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

ABSTRACT

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.

KEYWORDS:

• Acetylcholine
• acetylcholinesterase
• ACh
• AChE
• gravitropism
• rice (Oryza sativa)

Abbreviations

ACh	=	Acetylcholine
ASCh	=	Acetylthiocholine
AChE	=	Acetylcholinesterase
DTNB	=	5,5´-dithiobis-2-nitrobenzoic acid
UBQ	=	Ubiquitin

Acetylcholine (ACh) is well-known as the neurotransmitter in the mammalian central and peripheral cholinergic nervous systems.¹ ACh has been also identified in a number of non-neuronal animal tissues and in fungi, bacteria, and plants.^2,3 ACh may play a universal role as an elementary molecular agent of irritability in animal, plant and microbial cells.⁴ Although plants lack a nervous system, both ACh and AChE have been widely recognized in plants.⁵ The function of the plant ACh and AChE is still unclear, despite more than 30 years of research. To understand those functions, we have been studying about physiological roles of plant AChE under gravitropic stimulus. Indeed plants under gravitropic stimulus showed asymmetric distribution of hormones and AChE activity.^6,7 When a plant is moved from the vertical orientation to a horizontal position, the stem shows curvature as it returns to a vertical position by response acting against gravistimulus. On the other hands, maize seedlings that were treated first with neostigmine bromide (AChE inhibitor) did not respond to the gravistimulus.⁷ Furthermore, after the stimulus, the AChE activity was distributed asymmetrically at the interface between the stele and the cortex of the gravistimulated maize seedlings.⁶ The gravitropic bending occurs because of an asymmetric distribution of the growth hormone auxin (indole-3-acetic acid, IAA).⁸ We previously observed that gravistimulation leads to an asymmetric distribution of both IAA-inositol synthase and the AChE activities in maize and rice shoots.^9,10 In addition, its asymmetric distribution of both enzyme activities in maize and rice shoots was also inhibited by the treatment with neostigmine bromide.^9,10 The neostigmine bromide is an AChE inhibitor having no inhibitory effect on IAA metabolism in plants.¹¹ These findings suggest that AChE would play an important role in the gravitropic response of plants. To directly confirm our previous reports, the functionality of plant AChE under gravitropic stimulus was recently proved by overexpressing AChE gene in rice plants. AChE up-regulated plants showed an enhanced gravitropic response in rice seedlings.¹² In this report, in order to support our recent data, we silenced the AChE expression by RNAi in rice plants and assessed its impact on phenotypic characteristics under gravistimulation.

Generation and characterization of transgenic rice plants silencing rice AChE gene

We generated transgenic rice plants in which the expression of the AChE gene (accession no. AK073754) is suppressed by RNAi techniques. The RNAi plasmid was constructed and designated AChE-K_D. To generate inverted repeat regions from the gene-specific regions of AChE, 5′- and 3′-gene specific regions of rice AChE were connected. RNA-silencing construct carrying the inverted repeat of rice AChE fragments was made using the pANDA vector.^13,14 Transgenic rice plants were generated by Agrobacterium-mediated transformation of rice (cv. Nipponbare) calli as described in previous study.¹² Regenerated transgenic rice plants were grown in a large growth cabinet (Koitotron type KG: Koito Ind. Ltd. Co., Tokyo, Japan) at 28^oC under 16 h light/d. Five independent transgenic lines were selected by AChE activity assay (Fig. 1A) and 2 T2 lines (AChE-K_D No.3 and No.15) were chosen for RT-PCR. AChE-K_D No.3 and No.15 silencing AChE gene showed an 85.5 and 90% decrease in levels of AChE activities respectively, compared with wild-type plants (Fig. 1A). RNAi expression also caused reduced levels of gene expression compared with wild-type plants (Fig. 1B).

Figure 1. Generation and characterization of transgenic rice plants silencing the rice AChE gene. RNAi-silenced line: AChE-K_D. WT: wild type. (A) AChE activity in 4-d-old transgenic seedlings (T2 generation) measured by the DTNB method with acetylthiocholine (ASCh) as substrate according to our previous report.¹² Each textured bar represents the average of 3 replicates. Thin vertical bars represent standard errors. (B) RT-PCR analysis of mRNA expression. Designation to the left of each panel indicates primers used; UBQ indicates primers for the ubiquitin-encoding gene used as an internal control according to our previous report.¹²

Shoot gravitropism in AChE- silencing plants

We placed transgenic seeds of AChE-K_D No.3 and No.15 (T2 generation) in a tissue culture tube containing Murashige-Skoog nutrient medium and incubated in the dark at 28˚C for 4 d. For gravistimulation, the tube was oriented horizontally and curvature angles were measured on digital images taken 0, 30, 60, 90, 120, 150 and 180 min later as described in a previous study.¹² The shoot gravitropic response, relative to wild type, was inhibited when expression of the AChE gene was suppressed (Fig. 2A, B). After 180 min of gravistimulation, wild-type seedlings bent upward at approximately 52.4°, whereas suppression of AChE caused greatly reduced gravitropic responses; the curvature of gene-silencing plantlets was only approximately 12.8° in AChE-K_D No.3 and approximately 24.9° in AChE-K_D No.15 after 180 min gravistimulation. The higher concentration of ACh has already been shown to possess an inhibitory effect on cell elongation.¹⁵ In addition, Di Sansebastiano and colleagues recently reported that an ACh concentration beyond 50 mM appeared to negatively interfere with the auxin driven elongation process.¹⁶ It is conceivable, therefore, that an ACh concertation in AChE- silencing plants might be higher than that in wild-type plants and its higher level of ACh might negatively regulate shoot gravitropism in AChE- silencing plants. In a previous report, we have already showed AChE overexpression causes an enhanced gravitropic response.¹² Taken together, these results strongly suggest that the rice AChE plays an important role in the gravitropic response and is a positive regulator of shoot gravitropism in the seedlings.

Figure 2. Shoot gravitropic responses of transgenic rice plants suppressing the expression of rice AChE gene. RNAi-silenced lines: AChE-K_D No.3 and No.15. WT: wild type. (A) Quantification of shoot gravitropic responses in wild-type and transgenic rice plantlets silencing the rice AChE gene (T2 generation). Each data point represents measurement of 10 plantlets. Thin vertical bars represent standard errors. (B) Typical phenotypes of wild-type and transgenic rice plantlets silencing the rice AChE gene (T2 generation) under gravistimulation. Digital images were taken at designated time points to the upper of each panel.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. K. Shimamoto and Dr. D. Miki (Nara Institute of Science and Technology, Japan) for the kind gift of a pANDA vector. This research was supported by “Ground-based Research Program for Space Utilization (2006-2008)” (ID 77) promoted by Japan Space Forum.