Abstract
Auxin is one type of phytohormones that plays important roles in nearly all aspects of plant growth and developmental processes. The glycosylation of auxins is considered to be an essential mechanism to control the level of active auxins. Thus, the identification of auxin glycosyltransferases is of great significance for further understanding the auxin regulation. In this study, we biochemically screened the group L of Arabidopsis thaliana glycosyltransferase superfamily for enzymatic activity toward auxins. UGT74D1 was identified to be a novel auxin glycosyltransferase. Through HPLC and LC-MS analysis of reaction products in vitro by testing eight substrates including auxins and other compounds, we found that UGT74D1 had a strong glucosylating activity toward indole-3-butyric acid [IBA], indole-3-propionic acid [IPA], indole-3-acetic acid [IAA] and naphthaleneacetic acid [NAA], catalyzing them to form corresponding glucose esters. Biochemical characterization showed that this enzyme had a maximum activity in HEPES buffer at pH 6.0 and 37°C. In addition, the enzymatic activity analysis of crude protein and the IBA metabolite analysis from transgenic Arabidopsis plants overexpressing UGT74D1 gene were also carried out. Experimental results indicated that over-production of the UGT74D1 in plants indeed led to increased level of the glucose conjugate of IBA. Moreover, UGT74D1 overexpression lines displayed curling leaf phenotype, suggesting a physiological role of UGT74D1 in affecting the activity of auxins. Our current data provide a new target gene for further genetic studies to understand the auxin regulation by glycosylation in plants.
Highlights
Auxin is the first discovered phytohormone and is well-known for its regulatory role in virtually all aspects of plant growth and development, such as general root and shoot architecture, organ initiation and patterning, cell division and differentiation [1,2,3], plant responses to biotic and abiotic stresses, etc [4,5]
Purification of Recombinant UGT74D1 In order to explore more hormone-related UGTs, in this study, we put our focus on other members of group L whose activity and substrate have not been previously demonstrated. These UGTs were cloned into prokaryotic expression vector and expressed in Escherichia coli tagged with glutathione S-transferase (GST)
UGT74D1 was tested in vitro for its glycosyltransferase activity against each of the seven substrates used in this study: indole-3carboxylic acid [ICA], indole-3-acetic acid [Indole-3-acetic acid (IAA)], indole-3propionic acid [IPA], indole-3-butyric acid [Indole-3-butyric acid (IBA)], the synthetic auxin analogs naphthaleneacetic acid [NAA], 2,4-dichlorophenoxyacetic acid [2,4-D], and picloram
Summary
Auxin is the first discovered phytohormone and is well-known for its regulatory role in virtually all aspects of plant growth and development, such as general root and shoot architecture, organ initiation and patterning, cell division and differentiation [1,2,3], plant responses to biotic and abiotic stresses, etc [4,5]. Many Arabidopsis mutants unable to convert IBA to active IAA have reduced root growth sensitivity to IBA, but normal sensitivity to IAA, suggesting that IBA is an essential auxin precursor rather than active auxin [13,14,15,16,17]. The increase of free IBA, but not IAA level in UGT74E2OE plants indicated that the shoot morphogenesis might be directly affected by IBA rather than its conversion to IAA [23]. This partial independence of both auxins was further supported by accounted differences in IAA and IBA polar transport [18,19,24,25]
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