Browsing by Subject "Arabidopsis"
Now showing items 1-5 of 5
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(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.
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(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.
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(2022)Plant vascular cambium development involves dynamic changes in transcriptional profiles, which are mediated by key transcription factors. Understanding secondary growth through complex molecular processes of vascular cambium development has remarkable significance in maximizing carbon storage in trees and is crucial for the genetic improvement of trees for wood production. Here, we studied the functions of NO TRANSMITTING TRACT (NTT)/WIP DOMAIN PROTEIN2 (WIP2) zinc-finger transcription factor in regulating vascular cambium activity and xylem formation using Arabidopsis roots and tree stems as model organisms. Reporter lines carrying red fluorescent protein driven by the promoter of NTT/WIP2 showed cambium specific expression in Arabidopsis roots under confocal microscope. Inducible overexpression of NTT/WIP2 after cambium activation reduced vascular diameter, primary phloem pole distance and secondary vessel number in Arabidopsis roots. In loss of function ntt mutants, significant reductions of vascular diameter, phloem pole distance and number of secondary vessels were also evident. Our data suggests that, both increased expression and loss of function of NTT/WIP2 gene significantly reduce secondary growth in Arabidopsis roots. Simultaneous to our Arabidopsis experiments we also tested the possibility of increasing wood formation amounts through manipulation of PttNTT (putative Populus ortholog of Arabidopsis NTT/WIP2 gene) expression in hybrid aspen trees. But increased expression of NTT/WIP2 in Poplar significantly reduced plant height, stem girth and internode numbers. Altogether, we hypothesize that, appropriate spatiotemporal expression pattern and levels of NTT/WIP2 is required for cambial activity to proceed properly in plants. These findings represent a new foundation toward a more comprehensive understanding of the molecular mechanisms of NTT/WIP2 gene in regulation of cambium development and secondary growth in plants.
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(2022)Plant vascular cambium development involves dynamic changes in transcriptional profiles, which are mediated by key transcription factors. Understanding secondary growth through complex molecular processes of vascular cambium development has remarkable significance in maximizing carbon storage in trees and is crucial for the genetic improvement of trees for wood production. Here, we studied the functions of NO TRANSMITTING TRACT (NTT)/WIP DOMAIN PROTEIN2 (WIP2) zinc-finger transcription factor in regulating vascular cambium activity and xylem formation using Arabidopsis roots and tree stems as model organisms. Reporter lines carrying red fluorescent protein driven by the promoter of NTT/WIP2 showed cambium specific expression in Arabidopsis roots under confocal microscope. Inducible overexpression of NTT/WIP2 after cambium activation reduced vascular diameter, primary phloem pole distance and secondary vessel number in Arabidopsis roots. In loss of function ntt mutants, significant reductions of vascular diameter, phloem pole distance and number of secondary vessels were also evident. Our data suggests that, both increased expression and loss of function of NTT/WIP2 gene significantly reduce secondary growth in Arabidopsis roots. Simultaneous to our Arabidopsis experiments we also tested the possibility of increasing wood formation amounts through manipulation of PttNTT (putative Populus ortholog of Arabidopsis NTT/WIP2 gene) expression in hybrid aspen trees. But increased expression of NTT/WIP2 in Poplar significantly reduced plant height, stem girth and internode numbers. Altogether, we hypothesize that, appropriate spatiotemporal expression pattern and levels of NTT/WIP2 is required for cambial activity to proceed properly in plants. These findings represent a new foundation toward a more comprehensive understanding of the molecular mechanisms of NTT/WIP2 gene in regulation of cambium development and secondary growth in plants.
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(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.
Now showing items 1-5 of 5