Transcriptomic insights into the molecular aspects of salt stress responses in Kandelia candel roots

The mangrove plant Kandelia candel is a type of woody halophyte that grows in tropical and subtropical ocean intertidal zones and exhibits a high salt tolerance. In this study, 61,970 unigenes were obtained from the roots of 60-day-old K. candel seedlings treated with 0 (control), 200, 400, and 600 mM NaCl for 3 days with an N50 of 1510 bp. Moreover, 454, 311, and 2663 genes were differentially expressed under 200, 400, and 600 mM NaCl treatments, respectively. These differentially expressed genes were primarily involved in plant hormone signal transduction, carbohydrate and energy metabolism, amino acid metabolism, stress response, and defense. The levels of 12 important differentially expressed genes were confirmed by qRT-PCR, showing that the changing trend was generally consistent with the results of the transcriptomic analysis. In addition, physiological parameters involved in energy metabolism, amino acid metabolism, and the reactive oxygen species scavenging process were significantly increased under salt stress treatment, and the trend was consistent with the results of transcription and qRT-PCR. This study indicated that K. candel roots could tolerate high salt stress by enhancing ethylene signal transduction, maintaining a stable energy supply, increasing antioxidant capacity. Specially, we found that accumulation of ?-aminobutyric acid and glutamate, but not proline, might play an important role in salt tolerance in the roots of K. candel.

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  • Achard P, Cheng H, De Grauwe L, Decat J, Schoutteten H et al. (2006). Integration of plant responses to environmentally activated phytohormonal signals. Science 311: 91-94. doi: 10.1126/science.1118642
  • Akçay N, Bor M, Karabudak T, Ozdemir F, Türkan I (2012). Contribution of Gamma amino butyric acid (GABA) to salt stress responses of Nicotiana sylvestris CMSII mutant and wild type plants. Journal of Plant Physiology 169: 452-458. doi: 10.1016/j.jplph.2011.11.006
  • Alscher R G, Erturk N, Heath L S (2002). Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. Journal of Experimental Botany 53: 1331-1341. doi: 10.1093/ jxb/53.372.1331
  • Apweiler R, Bairoch A, Wu CH, Barker WC, Boeckmann B, Ferro S, Gasteiger E, Huang H, Lopez R, Magrane M (2004). UniProt: the universal protein knowledgebase. Nucleic Acids Research 32: D115-9. doi: 10.1093/nar/gkh131
  • Bates LS, Waldren RP, Teare ID (1973). Rapid determination of free proline for water-stress studies. Plant and Soil 39: 205-207. doi: 10.1007/BF00018060
  • Bleecker AB, Kende H (2000). Ethylene: a gaseous signal molecule in plants. Annual Review of Cell and Developmental Biology 16: 1-18. doi: 10.1146/annurev.cellbio.16.1.1
  • Campo S, Baldrich P, Messeguer J, Lalanne E, Coca M et al. (2014). Overexpression of a calcium-dependent protein kinase confers salt and drought tolerance in rice by preventing membrane lipid peroxidation. Plant Physiology 165: 688-704. doi: 10.1104/pp.113.230268
  • Cao WH, Liu J, Zhou QY, Cao YR, Zheng SF et al. (2006). Expression of tobacco ethylene receptor NTHK1 alters plant responses to salt stress. Plant, Cell & Environment 29: 1210-1219. doi: 10.1111/j.1365-3040.2006.01501.x
  • Colebrook EH, Thomas SG, Phillips AL, Hedden P (2014). The role of gibberellin signalling in plant responses to abiotic stress. Journal of Experimental Biology 217: 67-75. doi: 10.1242/ jeb.089938
  • Cram J W, Torr P G, Rose D A (2002). Salt allocation during leaf development and leaf fall in mangroves. Trees 16: 112-119. doi: 10.1007/s00468-001-0153-3
  • Deng Y, Li J, Wu S, Zhu Y, Chen Y et al. (2006). Integrated nr database in protein annotation system and its localization. Computer Engineering 32: 71-77 (in Chinese with an abstract in English). doi: 10.1109/INFOCOM.2006.241
  • Dubouzet J G, Sakuma Y, Ito Y, Kasuga M, Dubouzet E G et al. (2003). OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought, high salt and cold responsive gene expression. The Plant Journal 33: 751- 763. doi: 10.1046/j.1365-313X.2003.01661.x
  • Forde BG, Lea PJ (2007). Glutamate in plants: metabolism, regulation, and signaling. Journal of Experimental Botany 58: 2339-2358. doi: 10.1093/jxb/erm121
  • Gill SS, Tuteja N (2010). Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry 48: 909-930. doi: 10.1016/j. plaphy.2010.08.016
  • Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson, DA et al. (2011). Trinity: reconstructing a full-length transcriptome without a genome from RNA-Seq data. Nature Biotechnology 29: 644-652. doi: 10.1038/nbt.1883
  • Guo H, Ecker JR (2004). The ethylene signaling pathway: new insights. Current Opinion in Plant Biology 7: 40-49. doi: 10.1016/j.pbi.2003.11.011
  • Guo W, Wu H, Zhang Z, Yang C, Hu L et al. (2017). Comparative analysis of transcriptomes in Rhizophoraceae provides insights into the origin and adaptive evolution of mangrove plants in intertidal environments. Frontiers in Plant Science 8: 795. doi: 10.3389/fpls.2017.00795
  • Guo Y, Huang C, Xie Y, Song F, Zhou X (2010). A tomato glutaredoxin gene SlGRX1 regulates plant responses to oxidative, drought and salt stresses. Planta 232: 1499-1509. doi: 10.1007/s00425- 010-1271-1
  • Guri ASAF (1983). Variation in glutathione and ascorbic acid content among selected cultivars of Phaseolus vulgaris prior to and after exposure to ozone. Revue Canadienne De Phytotechnie 63: 10966-10974. doi: 10.4141/cjps83-090
  • Halliwell B, Foyer CH (1978). Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography. Planta 139: 9-17. doi: 10.2307/23373245
  • Holmgren F, Alkhalfioui F, Yano H, Vensel W H, Hurkman W J et al. (2009). Thioredoxintargets in plants: the first 30 years. Journal of Proteomics 72: 452-474. doi: 10.1016/j.jprot.2008.12.002
  • Huang Y, Li H, Gupta R, Morris P C, Luan S et al. (2000). AtMPK4, an Arabidopsis homolog of mitogen-activated protein kinase, is activated in vitro by AtMEK1 through threonine phosphorylation. Plant Physiology 122: 1301-1310. doi: 10.1104/pp.122.4.1301
  • Hwang I, Chen HC, Sheen J (2002). Two-component signal transduction pathways in Arabidopsis. Plant Physiology 129: 500-515. doi: 10.1104/pp.005504
  • Hyland K, Voisin E, Banoun H, Auclair C (1983). Superoxide dismutase assay using alkaline dimethylsulfoxide as superoxide anion-generating system, Analytical Biochemistry 135: 280- 287. doi: 10.1016/0003-2697(83)90684-X
  • Jordon-Thaden IE, Chanderbali AS, Gitzendanner MA, Soltis DE (2015). Modified CTAB and TRIzol protocols improve RNA extraction from chemically complex Embryophyta. Application in Plant Science 3: a1400105. doi: 10.3732/apps.1400105
  • Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M et al. (2008). KEGG for linking genomes to life and the environment. Nucleic Acids Research 36: 480-484. doi: 10.1093/nar/gkm882
  • Kekulandara DN, Nagi S, Seo H, Chow CS, Ahn YH (2018). Redoxinactive peptide disrupting trx1–ask1 interaction for selective activation of stress signaling, Biochemistry 57: 772-780. doi: 10.1021/acs.biochem.7b01083
  • Kiegerl S, Cardinale F, Siligan C, Gross A, Baudouin E et al. (2000). SIMKK, a mitogen-activated protein kinase (MAPK) kinase, is a specific activator of the salt stress–induced MAPK, SIMK. The Plant Cell 12: 2247-2258. doi: 10.2307/3871118
  • Krishnamurthy P, Mohanty B, Wijaya E (2017). Transcriptomics analysis of salt stress tolerance in the roots of the mangrove Avicennia officinalis. Scientific Reports 7: 10031. doi: 10.1038/ s41598-017-10730-2
  • Law M Y, Charles S A, Halliwell B (1983). Glutathione and ascorbic acid in spinach (Spinacia oleracea) chloroplasts. The effect of hydrogen peroxide and of paraquat. Biochemical Journal 210: 899-903. doi: 10.1042/bj2100899
  • Lei G, Shen M, Li ZG, Zhang B, Duan KX et al. (2011). EIN2 regulates salt stress response and interacts with a MA3 domain-containing protein ECIP1 in Arabidopsis. Plant, Cell & Environment 34: 1678-1692. doi: 10.1111/j.1365- 3040.2011.02363.x
  • Liang S, Fang L, Zhou R, Tang T, Deng S et al. (2012). Transcriptional homeostasis of a mangrove species, Ceriops tagal, in saline environments, as revealed by microarray analysis. PloS ONE 7: e36499. doi: 10.1371/journal.pone.0036499
  • Livak KJ, Schmittgen TD (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCTmethod. Methods 25: 402-408. doi: 10.1006/meth.2001.1262
  • Lü XM, Yang YF, Lu XY, Jin J, Fan XM (2016). Effects of NaCl stress on the AsA-GSH cycle in sour jujube seedlings. Plant Physiology Journal 52: 736-744(in Chinese with an abstract in English). doi: 10.13592/j.cnki.ppj.2015.0706
  • Mishra A, Tanna B (2017). Halophytes: potential resources for salt stress tolerance genes and promoters. Frontiers in Plant Science 8: 829. doi: 10.3389/fpls.2017.00829
  • Mittler R (2002). Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science 7: 405-410. doi: 10.1016/S1360- 1385(02)02312-9
  • Miyama M, Tada Y (2008). Transcriptional and physiological study of the response of Burma mangrove (Bruguiera gymnorhiza) to salt and osmotic stress. Plant Molecular Biology 68: 119-129. doi: 10.1007/s11103-008-9356-y
  • Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008). Mapping and quantifying mammalian transcriptomes by RNASeq. Nature Methods 5: 621-628. doi: 10.1038/nmeth.1226 Munns R (2002). Comparative physiology of salt and water stress. Plant, Cell & Environment 25: 239-250. doi: 10.1046/j.0016- 8025.2001.00808.x
  • Nakano Y, Asada K (1981). Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant and Cell Physiology 22: 867-880. doi: 10.1093/oxfordjournals.pcp. a076232
  • Patterson BD, MacRae EA, Ferguson IB (1984a). Estimation of hydrogen peroxide in plant extracts using titanium (IV). Analytical Biochemistry 139: 487-492. doi: 10.1016/0003- 2697(84)90039-3
  • Patterson BD, Payne LA, Chen YZ, Graham D (1984b). An inhibitor of catalase induced by cold in chilling-sensitive plants. Plant Physiology 76: 1014-1018. doi: 10.2307/4269048
  • Rouhier N, Gelhaye E, Jacquot JP (2002). Exploring the active site of plant glutaredoxin by site-directed mutagenesis. FEBS Letters 511: 145-149. doi: 10.1016/s0014-5793(01)03302-6
  • Roy S J, Negrão S, Tester M (2014). Salt resistant crop plants. Current Opinion in Biotechnology 26: 115-124. doi: 10.1016/j. copbio.2013.12.004
  • Saradhi PP, Mohanty P (1993). Proline in relation to free radical production in seedlings of Brassica juncea raised under sodium chloride stress. Plant Soil 155: 497-500. doi: 10.1007/bf00025092
  • Seki M. Narusaka M, Ishida J, Nanjo T, Fujita M (2002). Monitoring the expression profles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using afull-length cDNA microarray. Plant Journal 31: 279-292. doi: 10.1046/j.1365- 313X.2002.01359.x
  • Sharma R, Priya P, Jain M (2013). Modified expression of an auxinresponsive rice CC-type glutaredoxin gene affects multiple abiotic stress responses. Planta 238: 871-884. doi: 10.1007/ s00425-013-1940-y
  • Szabados L, Savouré A (2010). Proline: a multifunctional amino acid. Trends in Plant Science 15: 89-97. doi: 10.1016/j. tplants.2009.11.009
  • Tatusov RL, Galperin MY, Natale DA, Koonin EV. (2000). The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Research 28: 33-36. doi: 10.1093/ nar/28.1.33
  • Trapnell C, Pachter L, Salzberg SL (2009). TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25: 1105-1111. doi: 10.1093/bioinformatics/btp120
  • Walia H, Wilson C, Zeng L, Ismail AM, Condamine P et al. (2007). Genome-wide transcriptional analysis of salinity stressed japonica and indica rice genotypes during panicle initiation stage. Plant Molecular Biology 63: 609-623. doi: 10.1007/ s11103-006-9112-0
  • Wang AG, Luo GH (1990). Quantitative relation between the reaction of hydroxylamine and superoxide anion radicals in plants. Plant Physiology Communications 84: 2895-2898. doi: 10.1021/ja00874a010 (in Chinese with an abstract in English)
  • Wang L, Pan D, Lv X, Cheng C-L, Li J et al. (2016). A multilevel investigation to discover why Kandelia candel thrives in high salinity. Plant, Cell & Environment 39: 2486-2497. doi: 10.1111/pce.12804
  • Wang L, Pan D, Li J, Tan F, Hoffmann-Benning S et al. (2015). Proteomic analysis of changes in the Kandelia candel chloroplast proteins reveals pathways associated with salt tolerance. Plant Science 231: 159-172. doi: 10.1016/j.plantsci.2014.11.013
  • Wang NN, Shih M C, Li N (2005). The GUS reporter-aided analysis of the promoter activities of Arabidopsis ACC synthase genes AtACS4, AtACS5, and AtACS7 induced by hormones and stresses. Journal of Experimental Botany 56: 909-920. doi: 10.1093/jxb/eri083
  • Yang F, Tan H, Zhou Y, Lin X, Zhang S. (2011). High-Quality RNA Preparation from Rhodosporidium toruloides and cDNA Library Construction Therewith. Molecular Biotechnology, 47: 144-151. doi: 10.1007/s12033-010-9322-1
  • Yao D, Zhang X, Zhao X, Liu C, Wang C et al. (2011). Transcriptome analysis reveals salt-stress-regulated biological processes and key pathways in roots of cotton (Gossypium hirsutum L.). Genomics 98: 47-55. doi: 10.1016/j.ygeno.2011.04.007
  • Yasumoto E, Adachi K, Kato M, Sano H, Sasamoto H et al. (1999). Uptake of inorganic ions and compatible solutes in cultured mangrove cells during salt stress. In Vitro Cellular & Developmental Biology-Plant 35: 82-85. doi: 10.2307/4293165
  • Ye J, Fang L, Zheng H, Zhang Y, Chen J et al. (2006). WEGO: a web tool for plotting GO annotations. Nucleic Acids Research 34: 293-297. doi: 10.1093/nar/gkl031
  • Yu J , Chen S , Zhao Q , Wang T, Yang C et al. (2011). Physiological and proteomic analysis of salinity tolerance in Puccinellia tenuiflora. Journal of Proteome Research 10: 3852-3870. doi: 10.1021/pr101102p