Browsing by Subject "Arabidopsis"

Sort by: Order: Results:

Now showing items 1-15 of 15
  • Radhakrishnan, Dhanya; Shanmukhan, Anju Pallipurath; Kareem, Abdul; Aiyaz, Mohammed; Varapparambathu, Vijina; Toms, Ashna; Kerstens, Merijn; Valsakumar, Devisree; Landge, Amit N.; Shaji, Anil; Mathew, Mathew K.; Sawchuk, Megan G.; Scarpella, Enrico; Krizek, Beth A.; Efroni, Idan; Mähönen, Ari Pekka; Willemsen, Viola; Scheres, Ben; Prasad, Kalika (2020)
    Aerial organs of plants, being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaves and stems to study the reunion between mechanically disconnected tissues. We show that PLETHORA (PLT) and AINTEGUMENTA (ANT) genes, which encode stem cell-promoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. PLT proteins and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed-forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT-mediated regeneration response, leaf ground tissue cells can neither acquire the early vascular identity marker ATHB8, nor properly polarise auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaves. We reveal the mechanisms of vascular regeneration in plants and distinguish between the wound-repair ability of the tissue and its formation during normal development.
  • Valdebenito Alamar, Nerea (Helsingin yliopisto, 2021)
    Despite their immobile nature, their ability for adaptation allows plants to face harmful conditions from the environment to successfully survive and reproduce. Plant cells sense and integrate signals from the environment and activate response mechanisms. Participants in these mechanisms are the receptor-like protein kinases (RLKs) and a subgroup of RLKs, the cysteine-rich receptor-like kinases (CRKs). Members of this family have been associated with functions related to environmental stress responses in plants. CRK2 is one interesting member of the CRK clade of RLKs. While roles of CRK2 in the response to biotic and abiotic stimuli have been recently described, many aspects of the diverse functions of CRK2 remain elusive. The reduced size of the crk2 mutant suggests that developmental processes are affected by the absence of the protein. One of the objectives of this work was to analyse potential reasons for the smaller size of crk2. The difference in plant size could be due to a reduced number of cells. Results from the analysis of young cotyledons showed that the smaller plant size is not due to a reduced cell number in leaves when compared to Arabidopsis thaliana (Arabidopsis) ecotype Columbia (Col-0). Another way to understand the processes in which a protein is involved is to target possible interaction partners. Therefore, genotyping and analysis of growth phenotypes of T-DNA insertion mutant lines for candidate interaction partners for CRK2 was performed. The results revealed smaller phenotype for a nitrate transporter (NRT1.7) mutant in fresh weight and rosette area whereas for a protein kinase (QSK1) mutant, higher fresh weight but reduced rosette area was observed compared to Col-0. Generation of constructs for fusion protein expression and purification revealed the possibility of expressing tagged cytoplasmic regions of these proteins for further analysis of protein-protein interaction through kinase assays due to the kinase activity of CRK2. Generation of fluorescent-tagged proteins from the candidate interaction partners allowed for localization studies via confocal microscopy to determine the co-localization to the plasma membrane of these proteins with CRK2, which is located to plasma membrane under standard growth conditions. The co-localization results suggest that the proteins NRT1.7 and QSK1 colocalize with CRK2, which is a step forward in the verification of their possible interaction in planta. The smaller size of the nrt1.7 and qsk1 mutants indicates that the lack of these proteins affects plant development.
  • Valenzuela Averruz, Leticia Estereniel; Zou, Ling; Santanen, Arja Anelma; Mäkelä, Pirjo Sisko Anneli (2018)
  • Escamez, Sacha; Stael, Simon; Vainonen, Julia; Willems, Patrick; Jin, Huiting; Kimura, Sachie; Van Breusegem, Frank; Gevaert, Kris; Wrzaczek, Michael Alois; Tuominen, Hannele (2019)
    During plant vascular development, xylem tracheary elements (TEs) form water-conducting, empty pipes by genetically regulated cell death. Cell death is prevented from spreading to non-TEs by unidentified intercellular mechanisms, downstream of METACASPASE9 (MC9)-mediated regulation of autophagy in TEs. Here, we identified differentially abundant extracellular peptides in vascular-differentiating wild-type and MC9-down-regulated Arabidopsis cell suspensions. A peptide named Kratos rescued the abnormally high ectopic non-TE death resulting from either MC9 knockout or TE-specific overexpression of the ATG5 autophagy protein during experimentally induced vascular differentiation in Arabidopsis cotyledons. Kratos also reduced cell death following mechanical damage and extracellular ROS production in Arabidopsis leaves. Stress-induced but not vascular non-TE cell death was enhanced by another identified peptide, named Bia. Bia is therefore reminiscent of several known plant cell death-inducing peptides acting as damage-associated molecular patterns. In contrast, Kratos plays a novel extracellular cell survival role in the context of development and during stress response.
  • Pavicic, Mirko; Mouhu, Katriina; Wang, Feng; Bilicka, Marcelina Marta; Chovanček, Erik; Himanen, Kristiina Irma Helena (2017)
    Flowering time control integrates endogenous as well as environmental signals to promote flower development. The pathways and molecular networks involved are complex and integrate many modes of signal transduction. In plants ubiquitin mediated protein degradation pathway has been proposed to be as important mode of signaling as phosphorylation and transcription. To systematically study the role of ubiquitin signaling in the molecular regulation of flowering we have taken a genomic approach to identify flower related Ubiquitin Proteasome System components. As a large and versatile gene family the RING type ubiquitin E3 ligases were chosen as targets of the genomic screen. To this end the complete list of Arabidopsis RING E3 ligases were retrieved and verified in the Arabidopsis genome v11. Their differential expression was used for their categorization into flower organs or developmental stages. Known regulators of flowering time or floral organ development were identified in these categories through literature search and representative mutants for each category were purchased for functional characterization by growth and morphological phenotyping. To this end, a workflow was developed for high throughput phenotypic screening of growth, morphology and flowering of nearly a thousand Arabidopsis plants in one experimental round.
  • Pavicic, M.; Wang, F.; Mouhu, K.; Himanen, K. (2019)
    Seed quality is an important factor for seedling vigour as well as adult plant resilience. The key quality attributes are related to physical characteristics, physiological performance, genetic background and health status of the seeds. Many ways to address seed quality attributes have been developed and recently many of them have featured automated high throughput methods. In our study, we addressed two of the seed quality attributes, namely physiological performance and genetic background by analysing germination rates in our mutant collection. These mutants represent ubiquitin E3 ligases that transcriptionally respond to abscisic acid (ABA). This plant hormone is an important regulator of germination and seedling establishment. To facilitate in vitro germination screens of large seed collections a high throughput image-based assay was developed. As a read out of the germination on ABA treatment the cotyledon emergence was detected with top view chlorophyll fluorescence camera. By applying the ABA treatment during germination, RING-type ubiquitin E3 ligase mutants were identified, showing either resistant or sensitive responses to ABA. In conclusion, a scalable high throughput screen for in vitro germination assay was established that allowed fast screening of tens of mutants in a hormone supplemented media.
  • Pavicic, Mirko; Overmyer, Kirk; Rehman, Attiq ur; Jones, Piet; Jacobson, Daniel; Himanen, Kristiina (2021)
    Image-based symptom scoring of plant diseases is a powerful tool for associating disease resistance with plant genotypes. Advancements in technology have enabled new imaging and image processing strategies for statistical analysis of time-course experiments. There are several tools available for analyzing symptoms on leaves and fruits of crop plants, but only a few are available for the model plant Arabidopsis thaliana (Arabidopsis). Arabidopsis and the model fungus Botrytis cinerea (Botrytis) comprise a potent model pathosystem for the identification of signaling pathways confer- ring immunity against this broad host-range necrotrophic fungus. Here, we present two strategies to assess severity and symptom progression of Botrytis infection over time in Arabidopsis leaves. Thus, a pixel classification strategy using color hue values from red-green-blue (RGB) images and a random forest algorithm was used to establish necrotic, chlorotic, and healthy leaf areas. Secondly, using chlorophyll fluorescence (ChlFl) imaging, the maximum quantum yield of photosystem II (Fv/Fm) was determined to define diseased areas and their proportion per total leaf area. Both RGB and ChlFl imaging strategies were employed to track disease progression over time. This has provided a robust and sensitive method for detecting sensitive or resistant genetic backgrounds. A full methodological workflow, from plant culture to data analysis, is described.
  • Survila, Mantas; Davidsson, Pär R.; Pennanen, Ville; Kariola, Tarja; Broberg, Martin; Sipari, Nina; Heino, Pekka; Palva, Erkki T. (2016)
    Cuticular defects trigger a battery of reactions including enhanced reactive oxygen species (ROS) production and resistance to necrotrophic pathogens. However, the source of ROS generated by such impaired cuticles has remained elusive. Here, we report the characterization of Arabidopsis thaliana ohyl mutant, a Peroxidase 57 (PER57) - overexpressing line that demonstrates enhanced defense responses that result both from increased accumulation of ROS and permeability of the leaf cuticle. The ohyl mutant was identified in a screen of A. thaliana seedlings for oligogalacturonides (OGs) insensitive/hypersensitive mutants that exhibit altered growth retardation in response to exogenous OGs. Mutants impaired in OG sensitivity were analyzed for disease resistance/susceptibility to the necrotrophic phytopathogens Botrytis cinerea and Pectobacterium carotovorum. In the ohyl line, the hypersensitivity to OGs was associated with resistance to the tested pathogens. This PER57 overexpressing line exhibited a significantly more permeable leaf cuticle than wild-type plants and this phenotype could be recapitulated by overexpressing other class III peroxidases. Such peroxidase overexpression was accompanied by the suppressed expression of cutin biosynthesis genes and the enhanced expression of genes associated with OG-signaling. Application of ABA completely removed ROS, restored the expression of genes associated with cuticle biosynthesis and led to decreased permeability of the leaf cuticle, and finally, abolished immunity to B. cinerea. Our work demonstrates that increased peroxidase activity increases permeability of the leaf cuticle. The loss of cuticle integrity primes plant defenses to necrotrophic pathogens via the activation of DAMP-responses.
  • Kareem, Abdul; Radhakrishnan, Dhanya; Wang, Xin; Bagavathiappan, Subhikshaa; Trivedi, Zankhana B.; Sugimoto, Kaoru; Xu, Jian; Mähonen, Ari Pekka; Prasad, Kalika (2016)
    Background: Plants have the remarkable property to elaborate entire body plan from any tissue part. The conversion of lateral root primordium (LRP) to shoot is an ideal method for plant propagation and for plant researchers to understand the mechanism underlying trans-differentiation. Until now, however, a robust method that allows the efficient conversion of LRP to shoot is lacking. This has limited our ability to study the dynamic phases of reprogramming at cellular and molecular levels. Results: Here we present an efficient protocol for the direct conversion of LRP to a complete fertile shoot system. This protocol can be readily applied to the various ecotypes of Arabidopsis. We show that, the conversion process is highly responsive to developmental stages of LRP and changes in external environmental stimuli such as temperature. The entire conversion process can be adequately analyzed by histological and imaging techniques. As a demonstration, using a battery of cell fate specific markers, we show that confocal time-lapse imaging can be employed to uncover the early molecular events, intermediate developmental phases and relative abundance of stem cell regulators during the conversion of LRP to shoot. Conclusion: Our method is highly efficient, independent of genotypes tested and suitable to study the reprogramming of LRP to shoot in intact plants as well as in excised roots.
  • Grabsztunowicz, Magda; Rantala, Marjaana; Ivanauskaite, Aiste; Blomster, Tiina; Koskela, Minna M.; Vuorinen, Katariina; Tyystjarvi, Esa; Burow, Meike; Overmyer, Kirk; Mähönen, Ari P.; Mulo, Paula (2021)
    In Arabidopsis, two leaf-type ferredoxin-NADP(+) oxidoreductase (LFNR) isoforms function in photosynthetic electron flow in reduction of NADP(+), while two root-type FNR (RFNR) isoforms catalyse reduction of ferredoxin in non-photosynthetic plastids. As the key to understanding, the function of RFNRs might lie in their spatial and temporal distribution in different plant tissues and cell types, we examined expression of RFNR1 and RFNR2 genes using beta-glucuronidase (GUS) reporter lines and investigated accumulation of distinct RFNR isoforms using a GFP approach and Western blotting upon various stresses. We show that while RFNR1 promoter is active in leaf veins, root tips and in the stele of roots, RFNR2 promoter activity is present in leaf tips and root stele, epidermis and cortex. RFNR1 protein accumulates as a soluble protein within the plastids of root stele cells, while RFNR2 is mainly present in the outer root layers. Ozone treatment of plants enhanced accumulation of RFNR1, whereas low temperature treatment specifically affected RFNR2 accumulation in roots. We further discuss the physiological roles of RFNR1 and RFNR2 based on characterization of rfnr1 and rfnr2 knock-out plants and show that although the function of these proteins is partly redundant, the RFNR proteins are essential for plant development and survival.
  • Kalmbach, Lothar; Helariutta, Yrjö Eero (2019)
    Sieve pores of the sieve plates connect neighboring sieve elements to form the conducting sieve tubes of the phloem. Sieve pores are critical for phloem function. From the 1950s onwards, when electron microscopes became increasingly available, the study of their formation had been a pillar of phloem research. More recent work on sieve elements instead has largely focused on sieve tube hydraulics, phylogeny, and eco-physiology. Additionally, advanced molecular and genetic tools available for the model species Arabidopsis thaliana helped decipher several key regulatory mechanisms of early phloem development. Yet, the downstream differentiation processes which form the conductive sieve tube are still largely unknown, and our understanding of sieve pore formation has only moderately progressed. Here, we summarize our current knowledge on sieve pore formation and present relevant recent advances in related fields such as sieve element evolution, physiology, and plasmodesmata formation.
  • van den Bosch, Tijs J. M.; Niemi, Outi; Welte, Cornelia U. (2020)
    Plants of the Brassicales order, including Arabidopsis and many common vegetables, produce toxic isothiocyanates to defend themselves against pathogens. Despite this defence, plant pathogenic microorganisms like Pectobacterium cause large yield losses in fields and during storage of crops. The bacterial gene saxA was previously found to encode isothiocyanate hydrolase that degrades isothiocyanates in vitro. Here we demonstrate in planta that saxA is a virulence factor that can overcome the chemical defence system of Brassicales plants. Analysis of the distribution of saxA genes in Pectobacterium suggests that saxA from three different phylogenetic origins are present within this genus. Deletion of saxA genes representing two of the most common classes from P. odoriferum and P. versatile resulted in significantly reduced virulence on Arabidopsis thaliana and Brassica oleracea. Furthermore, expressing saxA from a plasmid in a potato-specific P. parmentieri strain that does not naturally harbour this gene significantly increased the ability of the strain to macerate Arabidopsis. These findings suggest that a single gene may have a significant role in defining the host range of a plant pathogen.
  • Smit, Margot E.; Llavata-Peris, Cristina; Roosjen, Mark; van Beijnum, Henriette; Novikova, Daria; Levitsky, Victor; Sevilem, Iris; Roszak, Pawel; Slane, Daniel; Juergens, Gerd; Mironova, Victoria; Brady, Siobhan M.; Weijers, Dolf (2020)
    Development of plant vascular tissues involves tissue identity specification, growth, pattern formation and cell-type differentiation. Although later developmental steps are understood in some detail, it is still largely unknown how the tissue is initially specified. We used the early Arabidopsis embryo as a simple model to study this process. Using a large collection of marker genes, we found that vascular identity was specified in the 16-cell embryo. After a transient precursor state, however, there was no persistent uniform tissue identity. Auxin is intimately connected to vascular tissue development. We found that, although an AUXIN RESPONSE FACTOR5/MONOPTEROS (ARF5/MP)-dependent auxin response was required, it was not sufficient for tissue specification. We therefore used a large-scale enhanced yeast one-hybrid assay to identify potential regulators of vascular identity. Network and functional analysis of candidate regulators suggest that vascular identity is under robust, complex control. We found that one candidate regulator, the G-class bZIP transcription factor GBF2, can modulate vascular gene expression by tuning MP output through direct interaction. Our work uncovers components of a gene regulatory network that controls the initial specification of vascular tissue identity.
  • Israel, David; Khan, Shandjida; Warren, Charles R; Zwiazek, Janusz J; Robson, T Matthew (2021)
    The roles of different plasma membrane aquaporins (PIPs) in leaf-level gas exchange of Arabidopsis thaliana were examined using knockout mutants. Since multiple Arabidopsis PIPs are implicated in CO2 transport across cell membranes, we focused on identifying the effects of the knockout mutations on photosynthesis, and whether they are mediated through the control of stomatal conductance of water vapour (g(s)), mesophyll conductance of CO2 (g(m)), or both. We grew Arabidopsis plants in low and high humidity environments and found that the contribution of PIPs to g s was larger under low air humidity when the evaporative demand was high, whereas any effect of a lack of PIP function was minimal under higher humidity. The pip2;4 knockout mutant had 44% higher g(s) than wild-type plants under low humidity, which in turn resulted in an increased net photosynthetic rate (A(net)). We also observed a 23% increase in whole-plant transpiration (E) for this knockout mutant. The lack of functional plasma membrane aquaporin AtPIP2;5 did not affect g(s) or E, but resulted in homeostasis of g(m) despite changes in humidity, indicating a possible role in regulating CO2 membrane permeability. CO2 transport measurements in yeast expressing AtPIP2;5 confirmed that this aquaporin is indeed permeable to CO2.
  • Ilomäki, Miika Valtteri (Helsingfors universitet, 2016)
    Ubiquitin is an important modifier in eukaryotic cells through many effects on the targeted protein. Ubiquitination is a reaction cascade, catalyzed by E1 – E2 and finally E3 enzyme which completes the ubiquitination. In this study preselected 61 RING type ubiquitin E3 proteins of Arabidopsis thaliana were classified, grouped and analysed to characterize what kind of domains and groups were included. Proteins which contain a RING domain, can either ubiquitinate substrates independently or function as part of a multi-subunit complex. RING E3s are known to act as a molecular adaptors for the E2s and the substrates. It is the E3 ligase that is responsible for selecting the target protein for ubiquitination and later for degradation in proteasome in to peptides. The same preselected 61 genes were also researched from the Betula pendula Roth genome. Web Apollo database was used to annotate genes from the recently sequenced silver birch genome. As a result 32 gene homologs that included RING domain were identified in silver birch.