Identification and molecular characterization of propionylcholinesterase, a novel pseudocholinesterase in rice.

Cholinesterase is consisting of acetylcholinesterase (AChE) and pseudocholinesterase in vertebrates and invertebrates. AChE gene has been identified in several plant species, while pseudocholinesterase gene has not yet been found in any plant species. In this study, we report that the AChE gene paralog encodes propionylcholinesterase (PChE), a pseudocholinesterase in rice. PChE was found to be located adjacent to AChE (Os07g0586200) on rice chromosome 7 and designated as Os07g0586100. Phylogenetic tree analysis showed a close relationship between rice AChE and PChE. PChE-overexpressing rice had higher hydrolytic activity toward propionylthiocholine than acetylthiocholine and showed extremely low activity against butyrylthiocholine. Therefore, the PChE gene product was characterized as a propionylcholinesterase, a pseudocholinesterase. The rice PChE displayed lower sensitivity to the cholinesterase inhibitor, neostigmine bromide, than electric eel, maize, and rice AChEs. The recombinant PChE functions as a 171 kDa homotetramer. PChE was expressed during the later developmental stage, and it was found be localized in the extracellular spaces of the rice leaf tissue. These results suggest that the rice plant possesses PChE, which functions in the extracellular spaces at a later developmental stage. To the best of our knowledge, this study provides the first direct evidence and molecular characterization of PChE in plants.

Altered expression of acetylcholinesterase gene in rice results in enhancement or suppression of shoot gravitropism.

Acetylcholinesterase (AChE), an acetylcholine-hydrolyzing enzyme, exists widely in plants, although its role in plant signal transduction is still unclear. We have hypothesized that the plant AChE regulates asymmetric distribution of hormones and substrates due to gravity stimulus, based on indirect pharmacological experiments using an AChE inhibitor. As a direct evidence for this hypothesis, our recent study has shown that AChE overexpression causes an enhanced gravitropic response in rice seedlings and suggested that the function of the rice AChE relates to the promotion of shoot gravitropism in the seedlings. Here, we report that AChE suppression inhibited shoot gravitropism in rice seedlings, as supportive evidence demonstrating the role of AChE as a positive regulator of shoot gravitropic response in plants.

Overexpression of acetylcholinesterase gene in rice results in enhancement of shoot gravitropism.

Acetylcholine (ACh), a known neurotransmitter in animals and acetylcholinesterase (AChE) exists widely in plants, although its role in plant signal transduction is unclear. We previously reported AChE in Zea mays L. might be related to gravitropism based on pharmacological study using an AChE inhibitor. Here we clearly demonstrate plant AChE play an important role as a positive regulator in the gravity response of plants based on a genetic study. First, the gene encoding a second component of the ACh-mediated signal transduction system, AChE was cloned from rice, Oryza sativa L. ssp. Japonica cv. Nipponbare. The rice AChE shared high homology with maize, siratro and Salicornia AChEs. Similar to animal and other plant AChEs, the rice AChE hydrolyzed acetylthiocholine and propionylthiocholine, but not butyrylthiocholine. Thus, the rice AChE might be characterized as an AChE (E.C.3.1.1.7). Similar to maize and siratro AChEs, the rice AChE exhibited low sensitivity to the AChE inhibitor, neostigmine bromide, compared with the electric eel AChE. Next, the functionality of rice AChE was proved by overexpression in rice plants. The rice AChE was localized in extracellular spaces of rice plants. Further, the rice AChE mRNA and its activity were mainly detected during early developmental stages (2 d-10 d after sowing). Finally, by comparing AChE up-regulated plants with wild-type, we found that AChE overexpression causes an enhanced gravitropic response. This result clearly suggests that the function of the rice AChE relate to positive regulation of gravitropic response in rice seedlings.

Tissue localization of maize acetylcholinesterase associated with heat tolerance in plants.

Our recent study reported that maize acetylcholinesterase (AChE) activity in the coleoptile node is enhanced through a post-translational modification response to heat stress and transgenic plants overexpressing maize AChE gene had an elevated heat tolerance, which strongly suggests that maize AChE plays a positive, important role in maize heat tolerance. Here we present (1) maize AChE activity in the mesocotyl also enhances during heat stress and (2) maize AChE mainly localizes in vascular bundles including endodermis and epidermis in coleoptile nodes and mesocotyls 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 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 SerPro(4) 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.

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.

Characterization of trimeric acetylcholinesterase from a legume plant, Macroptilium atropurpureum Urb.

We recently identified plant acetylcholinesterases (E.C.3.1.1.7; AChEs) homologous to the AChE purified from a monocotyledon, maize, that are distinct from the animal AChE family. In this study, we purified, cloned and characterized an AChE from a dicotyledon, siratro. The full-length cDNA of siratro AChE is 1,441 nucleotides, encoding a 382-residue protein that includes a signal peptide. This AChE is a disulfide-linked 125-kDa homotrimer consisting of 41-42 kDa subunits, in contrast to the maize AChE, which exists as a mixture of disulfide and non-covalently linked 88-kDa homodimers. The plant AChEs apparently consist of various quaternary structures, depending on the plant species, similar to the animal AChEs. We compared the enzymatic properties of the dimeric maize and trimeric siratro AChEs. Similar to electric eel AChE, both plant AChEs hydrolyzed acetylthiocholine (or acetylcholine) and propionylthiocholine (or propionylcholine), but not butyrylthiocholine (or butyrylcholine), and their specificity constant was highest against acetylcholine. There was no significant difference between the enzymatic properties of trimeric and dimeric AChEs, although two plant AChEs had low substrate turnover numbers compared with electric eel AChE. The two plant AChE activities were not inhibited by excess substrate concentrations. Thus, similar to some plant AChEs, siratro and maize AChEs showed enzymatic properties of both animal AChE and animal BChE. On the other hand, both siratro and maize AChEs exhibited low sensitivity to the AChE-specific inhibitor neostigmine bromide, dissimilar to other plant AChEs. These differences in enzymatic properties of plant AChEs may reflect the phylogenetic evolution of AChEs.

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.

Characterization and sequence of tomato 2S seed albumin: a storage protein with sequence similarities to the fruit lectin.

We found a 2S storage albumin from the seed of tomato ( Lycopersicon esculentum L. cv. Cherry) that cross-reacted with antiserum to the fruit lectin, and named it Lec2SA. According to its size and basicity, Lec2SA was classified into four isoforms. These isoforms have an M(r) of approximately 12,000, and are composed of a small subunit (M(r) 4,000) and a large subunit (M(r) 8,000) linked by disulfide bonds. The complete amino acid sequence of Lec2SA was determined. The small subunit was composed of 32 amino acids, whereas the large subunit contained 70 amino acids with a pyroglutamine as the N-terminal residue. The sequence of Lec2SA was similar to that of 2S albumins from different plants, such as Brazil nut and castor beans. Furthermore, a sequence similarity was found between the large subunit of Lec2SA and the peptide sequence from tomato lectin. Although these similarities were found, Lec2SA did not show hemagglutinating activity or sugar-chain-binding activity, indicating that Lec2SA lacks the carbohydrate-binding domain. These results suggest that tomato lectin is a chimeric lectin sharing the seed storage protein-like domain that is incorporated into the gene encoding tomato lectin through gene fusion.

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. ) 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.