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  • 1.
    Brunoni, Federica
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC). Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences (SLU), Umeå, Sweden; Present address: Laboratory of Growth Regulators, Faculty of Science, Palacký University & Institute of Experimental Botany, The Czech Academy of Sciences, Šlechtitelů 27, 78371 Olomouc, Czech Republic.
    Collani, Silvio
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Simura, Jan
    Schmid, Markus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Bellini, Catherine
    Ljung, Karin
    A bacterial assay for rapid screening of IAA catabolic enzymes2019Ingår i: Plant Methods, ISSN 1746-4811, E-ISSN 1746-4811, Vol. 15, nr 1, artikel-id 126Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: Plants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-L-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. Dioxygenase for auxin oxidation (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on producing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays.

    Results: In this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that produce the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC–ESI-MS/MS). Experimental procedures were optimized for testing the activity of different classes of IAA-modifying enzymes without the need to purify recombinant protein.

    Conclusions: This new method represents an alternative to existing in vitro assays. It can be applied to the analysis of IAA metabolites that are produced upon supplementation of substrate to engineered bacterial cultures and can be used for a rapid screening of orthologous candidate genes from non-model species.

  • 2.
    Brunoni, Federica
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden.
    Ljung, Karin
    Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden.
    Bellini, Catherine
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik.
    Control of root meristem establishment in conifers2019Ingår i: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 165, nr 1, s. 81-89Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The evolution of terrestrial plant life was made possible by the establishment of a root system, which enabled plants to migrate from aquatic to terrestrial habitats. During evolution, root organization has gradually progressed from a very simple to a highly hierarchical architecture. Roots are initiated during embryogenesis and branch afterward through lateral root formation. Additionally, adventitious roots can be formed post-embryonically from aerial organs. Induction of adventitious roots (ARs) forms the basis of the vegetative propagation via cuttings in horticulture, agriculture and forestry. This method, together with somatic embryogenesis, is routinely used to clonally multiply conifers. In addition to being utilized as propagation techniques, adventitious rooting and somatic embryogenesis have emerged as versatile models to study cellular and molecular mechanisms of embryo formation and organogenesis of coniferous species. Both formation of the embryonic root and the AR primordia require the establishment of auxin gradients within cells that coordinate the developmental response. These processes also share key elements of the genetic regulatory networks that, e.g. are triggering cell fate. This minireview gives an overview of the molecular control mechanisms associated with root development in conifers, from initiation in the embryo to post-embryonic formation in cuttings.

  • 3. Edwards, Kieron D.
    et al.
    Takata, Naoki
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Johansson, Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC). RNA Biology and Molecular Physiology, Bielefeld University, Bielefeld, Germany.
    Jurca, Manuela
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Novak, Ondrej
    Henykova, Eva
    Department of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Swedish University of Agricultural Sciences, Umeå, Sweden.
    Liverani, Silvia
    Kozarewa, Iwanka
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Strnad, Miroslav
    Millar, Andrew J.
    Ljung, Karin
    Department of Forest Genetics and Plant Physiology, Umeå Plant Science Centre, Swedish University of Agricultural Sciences, Umeå, Sweden.
    Eriksson, Maria E.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysiologisk botanik. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Umeå Plant Science Centre (UPSC).
    Circadian clock components control daily growth activities by modulating cytokinin levels and cell division-associated gene expression in Populus trees2018Ingår i: Plant, Cell and Environment, ISSN 0140-7791, E-ISSN 1365-3040, Vol. 41, nr 6, s. 1468-1482Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Trees are carbon dioxide sinks and major producers of terrestrial biomass with distinct seasonal growth patterns. Circadian clocks enable the coordination of physiological and biochemical temporal activities, optimally regulating multiple traits including growth. To dissect the clock's role in growth, we analysed Populus tremula x P. tremuloides trees with impaired clock function due to down-regulation of central clock components. late elongated hypocotyl (lhy-10) trees, in which expression of LHY1 and LHY2 is reduced by RNAi, have a short free-running period and show disrupted temporal regulation of gene expression and reduced growth, producing 30-40% less biomass than wild-type trees. Genes important in growth regulation were expressed with an earlier phase in lhy-10, and CYCLIN D3 expression was misaligned and arrhythmic. Levels of cytokinins were lower in lhy-10 trees, which also showed a change in the time of peak expression of genes associated with cell division and growth. However, auxin levels were not altered in lhy-10 trees, and the size of the lignification zone in the stem showed a relative increase. The reduced growth rate and anatomical features of lhy-10 trees were mainly caused by misregulation of cell division, which may have resulted from impaired clock function.

  • 4. Pencik, Ales
    et al.
    Casanova-Sáez, Ruben
    Pilarova, Veronika
    Zukauskaite, Asta
    Pinto, Rui
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Luis Micol, José
    Ljung, Karin
    Novák, Ondrej
    Ultra-rapid auxin metabolite profiling for high-throughput mutant screening in Arabidopsis2018Ingår i: Journal of Experimental Botany, ISSN 0022-0957, E-ISSN 1460-2431, Vol. 69, nr 10, s. 2569-2579Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Auxin (indole-3-acetic acid, IAA) plays fundamental roles as a signalling molecule during numerous plant growth and development processes. The formation of local auxin gradients and auxin maxima/minima, which is very important for these processes, is regulated by auxin metabolism (biosynthesis, degradation, and conjugation) as well as transport. When studying auxin metabolism pathways it is crucial to combine data obtained from genetic investigations with the identification and quantification of individual metabolites. Thus, to facilitate efforts to elucidate auxin metabolism and its roles in plants, we have developed a high-throughput method for simultaneously quantifying IAA and its key metabolites in minute samples (<10 mg FW) of Arabidopsis thaliana tissues by in-tip micro solid-phase extraction and fast LC-tandem MS. As a proof of concept, we applied the method to a collection of Arabidopsis mutant lines and identified lines with altered IAA metabolite profiles using multivariate data analysis. Finally, we explored the correlation between IAA metabolite profiles and IAA-related phenotypes. The developed rapid analysis of large numbers of samples (>100 samples d(-1)) is a valuable tool to screen for novel regulators of auxin metabolism and homeostasis among large collections of genotypes.

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