Skip to main content
Log in

Heterologous expression of two Medicago truncatula putative ERF transcription factor genes, WXP1 and WXP2, in Arabidopsis led to increased leaf wax accumulation and improved drought tolerance, but differential response in freezing tolerance

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

Cuticular waxes are the major components of plant cuticle and play an important role in protecting aerial organs from damage caused by biotic and abiotic stresses. Here we report the functional characterization of two putative ERF transcription factor genes WXP1 and its paralog WXP2 from Medicago truncatula. Transgenic expression of WXP1 and WXP2 in Arabidopsis (ecotype Columbia) led to significantly increased cuticular wax deposition on leaves of 4-week-old and 6-week-old transgenic plants, assessed based on fresh weight or based on surface area. Differences in the accumulation of various wax components as well as their chain length distributions were found in the WXP1 and WXP2 plants. The major wax component in Arabidopsis, n-alkanes, increased substantially in both WXP1 and WXP2 transgenics, however, another wax component, primary alcohols, increased in WXP1 plants but decreased in WXP2 plants. Cuticle properties of the transgenic leaves were analyzed by chlorophyll leaching assay; while the WXP1 plants had no change, the WXP2 plants showed more chlorophyll leaching. Analysis of fresh weight loss from detached leaves revealed that the transgenic leaves tend to retain more water than the control. Both WXP1 and WXP2 transgenic plants showed significantly enhanced whole plant drought tolerance. Analysis of freezing tolerance at the whole plant level and measurement of electrolyte leakage from detached leaves revealed that the WXP1 plants had increased freezing tolerance while the WXP2 plants were more sensitive to low temperature when compared to the control. Transgenic expression of WXP1 had no obvious effects on plant growth and development, however, the expression of WXP2 led to slower plant growth. These results indicate that WXP1 is a useful candidate gene for improving plant drought and freezing tolerance by genetic transformation.

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
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  • Aarts M, Keijzer CJ, Stiekema WJ, Pereira A (1995) Molecular characterization of the cer1 gene of Arabidopsis involved in epicuticular wax biosynthesis and pollen fertility. Plant Cell 7:2115–2127

    Article  PubMed  CAS  Google Scholar 

  • Aharoni A, Dixit S, Jetter R, Thoenes E, van Arkel G, Pereira A (2004) The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis. Plant Cell 16:2463–2480

    Article  PubMed  CAS  Google Scholar 

  • Bakker MI, Baas WJ, Sijm DTHM, Kolloffel C (1998) Leaf wax of Lactuca sativa and Plantago major. Phytochemistry 47:1489–1493

    Article  CAS  Google Scholar 

  • Barker DG, Bianchi S, Blondon F, Dattée Y, Duc G, Essad S, Flament P, Gallusci P, Génier G, Guy P, Muel X, Tourneur J, Dénarié J, Huguet T (1990) Medicago truncatula, a model plant for studying the molecular genetics of the Rhizobium-legume symbiosis. Plant Mol Biol Rep 8:40–49

    CAS  Google Scholar 

  • Bell CJ, Dixon RA, Farmer AD, Flores R, Inman J, Gonzales RA, Harrison MJ, Paiva NL, Scott AD, Weller JW, May GD (2001) The Medicago Genome Initiative: a model legume database. Nucleic Acids Res 29:114–117

    Article  PubMed  CAS  Google Scholar 

  • Broun P (2004) Transcription factors as tools for metabolic engineering in plants. Curr Opin Plant Biol 7:202–209

    Article  PubMed  CAS  Google Scholar 

  • Broun P, Poindexter P, Osborne E, Jiang C-Z, Riechmann JL (2004) WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis. PNAS 101:4706–4711

    Article  PubMed  CAS  Google Scholar 

  • Chen H, Nelson RS, Sherwood JL (1994) Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection. Biotechniques 16:664–670

    PubMed  CAS  Google Scholar 

  • Chen X, Goodwin SM, Boroff VL, Liu X, Jenks MA (2003) Cloning and characterization of the WAX2 gene of Arabidopsis involved in cuticle membrane and wax production. Plant Cell 15:1170–1185

    Article  PubMed  CAS  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  PubMed  CAS  Google Scholar 

  • Cook DR (1999) Medicago truncatula—a model in the making! Curr Opin Plant Biol 2:301–304

    Article  PubMed  CAS  Google Scholar 

  • Dove H, Mayes RW, Freer M (1996) Effects of species, plant part, and plant age on the n-alkane concentrations in the cuticular wax of pasture plants. Aust J Agric Res 47:1333–1347

    Article  CAS  Google Scholar 

  • Fiebig A, Mayfield JA, Miley NL, Chau S, Fischer RL, Preuss D (2000) Alterations in CER6, a gene identical to CUT1, differentially affect long-chain lipid content on the surface of pollen and stems. Plant Cell 12:2001–2008

    Article  PubMed  CAS  Google Scholar 

  • Geyer U, Schönherr J (1990) The effect of the environment on the permeability and composition of Citrus leaf cuticles. I. Water permeability of isolated cuticular membranes. Planta 180:147–153

    Article  Google Scholar 

  • Gilmour SJ, Sebolt AM, Salazar MP, Everard JD, Thomashow MF (2000) Overexpression of the Arabidopsis CBF3 transcriptional activator mimics multiple biochemical changes associated with cold acclimation. Plant Physiol 124:1854–1865

    Article  PubMed  CAS  Google Scholar 

  • Goodwin SM, Rashotte AM, Rahman M, Feldmann KA, Jenks MA (2005) Wax constituents on the inflorescence stems of double eceriferum mutants in Arabidopsis reveal complex gene interactions. Phytochemistry 66:771–780

    Article  PubMed  CAS  Google Scholar 

  • Griffith M, Lumb C, Wiseman SB, Wisniewski M, Johnson RW, Marangoni AG (2005) Antifreeze proteins modify the freezing process in planta. Plant Physiol 138:330–340

    Article  PubMed  CAS  Google Scholar 

  • Guo Y, Xiong L, Ishitani M, Zhu J-K (2002) An Arabidopsis mutation in translation elongation factor 2 causes superinduction of CBF/DREB1 transcription factor genes but blocks the induction of their downstream targets under low temperatures. PNAS 99:7786–7791

    Article  PubMed  CAS  Google Scholar 

  • Haake V, Cook D, Riechmann JL, Pineda O, Thomashow MF, Zhang JZ (2002) Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis. Plant Physiol 130:639–648

    Article  PubMed  CAS  Google Scholar 

  • Hannoufa A, Negruk V, Eisner G, Lemieux B (1996) The CER3 gene of Arabidopsis thaliana is expressed in leaves, stems, roots, flowers and apical meristems. Plant J 10:459–467

    Article  PubMed  CAS  Google Scholar 

  • Hansen JD, Pyee J, Xia Y, Wen TJ, Robertson DS, Kolattukudy PE, Nikolau BJ, Schnable PS (1997) The glossy1 locus of maize and an epidermis-specific cDNA from Kleinia odora define a class of receptor-like proteins required for the normal accumulation of cuticular waxes. Plant Physiol 113:1091–1100

    Article  PubMed  CAS  Google Scholar 

  • Jenks MA, Ashworth EN (1999) Plant epicuticular waxes: function, production, and genetics. In: Janick J (ed) Horticultural reviews. John Wiley & Sons, Inc., New York, USA, pp 1–68

    Google Scholar 

  • Jenks MA, Eigenbrode S, Lemeiux B (2002) Cuticular waxes of Arabidopsis. In: Somerville C, Meyerowitz E (eds) The Arabidopsis Book, American Society of Plant Biologists, Rockville, MD, USA, doi: 10.1199/tab.0016

  • Jenks MA, Joly RJ, Peters PJ, Rich PJ, Axtell JD, Ashworth EN (1994) Chemically induced cuticle mutation affecting epidermal conductance to water vapor and disease susceptibility in Sorghum bicolor (L.) Moench. Plant Physiol 105:1239–1245

    PubMed  CAS  Google Scholar 

  • Jenks MA, Tuttle HA, Eigenbrode SD, Feldmann KA (1995) Leaf epicuticular waxes of the eceriferum mutants in Arabidopsis. Plant Physiol 108:369–377

    PubMed  CAS  Google Scholar 

  • Jenks MA, Tuttle HA, Feldmann KA (1996) Change in epicuticular waxes on wildtype and Eceriferum mutants in Arabidopsis during development. Phytochemistry 42:29–34

    Article  CAS  Google Scholar 

  • Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1999) Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol 17:287–291

    Article  PubMed  CAS  Google Scholar 

  • Kerstiens G (1996) Cuticular water permeability and its physiologial significance. J Exp Bot 47:1813–1832

    Article  CAS  Google Scholar 

  • Koornneef M, Hanhart CJ, Thiel F (1989) A genetic and phenotypic description of Eceriferum (cer) mutants in Arabidopsis thaliana. J Hered 80:118–122

    Google Scholar 

  • Kunst L, Samuels AL (2003) Biosynthesis and secretion of plant cuticular wax. Prog Lipid Res 42:51–80

    Article  PubMed  CAS  Google Scholar 

  • Lolle SJ, Berlyn GP, Engstrom EM, Krolikowski KA, Reiter WD, Pruitt RE (1997) Developmental regulation of cell interactions in the Arabidopsis fiddlehead-1 mutant: a role for the epidermal cell wall and cuticle. Dev Biol 189:311–321

    Article  PubMed  CAS  Google Scholar 

  • Millar AA, Clemens S, Zachgo S, Giblin EM, Taylor DC, Kunst L (1999) CUT1, an Arabidopsis gene required for cuticular wax biosynthesis and pollen fertility, encodes a very-long-chain fatty acid condensing enzyme. Plant Cell 11:825–838

    Article  PubMed  CAS  Google Scholar 

  • Moose S, Sisco P (1996) Glossy15, an APETALA2-like gene from maize that regulates leaf epidermal cell identity. Genes Dev 10:3018–3027

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Naß R, Markstaedter C, Hauke V, Riederer M (1998) Quantitative gas chromatographic analysis of plant cuticular waxes containing long-chain aldehydes. Phytochem Anal 9:112–118

    Article  Google Scholar 

  • Nakano T, Suzuki K, Fujimura T, Shinshi H (2006) Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiol 140:411–432

    Article  PubMed  CAS  Google Scholar 

  • Negruk V, Yang P, Subramanian M, McNevin JP, Lemieux B (1996) Molecular cloning and characterization of the CER2 gene of Arabidopsis thaliana. Plant J 9:137–145

    Article  PubMed  CAS  Google Scholar 

  • Nunes MES, Smith GR (2003) Electrolyte leakage assay capable of quantifying freezing resistance in rose clover. Crop Sci 43:1349–1357

    Article  Google Scholar 

  • Pighin JA, Zheng H, Balakshin LJ, Goodman IP, Western TL, Jetter R, Kunst L, Samuels AL (2004) Plant cuticular lipid export requires an ABC transporter. Science 306:702–704

    Article  PubMed  CAS  Google Scholar 

  • Rashotte AM, Jenks MA, Thanh D. Nguyen, Feldmann KA (1997) Epicuticular wax variation in ecotypes of Arabidopsis thaliana. Phytochemistry 45:251–255

    Article  PubMed  CAS  Google Scholar 

  • Riederer M, Schreiber L (2001) Protecting against water loss: analysis of the barrier properties of plant cuticles. J Exp Bot 52:2023–2032

    Article  PubMed  CAS  Google Scholar 

  • Ristic Z, Jenks MA (2002) Leaf cuticle and water loss in maize lines differing in dehydration avoidance. J Plant Physiol 159:645–651

    Article  CAS  Google Scholar 

  • Rohde P, Hincha DK, Heyer AG (2004) Heterosis in the freezing tolerance of crosses between two Arabidopsis thaliana accessions (Columbia-0 and C24) that show differences in non-acclimated and acclimated freezing tolerance. Plant J 38:790–799

    Article  PubMed  CAS  Google Scholar 

  • Schnurr J, Shockey J, Browse J (2004) The Acyl-CoA synthetase encoded by LACS2 is essential for normal cuticle development in Arabidopsis. Plant Cell 16:629–642

    Article  PubMed  CAS  Google Scholar 

  • Shepherd T, Wynne Griffiths D (2006) The effects of stress on plant cuticular waxes. New Phytologist 171:469–499

    Article  PubMed  CAS  Google Scholar 

  • Shinozaki K, Yamaguchi-Shinozaki K, Seki M (2003) Regulatory network of gene expression in the drought and cold stress responses. Curr Opin Plant Biol 6:410–417

    Article  PubMed  CAS  Google Scholar 

  • Sturaro M, Hartings H, Schmelzer E, Velasco R, Salamini F, Motto M (2005) Cloning and characterization of GLOSSY1, a maize gene involved in cuticle membrane and wax production. Plant Physiol 138:478–489

    Article  PubMed  CAS  Google Scholar 

  • Tacke E, Korfhage C, Michel D, Maddaloni M, Motto M, Lanzini S, Salamini F, Doring H-P (1995) Transposon tagging of the maize Glossy2 locus with the transposable element En/Spm. Plant J 8:907–917

    PubMed  CAS  Google Scholar 

  • Todd J, Post BD, Jaworski JG (1999) KCS1 encodes a fatty acid elongase 3-ketoacyl-CoA synthase affecting wax biosynthesis in Arabidopsis thaliana. Plant J 17:119–130

    Article  PubMed  CAS  Google Scholar 

  • van Buskirk HA, Thomashow MF (2006) Arabidopsis transcription factors regulating cold acclimation. Physiol Plant 126:72–80

    Article  Google Scholar 

  • Verslues PE, Agarwal M, Katiyar-Agarwal S, Zhu JH, Zhu JK (2006) Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status. Plant J 45:523–539

    Article  PubMed  CAS  Google Scholar 

  • Vogg G, Fischer S, Leide J, Emmanuel E, Jetter R, Levy AA, Riederer M (2004) Tomato fruit cuticular waxes and their effects on transpiration barrier properties: functional characterization of a mutant deficient in a very-long-chain fatty acid beta-ketoacyl-CoA synthase. J Exp Bot 55:1401–1410

    Article  PubMed  CAS  Google Scholar 

  • Wisniewski M, Fuller M, Glenn D, Gusta L, Duman J, Griffith M (2002a). Extrinsic ice nucleation in plants: what are the factors involved and can they be manipulated? Kluwer Academic/Plenum Publishers, New York

    Google Scholar 

  • Wisniewski M, Glenn DM, Fuller MP (2002b) Use of a hydrophobic particle film as a barrier to extrinsic ice nucleation in tomato plants. J Am Soc Hort Sci 127:358–364

    Google Scholar 

  • Workmaster BAA, Palta JP, Wisniewski M (1999) Ice nucleation and propagation in cranberry uprights and fruit using infrared video thermography. J Am Soc Hort Sci 124:619–625

    Google Scholar 

  • Xia Y, Nikolau BJ, Schnable PS (1996) Cloning and characterization of CER2, an Arabidopsis gene that affects cuticular wax accumulation. Plant Cell 8:1291–1304

    Article  PubMed  CAS  Google Scholar 

  • Xu X, Dietrich CR, Delledonne M, Xia Y, Wen TJ, Robertson DS, Nikolau BJ, Schnable PS (1997) Sequence analysis of the cloned glossy8 gene of maize suggests that it may code for a β-ketoacyl reductase required for the biosynthesis of cuticular waxes. Plant Physiol 115:501–510

    Article  PubMed  CAS  Google Scholar 

  • Yanagisawa S, Akiyama A, Kisaka H, Uchimiya H, Miwa T (2004) Metabolic engineering with Dof1 transcription factor in plants: improved nitrogen assimilation and growth under low-nitrogen conditions. PNAS 101:7833–7838

    Article  PubMed  CAS  Google Scholar 

  • Young ND, Cannon SB, Sato S, Kim DJ, Cook DR, Town CD, Roe BA, Tabata S (2005) Sequencing the genespaces of Medicago truncatula and Lotus japonicus. Plant Physiol 137:1174–1181

    Article  PubMed  CAS  Google Scholar 

  • Zhang J-Y, Broeckling CD, Blancaflor EB, Sledge M, Sumner LW, Wang Z-Y (2005) Overexpression of WXP1, a putative Medicago truncatula AP2 domain-containing transcription factor gene, increases cuticular wax accumulation and enhances drought tolerance in transgenic alfalfa (Medicago sativa). Plant J 42:689–707

    Article  PubMed  CAS  Google Scholar 

  • Zhu J, Shi H, Lee B-h, Damsz B, Cheng S, Stirm V, Zhu J-K, Hasegawa PM, Bressan RA (2004) An Arabidopsis homeodomain transcription factor gene, HOS9, mediates cold tolerance through a CBF-independent pathway. PNAS 101:9873–9878

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Michael Udvardi for critical reading of the manuscript. This work was supported by the Samuel Roberts Noble Foundation.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Zeng-Yu Wang.

Electronic supplementary material

Below is the electronic supplementary material.

11103_2007_9150_MOESM1_ESM.doc

11103_2007_9150_MOESM2_ESM.ppt

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhang, JY., Broeckling, C.D., Sumner, L.W. et al. Heterologous expression of two Medicago truncatula putative ERF transcription factor genes, WXP1 and WXP2, in Arabidopsis led to increased leaf wax accumulation and improved drought tolerance, but differential response in freezing tolerance. Plant Mol Biol 64, 265–278 (2007). https://doi.org/10.1007/s11103-007-9150-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11103-007-9150-2

Keywords

Navigation