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. 2017 Jul 11:8:1206.
doi: 10.3389/fpls.2017.01206. eCollection 2017.

Antarctic Moss Multiprotein Bridging Factor 1c Overexpression in Arabidopsis Resulted in Enhanced Tolerance to Salt Stress

Hemasundar Alavilli  1 , Hyoungseok Lee  2 , Mira Park  1   2 , Byeong-Ha Lee  1
Affiliations

Antarctic Moss Multiprotein Bridging Factor 1c Overexpression in Arabidopsis Resulted in Enhanced Tolerance to Salt Stress

Hemasundar Alavilli et al. Front Plant Sci. .

Abstract

Polytrichastrum alpinum is one of the moss species that survives extreme conditions in the Antarctic. In order to explore the functional benefits of moss genetic resources, P. alpinum multiprotein-bridging factor 1c gene (PaMBF1c) was isolated and characterized. The deduced amino acid sequence of PaMBF1c comprises of a multiprotein-bridging factor (MBF1) domain and a helix-turn-helix (HTH) domain. PaMBF1c expression was induced by different abiotic stresses in P. alpinum, implying its roles in stress responses. We overexpressed PaMBF1c in Arabidopsis and analyzed the resulting phenotypes in comparison with wild type and/or Arabidopsis MBF1c (AtMBF1c) overexpressors. Overexpression of PaMBF1c in Arabidopsis resulted in enhanced tolerance to salt and osmotic stress, as well as to cold and heat stress. More specifically, enhanced salt tolerance was observed in PaMBF1c overexpressors in comparison to wild type but not clearly observable in AtMBF1c overexpressing lines. Thus, these results implicate the evolution of PaMBF1c under salt-enriched Antarctic soil. RNA-Seq profiling of NaCl-treated plants revealed that 10 salt-stress inducible genes were already up-regulated in PaMBF1c overexpressing plants even before NaCl treatment. Gene ontology enrichment analysis with salt up-regulated genes in each line uncovered that the terms lipid metabolic process, ion transport, and cellular amino acid biosynthetic process were significantly enriched in PaMBF1c overexpressors. Additionally, gene enrichment analysis with salt down-regulated genes in each line revealed that the enriched categories in wild type were not significantly overrepresented in PaMBF1c overexpressing lines. The up-regulation of several genes only in PaMBF1c overexpressing lines suggest that enhanced salt tolerance in PaMBF1c-OE might involve reactive oxygen species detoxification, maintenance of ATP homeostasis, and facilitation of Ca2+ signaling. Interestingly, many salt down-regulated ribosome- and translation-related genes were not down-regulated in PaMBF1c overexpressing lines under salt stress. These differentially regulated genes by PaMBF1c overexpression could contribute to the enhanced tolerance in PaMBF1c overexpressing lines under salt stress.

Keywords: Antarctic moss; MBF1c; Polytrichastrum alpinum; RNA sequencing; salt stress; stress tolerance.

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Figures

FIGURE 1
FIGURE 1
Sequence alignment and phylogenetic analysis of PaMBF1c. (A) Amino acid sequences of PaMBF1c protein (AJG41867) and homologs from Physcomitrella patens (XP_001771731), Oryza sativa (NP_001057974), Medicago truncatula (AES76734), Triticum aestivum (ACU43593), and Arabidopsis thaliana (NP_189093) were used for amino acid sequence alignment. The dotted lines below the sequence alignment indicate multiprotein bridging factor 1 (MBF1) domain and helix-turn-helix (HTH) domain. In the HTH domain, the amino acid residue glutamic acid (E) (arrow head) is conserved among all plant MBF1 proteins. (B) Phylogenetic tree of MBF1 proteins from diverse species. MBF1 proteins from mosses (Polytrichastrum alpinum; Phpat, Physcomitrella patens; Sphfalx, Sphagnum fallax; Mapoly, Marchantia polymorpha), Lycophyte (Selmo, Selaginella moellendorffii), algae (Chlre, Chlamydomonas reinhardtii; Cocsu, Coccomyxa subellipsoidea; Vocar, Volvox carteri), monocots (Zeama, Zea mays; Bradi, Brachypodium distachyon; Orysa, Oryza sativa; Sobic, Sorghum bicolor), and dicots (Arath, Arabidopsis thaliana; Vitvi, Vitis vinifera; Solyc, Solanum lycopersicum; Potri, Populus trichocarpa) were included. The phylogenetic tree was constructed using the Neighbor-Joining method.
FIGURE 2
FIGURE 2
Expression of PaMBF1c in P. alpinum under various abiotic stress conditions. The PaMBF1c expression levels were measured by quantitative real-time PCR with total RNA from P. alpinum gametophores under osmotic stress (150 mM or 300 mM mannitol for 6 h), salt stress (75 or 150 mM NaCl for 6 h) or heat stress (37 or 42°C for 2 h). The P. alpinum tubulin gene was used as an internal control for normalization. The expression level of PaMBF1c grown on normal BCD was used as a control (calibrator for quantification) and was assumed as 1. Error bars represents standard deviation of means (n = 3). Asterisks indicate statistical significance in LSD test (p < 0.05).
FIGURE 3
FIGURE 3
Growth and development of PaMBF1c-OE lines. (A) Higher accumulation of transgene transcripts in PaMBF1c-OE lines were confirmed by semi-quantitative RT-PCR. (B) The size of PaMBF1c-OE lines was bigger than WT. Pictures were taken 12 days after germination. (C) Measurement of fresh weights per seedling revealed a better growth of PaMBF1c-OE lines in comparison to WT. (D,E) PaMBF1c-OE lines bolted earlier than WT. Error bars represents standard deviation of means (n = 20). Asterisks indicate statistical significance in LSD test (p < 0.05).
FIGURE 4
FIGURE 4
Evaluation of PaMBF1c-OE lines under different abiotic stress conditions. (A) PaMBF1c-OE lines germinated better than did WT under salt stress (200 mM NaCl), ionic stress (15 mM LiCl), and osmotic stress (200 mM mannitol). The number of germinated seeds was expressed as a percentage of total number of seeds planted (n ≥ 100). Radicle emergence was considered germination and the germination was scored after 4 days of planting. (B) Roots of PaMBF1c-OE lines elongated longer than that of WT under salt stress (150 mM NaCl), ionic stress (15 mM LiCl), and osmotic stress (200 mM mannitol). Root growth was scored after 6 days of transfer of seedlings from normal medium to stress medium. Root elongation of seedlings under stress conditions was expressed as a percentage of each stress control grown on normal MS medium after 6 days of transfer (n ≥ 10). (C) PaMBF1c-OE lines survived better than WT under salt stress (200 mM NaCl), ionic stress (20 mM LiCl) and heat stress (45°C for 60 min) (n ≥ 15). Error bars represents standard deviation of the mean values of three independent experiments. Asterisks indicate statistical significance in LSD test (p < 0.05).
FIGURE 5
FIGURE 5
Chlorophyll, malondialdehyde (MDA), and anthocyanin levels in PaMBF1c-OE lines. (A,B) PaMBF1c-OE lines retained more chlorophyll content than WT at 75 and 150 mM NaCl. (C) PaMBF1c-OE lines showed lower MDA level than WT. MDA levels were examined with 21-day-old seedlings grown on MS plates with 0 or 120 mM NaCl. (D) PaMBF1c-OE lines accumulated higher amount of anthocyanin than WT. Anthocyanin content was measured with 10-day-old seedlings grown on MS plates with 0 or 150 mM NaCl. Error bars represents standard deviation of mean values of at least three independent experiments (n = 25 seedlings per each treatment). Asterisks indicate statistical significance in LSD test (p < 0.05).
FIGURE 6
FIGURE 6
Comparison of stress tolerance between PaMBF1c-OE lines and AtMBF1c-OE lines under various abiotic stress conditions. (A) Comparison of hypocotyl elongation between PaMBF1c-OE and AtMBF1c-OE lines under heat stress. Both OE lines showed similar levels of heat tolerance but higher than WT (n ≥ 10). (B) Comparison of germination between PaMBF1c-OE and AtMBF1c-OE lines under salt stress (200 mM NaCl), ionic stress (15 mM LiCl), and osmotic stress (200 mM mannitol). PaMBF1c-OE displayed higher germination ratio than WT and AtMBF1c-OE lines under salt and ionic stresses (n ≥ 100). (C) Comparison of root elongation between PaMBF1c-OE and AtMBF1c-OE lines under salt stress (100 mM NaCl), ionic stress (15 mM LiCl), and osmotic stress (200 mM mannitol). PaMBF1c-OE showed higher root elongation than AtMBF1c-OE and WT under salt and ionic stresses (n ≥ 10). Error bars represents standard deviation of the mean values of three independent experiments. Asterisks indicate statistical significance in LSD test (p < 0.05).
FIGURE 7
FIGURE 7
Transcriptome analysis of WT, PaMBF1c-OE, and AtMBF1c-OE lines. (A) Numbers of salt-regulated genes (p-value < 0.05, FDR corrected p-value < 0.05, absolute value of fold change > 1.5) in WT, PaMBF1c-OE, and AtMBF1c-OE lines. Shaded regions indicate genes commonly regulated by salt in all three lines. Numbers above the pink bars denote the total number of up-regulated genes and ones below the blue bars indicate the total number of down-regulated genes in each genotype. Numbers by the shaded regions in the middle of the bar are the total number of genes that were commonly up-/down-regulated in all variants. (B) Venn diagram showing 10 salt up-regulated genes that were already up-regulated in PaMBF1c-OE and 7 genes in AtMBF1c-OE plants under normal conditions.
FIGURE 8
FIGURE 8
Gene ontology enrichment analysis of salt regulated genes in WT, PaMBF1c-OE, and AtMBF1c-OE lines. (A) Gene Ontology (GO) terms with significance (corrected p-value of Bonferroni correction < 0.05) from the GO enrichment analysis with salt-regulated genes using Arabidopsis whole genome as a comparison reference. (B) Functional GO classification of genes salt up-regulated genes based on plant GO slim terms. (C) Functional GO classification of genes salt down-regulated genes based on plant GO slim terms. Only GO terms with significance (corrected p-value of Bonferroni correction < 0.05) from the GO enrichment analysis were presented. For suffix of each line in the graph legends, “-S” indicates “salt-treated,” “up” and “down” mean “up-regulated” and “down-regulated,” respectively. Asterisks indicate statistical significance.
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