Yoshie S. Momonoki

A gene encoding a protein modified by the phytohormone indoleacetic acid.

We show that the expression of an indole-3-acetic acid (IAA)-modified protein from bean seed, IAP1, is correlated to the developmental period of rapid growth during seed development. Moreover, this protein undergoes rapid degradation during germination. The gene for IAP1, the most abundant protein covalently modified by IAA (iap1, GenBank accession no. AF293023) was isolated and cloned from bush bean (Phaseolus vulgaris) seeds. The 957-bp sequence encodes a 35-kDa polypeptide. IAA-modified proteins represent a distinct class of conjugated phytohormones and appear in bean to be the major form of auxin in seeds. IAA proteins also are found at other stages of development in bean plants. Our immunological and analytical data suggest that auxin modification of a small class of proteins may be a feature common to many plants.

Molecular Characterization of Maize Acetylcholinesterase. A Novel Enzyme Family in the Plant Kingdom

Acetylcholinesterase (AChE) has been increasingly recognized in plants by indirect evidence of its activity. Here, we report purification and cloning of AChE from maize (Zea mays), thus providing to our knowledge the first direct evidence of the AChE molecule in plants. AChE was identified as a mixture of disulfide- and noncovalently linked 88-kD homodimers consisting of 42- to 44-kD polypeptides. The AChE hydrolyzed acetylthiocholine and propyonylthiocholine, but not S-butyrylthiocholine, and the AChE-specific inhibitor neostigmine bromide competitively inhibited its activity, implying that maize AChE functions in a similar manner as the animal enzyme. However, kinetic analyses indicated that maize AChE showed a lower affinity to substrates and inhibitors than animal AChE. The full-length cDNA of maize AChE gene is 1,471 nucleotides, which encode a protein having 394 residues, including a signal peptide. The deduced amino acid sequence exhibited no apparent similarity with that of the animal enzyme, although the catalytic triad was the same as in the animal AChE. In silico screening indicated that maize AChE homologs are widely distributed in plants but not in animals. These findings lead us to propose that the AChE family, as found here, comprises a novel family of the enzymes that is specifically distributed in the plant kingdom.

Asymmetric Distribution of Acetylcholinesterase in Gravistimulated Maize Seedlings

Acetylcholinesterase (AChE) activity has previously been studied by this laboratory and shown to occur at the interface between the stele and cortex of the mesocotyl of maize (Zea mays L.) seedlings. In this work we studied the distribution of AChE activity in 5-d-old maize seedlings following a gravity stimulus. After the stimulus, we found an asymmetric distribution of the enzyme in the coleoptile, the coleoptile node, and the mesocotyl of the stimulated seedlings using both histochemical and colorimetric methods for measuring the hydrolysis of acetylthiocholine. The hydrolytic capability of the esterase was greater on the lower side of the horizontally placed seedlings. Using the histochemical method, we localized the hydrolytic capability in the cortical cells around the vascular stele of the tissues. The hydrolytic activity was inhibited 80 to 90% by neostigmine, an inhibitor of AChE. When neostigmine was applied to the corn kernel, the gravity response of the seedling was inhibited and no enzyme-positive spots appeared in the gravity-stimulated seedlings. We believe these results indicate a role for AChE in the gravity response of maize seedlings.

Maize acetylcholinesterase is a positive regulator of heat tolerance in plants

We previously reported that native tropical zone plants showed high acetylcholinesterase (AChE) activity during heat stress, and that AChE activity in endodermal cells of maize seedlings was increased by heat treatment. However, the physiological role of AChE in heat stressed plants is still unclear. Here we report (1) tissue-specific expression and subcellular localization of maize AChE, (2) elevation of AChE activity and possible post-translational modifications of this enzyme under heat stress, and (3) involvement of AChE in plant heat stress tolerance. Maize AChE was mainly expressed in coleoptile nodes and seeds. Maize AChE fused with green fluorescent protein (GFP) was localized in extracellular spaces of transgenic rice plants. Therefore, in maize coleoptile nodes and seeds AChE mainly functions in the cell wall matrix. After heat treatment, enhanced maize AChE activity was observed by in vitro activity measurement and by in situ cytochemical staining; transcript and protein levels, however, were not changed. Protein gel blot analysis revealed two AChE isoforms (upper and lower); the upper-form gradually disappeared after heat treatment. Thus, maize AChE activity might be enhanced through a post-translational modification response to heat stress. Finally, we found that overexpression of maize AChE in transgenic tobacco plants enhanced heat tolerance relative to that of non-transgenic plants, suggesting AChE plays a positive role in maize heat tolerance.

Molecular cloning of acetylcholinesterase gene from Salicornia europaea L.

“Salicornia europaea” increases acetylcholinesterase (AChE) accompanied by salt accumulation during their growth. The plant acetylcholine (ACh)-mediated system in Salicornia could be responsible for transport of ions through channels in a manner similar to the animal systems. In this study, Salicornia AChE gene was identified by RT-PCR using degenerate primers designed based on previously cloned maize and siratro AChE genes. The full-length cDNA of Salicornia AChE was 1,536 nucleotides, encoding a 387-residue protein that includes a 28-residue signal sequence. In silico research presumed that Salicornia AChE is targeted to the secretory pathway via the endoplasmic reticulum.

Gravitropic Response of Acetylcholinesterase and IAA-Inositol Synthase in Lazy Rice.

Summary We previously observed that gravistimulation changes the localization of acetylcholinesterase (AChE) and IAA-inositol synthase in maize shoots. In the present study, we analyzed the localization patterns of these enzymes in a lazy strain of rice, which lacks gravitropic responses. AChE was detected by color development due to enzymic reaction in the coleoptile of dark-grown rice plants under a light microscope. IAA-inositol synthase was detected immunochemically using an anti-IAA-inositol synthase polyclonal antibody. Gravistimulation was given by moving 5-d-old rice seedlings from a vertical to a horizontal position. AChE and IAA-inositol synthase in both normal and lazy rice strains were both distributed asymmetrically in the vascular bundles in the lower half of the horizontally oriented coleoptile at the 90th min. At the 4th h, they were distributed asymmetrically in normal rice, but symmetrically in the lazy rice. The immunoreaction of IAA-inositol synthase in the lazy rice was weak compared to that of the normal rice. Neostigmine bromide inhibited AChE activity and asymmetric distribution of IAA-inositol synthase in both normal and lazy rice strains. The results showed that AChE responded to gravistimulus, and consequently controlled the distribution of IAA-inositol synthase in both normal and lazy rice strains.

Subcellular localization of overexpressed maize AChE gene in rice plant.

The ACh-mediated system consisting of acetylcholine (ACh), acetylcholine receptor (AChR) and acetylcholinesterase (AChE) is fundamental for nervous system function in animals and insects. Although plants lack a nervous system, both ACh and ACh-hydrolyzing activity have been widely recognized in the plant kingdom. The function of the plant ACh-mediated system is still unclear, despite more than 30 years of research. To understand ACh-mediated systems in plants, we previously purified maize AChE and cloned the corresponding gene from maize seedlings (Plant Physiology). In a recent paper in Planta, we also purified and cloned AChE from the legume plant siratro (Macroptilium atropurpureum). In comparison with electric eel AChE, both plant AChEs showed enzymatic properties of both animal AChE and animal butyrylcholinesterase. On the other hand, based on Pfam protein family analysis, both plant AChEs contain a consensus sequence of the lipase GDSL family, while the animal AChEs possess a distinct alpha/beta-hydrolase fold superfamily sequence, but no lipase GDSL sequence. Thus, neither plant AChE belongs to the well-known AChE family, which is distributed throughout the animal kingdom. To address the possible physiological roles of plant AChEs, we herein report our data from the immunological analysis of the overexpressed maize AChE gene in plants.

Molecular Structure and Properties of Lectin from Tomato Fruit

A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro4 motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin.

Kunitz soybean trypsin inhibitor is modified at its C-terminus by novel soybean thiol protease (protease T1).

Abstract Kunitz soybean trypsin inhibitor (KSTI) is hydrolyzed during seed germination to yield amino acids needed to support initial seedling growth. The type of KSTI from Glycine max (L.) Merrill cv. Toyokomachi is KSTI-Ti b. The KSTI-Ti b from 4-day-old post-germination cotyledons (KSTI-Ti b’) has 3 or 4 amino acid residues cleaved off at the C-terminus. This KSTI modification is important to understand the mechanism of degradation in seed reserve proteins by proteases. Protease K1 also cleaves amino acid residues at the C-terminus of KSTI but it removes 5 amino acid residues. Therefore, we presumed the KSTI-Ti b’ was produced by a protease other than protease K1. In this study, the protease T1 responsible for cleavage of KSTI-Ti b at the C-terminus was purified. The enzyme was estimated to have a molecular mass of 33 kDa from its mobility on SDS-PAGE gels. The N-terminal amino acid sequence of the purified protease T1 corresponded to amino acids Phe-73 to Phe-92 of both thiol protease isoforms A and B from the soybean leaf, and shared 83% identity with the partial amino acid sequence of the membrane-associated cysteine protease from mung bean seedlings, a protease known to perform post-translational cleavage of C-terminal peptides of target proteins. Finally, this enzyme was shown to convert KSTI-Ti b to KSTI-Ti b’.

Identification of Salicornia Populations : Comparison between Morphological Characterization and RAPD Fingerprinting

Abstract In Japan, there are two taxa of the genus Salicorniaplants; S. europaeaL. distributed in Hokkaido and S. herbaceaL. distributed on the coast of Inland Sea of Seto. To estimate the polymorphism of the Salicorniaplants, we statistically analyzed the morphological features and random amplified polymorphic DNA (RAPD) of five groups from three populations found at Lake Tofutsu and Lake Notori in Hokkaido and Okayama Prefecture on the coast of Inland Sea of Seto. The morphological features, such as plant length, segment number, length and number of branches, and incidence of the secondary branches showed variations among locations. The morphological plasticity of Salicorniaplants was also observed at different plant densities. Thereby these features were difficult to use for identifying the populations. On the other hand, the genotype based on the RAPD markers implied five groups : two groups from the Notori population, two groups from the Tofutsu population and one group from the Okayama population. Additionally the Notori and Tofutsu populations were identified as genotypically related, and different from the Okayama population. The RAPD method, which is one of the simplest and fastest molecular techniques, was found useful for identifying the type of Salicorniaplant.