Gibberellins and the cold requirement of tulip

Tulip bulbs (Tulipa gesneriana L.), with terminal buds containing a complete flower, require a period of low temperature to prepare the bud for floral stalk elongation and full flower development at subsequent higher temperatures. For the cultivar Apeldoorn, a dry- storage treatment of 12 weeks at 5°C prior to planting at 20°C, win lead to proper floral stalk elongation and full flower development. Shorter periods at 5°C usually result in slower shoot elongation and delayed flowering. Without any cold treatment, the growth of the shoot is strongly reduced and often flower abortion occurs. In these processes, the involvement of gibberellins (GAs) has been implicated, because application of GAs could partly replace the cold treatment. In addition, GA biosynthesis inhibitors could reduce the floral stalk elongation of cold-treated bulbs and this effect was reversed by simultaneous application of GA.In horticultural practice, there is a need for a practical assay to test whether a particular bulb has received a proper cold treatment. The amount of GAs or of one particular GA, might provide a suitable parameter in a test for properly cold-treated bulbs.In this study, the role of GAs in the cold requirement of tulip was investigated by analysing the GA levels in cooled and noncooled tulip bulbs, and by studying the effect and metabolism of applied GAs in combination with a' GA biosynthesis inhibitor.An inventory was made of GAs, in sprouts of cooled (12 weeks 5°C) and noncooled bulbs (12 weeks 17°C) (chapter 3). By combined gas chromatography-mass spectrometry (GC-MS) and GC-selected ion monitoring (SIM), GA 1 , GA 4 , GA 9 , GA 12 , GA 24 , GA 34 and three GA-related compounds were detected. They all occurred in sprouts of both cooled and noncooled bulbs. Most of them were found in the conjugated form as well. Among these GAs, GA 4 and/or GA 1 might be the active forms, the others being precursors (GA 9 , GA 12 , and GA 24 ), or an inactivation product (GA 34 ).Using GC-SIM and deuterated GAs as internal standards, the changes in endogenous GA levels were measured in sprouts and basal plates during cooled and noncooled bulb storage, as well as after planting these bulbs (chapter 4). GA 4 and GA 24 were the major occurring gibberellins, with levels up to ca. 10 ng per sprout or basal plate. GA 1 , GA 9 and GA 34 were present in much lower amounts. The levels of GA 12 and of the GA conjugates and GA-related compounds were not analysed.During bulb storage, the level of GA 4 per sprout increased, especially in noncooled bulbs. After 12 weeks, these sprouts contained more GA 4 and also more GA 1 than cooled sprouts. However, sprouts in noncooled bulbs did hardly show any development after planting and it is unlikely that the increased level at the end of bulb storage is correlated with floral stalk elongation at subsequent higher temperatures. In the basal plates no significant changes occurred in the GA levels during storage. Therefore, the GA content in sprouts or basal plates at the end of bulb storage, cannot be used as marker in a test for properly cold-treated bulbs.After planting cooled bulbs, the sprouts started to grow and within the first 11 days the level of GA 4 in the floral sulks increased. In planted noncooled bulbs, sprout growth was negligible and an increase in the level of GA 4 did not occur.The biological activity of GA 1 , GA 4 and GA 9 , was tested on isolated sprouts, cultivated on a liquid medium in vitro (chapter 5). To compare the sensitivity to exogenous GAs, sprouts from both cooled and noncooled bulbs were used. The growth retardant paclobutrazol was used to study the role of GA biosynthesis. The growth of these isolated sprouts, the response to GAs and the effect of paclobutrazol, appeared to be dependent not only on the pretreatment of the bulbs, but also on the time in the season that the sprouts were isolated and incubated.At early starting dates of incubation, floral stalks from both cooled and noncooled bulbs hardly showed any elongation in the absence of exogenous GA. Paclobutrazol had no effect on floral stalk elongation, and the response to GAs of sprouts from cooled bulbs was greater than the response of sprouts from noncooled bulbs. At later starts, considerable floral stalk elongation already occurred without GA application. Paclobutrazol inhibited this floral stalk elongation, and the growth of sprouts from both cooled and noncooled bulbs was stimulated by GA application. The three tested GAs were not significantly different in stimulating floral stalk elongation. The effect of paclobutrazol. was reversed by simultaneous application of GA. The results of these in vitro experiments demonstrated that, although depending on the time of the year, sprouts from both cooled and noncooled bulbs are responsive to exogenous GAs. Moreover, sprouts from both bulb treatments are capable of GA biosynthesis. The increasing performance of the isolated sprouts when incubated at later starting dates, and the increasing effect of paclobutrazol on these sprouts, suggested an increase in the availability of precursors for the synthesis of GAs. Apparently, low temperatures as well as bulb storage itself enhance GA biosynthesis and GA sensitivity, and consequently floral stalk elongation after planting when conditions are favourable for growth.The isolated sprouts did not develop a full-grown flower without the addition of GA. GA 4 was more effective than GA 9 in stimulating this flower development. GA 1 could also stimulate flower development, but was no more effective than GA 4 .The activity of applied GA 9 might be due to its conversion to GA 4 . GA 4 on its turn, might have to be converted to GA 1 before becoming biologically active. The metabolism of applied GA 9 was studied, with the purpose to investigate whether tulip sprouts are able to metabolize GA 9 to biologically active GA 4 or GA 1 , and whether sprouts from cooled and noncooled bulbs show differences in GA metabolism (chapter 6). [ 3H]GA 9 and [ 2H]GA 9 were applied to isolated sprouts by injection into the floral stalk and the metabolites were analysed in the sprouts after 24 h. According to HPLC analyses, [ 3H]GA 9 was converted to GA 4 -like and GA 34 -like compounds. The labelled metabolites of [ 2H]GA 9 were identified by GC-SIM, which demonstrated the conversion of [ 2H]GA 9 to [ 2H]GA 4 and [ 2H]GA 34 . Sprouts from both cooled and noncooled bulbs were able to convert GA 9 to GA 4 and GA 34in vitro . No evidence was found for the production of labelled GA 1 .In the presence of prohexadione (BX-112), known for its inhibiting effect on 2- and 3β-hydroxylations of GAs, the formation of [ 2H] metabolites was less or absent.In conclusion, there is no direct correlation between the cold-stimulated growth and a change in the endogenous GA status in sprouts or basal plates during cold bulb storage. Further, the sensitivity to GAs increases in cooled sprouts, but also noncooled sprouts are responsive to applied GAs, and GA sensitivity apparently is not limiting for the development of noncooled sprouts in vitro . After cooled bulb storage, GA biosynthesis is essential for floral stalk elongation to proceed. The increase in the level of GA 4 in the growing floral stalks of cooled bulbs, the response of isolated sprouts to GA 4 and the inability of isolated sprouts to produce detectable amounts of GA 1 from applied GA 9 , support the hypothesis that GA 4 is the major intrinsically active GA in the floral stalk elongation of tulip

Impact of Extraction Time during Donation after Circulatory Death Organ Procurement on Kidney Function after Transplantation in the Netherlands

Background:In The Netherlands, 60% of deceased-donor kidney offers are after donation after circulatory death. Cold and warm ischemia times are known risk factors for delayed graft function (DGF) and inferior allograft survival. Extraction time is a relatively new ischemia time. During procurement, cooling of the kidneys is suboptimal with ongoing ischemia. However, evidence is lacking on whether extraction time has an impact on DGF if all ischemic periods are included.Methods:Between 2012 and 2018, 1524 donation after circulatory death kidneys were procured and transplanted in The Netherlands. Donation and transplantation-related data were obtained from the database of the Dutch Transplant Foundation. The primary outcome parameter was the incidence of DGF. Results:In our cohort, extraction time ranged from 14 to 237 min, with a mean of 62 min (SD 32). In multivariate logistic regression analysis, extraction time was an independent risk factor for incidence of DGF (odds ratio per minute increase 1.008; 95% confidence interval, 1.003-1.013; P = 0.001). The agonal phase, hypoperfusion time, and anastomosis time were not independent risk factors for incidence of DGF. Conclusions:Considering all known ischemic periods during the donation after the circulatory death process, prolonged kidney extraction time increased the risk of DGF after kidney transplantation.</p

Antibody Labelling of Resilin in Energy Stores for Jumping in Plant Sucking Insects

The rubbery protein resilin appears to form an integral part of the energy storage structures that enable many insects to jump by using a catapult mechanism. In plant sucking bugs that jump (Hemiptera, Auchenorrhyncha), the energy generated by the slow contractions of huge thoracic jumping muscles is stored by bending composite bow-shaped parts of the internal thoracic skeleton. Sudden recoil of these bows powers the rapid and simultaneous movements of both hind legs that in turn propel a jump. Until now, identification of resilin at these storage sites has depended exclusively upon characteristics that may not be specific: its fluorescence when illuminated with specific wavelengths of ultraviolet (UV) light and extinction of that fluorescence at low pH. To consolidate identification we have labelled the cuticular structures involved with an antibody raised against a product of the Drosophila CG15920 gene. This encodes pro-resilin, the first exon of which was expressed in E. coli and used to raise the antibody. We show that in frozen sections from two species, the antibody labels precisely those parts of the metathoracic energy stores that fluoresce under UV illumination. The presence of resilin in these insects is thus now further supported by a molecular criterion that is immunohistochemically specific

Direct-Acting Antiviral Treatment for Hepatitis C Genotypes Uncommon in High-Income Countries:A Dutch Nationwide Cohort Study

Background. The majority of hepatitis C virus (HCV) infections are found in low- and middle-income countries, which harbor many region-specific HCV subtypes. Nevertheless, direct-acting antiviral (DAA) trials have almost exclusively been conducted in high-income countries, where mainly epidemically spread HCV subtypes are present. Recently, several studies have demonstrated suboptimal DAA efficacy for certain nonepidemic subtypes, which could hamper global HCV elimination. Therefore, we aimed to evaluate DAA efficacy in patients treated for a nonepidemic HCV genotype infection in the Netherlands. Methods. We performed a nationwide retrospective study including patients treated with interferon-free DAAs for an HCV genotype other than 1a/1b/2a/2b/3a/4a/4d. The genotype was determined by NS5B region phylogenetic analysis. The primary end point was SVR-12. If stored samples were available, NS5A and NS5B sequences were obtained for resistance-associated substitutions (RAS) evaluation. Results. We included 160 patients, mainly infected with nonepidemic genotype 2 (41%) and 4 (31%) subtypes. Most patients were from Africa (45%) or South America (24%); 51 (32%) were cirrhotic. SVR-12 was achieved in 92% (140/152) of patients with available SVR-12 data. Only 73% (8/11) genotype 3-infected patients achieved SVR-12, the majority being genotype 3b patients with 63% (5/8) SVR. Regardless of SVR, all genotype 3b patients had 30K and 31M RAS. Conclusions. (T)he DAA efficacy we observed in most nonepidemic genotypes in the Netherlands seems reassuring. However, the low SVR-12 rate in subtype 3b infections is alarming, especially as it is common in several HCV-endemic countries. Alongside earlier results, our results indicate that a remaining challenge for global HCV elimination is confirming and monitoring DAA efficacy in nonepidemic genotypes

High frequency of Polio-like Enterovirus C strains with differential clustering of CVA-13 and EV-C99 subgenotypes in a cohort of Malawian children

Enteroviruses (EVs) are among the most commonly detected viruses infecting humans worldwide. Although the prevalence of EVs is widely studied, the status of EV prevalence in sub-Saharan Africa remains largely unknown. The objective of our present study was therefore to increase our knowledge on EV circulation in sub-Saharan Africa. We obtained 749 fecal samples from a cross-sectional study conducted on Malawian children aged 6 to 60 months. We tested the samples for the presence of EVs using real time PCR, and typed the positive samples based on partial viral protein 1 (VP1) sequences. A large proportion of th

Direct-Acting Antiviral Treatment for Hepatitis C Genotypes Uncommon in High-Income Countries: A Dutch Nationwide Cohort Study (vol 8, ofab006, 2021)

Cellular mechanisms in basic and clinical gastroenterology and hepatolog

Rapid transcriptional plasticity of duplicated gene clusters enables a clonally reproducing aphid to colonise diverse plant species

Background: The prevailing paradigm of host-parasite evolution is that arms races lead to increasing specialisation via genetic adaptation. Insect herbivores are no exception and the majority have evolved to colonise a small number of closely related host species. Remarkably, the green peach aphid, Myzus persicae, colonises plant species across 40 families and single M. persicae clonal lineages can colonise distantly related plants. This remarkable ability makes M. persicae a highly destructive pest of many important crop species. Results: To investigate the exceptional phenotypic plasticity of M. persicae, we sequenced the M. persicae genome and assessed how one clonal lineage responds to host plant species of different families. We show that genetically identical individuals are able to colonise distantly related host species through the differential regulation of genes belonging to aphid-expanded gene families. Multigene clusters collectively upregulate in single aphids within two days upon host switch. Furthermore, we demonstrate the functional significance of this rapid transcriptional change using RNA interference (RNAi)-mediated knock-down of genes belonging to the cathepsin B gene family. Knock-down of cathepsin B genes reduced aphid fitness, but only on the host that induced upregulation of these genes. Conclusions: Previous research has focused on the role of genetic adaptation of parasites to their hosts. Here we show that the generalist aphid pest M. persicae is able to colonise diverse host plant species in the absence of genetic specialisation. This is achieved through rapid transcriptional plasticity of genes that have duplicated during aphid evolution

Gibberellin A1 Metabolism Contributes to the Control of Photoperiod-Mediated Tuberization in Potato

Some potato species require a short-day (SD) photoperiod for tuberization, a process that is negatively affected by gibberellins (GAs). Here we report the isolation of StGA3ox2, a gene encoding a GA 3-oxidase, whose expression is increased in the aerial parts and is repressed in the stolons after transfer of photoperiod-dependent potato plants to SD conditions. Over-expression of StGA3ox2 under control of constitutive or leaf-specific promoters results in taller plants which, in contrast to StGA20ox1 over-expressers previously reported, tuberize earlier under SD conditions than the controls. By contrast, StGA3ox2 tuber-specific over-expression results in non-elongated plants with slightly delayed tuber induction. Together, our experiments support that StGA3ox2 expression and gibberellin metabolism significantly contribute to the tuberization time in strictly photoperiod-dependent potato plants

Transcriptomics of the Bed Bug (Cimex lectularius)

BACKGROUND: Bed bugs (Cimex lectularius) are blood-feeding insects poised to become one of the major pests in households throughout the United States. Resistance of C. lectularius to insecticides/pesticides is one factor thought to be involved in its sudden resurgence. Despite its high-impact status, scant knowledge exists at the genomic level for C. lectularius. Hence, we subjected the C. lectularius transcriptome to 454 pyrosequencing in order to identify potential genes involved in pesticide resistance. METHODOLOGY AND PRINCIPAL FINDINGS: Using 454 pyrosequencing, we obtained a total of 216,419 reads with 79,596,412 bp, which were assembled into 35,646 expressed sequence tags (3902 contigs and 31744 singletons). Nearly 85.9% of the C. lectularius sequences showed similarity to insect sequences, but 44.8% of the deduced proteins of C. lectularius did not show similarity with sequences in the GenBank non-redundant database. KEGG analysis revealed putative members of several detoxification pathways involved in pesticide resistance. Lamprin domains, Protein Kinase domains, Protein Tyrosine Kinase domains and cytochrome P450 domains were among the top Pfam domains predicted for the C. lectularius sequences. An initial assessment of putative defense genes, including a cytochrome P450 and a glutathione-S-transferase (GST), revealed high transcript levels for the cytochrome P450 (CYP9) in pesticide-exposed versus pesticide-susceptible C. lectularius populations. A significant number of single nucleotide polymorphisms (296) and microsatellite loci (370) were predicted in the C. lectularius sequences. Furthermore, 59 putative sequences of Wolbachia were retrieved from the database. CONCLUSIONS: To our knowledge this is the first study to elucidate the genetic makeup of C. lectularius. This pyrosequencing effort provides clues to the identification of potential detoxification genes involved in pesticide resistance of C. lectularius and lays the foundation for future functional genomics studies