| 1. |
- Darwish, Essam, et al.
(författare)
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The dynamics of touch-responsive gene expression in cereals
- 2023
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Ingår i: The Plant Journal. - 0960-7412 .- 1365-313X. ; 116:1, s. 282-302
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Tidskriftsartikel (refereegranskat)abstract
- Wind, rain, herbivores, obstacles, neighbouring plants, etc. provide important mechanical cues to steer plant growth and survival. Mechanostimulation to stimulate yield and stress resistance of crops is of significant research interest, yet a molecular understanding of transcriptional responses to touch is largely absent in cereals. To address this, we performed whole-genome transcriptomics following mechanostimulation of wheat, barley, and the recent genome-sequenced oat. The largest transcriptome changes occurred ±25 min after touching, with most of the genes being upregulated. While most genes returned to basal expression level by 1–2 h in oat, many genes retained high expression even 4 h post-treatment in barley and wheat. Functional categories such as transcription factors, kinases, phytohormones, and Ca2+ regulation were affected. In addition, cell wall-related genes involved in (hemi)cellulose, lignin, suberin, and callose biosynthesis were touch-responsive, providing molecular insight into mechanically induced changes in cell wall composition. Furthermore, several cereal-specific transcriptomic footprints were identified that were not observed in Arabidopsis. In oat and barley, we found evidence for systemic spreading of touch-induced signalling. Finally, we provide evidence that both the jasmonic acid-dependent and the jasmonic acid-independent pathways underlie touch-signalling in cereals, providing a detailed framework and marker genes for further study of (a)biotic stress responses in cereals.
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| 2. |
- Hansson, Mattias, et al.
(författare)
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PCR-mediated deletion of plasmid DNA
- 2008
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 375:2, s. 373-375
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Tidskriftsartikel (refereegranskat)abstract
- The PCR-mediated plasmid DNA deletion method is a simple approach to delete DNA sequences from plasmids using only one round of PCR, with two primers, and without ligation or purification prior to in vivo recombination. By using only PCR, the method is sequence independent and, as shown in this study, is applicable to various sizes of plasmids and deletions using minimal primer design.
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| 3. |
- Kamal, Nadia, et al.
(författare)
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The mosaic oat genome gives insights into a uniquely healthy cereal crop
- 2022
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 606:7912, s. 113-119
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Tidskriftsartikel (refereegranskat)abstract
- Cultivated oat (Avena sativa L.) is an allohexaploid (AACCDD, 2n = 6x = 42) thought to have been domesticated more than 3,000 years ago while growing as a weed in wheat, emmer and barley fields in Anatolia1,2. Oat has a low carbon footprint, substantial health benefits and the potential to replace animal-based food products. However, the lack of a fully annotated reference genome has hampered efforts to deconvolute its complex evolutionary history and functional gene dynamics. Here we present a high-quality reference genome of A. sativa and close relatives of its diploid (Avena longiglumis, AA, 2n = 14) and tetraploid (Avena insularis, CCDD, 2n = 4x = 28) progenitors. We reveal the mosaic structure of the oat genome, trace large-scale genomic reorganizations in the polyploidization history of oat and illustrate a breeding barrier associated with the genome architecture of oat. We showcase detailed analyses of gene families implicated in human health and nutrition, which adds to the evidence supporting oat safety in gluten-free diets, and we perform mapping-by-sequencing of an agronomic trait related to water-use efficiency. This resource for the Avena genus will help to leverage knowledge from other cereal genomes, improve understanding of basic oat biology and accelerate genomics-assisted breeding and reanalysis of quantitative trait studies.
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| 4. |
- Lethin, Johanna, et al.
(författare)
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Development and characterization of an EMS-mutagenized wheat population and identification of salt-tolerant wheat lines
- 2020
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Ingår i: Bmc Plant Biology. - : Springer Science and Business Media LLC. - 1471-2229. ; 20:1
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Tidskriftsartikel (refereegranskat)abstract
- Background Triticum aestivum (wheat) is one of the world's oldest crops and has been used for >8000 years as a food crop in North Africa, West Asia and Europe. Today, wheat is one of the most important sources of grain for humans, and is cultivated on greater areas of land than any other crop. As the human population increases and soil salinity becomes more prevalent, there is increased pressure on wheat breeders to develop salt-tolerant varieties in order to meet growing demands for yield and grain quality. Here we developed a mutant wheat population using the moderately salt-tolerant Bangladeshi variety BARI Gom-25, with the primary goal of further increasing salt tolerance. Results After titrating the optimal ethyl methanesulfonate (EMS) concentration, ca 30,000 seeds were treated with 1% EMS, and 1676 lines, all originating from single seeds, survived through the first four generations. Most mutagenized lines showed a similar phenotype to BARI Gom-25, although visual differences such as dwarfing, giant plants, early and late flowering and altered leaf morphology were seen in some lines. By developing an assay for salt tolerance, and by screening the mutagenized population, we identified 70 lines exhibiting increased salt tolerance. The selected lines typically showed a 70% germination rate on filter paper soaked in 200 mM NaCl, compared to 0-30% for BARI Gom-25. From two of the salt-tolerant OlsAro lines (OA42 and OA70), genomic DNA was sequenced to 15x times coverage. A comparative analysis against the BARI Gom-25 genomic sequence identified a total of 683,201 (OA42), and 768,954 (OA70) SNPs distributed throughout the three sub-genomes (A, B and D). The mutation frequency was determined to be approximately one per 20,000 bp. All the 70 selected salt-tolerant lines were tested for root growth in the laboratory, and under saline field conditions in Bangladesh. The results showed that all the lines selected for tolerance showed a better salt tolerance phenotype than both BARI Gom-25 and other local wheat varieties tested. Conclusion The mutant wheat population developed here will be a valuable resource in the development of novel salt-tolerant varieties for the benefit of saline farming.
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| 5. |
- Lu, Qiongxian, et al.
(författare)
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Analysis of barley mutants ert-c.1 and ert-d.7 reveals two loci with additive effect on plant architecture
- 2021
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Ingår i: Planta. - : Springer Science and Business Media LLC. - 0032-0935 .- 1432-2048. ; 254:1
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Tidskriftsartikel (refereegranskat)abstract
- Main conclusion: Both mutant ert-c.1 and ert-d.7 carry T2-T3 translocations in the Ert-c gene. Principal coordinate analyses revealed the translocation types and translocation breakpoints. Mutant ert-d.7 is an Ert-cErt-d double mutant. Abstract: Mutations in the Ert-c and Ert-d loci are among the most common barley mutations affecting plant architecture. The mutants have various degrees of erect and compact spikes, often accompanied with short and stiff culms. In the current study, complementation tests, linkage mapping, principal coordinate analyses and fine mapping were conducted. We conclude that the original ert-d.7 mutant does not only carry an ert-d mutation but also an ert-c mutation. Combined, mutations in Ert-c and Ert-d cause a pyramid-dense spike phenotype, whereas mutations in only Ert-c or Ert-d give a pyramid and dense phenotype, respectively. Associations between the Ert-c gene and T2-T3 translocations were detected in both mutant ert-c.1 and ert-d.7. Different genetic association patterns indicate different translocation breakpoints in these two mutants. Principal coordinate analysis based on genetic distance and screening of recombinants from all four ends of polymorphic regions was an efficient way to narrow down the region of interest in translocation-involved populations. The Ert-c gene was mapped to the marker interval of 2_0801to1_0224 on 3HL near the centromere. The results illuminate a complex connection between two single genes having additive effects on barley spike architecture and will facilitate the identification of the Ert-c and Ert-d genes.
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| 6. |
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| 7. |
- Olsson, Ulf, et al.
(författare)
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Characterization of eight barley xantha-f mutants deficient in magnesium chelatase
- 2004
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Ingår i: Plant Physiology and Biochemistry. - : Elsevier BV. - 1873-2690 .- 0981-9428. ; 42:6, s. 557-564
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Tidskriftsartikel (refereegranskat)abstract
- Magnesium chelatase (EC 6.6.1.1) catalyses the insertion of magnesium into protoporphyrin IX, the first unique step of the chlorophyll biosynthetic pathway. The enzyme is composed of three different subunits of approximately 40, 70 and 140 kDa. In barley (Hordeum vulgare L.) the subunits are encoded by the genes Xantha-h, Xantha-g and Xantha-f. In the 1950s, eight induced xantha-f mutants were isolated. In this work we characterized these mutations at the DNA level and provided explanations for their phenotypes. The xantha-f10 mutation is a 3 bp deletion, resulting in a polypeptide lacking the glutamate residue at position 424. The leaky mutation xantha-f26 has a missense mutation leading to a M632R exchange. The xantha-f27 and -f40 are deletions of 14 and 2 bp, respectively, resulting in truncated polypeptides of 1104 and 899 amino acid residues, respectively. Mutation xantha-f41 is an in-frame deletion that removes A439, L440, Q441 and V442 from the resulting protein. Mutation xantha-f58 is most likely a deletion of the whole Xantha-f gene, as no DNA fragments could be detected by PCR or southern blot experiments. The slightly leaky xantha-f60 and non-leaky -f68 mutations each have a missense mutation causing a P393L and G794E exchange in the polypeptide, respectively.
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| 8. |
- Sirijovski, Nick, et al.
(författare)
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ATPase activity associated with the magnesium chelatase H-subunit of the chlorophyll biosynthetic pathway is an artefact
- 2006
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Ingår i: Biochemical Journal. - 0264-6021. ; 400, s. 477-484
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Tidskriftsartikel (refereegranskat)abstract
- Magnesium chelatase inserts Mg2+ into protoporphyrin IX and is the first unique enzyme of the chlorophyll biosynthetic pathway. It is a heterotrimeric enzyme, composed of I- (40 kDa), D- (70 kDa) and H- (140 kDa) subunits. The I- and D-proteins belong to the family of AAA(+) (ATPases associated with various cellular activities), but only I-subunit hydrolyses ATP to ADP. The D-subunits provide a platform for the assembly of the I-subunits, which results in a two-tiered hexameric ring complex. However, the D-subunits are unstable in the chloroplast unless ATPase active I-subunits are present. The H-subunit binds protoporphyrin and is suggested to be the catalytic subunit. Previous studies have indicated that the H-subunit also has ATPase activity, which is in accordance with an earlier suggested two-stage mechanism of the reaction. In the present study, we demonstrate that gel filtration chromatography of affinity-purified Rhodobacter capsulatus H-subunit produced in Escherichia coli generates a high- and a low-molecular-mass fraction. Both fractions were dominated by the H-subunit, but the ATPase activity was only found in the high-molecular-mass fraction and magnesium chelatase activity was only associated with the low-molecular-mass fraction. We demonstrated that light converted monomeric low-molecular-mass H-subunit into high-molecular-mass aggregates. We conclude that ATP utilization by magnesium chelatase is solely connected to the I-subunit and suggest that a contaminating E. coli protein, which binds to aggregates of the H-subunit, caused the previously reported ATPase activity of the H-subunit.
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| 9. |
- Sirijovski, Nick
(författare)
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Magnesium Chelatase: Insights into the first Step of Chlorophyll Biosynthesis
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The enzyme magnesium chelatase inserts magnesium into protoporphyrin IX (Proto) to produce to magnesium protoporphyrin IX, the first unique intermediate of the chlorophyll biosynthetic pathway. Magnesium chelatase is composed of three distinct proteins termed I (molecular weight ~40 kDa), D (~70 kDa) and H (~140 kDa). Defining the individual properties and structure of the magnesium chelatase components and their role in the reaction mechanism is important for a full understanding of the first step in magnesium tetrapyrrole biosynthesis. The three components of magnesium chelatase show significant conservation at the protein sequence level, which extends from bacteriochlorophyll synthesising purple non-sulfur bacteria and green sulfur bacteria to chlorophyll synthesising eukaryotes and cyanobacteria. In Paper I, eight mutants of the H gene (Xantha-f) from barley were characterised at the molecular level and provide explanations for the yellow phenotypes of germinating mutant seedlings. In this thesis magnesium chelatase from the photosynthetic bacterium Rhodobacter capsulatus has been used as a model system since much of the pioneering work has been conducted on this organism. Magnesium chelatase requires ATP to insert magnesium into Proto. There has been conflicting results as to the ATPase activity of the H subunit. In Paper II it was demonstrated that ATP hydrolysis can be attributed the I subunit and not the H. The unprecedented discovery of an iron-sulfur cluster in the H subunit of R. capsulatus is described in Paper III. The cysteine motif that coordinates this iron-sulfur cluster is only present in five other facultative proteobacteria and absent in all oxygenic or anaerobic species. The function of this cluster is yet to be established. In Paper IV the first insights into the structure of an H subunit is presented. Electron microscopy and single-particle reconstruction was used to solve the structure in the apo and substrate bound conformations at a resolution of 25 Å, and revealed a conformational change upon Proto binding. Limited proteolysis and construction of truncated H polypeptides provided supporting information to propose a cooperative substrate binding model. The binding of porphyrin to the H subunit was further investigated in Paper V using tryptophan fluorescence quenching to detect a high affinity porphyrin binding site in the nanomolar range. Alanine mutagenesis of the H subunit implicated key residues involved in porphyrin binding and catalysis. The work presented in this thesis has expanded our understanding of the magnesium chelatase, particularly in respect to the H subunit. With three different proteins and three substrates, it is clear that magnesium chelatase is an elaborate molecular machine that is proving to be far more complicated than ever expected.
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| 10. |
- Sirijovski, Nick, et al.
(författare)
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Rhodobacter capsulatus magnesium chelatase subunit BchH contains an oxygen sensitive iron-sulfur cluster
- 2007
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Ingår i: Archives of Microbiology. - : Springer Science and Business Media LLC. - 0302-8933 .- 1432-072X. ; 188:6, s. 599-608
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Tidskriftsartikel (refereegranskat)abstract
- Magnesium chelatase is the first unique enzyme of the bacteriochlorophyll biosynthetic pathway. It consists of three subunits (BchI, BchD, and BchH). Amino acid sequence analysis of the Rhodobacter capsulatus BchH revealed a novel cysteine motif ((393)CX(2)CX(3)CX(14)C) that was found in only six other proteobacteria (CX(2)CX(3)CX(11-14)C). The cysteine motif is likely to coordinate an unprecedented [Fe-S] cluster. Purified BchH demonstrated absorbance in the 460 nm region. This absorbance was abolished in BchH proteins with alanine substitutions at positions Cys396 and Cys414. These modified proteins were also EPR silent. In contrast, wild type BchH protein in the reduced state showed EPR signals resembling those of a [4Fe-4S] cluster with rhombic symmetry and g values at 1.90, 1.93, and 2.09, superimposed with a [3Fe-4S] cluster centered at g = 2.02. The [3Fe-4S] signal was observed independently of the [4Fe-4S] signal under oxidizing conditions. Mg-chelatase activity assays showed that the cluster is not catalytic. We suggest that the [4Fe-4S] and [3Fe-4S] signals originate from a single coordination site on the monomeric BchH protein and that the [4Fe-4S] cluster is sensitive to oxidation. It is speculated that the cluster participates in the switching between aerobic and anaerobic life of the proteobacteria.
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