Ethylene Is Not Responsible for Phytochrome-Mediated Apical Hook Exaggeration in Tomato.

The apical hook of tomato seedlings is exaggerated by phytochrome actions, while in other species such as bean, pea and Arabidopsis, the hook is exaggerated by ethylene and opens by phytochrome actions. The present study was aimed to clarify mainly whether ethylene is responsible for the phytochrome-mediated hook exaggeration of tomato seedlings. Dark-grown 5-day-old seedlings were subjected to various ways of ethylene application in the dark as well as under the actions of red (R) or far-red light (FR). The ethylene emitted by seedlings was also quantified relative to hook exaggeration. The results show: Ambient ethylene, up-to about 1.0 µL L-1, suppressed (opened) the hooks formed in the dark as well as the ones exaggerated by R or FR, while at 3.0-10 µL L-1 it enhanced (closed) the hook only slightly as compared with the most-suppressed level at about 1.0 µL L-1. Treatment with 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene biosynthesis, did not enhance the hook, only mimicking the suppressive effects of ambient ethylene. The biosynthesis inhibitor, CoCl2 or aminoethoxyvinylglycine, enhanced hook curvature, and the enhancement was canceled by supplement of ethylene below 1.0 µL L-1. Auxin transport inhibitor, N-1-naphthylphthalamic acid, by contrast, suppressed curvature markedly without altering ethylene emission. The effects of the above-stated treatments did not differentiate qualitatively among the R-, FR-irradiated seedlings and dark control so as to explain phytochrome-mediated hook exaggeration. In addition, ethylene emission by seedlings was affected neither by R nor FR at such fluences as to cause hook exaggeration. In conclusion, (1) ethylene suppresses not only the light-exaggerated hook, but also the dark-formed one; (2) ethylene emission is not affected by R or FR, and also not correlated with the hook exaggerations; thus ethylene is not responsible for the hook exaggeration in tomato; and (3) auxin is essential for the maintenance and development of the hook in tomato as is the case in other species lacking phytochrome-mediated hook exaggeration. A possible mechanism of phytochrome action for hook exaggeration is discussed.

Gravitropism of Arabidopsis thaliana roots requires the polarization of PIN2 toward the root tip in meristematic cortical cells.

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.

Light exaggerates apical hook curvature through phytochrome actions in tomato seedlings.

Contrary to the established notion that the apical hook of dark-grown dicotyledonous seedlings opens in response to light, we found in tomato (Solanum lycopersicum L.) that the apical hook curvature is exaggerated by light. Experiments with several tomato cultivars and phytochrome mutants, irradiated with red and far-red light either as a brief pulse (Rp, FRp) or continuously (Rc, FRc), revealed: the hook-exaggeration response is maximal at the emergence of the hypocotyl from the seed; the effect of Rp is FRp-reversible; fluence-response curves to a single Rp or FRp show an involvement of low and very low fluence responses (LFR, VLFR); the effect of Rc is fluence-rate dependent, but that of FRc is not; the phyA mutant (phyA hp-1) failed to respond to an Rp of less than 10(-2) micromol m(-2) and to an FRp of all fluences tested as well as to FRc, thus indicating that the hook-exaggeration response involves phyA-mediated VLFR. The Rp fluence-response curve with the same mutant also confirmed the presence of an LFR mediated by phytochrome(s) other than phyA, although the phyB1 mutant (phyB1 hp-1) still showed full response probably due to other redundant phytochrome species (e.g., phyB2). Simulation experiments led to the possible significance of hook exaggeration in the field that the photoresponse may facilitate the release of seed coat when seeds germinate at some range of depth in soil. It was also observed that seed coat and/or endosperm are essential to the hook exaggeration.

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 degrees C inhibited root growth and gravity response by approximately 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.

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.

Saturated humidity accelerates lateral root development in rice (Oryza sativa L.) seedlings by increasing phloem-based auxin transport.

Auxin transport plays a significant role modifying plant growth and development in response to environmental signals such as light and gravity. However, the effect of humidity on auxin transport is rarely documented. It is shown here that the transport of labelled indole-3-acetic acid (IAA) from the shoot to the root is accelerated in rice (Oryza sativa L. ssp. indica cv. IR8) seedlings grown under saturated humidity (SH-seedlings) compared with plants grown under normal humidity (NH-seedlings). The development of lateral roots in SH-seedlings was greatly enhanced compared with NH-seedlings. Removal of the shoot from SH-seedlings reduced the density of lateral roots, and the application of IAA to the cut stem restored the lateral root density, while the decapitation of NH-seedlings did not alter lateral root development. Phloem-based auxin transport appeared responsible for enhanced lateral root formation in SH-seedlings since (i) the rate of IAA transport from the shoot to the root tip was greater than 3.5 cm h-1 and (ii) naphthylphthalamic acid (NPA)-induced reduction of polar auxin transport in the shoot did not influence the number of lateral roots in SH-seedlings. It is proposed that high humidity conditions accelerate the phloem-based transport of IAA from the leaf to the root, resulting in an increase in the number of lateral roots.

A small acidic protein 1 (SMAP1) mediates responses of the Arabidopsis root to the synthetic auxin 2,4-dichlorophenoxyacetic acid.

2,4-dichlorophenoxyacetic acid (2,4-D), a chemical analogue of indole-3-acetic acid (IAA), is widely used as a growth regulator and exogenous source of auxin. Because 2,4-D evokes physiological and molecular responses similar to those evoked by IAA, it is believed that they share a common response pathway. Here, we show that a mutant, antiauxin resistant1 (aar1), identified in a screen for resistance to the anti-auxin p-chlorophenoxy-isobutyric acid (PCIB), is resistant to 2,4-D, yet nevertheless responds like the wild-type to IAA and 1-napthaleneacetic acid in root elongation and lateral root induction assays. That the aar1 mutation alters 2,4-D responsiveness specifically was confirmed by analysis of GUS expression in the DR5:GUS and HS:AXR3NT-GUS backgrounds, as well as by real-time PCR quantification of IAA11 expression. The two characterized aar1 alleles both harbor multi-gene deletions; however, 2,4-D responsiveness was restored by transformation with one of the genes missing in both alleles, and the 2,4-D-resistant phenotype was reproduced by decreasing the expression of the same gene in the wild-type using an RNAi construct. The gene encodes a small, acidic protein (SMAP1) with unknown function and present in plants, animals and invertebrates but not in fungi or prokaryotes. Taken together, these results suggest that SMAP1 is a regulatory component that mediates responses to 2,4-D, and that responses to 2,4-D and IAA are partially distinct.

Defects in root development and gravity response in the aem1 mutant of rice are associated with reduced auxin efflux.

The phytohormone auxin is involved in the regulation of a variety of developmental processes. In this report, we describe how the processes of lateral root and root hair formations and root gravity response in rice are controlled by auxin. We use a rice mutant aem1 (auxin efflux mutant) because the mutant is defective in these characters. The aem1 line was originally isolated as a short lateral root mutant, but we found that the mutant has a defect in auxin efflux in roots. The acropetal and basipetal indole-3-acetic acid (IAA) transports were reduced in aem1 roots compared to wild type (WT). Furthermore, gravitropic bending as well as efflux of radioactive IAA was impaired in the mutant roots. We also propose a unique distribution of endogenous IAA in aem1 roots. An immunoassay revealed a 4-fold-endogenous IAA content in the aem1 roots compared to WT, and the application of IAA to the shoot of WT seedlings mimicked the short lateral root phenotype of aem1, suggesting that the high content of IAA in aem1 roots impaired the elongation of lateral roots. However, the high level of IAA in aem1 roots contradicts the auxin requirement for root hair formation in the epidermis of mutant roots. Since the reduced development in root hairs of aem1 roots was rescued by exogenous auxin, the auxin level in the epidermis is likely to be sub-optimum in aem1 roots. This discrepancy can be solved by the ideas that IAA level is higher in the stele and lower in the epidermis of aem1 roots compared to WT and that the unique distribution of IAA in aem1 roots is induced by the defect in auxin efflux. All these results suggest that AEM1 may encode a component of auxin efflux carrier in rice and that the defects in lateral roots, root hair formation and root gravity response in aem1 mutant are due to the altered auxin efflux in roots.

Involvement of ARM2 in the uptake of indole-3-butyric acid in rice (Oryza sativa L.) roots.

Auxin influx carriers are involved in auxin transport and plant development. Here we show that the mutant of rice (Oryza sativa L. ssp. indica cv IR8) arm2 is defective in the uptake of the naturally occurring auxin indole-3-butyric acid (IBA). The acropetal and basipetal transport of IBA is reduced in arm2 roots compared with wild type. In contrast, arm2 roots are normal with respect to uptake and transport of indole-3-acetic acid (IAA). Furthermore, arm2 roots are resistant to IBA but respond normally to IAA. The mutant analysis of arm2 indicates the presence of an influx carrier system for IBA in rice roots.

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.

Interaction between two auxin-resistant mutants and their effects on lateral root formation in rice (Oryza sativa L.).

Since root elongation is very sensitive to auxin, screening for reduced inhibition in root elongation has been an important method for the detection of auxin-resistant mutants. Two recessive auxin-resistant lines of rice (Oryza sativa L. ssp. indica cv. IR8), arm1 and arm2, have been isolated by screening for resistance to 2,4-dichlorophenoxyacetic acid (2,4-D). arm1 displays a variety of morphological defects including reduced lateral root formation, increased seminal root elongation, reduced root diameter, and impaired xylem development in roots, while the arm2 phenotype is almost similar to wild-type IR8 except for a slightly reduced lateral root formation, impaired xylem development in roots and an enhanced plant height. Although the growth of arm2 roots exhibited a resistance to 2,4-D, it was sensitive to 1-naphthaleneacetic acid (NAA) as the wild type. At the same time, the arm2 roots showed a reduced [14C]2,4-D uptake while uptake of [3H]NAA was normal, suggesting that the resistance to 2,4-D of arm2 roots is due to a defect in 2,4-D uptake. To investigate the possible interaction between arm1 and arm2 genes, a double mutant has been constructed. The roots of arm1 arm2 double mutant were more resistant to 2,4-D and formed fewer lateral roots than those of either single mutant, suggesting that the two genes show synergistic effects with respect to both auxin response and lateral root formation. By contrast, all these mutants displayed the normal gravitropic response in roots, as did the wild-type plants. Taken together, Arm1 and Arm2 genes seem to function in different processes in the auxin-response pathways leading to lateral root formation.

Isolation of chromosaponin I-specific antibody by affinity chromatography.

Chromosaponin I (CSI), a gamma-pyronyl-triterpenoid saponin isolated from pea and other leguminous plants, modulates several developmental processes of plant roots and activates the sugar taste receptor cells in blowflies. CSI is a unique saponin for its reducing power and biological activities in both plants and insects. In the present paper, we described the method of preparation for CSI-specific antibody using CSI-affinity and soyasaponin I-affinity columns. The antibody's-specific binding activity to CSI was confirmed by a bioassay using Arabidopsis roots and a ligand-molecule interaction analysis using BIAcore 3000. Because of the lability of CSI, the CSI-affinity column was made only by a moderate reaction condition in which CSI was coupled to EAH Sepharose 4B in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). The special control of the reaction temperature was essential to complete the coupling reaction; the reaction with EDC at 0 degrees C followed by a gradual increase in temperature.

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 mutant ein2-1, and auxin influx mutants aux1-7, aux1-22, and double mutant aux1-7 ein2. Beta-glucuronidase (GUS) expression analysis in the BA-GUS transgenic line, consisting of auxin-responsive domains of PS-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 ein2-1 roots 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 of aux1 mutants to wild-type level, whereas 100 nM NAA was needed for ein2-1 and aux1-7 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 and ein2-1 roots, while stimulating these traits in aux1-7and aux1-7ein2 roots, confirming that CSI is a unique modulator of AUX1.

Triterpenoid saponins stimulate the sugar taste receptor cell through a G protein-mediated mechanism in the blowfly, Phormia regina.

The blowfly has taste chemosensilla on the labellum. The sensory receptor cells in the chemosensillum are highly specialized for the tastes of sugar, salt and water, respectively. Previously we introduced chromosaponin I (CSI) and glycyrrhizin (GL), as sweet substances for the blowfly, Phormia regina. Application of these triterpenoid saponins induced feeding responses as well as impulses of the sugar taste receptor cell in the LL-type sensillum at a much lower concentration than that of sucrose. In the present paper, we show the involvement of G protein-mediated cascade in the CSI- and GL-responses as well as in sugar responses. CSI activates the sugar signal transduction cascade after penetrating through the membrane. On the other hand, GL exerts dual effects to stimulate the sugar signal transduction possibly by activating it inside the cell and also by interacting with the pyranose sugar receptor site. A non hydrolyzable G protein inhibitor guanosine 5'-O-(2-thiodiphosphate), GDPbetaS, markedly decreased the responses of the sugar receptor cell to the two triterpenoid saponins as well as the response to sucrose and fructose. These results suggest that CSI and GL are direct activators of G protein.