Seiji Tsurumi

Structure-Function Analysis of the Presumptive Arabidopsis Auxin Permease AUX1

We have investigated the subcellular localization, the domain topology, and the amino acid residues that are critical for the function of the presumptive Arabidopsis thaliana auxin influx carrier AUX1. Biochemical fractionation experiments and confocal studies using an N-terminal yellow fluorescent protein (YFP) fusion observed that AUX1 colocalized with plasma membrane (PM) markers. Because of its PM localization, we were able to take advantage of the steep pH gradient that exists across the plant cell PM to investigate AUX1 topology using YFP as a pH-sensitive probe. The YFP-coding sequence was inserted in selected AUX1 hydrophilic loops to orient surface domains on either apoplastic or cytoplasmic faces of the PM based on the absence or presence of YFP fluorescence, respectively. We were able to demonstrate in conjunction with helix prediction programs that AUX1 represents a polytopic membrane protein composed of 11 transmembrane spanning domains. In parallel, a large aux1 allelic series containing null, partial-loss-of-function, and conditional mutations was characterized to identify the functionally important domains and amino acid residues within the AUX1 polypeptide. Whereas almost all partial-loss-of-function and null alleles cluster in the core permease region, the sole conditional allele aux1-7 modifies the function of the external C-terminal domain.

Auxin and Ethylene Response Interactions during Arabidopsis Root Hair Development Dissected by Auxin Influx Modulators

The plant hormones auxin and ethylene have been shown to play important roles during root hair development. However, cross talk between auxin and ethylene makes it difficult to understand the independent role of either hormone. To dissect their respective roles, we examined the effects of two compounds, chromosaponin I (CSI) and 1-naphthoxyacetic acid (1-NOA), on the root hair developmental process in wild-type Arabidopsis, ethylene-insensitive mutantein2-1, and auxin influx mutants aux1-7,aux1-22, and double mutant aux1-7 ein2. β-Glucuronidase (GUS) expression analysis in the BA-GUS transgenic line, consisting of auxin-responsive domains ofPS-IAA4/5 promoter and GUS reporter, revealed that 1-NOA and CSI act as auxin uptake inhibitors in Arabidopsis roots. The frequency of root hairs in ein2-1roots was greatly reduced in the presence of CSI or 1-NOA, suggesting that endogenous auxin plays a critical role for the root hair initiation in the absence of an ethylene response. All of these mutants showed a reduction in root hair length, however, the root hair length could be restored with a variable concentration of 1-naphthaleneacetic acid (NAA). NAA (10 nm) restored the root hair length ofaux1 mutants to wild-type level, whereas 100 nm NAA was needed for ein2-1 andaux1-7 ein2 mutants. Our results suggest that insensitivity in ethylene response affects the auxin-driven root hair elongation. CSI exhibited a similar effect to 1-NOA, reducing root hair growth and the number of root hair-bearing cells in wild-type andein2-1 roots, while stimulating these traits inaux1-7and aux1-7ein2 roots, confirming that CSI is a unique modulator of AUX1.

Auxin Response in Arabidopsis under Cold Stress: Underlying Molecular Mechanisms

To understand the mechanistic basis of cold temperature stress and the role of the auxin response, we characterized root growth and gravity response of Arabidopsis thaliana after cold stress, finding that 8 to 12 h at 4°C inhibited root growth and gravity response by ∼50%. The auxin-signaling mutants axr1 and tir1, which show a reduced gravity response, responded to cold treatment like the wild type, suggesting that cold stress affects auxin transport rather than auxin signaling. Consistently, expression analyses of an auxin-responsive marker, IAA2-GUS, and a direct transport assay confirmed that cold inhibits root basipetal (shootward) auxin transport. Microscopy of living cells revealed that trafficking of the auxin efflux carrier PIN2, which acts in basipetal auxin transport, was dramatically reduced by cold. The lateral relocalization of PIN3, which has been suggested to mediate the early phase of root gravity response, was also inhibited by cold stress. Additionally, cold differentially affected various protein trafficking pathways. Furthermore, the inhibition of protein trafficking by cold is independent of cellular actin organization and membrane fluidity. Taken together, these results suggest that the effect of cold stress on auxin is linked to the inhibition of intracellular trafficking of auxin efflux carriers.

Gravitropism of Arabidopsis thaliana Roots Requires the Polarization of PIN2 toward the Root Tip in Meristematic Cortical Cells

Gravitropism of roots depends on a flow of auxin from the root cap to the zone of elongation via the auxin efflux carrier PIN2. While PIN2 in epidermis and lateral root cap is positioned appropriately, PIN2 in the cortex has the opposite polarity. We report that, despite this, PIN2 functions in the root cortex for optimal gravitropism, apparently by limiting the auxin flow. In the root, the transport of auxin from the tip to the elongation zone, referred to here as shootward, governs gravitropic bending. Shootward polar auxin transport, and hence gravitropism, depends on the polar deployment of the PIN-FORMED auxin efflux carrier PIN2. In Arabidopsis thaliana, PIN2 has the expected shootward localization in epidermis and lateral root cap; however, this carrier is localized toward the root tip (rootward) in cortical cells of the meristem, a deployment whose function is enigmatic. We use pharmacological and genetic tools to cause a shootward relocation of PIN2 in meristematic cortical cells without detectably altering PIN2 polarization in other cell types or PIN1 polarization. This relocation of cortical PIN2 was negatively regulated by the membrane trafficking factor GNOM and by the regulatory A1 subunit of type 2-A protein phosphatase (PP2AA1) but did not require the PINOID protein kinase. When GNOM was inhibited, PINOID abundance increased and PP2AA1 was partially immobilized, indicating both proteins are subject to GNOM-dependent regulation. Shootward PIN2 specifically in the cortex was accompanied by enhanced shootward polar auxin transport and by diminished gravitropism. These results demonstrate that auxin flow in the root cortex is important for optimal gravitropic response.

A γ-pyronyl-triterpenoid saponin from Pisum sativum

Abstract A new triterpenoid saponin was isolated from Pisum sativum and characterized as 3- O -[α- l -rhamnopyranosyl-(1 → 2)-β- d -galactopyranosyl(1 → 2)-β- d -glucuronopyranosyl(1 →)]-22- O -[3′-hydroxy-2′-methyl-5′,6′-dihydro-4′-pyrone(6′ →)]-3β,22β,24-trihydroxyolean-12-ene. The name chromosaponin I is proposed. Chromosaponin I yielded soyasaponin I, known as phytochrome inhibitor, during extraction, but the latter was not found in the free form in this plant.

The effects of auxin on lateral root initiation and root gravitropism in a lateral rootless mutant Lrt1 of rice (Oryza sativa L.)

Auxins control growth and development in plants, including lateral rootinitiation and root gravity response. However, how endogenous auxin regulatesthese processes is poorly understood. In this study, the effects of auxins onlateral root initiation and root gravity response in rice were investigatedusing a lateral rootless mutant Lrt1, which fails to formlateral roots and shows a reduced root gravity response. Exogenous applicationof IBA to the Lrt1 mutant restored both lateral rootinitiation and root gravitropism. However, application of IAA, a major form ofnatural auxin, restored only root gravitropic response but not lateral rootinitiation. These results suggest that IBA is more effective than IAA in lateralroot formation and that IBA also plays an important role in root gravitropicresponse in rice. The application of NAA restored lateral root initiation, butdid not completely restore root gravitropism. Root elongation assays ofLrt1 displayed resistance to 2,4-D, NAA, IBA, and IAA.This result suggests that the reduced sensitivity to exogenous auxins may be due tothe altered auxin activity in the root, thereby affecting root morphology inLrt1.

Genetic Dissection of Hormonal Responses in the Roots of Arabidopsis Grown under Continuous Mechanical Impedance

We investigated the role of ethylene and auxin in regulating the growth and morphology of roots during mechanical impedance by developing a new growing system and using the model plant Arabidopsis (Arabidopsis thaliana). The Arabidopsis seedlings grown horizontally on a dialysis membrane-covered agar plate encountered adequate mechanical impedance as the roots showed characteristic ethylene phenotypes: 2-fold reduction in root growth, increase in root diameter, decrease in cell elongation, and ectopic root hair formation. The root phenotype characterization of various mutants having altered response to ethylene biosynthesis or signaling, the effect of ethylene inhibitors on mechanically impeded roots, and transcription profiling of the ethylene-responsive genes led us to conclude that enhanced ethylene response plays a primary role in changing root morphology and development during mechanical impedance. Further, the differential sensitivity of horizontally and vertically grown roots toward exogenous ethylene suggested that ethylene signaling plays a critical role in enhancing the ethylene response. We subsequently demonstrated that the enhanced ethylene response also affects the auxin response in roots. Taken together, our results provide a new insight into the role of ethylene in changing root morphology during mechanical impedance.

Ion Efflux from Pulvinar Cells during Slow Downward Movement of the Petiole of Mimosa pudica L. Induced by Photostimulation

Change of extracellular ion concentration in the main pulvinus of Mimosa pudica L. during a slow downward movement induced by white light (0.24 mW cm(-2)) was investigated using K(+)- and Cl(-)-selective electrodes and a pH electrode. The photostimulation induced a transient and concurrent efflux of K(+) and Cl(-) from the motor cells and acidification of extracellular pH in the main pulvinus.

Different Behaviour of Indole-3-Acetic Acid and Indole-3-Butyric Acid in Stimulating Lateral Root Development in Rice (Oryza sativa L.)

The plant hormone auxin has been shown to be involved in lateral root development and application of auxins, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), increases the number of lateral roots in several plants. We found that the effects of two auxins on lateral root development in the indica rice (Oryza sativa L. cv. IR8) were totally different from each other depending on the application method. When the roots were incubated with an auxin solution, IAA inhibited lateral root development, while IBA was stimulatory. In contrast, when auxin was applied to the shoot, IAA promoted lateral root formation, while IBA did not. The transport of [3H]IAA from shoot to root occurred efficiently (% transported compared to supplied) but that of [3H]IBA did not, which is consistent with the stimulatory effect of IAA on lateral root production when applied to the shoot. The auxin action of IBA has been suggested to be due to its conversion to IAA. However, in rice IAA competitively inhibited the stimulatory effect of IBA on lateral root formation when they were applied to the incubation solution, suggesting that the stimulatory effect of IBA on lateral root development is not through its conversion to IAA.

Inhibitory effects on Epstein-Barr virus activation of anthraquinones: correlation with redox potentials

The redox potentials have been determined for nine anthraquinones in phosphate buffer at pH 7.2 by means of cyclic voltammetry. A definite correlation has been found between the redox potentials and the inhibitory effects of the anthraquinones on the EBV-EA activation. It has further been shown that the correlation can be made better by introducing an electronic property, i.e. the atomic charge at O12 as an additional parameter.