Skip to main content
SpringerLink
Log in

A method to identify early-stage transgenic Medicago truncatula with improved physiological response to water deficit

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

Abstract

Phenotypic screening after transformation experiments aiming to identify lines with the enhanced/desired trait is still a time consuming process for most agricultural crops, especially when dealing with complex physiological responses such as water deficit. In this study we evaluated the suitability of non-destructive leaf gas-exchange analysis and imaging-PAM chlorophyll a fluorescence to select transgenic lines of Medicago truncatula expressing the Trehalose-6-Phosphate Synthase 1 (AtTPS1) from Arabidopsis thaliana with altered response to water deficit (WD) and WD recovery (WDR) in the early stages of the transformation process (T0). Primary transformants (T0) with different expression levels of a constitutive AtTPS1 construct were used. Additionally, we evaluated if the expression of the transgene could be correlated with the phenotype assessed. Among tested techniques and parameters measured, the net carbon assimilation (A) from gas-exchange analysis was the best parameter to early detect lines with WD and WDR improved performance, at the earliest stages of the transformation process. With this multidisciplinary approach, we selected 3 transgenic lines TPS7, TPS10 and TPS16 for further studies, which have higher or intermediate expression levels of the transgene and improved response to WD and WDR. This work will contribute to speed-up the identification of elite lines with confidence within a large number of individuals, thus reducing time, cost and labor associated with this plant improvement strategy.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

A:

Net photosynthesis rate

Chl a :

Chlorophyll a

Chl b :

Chlorophyll b

MWD:

Moderate water deficit

PAR:

Photosynthetic active radiation

RT-qPCR:

Reverse transcription quantitative PCR

RWC:

Relative water content

SWC:

Soil water content

SWD:

Severe water deficit

WD:

Water deficit

WDR:

Water deficit recovery

WW:

Well watered

T0 :

Primary transformants

T6P:

Trehalose-6-phosphate

ΦPSII :

Effective quantum yield of the photosystem II

Ψw :

Leaf water potential

References

  • Almeida AM, Villalobos E, Araújo SS, Leyman B, van Dijk P, Alfaro-Cardoso L, Fevereiro P, Torné JM, Santos DM (2005) Transformation of tobacco with an Arabidopsis thaliana gene involved in trehalose biosynthesis increases tolerance to several abiotic stresses. Euphytica 146:165–176. doi:10.1007/s10681-005-7080-0

    Article  CAS  Google Scholar 

  • Almeida AM, Bernardes da Silva AR, Araújo SS, Cardoso AC, Santos DM, Torné JM, Marques da Silva J, Paul MJ, Fevereiro P (2007) Responses to water withdrawal of tobacco plants genetically engineered with the AtTPS1 gene: a special reference to photosynthetic parameters. Euphytica 154:113–126. doi:10.1007/s10681-006-9277-2

    Article  CAS  Google Scholar 

  • Andersen CL, Jensen JL, Ørntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245–5250. doi:10.1158/0008-5472.CAN-04-0496

    Article  CAS  PubMed  Google Scholar 

  • Araújo SS, Duque ASRLA, Santos DMMF, Fevereiro MPS (2004) An efficient transformation method to regenerate a high number of transgenic plants using a new embryogenic line of Medicago truncatula cv. Jemalong. Plant Cell Tissue Organ Cult 78:123–131. doi:10.1023/B:TICU.0000022540.98231.f8

    Article  Google Scholar 

  • Araújo SS, Duque A, Silva J, Santos D, Silva AB, Fevereiro P (2013) Water deficit and recovery response of Medicago truncatula plants expressing the ELIP-like DSP22. Biol Plant 57:159–163

    Article  Google Scholar 

  • Araújo SS, Beebe S, Crespi M, Delbreil B, González EM, Gruber V, Lejeune-Henaut I, Link W, Monteros MJ, Prats E, Rao I, Vadez V, Vaz Patto MC (2015) Abiotic stress responses in legumes: strategies used to cope with environmental challenges. Crit Rev Plant Sci 34:237–280. doi:10.1080/07352689.2014.898450

    Article  Google Scholar 

  • Bhat S, Srinivasan S (2002) Molecular and genetic analyses of transgenic plants. Plant Sci 163:673–681. doi:10.1016/S0168-9452(02)00152-8

    Article  CAS  Google Scholar 

  • Birch RG (1997) Plant transformation: problems and strategies for practical application. Annu Rev Plant Physiol Plant Mol Biol 48:297–326. doi:10.1146/annurev.arplant.48.1.297

    Article  CAS  PubMed  Google Scholar 

  • Butaye KMJ, Cammue BPA, Delauré SL, De Bolle MFC (2005) Approaches to minimize variation of transgene expression in plants. Mol Breed 16:79–91. doi:10.1007/s11032-005-4929-9

    Article  Google Scholar 

  • Capitão C, Paiva JAP, Santos DM, Fevereiro P (2011) In Medicago truncatula, water deficit modulates the transcript accumulation of components of small RNA pathways. BMC Plant Biol 11:79. doi:10.1186/1471-2229-11-79

    Article  PubMed Central  PubMed  Google Scholar 

  • Čatský J (1960) Determination of water deficit in disks cut out from leaf blades. Biol Plant 2:76–78. doi:10.1007/BF02920701

    Article  Google Scholar 

  • Confalonieri M, Cammareri M, Biazzi E, Pecchia P, Fevereiro MPS, Balestrazzi A, Tava A, Conicella C (2009) Enhanced triterpene saponin biosynthesis and root nodulation in transgenic barrel medic (Medicago truncatula Gaertn.) expressing a novel beta-amyrin synthase (AsOXA1) gene. Plant Biotechnol J 7:172–182. doi:10.1111/j.1467-7652.2008.00385.x

    Article  CAS  PubMed  Google Scholar 

  • Confalonieri M, Faè M, Balestrazzi A, Donà M, Macovei A, Valassi A, Giraffa G, Carbonera D (2014) Enhanced osmotic stress tolerance in Medicago truncatula plants overexpressing the DNA repair gene MtTdp2α (tyrosyl-DNA phosphodiesterase 2). Plant Cell Tissue Organ Cult 116:187–203. doi:10.1007/s11240-013-0395-y

    Article  CAS  Google Scholar 

  • Coombs J, Hall D, Long S, Scurlock J (1985) Techniques in bioproductivity and photosynthesis, 2nd edn. Tech Bioprod Photosynth. doi:10.1016/B978-0-08-031999-5.50001-7

    Google Scholar 

  • Da Silva JM, Arrabaça MC (2004) Contributions of soluble carbohydrates to the osmotic adjustment in the C4 grass Setaria sphacelata: a comparison between rapidly and slowly imposed water stress. J Plant Physiol 161:551–555. doi:10.1078/0176-1617-01109

    Article  PubMed  Google Scholar 

  • Debast S, Nunes-Nesi A, Hajirezaei MR, Hofmann J, Sonnewald U, Fernie AR, Börnke F (2011) Altering trehalose-6-phosphate content in transgenic potato tubers affects tuber growth and alters responsiveness to hormones during sprouting. Plant Physiol 156:1754–1771. doi:10.1104/pp.111.179903

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Delorge I, Janiak M, Carpentier S, Van Dijck P (2014) Fine tuning of trehalose biosynthesis and hydrolysis as novel tools for the generation of abiotic stress tolerant plants. Front Plant Sci 5:147. doi:10.3389/fpls.2014.00147

    Article  PubMed Central  PubMed  Google Scholar 

  • Goddijn OJ, Verwoerd TC, Voogd E, Krutwagen RW, de Graaf PT, van Dun K, Poels J, Ponstein AS, Damm B, Pen J (1997) Inhibition of trehalase activity enhances trehalose accumulation in transgenic plants. Plant Physiol 113:181–190

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, Mitros T, Dirks W, Hellsten U, Putnam N, Rokhsar DS (2012) Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res 40:D1178–D1186. doi:10.1093/nar/gkr944

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218. doi:10.1007/BF01977351

    Article  CAS  Google Scholar 

  • Imai MI (2011) Abiotic stress in plants—mechanisms and adaptations. doi:10.5772/895

  • Jang I-C, Oh SJ, Seo JS, Choi WB, Song SI, Kim CH, Kim YS, Seo HS, Choi YD, Nahm BH, Kim JK (2003) Expression of a bifunctional fusion of the Escherichia coli genes for trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase in transgenic rice plants increases trehalose accumulation and abiotic stress tolerance without stunting growth. Plant Physiol 131:516–524. doi:10.1104/pp.007237

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Li H-W, Zang B-S, Deng X-W, Wang X-P (2011) Overexpression of the trehalose-6-phosphate synthase gene OsTPS1 enhances abiotic stress tolerance in rice. Planta 234:1007–1018. doi:10.1007/s00425-011-1458-0

    Article  CAS  PubMed  Google Scholar 

  • Lichtenthaler H (1987) Chlorophylls and carotenoids—pigments of photosynthetic biomembranes. Methods Enzymol 148:350–382. doi:10.1016/0076-6879(87)48036-1

    Article  CAS  Google Scholar 

  • Lunn JE, Delorge I, Figueroa CM, van Dijck P, Stitt M (2014) Trehalose metabolism in plants. Plant J 79:544–567. doi:10.1111/tpj.12509

    Article  CAS  PubMed  Google Scholar 

  • Martínez-Barajas E, Delatte T, Schluepmann H, de Jong GJ, Somsen GW, Nunes C, Primavesi LF, Coello P, Mitchell RA, Paul MJ (2011) Wheat grain development is characterized by remarkable trehalose 6-phosphate accumulation pregrain filling: tissue distribution and relationship to SNF1-related protein kinase1 activity. Plant Physiol 156:373–381. doi:10.1104/pp.111.174524

    Article  PubMed Central  PubMed  Google Scholar 

  • Mieog JC, Howitt CA, Ral J-P (2013) Fast-tracking development of homozygous transgenic cereal lines using a simple and highly flexible real-time PCR assay. BMC Plant Biol 13:71. doi:10.1186/1471-2229-13-71

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Miranda JA, Avonce N, Suárez R, Thevelein JM, Van Dijck P, Iturriaga G (2007) A bifunctional TPS-TPP enzyme from yeast confers tolerance to multiple and extreme abiotic-stress conditions in transgenic Arabidopsis. Planta 226:1411–1421. doi:10.1007/s00425-007-0579-y

    Article  CAS  PubMed  Google Scholar 

  • Neves LO, Tomaz L, Fevereiro MPS (2001) Micropropagation of Medicago truncatula Gaertn. cv. Jemalong and Medicago truncatula ssp. Narbonensis. Plant Cell Tissue Organ Cult 67:81–84. doi:10.1023/A:1011699608494

    Article  CAS  Google Scholar 

  • Nunes CMJ, Araújo SS, Marques da Silva J, Fevereiro P, Bernandes da Silva AR (2008) Physiological responses of the legume model Medicago truncatula cv. Jemalong to water deficit. Environ Exp Bot 63:289–296. doi:10.1016/j.envexpbot.2007.11.004

    Article  CAS  Google Scholar 

  • Nunes CMJ, Araújo SS, Marques da Silva J, Fevereiro P, Bernandes da Silva AR (2009) Photosynthesis light curves: a method for screening water deficit resistance in the model legume Medicago truncatula. Ann Appl Biol 155:321–332. doi:10.1111/j.1744-7348.2009.00341.x

    Article  Google Scholar 

  • Nunes C, O’Hara LE, Primavesi LF, Delatte TL, Schluepmann H, Somsen GW, Silva AB, Fevereiro PS, Wingler A, Paul MJ (2013) The trehalose 6-phosphate/SnRK1 signaling pathway primes growth recovery following relief of sink limitation. Plant Physiol 162:1720–1732. doi:10.1104/pp.113.220657

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • O’Hara LE, Paul MJ, Wingler A (2012) How do sugars regulate plant growth and development? New insight into the role of trehalose-6-phosphate. Mol Plant. doi:10.1093/mp/sss120

    PubMed  Google Scholar 

  • Paul MJ, Primavesi LF, Jhurreea D, Zhang Y (2008) Trehalose metabolism and signaling. Annu Rev Plant Biol 59:417–441. doi:10.1146/annurev.arplant.59.032607.092945

    Article  CAS  PubMed  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res 29:e45. doi:10.1093/nar/29.9.e45

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ponnu J, Wahl V, Schmid M (2011) Trehalose-6-phosphate: connecting plant metabolism and development. Front Plant Sci 2:70. doi:10.3389/fpls.2011.00070

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Google Scholar 

  • Trindade I, Capitão C, Dalmay T, Fevereiro MP, Santos DM (2010) miR398 and miR408 are up-regulated in response to water deficit in Medicago truncatula. Planta 231:705–716. doi:10.1007/s00425-009-1078-0

    Article  CAS  PubMed  Google Scholar 

  • Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:RESEARCH0034. doi:10.1186/gb-2002-3-7-research0034

  • Wingler A (2002) The function of trehalose biosynthesis in plants. Phytochemistry 60:437–440. doi:10.1016/S0031-9422(02)00137-1

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The financial support from Fundação para a Ciência e a Tecnologia (Lisbon, Portugal) is acknowledged through research projects PTDC/AGR-GPL/099866/2008, PTDC/AGR-GPL/110224/2009 and research unit GREEN-it “Bioresources for Sustainability” (UID/Multi/04551/2013). SSA acknowledges a grant by the CARIPLO Foundation (Milan, Italy), in scope of the Integrated Project ‘Advanced Priming Technologies for the Lombardy Agro-Seed Industry-PRIMTECH’ (Action 3, Code 2013-1727). The authors would like to thank to Prof. André Almeida (Ross University, St. Kitts and Nevis) for kindly reviewing the English language of this manuscript.

Author information

Author notes

  1. S. S. Araújo

    Present address: Plant Biotechnology Laboratory, Department of Biology and Biotechnology ‘L. Spallanzani’, Università degli Studi di Pavia, via Ferrata 1, 27100, Pavia, Italy

Authors and Affiliations

  1. Biosystems and Integrative Sciences Institute (BioISI), Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016, Lisbon, Portugal

    A. Alcântara, S. Silvestre, J. Marques da Silva & A. Bernardes da Silva

  2. Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016, Lisbon, Portugal

    A. Alcântara, S. Silvestre, J. Marques da Silva, A. Bernardes da Silva & P. Fevereiro

  3. Plant Cell Biotechnology Laboratory, Instituto de Tecnologia Química e Biológica António Xavier (ITQB), Universidade Nova de Lisboa (UNL), Apartado 127, 2781-901, Oeiras, Portugal

    R. S. Morgado, P. Fevereiro & S. S. Araújo

  4. Unidade de Veterinária e Zootecnia, Instituto de Investigação Científica e Tropical (BIOTROP-CVZ), Avenida da Universidade Técnica, 1300-477, Lisbon, Portugal

    S. S. Araújo

Authors
  1. A. Alcântara
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R. S. Morgado
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S. Silvestre
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. J. Marques da Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. A. Bernardes da Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. P. Fevereiro
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. S. S. Araújo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to S. S. Araújo.

Electronic supplementary material

Below is the link to the electronic supplementary material.

11240_2015_793_MOESM1_ESM.ppt

Supplementary material 1 Figure S.1 – Representative PCR for the presence of the AtTPS1 fragment in regenerated transgenic M. truncatula lines. A 918 bp fragment was amplified in tested lines (TPS4, TPS7, TPS10, TPS14 and TPS 16) and positive control (C+, plasmid pBIN-2x35S-AtTPS1-t35S). No amplification was detected in non-transformed control (M910a) and non-template control (C-). M stands for molecular weight marker (1 kb DNA ladder) (PPT 141 kb)

Supplementary material 2 (DOC 39 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Alcântara, A., Morgado, R.S., Silvestre, S. et al. A method to identify early-stage transgenic Medicago truncatula with improved physiological response to water deficit. Plant Cell Tiss Organ Cult 122, 605–616 (2015). https://doi.org/10.1007/s11240-015-0793-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-015-0793-4

Keywords

Search

Navigation