ABSTRACT
The phytohormone auxin is critical for plant growth and many developmental processes. Members of the P-glycoprotein (PGP/ABCB) subfamily of ATP-binding cassette (ABC) transporters have been shown to function in the polar movement of auxin by transporting auxin over the plasma membrane in both monocots and dicots. Here, we characterize a new Arabidopsis member of the ABCB subfamily, ABCB21/PGP21, a close homolog of ABCB4, for which conflicting transport directionalities have been reported. ABCB21 is strongly expressed in the abaxial side of cotyledons and in junctions of lateral organs in the aerial part, whereas in roots it is specifically expressed in pericycle cells. Membrane fractionation by sucrose density gradient centrifugation followed by Western blot showed that ABCB21 is a plasma membrane-localized ABC transporter. A transport assay with Arabidopsis protoplasts suggested that ABCB21 was involved in IAA transport in an outward direction, while naphthalene acetic acid (NAA) was a less preferable substrate for ABCB21. Further functional analysis of ABCB21 using yeast import and export assays showed that ABCB21 mediates the 1-N-naphthylphthalamic acid (NPA)-sensitive translocation of auxin in an inward direction when the cytoplasmic IAA concentration is low, whereas this transporter mediates outward transport under high internal IAA. An increase in the cytoplasmic IAA concentration by pre-loading of IAA into yeast cells abolished the IAA uptake activity by ABCB21 as well as ABCB4. These findings suggest that ABCB21 functions as a facultative importer/exporter controlling auxin concentrations in plant cells.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Arabidopsis/metabolism , Indoleacetic Acids/metabolism , Plant Growth Regulators/metabolism , ATP-Binding Cassette Transporters/genetics , Arabidopsis/cytology , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Biological Transport , Cell Membrane/genetics , Cell Membrane/metabolism , Cytoplasm/metabolism , Gene Expression Regulation, Plant , Gene Knockdown Techniques , Mutation , Naphthaleneacetic Acids/metabolism , Organ Specificity , Phenotype , Phylogeny , Plant Roots/genetics , Plant Roots/growth & development , Plant Roots/metabolism , Plants, Genetically Modified , Protoplasts , RNA Interference , Substrate SpecificityABSTRACT
Yeast functional screening with a Sophora flavescens cDNA library was performed to identify the genes involved in the tolerant mechanism to the self-producing prenylated flavonoid sophoraflavanone G (SFG). One cDNA, which conferred SFG tolerance, encoded a regulatory particle triple-A ATPase 2 (SfRPT2), a member of the 26S proteasome subunit. The yeast transformant of SfRPT2 showed reduced SFG accumulation in the cells.
Subject(s)
Flavanones/metabolism , Prenylation , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/genetics , Sophora/genetics , Sophora/physiology , Flavanones/biosynthesis , Phylogeny , Proteasome Endopeptidase Complex/metabolism , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Sophora/enzymology , Sophora/metabolismABSTRACT
Many plant secondary metabolites show strong biological activities and are potentially also toxic to plants, while plants producing such active compounds are usually insensitive to their own metabolites, suggesting that they have species-specific detoxification mechanisms. In order to clarify the detoxification mechanism of alkaloids, we used cultured cells of Coptis japonica, which are capable of producing a yellow benzylisoquinoline alkaloid, berberine, and accumulate it in the vacuole. Unlike other plant cells that do not produce berberine, C. japonica shows strong tolerance to this alkaloid. We established a fission yeast strain that was sensitive to berberine and performed functional screening using a C. japonica cDNA library. One cDNA clone, which conferred clear berberine tolerance, encoded galactinol synthase (CjGolS). The possible role of CjGolS in berberine tolerance is discussed.
