Skip to main content
Log in

Secretory peptide PdEPF2 enhances drought tolerance by modulating stomatal density and regulates ABA response in transgenic Arabidopsis thaliana

  • Original Article
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

Water deficit limits the growth and productivity of plants worldwide. Improved water use efficiency (WUE) and drought tolerance are important adaptations to address these limitations. In this study, an epidermal patterning factor (EPF), PdEPF2, from a fast-growing poplar clone NE-19 (Populus nigra × (Populus deltoids × Populus nigra)) was isolated. Quantitative reverse transcription polymerase chain reaction showed that transcription of this gene was induced by drought and abscisic acid (ABA). To study the biological functions of PdEPF2, transgenic Arabidopsis plants harboring (35S:PdEPF2) in which PdEPF2 was constitutively expressed were generated. Compared with the wild type and epf2-3 mutant, the transgenic plants ectopically expressing PdEPF2 showed favorable osmotic parameters, such as seed germination rate, primary root length, proline and chlorophyll content, Fv/Fm, photosynthetic rate, and instantaneous leaf WUE, under drought stress. In addition, the transgenic Arabidopsis plants displayed enhanced drought tolerance as a result of decreased stomatal density, which would limit transpiration and reduce water loss. Compared with the wild-type, plants that overexpressed PdEPF2 had decreased sensitivity to exogenous ABA during germination and seedling development, whereas the epf2-3 mutant showed increased sensitivity to ABA. Furthermore, PdEPF2 positively regulated expression of two ABA signaling-related genes, ABI1 and ABI2. These findings indicate that PdEPF2 may enhance drought tolerance by regulating stomatal density and the response to the ABA signaling pathway.

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

References

  • Bhatnagar-Mathur P, Devi MJ, Reddy DS, Lavanya M, Vadez V, Serraj R, Yamaguchi-Shinozaki K, Sharma KK (2007) Stress-inducible expression of At DREB1A in transgenic peanut (Arachis hypogaea L.) increases transpiration efficiency under water-limiting conditions. Plant Cell Rep 26(12):2071–2082. doi:10.1007/s00299-007-0406-8

    Article  CAS  PubMed  Google Scholar 

  • Bhave NS, Veley KM, Nadeau JA, Lucas JR, Bhave SL, Sack FD (2008) TOO MANY MOUTHS promotes cell fate progression in stomatal development of Arabidopsis stems. Planta 229(2):357–367. doi:10.1007/s00425-008-0835-9

    Article  PubMed  Google Scholar 

  • Boccalandro HE, Rugnone ML, Moreno JE, Ploschuk EL, Serna L, Yanovsky MJ, Casal JJ (2009) Phytochrome B enhances photosynthesis at the expense of water-use efficiency in Arabidopsis. Plant Physiol 150(2):1083–1092. doi:10.1104/pp.109.135509

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cao WH, Liu J, He XJ, Mu RL, Zhou HL, Chen SY, Zhang JS (2007) Modulation of ethylene responses affects plant salt-stress responses. Plant Physiol 143(2):707–719. doi:10.1104/pp.106.094292

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Casson SA, Hetherington AM (2010) Environmental regulation of stomatal development. Curr Opin Plant Biol 13(1):90–95. doi:10.1016/j.pbi.2009.08.005

    Article  CAS  PubMed  Google Scholar 

  • Casson SA, Franklin KA, Gray JE, Grierson CS, Whitelam GC, Hetherington AM (2009) phytochrome B and PIF4 regulate stomatal development in response to light quantity. Curr Biol: CB 19(3):229–234. doi:10.1016/j.cub.2008.12.046

    Article  CAS  PubMed  Google Scholar 

  • Chaerle L, Saibo N, Van Der Straeten D (2005) Tuning the pores: towards engineering plants for improved water use efficiency. Trends Biotechnol 23(6):308–315

    Article  CAS  PubMed  Google Scholar 

  • Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11(2):113–116

    Article  CAS  Google Scholar 

  • Chater CC, Oliver J, Casson S, Gray JE (2014) Putting the brakes on: abscisic acid as a central environmental regulator of stomatal development. New phytol 202(2):376–391. doi:10.1111/nph.12713

    Article  CAS  PubMed  Google Scholar 

  • Chaves MM, Maroco JP, Pereira JS (2003) Understanding plant responses to drought—from genes to the whole plant. Funct Plant Biol 30(3):239–264

    Article  CAS  Google Scholar 

  • Chen ZH, Hills A, Lim CK, Blatt MR (2010) Dynamic regulation of guard cell anion channels by cytosolic free Ca2+ concentration and protein phosphorylation. Plant J Cell Mol Biol 61(5):816–825. doi:10.1111/j.1365-313X.2009.04108.x

    Article  CAS  Google Scholar 

  • Confalonieri M, Balestrazzi A, Bisoffi S, Carbonera D (2003) In vitro culture and genetic engineering of Populus spp. synergy for forest tree improvement. Plant Cell Tissue Organ Cult 72:109–138

    Article  CAS  Google Scholar 

  • Han KH, Meilan R, Ma C, Strauss SH (2000) An Agrobacterium tumefaciens transformation protocol effective on a variety of cottonwood hybrids. Plant Cell Rep 19:315–320

    Article  CAS  Google Scholar 

  • Han X, Tang S, An Y, Zheng DC, Xia XL, Yin WL (2013) Overexpression of the poplar NF-YB7 transcription factor confers drought tolerance and improves water-use efficiency in Arabidopsis. J Exp Bot 64(14):4589–4601. doi:10.1093/jxb/ert262

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hao S, Zhao T, Xia X, Yin W (2011) Genome-wide comparison of two poplar genotypes with different growth rates. Plant Mol Biol 76(6):575–591. doi:10.1007/s11103-011-9790-0

    Article  CAS  PubMed  Google Scholar 

  • Hara K, Yokoo T, Kajita R, Onishi T, Yahata S, Peterson KM, Torii KU, Kakimoto T (2009) Epidermal cell density is autoregulated via a secretory peptide, EPIDERMAL PATTERNING FACTOR 2 in Arabidopsis leaves. Plant Cell Physiol 50(6):1019–1031. doi:10.1093/pcp/pcp068

    Article  CAS  PubMed  Google Scholar 

  • Hetherington AM, Woodward FI (2003) The role of stomata in sensing and driving environmental change. Nature 424(6951):901–908

    Article  CAS  PubMed  Google Scholar 

  • Huang P, Ju HW, Min JH, Zhang X, Chung JS, Cheong HS, Kim CS (2012) Molecular and physiological characterization of the Arabidopsis thaliana oxidation-related zinc finger 2, a plasma membrane protein involved in ABA and salt stress response through the ABI2-mediated signaling pathway. Plant Cell Physiol 53(1):193–203. doi:10.1093/pcp/pcr162

    Article  CAS  PubMed  Google Scholar 

  • Jin YM, Jung J, Jeon H, Won SY, Feng Y, Kang JS, Lee SY, Cheong JJ, Koiwa H, Kim M (2011) AtCPL5, a novel Ser-2-specific RNA polymerase II C-terminal domain phosphatase, positively regulates ABA and drought responses in Arabidopsis. New Phytol 190(1):57–74. doi:10.1111/j.1469-8137.2010.03601.x

    Article  CAS  PubMed  Google Scholar 

  • Jung C, Seo JS, Han SW, Koo YJ, Kim CH, Song SI, Nahm BH, Choi YD, Cheong JJ (2008) Overexpression of AtMYB44 enhances stomatal closure to confer abiotic stress tolerance in transgenic Arabidopsis. Plant Physiol 146(2):623–635. doi:10.1104/pp.107.110981

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Karaba A, Dixit S, Greco R, Aharoni A, Trijatmiko KR, Marsch-Martinez N, Krishnan A, Nataraja KN, Udayakumar M, Pereira A (2007) Improvement of water use efficiency in rice by expression of HARDY, an Arabidopsis drought and salt tolerance gene. Proc Natl Acad Sci USA 104(39):15270–15275. doi:10.1073/pnas.0707294104

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kim TH, Bohmer M, Hu H, Nishimura N, Schroeder JI (2010) Guard cell signal transduction network: advances in understanding abscisic acid, CO2, and Ca2+ signaling. Annu Rev Plant Biol 61:561–591. doi:10.1146/annurev-arplant-042809-112226

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lee JS, Kuroha T, Hnilova M, Khatayevich D, Kanaoka MM, McAbee JM, Sarikaya M, Tamerler C, Torii KU (2012) Direct interaction of ligand-receptor pairs specifying stomatal patterning. Genes Dev 26(2):126–136. doi:10.1101/gad.179895.111

    Article  PubMed  PubMed Central  Google Scholar 

  • Liu J, Zhu J-K (1997) Proline accumulation and salt-stress-induced gene expression in a salt-hypersensitive mutant of Arabidopsis. Plant Physiol 114(2):591–596

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu L, Hu X, Song J, Zong X, Li D, Li D (2009) Over-expression of a Zea mays L. protein phosphatase 2C gene (ZmPP2C) in Arabidopsis thaliana decreases tolerance to salt and drought. J Plant Physiol 166(5):531–542

    Article  CAS  PubMed  Google Scholar 

  • Luo X, Bai X, Sun X, Zhu D, Liu B, Ji W, Cai H, Cao L, Wu J, Hu M (2013) Expression of wild soybean WRKY20 in Arabidopsis enhances drought tolerance and regulates ABA signalling. J Exp Bot 64:2155–2169

    Article  CAS  PubMed  Google Scholar 

  • Ma HS, Liang D, Shuai P, Xia XL, Yin WL (2010) The salt- and drought-inducible poplar GRAS protein SCL7 confers salt and drought tolerance in Arabidopsis thaliana. J Exp Bot 61(14):4011–4019. doi:10.1093/jxb/erq217

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • MacAlister CA, Ohashi-Ito K, Bergmann DC (2007) Transcription factor control of asymmetric cell divisions that establish the stomatal lineage. Nature 445(7127):537–540. doi:10.1038/nature05491

    Article  CAS  PubMed  Google Scholar 

  • Merlot S, Gosti F, Guerrier D, Vavasseur A, Giraudat J (2001) The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway. Plant J 25(3):295–303

    Article  CAS  PubMed  Google Scholar 

  • Monclus R, Dreyer E, Villar M, Delmotte FM, Delay D, Petit JM, Barbaroux C, Le Thiec D, Brechet C, Brignolas F (2006) Impact of drought on productivity and water use efficiency in 29 genotypes of Populus deltoides × Populus nigra. New Phytol 169(4):765–777. doi:10.1111/j.1469-8137.2005.01630.x

    Article  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Nakashima K, Ito Y, Yamaguchi-Shinozaki K (2009) Transcriptional regulatory networks in response to abiotic stresses in Arabidopsis and grasses. Plant Physiol 149(1):88–95. doi:10.1104/pp.108.129791

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nilson SE, Assmann SM (2007) The control of transpiration. Insights from Arabidopsis. Plant Physiol 143(1):19–27. doi:10.1104/pp.106.093161

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nilson SE, Assmann SM (2010) The alpha-subunit of the Arabidopsis heterotrimeric G protein, GPA1, is a regulator of transpiration efficiency. Plant Physiol 152(4):2067–2077. doi:10.1104/pp.109.148262

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ohashi-Ito K, Bergmann DC (2006) Arabidopsis FAMA controls the final proliferation/differentiation switch during stomatal development. Plant Cell 18(10):2493–2505. doi:10.1105/tpc.106.046136

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pillitteri LJ, Sloan DB, Bogenschutz NL, Torii KU (2007) Termination of asymmetric cell division and differentiation of stomata. Nature 445(7127):501–505. doi:10.1038/nature05467

    Article  CAS  PubMed  Google Scholar 

  • Quarrie S, Jones H (1977) Effects of abscisic acid and water stress on development and morphology of wheat. J Exp Bot 28(1):192–203

    Article  CAS  Google Scholar 

  • Ren X, Chen Z, Liu Y, Zhang H, Zhang M, Liu Q, Hong X, Zhu JK, Gong Z (2010) ABO3, a WRKY transcription factor, mediates plant responses to abscisic acid and drought tolerance in Arabidopsis. Plant J Cell Mol Biol 63(3):417–429. doi:10.1111/j.1365-313X.2010.04248.x

    Article  CAS  Google Scholar 

  • Royer D (2001) Stomatal density and stomatal index as indicators of paleoatmospheric CO2 concentration. Rev Palaeobot Palynol 114(1):1–28

    Article  PubMed  Google Scholar 

  • Seo YJ, Park JB, Cho YJ, Jung C, Seo HS, Park SK, Nahm BH, Song JT (2010) Overexpression of the ethylene-responsive factor gene BrERF4 from Brassica rapa increases tolerance to salt and drought in Arabidopsis plants. Mol Cells 30(3):271–277. doi:10.1007/s10059-010-0114-z

    Article  CAS  PubMed  Google Scholar 

  • Shimada T, Sugano SS, Hara-Nishimura I (2011) Positive and negative peptide signals control stomatal density. Cell Mol Life Sci CMLS 68(12):2081–2088. doi:10.1007/s00018-011-0685-7

    Article  CAS  PubMed  Google Scholar 

  • Shinozaki K, Yamaguchi-Shinozaki K (2007) Gene networks involved in drought stress response and tolerance. J Exp Bot 58(2):221–227. doi:10.1093/jxb/erl164

    Article  CAS  PubMed  Google Scholar 

  • Shpak ED, McAbee JM, Pillitteri LJ, Torii KU (2005) Stomatal patterning and differentiation by synergistic interactions of receptor kinases. Science 309(5732):290–293

    Article  CAS  PubMed  Google Scholar 

  • Shu Z, Zhang XS, Chen J, Chen GY, Xu DQ (2010) The simplification of chlorophyll content measurement. Plant Physiol Commun 46(4):399–402. doi:10.13592/j.cnki.ppj.2010.04.001

    CAS  Google Scholar 

  • Silva EC, Nogueira RJ, Vale FH, Araújo FPd, Pimenta MA (2009) Stomatal changes induced by intermittent drought in four umbu tree genotypes. Braz J Plant Physiol 21(1):33–42

    Article  Google Scholar 

  • Tricker PJ, Gibbings JG, López CMR, Hadley P, Wilkinson MJ (2012) Low relative humidity triggers RNA-directed de novo DNA methylation and suppression of genes controlling stomatal development. J Exp Bot 63(10):3799–3813

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tschaplinski T, Blake T (1989) Water relations, photosynthetic capacity, and root/shoot partitioning of photosynthate as determinants of productivity in hybrid poplar. Can J Bot 67(6):1689–1697

    Article  Google Scholar 

  • Wang WH, Chen J, Liu TW, Chen J, Han AD, Simon M, Dong XJ, He JX, Zheng HL (2014a) Regulation of the calcium-sensing receptor in both stomatal movement and photosynthetic electron transport is crucial for water use efficiency and drought tolerance in Arabidopsis. J Exp Bot 65(1):223–234. doi:10.1093/jxb/ert362

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang HL, Chen J, Tian Q, Wang S, Xia X, Yin W (2014b) Identification and validation of reference genes for Populus euphratica gene expression analysis during abiotic stresses by quantitative real-time PCR. Physiol Plant 152(3):529–545. doi:10.1111/ppl.12206

    Article  CAS  PubMed  Google Scholar 

  • Wang C, Liu S, Dong Y, Zhao Y, Geng A, Xia X, Yin W (2015) PdEPF1 regulates water-use efficiency and drought tolerance by modulating stomatal density in poplar. Plant Biotechnol J. doi:10.1111/pbi.12434

    Google Scholar 

  • Willems E, Leyns L, Vandesompele J (2008) Standardization of real-time PCR gene expression data from independent biological replicates. Anal Biochem 379(1):127–129. doi:10.1016/j.ab.2008.04.036

    Article  CAS  PubMed  Google Scholar 

  • Xiang Y, Huang Y, Xiong L (2007) Characterization of stress-responsive CIPK genes in rice for stress tolerance improvement. Plant Physiol 144(3):1416–1428. doi:10.1104/pp.107.101295

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xing HT, Guo P, Xia XL, Yin WL (2011) PdERECTA, a leucine-rich repeat receptor-like kinase of poplar, confers enhanced water use efficiency in Arabidopsis. Planta 234(2):229–241. doi:10.1007/s00425-011-1389-9

    Article  CAS  PubMed  Google Scholar 

  • Xiong L, Schumaker KS, Zhu J-K (2002) Cell signaling during cold, drought, and salt stress. Plant Cell Online 14(suppl 1):S165–S183

    CAS  Google Scholar 

  • Xu Z, Zhou G (2008) Responses of leaf stomatal density to water status and its relationship with photosynthesis in a grass. J Exp Bot 59(12):3317–3325. doi:10.1093/jxb/ern185

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yoo CY, Pence HE, Hasegawa PM, Mickelbart MV (2009) Regulation of transpiration to improve crop water use. Crit Rev Plant Sci 28(6):410–431. doi:10.1080/07352680903173175

    Article  CAS  Google Scholar 

  • Yoo CY, Pence HE, Jin JB, Miura K, Gosney MJ, Hasegawa PM, Mickelbart MV (2010) The Arabidopsis GTL1 transcription factor regulates water use efficiency and drought tolerance by modulating stomatal density via transrepression of SDD1. Plant Cell 22(12):4128–4141. doi:10.1105/tpc.110.078691

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yu H, Chen X, Hong YY, Wang Y, Xu P, Ke SD, Liu HY, Zhu JK, Oliver DJ, Xiang CB (2008) Activated expression of an Arabidopsis HD-START protein confers drought tolerance with improved root system and reduced stomatal density. Plant Cell 20(4):1134–1151. doi:10.1105/tpc.108.058263

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zeiger E, Field C (1982) Photocontrol of the functional coupling between photosynthesis and stomatal conductance in the intact leaf blue light and par-dependent photosystems in guard cells. Plant Physiol 70(2):370–375

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang X, Henriques R, Lin S-S, Niu Q-W, Chua N-H (2006) Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc 1(2):641–646

    Article  CAS  PubMed  Google Scholar 

  • Zhu JK (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53:247–273. doi:10.1146/annurev.arplant.53.091401.143329

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhu J, Dong C-H, Zhu J-K (2007) Interplay between cold-responsive gene regulation, metabolism and RNA processing during plant cold acclimation. Curr Opin Plant Biol 10(3):290–295

    Article  CAS  PubMed  Google Scholar 

  • Zsuffa L, Giordano E, Pryor L, Stettler R (1996) Trends in poplar culture: some global and regional perspectives. In: Stettler RF, Bradshaw HD Jr, Heilman PE, Hinckley TM (eds) Biology of Populus and its implications for management and conservation. NRC Research Press, Ottawa, pp 515–539

    Google Scholar 

Download references

Acknowledgments

This research was supported by the Special Fund for forestry scientific Research in the Public Interests (201304301), the Hi-Tech Research and Development Program of China (2013AA102701), the National Natural Science Foundation of China (31270656), Program for Changjiang Scholars and Innovative Research Team in University (IRT13047) and the 111 Project (B13007). We thank Dun Zhang for his helpful comments on the manuscript and technical assistance. We also thank Junna Shi for her technical assistance with scanning electron microscopy.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Xinli Xia.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liu, S., Wang, C., Jia, F. et al. Secretory peptide PdEPF2 enhances drought tolerance by modulating stomatal density and regulates ABA response in transgenic Arabidopsis thaliana . Plant Cell Tiss Organ Cult 125, 419–431 (2016). https://doi.org/10.1007/s11240-016-0957-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-016-0957-x

Keywords

Navigation