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Arabidopsis small molecule glucosyltransferase UGT76B1 conjugates both ILA and SA and is essential for the root-driven control of defense marker genes in leaves
Arabidopsis small molecule glucosyltransferase UGT76B1 conjugates both ILA and SA and is essential for the root-driven control of defense marker genes in leaves
Plants as sessile organisms evolved different, sophisticated mechanisms to defend themselves against plethora of environmental stress factors. Pathogen defense is regulated by the mostly antagonistic salicylic acid (SA)- and jasmonic acid (JA)-mediated signaling pathways. The small molecule glucosyltransferase UGT76B1 was identified as a regulator of SA-JA crosstalk, positively stimulating SA-dependent defense, whereas suppressing JA pathway. UGT76B1 is able to glucosylate SA and a new signaling molecule, isoleucic acid (ILA). Thus, SA glucosylation could be catalyzed by UGT76B1 in addition to the previously identified SA glucosyltransferases UGT74F1 and UGT74F2. Therefore, lines with impaired expression of UGT74F1, UGT74F2 and UGT76B1 were applied to study whether UGT76B1 can be integrated in the homeostasis of SA and its conjugates. SA glucosides were not reduced in single ugt76b1 mutants in three different accessions Col-0, Ler and Ws-4 as it was previously shown for the Ws-4-based mutant ugt74f1 amiugt74f2. In the Ws-4 background, the introgression of ugt76b1-3 into ugt74f1 amiugt74f2 led to a strong repression of SA glucoside levels indicating that all three enzymes are involved in SA glucosylation. The root growth inhibition by exogenously added SA was employed as another assay to study the involvement of UGTs in SA glucosylation, since this reaction can be regarded as an inactivation of the inhibitor. ugt74f1-1, ugt74f2-1 and ugt74f1 amiugt74f2 were not differently affected than wild type, whereas ugt76b1 single mutants demonstrated stronger root growth inhibition than wild type. The latter was strongly enhanced by the introgression of ugt74f1 amiugt74f2. Thus, UGT76B1 might have a specific role in SA conjugation in roots, although again there is an interaction with the two other glucosyltransferases. ILA is known to stimulate SA-mediated defense and the abundance of ILA conjugate is positively related with UGT76B1 expression. However, the endogenous abundance of ILA aglycon has never been determined and monitored in response to environmental stresses. An optimized GC-MS based method demonstrated that ILA was dependent on UGT76B1 expression level in contrast to its chemically closely related compound LA (leucic acid). Both compounds showed also a different accumulation during the infection with Pseudomonas syringae and during the growth and development, suggesting their distinct role in plants. Exogenously applied ILA was shown to inhibit root growth in a concentration-dependent manner. Nevertheless, the mechanism behind this process is still not known. Therefore, two different screening approaches involving root growth response of T-DNA insertion lines and A. thaliana accessions were used in this project. The analysis of 159 Arabidopsis accessions revealed a region on the chromosome 1 as being weakly associated with root growth response to ILA. Further sequence analysis suggested that polymorphisms in SRX gene that is involved in regulation of intracellular ROS levels may cause ILA hypersensitivity. The role of ROS in ILA-mediated root growth inhibition was also supported by one T-DNA insertion line. Furthermore, the study on T-DNA insertion lines suggested that the ABC transporter PDR3/ABCG31 is involved in ILA export. To assess whether the relatively high expression of UGT76B1 in root has an impact on shoot defense status, reciprocal grafting experiments of ugt76b1 and Col-0 were employed. This approach clearly demonstrated that UGT76B1 is essential for a root-driven control of SA-dependent defense marker genes in leaves.
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Maksym, Rafał Paweł
2018
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Maksym, Rafał Paweł (2018): Arabidopsis small molecule glucosyltransferase UGT76B1 conjugates both ILA and SA and is essential for the root-driven control of defense marker genes in leaves. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Plants as sessile organisms evolved different, sophisticated mechanisms to defend themselves against plethora of environmental stress factors. Pathogen defense is regulated by the mostly antagonistic salicylic acid (SA)- and jasmonic acid (JA)-mediated signaling pathways. The small molecule glucosyltransferase UGT76B1 was identified as a regulator of SA-JA crosstalk, positively stimulating SA-dependent defense, whereas suppressing JA pathway. UGT76B1 is able to glucosylate SA and a new signaling molecule, isoleucic acid (ILA). Thus, SA glucosylation could be catalyzed by UGT76B1 in addition to the previously identified SA glucosyltransferases UGT74F1 and UGT74F2. Therefore, lines with impaired expression of UGT74F1, UGT74F2 and UGT76B1 were applied to study whether UGT76B1 can be integrated in the homeostasis of SA and its conjugates. SA glucosides were not reduced in single ugt76b1 mutants in three different accessions Col-0, Ler and Ws-4 as it was previously shown for the Ws-4-based mutant ugt74f1 amiugt74f2. In the Ws-4 background, the introgression of ugt76b1-3 into ugt74f1 amiugt74f2 led to a strong repression of SA glucoside levels indicating that all three enzymes are involved in SA glucosylation. The root growth inhibition by exogenously added SA was employed as another assay to study the involvement of UGTs in SA glucosylation, since this reaction can be regarded as an inactivation of the inhibitor. ugt74f1-1, ugt74f2-1 and ugt74f1 amiugt74f2 were not differently affected than wild type, whereas ugt76b1 single mutants demonstrated stronger root growth inhibition than wild type. The latter was strongly enhanced by the introgression of ugt74f1 amiugt74f2. Thus, UGT76B1 might have a specific role in SA conjugation in roots, although again there is an interaction with the two other glucosyltransferases. ILA is known to stimulate SA-mediated defense and the abundance of ILA conjugate is positively related with UGT76B1 expression. However, the endogenous abundance of ILA aglycon has never been determined and monitored in response to environmental stresses. An optimized GC-MS based method demonstrated that ILA was dependent on UGT76B1 expression level in contrast to its chemically closely related compound LA (leucic acid). Both compounds showed also a different accumulation during the infection with Pseudomonas syringae and during the growth and development, suggesting their distinct role in plants. Exogenously applied ILA was shown to inhibit root growth in a concentration-dependent manner. Nevertheless, the mechanism behind this process is still not known. Therefore, two different screening approaches involving root growth response of T-DNA insertion lines and A. thaliana accessions were used in this project. The analysis of 159 Arabidopsis accessions revealed a region on the chromosome 1 as being weakly associated with root growth response to ILA. Further sequence analysis suggested that polymorphisms in SRX gene that is involved in regulation of intracellular ROS levels may cause ILA hypersensitivity. The role of ROS in ILA-mediated root growth inhibition was also supported by one T-DNA insertion line. Furthermore, the study on T-DNA insertion lines suggested that the ABC transporter PDR3/ABCG31 is involved in ILA export. To assess whether the relatively high expression of UGT76B1 in root has an impact on shoot defense status, reciprocal grafting experiments of ugt76b1 and Col-0 were employed. This approach clearly demonstrated that UGT76B1 is essential for a root-driven control of SA-dependent defense marker genes in leaves.