Structural and Functional Characteristics of Two Molecular Variants of the Nitrogen Sensor PII in Maritime Pine.
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ABSTRACT:
SUBMITTER: Llebres MT
PROVIDER: S-EPMC7308587 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Nitrate and ammonium are the main forms of inorganic nitrogen available to plants. The present study aimed to investigate the metabolic changes caused by ammonium and nitrate nutrition in maritime pine (Pinus pinaster Ait.). Seedlings were grown with five solutions containing different proportions of nitrate and ammonium. Their nitrogen status was characterized through analyses of their biomass, different biochemical and molecular markers as well as a metabolite profile using 1H-NMR. Ammonium-fed seedlings exhibited higher biomass than nitrate-fed-seedlings. Nitrate mainly accumulated in the stem and ammonium in the roots. Needles of ammonium-fed seedlings had higher nitrogen and amino acid contents but lower levels of enzyme activities related to nitrogen metabolism. Higher amounts of soluble sugars and L-arginine were found in the roots of ammonium-fed seedlings. In contrast, L-asparagine accumulated in the roots of nitrate-fed seedlings. The differences in the allocation of nitrate and ammonium may function as metabolic buffers to prevent interference with the metabolism of photosynthetic organs. The metabolite profiles observed in the roots suggest problems with carbon and nitrogen assimilation in nitrate-supplied seedlings. Taken together, this new knowledge contributes not only to a better understanding of nitrogen metabolism but also to improving aspects of applied mineral nutrition for conifers.
| S-EPMC7238028 | biostudies-literature
Project description:BackgroundPredicting the adaptability of forest tree populations under future climates requires a better knowledge of both the adaptive significance and evolvability of measurable key traits. Phenotypic plasticity, standing genetic variation and degree of phenotypic integration shape the actual and future population genetic structure, but empirical estimations in forest tree species are still extremely scarce. We analysed 11 maritime pine populations covering the distribution range of the species (119 families and 8 trees/family, ca. 1300 trees) in a common garden experiment planted at two sites with contrasting productivity. We used plant height as a surrogate of fitness and measured five traits (mean and plasticity of carbon isotope discrimination, specific leaf area, needle biomass, Phenology growth index) related to four different strategies (acquisitive economics, photosynthetic organ size, growth allocation and avoidance of water stress).ResultsEstimated values of additive genetic variation would allow adaptation of the populations to future environmental conditions. Overall phenotypic integration and selection gradients were higher at the high productivity site, while phenotypic integration within populations was higher at the low productivity site. Response to selection was related mainly to photosynthetic organ size and drought-avoidance mechanisms rather than to water use efficiency. Phenotypic plasticity of water use efficiency could be maladaptive, resulting from selection for height growth.ConclusionsContrary to the expectations in a drought tolerant species, our study suggests that variation in traits related to photosynthetic organ size and acquisitive investment of resources drive phenotypic selection across and within maritime pine populations. Both genetic variation and evolvability of key adaptive traits were considerably high, including plasticity of water use efficiency. These characteristics would enable a relatively fast micro-evolution of populations in response to the ongoing climate changes. Moreover, differentiation among populations in the studied traits would increase under the expected more productive future Atlantic conditions.
| S-EPMC10775667 | biostudies-literature
Project description:Posttranslational regulation of nitrogenase, or switch-off, in the methanogenic archaeon Methanococcus maripaludis requires both nifI(1) and nifI(2), which encode members of the PII family of nitrogen-regulatory proteins. Previous work demonstrated that nitrogenase activity in cell extracts was inhibited in the presence of NifI(1) and NifI(2), and that 2-oxoglutarate (2OG), a potential signal of nitrogen limitation, relieved this inhibition. To further explore the role of the NifI proteins in switch-off, we found proteins that interact with NifI(1) and NifI(2) and determined whether 2OG affected these interactions. Anaerobic purification of His-tagged NifI(2) resulted in copurification of NifI(1) and the dinitrogenase subunits NifD and NifK, and 2OG or a deletion mutation affecting the T-loop of NifI(2) prevented copurification of dinitrogenase but did not affect copurification of NifI(1). Similar results were obtained with His-tagged NifI(1). Gel-filtration chromatography demonstrated an interaction between purified NifI(1,2) and dinitrogenase that was inhibited by 2OG. The NifI proteins themselves formed a complex of approximately 85 kDa, which appeared to further oligomerize in the presence of 2OG. NifI(1,2) inhibited activity of purified nitrogenase when present in a 1:1 molar ratio to dinitrogenase, and 2OG fully relieved this inhibition. These results suggest a model for switch-off of nitrogenase activity, where direct interaction of a NifI(1,2) complex with dinitrogenase causes inhibition, which is relieved by 2OG. The presence of nifI(1) and nifI(2) in the nif operons of all nitrogen-fixing Archaea and some anaerobic Bacteria suggests that this mode of nitrogenase regulation may operate in a wide variety of diazotrophs.
| S-EPMC1502530 | biostudies-literature
Project description:Maritime pine (Pinus pinaster Aiton) is a coniferous native of the Mediterranean basin. Because of its adaptability to a wide range of environmental conditions, the species have become a model for studies in coniferous forest management and functional genomics. Somatic embryogenesis (SE) has been so far, the preferred biotechnological strategy for maritime pine breeding programs initiated at the middle-end of the 20th century. To overcome the limitations of the induction and maturation phases in maritime pine SE, we analyzed the possible maternal influence on the embryogenic capability of megagametophytes from controlled crosses, as well as the effect of the temperature and water availability during SE process on the production of plants. A strong maternal effect on the embryogenic potential of maritime pine megagametophytes was observed in our experiments using half-sib and full-sib progenies, while paternal effect was almost undetectable. Besides, it seems possible to improve somatic embryo production of maritime pine megagametophytes by adjusting optimal temperature throughout the process: 28°C during induction and proliferation, and 23°C during the maturation phase. Using induction and proliferation media with reduced water availability (6 g/L Gelrite) can also increase embryo production. Since other limitation of maritime pine SE is culture decline of embryogenic masses (EMs), that reduces embryo yield and germination, we assessed the profile of ABA and IAA and the expression of two embryogenesis-related genes (LEC1 and WOX2) during maturation of EMs of two morphotypes that differed in their maturation capability. Spiky morphotype (SK), with high maturation capability, had a steady increase in both hormones along the 12 weeks of the maturation, whereas ABA content in smooth morphotype picked at the 4th week and dropped. EMs with this morphotype also had a higher IAA content at the beginning of the maturation. A decrease of LEC1 and WOX2 gene expression over the course of embryo development was found to be characteristic of the SK with high maturation capability.
| S-EPMC6389691 | biostudies-literature
Project description:Pines are the dominant conifers in Mediterranean forests. As long-lived sessile organisms that seasonally have to cope with drought periods, they have developed a variety of adaptive responses. However, during last decades, highly intense and long-lasting drought events could have contributed to decay and mortality of the most susceptible trees. Among conifer species, Pinus pinaster Ait. shows remarkable ability to adapt to different environments. Previous molecular analysis of a full-sib family designed to study drought response led us to find active transcriptional activity of stress-responding genes even without water deprivation in tolerant genotypes. To improve our knowledge about communication between above- and below-ground organs of maritime pine, we have analyzed four graft-type constructions using two siblings as rootstocks and their progenitors, Gal 1056 and Oria 6, as scions. Transcriptomic profiles of needles from both scions were modified by the rootstock they were grafted on. However, the most significant differential gene expression was observed in drought-sensitive Gal 1056, while in drought-tolerant Oria 6, differential gene expression was very much lower. Furthermore, both scions grafted onto drought-tolerant rootstocks showed activation of genes involved in tolerance to abiotic stress, and is most remarkable in Oria 6 grafts where higher accumulation of transcripts involved in phytohormone action, transcriptional regulation, photosynthesis and signaling has been found. Additionally, processes, such as those related to secondary metabolism, were mainly associated with the scion genotype. This study provides pioneering information about rootstock effects on scion gene expression in conifers.
| S-EPMC8173007 | biostudies-literature
Project description:BACKGROUND: Pinus pinaster is an economically and ecologically important species that is becoming a woody gymnosperm model. Its enormous genome size makes whole-genome sequencing approaches are hard to apply. Therefore, the expressed portion of the genome has to be characterised and the results and annotations have to be stored in dedicated databases. DESCRIPTION: EuroPineDB is the largest sequence collection available for a single pine species, Pinus pinaster (maritime pine), since it comprises 951 641 raw sequence reads obtained from non-normalised cDNA libraries and high-throughput sequencing from adult (xylem, phloem, roots, stem, needles, cones, strobili) and embryonic (germinated embryos, buds, callus) maritime pine tissues. Using open-source tools, sequences were optimally pre-processed, assembled, and extensively annotated (GO, EC and KEGG terms, descriptions, SNPs, SSRs, ORFs and InterPro codes). As a result, a 10.5× P. pinaster genome was covered and assembled in 55 322 UniGenes. A total of 32 919 (59.5%) of P. pinaster UniGenes were annotated with at least one description, revealing at least 18 466 different genes. The complete database, which is designed to be scalable, maintainable, and expandable, is freely available at: http://www.scbi.uma.es/pindb/. It can be retrieved by gene libraries, pine species, annotations, UniGenes and microarrays (i.e., the sequences are distributed in two-colour microarrays; this is the only conifer database that provides this information) and will be periodically updated. Small assemblies can be viewed using a dedicated visualisation tool that connects them with SNPs. Any sequence or annotation set shown on-screen can be downloaded. Retrieval mechanisms for sequences and gene annotations are provided. CONCLUSIONS: The EuroPineDB with its integrated information can be used to reveal new knowledge, offers an easy-to-use collection of information to directly support experimental work (including microarray hybridisation), and provides deeper knowledge on the maritime pine transcriptome.
| S-EPMC3152544 | biostudies-literature
Project description:The widespread PII signal transduction proteins are known for integrating signals of nitrogen and energy supply and regulating cellular behavior by interacting with a multitude of target proteins. The PII protein of the cyanobacterium Synechococcus elongatus forms complexes with the controlling enzyme of arginine synthesis, N-acetyl-L-glutamate kinase (NAGK) in a 2-oxoglutarate- and ATP/ADP-dependent manner. Fusing NAGK and PII proteins to either CFP or YFP yielded a FRET sensor that specifically responded to 2-oxoglutarate. The impact of the fluorescent tags on PII and NAGK was evaluated by enzyme assays, surface plasmon resonance spectroscopy and isothermal calorimetric experiments. The developed FRET sensor provides real-time data on PII - NAGK interaction and its modulation by the effector molecules ATP, ADP and 2-oxoglutarate in vitro. Additionally to its utility to monitor 2-oxoglutarate levels, the FRET assay provided novel insights into PII - NAGK complex formation: (i) It revealed the formation of an encounter-complex between PII and NAGK, which holds the proteins in proximity even in the presence of inhibitors of complex formation; (ii) It revealed that the PII T-loop residue Ser49 is neither essential for complex formation with NAGK nor for activation of the enzyme but necessary to form a stable complex and efficiently relieve NAGK from arginine inhibition; (iii) It showed that arginine stabilizes the NAGK hexamer and stimulates PII - NAGK interaction.
| S-EPMC3861474 | biostudies-literature
Project description:Intra-annual density fluctuations (IADFs) are anatomical features formed in response to changes in the environmental conditions within the growing season. These anatomical features are commonly observed in Mediterranean pines, being more frequent in younger and wider tree rings. However, the process behind IADF formation is still unknown. Weekly monitoring of cambial activity and wood formation would fill this void. Although studies describing cambial activity and wood formation have become frequent, this knowledge is still fragmentary in the Mediterranean region. Here we present data from the monitoring of cambial activity and wood formation in two diameter classes of maritime pine (Pinus pinaster Ait.), over two years, in order to test: (i) whether the differences in stem diameter in an even-aged stand were due to timings and/or rates of xylogenesis; (ii) if IADFs were more common in large trees; and (iii) if their formation is triggered by cambial resumption after the summer drought. Larger trees showed higher rates of cell production and longer growing seasons, due to an earlier start and later end of xylogenesis. When a drier winter occurs, larger trees were more affected, probably limiting xylogenesis in the summer months. In both diameter classes a latewood IADF was formed in 2012 in response to late-September precipitation, confirming that the timing of the precipitation event after the summer drought is crucial in determining the resumption of cambial activity and whether or not an IADF is formed. It was the first time that the formation of a latewood IADF was monitored at a weekly time scale in maritime pine. The capacity of maritime pine to adjust cambial activity to the current environmental conditions represents a valuable strategy under the future climate change conditions.
| S-EPMC4427410 | biostudies-literature
Project description:An efficient transformation protocol based on kanamycin selection was developed for Agrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which contained neomycin phosphotransferase II (nptII) as a selectable marker gene and β -glucuronidase (uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15 mgL(-1) kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated with Agrobacterium strain AGL1 at the optical density (OD(600 nm)) of 0.3 for 72 h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for the nptII and uidA genes and expression of the uidA gene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth.
| S-EPMC3859213 | biostudies-literature
Project description:NAC transcription factors comprise a large plant-specific gene family involved in the regulation of diverse biological processes. Despite the growing number of studies on NAC transcription factors in various species, little information is available about this family in conifers. The goal of this study was to identify the NAC transcription family in maritime pine (Pinus pinaster), to characterize ATAF-like genes in response to various stresses and to study their molecular regulation.We have isolated two maritime pine NAC genes and using a transient expression assay in N. benthamiana leaves estudied the promoter jasmonate response.In this study, we identified 37 NAC genes from maritime pine and classified them into six main subfamilies. The largest group includes 12 sequences corresponding to stress-related genes. Two of these NAC genes, PpNAC2 and PpNAC3, were isolated and their expression profiles were examined at various developmental stages and in response to various types of stress. The expression of both genes was strongly induced by methyl jasmonate (MeJA), mechanical wounding, and high salinity. The promoter regions of these genes were shown to contain cis-elements involved in the stress response and plant hormonal regulation, including E-boxes, which are commonly found in the promoters of genes that respond to jasmonate, and binding sites for bHLH proteins. Using a transient expression assay in N. benthamiana leaves, we found that the promoter of PpNAC3 was rapidly induced upon MeJA treatment, while this response disappeared in plants in which the transcription factor NbbHLH2 was silenced.Our results suggest that PpNAC2 and PpNAC3 encode stress-responsive NAC transcription factors involved in the jasmonate response in pine. Furthermore, these data also suggest that the jasmonate signaling pathway is conserved between angiosperms and gymnosperms. These findings may be useful for engineering stress tolerance in pine via biotechnological approaches.
| S-EPMC4619436 | biostudies-literature