Tissue localization of maize acetylcholinesterase associated with heat tolerance in plants

Abstract

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.

Response of Maize AChE During Heat Stress

The neurotransmitters such as acetylcholine (ACh), catecholamines, histamines, serotonin and dopamine are commonly found in animal nervous systems and play important roles in sensing, locomotion, vision, information processing and development.¹ Based on a number of reports indicating these compounds are also present in plants,² a novel field of research called “plant neurobiology” has been established.^1,3 The functions of neurotransmitters in plant signaling remain unclear, however, despite numerous studies.

AChE is an enzyme that terminates ACh mediated neurotransmission.⁴ Both AChE and ACh have been widely recognized in higher plants,² as has choline acetyltransferase, which is involved in ACh synthesis by many plant species.^5,6 It is conceivable that in plants the ACh-mediated system, composed of ACh, ACh receptor (AChR), and AChE, plays a significant role in signal transduction, as it does in animals.

We have focused on the ACh-mediated system as a potential gating regulator that causes asymmetric distribution of hormones and substrates in response to gravity, heat, and changes in ACh content, AChE activity and Ca²⁺ concentration.^7,8 We identified AChE genes from maize, siratro (Macroptilium atropurpureum) and Salicornia europaea plants.^9-11 Thus, transgenic analyses of plant AChEs are now possible.

In our latest publication,¹² we found that AChE activity in coleoptile nodes of maize seedlings was enhanced by heat treatment. As additional analysis, effect of heat treatment on AChE activity was measured in coleoptiles and mesocotyls (Fig. 1). Similar to our recent publication,¹² AChE activity in coleoptile nodes increased 13–19% (acetylthiocholine as a substrate) or 10–20% (propionylthiocholine as substrate) after 10–60 min of heat stress, compared with control (before heat treatment at 0 min). Not only increase of AChE activity in coleoptile nodes, the activity in mesocotyls also increased 30–50% (acetylthiocholine as a substrate) or 20–40% (propionylthiocholine as a substrate) after 10–60 min of heat stress, relative to control. This activity increase in mesocotyls after heat treatment was greater than that in coleoptile nodes. On the other hands, AChE activity in coleoptiles did not change during the treatment (Fig. 1). These results suggest that AChE in coleoptile nodes and mesocotyls plays an important role in heat stress response. In our recent publication,¹² we concluded that increased maize AChE activity is accompanied by post-translational modifications but not increased levels of AChE mRNA and protein. To confirm whether mature maize AChE is glycosylated, purified AChE was applied to non-reducing SDS-PAGE, and then stained with Coomassie brilliant blue (CBB) and periodic acid-Schiff’s (PAS) reagent. On a CBB stained gel, AChE appeared as a dimer with a molecular mass of 88 kDa consisting of disulfide-linked 42- to 44-kDa polypeptides and a 42- to 44-kD monomer as described by Sagane et al.⁹ Both dimer- and monomer-forms of maize AChE were stained with PAS reagent, indicating that both forms of maize AChE are certainly glycosylated (Fig. 2). In the future, we will determine the activation mechanisms of plant AChEs by post-translational modifications, which may consist of amino acid deletions from the C terminus and/or different degrees of glycosylation.

Figure 1.

Enhancement of in vitro AChE activities in maize coleoptiles, coleoptile nodes and mesocotyls by heat treatment. Total proteins were extracted from coleoptiles, coleoptile nodes and mesocotyls at 0, 10, 30, 60 and 120 min after the start of heat treatment (45°C). Total protein content in each extract was determined by the Bradford method, as described previously.¹² AChE activity was determined by the DTNB method with acetylthiocholine (closed circle) and propionylthiocholine (open square) as substrates, as described previously.¹²

Figure 2.

SDS-PAGE profiles stained for purified maize AChE with Coomassie brilliant blue and periodic acid-Schiff’s reagent. The purified maize AChE was applied into 12.5% SDS-gel under non-reduced condition without dithiothreitol (DTT). CBB, Coomassie brilliant blue stained gel; PAS, periodic acid-Schiff stained gel; D, dimer-form of maize AChE; M, monomer-form of maize AChE.

To determine whether increased AChE activity after heat stress has a functional role in heat tolerance, maize AChE cDNA was overexpressed in tobacco plants. As shown in our recent publication,¹² all wild type (WT) plants were dead, whereas most of transgenic tobacco seedlings (three T1 lines, T1-1, 3 and 5) survived, within 7 d after heat (48°C for 45 min) treatment. We also obtain similar results from T₀ generations. Transgenic tobacco plants (T₀-1) carrying maize AChE had approximately 3.5-fold higher AChE activity than WT plants (Fig. 3A). The transgenic plant survived, within 18 d under continuous heat-stress condition (40°C with an 8 h dark/16 h light photoperiod), whereas WT plant was completely dead (Fig. 3B). These results suggest that AChE plays a positive role in maize heat tolerance.

Figure 3.

Heat tolerance of transgenic tobacco plants expressing the maize AChE gene. (A) AChE activity (measured by the DTNB method) in leaves of wild type and T₀ generation transgenic tobacco plants (T₀-1). Each textured bar represents the average of three replicates. Thin bars represent standard errors. (B) Phenotypes of wild type and transgenic tobacco plants T₀-1 before and after heat treatment. Plants were continuously exposed to heat (40°C, 8 h dark/16 h light photoperiod) for 18 d.

Tissue Localization of Maize AChE in the Seedlings

To further investigate the biological role of AChE in maize seedlings, we analyzed tissue localization of AChE in coleoptile nodes and mesocotyls of maize seedlings using anti-maize AChE antibody. AChE localized in vascular bandles including endodermis and epidermis in coleoptile nodes and mesocotyls (Fig. 4). Additionally, AChE mainly localized at extracellular space in these tissues (see the arrows in Fig. 4). This result supports our previous results, in which maize AChE localizes to and functions in the cell wall matrix.^12,13 We previously reported that AChE activity in coleoptile nodes was detected in cortical and endodermal cells surrounding the vascular system.¹⁴ In this study, AChE localized not only in endodermis, but also in vacluar bandles. Interestingly, AChE was also observed in epidermis of coleoptile nodes and mesocotyls. In our previous work,¹⁵ AChE activity was localized in epidermis of Salicornia roots. Similar evidence has been obtained in the case of human AChE.¹⁶ As additional supportive evidence, we recently cloned AChE gene from rice and analyzed AChE gene expression in a tissue-dependent manner by promoter-reporter gene assay. Rice AChE expressed in epidermis of leaves of transgenic rice plants (Yamamoto et al., unpublished results). Taken together, AChE activities in coleoptile nodes and mesocotyls enhance at extracellular space around vacluar bandles and epidermis by heat stress. Wessler et al.¹⁷ have demonstrated that the cholinergic system in Urtica dioica is involved in the regulation of water homeostasis. Therefore, enhanced AChE activity might relate to the regulation of water homeostasis during heat stress, such as prevention of epidermal transpiration and/or control of water and ionic balance through vacluar bandles.

Figure 4.

Tissue-localization of maize AChE in the coleoptile node and the mesocotyl. Immunofluorescence images of cross sections (6 μm) of the paraffin-embedded coleoptile node (A–F) and mesocotyl (G–L). (A, C, G and I) secondary antibody alone (negative control). (B, D–F, H and J–L) anti-maize AChE staining. (E, F, K and L) magnified images of the regions highlighted by dashed line boxes in (B, D, H and J) respectively. Arrowheads indicate localization of maize AChE. En, endodermis; Ph, phloem; Xy, xylem. Bars = 100 μm for (A–D and G–J); 10 μm for (E, F, K and L).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.