Skip to main content
Log in

Overexpression of HVA1 gene from barley generates tolerance to salinity and water stress in transgenic mulberry (Morus indica)

  • Original Paper
  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic proteins found primarily in plants. The barley hva1 gene encodes a group 3 LEA protein and is induced by ABA and water deficit conditions. We report here the over expression of hva1 in mulberry under a constitutive promoter via Agrobacterium-mediated transformation. Molecular analysis of the transgenic plants revealed the stable integration and expression of the transgene in the transformants. Transgenic plants were subjected to simulated salinity and drought stress conditions to study the role of hva1 in conferring tolerance. The transgenic plants showed better cellular membrane stability (CMS), photosynthetic yield, less photo-oxidative damage and better water use efficiency as compared to the non-transgenic plants under both salinity and drought stress. Under salinity stress, transgenic plants show many fold increase in proline concentration than the non-transgenic plants and under water deficit conditions proline is accumulated only in the non-transgenic plants. Results also indicate that the production of HVA1 proteins helps in better performance of transgenic mulberry by protecting membrane stability of plasma membrane as well as chloroplastic membranes from injury under abiotic stress. Interestingly, it was observed that hva1 conferred different degrees of tolerance to the transgenic plants towards various stress conditions. Amongst the lines analysed for stress tolerance transgenic line ST8 was relatively more salt tolerant, ST30, ST31 more drought tolerant, and lines ST11 and ST6 responded well under both salinity and drought stress conditions as compared to the non-transgenic plants. Thus hva1 appears to confer a broad spectrum of tolerance under abiotic stress in mulberry.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

ABA:

Abscisic acid

CMS:

Cellular membrane stability

DW:

Dry weight

FW:

Fresh weight

IRMS:

Isotope ratio mass spectrometry

LEA:

Late embryogenesis abundant protein

MS:

Murashige and Skoog

RBD:

Randomized block design

RWC:

Relative water content

TDZ:

Thidiazuron

TW:

Turgid weight

WUE:

Water use efficiency

YEM:

Yeast extract mannitol

References

  • Ansari R, Naqvi MS, Khanzada NA, Hubick KT (1998) Carbon isotope discrimination in wheat cultivars under saline conditions. Pak J Bot 30:87–93

    Google Scholar 

  • Ashraf M, Harris PJC (2004) Potential biochemical indicators of salinity tolerance in plants. Plant Sci 166:3–16

    Article  CAS  Google Scholar 

  • Babu RC, Zhang J, Blum A, Ho DT-H, Wu R, Nguyen HT (2004) HVA1, a LEA gene from barley confers dehydration tolerance in transgenic rice (Oryza sativa L.) via cell membrane protection. Plant Sci 166:855–862

    Article  CAS  Google Scholar 

  • Bajii M, Kinet JM, Luts S (2002) The use of electrolyte leakage method for assessing the cell membrane stability as a water stress tolerance test in durum wheat. Plant Growth Regul 36:61–70

    Article  Google Scholar 

  • Baker NR (1991) Possible role of photosystem II in environmental perturbations of photosynthesis. Physiol Plant 81:563–570

    Article  CAS  Google Scholar 

  • Bates LS, Waldren RP, Teare ID (1973) Rapid determination of proline for water stress studies. Plant Soil 39:205–207

    Article  CAS  Google Scholar 

  • Bartels D, Salamini F (2001) Dessication tolerance in the resurrection plant Craterostigma plantagineum. A contribution to the study of drought tolerance at the molecular level. Plant Physiol 127:1346–1353

    Article  PubMed  CAS  Google Scholar 

  • Bhatnagar S, Kapur A, Khurana P (2000) TDZ mediated differentiation in commercially valuable Indian mulberry Morus indica cultivars K2 and DD. Plant Biotechnol 18:61–65

    Google Scholar 

  • Bhatnagar S, Kapur A, Khurana P (2002) Evaluation of parameters for high efficiency gene transfer via particle bombardment in Indian mulberry. Indian J Exp Biol 40:1387–1393

    PubMed  CAS  Google Scholar 

  • Bhatnagar S, Khurana P (2003) Agrobacterium tumefaciens-mediated transformation of Indian mulberry, Morus indica cv. K2: a time-phased screening strategy. Plant Cell Rep 21:669–675

    PubMed  CAS  Google Scholar 

  • Bowler C, Montagu M V, Inze D (1992) Superoxide dismutase and stress tolerance. Annu Rev Plant Physiol Plant Mol Biol 43:83–116

    Article  CAS  Google Scholar 

  • Chaves MM, Maroco JP, Pereira JS (2003) Understanding plant responses to drought––from genes to the whole plant. Funct Plant Biol 30:239–264

    Article  CAS  Google Scholar 

  • Close TJ (1996) Dehydrins: emergence of a biochemical role of a family of plant dehydration proteins. Physiol Plant 97:795–803

    Article  CAS  Google Scholar 

  • Dure L (1993) A repeating 11-mer amino acid motif and plant desiccation. Plant J 3(3):363–369

    Article  PubMed  CAS  Google Scholar 

  • Dure L (2001) Occurrence of a repeating 11-mer amino acid sequence motif in diverse organisms. Protein Pept Lett 8:15–122

    Article  Google Scholar 

  • Farquhar GD, Ehleringer JR, Hubick KT (1989) Carbon isotope discrimination and photosynthesis. Annu Rev Plant Physiol Plant Mol Biol 40:503–537

    Article  CAS  Google Scholar 

  • Foyer CH, Noctor G (2000) Oxygen processing in photosynthesis: regulation and signaling. New Phytol 146:359–388

    Article  CAS  Google Scholar 

  • Frederick CM, Plaut Z, Saliendra ZN (1994) Carbon isotope discrimination, gas exchange and growth of sugar cane cultivars under salinity. Plant Physiol 104:521–526

    Google Scholar 

  • Fu D, Huang B, Xiao Y, Muthukrishnan S, Liang GH (2007) Overexpression of barley HVA1 gene in creeping bent grass for improving drought tolerance. Plant Cell Rep 26:467–477

    Article  PubMed  CAS  Google Scholar 

  • Goday A, Jensen AB, Culianez-Macia FA, Mar Alba M, Figueras M, Serratosa J, Torrent M, Pages M (1994) The maize abscisic acid-responsive protein Rab17 is located in the nucleus and interacts with nuclear localization signals. Plant Cell 6:351–60

    Article  PubMed  CAS  Google Scholar 

  • Hanson AD, Burnet M (1994) Evolution and metabolic engineering of osmoprotectant accumulation in higher plants. In: Cherry JH (ed) Cell biology: biochemical and cellular mechanism of stress tolerance in Plants. NATO ASI Series H, Springer, Berlin, pp 291–302

  • Harbinson J, Genty B, Baker NR (1989) Relationship between the quantum efficiencies of photosystems I and II in pea leaves. Plant Physiol 90:1029–1034

    Article  PubMed  CAS  Google Scholar 

  • Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ (2000) Plant cellular and molecular responses to high salinity. Annu Rev Plant Physiol Plant Mol Biol 51:463–499

    Article  PubMed  CAS  Google Scholar 

  • Hong B, Uknes SJ, Ho T-HD (1988) Cloning and characterization of a cDNA encoding an mRNA rapidly induced by ABA in barley aleurone layers. Plant Mol Biol 11:495–506

    Article  CAS  Google Scholar 

  • Ingram J, Bartels D (1996) The molecular basis of dehydration tolerance in plants. Annu Rev Plant Physiol Plant Mol Biol 47:377–403

    Article  PubMed  CAS  Google Scholar 

  • Isla R, Aragues R, Royo A (1998) Validity of various physiological traits as screening criteria for salt tolerance in barley. Field Crops Res 58:97–107

    Article  Google Scholar 

  • Kapur A, Bhatnagar S, Khurana P (2001) Efficient regeneration from mature leaf explants of Indian mulberry via organogenesis. Sericologia 41:207–214

    Google Scholar 

  • Kawakami K, Yanagawa H, (2003) Illustrated working process of new bivoltine silkworm rearing technology. JICA, India

    Google Scholar 

  • Kim CS, Lee CH, Shin JS, Chung YS, Hyung NI (1997) A simple and rapid method for isolation of high quality genomic DNA from fruit trees and conifers using PVP. Nuc Acids Res 25:1085–1086

    Article  CAS  Google Scholar 

  • Koag MC, Fenton RD, Wilkens S, Close TJ (2003) The binding of Maize DHN1 to lipid vesicles. Gain of structure and lipid specificity. Plant Physiol 131:309–316

    Article  PubMed  CAS  Google Scholar 

  • Kumar SG, Madhusudan KV, Sreenivasulu N, Sudhakar C (2000) Stress responses in two genotypes of mulberry (Morus alba L.) under NaCl salinity. Indian J Exp Biol 38:192–195

    PubMed  CAS  Google Scholar 

  • Kumar SG, Reddy AM, Sudhakar C (2003) NaCl effects on proline metabolism in two high yielding genotypes of mulberry (Morus alba L.) with contrasting salt tolerance. Plant Sci 165:1245–1251

    Article  CAS  Google Scholar 

  • Lal S, Bhatnagar S, Khurana P (2006) Screening of Indian mulberry for abiotic stress tolerance and ameliorative effect of calcium on salinity stress. Physiol Mol Biol Plants 12:193–199

    CAS  Google Scholar 

  • Maqbool B, Zhong H, El-Maghraby Y, Ahmad A, Chai B, Wang W, Sabzikar R, Sticklen MB (2002) Competence of oat (Avena sativa L.) shoot apical meristems for integrative transformation, inherited expression, and osmotic tolerance of transgenic lines containing HVA1. Theor Appl Genet 105:201–208

    Article  PubMed  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Oraby HF, Ransom CB, Kravchenko AN, Sticklen MB (2005) Barley HVA1 gene confers salt tolerance in r3 transgenic oat. Crop Sci 45:2218–2227

    Article  CAS  Google Scholar 

  • Patnaik D, Khurana P (2003) Genetic transformation of Indian bread (T. aestivum) and pasta (T. durum) wheat by particle bombardment of mature embryo-derived calli. BMC Plant Biol 3:3–5

    Article  Google Scholar 

  • Ramanjulu S, Sudhakar C (2000) Proline metabolism during dehydration in two mulberry genotypes with contrasting drought tolerance. J Plant Physiol 157:81–85

    CAS  Google Scholar 

  • Ramanjulu S, Sudhakar C (2001) Allevation of NaCl salinity stress by calcium is partly related to the increase in proline accumulation in mulberry (Morus alba L.) callus. J Plant Biol 28:203–206

    Google Scholar 

  • Rao AA (2002) Conservation status of mulberry genetic resources in India. Paper contributed to Expert Consultation on Promotion of Global Exchange of Sericultural Germplasm Resources, pp 21–25, Satellite session of XIXth ISC Congress, Bangkok, Thailand

  • Rohila JS, Jain RK, Wu R (2002) Genetic improvement of Basmati rive for salt and dought tolerance by regulated gene expression of a barley HVA1 cDNA. Plant Sci 163:525–532

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Schneider K, Wells B, Schmeizer F, Bartels D (1993) Dessication leads to rapid accumulation of both cytosolic and chloroplastic proteins in the resurrection plant Craterostigma plantagineum Hochst. Planta 189:120–131

    Article  CAS  Google Scholar 

  • Sivamani E, Bahieldin1 A, Wraith JM, Al-Niemi T, Dyer WE, Ho TD, Qu R (2000) Improved biomass productivity and water use efficiency under water deficit conditions in transgenic wheat constitutively expressing the barley HVA1 gene. Plant Sci 155:1–9

    Article  PubMed  CAS  Google Scholar 

  • Sullivan CY, Ross WM (1979) Selecting for drought and heat resistance in grain sorghum. In: Mussell H, Staples RC (eds) Stress physiology in crop plants. John Wiley and Sons, New York, pp 263–281

    Google Scholar 

  • Tewari A, Bhatnagar S, Khurana P (1999) In vitro response of commercially valuable varieties of Morus sp. to Thidizuron and activated charcoal. Plant Biotechnol 16:413–417

    CAS  Google Scholar 

  • Xu D, Duan X, Wang B, Hong B, Ho T, Wu R (1996) Expression of a late embryogenesis abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in transgenic rice. Plant Physiol 110:249–257

    PubMed  CAS  Google Scholar 

  • Wise MJ (2003) LEAping to conclusion: A computational reanalysis of late embryogenesis abundant proteins and their possible roles. BMC Bioinformatics 4:52

    Article  PubMed  Google Scholar 

  • Zhu JK (2002) Salt and drought stress signal transduction in plants. Ann Rev of Plant Biol 53:247–273

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was financially supported by the Department of Biotechnology, Govt. of India. SL and VG acknowledge CSIR and UGC for Research Fellowships. Help of Ms Manaswini Das in rearing of silkworms for the biotic assay is also acknowledged.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Paramjit Khurana.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lal, S., Gulyani, V. & Khurana, P. Overexpression of HVA1 gene from barley generates tolerance to salinity and water stress in transgenic mulberry (Morus indica). Transgenic Res 17, 651–663 (2008). https://doi.org/10.1007/s11248-007-9145-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11248-007-9145-4

Keywords

Navigation