at 2.7 mbar drift pressure, 600 V drift voltage, at an E/N of 120 townsend (Td), and drift tube heated to 80 °C. The recording frequency of the PTR-TOF-MS was 1 second, and for better graphic representation, hourly averages were calculated for each measured plant.In the laboratory studies, real-time measurements of isoprenoids were performed with a 4000ultra PTR-TOF-MS . The PTR-MS technique uses soft chemical ionization allowing for low fragmentation levels. This instrument uses an orthogonal acceleration electron time-of-flight mass spectrometer as detector, capable of measuring VOCs with high sensitivity (up to tens of cps/ppb), low detection limit (to ppt range), fast response time (less than 100 ms) and high mass resolution  normalization of the data to primary ions and water; (iii) background subtraction from the signal with the measurement of VOC-free air, due to impurities that originate from the system; and (iv) application of the calibration factors.The PTR-TOF-MS data processing consisted in: i) correction for non-extending and extending dead times as well as the correction for Poisson statisticsm/z 69.07 (isotopologue m/z 70.07), m/z 137.13 (isotopologue m/z 138.13), m/z 205.20 (isotopologue m/z 206.20), which represent the compounds isoprene, monoterpenes and sesquiterpenes. We also detected substantial signals at m/z 273.20 and its isotopologue m/z 274.20 , a tetracyclic diterpene, that exists in solid form and sublimes at room temperature  was purchased and used to fully proof and quantify kaurene emission rates. The calibration for the PTR-TOF-MS including the calibration of the 12C molecule of kaurene and its isotopologue, is shown in Fig. In the present study, we focused on .20 Fig. . By applure Fig. 19. Once 61, however, the regression between m/z 137.13 and m/z 69.07 revealed a r2 of 0.17, evidencing no fractionation of myrcene on this mass. The total uncertainty of the PTR-TOF-MS determination was calculated according to the error propagation method taking into account the uncertainty of the calibration (including multicomponent gas standard and mass flow controller errors), the instrument error and the background error. The uncertainty of the measurements was determined to be 44% for isoprene, 24% for monoterpenes, 35% sesquiterpenes, and 46% for kaurene. In addition, detection limits were calculated as 3σ of the background averages. They were determined as 0.18 ppb for isoprene, 0.09 ppb for monoterpenes, 0.20 ppb for sesquiterpenes and 0.51 ppb for kaurene.The calibration factors of isoprene and α-pinene were obtained from humid calibrations from a gravimetrically prepared multicomponent gas standard . For β-caryophyllene  and kaurene liquid calibrations were performed. Both calibrations were done using the Liquid Calibration Unit , and since solutions were hexanal based, which is not soluble in water, humidity dependency could not be characterized for β-caryophyllene and kaurene. On the other hand, the comparability with the TD-GC-MS magnitudes gives us confidence on the accuracy of the calibration Fig. . During 62. The fragmentation spectra and available external standards of known concentrations were used for peak identification and quantification, respectively. Both, in field and laboratory studies, emission rates were calculated by subtracting isoprenoid mixing ratios of an empty control cuvette from plant cuvette concentrations and by accounting for the flow rate through the system and the leaf area in the cuvette.All adsorbent tubes were analysed for isoprenoid content using a gas chromatograph  equipped with a mass-selective detector  and a thermodesorption-cold injection system (TDU-CIS) . Details are provided elsewhere−2 s−1 to µg gdw−1 h−1 using a dry weight-to-leaf area ratio of 63.28 obtained from the measurement of leaf areas and posterior dry weighting of such leaves was used.The leaf areas of the branches in the cuvettes was determined using a scanner  and the GSA Imagine Analyser v4.09 . At the field site in Portugal, leaf area was determined with a customary scanner (EPSON EXPRESSION 1680) and analysed with the software WinSEEDLE  in the laboratory. For comparability with other values reported in literature a conversion from nmol mui is the molar flux in the cuvette inlet in mol s−1, s is the leaf area of measured branch in m2, co is the mixing ratio at the outlet of the cuvette and ci is the mixing ratio at the cuvette inlet, both in mol mol−1.The equation used for the calculation of the fluxes was:Statistical significant differences between high and low emitter groups were assessed with a Kruskal-Wallies test as data failed the to meet the assumptions of normality and homeostasis by Shapiro-Wilk. In order to test if the isopreneoid emission rates and eco-physiological parameters for the control condition measurements were statistically significant regression analysis with a p-value of 0.05 were also performed. All statistical analyses were performed by the Sigma plot 2017 software .Data is available upon request to the main author.