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Molecular Biology Reports

, Volume 46, Issue 2, pp 1985–2002 | Cite as

Ectopic expression of SOD and APX genes in Arabidopsis alters metabolic pools and genes related to secondary cell wall cellulose biosynthesis and improve salt tolerance

  • Amrina ShafiEmail author
  • Tejpal Gill
  • Insha Zahoor
  • Paramvir Singh Ahuja
  • Yelam Sreenivasulu
  • Sanjay Kumar
  • Anil Kumar SinghEmail author
Original Article

Abstract

Hydrogen peroxide (H2O2) is known to accumulate in plants during abiotic stress conditions and also acts as a signalling molecule. In this study, Arabidopsis thaliana transgenics overexpressing cytosolic CuZn-superoxide dismutase (PaSOD) from poly-extremophile high-altitude Himalayan plant Potentilla atrosanguinea, cytosolic ascorbate peroxidase (RaAPX) from Rheum australe and dual transgenics overexpressing both the genes were developed and analyzed under salt stress. In comparison to wild-type (WT) or single transgenics, the performance of dual transgenics under salt stress was better with higher biomass accumulation and cellulose content. We identified genes involved in cell wall biosynthesis, including nine cellulose synthases (CesA), seven cellulose synthase-like proteins together with other wall-related genes. RNA-seq analysis and qPCR revealed differential regulation of genes (CesA 4, 7 and 8) and transcription factors (MYB46 and 83) involved in secondary cell wall cellulose biosynthesis, amongst which most of the cellulose biosynthesis gene showed upregulation in single (PaSOD line) and dual transgenics at 100 mM salt stress. A positive correlation between cellulose content and H2O2 accumulation was observed in these transgenic lines. Further, cellulose content was 1.6–2 folds significantly higher in PaSOD and dual transgenic lines, 1.4 fold higher in RaAPX lines as compared to WT plants under stress conditions. Additionally, transgenics overexpressing PaSOD and RaAPX also displayed higher amounts of phenolics as compared to WT. The novelty of present study is that H2O2 apart from its role in signalling, it also provides mechanical strength to plants and aid in plant biomass production during salt stress by transcriptional activation of cellulose biosynthesis pathway. This modulation of the cellulose biosynthetic machinery in plants has the potential to provide insight into plant growth, morphogenesis and to create plants with enhanced cellulose content for biofuel use.

Keyword

APX CuZn-SOD Cell wall biosynthesis Flavonoids H2O2 signalling Salinity 

Notes

Acknowledgements

This research was supported by grants from the Council of Scientific and Industrial Research (CSIR), New Delhi, India under CSIR Network Projects BSC107 and BSC109.

Author contributions

AKS, AS and PSA conceived and designed the experiments. AS and TG performed the experiments. PSA, YS, SK and AKS analyzed the data. IZ did statistical analysis. AS and AKS wrote the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11033_2019_4648_MOESM1_ESM.pdf (501 kb)
Hierarchical tree graph of over-represented GO terms in down-regulated genes by singular enrichment analysis generated by agriGO. Boxes in the graph show GO terms labelled by their GO ID, term definition and statistical information. The significant terms (adjusted p  < 0.05) are marked with colour, while non-significant terms are shown as white boxes. The degree of colour saturation of a box correlates positively with the enrichment level of the term. Solid, dashed and dotted lines represent two, one and zero enriched terms at both ends connected by the line, respectively. The rank direction of the graph runs from top to bottom. (PDF 500 KB)
11033_2019_4648_MOESM2_ESM.pdf (268 kb)
UPLC Analysis of Soluble Phenolics in WT (a), S26 (b), APX20 (c) and 18O (d) lines of Arabidopsis thaliana. Phenolic profiles of transgenic (b, c, d) and wild type (a) lines were compared at 270 nm under control and stress conditions. Key to peak Peaks was confirmed by mass spectrometry. (PDF 267 KB)
11033_2019_4648_MOESM3_ESM.docx (29 kb)
Primer sequence and PCR conditions for genes used for semi-quantitative and real-time expression analysis. (DOCX 28 KB)
11033_2019_4648_MOESM4_ESM.xlsx (22 kb)
RNA seq FPKM values for the genes and transcription factors involved in secondary cell wall cellulose biosynthesis. (XLSX 22 KB)

References

  1. 1.
    Qin H, Gu Q, Zhang J, Sun L, Kuppu S, Zhang Y et al (2011) Regulated expression of an isopentenyltransferase gene (IPT) in peanut significantly improves drought tolerance and increases yield under field conditions. Plant Cell Physiol 52:1904–1914CrossRefPubMedGoogle Scholar
  2. 2.
    Mittler R, Vanderauwera S, Suzuki N, Miller G, Tognetti VB, Vandepoele K, Gollery M, Shulaev V, Van Breusegem F (2011) ROS signaling: the new wave? Trends Plant Sci 16:300–309CrossRefPubMedGoogle Scholar
  3. 3.
    Wrzaczek M, Brosche M, Kangasjarvi J (2013) ROS signaling loops: production, perception, regulation. Curr Opin Plant Biol 16:575–582CrossRefPubMedGoogle Scholar
  4. 4.
    Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactive oxygen gene network of plants. Trends Plant Sci 9:490–498CrossRefPubMedGoogle Scholar
  5. 5.
    Asada K (1999) The water-water cycle in chloroplasts: scavenging of active oxygen and dissipation of excess photons. Ann Rev Plant Physiol Plant Mol Biol 50:601–639CrossRefGoogle Scholar
  6. 6.
    Bienert GP, Møller AL, Kristiansen KA et al (2007) Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes. J Biol Chem 282:1183–1192CrossRefPubMedGoogle Scholar
  7. 7.
    Somerville C, Youngs H, Taylor C, Davis SC, Long SP (2010) Feedstocks for lignocellulosic biofuels. Science 329:790–792CrossRefPubMedGoogle Scholar
  8. 8.
    Demura T, Ye ZH (2010) Regulation of plant biomass production. Curr Opin Plant Biol 13:299–304CrossRefPubMedGoogle Scholar
  9. 9.
    Richmond TA, Somerville CR (2001) Integrative approaches to determining Csl function. Plant Mol Biol 47:131–143CrossRefPubMedGoogle Scholar
  10. 10.
    McFarlane HE, Do¨ring A, Persson S (2014) The cell biology of cellulose synthesis. Annu Rev Plant Biol 65:69–94CrossRefPubMedGoogle Scholar
  11. 11.
    Carpita NC, McCann M (2000) The cell wall. In: Buchanan B, Gruissem W, Jones RL (eds) Biochemistry and molecular biology of plants. American Society of Plant Physiologists, Rockville, pp 52–108Google Scholar
  12. 12.
    Ueda A, Yamamoto-Yamane Y, Takabe T (2007) Salt stress enhances proline utilization in the apical region of barley roots. Biochem Biophys Res Commun 355:61–66CrossRefPubMedGoogle Scholar
  13. 13.
    Muszyńska A, Jarocka K, Kurczynska E (2014) Plasma membrane and cell wall properties of an aspen hybrid (Populus tremula × tremuloides) parenchyma cells under the influence of salt stress. Acta Physiol Plant 36:1155–1165CrossRefGoogle Scholar
  14. 14.
    Shafi A, Chauhan R, Gill T, Swarnkar MK, Sreenivasulu Y, Kumar S, Kumar N, Shankar R, Ahuja PS, Singh AK (2015) 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 87:615–631CrossRefPubMedGoogle Scholar
  15. 15.
    Shafi A, Gill T, Sreenivasulu Y, Kumar S, Ahuja PS, Singh AK (2015) Improved callus induction, shoot regeneration, and salt stress tolerance in Arabidopsis overexpressing superoxide dismutase from Potentilla atrosanguinea. Protoplasma 252:41–51CrossRefPubMedGoogle Scholar
  16. 16.
    Shafi A, Pal AK, Sharma V, Kalia S, Kumar S, Ahuja PS, Singh AK (2017) Transgenic potato plants Overexpressing SOD and APX exhibit enhanced lignification and starch biosynthesis with improved salt stress tolerance. Plant Mol Biol Rep 35:504–518CrossRefGoogle Scholar
  17. 17.
    Heyndrickx KS, Van de Velde J, Wang X, Weigel D, Vandepoele K (2014) A functional and evolutionary perspective on transcription factor binding in Arabidopsis thaliana. Plant Cell 26:3894–3910CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Kurek I, Kawagoe Jacob-Wilk YD, Doblin Delmer MD (2002) Dimerization of cotton fiber cellulose synthase catalytic subunits occurs via oxidation of the zinc-binding domains. Proc Natl Acad Sci USA 99:11109–11114CrossRefPubMedGoogle Scholar
  19. 19.
    Kim HJ, Barbara T (2008) Involvement of extracellular Cu/Zn superoxide dismutase in cotton fiber primary and secondary cell wall biosynthesis. Plant Signaling Behavior 3:1119–1121CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Xiong J, Yang Y, Fu G, Tao L (2015) Novel roles of hydrogen peroxide (H2O2) in regulating pectin synthesis and demethylesterification in the cell wall of rice (Oryza sativa) root tips. New Phytol 206:118–126CrossRefPubMedGoogle Scholar
  21. 21.
    Shafi A, Dogra V, Gill T, Ahuja PS, Sreenivasulu Y (2014) Simultaneous over-expression of PaSOD and RaAPX in transgenic Arabidopsis thaliana confers cold stress tolerance through increase in vascular lignifications. PLoS One 9:e110302CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Gill T, Kumar S, Ahuja PS, Sreenivasulu Y (2010) Over-expression of Potentilla superoxide dismutase improves salt stress tolerance during germination and growth in Arabidopsis thaliana. J Plant Genet Transgenics 1:1–10Google Scholar
  23. 23.
    Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  24. 24.
    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 Research Notes 4:85CrossRefGoogle Scholar
  25. 25.
    Gill T, Sreenivasulu Y, Kumar S, Ahuja PS (2010) Over-expression of superoxide dismutase exhibits lignification of vascular structures in Arabidopsis thaliana. J Plant Physiol 167:757–760CrossRefPubMedGoogle Scholar
  26. 26.
    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 Bioch 40:501–507CrossRefGoogle Scholar
  27. 27.
    Updegraff DM (1969) Semimicro determination of cellulose in biological materials. Anal Biochem 32:420–424CrossRefPubMedGoogle Scholar
  28. 28.
    Dubois M, Gilles K, Hamilton JK, Rebers PA, Smith F (1956)) Colorimetric method for determination of sugars and related substances. Anal Chem 38:350–361CrossRefGoogle Scholar
  29. 29.
    Bates L, Waldren R, Teare ID (1973) Rapid determination of free proline for water-stress studies. Plant Soil 39:205–207CrossRefGoogle Scholar
  30. 30.
    Orozco-Cárdenas ML, Ryan CA (1999) Hydrogen peroxide is generated systematically in plant leaves by wounding and systemin via the octadecanoid pathway. Proc Natl Acad Sci USA 96:6553–6557CrossRefPubMedGoogle Scholar
  31. 31.
    Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386Google Scholar
  32. 32.
    Mathur J, Koncz C, Szabados L (2005) A simple method for isolation, liquid culture, transformation and regeneration of Arabidopsis thaliana protoplasts. Plant Cell Rep 14:221–226Google Scholar
  33. 33.
    Nagata T, Takebe I (1970) Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts. Planta 92:301–308CrossRefPubMedGoogle Scholar
  34. 34.
    Fu ZQ, Yan S, Saleh A, Wang W, Ruble J, Oka N et al (2012) NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants. Nature 486:228–232CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Faize M, Burgos L, Faize L, Piqueras A, Nicolas E, Barba-Espin G, Clemente-Moreno MJ, Alcobendas R, Artlip T, Hernandez JA (2011) Involvement of cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase for improved tolerance against drought. J Exp Bot 62:2599–2613CrossRefPubMedGoogle Scholar
  36. 36.
    Bhaskaran S, Savithramma DL (2011) Co-expression of Pennisetum glaucum vacuolar Na+/H+antiporter and Arabidopsis H+-pyrophosphatase enhances salt tolerance in transgenic tomato. J Exp Bot 62:5561–5570CrossRefPubMedGoogle Scholar
  37. 37.
    Abogadallah GM, Serag MM, El-Katouny TM, Quick PW (2010) Salt tolerance at germination and vegetative growth involves different mechanisms in barnyard grass (Echinochloa crusgalli L.) mutants. Plant Growth Regul 60:1–12CrossRefGoogle Scholar
  38. 38.
    Waditee R, Bhuiyan MNH, Rai V, Aoki K, Tanaka Y, Hibino T, Suzukim S, Takanom J, Jagendorf AT, Takabe T, Takabe T (2005) Genes for direct methylation of glycine provide high levels of glycinebetaine and abiotic-stress tolerance in Synechococcus and Arabidopsis. Proc Natl Acad Sci USA 102:1318–1323CrossRefPubMedGoogle Scholar
  39. 39.
    Koster KL, Lynch DV (1992) Solute accumulation and compartmentation during the cold acclimation of puma rye. Plant Physiol 98:108–113CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Harris DM, Corbin K, Wang T, Gutierrez R, Bertolo AL, Petti C, Smilgies DM, Estevez JM, Bonetta D, Urbanowicz BR et al (2012) Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1A903V and CESA3T942I of cellulose synthase. Proc Natl Acad Sci USA 109:4098–4103CrossRefPubMedGoogle Scholar
  41. 41.
    Fujita M, Himmelspach R, Ward J, Whittington A, Hasenbein N, Liu C, Truong TT, Galway ME, Mansfield SD, Hocart CH et al (2013) The anisotropy1 D604N mutation in the Arabidopsis cellulose synthase1 catalytic domain reduces cell wall crystallinity and the velocity of cellulose synthase complexes. Plant Physiol 162:74–85CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Wang G, Gao Y, Wang J, Yang L, Song R et al (2011) Overexpression of two cambium-abundant Chinese fir (Cunninghamia lanceolata) alpha-expansin genesClEXPA1 and ClEXPA2 affect growth and development in transgenic tobacco and increase the amount of cellulose in stem cell walls. Plant Biotechnol J 9:486–502CrossRefPubMedGoogle Scholar
  43. 43.
    Zenoni S, Fasoli M, Tornielli GB, Dal Santo S, Sanson A et al (2011) Overexpression of PhEXPA1increases cell size, modifies cell wall polymer composition and affects the timing of axillary meristem development in Petunia hybrida. New Phytol 191:662–677CrossRefPubMedGoogle Scholar
  44. 44.
    Yokoyama R, Nishitani K (2006) Identification and characterization of Arabidopsis thaliana genes involved in xylem secondary cell walls. J Plant Res 119:189–194CrossRefPubMedGoogle Scholar
  45. 45.
    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 19:1855–1860CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    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 17:2993–3006CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    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 19:270–280CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    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 18:3158–3170CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Zhong R, Richardson EA, Ye ZH (2007) Two NAC domain transcription factors, SND1 and NST1, function redundantly in regulation of secondary wall synthesis in fibers of Arabidopsis. Planta 225:1603–1611CrossRefPubMedGoogle Scholar
  50. 50.
    Taylor NG, Howells RM, Huttly AK, Vickers K, Turner SR (2003) Interactions among three distinct CesA proteins essential for cellulose synthesis. Proc Natl Acad Sci USA 100:1450–1455CrossRefPubMedGoogle Scholar
  51. 51.
    Zhong R, Ye ZH (2007) Regulation of cell wall biosynthesis. Curr Opin Plant Biol 10:564–572CrossRefPubMedGoogle Scholar
  52. 52.
    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 20:2763–2782CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Kim WC, Ko JH, Kim JY, Kim JM, Bae HJ, Han KH (2012) MYB46 directly regulates the gene expression of secondary wall-associated cellulose synthases in Arabidopsis. Plant J 73:26–36CrossRefPubMedGoogle Scholar
  54. 54.
    Passardi F, Penel C, Dunand C (2004) Performing the paradoxical: how plant peroxidases modify the cell wall. Trends Plant Sci 9:534–540CrossRefPubMedGoogle Scholar
  55. 55.
    Cosgrove DJ (2001) Wall structure and wall loosening: a look backwards and forwards. Plant Physiol 125:131–134CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Gechev TS, Hille J (2005) Hydrogen peroxide as a signal controlling plant programmed cell death. J Cell Biol 168:17–20CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Olson PD, Varner JE (1993) Hydrogen peroxide and lignification. Plant J 4:887–892CrossRefGoogle Scholar
  58. 58.
    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. J Plant Physiol 126:1668–1677CrossRefGoogle Scholar
  59. 59.
    Saslowsky DE, Warek U, Winkel BSJ (2005) Nuclear localization of flavonoid enzymes in Arabidopsis. J Biol Chem 25:23735–23740CrossRefGoogle Scholar
  60. 60.
    Winkel-Shirley B (2002) Biosynthesis of flavonoids and effects of stress. Curr Opin Plant Biol 5:218–223CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Amrina Shafi
    • 1
    • 5
    Email author
  • Tejpal Gill
    • 2
  • Insha Zahoor
    • 3
  • Paramvir Singh Ahuja
    • 1
  • Yelam Sreenivasulu
    • 1
  • Sanjay Kumar
    • 1
  • Anil Kumar Singh
    • 1
    • 4
    Email author
  1. 1.Department of BiotechnologyCSIR-Institute of Himalayan Bioresource TechnologyPalampurIndia
  2. 2.National Institute of Arthritis and Musculoskeletal and Skin DiseasesNational Institute of HealthBethesdaUSA
  3. 3.Bioinformatics CentreUniversity of KashmirSrinagarIndia
  4. 4.ICAR-Indian Institute of Agricultural BiotechnologyRanchiIndia
  5. 5.Biotechnology DepartmentUniversity of KashmirSrinagarIndia

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