Functional characterization of Mitogen-Activated Protein Kinase Kinase (MAPKK) gene in Halophytic Salicornia europaea against salt stress

Highlights

• Functional characterization of 1023bp SeMAPKK gene was done from Salicornia europaea.

• We identified TVY and MEY as well as LKY, CAY and SKY motifs at both termini.

• Phylogenetic relationship revealed that SeMAPKK belonged to group D.

• Overexpression of SeMAPKK gene conferred to salt tolerance, here, down-regulation was observed at 1.0 M salt concentration.

• Transgenic plants were more tolerant, along with better growth than non-transgenic plants.

Abstract

Mitogen-activated protein kinase (MAPK) cascade is conserved in eukaryotic organisms and plays a crucial role in signal transduction pathways, related to biotic and abiotic stress responses. The current study was aimed to target at cloning, structural and functional characterization of 1023bp SeMAPKK gene from Salicornia europaea. A SeMAPKK polypeptide chain of 340aa has eleven sub-domains, and S/TXXXXXS/T residues are conserved in the activation loop site between VII-VIII sub-domains. We identified TVY and MEY motifs at N-terminus and LKY, CAY and SKY motifs at C-terminus. Phylogenetic relationship revealed that SeMAPKK belongs to group D. Highest transcript signals were recorded in 0.75 M salt level stress, and down-regulation was observed in 1.0 M salt concentration. Overexpression of SeMAPKK gene in Arabidopsis conferred salt tolerance. Results of different salt tolerance assays revealed that transgenic plants were more tolerant and exhibited better growth than non-transgenic plants under salt stress.

Introduction

Plants are directly influenced by abiotic and biotic stresses due to a sessile lifestyle. Plants have developed intricate mechanisms for perception of external stimuli resulting in the initiation of responses at the cellular, biochemical, physiological and molecular levels for adaptation to the changing environment (Zaynab et al., 2017). To withstand adverse environmental constraints, the perception of stress and transmission of signals are essential for triggering the adaptive responses. Mitogen-Activated Protein Kinase (MAPK) cascades have significant importance for decoding external stimuli into adaptive responses and signal transduction in plants (Teige et al., 2004). In plants, the versatile MAPK cascade involved in the growth and development, as well as responses to environmental stresses [reviewed in (Cristina et al., 2010; Ligterink and Hirt, 2001)]. MAPK cascade is a universal downstream three-kinase module conserved in all eukaryotes. The protein kinases module is constituted by MAPKKK (MAPK kinase kinase), MAPKKs (MAPK kinase) and MAPKs, which are functionally interlinked. A MAPKKK phosphorylates MAPKK which successively phosphorylates a MAPK. For induction of cellular responses, the stimulated MAPK is often transported to the nucleus for phosphorylation and activation of downstream signaling apparatus, like transcription factors (Wimalasekera and Scherer, 2018; Xiong and Yang, 2003).

The MAPKKs are triggered by MAPKKKs through phosphorylation on serine/threonine residues, conserved S/TxxxxxS/T in plants (Wang et al., 2015; Yang et al., 2001). The activated MAPK involved in the phosphorylation of transcription factors and other signaling machinery, which are crucial for the regulation of downstream genes expression (Colcombet and Hirt, 2008).

By wide-genome identification of various plants, numerous genes encoding proteins of MAPK cascade have been identified. In Arabidopsis, 80 MAPKKK, 10 MAPKK and 20 MAPK have been recorded (Benhamman et al., 2017; Ichimura et al., 2002; Xu and Zhang, 2015), while rice genome holds 75 MAPKKK, 8 MAPKK and 17 MAPK (Liu et al., 2019; Rao et al., 2010; Rohila and Yang, 2007). Likewise, 74 MAPKKK, 9 MAPKK and 9 MAPK genes have been identified from the maize genome (Kong et al., 2013). Recently, Wang et al. (2015) as well as Singh et al. (2018) identified 59 MAPKKK, 6 MAPKK and more than 14 MAPK genes from cucumber and chickpea genomes.

Numerous studies have been conducted on MAPKK gene isolation from various plants and its characterization to improve signaling pathways for biotic and abiotic stress tolerance (Wang et al., 2017). Recently MAPKK gene was isolated from Salicornia brachiata, and its expression pattern in abiotic stresses was determined (Agarwal et al., 2010), but its structural and functional characterization remained obscure. Therefore, the current research work was conducted to explore the molecular traits of leafless succulent S. europaea plant, like isolation of MAPKK gene; MAPKK expression pattern; structural features of MAPKK; the potential of MAPKK against salt stress tolerance and to unravel the phylogenetic association with other orthologs.