Dissecting the potential role of BAX Inhibitor 1 in salt tolerance using bioinformatics and experimental tools

Salinity stress is one of the most important factors causing yield loss in crop worldwide. In order to improve salt tolerance in crop, it is important to understand salt-tolerance mechanism. Ongoing researches have been directed toward understanding the effects of salt stress, with the eventual goal of discovering molecular and cellular mechanisms used by stress-tolerant species and the elements that might contribute to enhanced salt tolerance to sensitive plants. An active process of cellular suicide termed programmed cell death (PCD) is crucial for development and immune responses in eukaryotes. In plants, PCD is involved in plant development and survival. Recent studies have revealed that diverse environmental stresses, such as salt stress, nutrient starvation and drought, are able to induce PCD in plant root tips. This findings indicate that this active process is highly conserved and has vital roles in development and response to external stimuli. PCD plays an important role in adapting to environmental stress. Understanding the molecular basis of PCD mechanism makes possible genetically manipulation of plants to improve environmental stress tolerance. BAX inhibitor-1 (BI-1) located in the Endoplasmic reticulum(ER) was found to be a key cell death attenuator in eukaryotes.

In this study, the potential role of a gene which encodes BAX Inhibitor 1-like protein (BI_85) in salt tolerance was evaluated using bioinformatics and experimental approaches such as promoter and gene regulatory network analysis, as well as Real-Time PCR. Two salt-tolerant (Arg) and salt-sensitive (Alamut) cultivated wheat genotypes and Aegilops crassa, as a wild wheat relative, were materials used in this experiment. Seeds imbibing in the dark for 24h at 4°c germinated for 3d at 25°C and were grown hydroponically in half-strength Hoagland solution circulated by air pumps in a stabilized greenhouse at 25oC, with a 16h light/8h dark photoperiod. To distinguish salt stress response from developmental changes in gene expression, an experiment was designed to monitor changes in transcripts in the absence of stress. Three-week-old seedlings were treated with a 0 and 150-mM NaCl solutions in combination with Hoagland solution. Sampling was carried out after treatment at 0h, 12h, and 3w. RNA extraction (Denazist, Mashhad, Iran, S-1020-1) and cDNA synthesis (Fermentas, Ontario, Canada, EP0441) were carried out for real-time RT-PCR according to the manufacturer’s instruction. Normalization of the target gene (BI_85) was carried out based on Actin reference gene. The Pfaffl formula (ratio=2-ΔΔCt) was used to calculate relative expression. Building a network using Pathway studio software was carried to discover another components that have relationships with differentially expression gene (BI-1), which probably are involved in stress-related responses.

BAX Inhibitor was shown to be part of an interaction network that included 26 relationships. For example TED4 which has a relationship with BAX Inhibitor like-1 is implicated in salt acclimation signaling. Some evidence has been offered for the hypothesis that BI-1 probably can be used as a pore or ion channel in the endoplasmic reticulum for calcium handling. The Salt Overly Sensitive (SOS) signaling pathway is a well-recognized signaling pathway known to be essential for acquisition of ion homeostasis. In response to salt stress, a calcium signal activates the SOS pathway by binding to the calcium binding proteins, SOS3 and SCaBP8/CBL10, which in turn activate the protein kinase protein kinase SOS2 to regulate the plasma membrane Na+/H+ antiporter SOS1. According to the regulatory network, this gene may act upstream of the SOS signaling pathway. Promoter analysis were applied using plantcare database to shed light on underlying regulatory mechanism of the BI_85 expression. According to promoter analysis, the presence of stress-responsive regulatory elements such as ABRE (abscisic acid responsive element), LTR (low-temperature responsive element), MBS (MYB binding site involved in drought-inducibility), CGTCA-motif (MeJA- responsive element), TGACG-motif (MeJA- responsive element), ERE (ethylene-responsive element), and GT-1 motif (salt responsive element) in the promoter confirms the role of this gene in environmental stresses tolerance including salinity. It was also figured out that the expression patterns of BI_85 was significantly different between susceptible and salt resistant cultivars in response to salt stress. In more details, after 12h, salt stress induced BI-1 expression in the shoots of Arg and roots of Ae. crassa and reduced it in shoots of Alamut. After 3 weeks, salt stress induced BI-1 expression in the shoots of Arg and reduced it in shoots of Alamut and Ae. crassa.

According to bioinformatics and experimental results, it can be concluded that BI_85 can contribute to salt tolerance in wheat and can be used for genetic manipulation to improve tolerance to stress.