Gamma-aminobutyric acid (GABA) and acetylcholine (ACh) alleviate water deficit effects in soybean: From gene expression up to growth performance

Highlights

• The application of ACh and GABA bioregulators in soybean promoted the better water-use efficiency (WUE) even under water déficit, hence contributing for a more stable CO₂ assimilation with less water vapor molecule lost. Maintaining transpiration was in conjunction with greater Pn leading to significant improved WUE, due to the improvement of cell function. The physiological analysis suggested that ACh:GABA could alleviate drought damages in photosynthesis by balancing water relations through improving leaf water potential, WUE and osmotic adjustment.

• The mitigation effects of ACh and GABA in soybean under water deficit was indirectly assessed by reducing the expression of P5CS1, LEA3 and ABA2 genes. However, we can not discard the possibility that the exogenous ACh application interferes in ABA biosynthesis pathway, resulting in a decrease in the expression levels of ABA2 and, therefore, lower levels of ABA content in the cells. The P5CS1 gene showed lower expression in plants that received application of the bioregulators, which resulted in lower proline accumulation in the leaves of plants, indicating that these plants were less stressed compared to control plants without application of ACh and GABA.

• Soybean plants under water supplied with the ACh:GABA solution showed an increased antioxidant enzymes activity SOD, CAT e APX, indicating its protective role against the effects of ROS, along with a reduction in oxidative damage to the membranes of organelles and cells., suggesting that ACh:GABA has a role is a key adaptation of plants at the cellular level to minimize the effects of drought stress.

• In plants treated with ACh:GABA showed remarkable responses on the growth and yield parameters.The improvement in the transport of photoassimilates, observed by the increase of photosynthetic efficiency in gas exchange analyses, resulted in better seed establishment, as observed in the production results. Grain yield was positively correlated to the net CO₂ assimilation rate. Thus, the treatment with ACh: GABA priming was effective in mitigating production losses and reductions in net CO₂ assimilation, under conditions of water deficit stress, through metabolic homeostasis. Thus, the bioregulators application in soybean has potential use in management practices in areas subject to water deficit.

Abstract

Bioregulators act as priming agents under abiotic stress conditions. Bioregulators such as gamma-aminobutyric acid (GABA) and acetylcholine (ACh) affect the efficiency of the antioxidant system and the regulation of the stomatal aperture, respectively. The aim of this study was to verify a possible synergistic effect among these bioregulators as attenuators of the effects of water deficiency in Glycine max. We combined the application of GABA and ACh at 2.0 mM in soybean plants under different water regimes. The factors studied were: 1) application of Gaba and ACh in seeds (S); in leaves (L); in seeds and leaves (SL); control without application (C); and (2) water regimes at 100 % field capacity (FC) and water deficit (WD). When the application of bioregulators were removed from the process, a severe decrease in photosynthesis capacity (93 %) was observed on the sixth day (after withholding water), as well as a higher expression of the genes known to be induced by water deficit. The combination of GABA and ACh applied to seeds and leaves under water deficit resulted in a lower decline in photosynthesis, as well as better water-use efficiency and biomass production. Soybean plants subjected to this treatment also showed lower expression of GmABA2, GmLEA3 and GmP5CS genes, lower proline content and increased activity of SOD, CAT and APX compared to the control treatment. The results indicate that the combined exogenous application of GABA and ACh in soybean plants acted to promote increased tolerance to water deficit, showing their potential for use on agricultural areas which are prone to droughts.

Introduction

Environmental constrains severely affect both plant growth and productivity (Shao et al., 2009). Water deficiency is one of the most important factors limiting crop yields and the capability to reach maximum yield potential, adversely affecting many basic physiological processes (Boyer, 1982; Seeve et al., 2017).

In plants, water deficit induces complex responses at morphological, physiological, biochemical and molecular levels (Shinozaki and Yamaguchi-Shinozaki, 2007; Park et al., 2012; George et al., 2017). Among physiological responses to water deficit, the reduction of stomata opening to decrease water loss through transpiration, is one of the first measurable effects, which in turn leads to a reduced CO₂ absorption and photosynthetic rate (Souza et al., 2013). In addition, various metabolites are accumulated that can act as osmolytes for maintaining cellular water balance, including sugars (e.g., raffinose family oligosaccharides, sucrose, trehalose and sorbitol), polyols (e.g., mannitol), amino acids (e.g., proline, glycine, betaine), proteins (e.g., chaperones) and amines (Hasanuzzaman et al. 2012; Mahmud et al., 2017).

An emerging approach that has been took into account as an attempt to increase water deficit tolerance in crops is the use of bioregulators (Srivastava et al., 2016; Wakchaure et al., 2016). Bioregulators can modify plant physiology through changes in protein, carbohydrate and lipid metabolisms (Srivastava et al., 2016), wich may result in improved crop yield even under adverse conditions (Kumar et al., 2014; Pasala et al., 2017).

Most bioregulators stimulate the redox metabolism signaling under abiotic stress conditions (Pasala et al., 2017) and may have priming effects allowing plants to be prepared to increase their tolerance to future environmental challenges. Plants subjected to priming stimuli often activate faster and stronger responses when exposed to stress compared to naïve plants (Conrath, 2009; Savvides et al., 2016; Vijayakumari and Puthur, 2016). When applied exogenously at appropriate concentrations, bioregulators such as gamma-aminobutyric acid (GABA), a non-protein amino acid that accumalate in plants in response to biotic and abiotic stress, can act in signaling cascades to increase the efficiency of the antioxidant systems, osmolyte production and expression of stress-responsive genes (Srivastava et al., 2016). The role of GABA to elicit antioxidant enzymes was observed in several species, such as Capsicum annuum under low light (Li et al., 2017), peach fruits and tomato seedlings under low temperature stress (Yang et al., 2011; Malekzadeh et al., 2014) and rice plants under hypoxia stress (Ding et al., 2016).

Other bioregulators may also impact on plant development, accelerating root and shoot growth, and improving the fruit set (Pasala et al., 2017). For example, the action of acetylcholine (ACh) promoted the root growth in Raphanus sativus (Sugiyama and Tezuka, 2011), as well as of coleoptiles in Avena sativa (Evans, 1972). Using protoplasts as a model system, it was possible to demonstrate that addition of auxin and acetylcholine leads to an increased expression of LeEXPA2, a gene involved in elongating tomato hypocotyl segments (Di-Sansebastiano et al., 2014). Additionally, it has been suggested that ACh may play an important role in regulating stomatal movement (Lou, 1998; Wang et al., 1999). It has been found that the interaction of Ca^{2 +} with calmodulin involved in the signal transduction in stomatal cells influences the response to ACh (Meng et al., 2004).

Considering the aforementioned studies on the isolated effects of GABA or ACh in enhancing plant tolerance/resistance to different abiotic stresses, in this study we examined the possible synergistic action of these bioregulators as attenuators of the harmful effects of water deficiency on Glycine max, taking into account likely effects from gene expression up to growth performance.