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1.
Plant Mol Biol ; 73(4-5): 391-7, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20300806

ABSTRACT

MicroRNAs (miRNAs) are key regulatory molecules in plants. These small RNAs are processed in the nucleus from longer precursor transcripts that form distinct secondary structures. The miRNAs target specific messenger RNAs (mRNAs) and consequently down-regulate gene expression. The importance of these regulatory molecules is wide-ranging, however, few loss-of-function mutants have been identified in miRNA genes and understanding the biology of miRNA-target pairings has largely depended upon creating alterations in the sequences of the target genes. Here we demonstrate using Arabidopsis thaliana, that it is possible to use RNA interference (RNAi) to suppress accumulation of miRNAs. Significantly reduced accumulation of miR163 and miR171a was achieved using hairpin RNAi constructs that were designed to target both the primary miRNA transcripts and their promoters. The presence of DNA methylation in the targeted promoter regions suggests that inhibition of transcription of the miRNA precursors is responsible. Reduction of miRNA accumulation resulted in an increase in accumulation of the mRNA targets of these miRNAs. This demonstrates that knock-down of miRNA expression can be achieved, thereby providing a straightforward approach for disrupting miRNA-target pairings and studying miRNA functions.


Subject(s)
Arabidopsis/genetics , MicroRNAs/metabolism , RNA Interference , DNA Methylation/genetics , MicroRNAs/genetics , Plants, Genetically Modified , Promoter Regions, Genetic/genetics
2.
Plant J ; 48(2): 286-95, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16995900

ABSTRACT

The phenylpropanoid pathway in plants leads to the synthesis of a wide range of soluble secondary metabolites, many of which accumulate as glycosides. In Arabidopsis, a small cluster of three closely related genes, UGT72E1-E3, encode glycosyltransferases shown to glucosylate several phenylpropanoids in vitro, including monolignols, hydroxycinnamic acids and hydroxycinnamic aldehydes. The role of these genes in planta has now been investigated through genetically downregulating the expression of individual genes or silencing the entire cluster. Analysis of these transgenic Arabidopsis plants showed that the levels of coniferyl and sinapyl alcohol 4-O-glucosides that accumulate in light-grown roots were significantly reduced. A 50% reduction in both glucosides was observed in plants in which UGT72E2 was downregulated, whereas silencing the three genes led to a 90% reduction, suggesting some redundancy of function within the cluster. The gene encoding UGT72E2 was constitutively overexpressed in transgenic Arabidopsis to determine whether increased glucosylation of monolignols could influence flux through the soluble phenylpropanoid pathway. Elevated expression of UGT72E2 led to increased accumulation of monolignol glucosides in root tissues and also the appearance of these glucosides in leaves. In particular, coniferyl alcohol 4-O-glucoside accumulated to massive amounts (10 micromol g(-1) FW) in root tissues of these plants. Increased glucosylation of other phenylpropanoids also occurred in plants overexpressing this glycosyltransferase. Significantly changing the pattern of glycosides in the leaves also led to a pronounced change in accumulation of the hydroxycinnamic ester sinapoyl malate. The data demonstrate the plasticity of phenylpropanoid metabolism and the important role that glucosylation of secondary metabolites can play in cellular homeostasis.


Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/enzymology , Glucosides/biosynthesis , Glucosyltransferases/physiology , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Down-Regulation , Gene Silencing , Glucosides/chemistry , Glucosyltransferases/genetics , Glucosyltransferases/metabolism , Models, Biological , Multigene Family , Plant Roots/enzymology , Plant Roots/genetics , Plant Roots/metabolism , Plants, Genetically Modified/metabolism
3.
Proc Natl Acad Sci U S A ; 102(42): 15253-8, 2005 Oct 18.
Article in English | MEDLINE | ID: mdl-16214889

ABSTRACT

Steroid hormones are essential for development, and the precise control of their homeostasis is a prerequisite for normal growth. UDP-glycosyltransferases (UGTs) are considered to play an important regulatory role in the activity of steroids in mammals and insects. This study provides an indication that a UGT accepting plant steroids as substrates functions in brassinosteroid (BR) homeostasis. The UGT73C5 of Arabidopsis thaliana catalyses 23-O-glucosylation of the BRs brassinolide (BL) and castasterone. Transgenic plants overexpressing UGT73C5 displayed BR-deficient phenotypes and contained reduced amounts of BRs. The phenotype, which was already apparent in seedlings, could be rescued by application of BR. In feeding experiments with BL, wild-type seedlings converted BL to the 23-O-glucoside; in the transgenic lines silenced in UGT73C5 expression, no 23-O-glucoside was detected, implying that this UGT is the only enzyme that catalyzes BL-23-O-glucosylation in seedlings. Plant lines in which UGT73C5 expression was altered also displayed hypocotyl phenotypes previously described for seedlings in which BR inactivation by hydroxylation was changed. These data support the hypothesis that 23-O-glucosylation of BL is a function of UGT73C5 in planta, and that glucosylation regulates BR activity.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Cholestanols/metabolism , Glucosyltransferases/metabolism , Steroids, Heterocyclic/metabolism , Animals , Arabidopsis Proteins/genetics , Brassinosteroids , Glucosyltransferases/genetics , Phenotype , Plant Growth Regulators/metabolism , Promoter Regions, Genetic , RNA Interference
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