Abstract
Abiotic stresses cause accumulation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) in plants. Sophisticated mechanisms are required to maintain optimum level of H2O2 that acts as signalling molecule regulating adaptive response to salt stress. CuZn-superoxide dismutase (CuZn-SOD) and ascorbate peroxidase (APX) constitute first line of defence against oxidative stress. In the present study, PaSOD and RaAPX genes from Potentilla atrosanguinea and Rheum australe, respectively were overexpressed individually as well as in combination in Arabidopsis thaliana. Interestingly, PaSOD and dual transgenic lines exhibit enhanced lignin deposition in their vascular bundles with altered S:G ratio under salt stress. RNA-seq analysis revealed that expression of PaSOD gene in single and dual transgenics positively regulates expression of lignin biosynthesis genes and transcription factors (NACs, MYBs, C3Hs and WRKY), leading to enhanced and ectopic deposition of lignin in vascular tissues with larger xylem fibres and alters S:G ratio, as well. In addition, transgenic plants exhibit growth promotion, higher biomass production and increased yield under salt stress as compared to wild type plants. Our results suggest that in dual transgenics, ROS generated during salt stress gets converted into H2O2 by SOD and its optimum level was maintained by APX. This basal level of H2O2 acts as messenger for transcriptional activation of lignin biosynthesis in vascular tissue, which provides mechanical strength to plants. These findings reveal an important role of PaSOD and RaAPX in enhancing salt tolerance of transgenic Arabidopsis via increased accumulation of compatible solutes and by regulating lignin biosynthesis.
Similar content being viewed by others
References
Adler E, Bjorkquist KJ, Haggroth S (1948) Über die Ursache der Farbreaktionen des Holzes. Acta Chem Scand 2:93–94
Akin DE, Morrison WH, Himmelsbach DS (1993) Characterization of digestion residues of alfalfa and orchardgrass leaves by microscopic, spectroscopic and chemical analysis. J Sci Food Agric 63:339–347
Almagro L, Ros LVG, Bekchi-Navarro S, Bru R, Barcelo AR, Predeno MA (2009) Class III peroxidases in plant defence reactions. J Exp Bot 60:377–390
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Aoyama W, Matsumura A, Tsutsumi Y, Nishida T (2001) Lignification and peroxidase in tension wood of Eucalyptus viminalis seedlings. J Wood Sci 47:419–424
Asada K (1999) The water–water cycle in chloroplasts: scavenging of active oxygen and dissipation of excess photons. Annu Rev Plant Physiol Plant Mol Biol 50:601–639
Barrs HD, Weatherley PE (1962) A re-examination of the relative turgidity technique for estimating water deficits in leaves. Aust J Biol Sci 15:413–428
Bates L, Waldren R, Teare ID (1973) Rapid determination of free proline for water–stress studies. Plant Soil 39:205–207
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Czechowski T, Stitt M, Altman T, Udvardi MK, Scheible WR (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17
Desikan R, Clarke A, Hancock JT, Neill SJ (1999) H2O2 activates a MAP kinase-like enzyme in Arabidopsis thaliana suspension cultures. J Exp Bot 50:1863–1866
Fujita M, Fujita Y, Noutoshi Y, Takahashi F, Narusaka Y, Yamaguchi-Shinozaki K, Shinozaki K (2006) Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signalling networks. Curr Opin Plant Biol 9:436–442
Gahlan P, Singh HR, Shankar R, Sharma N, Kumari A, Chawla V, Ahuja PS, Kumar S (2012) De novo sequencing and characterization of Picrorhiza kurrooa transcriptome at two temperatures showed major transcriptome adjustments. BMC Genom 13:126
Gechev TS, Hille J (2005) Hydrogen peroxide as a signal controlling plant programmed cell death. J Cell Biol 168:17–20
Ghawana S, Paul A, Kumar H, Kumar A, Singh H, Bhardwaj PK, Rani A, Singh RS, Raizada J, Singh K, Kumar S (2007) An RNA isolation system for plant tissues rich in secondary metabolites. BMC Res Notes 4:85
Gill T, Kumar S, Ahuja PS, Sreenivasulu Y (2010a) Over-expression of Potentilla superoxide dismutase improves salt stress tolerance during germination and growth in Arabidopsis thaliana. J Plant Genet Transgenics 1:1–10
Gill T, Sreenivasulu Y, Kumar S, Ahuja PS (2010b) Over-expression of superoxide dismutase exhibits lignification of vascular structures in Arabidopsis thaliana. J Plant Physiol 167:757–760
Hauffe KD, Paszkowski U, Schulze-Lefert P, Hahlbrock K, Dangl JL, Douglas CJ (1991) A parsley 4CL-1 promoter fragment specifies complex expression patterns in transgenic tobacco. Plant Cell 3:435–443
Hilal M, Zenoff AM, Ponessa G, Moreno H, Massa ED (1998) Saline stress alters the temporal patterns of xylem differentiation and alternative oxidase expression in developing soybean roots. Plant Physiol 117:695–701
Irigoyen JJ, Emerich DW, Sanchez-Diaz M (1992) Water stress induced changes in concentrations of proline and total soluble sugars in nodulated alfalfa (Medicago sativa) plants. Physiol Plant 84:55–60
Jbir N, Chaïbi W, Ammar S, Jemmali A, Ayadi A (2001) Root growth and lignification of two wheat species differing in their sensitivity to NaCl, in response to salt stress. Compt Rendus Acad Sci III Sci Vie 324:863–868
Karahara I, Ikeda A, Kondo T, Uetake Y (2004) Development of the Casparian strip in primary roots of maize under salt stress. Planta 219:41–47
Karpinska B, Karlsson M, Schinkel H, Streller S, Suss KH, Melzer M, Wingsle G (2001) A novel superoxide dismutase with a high isoelectric point in higher plants: expression, regulation and protein localisation. Plant Physiol 126:1668–1677
Kim HJ, Barbara T (2008) Involvement of extracellular Cu/Zn superoxide dismutase in cotton fiber primary and secondary cell wall biosynthesis. Plant Signal Behav 3:1119–1121
Kirk TK, Obst JR (1988) Lignin Determination. In: Wood WA, Kellogg ST (eds) Methods in enzymology, volume 161, biomass, Part B: lignin, pectin, and chitin. Academic Press Inc, New York, pp 87–101
Koster KL, Lynch DV (1992) Solute accumulation and compartmentation during the cold acclimation of puma rye. Plant Physiol 98:108–113
Kovtun Y, Chiu WL, Tena G, Sheen J (2000) Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc Natl Acad Sci USA 97:2940–2945
Kubo M, Udagawa M, Nishikubo N, Horiguchi G, Yamaguchi M, Ito J, Mimura T, Fukuda H, Demura T (2005) Transcription switches for protoxylem and metaxylem vessel formation. Genes Dev 19:1855–1860
Kumar S, Sahoo R, Ahuja PS (2002) Isozyme of autoclavable superoxide dismutase (SOD), a process for the identification and extraction of the SOD in cosmetic, food and pharmaceutical compositions. US Patent No. 6,485950 B1
Levya A, Liang X, Pintor-Toro JA, Dixon RA, Lamb CJ (1992) cis-Element combinations determine phenylalanine ammonia-lyase gene tissue-specific expression patterns. Plant Cell 4:263–271
Lewis NG, Yamamoto E (1990) Lignin: occurrence, biogenesis, and biodegradation. Annu Rev Plant Physiol Plant Mol Biol 41:455–496
Liyama K, Wallis PJ (1990) Determination of lignins in herbaceous plants by improved acetylbromide procedure. J Sci Food Agric 51:145–161
Lutts S, Kinet JM, Bouharmont J (1996) NaCl-induced senescence in leaves of rice (Oryza sativa L.) cultivars differing in salinity resistance. Ann Bot 78:389–398
Miller G, Shulaev V, Mittler R (2008) Reactive oxygen signalling and abiotic stress. Physiol Plantarum 133:481–489
Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R (2010) Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant Cell Environ 33:453–467
Mitsuda N, Seki M, Shinozaki K, Ohme-Takagi M (2005) The NAC transcription factors NST1 and NST2 of Arabidopsis regulate secondary wall thickenings and are required for anther dehiscence. Plant Cell 17:2993–3006
Mitsuda N, Iwase A, Yamamoto H, Yoshida M, Seki M, Shinozaki K, Ohme-Takagi M (2007) NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis. Plant Cell 19:270–280
Moura JCMS, Bonine CAV, Viana JOF, Dornelas MC, Mazzafera P (2010) Abiotic and biotic stresses and changes in the lignin content and composition in plants. J Integr Plant Biol 52:360–376
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plantarum 15:473–497
Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880
Neill SJ, Desikan R, Hancock J (2002) Hydrogen peroxide signalling. Curr Opin Plant Biol 5:388–395
Ogawa K, Kanematsu S, Asada K (1996) Intra- and extra-cellular localization of “cytosolic” CuZn-superoxide dismutase in spinach leaf and hypocotyl. Plant Cell Physiol 37:790–799
Ogawa K, Kanematsu S, Asada K (1997) Generation of superoxide anion and localization of CuZn-superoxide dismutase in the vascular tissue of spinach hypocotyls: their association with lignification. Plant Cell Physiol 38:1118–1126
Olson PD, Varner JE (1993) Hydrogen peroxide and lignification. Plant J 4:887–892
Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:36
Potikha TS, Collins CC, Johnson DI, Delmer DP, Levine A (1999) The involvement of hydrogen peroxide in the differentiation of secondary walls in cotton fibers. Plant Physiol 119:849–858
Quiroga M, Guerrero C, Botella MA, Barcelo A, Amaya I, Medina MI, Alonso FJ, de Forchetti SM, Tigier H, Valpuesta V (2000) A tomato peroxiase involved in the synthesis of lignin and suberin. Plant Physiol 122:1119–1127
Ros-Barceló A (1995) Peroxidase and not laccase is the enzyme responsible for cell wall lignification in the secondary thickening of xylem vessels in Lupinus. Protoplasma 186:41–44
Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386
Ruth GA, Neval E, Lenwood SH (2002) Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J Exp Bot 372:1331–1341
Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor laboratory Press, New York
Sánchez-Aguayo I, Rodrigues-Galán JM, Garcia R, Torreblanca J, Pardo JM (2004) Salt stress enhances xylem development and expression of S-adenosyl-l-methionine synthase in lignifying tissues of tomato plants. Planta 220:278–285
Schmid R, Blaxter ML (2008) annot8r: GO, EC and KEGG annotation of EST datasets. BMC Bioinformatics 9:180
Sonja V, Noctor G, Foyer CH (2002) Are leaf hydrogen peroxide concentrations commonly overestimated? The potential influence of artefactual interference by tissue phenolics and ascorbate. Plant Physiol Biochem 40:501–507
Tang L, Kwon SY, Kim SY, Kim JS, Choi JS, Kwang YC, Sung CK, Kwak SS, Lee HS (2006) Enhanced tolerance of transgenic potato plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against oxidative stress and high temperature. Plant Cell Rep 25:1380–1386
Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7:562–578
Vierling E, Kimpel JA (1992) Plant responses to environmental stress. Curr Opin Biotechnol 3:164–170
Zhong R, Ye ZH (2007) Regulation of cell wall biosynthesis. Curr Opin Plant Biol 10:564–572
Zhong R, Demura T, Ye ZH (2006) SND1, a NAC domain transcription factor, is a key regulator of secondary wall synthesis in fibers of Arabidopsis. Plant Cell 18:3158–3170
Zhong R, Lee C, Zhou J, McCarthy RL, Ye ZH (2008) A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. Plant Cell 20:2763–2782
Zhou J, Lee C, Zhong R, Ye ZH (2009) MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis. Plant cell 121:248–266
Zhu JK (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53:247–273
Acknowledgments
This work was supported by Grants from the Council of Scientific and Industrial Research (CSIR), New Delhi, India under CSIR Network Projects: SIMPLE (BSC0109) and PlaGen (BSC0107) and Indo-German Science and Technology Centre (IGSTC), India. A.S. and T.G. acknowledge fellowships awarded by the CSIR, India. Mr. Sanjoy Chanda is acknowledged for technical help in confocal microscopy. We thank Dr. Ivelin Pantchev, Associate Professor, Sofia University, Sofia, Bulgaria for critically reading the manuscript. This paper represents CSIR-IHBT communication number 3567.
Author information
Authors and Affiliations
Corresponding authors
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Shafi, A., Chauhan, R., Gill, T. et al. Expression of SOD and APX genes positively regulates secondary cell wall biosynthesis and promotes plant growth and yield in Arabidopsis under salt stress. Plant Mol Biol 87, 615–631 (2015). https://doi.org/10.1007/s11103-015-0301-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11103-015-0301-6