Taisaku Amakawa

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.

Light-mediated breakdown of phosphatidylinositol-4,5-bisphosphate in isolated rod outer segments of frog photoreceptor

When isolated frog (Rana catesbeiana) rod outer segment (ROS) fragments were incubated with [gamma-32P]ATP in the dark, only two of phospholipids, i.e., phosphatidylinositol-4-phosphate (DPI) and phosphatidic acid (PA) incorporated 32P. Upon addition of DPI (100 microM), considerable amount of 32P was incorporated into phosphatidylinositol-4,5-bisphosphate (TPI) as well as DPI and PA. Exposure of the ROS membranes to 5 sec flash of light resulted in approx. 20% decrease in the labeled TPI, while no significant effect was observed on DPI and PA. It was also observed that Ca2+ markedly accelerated the production of PA in the dark, while it reduced the 32P-incorporation into TPI. These results suggest that there is light- and/or Ca2+-dependent TPI-specific phospholipase C in ROS of vertebrate photoreceptors.

Effects of cyclic GMP on the sugar taste receptor cell of the fly Phormia regina

Abstract In some sensory receptors of vertebrates, cyclic GMP (cGMP) and/or cyclic AMP (cAMP) is demonstrated to mediate information from the receptor molecule to the ion channel. The labellar chemosensillum of the fly, Phormia regina, is a hair-like organ which contains four taste cells, i.e. the sugar, the salt, the water receptor cells and the fifth cell. They generate spike potentials of different sizes which are easily discriminated from each other. When a membrane-permeable cGMP analogue, dibutyryl cyclic GMP, was applied to the tip of sensillum, large spikes were evoked. Membrane-impermeable nucleotides also evoked the same spikes but their stimulating effect was less than that of dibutyryl cyclic GMP. Judging from the size of the spikes and feeding behaviour of flies to the nucleotides, the spikes were identified as those from the sugar receptor cells. When the mixture of dibutyryl cyclic GMP and phosphodiesterase inhibitor, both of which are membrane-permeable, was repeatedly applied to the sensillum tip, “remaining spikes” were observed after the removal of the mixture. Furthermore, application of dibutyryl cyclic GMP to the sensillum induced extremely slow adaptation of the sugar receptor cell while adaptation of the cell by sugar stimulation is usually rapid. Based upon these results, we discuss the possibility that cGMP works as a second messenger for the sugar receptor excitation of the fly.

The effect of isobutylmethylxanthine on the photoresponse and phosphorylation of phosphatidylinositol in octopus retina

The effect of isobutylmethylxanthine on the photoresponse and the phosphorylation of phosphatidylinositol was studied. The recovery of photoresponse after a bright flash was greatly altered by the addition of isobutylmethylxanthine. Phosphatidylinositol phosphorylation, assessed by the incorporation of label from [gamma-32P]ATP, was influenced by isobutylmethylxanthine. Incorporation of 32P was decreased into diphosphoinositide and triphosphoinositide, and increased into phosphatidic acid. These changes were interpreted by introducing a parameter, phi, calcium charge of the plasma membrane.

Nucleotide receptor-site on the labellar sugar receptor cell of the blowfly Phormia regina

Abstract Previously we reported that, among nucleotides applied extracellularly to the chemosensillum of the blowfly Phormia regina, a membrane-permeable cyclic GMP analogue, dibutyryl cyclic GMP, was particularly effective, inducing much impulse-discharge. From the results obtained, we discussed the possibility that a nucleotide site exists on the sugar receptor cell. Here we report results of our electrophysiological experiments to investigate this putative nucleotide receptor-site. As previously reported, when dibutyryl cyclic GMP was applied with sucrose, the impulse-discharge was depressed. Cyclic GMP or another membrane-permeable cyclic GMP analogue, 8-bromo cyclic GMP, enhanced the impulse frequency when applied to a chemosensillum with sucrose. However, 8-bromo cyclic GMP partly depressed the impulse-discharge evoked by dibutyryl cyclic GMP. If we hypothesize that the sugar receptor-site has a nucleotide receptor-site separated from the receptor site for sucrose (P-site) on the membrane and that the cell uses cyclic GMP as an intrinsic intracellular messenger mediating the taste transduction, then, these results can be explained as follows. Dibutyryl cyclic GMP inhibits the P-site with its butyryl groups. On the other hand, it binds the nucleotide receptor-site and stimulates the cell like other nucleotides. However, 8-bromo cyclic GMP, which does not affect the P-site, binds the nucleotide receptor-site like a competitive inhibitor. Thus, the impulses induced by 8-bromo cyclic GMP are due to the 8-bromo cyclic GMP acting in place of cyclic GMP as the intracellular messenger. As for the responses induced by dibutyryl cyclic GMP, a part which can be reduced by 8-bromo cyclic GMP is due to the dibutyryl cyclic GMP binding the nucleotide receptor-site, and other part is due to the dibutyryl cyclic GMP acting as the intracellular messenger.

Protein kinase C-promoted adaptation of the sugar receptor cell of the blowfly Phormia regina

Abstract We applied protein kinase C activators (phorbol esters) or protein kinase inhibitors (H series inhibitors) inside the sugar taste receptor cell of the fly, Phormia regina , and investigated the impulse-discharge pattern generated by the cell. Protein kinase C activators accelerated but its inhibitor (H-7) reduced adaptation (gradual decrease of impulse frequency during constant stimulation) to sucrose. EGTA (a Ca 2+ chelator) applied with DOC reduced adaptation. These results strongly suggest that protein kinase C takes part in the adaptation process. Effects of H-8 and HA1004, which have a high affinity for protein kinase A or G, were similar to each other but different from that of H-7. They depressed the impulse frequency especially at the beginning of stimulation. This suggests that protein kinase A or G is strongly related with some mechanism essential for transduction rather than for adaptation.

Stable and unstable phase of memory in classically conditioned fly, Phormia regina: Effects of nitrogen gas anaesthesia and cycloheximide injection

Abstract This study examined the effects of nitrogen anaesthesia and cycloheximide injection on memory of the classically-conditioned fly, Phormia . 1 M NaCl solution was given to each fly as a conditioning stimulus and 0.5 M sucrose solution was the unconditioned stimulus that induced the proboscis extension response. The training period was as short as 2 min and testing was usually carried out 2 hr later. At varying times (0–60 min) between training and testing, flies were anaesthetized with nitrogen gas for 25 sec. When flies were anaesthetized immediately after training the effect of nitrogen gas was the greatest and few flies showed any conditioned response, but the sensitivity of memory to nitrogen gas declined as the interval between training and nitrogen treatment became longer, and such treatment had no effect on memory when the interval was longer than 30 min. The effect on memory of cycloheximide, an inhibitor of peptide bond synthesis, was also investigated. The injection of cycloheximide (0.37 μg) immediately after training diminished the memory, but when given 1 hr after training it had no effect on memory. These results show that the memory in Phormia has two phases, stable and an unstable phase, like long-term and short-term memory in vertebrates.

Phosphatidylinositol stimulates phosphorylation of protein components I and II in rod outer segments of frog photoreceptors

We studied the effect of phosphoinositides on the phosphorylation of endogenous proteins in the soluble fraction of the frog photoreceptor rod outer segments (ROS). Phosphatidylinositol (PI) stimulated the phosphorylation of two low molecular weight proteins, components I and II (12 and 11 kDa) which are known to be the preferential substrates of the cyclic GMP (cGMP)-dependent protein kinase in the ROS. Polyphosphoinositides (PPI) specifically inhibited the PI-dependent phosphorylation of these two components. On the other hand, PPI stimulated the phosphorylation of 38, 48 and 52 kDa proteins in the absence of PI. These data suggest that PI and PPI may function in the ROS by regulating the phosphorylation of some enzymes or regulator proteins in the transduction mechanism in the ROS.

Properties of membrane-bound α-glucosidases: Possible sugar receptor protein of the blowfly, Phormia regina

Abstract Previously reported PII-type α-glucosidase located in the precipitate of the labellar homogenate of the blowfly Phormia regina was solubilized by sodium deoxycholate (DOC) and further separated into three isozymes with different molecular weight: PII-M (mol. wt 9 × 10 4 ). PII-D (mol. wt 2 × 10 5 ) and PII-T (mol. wt 8 × 10 5 ) by molecular sieve chromatography on Biogel P-300 or Ultragel AcA-34. These three isozymes had almost the same K m s and relative values of V m s for several substrates, suggesting that they had the same common active site. PII-D and PII-T are more strongly embedded in the membrane than PII-M, because the proportion of PII-D and PII-T was much increased when the remaining glucosidase in the precipitate after the first solubilization was reextracted by DOC. A large peak of α-glucosidase isozyme P-IV which preferentially hydrolyze sucrose eluted just after P-II (soluble P-II) when the supernatant fraction of the labellar homogenate was chromatographed on DEAE-Sephadex A-50. P-IV was scarcely present in the precipitate fraction. Soluble P-II had the same mol. wt as PII-M and had similar properties to PII-M except for the ratio of V m s. A large proportion of PII-D was contained in the well washed labellar integuments, a preparation rich in labellar chemosensilla. It suggests that most of the insoluble α-glucosidase contained in the dendrite in labellar chemosensilla is PII-D. PII-D (and PII-T) are possible sites of the pyranose receptor molecule because their properties and localization agree well with those of the receptor.

Insoluble α-glucosidase: Possible pyranose site of the sugar receptor of the labellar of the blowfly, Phormia regina

Abstract The insoluble α-glucosidase activity was examined in three kinds of preparations of the fly labella: labellar integuments with intact chemosensilla, fragments of nerve bundles, and sensilla cut off from the labella. α-Glucosidases having a similar affinity for substrates to isozyme P-II were found in each preparation. Kinetics, inhibition by tris(hydroxymethyl)amino methane (Tris), and pH dependence were examined on the activity of both the membrane-fixed and solublized glucosidase isozymes. Most of the insoluble activity was observed in the nerve bundle fractions, which showed the same properties as P-II in the soluble fraction but were not solubilized by chaotropic anion or by addition of EDTA (insoluble P-II). A different type of insoluble α-glucosidase has been suggested to exist in the labellar chemosensilla in addition to insoluble P-II. It showed a very broad pH dependence and had a larger inhibition constant for Tris. It was inferred to be identical with the enzyme found at the tip of the intact chemosensilla. The hypothesis that the above isozyme might be the pyranose site of the sugar receptor was supported.