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.Key words: acetylcholinesterase activity, maize AChE gene, overexpression, rice, subcellular localizationIn the animal ACh-mediated system, ACh serves to propagate an electrical stimulus across the synaptic junction. At the presynaptic neuron end, an electrical impulse triggers the release of ACh, which accumulates in vesicles into the synaptic cleft via exocytosis. ACh then binds to an ACh receptor (AChR) on the postsynaptic neuron surface, and the ACh-AChR binding induces subsequent impulses to the postsynaptic neuron. Finally, ACh, which is released again by the receptor into the synaptic cleft, is rapidly degraded by acetylcholinesterase (AChE; E.C.3.1.1.7).^1,2 ACh and AChE,^3,4,5 and choline acetyltransferase activity that takes part during synthesis of ACh^6,7 have been recognized in plants. AChR has not been identified in plant cells so far. However, so-called “ACh-binding sites” were detected in membrane fractions from some bean seedlings^8,9 and evidence was also detected in plant organelles, such as chloroplasts¹⁰ and tonoplasts¹¹ using pharmacological methods.Concerning the function of the ACh-mediated system in plants, Momonoki^12,13 has proposed that it results in an asymmetric distribution of hormones and substances due to gravity stimuli, as well as changes in ACh content, AChE activity and Ca²⁺ level in response to heat stress. However, all these phenomena have been investigated using indirect techniques. Thus, to understand the plant ACh-mediated system, we purified AChEs and cloned the AChE genes from maize¹⁴ and siratro¹⁵ seedlings. The maize AChE was found to exist as two types of 88-kDa homodimers, which in turn consisted of disulfide-linked and noncovalently-linked 42- to 44-kDa subunits.¹⁴ The siratro AChE might exist as a disulfide-linked 125-kDa homotrimer consisting of 41- or 42-kDa subunits.¹⁵ The plant AChEs apparently from various quaternary structures, depending on the plant species, similar to animal AChEs. Furthermore, maize and siratro AChEs showed enzymatic properties of both animal AChE and animal butyrylcholinesterase, compared with electric eel AChE.¹⁵In this addendum, we report our recent immunohistochemical study using an antibody against maize AChE. In order to overexpress the maize AChE gene in rice plants, we constructed a plasmid for the sense expression of the AChE gene by cloning it into the pT7 Blue vector. The maize AChE gene¹⁴ was introduced behind the maize ubiquitin 1 promoter (Ubi) in the p2K-1⁺ plant expression vector. The Ubi::maize AChE and control (p2K-1⁺ only) plasmid were introduced into Agrobacterium tumefaciens EHA 101, which was transformed into rice (Nihonbare) via Agrobacterium-mediated transformation methods.¹⁶ The maize AChE transgenic plants exhibited approximately 650-fold higher AChE activity than was observed in the control plants but no phenotypic changes between transgenic and control plants. The subcellular localization of AChE was observed by immunofluorescence in paraffin-embedded leaf and stem tissues of transgenic rice plants. The maize AChE protein was detected in extracellular spaces in the leaf and stem of the plants (Fig. 1). Therefore, plant AChEs may function in the extracellular space, similar to some isoforms of animal AChE.^2,17Open in a separate windowFigure 1Subcellular localization of maize AChE in leaf and stem of transgenic rice. (A) Leaf cross-section of transgenic rice; (B) leaf cross-section of control; (C) stem cross-section of transgenic rice; (D) stem cross-section of control. Each section was probed with maize AChE antibody and then visualized with Alexa Fluor 488-conjugated secondary antibody. Control indicates rice plants transfected with p2K-1⁺ vector only. Arrowheads indicate localization of maize AChE.Most of the AChE activity in the root was associated with cell wall materials.¹⁸ The computer-assisted cellular sorting prediction program TargetP presumed that our purified maize AChE¹⁴ is targeted to the secretory pathway via the endoplasmic reticulum. Furthermore, the SOSUI program (http://sosui.proteome.bio.tuat.ac.jp / sosuiframe0.html), which discriminates between membrane and soluble proteins, showed that the maize AChE does not contain any likely transmembrane helical regions, which are features of proteins that associate with the lipid bilayers of the cell membrane. These findings suggested that the maize AChE might be localized at the cell wall. However, in an earlier work,¹³ we speculated that AChE is localized at the plasmodesmatal cell-to-cell interface and that it plays a role in regulation of the plasmodesmatal channel as a constituent of the ACh-mediated system. We improved our hypothesis of the role of the ACh-mediated system in a paper in Plant Physiol.¹⁴The results based on fluorescence-immunohistochemistry in transgenic rice plants reported in this paper confirmed that the maize AChE is localized at the cell wall. Here we propose again our hypothesis of an ACh-mediated system including this new finding; the system might be localized to the extracellular region around the plasmodesmatal channel and might conduct cell-to-cell trafficking by channel gating regulation. Adjoining cells in plant tissues are interconnected via plasmodesmata, which allow the trafficking of low-molecular-mass materials across the cell wall between two cells. According to a recent model,¹⁹ transport of these substances could be regulated by the opening and/or closing of conductive channels to prevent infection by pathogens and to selectively control trafficking through the plasmodesmata. Furthermore, it has been speculated that morphoregulatory proteins around the plasmodesmata could be involved in channel regulation.²⁰ Therefore, the ACh-mediated system might regulate the opening and/or closing of channels by interactions with morphoregulatory proteins at the cell wall matrix surrounding the plasmodesmata. Further research will be required to clarify the precise physiological roles of plant AChEs.