Moringa oleifera leaf extract against aspirin-induced ulcers was investigated in rats. Thirty (30) rats under starvation but with access to drinking water for 48 h were divided into 6 groups of 5 animals each. Animals in groups 1 and 2 were pretreated with 0.2 ml normal saline via the oral route. Group 3 received 32 mg/kg cimetidine while those in groups 4, 5 and 6 received oral Moringa leaf extract treatments at doses 200, 400 and 800 mg/kg body weight respectively. Thirty minutes after treatment, all animals in groups 2 to 6 were given 800 mg/kg Aspirin to induce ulcer. Results obtained showed complete erosion of the superficial epithelium with complete loss of the mucus globules and sloughing off of immediate underlying cells and sparsely distributed intraepithelial lymphocytes in the stomach of rats in which no treatment was given and significantly differed from those of the normal control animals which were essentially intact. No significant gastroprotection was observed in rats pretreated with the lowest dose of the extract (200 mg/kg) as a high degree of intestinal mucosal lesions and complete erosion of the surface epithelium with intraepithelial haemorrhage, moderate inflammation and tissue oedema were observed. Pretreatment with 400 mg/kg, however, offered a mild degree of protection with patches of surface epithelial protection and mucus globules, even though there was still predominant disintegration and sloughing off of superficial and underlying epithelial cells. The level of protection was sufficiently increased in animals treated with 800 mg/kg Moringa extract as there was increased protection of surface epithelium with more mucus globules and compared favourably with the effect of Cimetidine in which patches of intact superficial cells were observed. Moringa leaf extract may contain active agents with gastroprotective and mucus enhancing activities and could be harnessed into safe and potent treatment agents for ulcer in addition to providing template for the development of new antiulcer agents.The gastroprotective activity of  Helicobacter pylori  injury and bleeding in more than 50% of its users  has shown that the plant has antioxidant, antimicrobial, anti-inflammatory, antipyretic, antidiabetic, antiulcer, antitumor antidiarrheal and hypocholesteromic properties  rats under starvation but with access to drinking water for 48 h were divided into 6 groups of 5 animals each. Animals in groups 1 and 2 were pretreated with 0.2 ml normal saline via the oral route. Group 3 received 32 mg/kg cimetidine. Groups 4, 5 and 6 received oral Moringa extract treatments at doses of 200, 400 and 800 mg/kg body weight respectively. Thirty minutes after treatment, all animals in groups 2 to 6 were given 800 mg/kg Aspirin to induce ulcer. Ulcer was not induced in group 1 rats. All administrations were done via the oral route. Two hours after Aspirin administration, all animals were sacrificed by cervical dislocation. The stomach of each animal was carefully isolated, incised along the greater curvature, rinsed in normal saline and transferred into 10% formalin solution for histological study.Slices of the isolated stomach were fixed in 10% formal saline for 48 h and were processed by placing them in ascending grades of alcohol in the following order, 50% alcohol for 1 h, 70% alcohol for 1 h, first 95% alcohol for 1 h and second 95% alcohol for 1 h 15 min, first absolute alcohol for one and half hours and second absolute alcohol for 2 h to ensure proper dehydration of the tissues. The dehydrated tissues were then transferred to a mixture of equal volumes of alcohol and xylene where they were left overnight and later cleared with two changes of xylene for 1 h each. They were then infiltrated twice for 1 h each with molten paraffin wax in the oven at 60 °C. The tissues were then embedded in paraffin wax, trimmed and mounted on wooden chuck, and then taken to the microtome for sectioning at 5 μm thickness. The sections were floated in floating-out bath from where they were picked with clean albuminized slides. The slides were placed in a staining dish and excess wax was removed by two changes of xylene, hydrated by descending grades of alcohol in the order absolute alcohol, 95% alcohol and 70% alcohol for 2 min each. The slides were taken to water and then stained by infiltrated Ehrlich haematoxylin for 15 min, and then washed in water for 5 min, differentiated in 10% acid alcohol and blued in running tap for 10 min. They were then counter stained with filtered eosine for 2 min. Excess eosine was removed in ascending grades of alcohol in the order 75% alcohol, 95% alcohol and absolute alcohol for 2 min each. They were then cleared in two changes of xylene and each was cover slipped with depex mountant Clayden . The sliMoringa oleifera extract showed that Aspirin (800 mg/kg) could successfully induce gastric ulcerations in rats as the stomach of rats in group 2 treated with this induction agent alone had complete erosion of the superficial epithelium with complete loss of the mucus globules and sloughing off of immediate underlying cells and sparsely distributed intraepithelial lymphocytes .The reported antibacterial activity of Moringa leaf extract may suggest another mechanism of antiulcer effects but leaves room for further evaluation of this antibacterial effect in relation to inhibiting the growth of Moringa leaf extract may contain active agents with gastroprotective and mucus-enhancing activities and could be harnessed into safe and potent treatment agents for ulcers and may provide template for the development of new antiulcer agents.


Proton-pump inhibitors (PPIs) seem to increase the incidence of cardiovascular events in patients with coronary artery disease (CAD), mainly in those using clopidogrel. We analysed the impact of PPIs on the prognosis of patients with stable CAD.We followed 706 patients with CAD. Primary outcome was the combination of secondary outcomes. Secondary outcomes were 1) acute ischaemic events  and 2) heart failure (HF) or death.Patients on PPIs were older  and had a more frequent history of stroke  than those from the non-PPI group, and presented no differences in any other clinical variable, including cardiovascular risk factors, ejection fraction, and therapy with aspirin and clopidogrel. Follow-up was 2.2±0.99 years. Seventy-eight patients met the primary outcome, 53 developed acute ischaemic events, and 33 HF or death. PPI use was an independent predictor of the primary outcome , along with hypertension, body-mass index, glomerular filtration rate, atrial fibrillation, and nitrate use. PPI use was also an independent predictor of HF/death , but not of acute ischaemic events. A propensity score showed similar results.In patients with CAD, PPI use is independently associated with an increased incidence of HF and death but not with a high rate of acute ischaemic events. Further studies are needed to confirm these findings. These antiplatelet agents, however, may favour the development of gastrointestinal (GI) complications. Prolonged aspirin therapy is associated with GI ulceration and bleeding, which have been attributed to mucosal injury caused by inhibition of prostaglandin and to systemic inhibition of thromboxane A2 production, respectively. In addition, clopidogrel may impair the healing of gastric erosions, exacerbating GI complications associated with the concomitant administration of aspirin [In patients with coronary artery disease (CAD), aspirin is used to decrease the incidence of cardiovascular events, and in patients who have undergone stent placement or have suffered an acute coronary syndrome, a PClostridium difficile infection, acute interstitial nephritis, and micronutrient deficiencies [PPIs are indicated in CAD patients to decrease the risk of upper GI haemorrhage due to antiplatelet therapy . Howeverciencies ,6. In adciencies –11. Althciencies , clinicaciencies ,14.In this study we assessed the potential association between the use of PPIs and adverse outcome in patients with stable CAD who had developed an acute coronary syndrome 6–12 months before.The research protocol complies with the Declaration of Helsinki and was approved by the ethics committees of the participating hospitals. All patients included in the study signed informed consent documents. As described in detail previously, the BACS & BAMI  studies included patients admitted to 4 hospitals in Madrid with either non-ST elevation acute coronary syndrome (NSTEACS) or ST elevation myocardial infarction (STEMI) . DetaileBetween July 2006 and April 2010, 1,898 patients were discharged from the study hospitals with a diagnosis of NSTEACS or STEMI . Of thesAs explained previously, at baseline, clinical variables were recorded and twelve-hour fasting venous blood samples were withdrawn and collected in EDTA. Blood samples were centrifuged at 2,500 g for 10 minutes and plasma was stored at –80°C. Patients were seen every year at their hospital. At the end of follow-up (maximum 4.6 years) medical records were reviewed and patient status was confirmed by telephone contact.The primary outcome was the composite of the secondary end points. The secondary outcomes were 1) recurrence of acute ischaemic events such as NSTEACS, STEMI, stroke, or transient ischaemic attack, and 2) incidence of heart failure (HF) or death from any cause. NSTEACS was defined as rest angina lasting more than 20 minutes in the previous 24 hours or new-onset class III-IV angina, along with transient ST-segment depression or T-wave inversion in the electrocardiogram considered diagnostic by the attending cardiologist and/or troponin elevation. STEMI was defined as symptoms compatible with angina lasting more than 20 minutes and ST elevation in 2 adjacent leads in the electrocardiogram without response to nitroglycerin, and troponin elevation. Stroke was defined as rapid onset of a persistent neurologic deficit attributable to a focal vascular cause and lasting more than 24 hours, supported in most cases by imaging studies. A transient ischaemic attack was defined as a stroke with signs and symptoms resolving before 24 hours without acute ischaemic lesions as assessed by imaging studies. The HF end point was assigned to patients hospitalised for this reason. Events were adjudicated by 2 investigators. Cerebrovascular events were adjudicated with the assistance of a neurologist.Laboratory analyses were carried out in the Clinical Biochemistry Laboratory of the IIS-Fundación Jiménez Díaz by investigators who were unaware of the clinical data. As in previous papers studying this population, high-sensitivity C-reactive protein was determined by latex-enhanced immunoturbidimetry  . Lipid, 2 or Fisher exact test when appropriate. Kaplan-Meier curves and the log-rank test were used to compare time to outcome according to therapy with PPIs. Cox proportional hazards modelling was used with forward stepwise selection to assess the variables associated with the primary and secondary outcomes. In model 1, clinical and analytical variables were studied: age, sex, diabetes, smoking status, hypertension, body-mass index, LDL, HDL, triglycerides, previous history of peripheral artery disease, cerebrovascular events, atrial fibrillation or coronary artery by-pass graft; ejection fraction<40%, glomerular filtration rate assessed as Chronic Kidney Disease Epidemiology Collaboration method (CKD-EPI), high-sensitivity C-reactive protein, type of last acute coronary event, number of diseased vessels, percutaneous or surgical revascularisation, use of drug-eluting stents and existence of complete revascularisation at that event. In model 2, treatment with PPIs and other therapies were added: aspirin, clopidogrel, statins, acenocumarol, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, betablockers, nitrates/nitroglycerin and diuretics. We considered significant when “p” value was lower than 0.05 (two-tailed). Analyses were carried out with SPSS 19.0 .Quantitative data following a normal distribution are presented as mean ± standard deviation and compared using the Student “t” test. Data that were not normally distributed are displayed as median (interquartile range) and compared using the Mann-Whitney test. Qualitative variables are displayed as percentages and were compared by χA propensity score adjustment was performed using a multivariable logistic regression model in 2 steps, including clinical variables in the first one and adding the therapies used in the second step.In conclusion, in this study we have followed 706 patients with chronic CAD, assessing the potential relationship of PPI use with the development of acute ischaemic events, heart failure (HF), or death.Mean follow-up was 2.2±0.99 years. Time from the previous acute coronary event was 7.5 ± 3.0 months.2 receptor antagonists, and the remaining patients did not receive gastric protectors. Patients receiving these drugs were older and had a more frequent history of cerebrovascular events than those not receiving them. No differences were observed in cardiovascular therapy or any other clinical or analytical variables  were receiving PPIs. Most of them ) were taking omeprazole, 19 (2.7%) pantoprazole, 6 (0.8%) lansoprazole, and 1 patient (0.1%) was taking rabeprazole. There were 72 (10.2%) patients treated with histamine Hariables .Seventy-eight patients developed the primary outcome of acute ischaemic events, HF, or death. Sixty-three (14.61%) patients taking PPIs and 15 (5.45%) not on PPIs met this outcome. Twelve patients developed 2 events, 5 patients experienced 3 events and, the remainder, one event with a total of 100 events. At multivariable analysis we included the variables displayed in When multivariable Cox regression analysis was performed comparing patients with omeprazol with those no taking this drug (then including 26 patients using other PPIs in this group) omeprazol was an independent risk predictor  along with hypertension , body-mass index , atrial fibrillation , glomerular filtration rate , and use of nitrates . Comparing patients with omeprazol with the remaining cases not on PPIs confirmed that omeprazol  was an independent predictor of the primary outcome along with the same other described variables. Given that the number of cases on anti-H2 and without gastric protectors was very low it was not possible to perform other comparisons.Fifty-three patients developed acute ischaemic events. Forty-two (9.74%) of the patients on PPIs and 11 (4%) of those not receiving these drugs met this end point. There were 4 episodes of STEMI, 22 of NSTEACS, 17 of unstable angina, 8 strokes, and 10 transient ischaemic attacks. Four patients experienced 2 events and 2 patients experienced 3 events. By multivariable analysis, age, body-mass index, and treatment with nitrates, but not with PPIs, were independent predictors of this end point .Thirty-three patients developed HF or death. This end point was met by 28 patients (6.49%) on PPIs, and by 5 patients (1.81%) not receiving this therapy. There were 16 episodes of HF and 23 deaths, with 6 patients experiencing 2 events. Nine deaths were due to cardiovascular causes (3 of them were sudden death) and 4 were due to malignancies. Infection, renal failure, bowel ischaemia, GI bleeding, and pancreatitis accounted for 1 death each. Five deaths were of unknown cause. Therapy with PPIs was also an independent predictor of this outcome .“p” value (p = 0.050) for the secondary outcome of heart failure or death   and a boor death .Finally, we performed a propensity score analysis. In the first step, we included clinical variables, and treatment with PPIs significantly increased the incidence of the primary outcome . The combined outcome of HF or death showed a result in the limit of statistical significance . In the second step, we added concomitant drug therapy, showing that PPI treatment increased the incidence of the primary outcome , although the combination of HF or death lost the statistical significance .It has been suggested that treatment with PPIs are associated with an increased incidence of cardiovascular events in patients with CAD, mainly in those receiving clopidogrel .In the present study, PPI treatment in patients with CAD was associated with a significant increase in the incidence of HF or death. The distribution of clinical and analytical variables across patients taking PPIs was similar to that of patients not taking these drugs, though with 2 exceptions. First, patients on PPIs were older. This is logical, since age is a risk factor for GI bleeding . Second,More than 90% of patients were taking aspirin. Chronic treatment with aspirin is a risk factor for GI bleeding regardless of the dose used . Further-/K+-adenosine triphosphatase (ATPase). This pump is also present in the myocardium of rats [-/K+ ATPase might therefore induce cellular acidosis and a secondary depression in myocardial contractility. However, this was not the mechanism of action of pantoprazole, as no significant changes in intracellular pH could be detected, and all effects occurred at pH 7.3–7.4. Two underlying mechanisms for the pantoprazole-dependent inhibition of contractile force have been described [2+ transients due to an impaired sarcoplasmic reticulum Ca2+ uptake and diminished Ca2+ influx via ICa L, and (2) reduced Ca2+ responsiveness of the myofilaments as a consequence of decreased maximal active tension and mildly lower Ca2+ sensitivity. Similar results have been obtained with esomeprazole [The present work shows that PPI use is an independent predictor of HF or death. Although there are no previous studies reporting this association, it is known that pantoprazole may exert negative inotropic effects on isolated myocardium from humans and rabbits . This ef of rats . Inhibitescribed : (1) a deprazole  and omepeprazole , suggestet al did not observe any effect of high doses of intravenous pantoprazole on left ventricular ejection fraction or several hemodynamic parameters in healthy volunteers [et al showed that chronic administration of PPIs in patients with stable angina could be associated with a decrease in ejection fraction and an increase in end-systolic volume index [Although the findings from laboratory studies have been promising, there is controversy regarding the clinical effects of PPIs on myocardial function. Using echocardiography, Schillinger lunteers . On the me index .The increase in mortality associated with PPI use described in the present paper is consistent with previous data. The use of high doses of PPIs has been associated with increased mortality in 491 older patients discharged from acute care hospitals, even when multivariable analysis including predictors of adverse outcomes was carried out . SimilarClostridium difficile infections and community-acquired pneumonia described in long-term PPI users [12 deficiency, thus leading to a poor nutritional status [Several potential mechanisms have been suggested to explain the relationship between PPIs and the risk of death . First, PI users ,27,28. Sl status . In factl status . Third, l status ,32. Thenl status ,34. Nevel status . Althouget al found that PPI use was not associated with all-cause mortality in a cohort of 22,107 patients over age 65 with HF [In contrast to our data, Oudit  with HF . Howeveret al reported an increased incidence of hospitalisation for acute coronary syndromes or death in patients with acute coronary syndrome receiving clopidogrel and PPIs [vs. non-users. What is more, treatment with clopidogrel did not affect the association between therapy with PPIs and the incidence of the primary outcome. It therefore seems reasonable to conclude that PPIs had no effect on the incidence of acute ischaemic events and that clopidogrel use did not affect the association observed with the primary outcome.Use of PPIs was not an independent predictor of acute ischaemic events. Pharmacokinetic and pharmacodynamic studies suggest that use of PPIs may reduce the antiplatelet effects of clopidogrel. The strongest evidence for such an interaction has been found between omeprazole and clopidogrel ,38. Ho eand PPIs . Howeverand PPIs . In addi2 or, alternatively, if this effect was also present when comparing PPI patients with those receiving no gastric protectors. The reason for this is that, in accordance with the current clinical practice, the number of patients without any gastric protector was very low.Finally, we were unable to explore whether adverse prognosis was associated with all PPIs or only with omeprazol, given that the number of patients with other PPIs was low. Similarly, our study did not allow us to analyse whether the adverse effect of PPIs was evident only when comparing these patients to those taking antiHThis work has certain limitations. First, this is a non-randomised study with some significant differences at baseline between patients receiving PPI and those who did not. Although we included these variables in the multivariate and propensity score analysis, the small sample size may have limited the statistical power. Second, excluding patients with clinical instability in the first days after the index event may have introduced a bias, as these patients would probably have evidenced a worse prognosis. Nevertheless, only 9 percent of cases were excluded for this reason and the remaining exclusion criteria were designed to prevent the inclusion of cases that could have yielded confounding information, such as patients with major additional disorders. Third, the number of total events was small, thus limiting the strength of the statistical analysis. Fourth, there were few deaths, making it impossible to test whether there was an association between use of PPIs and some specific cause of death. Fifth, the study quality is modest, as more than 50% of patients discharged from the hospitals with a diagnosis of NSTEACS or STEMI were not included in the study based on the exclusion criteria described in the Methods section. Finally, adherence to medication regimens during follow-up was not addressed.In conclusion, our results suggest an association between use of PPIs and the incidence of death or HF but do not point to an association with incidence of acute ischaemic events. More studies are needed to confirm these data.S1 File(XLSX)Click here for additional data file.


Integrating these data with other bodies of archaeological information, we suggest that the arrival of Baltic amber was part of broader Mediterranean exchange networks, and not necessarily the result of direct trade with the North. From a methodological perspective, thanks to the analyses carried out on both the vitreous core and the weathered surfaces of objects made of Sicilian simetite, we define the characteristic FTIR bands that allow the identification of Sicilian amber even in highly deteriorated archaeological samples.Provenancing exotic raw materials and reconstructing the nature and routes of exchange is a major concern of prehistoric archaeology. Amber has long been recognised as a key commodity of prehistoric exchange networks in Europe. However, most science-based studies so far have been localised and based on few samples, hence making it difficult to observe broad geographic and chronological trends. This paper concentrates on the nature, distribution and circulation of amber in prehistoric Iberia. We present new standardised FTIR analyses of 22 archaeological and geological samples from a large number of contexts across Iberia, as well as a wide scale review of all the legacy data available. On the basis of a considerable body of data, we can confirm the use of local amber resources in the Northern area of the Iberian Peninsula from the Palaeolithic to the Bronze Age; we push back the arrival of Sicilian amber to at least the 4 Amber and other unusual materials such as jade, obsidian and rock crystal, have attracted interest as raw materials for the manufacture of decorative items since Late Prehistory, and indeed amber retains a high value in present-day jewellery. There are many aspects that come into play when assessing the social value of these raw materials, ranging from intrinsic material properties  that can render them appealing for the manufacture of adornments, to other environmental or cultural aspects that may relate, for example, to their relative scarcity and subsequent added value as exotic or 'exclusive'. As such, the circulation of unusual raw materials can be traced back to Prehistory, and the Iberian Peninsula is no exception.th and the 2nd Millennia BC [th Millennium BC [th Millennium BC [For obsidian and jade, this movement is known to have taken place since early dates in the Late Prehistory. For instance, we can mention the Lipari and Pantelleria obsidian circuits in the Central Mediterranean that reached North Africa between the 6ennia BC –3; the Annium BC –6; or thnnium BC .As for amber, there is evidence of use since the Late Palaeolithic (e.g. ). In a pHere we present a new set of analyses of archaeological and geological amber that greatly expands previous coverage. Taking advantage of the growing body of data, and synthesising all the information available, this paper sets out to provide an up-to-date review of the evidence concerning the nature, distribution and circulation of amber in Prehistoric Iberia. This holistic perspective is contextualised more broadly in current discussion about the circulation of materials, knowledge and people in European Prehistory.In the last five years, there have been major developments in our understanding of Iberian amber. From a geological point of view, we highlight the project 'Iberian Amber: An Exceptional Record of Cretaceous Forests at the Rise of Modern Terrestrial Ecosystems', with the involvement of one of us (E.P.), focused on the palaeoenvironmental reconstruction of the ecosystems that generated the large Cretaceous amber deposits known in the Iberian Peninsula (e.g. ). From a-1, with a resolution of 4 cm-1. All of our raw spectra are included in full as Supporting Information , which is well-established as a reliable technique for the characterisation and sourcing of amber. The spectra obtained were compared with both reference spectra of geological samples previously available, and the new geological spectra presented in this article. The new specimens were analysed using a Perkin Elmer Spectrum Two FTIR spectrometer at the Wolfson Archaeological Science Laboratories of the UCL Institute of Archaeology (UK). This instrument allows for Attenuated Total Reflection (ATR) analyses and hence potentially totally non-invasive analyses. However, most of the archaeological objects studied here are held or exhibited at various museums in Spain and Portugal, and for curatorial concerns the extraction of a very small sample was preferable to moving the whole objects. Therefore, pellets were made using approximately 2 mg of analyte which was ground by hand in an agate mortar and mixed with a small amount of KBr, before pressing the mixture in a 13 mm diameter mould in order to produce 1 mm thick discs. The data were collected as infrared transmission spectra after scanning each specimen 50 times in the range 4000–370 cmormation  to facilthcentury Mr. S. Calderón [Around 150 localities with Cretaceous amber have been documented in the Iberian Peninsula , 13. MosCalderón  referredCalderón .Some of the amber deposits have undergone intense palaeobotanic and geochemical study. There have been systematic studies using FTIR of the most important localities such as Sant Just in the Maestrazgo Basin, or Peñacerrada and El Soplao in the Basque-Cantabrian Basin –22, to wGeological samples of the main deposits have been selected as references in the present work. One sample from Jaén province  has also been included due to its closer proximity to some of the archaeological sites considered. The geological samples analysed are presented in the following sections .There is a large sedimentary basin that developed during the Cretaceous period between the Iberian and European tectonic plates. The main deposits of amber in this area are those of El Soplao and Peñacerrada , 21, whiAmber is documented in clayey rocks rich in organic matter from sediments deposited mainly in coastal areas and fluvial channels. There is a large quantity of plant remains, coal and other continental organic material transported by rivers. Some deposits contain remains of marine life as molluscs, which indicate a coastal environment in the area in which the resin was buried .-1, bands at 1460 and 1380 cm-1 because of simple C-H bonds, and bands from the group of carbonyls around 1700 cm-1, with the absence of bands at 3070 cm-1 being consistent with the amber's high degree of maturity.The FTIR spectra for amber from this area had been previously determined through the study of the Peñacerrada and El Soplao deposits in particular , 21, and-1, a band with greater or lesser intensity at 1270 ±5 cm-1 preceded sometimes by a secondary peak and two secondary bands at 1180 and 1025 ±5 cm-1 followed in most instances by a band at 970 ±5 cm-1.We selected amber samples from six other localities  to explore variability across the entire area. In general, the spectra have the same characteristics as those already published for El Soplao and Peñacerrada, with the sample from Ajo presenting a spectrum that is slightly different from the rest : all theIn the Central Asturian Depression, the main known amber deposits are those documented in the El Caleyu and Ullaga formations. In Pola de Siero, amber occurs, along with pyrite and fossilised plants, in lenses of grey silt within a 50-metre-deep layer of siliceous conglomerates, with a silt-sand matrix deposited on the Palaeozoic levels . In the There are some studies of the bioinclusions of these amber deposits, as well as gemmological studies, although prior to this work no FTIR spectra have been published. For the present research, we selected samples of amber from El Caleyu, whose FTIR spectra were similar to those already published for other Cretaceous Peninsular amber .The Maestrazgo Basin, which reached a sediment thickness of 6.5 km during the Mesozoic, is an intra-continental basin located in the Iberian Range that underwent two significant stages of rift during the Late Jurassic and Early Cretaceous; the rift structure is characterised by a system of faults that divide the Maestrazgo Basin into different blocks . In geneThe San Just deposit has grey-black clay stones with abundant plant remains and fusinised wood. Palynological studies suggest a subtropical hot-humid environment with hot and dry areas .-1 corresponding to the carbonyl group and the simple C-H bond respectively, with another band at 856 cm-1. The absence of the band of the exocyclic methylene group at 1640 and 880 cm-1 is significant, as it indicates a high level of maturity (including diagenetic effects) which would be consistent with its Cretaceous age.The FTIR spectrum from San Just was published by Peñalver et al.  and is cThe La Hoya deposit, in Cortes de Arenoso municipality, contains abundant amber combined with coals and other vegetal remains deposited in deltaic environments. Insects have been documented in the amber as bioinclusions . The samIn a stratigraphic cross-section of Navalperal (Jaén), near the town of Orcera, in the Prebetic Domain, a few pieces of amber have also been identified, a few centimetres in diameter, in a level of dark limestone with lignite from the Ci3 sequence ; accordTo this day, such discoveries of amber in small quantities are unheard of. During the present research, we carried out FTIR analysis of this locality given its greater proximity to many of the archaeological sites studied, compared to the geological amber deposits previously discussed. However, the FTIR spectrum obtained do not differ qualitatively from those already known for the geological deposits of the Northern half of the Iberian Peninsula  and haveAll in all, therefore, any of these spectra may be taken as characteristic reference spectra of Cretaceous Peninsular amber.The following sections provide some archaeological background to the samples reported here, while our discussion will integrate these new data with results published previously. Further details of archaeological samples analysed previously, and relevant bibliographic references, can be found in our previous publication .The burial monument of La Velilla is a circular chamber of about 9.5 m in diameter in which a minimum of 71 individuals were buried (56 adults and 15 sub-adults) of which only 9 retained anatomical connection . Five amThe five amber beads have a similar barrel shape, ranging between 1.6 and 2.5 cm in length and with a maximum diameter of 1.2–1.9 cm . We analth Millennium and the first half of the 3rd Millennium BC  [A recent series of 13 radiocarbon dates  place the use of this monument between the second half of the 4The site of Valencina de la Concepción  holds the greatest number of amber objects known for a single site for all the Late Prehistory of the Iberian Peninsula, and it stands out both for its size and for the uniqueness of some of its graves and materials . The amber object from this monument is a large bead, 3.3 cm in length and 0.9 cm wide, currently on permanent display at the National Archaeological Museum (Madrid) together with 14 green beads . Until tin situ around a cinnabar-covered stele, and a further two individuals in the small chamber whose sex could not be identified. All the women in the large chamber were dressed with costumes made up of more than a million beads of shell (and 251 of amber), covered with red cinnabar and accompanied by impressive grave goods consisting of gold-embossed sheets, carved ivory objects, ostrich eggs, large flint sheets and 2–4 mm thick flint arrowheads, rock crystal and amber. Similarly, the orthostats of the tholos were lavishly decorated in shades of red and black on their inner surface [The tholos of Montelirio . This is a huge megalithic structure with a large corridor, 39 m in length and only 1.3 m high, which forces visitors to access the chamber by crawling. At the end of the corridor there is a chamber with a 4.75 m diameter and a second smaller chamber, 2.7 m in diameter. In total, 19 middle-aged women have been documented in the large chamber, recovered  surface . Most of surface  (for a d surface ).Grave 10042–10049 . About 200 m North of the tholos of Montelirio is an area called PP4 Montelirio where more than 130 negative structures  have been documented, of which 61 contained human remains and 73 did not . This is a tholos with a circular chamber that is 4.3 m in diameter and with a 3 m long corridor where the remains of 50 individuals were documented. Amongst other grave goods, three amber beads were recovered , althougMound 8(IV). This is a mound with a trapezoidal chamber and a 2.9 m long corridor with burned remains of 20 individuals. The Leisners  do not mMound 12. This is a tholos with a 3.8–4 m diameter chamber and a 2.4 m long corridor in which 12 individuals and five pieces of amber were recovered: a fragment of a cylindrical bead; a drop or tear drop-shaped bead with a "V" perforation; a smaller cylindrical bead with reddish pigmentation; a disc with an arched surface, and a barrel bead . RegrettMound 63(III). Architecturally, Mound 63(III) is a 4 m long mound with a trapezoidal chamber and a 3.5 m long corridor in which several individuals were documented . Here a Mound 74(XIII). This is a tholos with a chamber 1.6 m high and a diameter of 4 m; the corridor has not been preserved . AmongstAt the set of graves at Llano de la Sabina, the appearance of amber beads is reported for two of them: graves 97 and 99.Llano de la Sabina 97 is a mound with a 2 x 1.7 m quadrangular chamber in which 'remains' of a skeleton were documented . A circuLlano de la Sabina 99 is a mound with a 2.7 m long trapezoidal chamber and a 4 m long corridor in which up to 20 individuals could have been buried . Inside,Although both graves were considered Chalcolithic by the Leisners , accordiThis is a mound with a quadrangular chamber with dimensions 1.8 x 1.7 m and a 2 m long corridor in which the remains of an individual and an amber bead are documented . These aAlthough the Leisners place this tomb within the Chalcolithic period, Siret places it within the Iron Age because of the metallic materials found '0.5 m from the floor', which leads us to think that a subsequent reuse process took place, as might be further evidenced by the presence of bronze bracelets (8–9% Sn) and silver beads . The ambTrivia arctica in the whole of the interior Iberian Peninsula.The site of Valle de las Higueras is the largest necropolis of hypogea found so far in the interior part of the Iberian Peninsula. With a C14 series between 2831–1511 cal. BC, eight funerary caves with collective burials have been documented . In thesCave 1 has a chamber with a diameter of more than 3 m, with a small passageway on the side that gives access to another smaller chamber. To the West of the main chamber, a niche was dug in the ground where variscite necklace beads and ceramic remains were located. Also in the main chamber variscite and amber beads were recovered, along with Bell Beaker ceramic and two points of flint with cinnabar remains. The radiocarbon date from bones yielded a date between 2470–2030 cal. BC . In the rd Millennium BC, between 2460–2190 and 2180–2140 cal. BC [Cave 3 is the most central one in the necropolis. It has a circular chamber with a narrow corridor that connects it to a rectangular antechamber with rounded sides. At the back of the chamber three niches were documented—they had been dug into the rock and had the most significant grave goods. In the West niche, two children were buried, in the Central niche an adult woman with two children in her arms, and in the East niche, a young adult and another older adult. In front of the niches a dozen individuals were buried, of which only one woman had grave goods, which comprised of a variscite necklace. At least twelve burial sites were documented in the antechamber. A cylindrical amber bead 1.3 cm in length and with a 0.2 cm perforation comes from this set  and was  cal. BC .On the basis of his analysis, Domínguez-Bella ruled out a Baltic source but did not go on to propose an alternative origin . In thisrd Millennium BC, although at higher levels there is documented evidence of reuse in the Iron Age and even Roman times. Dating carried out twice on human bone [The artificial cave of Sao Paulo is located in the Portuguese municipality of Almada, on the left bank of the Tagus River. This is a monument dug into the rock, and is part of a necropolis of which only two graves are known to be in artificial caves, although it is possible that the pressure of urban development has destroyed other similar graves. The artificial cave was discovered in 1988 and was excavated by the Department of Archaeology and History of the Municipal Museum of Almada. The hypogeum was dug in calcareous rock and consists of an elliptical chamber, almost circular, 7–7.5 m in diameter preceded by a corridor about 2.5 m long . Inside man bone , directlrd Millennium BC. They can be found at the Municipal Museum of Almada.In the course of the excavation, two amber beads were identified in the lower strata, corresponding to the 3The first bead is cylindrical and flattened, about 1.5 cm in diameter with a fine central perforation . The secth and 8th centuries BC [The Quinta do Marcelo archaeological site is located next to the mouth of the Tagus and has been interpreted as one of the first settlements established for exchanges with the Phoenicians . Howeveruries BC , raises uries BC , 52. Theuries BC , 54.The materials are held at the Municipal Museum of Almada, largely unpublished, with partial references in more general works. The amber bead that we are concerned with appeared in a waste pit or "bag 2", together with very high-quality materials characteristic of the Late Bronze Age: fine ceramics with internal and external decoration , 54, twoThe amber object is a circular bead of approximately 1.1 cm in diameter, with a central perforation. Although it has light oxidation on the surface, its state of conservation is considerably good .The FTIR spectra obtained for the different samples allow us to confirm that, in all cases, the raw material used is amber. At the same time, they present significantly different patterns, which allow us to note the use of amber with different origins, and exposed to different postdepositional conditions. At least three different geological sources can be identified, which are discussed in turn:-1). The amber spectrum from La Velilla is characterised by five well-defined bands: at 1570 ±5 cm-1 by the aromatic double bonds C = C, at 1457 ±5 and 1381 ±5 cm-1 attributable to alkyl groups, the first the -CH2 and -CH3 flexures  and the second due only to the -CH3 flexure; a band at 1158 ±5 cm-1 which can be attributed to the tension of the C-O simple ester bond, and one last band at 1034 ±5 cm-1 as well as three secondary peaks at 1411 ±5, 1277 ±5 and 1230 ±5 cm-1 , the archaeological site with amber that is closest to some of the Iberia’s geological amber deposits. In this case, we can see that both the spectrum of the bead and the characteristic spectra of Cretaceous Iberian amber display similar patterns in the diagnostic area of the spectrum (i.e. c. 1600–900 cm-1 (e.g. ) Fig 10-1. The a-1 as well as a secondary peak at 1226 ±5 cm-1. The most significant differences in the spectra are the band at 1570 ±5 cm-1 and the secondary peak at 1411 ±5 cm-1 in the sample from La Velilla, which do not appear in the geological samples, and may be due to oxidation processes . In fact, the spectrum from La Velilla sample is almost identical to the archaeological amber samples from the Palaeolithic settings of La Garma A [This spectrum is similar to the reference spectrum of the Cretaceous Peninsular amber described above, which is characterised by bands at 1457, 1376, 1155, and 1030 ±5 cm-1 preceded by a horizontal band between 1250 and 1180 cm-1 –the so-called ‘Baltic shoulder’. This feature has been documented in only three of the samples studied for this paper: the beads from Llano de la Sabina 97 and 99, and Quinta do Marcelo. In all three cases, the Baltic shoulder can be clearly identified, so we can be confident about their attribution to an exogenous origin  from Los Millares and the artificial cave of Sao Paulo. Guiliano et al. [The spectra of the weathered surface of these same samples present significantly different features in the area between 1800 and 900 cm-1 . In theser (e.g. ), althouo et al.  proposedth Millennium BC [As a matter of fact, this same pattern is also observed in some of the most degraded samples elsewhere, such as those from Valle de las Higueras, Llano de la Teja 18 and Mound 12 from Los Millares ; Table 5nnium BC .All in all, we therefore assume that in all of these cases the original amber would also give spectra similar to those observed in the samples of the vitreous amber cores from the tholos of Montelirio, Mound 74(XIII) from Los Millares and the artificial cave of Sao Paulo. These spectra differ significantly from those of Cretaceous Iberian amber, and they lack the characteristic Baltic shoulder observed–while they retain the typical bands of Sicilian simetite . At pres-1 and they are both characterised by peaks at 1455 and 1392 ±5 cm-1 followed by a secondary peak at 1375 ±5 cm-1, as well as bands at 1240 and 1043 ±5 cm-1. Notably, the characteristic bands of Cretaceous Iberian amber are not present.Lastly, the analysed samples from the Dolmen of La Pastora in Valencina de la Concepción and the beads from burial Mound 63(III) from Los Millares produced very similar spectra, suggesting that they were made of amber of the same origin . None of-1 obliges us to be cautious when proposing Sicily as the source: this band does not appear in other degraded samples, so we cannot confident dismiss it as a result of postdepositional alteration. However, at present, Sicily offers the most similar spectrum we know of. What we can be sure of is that it is exotic material, since it does not resemble the Cretaceous amber spectra of Iberia, where no workable amber deposits of other geological age have been discovered  were the first to be explicitly identified as simetite in several publications [Of these samples, the beads from the Dolmen of Alberite , the loications  does notth Millennium BC) [-1. Although it is true that the peak to which they refer appears displaced (at 1019 cm-1 instead of 1041 ±5cm-1 as it is in the reference spectra of simetite), all the bands described coincide with those of simetite . The second peak (887cm-1) cannot be considered a marker of provenance but of postdepositional alteration since, as noted above, this band disappears following thermal exposure [In any case, FTIR spectra matching those of simetite have also been published in the studies of the Mamoa V of Chã de Arcas and Chousa Nova (5nium BC) . The latnium BC)  because simetite , and it exposure .th–4th Millennia BC); the results of these analyses have not been published, although it has been confirmed that the amber is not Baltic succinite [th–3rd Millennia BC, such as those from the tholos of Montelirio or the artificial cave of Sao Paulo, whose unaltered cores also give the same spectrum as Sicilian simetite. We therefore propose that those samples constitute degraded simetite [Other deposits of Late Neolithic chronology and bearing amber include the Campo de Hockey analyses  points to an Iberian source and thus confirms the strength of regional networks. The use of regional raw materials in the North Peninsular area continued until the 2 Lagos I .The use of these local resources, however, is not documented in the Southern part of the Iberian Peninsula. Outside the Northern region, where geological amber deposits are abundant, the amber objects analysed are, in all cases, foreign. This preference for exogenous raw materials could respond to different causes that are not mutually exclusive. From a technological point of view, the greater age of Iberian amber makes it more fragile and therefore more difficult to carve than succinite or Cenozoic simetite. From a more social perspective, it is worth considering that amber objects may have arrived in Iberia already manufactured; in this case, the choice of foreign objects would be driven not so much (or not just) by the quality of the material, but by the role played by these amber objects in establishing social relations or alliances through exchange or gift policies.rd Millennium BC [rd Millennium BC contexts for the objects from Valencina de la Concepción and those from the artificial cave of Sao Paulo, Valle de las Higueras, Llano de la Teja 18 and Los Millares.This presence of foreign amber materialises in a much more evident way during the 3nnium BC , 70. ThiTrivia arctica is from the Atlantic and the amber from Sicily, an image that is fairly coherent with the central location of this necropolis in the Iberian Peninsula.In the South Plateau, the necropolis of Valle de las Higueras is an exception because of the presence of amber associated to Bell Beaker, something not found in the area’s hypogea , nor in th Millennium BC at least; in parallel, the presence of variscite from the Iberian West in the megaliths of Luffang (North-West France) dated to the 4th Millennium BC [rd Millennium BC is notable and reflected in the greater participation of this region in trans-regional circuits of exchange, as manifest in the fact that all the sites with more abundant amber and other ‘exotic’ objects are located in this area.Overall, while the sites in the North of the Iberian Peninsula show continuity and the survival of the local networks of exchange, the identification of Sicilian amber in Andalusia demonstrates the beginning of Mediterranean exchange circuits dating back to the 4nnium BC  confirmsth Millennium BC, and possibly earlier. Interestingly, the first amber objects recovered in Sicily and assigned a local origin also date from the 4th Millennium BC [th and 3rd Millennia BC is similar to that of ivory objects [Thus we can confirm the presence of simetite in the Iberian Peninsula as early as the 4nnium BC , 72. Hownnium BC . Even th objects , which cth Millennium BC seems to be interrupted abruptly in the 2nd Millennium BC. With the exception of very late examples addressed below, there are only a few contexts from the 2ndMillennium BC in which amber is documented, and these are more or less restricted to the North-Eastern part of Iberia. It is in this period, however, when we first document Baltic succinite, which from then on will become virtually the only raw material used. A similar situation, namely simetite being the only type of amber deposited, has been described for Italy and Sicily from the Eneolitic to the Middle Bronze Age [nd Millennium BC , although most of the objects, especially in Early Mycenaean contexts, are concentrated in a few of the richest graves [nd Millennium BC samples from Greece analysed by Beck and Hartnett [th century BC [nd Millennium BC is Baltic in origin, evidencing connections between Nordic/Atlantic Europe and the Mediterranean (e.g. [The steady increase in amber consumption we had been observing since the 5onze Age . It shout graves –76. SiciHartnett  and the ntury BC . Howeveran e.g. –77)..th MilleUnfortunately, the lack of absolute dates in the Bronze Age settings of the North-East part of the Iberian Peninsula where amber is documented prevents us from accurately pinpointing the moment when succinite begins to reach Iberia. The earliest known evidence of Baltic amber may be that of the Dolmen of Larrarte (Guipúzcoa). Published dates for this site range between 5345–4051 cal. BC and 4236–3542 cal. BC . HoweverIn the Iberian North-East, Baltic amber has been documented in Pedra Cabana, Cabana del Moro de Colomera and Muricecs , 24. Altrd and the 2nd Millennia BC. Archaeologically it is impossible to assign the pieces of amber to any particular chronology but their Baltic origin suggests that they correspond to the later reutilisations. The sample from Llano de la Teja 18, another monument with evidence of reuse, raises more doubts, as the FTIR spectrum for the amber is consistent with Sicilian simetite and hence would suggest a 3rd Millennium BC chronology. We acknowledge, however, the risks of circularity in our argument.In the South-East, we have the samples from Llano de la Sabina 97 and 99, recovered in megalithic monuments with evidence of reuse, with mixed materials from both the 3nd Millennium BC is more significant when placed against the backdrop of other changes: during the 4th and 3rd Millennia BC, amber had been closely linked to megalithic monuments of undeniable symbolic significance, and often accompanied by other special raw materials such as ostrich eggs, ivory, cinnabar or gold; all of these exotic raw materials appear to vanish concurrently [The decline in amber consumption during the 2urrently . This raurrently ). Similaurrently .th-8th centuries BC), Moreirinha (13rd-9th centuries BC) and Senhora da Guia (10th-8thcenturies BC) in Portugal [this paper, st-6th centuries BC) and Herrerías II (10th century BC) in Spain [th century BC is concentrated close to coastal sites, and the earliest evidence of Baltic succinite in Southern and Western Iberia occurs in association with some of the earliest iron objects, and is hence connected to a technology that likely arrived via the Mediterranean. This is the case of Quinta do Marcelo, Moreirinha, Senhora da Guía and Palacio III. Moreover, there are other objects which point to Mediterranean pre-colonial contacts: metal artefacts recovered at Quinta do Marcelo have been related to the Mediterranean [th-13th centuries BC or the wheeled and Cogotas I pottery from Cuesta del Negro (Granada) dated to the 14th century BC, also with a proposed Mycenaean origin , amber will reappear in Iberia in the Late Bronze Age and the Early Iron Age , when Mein Spain , 82. Datin Spain –85. Howein Spain , 86. It erranean ; bronze erranean ; and siles Ortiz  for disces Ortiz  for genend Millennium BC, in a phenomenon that is usually connected with Mediterranean rather than Atlantic networks.In the North-Eastern Iberian region, continental contacts through the Pyrenees continued during the Bronze Age  as well as Mediterranean ones, although the latter being seemingly less important. This panorama will change during the Late Bronze Age/Early Iron Age, when Mediterranean contacts become more intense . Based oThe new evidence presented in this paper, brought together with information published previously, has allowed the most comprehensive review to date on the provision and exchange of amber in the Prehistory of Iberia. Several conclusions and priorities for future work can be highlighted:From a methodological perspective, thanks to the analyses carried out on both the core of vitreous amber and the oxidation layers of the objects from the tholos of Montelirio, Mound 74(XIII) from Los Millares and the artificial cave of Sao Paulo, we have been able to define the characteristic FTIR spectrum resulting from degraded Sicilian simetite. These spectra can be used as a reference to identify Sicilian amber even in highly deteriorated archaeological samples.The local origin of the amber from La Velilla emphasises the continued use of local resources from the Palaeolithic to the Bronze Age in the Northern region of Iberia, in stark contrast with the picture obtained from the South.th Millennium BC at least, and that it was likely integrated in broader Mediterranean exchange networks that intensified during the 3rd Millennium BC, as attested to by amber finds as well as other exotic materials.We can verify that the arrival of Sicilian amber in the Iberian Peninsula started in the 4nd Millennium BC, as evidenced by the analysis of Quinta do Marcelo and other sites; the more significant influx of Baltic amber will take place from the turn of the Millennium.After an apparent decline in the use of amber, Baltic succinite appears to replace Sicilian simetite in the Iberian Peninsula in the second half of the 2th–3rd Millennia BC and its archaeometric characterisation, not least considering the possible involvement of this region in the Mediterranean exchange network. Relatedly, a systematic exploration of North African amber resources should be warranted; there are currently no known geological deposits of amber in North Africa, except for a locality with amber in Tunisia that remains to be confirmed [The new results thus fill some of the research gaps highlighted in our 2012 publication , while tonfirmed . AnotherS1 File(XLSX)Click here for additional data file.


Mother of FT and TFL1 (MFT) belongs to phosphatidylethanolamine-binding protein (PEBP) family, which plays an important role in flowering time regulation, seed development, and germination. To gain insight into the molecular function of DlMFT in Dimocarpus longan Lour., we isolated DlMFT and its promoter sequence from longan embryogenic callus (EC). Bioinformatic analysis indicated that the promoter contained multiphytohormones and light responsive regulatory elements. Subcellular localization showed that the given the DlMFT signal localized in the nucleus, expression profiling implied that DlMFT showed significant upregulation during somatic embryogenesis (SE) and zygotic embryogenesis (ZE), and particular highly expressed in late or maturation stages. The accumulation of DlMFT was mainly detected in mature fruit and seed, while it was undetected in abortive seeds, and notably decreased during seed germination. DlMFT responded differentially to exogenous hormones in longan EC. Auxins, salicylic acid (SA) and methyl jasmonate (MeJa) suppressed its expression, however, abscisic acid (ABA), brassinosteroids (BR) showed the opposite function. Meanwhile, DlMFT differentially responded to various abiotic stresses. Our study revealed that DlMFT might be a key regulator of longan somatic and zygotic embryo development, and in seed germination, it is involved in complex plant hormones and abiotic stress signaling pathways. Dimocarpus longan Lour.) is a famous subtropical economic fruit tree in Southeast Asia. Embryogenesis is an important factor associated with fruit output quantity and quality in longan [Longan -like, TERMINAL FLOWER1 (TFL1)-like, and MFT-like [MFT-like clade seems to be the phylogenetically ancestral to other clades [FT and TFL1 are two regulators with antagonistic functions in controlling flowering time and plant architecture, FT acts as the floral activator, but TFL1 acts as inhibitor [MFT shows high similarity to both FT and TFL1, the function of MFT in controlling flowering time might be different from FT/TFL1. Overexpression of MFT in Arabidopsis caused slightly early flowering under long days, but its function in determination of flowering time was redundant [PopMFT did not identify a function in controlling flowering time [Arabidopsis indicates MFT was a potential candidate gene for altered flowering time at an increased atmospheric (CO2) [The plant phosphatidylethanolamine binding protein (PEBP) family was divided into three main clades, MFT-like ,12,13,14r clades ,15,16. Fnhibitor ,18,19. Inhibitor . Althougedundant , whereasing time ,22. Idenic (CO2) .MFT in several plants were very similar; Arabidopsis thaliana MFT [Vitis vinifera MFT [Populus nigra MFT [Citrus unshiu MFT [Zea mays MFT [Triticum aestivum MFT [Jatropha curcas MFT [Glycine max MFT [Hevea brasiliensis MFT1 [Gossypium hirsutum MFT1 and MFT2 were mainly expressed in flowers and low-transcribed in other tissues [SrMFT were constitutively expressed in various tissues and developmental stages in Symplocarpus renifolius, although the expression of SrMFT in female-stage spadices was higher than in other stages, whereas overexpression SrMFT in Arabidopsis did not show the function in earlier flowering [AcMFT showed a distinct decrease from the young frond stage to the sori development and maturation stages, and was regulated by photoperiod in Adiantum capillus-veneris, introduced AcMFT in Arabidopsis promoted flowering [Arabidopsis, MFT promoted embryonic growth during seed germination by directly repressing ABI5 expression, it played a critical role in seed germination via a negative regulation of abscisic acid (ABA) signaling pathway [MFT promoted primary dormancy during seed development, and enhanced germination in after-ripened imbibed seeds with exogenous ABA in Arabidopsis [TaMFT, an MFT homolog in wheat (Triticum aestivum) acted as a positive regulator of seed dormancy, while a negative regulator of seed germination [MFT in soybean responded to exogenous applied ABA and gibberellin (GA), ectopic expression of GmMFT in Arabidopsis did not affect the flowering time but suppressed seed germination [HbMFT1 in Arabidopsis inhibited seed germination, seedling growth, different with previous studies, it delayed flowering time [FvMFT located in the nucleus and cytoplasmic membranes, ectopic expression of FvMFT in Arabidopsis also inhibited seed germination through integrating ABA and GA signaling, FvMFT could notably promote the primary root growth under sugar-deficient conditions [The expression pattern of iana MFT , Vitis vfera MFT , Populusigra MFT , Citrus shiu MFT , Zea maymays MFT , Triticuivum MFT , Jatrophrcas MFT , Glycine max MFT  and Hevesis MFT1  were mai tissues . SrMFT wlowering . In addilowering . In Arab pathway , it also pathway . Meanwhibidopsis . Conversmination . The hommination . Ectopicing time . FvMFT lnditions .MFT during SE and ZE, and its signaling pathways involved in the response to hormones and environmental cues in embryogenic calli are still fragmented. In addition, the function of MFT homolog in longan has not been investigated. In our study, we isolated DlMFT and its promoter, and found that DlMFT was a seed-specific gene, and might play an important role in longan embryogenesis, seed development, and germination. DlMFT also involved in complex hormones and stress responses. The information provided by our study may help to illuminate the biological functions of DlMFT in longan.At present, the expression profile of DlMFT from embryogenic callus of Dimocarpus longan. The amplified 866 bp region of DlMFT cDNA contained a 519 bp protein coding sequence flanked by a 54 bp 5′-untranslated region and a 312 bp 3′-untranslated region (GenBank accession No. KP861622). The gDNA sequence of DlMFT was cloned from longan EC DAN with specific primer and submitted to GenBank (GenBank accession No. KY968646), the comparative analysis of MFT genomic organization indicated that the MFT contained three introns and four exons, and owned the same number of nucleotides in exons (The RT-PCR (reverse transcription polymerase chain reaction) combined with RACE (rapid amplification of cDNA ends) strategy was used to obtain the complete cDNA (complementary DNA) and gDNA (genomic DNA) sequences of in exons a.Arabidopsis MFT, Malus domestica MFT and Citrus clementina MFT, respectively (http://blast.ncbi.nlm.nih.gov). Two highly conserved DPDxP and GIHR motifs between the PEBP families were identified  and H (His-88) were possibly the most critical for distinguishing FT and TFL1 function on flowering regulation [DlMFT does not play a central role in flowering time regulation. A phylogenetic tree for the PEBP family was constructed using several amino acid sequences of other MFT orthologs  was isolated from embryogenic callus gDNA by genome walking. The PLACE database was used to analysis the putative cis-acting elements in this promoter, and the sequences on both strands were considered. The putative regulatory elements are show in DlMFT promoter contained multiple regulatory elements, including the AACA core (AACAAAC), (CA)n element (CNAACAC), DOF core (AAAG), E box (CANNTG), RY repeat (CATGCA) and SEF1, SEF3, SEF4 motifs , which were involved in seed-specific transcriptional regulation [DlMFT may be regulated by ABA, GA3, SA and MeJa. Furthermore, there were 22 light-responsiveness elements, including GT1-motif (3), SP1 (3), GA-motif (3), GAG-motif (3), Box-I (2), G-box (2), ACE (1), AE-box (1), ATCT-motif (1), GATA-motif (1), I-box (1), and TCT-motif (1).Base on the gDNA sequence, a 1.85-kb gulation . MeanwhiDlMFT pro:GUS fusion  scale leaf epidermis cells, 4′,6-diamidino-2-phenylindol (DAPI) was used as a maker of nuclear localization. As shown in The subcellular localization of DlMFT:mGFP was examined through a transient expression of DlMFT:mGFP in onion  during SE and ZE . As shown in Figure 7B, DlMFT was minimally expressed in non-embryogenic callus (NEC), embryogenic callus (EC) and incomplete compact pro-embryogenic cultures (ICpEC), but highly expressed in latter stages, and showed continuous up-regulation from ICpEC stage to cotyledonary embryos (CE) stages, and peaked at CE stage. Meanwhile, DlMFT mRNA was detected in all examined zygotic embryo development stages of ‘Honghezi’ longan , its expression level was very low in S1 and S2 stages, and continuously increased during the embryo development stages, and showed a peak in mature zygotic embryo stage (S7). The results showed that DlMFT exhibited a similar expression pattern during longan SE and ZE, the expression profile implied a promotion role of DlMFT during longan SE and ZE, especially, at the late embryogenesis stages.To better understand the transcription regulation of DlMFT in longan, we examined the levels of DlMFT transcript in different tissues, including roots, stems, leaves, vegetative buds, floral buds, inflorescence, flower buds, male flowers, filaments, anthers, female flowers, young fruits, ripe fruits, pericarp, pulp and seeds of ‘Honghezi’ longan  after flowering, and checked the expression of DlMFT in these seeds. The result showed that DlMFT was highly expressed in normal development seeds, but was undetected in abortive seeds of ‘Quanlong baihe’ longan  with quantitative real-time PCR. The expression level of DlMFT was significantly decreased at 4-day and 8-day periods, and then kept at a relatively stable and low level at 8, 12 and 16 day periods, finally markedly decreased at 20-day period , auxin transport inhibitor (NPA), PP333, MeJa, SA, BR, ABA, kinetin (KT) and ethylene (ETH) for 24 h. Among the treatments, exogenous 2,4-D inhibited its expression, and higher concentration of 2,4-D induced a more significantly expression decline , ABA, BR and PP333 did the opposite function, and KT and ETH showed no significantly function on DlMFT expression.Taken together, our results revealed the transcription mechanism of DlMFT is affected by abiotic stress, we detected the mRNA level of DlMFT in longan EC exposed to 150 mM NaCl and PEG-4000 for different time, and treated with different concentration of sucrose, KClO3 at 110 rpm at 25 °C under dark conditions for 24 h. In addition, longan EC was treated under different temperature and light qualities conditions for 24 h to define whether DlMFT responded to temperature and light. The results revealed that treatment with 150 mM NaCl for 1, 2, and 4 h induced a significant expression increase as the treatment time increased, and the highest transcript level (~32.4-fold of the control) of DlMFT was observed at 4 h; then, as the treatment time increased, the transcript level declined, but still higher than the control . However, it showed an arresting decline in the 40 °C treatment  and plant growth inhibitor (PP333) in longan EC. During longan SE, high dose of 2,4-D was essential for SE induction [DlMFT accumulation facilitated the morphogenesis in the middle and late SE and ZE stages, which suggested that DlMFT might involved in auxin signaling pathway during longan SE. ABA is an important plant growth regulator that is involved in embryo formation and maturation [DlMFT was promoted by ABA, consistent with the study that the expression of AtMFT was induced by ABA during seed germination [DlMFT was promoted by exogenous GA3, and inhibited by SA and MeJa. In addition, DlMFT promoter contained multiple elements related to hormone signals, including ABRE for ABA responsiveness, CGTCA-motif for MeJa responsiveness, TCA-element for SA responsiveness, TATC-box and GARE-motif for GA responsiveness. Thus, it was suggested that DlMFT was involved in various hormones signaling during longan SE.In our study, ibitor PP33 in lonturation ,39,40. Dturation ,42, but turation ,44,45,46mination . Plus th3 could increase the accumulation of DlMFT. Relatively high temperature was effective to induce secondary somatic embryos, while a low temperature was more suitable for further embryo development [DlMFT was inhibited with 40 °C treatment, while no significant change of the expression was found when treated with 32, 34 and 36 °C.Stress acts as an embryogenic stimulus, which plays an important role in plant SE . In citrelopment . The expDlMFT were observed, which might involve in transcriptional regulations of DlMFT gene. We found that DlMFT responded differently to various light treatments, all the light treatments up-regulated the expression of DlMFT, especially, with the yellow and blue light treatments. These results indicated that DlMFT might be involved in complicated light signaling pathways during longan SE.Light and temperature are the key abiotic modulators of plant gene expression . Light qFvMFT in Arabidopsis notably promoted the primary root growth under sugar-deficient condition [MFT might be involved in sugar signaling. In the present research, compared with 2% sucrose (control), the transcription level of DlMFT was promoted at higher and lower concentration of sucrose, while inhibited under sucrose-deficient condition, which indicated that DlMFT was included in sugar signaling pathway. All these results indicate that DlMFT, at least in part, take part in the abiotic stress response.In addition, sugars have important hormone-like functions as primary messengers in signal transduction, sugar signaling is yet another potential source for regulation of nuclear genes . High coondition , which sThe synchronized embryogenic cultures at different developmental stages, including the non-embryogenic callus (NEC), friable-embryogenic callus (EC), incomplete compact pro-embryogenic cultures (ICpEC), globular embryos (GE), heart-shape embryos (HE), cotyledonary embryos (CE) were obtained by the following previously published methods ,4. ‘HongN-1-Naphthylphthalamic acid  , Paclobutrazol  , KClO3   with agitation at 110 rpm at 25 °C under dark conditions for 24 h, with 3 replicates. EC culture in MS liquid medium as control. EC treated with 1 mg/L IAA for different time , treated with 150 mM NaCl for different time  and treated with PEG-4000 for different time  in liquid MS medium with agitation at 110 rpm at 25 °C under dark conditions, EC culture in MS liquid medium as control. EC treated with different light qualities  was carried out at 25 °C for 24 h. Frozen all samples in liquid nitrogen immediately for 5 min after collecting, then stored at −80 °C for isolating the total RNA.Transferred 0.2 g 18-day subculture longan EC to MS liquid basal medium (2% sucrose) supplemented with ABA  , 2,4-D  , BR  , IAA  , SA  , MeJa  , GA3  , MFT from longan embryo callus transcriptome (SRR accession number: SRA050205), specific primers DlMFT-GSP1 and DlMFT-GSP2 couple with 3′-primer and 3′-Nest primer, respectively, were used for 3′-RACE, DlMFT-GSPa and DlMFT-GSPb couple with 5′-primer and 5′-Nest primer, respectively, were used for 5′-RACE, DlMFT-F/R were used for Splice verification and gDNA amplification, all primers are list in The cDNA was synthesized from total RNA of embryo callus using GeneRacer™ Kit (RLM-RACE)  according to the manufacturer’s protocol. Base on the sequence of DlMFT, specific primers SP1, SP2 and SP3 and the adaptor primers AP1 and AP2 were used for cloning the promoter by Tail-RCR  protocol of genome walking Kit (Takara) from embryogenic callus DNA. The PLACE (https://sogo.dna.affrc.go.jp/cgi-bin/sogo.cgi?lang=en&pj=640&action=page&page=newplace) database and the PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) database were used to analyze the putative cis-acting regulatory elements. The primers used in promoter cloning are list in Based on the gDNA sequence of DlMFT without a terminator codon (TAA) was sub-cloned in BglII sites of pCAMBIA1302-GFP vector for investigating the subcellular localization of DlMFT in Allium cepa. The pCAMBIA1302-GFP vector (served as the positive control) was co-transformed into Allium cepa. 4′,6-diamidino-2-phenylindole (DAPI) was used as a maker of nuclear localization. Transient transformation of onion (Allium cepa) scale leaf epidermis cells was performed using the agrobacterium-mediated gene expression system and analyzed by laser scanning con-focal microscopy . The protocols were similar to transient transformation of onion scale leaf epidermal cells described previously [In order to generate DlMFT: GFP fusion protein, the coding sequence (CDS) of eviously , with soN. Benthamiana were extracted with TransZol Up Plus RNA Kit . Samples were then treated with DNase I to remove any genomic DNA. RNA was quantified by NanoDrop Lite spectrophotometer  and checked the integrality using 1.0% agarose gel electrophoresis.Total RNA of longan embryogenesis cultures and EC under various treatments were extracted by means of TriPure Isolation Reagent . Total RNA was isolated from longan tissues and different germination stages with the Universal Plant Total RNA Extraction Maxi Kit (Spin-column) . Total RNA of TM (Takara), 1 μL of 10× diluted cDNA, and 0.8 μL specific primer pairs , the amount of RNA was 500 ng in 10 μL reaction system. qRT-PCR was performed on the Lightcycler 480 system  in a 20 μL final volume containing 10 μL of 2× SYBR Premix Ex Taqes of SE , differees of SE . The expDlMFT pro:GUS plasmid, a 1.85-kb BamHI (5′)-NcoI (3′) DlMFT promoter fragment was subcloned into the BamHI-NcoI site of pCAMBIA1301, and designated this recombinant plasmid as DlMFT pro:GUS, used pCAMBIA1301 as control, then transferred DlMFT pro:GUS and pCAMBIA1301 into A. tumefaciens EHA105, respectively. Transient transformed the resulting A. Tumefaciens EHA105 in N. Benthamiana leaves by the injection method, then cultured the transformation plant in the greenhouse at 25 °C under dark conditions for three days. The injected part of the leaves was then harvested for verifying the activity of 35S and DlMFT promoter by qRT-PCR.To generate the


The time-dependent oxygen absorption of aqueous GA solutions (pH 2.9–13.8) was determined and the results were described using a second-order kinetic model. We calculated the degree of deprotonation (DoD) of GA in the solutions from the species distribution curves, and assessed the effect of the DoD on the reaction kinetics. This revealed that both the reaction rate coefficient k and the scavenger capacity n were significantly affected by the DoD. If the DoD fell below 0.25, there was no significant reaction. Although k increased with the DoD, n reached a maximum at DoD = 0.6–0.7. In principle, target DoD values can be achieved using any base, but a strong base is more efficient because lower quantities are required. In our experiments, the amount of Na2CO3 required to reach the maximum DoD was more than twice that of NaOH. Our results provide the basis for the functional design of active packaging systems incorporating optimized GA-based oxygen scavengers.Oxygen scavengers based on gallic acid (GA) usually contain a base that establishes the alkaline conditions necessary for the humidity-induced scavenger reaction. Here we measured the effect of sodium carbonate (Na Water-free sodium carbonate  was used as a reference.To prepare aqueous GA solutions at different pH values, samples of 0.040 g GA powder and the appropriate amount of base were mixed with 5 mL destilled water in a Petri dish at 23 °C. Solutions were prepared with NaOH at pH 5.7, 7.9, 9.1, 11.1, 13.0, 13.8. Solutions were prepared with NaK, as shown in Equations (1)–(5).An aqueous solution of GA includes several acid-base equilibria, each characterized by its corresponding equilibrium constant 4A, 3−, and A4− denote the GA species with 4, 3, 2, 1, and 0 acidic protons, respectively. Equations (1)–(4) describe the GA deprotonation equilibria characterized by the equilibrium constants pKa1 = 4.39 which is related to the carboxylic proton and pKa2 = 8.50, pKa3 = 10.38, pKa4 = 13  and the charge balance (Equation 7):A is the initial GA concentration and [A4−], [HA3−], 4A], [H3O+], [OH−], [Na+] are the equilibrium concentrations of the different GA species and the hydronium, hydroxide and sodium ions, respectively.where cfsolve function. The pH –(7) was solved in MATLAB R2014a  using the + refers to the total amount of acidic H+ available from the GA molecules in the solution, and donated H+ refers to the total amount of acidic H+ transferred to the solution at a certain pH.where total acidic H2CO3, a biprotic base, is added to the solution, additional equilibria have to be considered as shown in Equations (9) and (10).If Na2. The equilibrium constants we used were pKb1 = 10.3 and pKb2 = 6.3  was monitored for 30 days at 23°C using Fibox 4 Trace, a luminescence-based O2 detection system . To minimize the effect of O2 transport limits in the solution, the Petri dishes were only partially filled.The absorption of Of 88 cm3 . The Petp2O absorption data in hPa were converted to concentrations c2O using the ideal gas law (Equation 11).The n2O is the amount of substance of O2, VHS is the headspace volume of the cell, R = 8.314 J mol−1K−1 is the ideal gas constant, and T is the temperature in Kelvin.where Each experiment was carried out at least three times and the results are presented as the arithmetic mean with standard deviations.exp) of the observed O2 concentrations was calculated as shown in Equation (12):The mean square error   we do not differentiate between the different GA species; (ii) the oxygen uptake is irreversible; and (iii) all oxidation products are summarized as GAox.where GA represents all non-oxidized GA species, r was expressed as shown in Equation (14):The kinetic law of this reaction was approximated as a second-order elementary reaction and the reaction rate 2] denote the concentrations of GA and O2, respectively [mol m−3] and k is the reaction rate coefficient [m3 mol−1 s−1]. Based on this kinetic law, the net consumption rates of GA and O2 were described with the system of ordinary differential equations (ODEs) shown as Equations (15) and (16)where [GA] and [Oode15s. The model was fitted to the experimental data to determine the kinetic parameters k and n. The fit was optimized using a downhill simplex method as described by Pant and Reinelt  was described using Equations (1)–(10). GA is present as a range of deprotonated GA species that depend on the pH of the solution. The equilibrium concentrations of the different GA species at a given pH are determined by the acidity constants of the acidic protons. on of GA . The DoD2CO3 is necessary to achieve a certain DoD according to the simulation. Due to the different strength of the two bases, the amounts differ significantly and the difference in the quantity of each base required becomes larger as the DoD increases. For example, to achieve a DoD of 0.75 using Na2CO3, base:GA weight ratio is 2, but this falls to only 0.8 when using NaOH.2 scavengers can absorb O2 if water is present, i.e., in aqueous solutions comprising GA and a base. We assessed the effect of the base on the ability of GA to absorb O2 in solutions at different pH values, prepared by adding either NaOH : The necessary quantities to achieve higher pH values would not dissolve in the GA solution.The reference solution with no base added (pH = 2.9) did not absorb any On et al.  also rep2 absorption must reflect a pH-dependent effect on the mechanism of the oxidation reaction, leading to different ways of incorporating O2 and thereby to different oxidation products. This was confirmed by the color development of the samples. t = 0), the solutions were almost colorless with a slight yellow tint. Between days 1 and 30, the solutions at pH 7.2 and 11.1 changed from green to brown, whereas the solution at pH 13.0 change from organge to yellow. For autoxidation experiments in the pH range 2.6–11, Tóth et al. (The experiment was stopped after 30 days, but not all samples reached their maximum absorption capacity during this period because the scavenging reaction was too slow. The observed differences in Oh et al.  reportedh et al. . Further2 absorption measurements clearly demonstrated that pH has an effect on scavenger kinetics. However, the pH only provides information about conditions external to the reaction and, strictly speaking, the concept of pH can only be used to describe dilute aqueous solutions. For the design of O2 scavenger mixtures for packaging applications, it therefore seems more reasonable to describe the autoxidation process as a function of an intrinsic property of GA, the DoD.The Ok and n were determined for each experimental dataset by fitting a second-order kinetic model (exp. The fitted curves are shown in 2 uptake by GA under diverse conditions.The kinetic parameters ic model . For mosk and the absorption capacity n in the presence of each base. We found that k increased in line with the DoD, but that n reached a maximum at a DoD value of 0.6–0.7 equating to a pH of 10–11 under our experimental conditions. When the DoD fell below 0.2–0.3 there was no significant O2 absorption. At a DoD value of 0.6–0.7 the predominant species in the solution are HA3− and 4− is likely to involve a different reaction pathway leading to a lower absorption capacity.nd H2A2- . These s2CO3 required to achieve a certain DoD in the solution and this relationship is the basis for the development of an optimized scavenger composition. For maximum O2 scavenger capacity, the DoD should be 0.6–0.7. This requires a base:GA mass ration of ~1.8:1 for Na2CO3 and ~0.8:1 for NaOH. As expected, a much smaller amount of the strong base NaOH is required to reach any given DoD compared to the weaker base Na2CO3. In terms of the design and processing of packaging materials incorporating scavengers based on GA, the amount of base required is important in terms of the precessability of the packaging material and the cost efficiency of the process.2 absorption properties of the system in terms of the reaction rate constant and also the maximum absorption capacity. The addition of a base changes the acid-base equilibrium of the solution and therefore the type and amount of GA species that are present, which in turn influences the autoxidation reaction and ultimately the absorption of O2. The key property that must be targeted when designing O2 scavengers based on GA is therefore the DoD. We found that no reaction occurred at DoD values below 0.25. The absorption capacity n reaches its maximum value when the DoD is 0.6–0.7 whereas the reaction rate constant k increases with the DoD. Accordingly, the optimum DoD can be achieved by adding either NaOH or Na2CO3, but a strong base is more efficient because a lower quantity is required to reach the desired DoD value. These results, together with the models describing the GA solution and the autoxidation reaction, provide guidance for the development of packaging systems incorporating O2 scavengers based on GA.In this study, we found that the amount and type of base added to an aqueous GA solution changes the OThe datasets generated for this study are available on request to the corresponding author.AP conceived and designed the experiments, performed some of the oxygen absorption measurements, conducted the simulations and parameter estimations, and wrote the paper. DÖ contributed to the experimental design and performed some of the oxygen absorption measurements. SF contributed to the analysis and interpretation of the results and helped to write and revise the paper. MR contributed to the experimental design as well as modeling and simulation techniques and revised the paper.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.