Kazuyoshi Terasaka

Trafficking and Subcellular Localization of Multiwalled Carbon Nanotubes in Plant Cells

Major barriers to delivery of biomolecules are crossing the cellular membranes and achieving a high cytoplasmic concentration by circumventing entrapment into endosomes and other lytic organelles. Motivated by such aim, we have investigated the capability of multiwalled carbon nanotubes (MWCNTs) to penetrate the cell membrane of plant protoplasts (plant cells made devoid of their cell walls via enzymatic treatment) and studied their internalization mechanism via confocal imaging and TEM techniques. Our results indentified an endosome-escaping uptake mode of MWCNTs by plant protoplasts. Moreover, short MWCNTs (<100 nm) were observed to target specific cellular substructures including the nucleus, plastids, and vacuoles. These findings are expected to have a significant impact on plant cell biology and transformation technologies.

A 7-Deoxyloganetic Acid Glucosyltransferase Contributes a Key Step in Secologanin Biosynthesis in Madagascar Periwinkle

The biosynthesis of secologanin in Madagascar periwinkle involves iridoid assembly in specialized internal phloem-associated parenchyma (IPAP) and final elaboration in leaf epidermal cells. We report the molecular cloning, functional characterization, and IPAP-associated localization of 7-deoxyloganetic acid glucosyltransferase. Iridoids form a broad and versatile class of biologically active molecules found in thousands of plant species. In addition to the many hundreds of iridoids occurring in plants, some iridoids, such as secologanin, serve as key building blocks in the biosynthesis of thousands of monoterpene indole alkaloids (MIAs) and many quinoline alkaloids. This study describes the molecular cloning and functional characterization of three iridoid glucosyltransfeases (UDP-SUGAR GLYCOSYLTRANSFERASE6 [UGT6], UGT7, and UGT8) from Madagascar periwinkle (Catharanthus roseus) with remarkably different catalytic efficiencies. Biochemical analyses reveal that UGT8 possessed a high catalytic efficiency toward its exclusive iridoid substrate, 7-deoxyloganetic acid, making it better suited for the biosynthesis of iridoids in periwinkle than the other two iridoid glucosyltransfeases. The role of UGT8 in the fourth to last step in secologanin biosynthesis was confirmed by virus-induced gene silencing in periwinkle plants, which reduced expression of this gene and resulted in a large decline in secologanin and MIA accumulation within silenced plants. Localization studies of UGT8 using a carborundum abrasion method for RNA extraction show that its expression occurs preferentially within periwinkle leaves rather than in epidermal cells, and in situ hybridization studies confirm that UGT8 is preferentially expressed in internal phloem associated parenchyma cells of periwinkle species.

Arabidopsis ABCB21 is a facultative auxin importer/exporter regulated by cytoplasmic auxin concentration.

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.

An efficient chemoenzymatic production of small molecule glucosides with in situ UDP‐glucose recycling

A one‐pot system for efficient enzymatic synthesis of curcumin glucosides is described. The method couples the activities of two recombinant enzymes, UDP‐glucose: curcumin glucosyltransferase from Catharanthus roseus (CaUGT2) and sucrose synthase from Arabidopsis thaliana (AtSUS1). UDP, a product inhibitor of UDP‐glucosyltransferase, was removed from the system and used for regeneration of UDP‐glucose by the second enzyme, AtSUS1. The productivity was increased several‐fold and UDP‐glucose initially added to the reaction mixture could be reduced to one‐tenth of the normal level. The concept of enhancing glucosylation efficiency by coupling a UDP‐glucose regeneration system with glucosyltransferases should be applicable to enzymatic production of a wide range of glucosides.

Functional platform for controlled subcellular distribution of carbon nanotubes.

As nanoparticles can cross different cellular barriers and access different tissues, control of their uptake and cellular fate presents a functional approach that will be broadly applicable to nanoscale technologies in cell biology. Here we show that the trafficking of single-walled carbon nanotubes (SWCNTs) through various subcellular membranes of the plant cell is facilitated or inhibited by attaching a suitable functional tag and controlling medium components. This enables a unique control over the uptake and the subcellular distribution of SWCNTs and provides a key strategy to promote their cellular elimination to minimize toxicity. Our results also demonstrate that SWCNTs are involved in a carrier-mediated transport (CMT) inside cells; this is a phenomenon that scientists could use to obtain novel molecular insights into CMT, with the potential translation to advances in subcellular nanobiology.

Iridoid-specific Glucosyltransferase from Gardenia jasminoides

Iridoids are one of the most widely distributed secondary metabolites in higher plants. They are pharmacologically active principles in various medicinal plants and key intermediates in the biosynthesis of monoterpenoid indole alkaloids as well as quinoline alkaloids. Although most iridoids are present as 1-O-glucosides, the glucosylation step in the biosynthetic pathway has remained obscure. We isolated a cDNA coding for UDP-glucose:iridoid glucosyltransferase (UGT85A24) from Gardenia jasminoides. UGT85A24 preferentially glucosylated the 1-O-hydroxyl group of 7-deoxyloganetin and genipin but exhibited only weak activity toward loganetin and no activity toward 7-deoxyloganetic acid. This suggests that, in the biosynthetic pathway of geniposide, a major iridoid compound in G. jasminoides, glucosylation occurs after methylation of 7-deoxyloganetic acid. UGT85A24 showed negligible activity toward any acceptor substrates other than iridoid aglycones. Thus, UGT85A24 has a remarkable specificity for iridoid aglycones. The mRNA level of UGT85A24 overlaps with the marked increase in genipin glucosylation activity in the methyl jasmonate-treated cell cultures of G. jasminoides and is related to iridoid accumulation in G. jasminoides fruits.

Functional and Structural Characterization of a Flavonoid Glucoside 1,6-Glucosyltransferase from Catharanthus roseus

Sugar-sugar glycosyltransferases play an important role in structural diversity of small molecule glycosides in higher plants. We isolated a cDNA clone encoding a sugar-sugar glucosyltransferase (CaUGT3) catalyzing 1,6-glucosylation of flavonol and flavone glucosides for the first time from Catharanthus roseus. CaUGT3 exhibited a unique glucosyl chain elongation activity forming not only gentiobioside but also gentiotrioside and gentiotetroside in a sequential manner. We investigated the functional properties of CaUGT3 using homology modeling and site-directed mutagenesis, and identified amino acids positioned in the acceptor-binding pocket as crucial for providing enough space to accommodate flavonoid glucosides instead of flavonoid aglycones. These results provide basic information for understanding and engineering the catalytic functions of sugar-sugar glycosyltransferases involved in biosynthesis of plant glycosides.

In situ UDP‐glucose regeneration unravels diverse functions of plant secondary product glycosyltransferases

The catalytic function of plant secondary product glycosyltransferases (PSPGs) was investigated by coupling the activities of recombinant flavonoid glucosyltransferases having different regiospecificities with sucrose synthase from Arabidopsis thaliana. In the present system, UDP, a product inhibitor of PSPGs, was removed from the reaction mixture and used for regeneration of UDP‐glucose by AtSUS1. The in situ UDP‐glucose regeneration system not only enhanced the glucosylation efficiency but also unraveled the novel regioselectivity of PSPGs. The effect of the system was shown to be because of the removal of UDP from the reaction system and not because of the additional supply of UDP‐glucose.

A single amino acid in the PSPG-box plays an important role in the catalytic function of CaUGT2 (Curcumin glucosyltransferase), a Group D Family 1 glucosyltransferase from Catharanthus roseus

Curcumin glucosyltransferase (CaUGT2) isolated from cell cultures of Catharanthus roseus exhibits unique substrate specificity. To identify amino acids involved in substrate recognition and catalytic activity of CaUGT2, a combination of domain swapping and site‐directed mutagenesis was carried out. Exchange of the PSPG‐box of CaUGT2 with that of NtGT1b (a phenolic glucosyltransferase from tobacco) led to complete loss of enzyme activity in the resulting recombinant protein. However, replacement of Arg378 of the NtGT1b PSPG‐box with cysteine, the corresponding amino acid in CaUGT2, restored the catalytic activity of the chimeric enzyme. Further site‐directed mutagenesis revealed that the size of the amino acid side‐chain in that particular site is critical to the catalytic activity of CaUGT2.

UGT75L6 and UGT94E5 mediate sequential glucosylation of crocetin to crocin in Gardenia jasminoides

Crocin is an apocarotenoid glycosyl ester accumulating in fruits of Gardenia jasminoides and used as a food coloring and nutraceutical. For the first time, the two glucosyltransferases UGT75L6 and UGT94E5 that sequentially mediate the final glucosylation steps in crocin biosynthesis in G. jasminoides have been identified and functionally characterized. UGT75L6 preferentially glucosylates the carboxyl group of crocetin yielding crocetin glucosyl esters, while UGT94E5 glucosylates the 6′ hydroxyl group of the glucose moiety of crocetin glucosyl esters. The expression pattern of neither UGT75L6 nor UGT94E5 correlated with the pattern of crocin accumulation in G. jasminoides.