Shoot branching is a highly plastic developmental process in which axillary buds are formed in the axil of each leaf and may subsequently be activated to give branches. Three classes of plant ...hormones, auxins, cytokinins and strigolactones (or strigolactone derivatives) are central to the control of bud activation. These hormones move throughout the plant forming a network of systemic signals. The past decade brought great progress in understanding the mechanisms of shoot branching control. Biological and computational studies have led to the proposal of two models, the auxin transport canalization-based model and the second messenger model, which provide mechanistic explanations for apical dominance.
The biosynthesis of the recently identified novel class of plant hormones, strigolactones, is up-regulated upon phosphate deficiency in many plant species. It is generally accepted that the ...evolutionary origin of strigolactone up-regulation is their function as a rhizosphere signal that stimulates hyphal branching of arbuscular mycorrhizal fungi. In this work, we demonstrate that this induction is conserved in Arabidopsis (Arabidopsis thaliana), although Arabidopsis is not a host for arbuscular mycorrhizal fungi. We demonstrate that the increase in strigolactone production contributes to the changes in shoot architecture observed in response to phosphate deficiency. Using high-performance liquid chromatography, column chromatography, and multiple reaction monitoring-liquid chromatography-tandem mass spectrometry analysis, we identified two strigolactones (orobanchol and orobanchyl acetate) in Arabidopsis and have evidence of the presence of a third (5-deoxystrigol). We show that at least one of them (orobanchol) is strongly reduced in the putative strigolactone biosynthetic mutants more axillary growth1 (max1) and max4 but not in the signal transduction mutant max2. Orobanchol was also detected in xylem sap and up-regulated under phosphate deficiency, which is consistent with the idea that root-derived strigolactones are transported to the shoot, where they regulate branching. Moreover, two additional putative strigolactone-like compounds were detected in xylem sap, one of which was not detected in root exudates. Together, these results show that xylem-transported strigolactones contribute to the regulation of shoot architectural response to phosphate-limiting conditions.
Genetic interactions between phytohormones in the control of flowering time in Arabidopsis thaliana have not been extensively studied. Three phytohormones have been individually connected to the ...floral-timing program. The inductive function of gibberellins (GAs) is the most documented. Abscisic acid (ABA) has been demonstrated to delay flowering. Finally, the promotive role of brassinosteroids (BRs) has been established. It has been reported that for many physiological processes, hormone pathways interact to ensure an appropriate biological response.
We tested possible genetic interactions between GA-, ABA-, and BR-dependent pathways in the control of the transition to flowering. For this, single and double mutants deficient in the biosynthesis of GAs, ABA, and BRs were used to assess the effect of hormone deficiency on the timing of floral transition. Also, plants that over-express genes encoding rate-limiting enzymes in each biosynthetic pathway were generated and the flowering time of these lines was investigated.
Loss-of-function studies revealed a complex relationship between GAs and ABA, and between ABA and BRs, and suggested a cross-regulatory relation between GAs to BRs. Gain-of-function studies revealed that GAs were clearly limiting in their sufficiency of action, whereas increases in BRs and ABA led to a more modest phenotypic effect on floral timing. We conclude from our genetic tests that the effects of GA, ABA, and BR on timing of floral induction are only in partially coordinated action.
Aneuploidy causes system-wide disruptions in the stochiometric balances of transcripts, proteins, and metabolites, often resulting in detrimental effects for the organism. The protozoan parasite ...Leishmania has an unusually high tolerance for aneuploidy, but the molecular and functional consequences for the pathogen remain poorly understood. Here, we addressed this question in vitro and present the first integrated analysis of the genome, transcriptome, proteome, and metabolome of highly aneuploid Leishmania donovani strains. Our analyses unambiguously establish that aneuploidy in Leishmania proportionally impacts the average transcript- and protein abundance levels of affected chromosomes, ultimately correlating with the degree of metabolic differences between closely related aneuploid strains. This proportionality was present in both proliferative and non-proliferative in vitro promastigotes. However, as in other Eukaryotes, we observed attenuation of dosage effects for protein complex subunits and in addition, non-cytoplasmic proteins. Differentially expressed transcripts and proteins between aneuploid Leishmania strains also originated from non-aneuploid chromosomes. At protein level, these were enriched for proteins involved in protein metabolism, such as chaperones and chaperonins, peptidases, and heat-shock proteins. In conclusion, our results further support the view that aneuploidy in Leishmania can be adaptive. Additionally, we believe that the high karyotype diversity in vitro and absence of classical transcriptional regulation make Leishmania an attractive model to study processes of protein homeostasis in the context of aneuploidy and beyond.
Strigolactones (SLs), or their derivatives, were recently demonstrated to act as endogenous shoot branching inhibitors, but their biosynthesis and mechanism of action are poorly understood. Here we ...show that the branching phenotype of mutants in the Arabidopsis P450 family member, MAX1, can be fully rescued by strigolactone addition, suggesting that MAX1 acts in SL synthesis. We demonstrate that SLs modulate polar auxin transport to control branching and that both the synthetic SL GR24 and endogenous SL synthesis significantly reduce the basipetal transport of a second branch-regulating hormone, auxin. Importantly, GR24 inhibits branching only in the presence of auxin in the main stem, and enhances competition between two branches on a common stem. Together, these results support two current hypotheses: that auxin moving down the main stem inhibits branch activity by preventing the establishment of auxin transport out of axillary branches; and that SLs act by dampening auxin transport, thus enhancing competition between branches.
Abstract
Leishmania, a unicellular eukaryotic parasite, is a unique model for aneuploidy and cellular heterogeneity, along with their potential role in adaptation to environmental stresses. Somy ...variation within clonal populations was previously explored in a small subset of chromosomes using fluorescence hybridization methods. This phenomenon, termed mosaic aneuploidy (MA), might have important evolutionary and functional implications but remains under-explored due to technological limitations. Here, we applied and validated a high throughput single-cell genome sequencing method to study for the first time the extent and dynamics of whole karyotype heterogeneity in two clonal populations of Leishmania promastigotes representing different stages of MA evolution in vitro. We found that drastic changes in karyotypes quickly emerge in a population stemming from an almost euploid founder cell. This possibly involves polyploidization/hybridization at an early stage of population expansion, followed by assorted ploidy reduction. During further stages of expansion, MA increases by moderate and gradual karyotypic alterations, affecting a defined subset of chromosomes. Our data provide the first complete characterization of MA in Leishmania and pave the way for further functional studies.
Graphical Abstract
Graphical Abstract
Overview of the methodological approach (top) and the main results (bottom).
Graphical Movie Abstract
10.1093/nar/gkab1203
Graphical Movie Abstract
gkab1203Media1
6286118383001
Proline (Pro) is a versatile metabolite playing a role in the protection of plants against environmental stresses. To gain a deeper understanding of the regulation of Pro metabolism under predicted ...future climate conditions, including drought stress, elevated temperature and CO2, we combined measurements in contrasting grassland species (two grasses and two legumes) at multiple organisational levels, that is, metabolite concentrations, enzyme activities and gene expression.
Drought stress (D) activates Pro biosynthesis and represses its catabolism, and elevated temperature (DT) further elevated its content. Elevated CO2 attenuated the DT effect on Pro accumulation.
Computational pathway control analysis allowed a mechanistic understanding of the regulatory changes in Pro metabolism. This analysis indicates that the experimentally observed coregulation of multiple enzymes is more effective in modulating Pro concentrations than regulation of a single step. Pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) play a central role in grasses (Lolium perenne, Poa pratensis), and arginase (ARG), ornithine aminotransferase (OAT) and P5CR play a central role in legumes (Medicago lupulina, Lotus corniculatus).
Different strategies in the regulation of Pro concentrations under stress conditions were observed. In grasses the glutamate pathway is activated predominantly, and in the legumes the ornithine pathway, possibly related to differences in N-nutritional status.
Under stressful conditions some microorganisms adopt a quiescent stage characterized by a reversible non or slow proliferative condition that allows their survival. This adaptation was only recently ...discovered in Leishmania. We developed an in vitro model and a biosensor to track quiescence at population and single cell levels. The biosensor is a GFP reporter gene integrated within the 18S rDNA locus, which allows monitoring the expression of 18S rRNA (rGFP expression). We showed that rGFP expression decreased significantly and rapidly during the transition from extracellular promastigotes to intracellular amastigotes and that it was coupled in vitro with a decrease in replication as measured by BrdU incorporation. rGFP expression was useful to track the reversibility of quiescence in live cells and showed for the first time the heterogeneity of physiological stages among the population of amastigotes in which shallow and deep quiescent stages may coexist. We also validated the use of rGFP expression as a biosensor in animal models of latent infection. Our models and biosensor should allow further characterization of quiescence at metabolic and molecular level.
The aim of the study was to recognise what participant-, training- and post-injury-related factors are associated with an injury and re-injury occurrence in female pole dancers (PDs). 320 female PDs ...fulfilled a custom survey. 1050 injuries were reported by 276 PDs, 59% of injuries were related to lower extremity, 39% to upper extremity and 10% to spine and trunk. 156 PDs reported sustaining a re-injury, and overall, 628 re-injuries were reported. The median weekly pole-specific training session volume was 90 min and 240 min in the low and high qualified group, respectively. The total training volume was 180 min in the low qualified PDs and 240 min in the high qualified group. PDs with higher height and spending more time on pole-specific training in studio and on other forms of training have higher odds of sustaining an injury. PDs with lower level of experience in training, who sustained an injury, and who had a shorter pause between the moment of injury and the return to performance, and thus who did not fully recover, have higher odds of sustaining a re-injury. Sport-specific injury prevention strategies should be developed and implemented in this cohort, since over 85% of pole dancers reported sustaining some kind of injury.
Whole genome sequencing (WGS) is increasingly used for molecular diagnosis and epidemiology of infectious diseases. Current Leishmania genomic studies rely on DNA extracted from cultured parasites, ...which might introduce sampling and biological biases into the subsequent analyses. Up to now, direct analysis of Leishmania genome in clinical samples is hampered by high levels of human DNA and large variation in parasite load in clinical samples. Here, we present a method, based on target enrichment of Leishmania donovani DNA with Agilent SureSelect technology, that allows the analysis of Leishmania genomes directly in clinical samples. We validated our protocol with a set of artificially mixed samples, followed by the analysis of 63 clinical samples (bone marrow or spleen aspirates) from visceral leishmaniasis patients in Nepal. We were able to identify genotypes using a set of diagnostic SNPs in almost all of these samples (97%) and access comprehensive genome-wide information in most (83%). This allowed us to perform phylogenomic analysis, assess chromosome copy number and identify large copy number variants (CNVs). Pairwise comparisons between the parasite genomes in clinical samples and derived in vitro cultured promastigotes showed a lower aneuploidy in amastigotes as well as genomic differences, suggesting polyclonal infections in patients. Altogether our results underline the need for sequencing parasite genomes directly in the host samples.
PDF
