Skip to main content
SpringerLink
Log in

Dual effects of transgenic Brassica napus overexpressing CS gene on tolerances to aluminum toxicity and phosphorus deficiency

  • Regular Article
  • Published:
Plant and Soil Aims and scope Submit manuscript

Abstract

Background and aims

Low phosphorus (P) bioavailability and aluminum (Al) toxicity are two major constraints to plant growth in acid soil. To improve the tolerance of Brassica napus to Al toxicity and P deficiency, we generated transgenic canola (Brassica napus cv Westar) lines overexpressing a Pseudomonas aeruginosa citrate synthase (CS) gene and then investigated the effects of CS gene overexpressing in canola on enhancing tolerance to the two constraints.

Methods

The vector construction and plant transformation, molecular identification, estimation of extracellular and cellular citrate and malate concentrations, enzyme activity and gene expression analyse and Al tolerance and P acquisition assays were conducted using both hydroponics and soil culturing in the study.

Results

Both the root citrate and malate concentrations and their exudations in the two transgenic lines significantly increased compared with wild type (WT) following exposure to Al. These increases may be attributed to higher activities of the CS, malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC) enzymes in the TCA cycle and the expression of BnALMT and BnMATE in the transgenic plants following Al exposure. The primary root elongation and prolonged Al treatment (10 days) experiments revealed that the transgenic lines displayed enhanced levels of Al tolerance. In addition, they showed enhanced citrate and malate exudation when grown in P-deficient conditions. Moreover, the enzyme activities of the transgenic lines were significantly higher compared with WT in response to P-deficient stress. The soil culture experiment showed that the transgenic lines possessed improved P uptake from the soil and accumulated more P in their shoots and seeds when FePO4 was used as the sole P source.

Conclusions

These results indicate that the overexpression of the CS gene in B. napus not only leads to increased citrate synthesis and exudation but also changes malate metabolism, which confers improved tolerances to Al toxicity and P deficiency in the transgenic plants. These findings provide further insight into the dual effects of CS gene overexpression on Al toxicity and P deficiency in plants.

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
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

35S:

Cauliflower mosaic virus 35S promoter

CS:

Citrate synthase

MDH:

Malate dehydrogenase

PEPC:

Phosphoenolpyruvate carboxylase

NPT II:

Neomycin phosphotransferase

RT-PCR:

Reverse transcription-polymerase chain reaction

ALMT:

Al-activated malate transporter

MATE:

Multidrug and toxic compound extrusion

References

  • Anoop VM, Basu U, McCammon MT, McAlister-Henn L, Taylor GJ (2003) Modulation of citrate metabolism alters aluminum tolerance in yeast and transgenic canola overexpressing a mitochondrial citrate synthase. Plant Physiol 132:2205–2217

    Article  PubMed  CAS  Google Scholar 

  • Barcelo J, Poschenrieder C (2002) Fast root growth responses, root exudates, and internal detoxification as clues to the mechanisms of aluminium toxicity and resistance: a review. Environ Exp Bot 48:75–92

    Article  CAS  Google Scholar 

  • Barone P, Rosellini D, LaFayette P, Bouton J, Veronesi F, Parrott W (2008) Bacterial citrate synthase expression and soil aluminum tolerance in transgenic alfalfa. Plant Cell Rep 27:893–901

    Article  PubMed  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Clune T, Copeland L (1999) Effects of aluminium on canola roots. Plant Soil 216:27–33

    Article  CAS  Google Scholar 

  • de la Fuente JM, Ramírez-Rodríguez V, Cabrera-Ponce JL, Herrera-Estrella L (1997) Aluminum tolerance in transgenic plants by alteration of citrate synthesis. Science 276:1566–1568

    Article  PubMed  Google Scholar 

  • Delhaize E, Ryan PR, Randall PJ (1993) Aluminum tolerance in wheat (Triticum aestivum L.). II. Aluminum-stimulated excretion of malic acid from root apices. Plant Physiol 103:695–702

    PubMed  CAS  Google Scholar 

  • Delhaize E, Hebb DM, Ryan PR (2001) Expression of a Pseudomonas aeruginosa citrate synthase gene in tobacco is not associated with either enhanced citrate accumulation or efflux. Plant Physiol 125:2059–2067

    Article  PubMed  CAS  Google Scholar 

  • Delhaize E, Ryan PR, Hocking PJ, Richardson AE (2003) Effects of altered citrate synthase and isocitrate dehydrogenase expression on internal citrate concentrations and citrate efflux from tobacco (Nicotiana tabacum L.). Plant Soil 248:137–144

    Article  CAS  Google Scholar 

  • Delhaize E, Ryan PR, Hebb DM, Yamamoto Y, Sasaki T, Matsumoto H (2004) Engineering high-level aluminum tolerance in barley with the ALMT1 gene. Proc Natl Acad Sci USA 101:15249–15254

    Google Scholar 

  • Deng W, Luo KM, Li ZG, Yang YW, Hu N, Wu Y (2009) Overexpression of Citrus junos mitochondrial citrate synthase gene in Nicotiana benthamiana confers aluminum tolerance. Planta 230:355–365

    Article  PubMed  CAS  Google Scholar 

  • Donald LJ, Molgat GF, Duckworth HW (1989) Cloning, sequencing, and expression of the gene for NADH-sensitive citrate synthase of Pseudomonas aeruginosa. J Bacteriol 171:5542–5550

    PubMed  CAS  Google Scholar 

  • Fontecha G, Silva-Navas J, Benito C, Mestres MA, Espino FJ, Hernández-Riquer MV, Gallego FJ (2007) Candidate gene identification of an aluminum-activated organic acid transporter gene at the Alt4 locus for aluminum tolerance in rye (Secale cereale L.). Theor Appl Genet 114:249–260

    Article  PubMed  CAS  Google Scholar 

  • Furukawa J, Yamaji N, Wang H, Mitani N, Murata Y, Sato K, Katsuhara M, Takeda K, Ma JF (2007) An aluminum-activated citrate transporter in barley. Plant Cell Physiol 48:1081–1091

    Article  PubMed  CAS  Google Scholar 

  • Han YY, Zhang WZ, Zhang BL, Zhang SS, Wang W, Ming F (2009) One novel mitochondrial citrate synthase from Oryza sativa L. can enhance aluminum tolerance in transgenic tobacco. Mol Biotechnol 42:299–305

    Article  PubMed  CAS  Google Scholar 

  • Hinsinger P (2001) Bioavailability of soil inorganic P in the rhizosphere as affected by root-induced chemical changes: a review. Plant Soil 237:173–195

    Article  CAS  Google Scholar 

  • Hoagland DR, Arnon DI (1950) The water-culture method for growing plants without soil. California Department of Agriculture experimental station circular 347

  • Hoekenga OA, Maron LG, Piñeros MA, Canςado GMA, Shaff J, Kobayashi Y, Ryan PR, Dong B, Delhaize E, Sasaki T, Matsumoto H, Yamamoto Y, Koyama H, Kochian LV (2006) AtALMT1, which encodes a malate transporter, is identified as one of several genes critical for aluminum tolerance in Arabidopsis. Proc Natl Acad Sci USA 103:9738–9743

    Article  PubMed  CAS  Google Scholar 

  • Hoffland E, Van de Boogaard R, Nelemans J, Findenegg G (1992) Biosynthesis and root exudation of citric and malic acids in phosphate-starved rape plants. New Phytol 122:675–680

    Article  CAS  Google Scholar 

  • Johnson JF, Allan DL, Vance CP (1994) Phosphorus stress-induced proteoid roots show altered metabolism in Lupinus albus. Plant Physiol 104:657–665

    PubMed  CAS  Google Scholar 

  • Johnson JF, Allan DL, Vance CP, Weiblen G (1996) Root carbon dioxide fixation by phosphorus-deficient Lupinus albus (Contribution to organic acid exudation by proteoid roots). Plant Physiol 112:19–30

    Article  PubMed  CAS  Google Scholar 

  • Kochian LV, Hoekenga OA, Pineros MA (2004) How do crop plants tolerate acid soils? Mechanisms of aluminum tolerance and phosphorous efficiency. Annu Rev Plant Biol 55:459–493

    Article  PubMed  CAS  Google Scholar 

  • Koyama H, Takita E, Kawamura A, Hara T, Shibata D (1999) Over expression of mitochondrial citrate synthase gene improves the growth of carrot cells in Al-phosphate medium. Plant Cell Physiol 40:482–488

    Article  PubMed  CAS  Google Scholar 

  • Koyama H, Kawamura A, Kihara T, Hara T, Takita E, Shibata D (2000) Overexpression of mitochondrial citrate synthase in Arabidopsis thaliana improved growth on a phosphorus-limited soil. Plant Cell Physiol 41:1030–1037

    Article  PubMed  CAS  Google Scholar 

  • Li XF, Ma JF, Matsumoto H (2002) Aluminum-induced secretion of both citrate and malate in rye. Plant Soil 242:235–243

    Article  CAS  Google Scholar 

  • Ligaba A, Shen H, Shibata K, Yamamoto Y, Tanakamaru S, Matsumoto H (2004) The role of phosphorus in aluminium-induced citrate and malate exudation from rape (Brassica napus). Physiol Plant 120:575–584

    Article  PubMed  CAS  Google Scholar 

  • Ligaba A, Katsuhara M, Ryan PR, Shibasaka M, Matsumoto H (2006) The BnALMT1 and BnALMT2 genes from rape encode aluminum-activated malate transporters that enhance the aluminum resistance of plant cells. Plant Physiol 142:1294–1303

    Article  PubMed  CAS  Google Scholar 

  • Lipton DS, Blanchar RW, Blevins DG (1987) Citrate, malate, and succinate concentration in exudates from P-sufficient and P-stressed Medicago sativa L. seedlings. Plant Physiol 85:315–317

    Article  PubMed  CAS  Google Scholar 

  • Liu J, Magalhaes JV, Shaff J, Kochian LV (2009) Aluminum-activated citrate and malate transporters from the MATE and ALMT families function independently to confer Arabidopsis aluminum tolerance. Plant J 57:389–399

    Article  PubMed  CAS  Google Scholar 

  • Lόpez-Bucio J, de La Vega OM, Guevara-García A, Herrera-Estrella L (2000) Enhanced phosphorus uptake in transgenic tobacco plants that overproduce citrate. Nat Biotechnol 18:450–453

    Article  Google Scholar 

  • Ma JF, Furukawa J (2003) Recent progress in the research of external Al detoxification in higher plants: a minireview. J Inorg Biochem 97:46–51

    Article  PubMed  CAS  Google Scholar 

  • Ma JF, Ryan PR, Delhaize E (2001) Aluminium tolerance in plants and the complexing role of organic acids. Trends Plant Sci 6:273–278

    Article  PubMed  CAS  Google Scholar 

  • Magalhaes JV, Lui J, Guimarães CT, Lana U, Alves V, Wang YH, Schaffert RE, Hoekenga OA, Piñeros MA, Shaff JE, Klein PE, Carneiro NP, Coello CM, Trick HN, Kochian LV (2007) A gene in the multidrug and toxic compound extrusion (MATE) family confers aluminum tolerance in sorghum. Nat Genet 39:1156–1161

    Article  PubMed  CAS  Google Scholar 

  • Maron LG, Piñeros MA, Guimarães CT, Magalhaes J, Pleiman JK, Mao C, Shaff J, Belicuas SNJ, Kochian LV (2010) Two functionally distinct members of the MATE (multi-drug and toxic compound extrusion) family of transporters potentially underlie two major aluminum tolerance QTLs in maize. Plant J 61:728–740

    Article  PubMed  CAS  Google Scholar 

  • Martins N, Gonçalves S, Palma T, Romano A (2011) The influence of low pH on in vitro growth and biochemical parameters of Plantago almogravensis and P. algarbiensis. Plant Cell Tiss Org 107:113–121

    Article  CAS  Google Scholar 

  • Moloney MM, Walker JM, Sharma KK (1989) High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Rep 8:238–242

    Article  CAS  Google Scholar 

  • Osawa H, Matsumoto H (2001) Possible involvement of protein phosphorylation in aluminum-responsive malate efflux from wheat root apex. Plant Physiol 126:411–420

    Article  PubMed  CAS  Google Scholar 

  • Pal’ove-Balang P, Mistrík I (2007) Impact of low pH and aluminium on nitrogen uptake and metabolism in roots of Lotus japonicus. Biologia 62:715–719

    Article  Google Scholar 

  • Polle E, Konzak CF, Kittrick JA (1978) Visual detection of aluminum tolerance levels in wheat by hematoxylin staining of seedling roots. Crop Sci 18:823–827

    Article  CAS  Google Scholar 

  • Raghothama KG (1999) Phosphate acquisition. Annu Rev Plant Physiol Plant Mol Biol 50:665–693

    Article  PubMed  CAS  Google Scholar 

  • Ryan PR, Delhaize E, Jones DL (2001) Function and mechanism of organic anion exudation from plant roots. Annu Rev Plant Physiol Plant Mol Biol 52:527–560

    Article  PubMed  CAS  Google Scholar 

  • Ryan PR, Raman H, Gupta S, Horst WJ, Delhaize E (2009) A second mechanism for aluminum resistance in wheat relies on the constitutive efflux of citrate from roots. Plant Physiol 149:340–351

    Article  PubMed  CAS  Google Scholar 

  • Sasaki T, Yamamoto Y, Ezaki B, Katsuhara M, Ahn SJ, Ryan PR, Delhaize E, Matsumoto H (2004) A wheat gene encoding an aluminum-activated malate transporter. Plant J 37:645–653

    Article  PubMed  CAS  Google Scholar 

  • Sasaki T, Ryan PR, Delhaize E, Hebb DM, Ogihara Y, Kawaura K, Noda K, Kojima T, Toyoda A, Matsumoto M, Yamamoto Y (2006) Sequence upstream of the wheat (Triticum aestivum L.) ALMT1 gene and its relationship to aluminum resistance. Plant Cell Physiol 47:1343–1354

    Article  PubMed  CAS  Google Scholar 

  • Shen H, He LF, Sasaki T, Yamamoto Y, Zheng SJ, Ligaba A, Yan XL, Ahn SJ, Yamaguchi M, Hideo S, Matsumoto H (2005) Citrate secretion coupled with the modulation of soybean root tip under aluminum stress. Up-regulation of transcription, translation, and threonine-oriented phosphorylation of plasma membrane H+-ATPase. Plant Physiol 138:287–296

    Article  PubMed  CAS  Google Scholar 

  • Srere PA, Brazil H, Gonen L (1963) The citrate condensing enzyme of pigeon breast muscle and moth flight muscle. Acta Chem Scand 17:129–134

    Article  Google Scholar 

  • Taylor GJ (1991) Current views of the aluminum stress response: the physiological basis of tolerance. Curr Top Plant Biochem Physiol 10:57–93

    CAS  Google Scholar 

  • Vance CP, Stade S, Maxwell CA (1983) Alfalfa root nodule carbon dioxide fixation. I. Association with nitrogen fixation and incorporation into amino acids. Plant Physiol 72:469–473

    Article  PubMed  CAS  Google Scholar 

  • Vance CP, Uhde-Stone C, Allan DL (2003) Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. New Phytol 157:423–447

    Article  CAS  Google Scholar 

  • Wang J, Raman H, Zhou M, Ryan PR, Delhaize E, Hebb DM, Coombes N, Mendham N (2007) High-resolution mapping of the Alp locus and identification of a candidate gene HvMATE controlling aluminium tolerance in barley (Hordeum vulgare L.). Theor Appl Genet 115:265–276

    Article  PubMed  CAS  Google Scholar 

  • Westerman RL (1990) Soil testing and plant analysis, 3rd edn. Soil Science Society of America, Madison, WI

    Google Scholar 

  • Yang XY, Yang JL, Zhou Y, Piñeros MA, Kochian LV, Li GX, Zheng SJ (2011) A de novo synthesis citrate transporter, Vigna umbellata multidrug and toxic compound extrusion, implicates in Al-activated citrate efflux in rice bean (Vigna umbellata) root apex. Plant Cell Environ 34:2138–2148

    Article  PubMed  CAS  Google Scholar 

  • Yokosho K, Yamaji N, Ma JF (2010) Isolation and characterisation of two MATE genes in rye. Funct Plant Biol 37:296–303

    Article  CAS  Google Scholar 

  • Yokosho K, Yamaji N, Ma JF (2011) An Al-inducible MATE gene is involved in external detoxification of Al in rice. Plant J 68:1061–1069

    Article  PubMed  CAS  Google Scholar 

  • Zhang FS, Ma J, Cao YP (1997) Phosphorus deficiency enhances root exudation of low-molecular weight organic acids and utilization of sparingly soluble inorganic phosphates by radish (Raghanus satiuvs L.) and rape (Brassica napus L.) plants. Plant Soil 196:261–264

    Article  CAS  Google Scholar 

  • Zhang HW, Huang Y, Ye XS, Shi L, Xu FS (2009) Genotypic differences in phosphorus acquisition and the rhizosphere properties of Brassica napus in response to low phosphorus stress. Plant Soil 320:91–102

    Article  CAS  Google Scholar 

  • Zhao ZQ, Ma JF, Sato K, Takeda K (2003) Differential Al resistance and citrate secretion in barley (Hordeum vulgare L.). Planta 217:794–800

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Professor Yongjun Lin (Huazhong Agricultural University, China) for providing with the vector p3300-CSb containing P aeruginosa CS expression cassette. This work was supported by the grant from National Basic Research and Development Program (2011CB100301) and National Natural Science Foundation of China (31172019), China.

Author information

Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, China

    Yi Wang, Heng Xu, Jiaojiao Kou, Lei Shi, Chunyu Zhang & Fangsen Xu

  2. Microelement Research Center, Huazhong Agricultural University, Wuhan, 430070, China

    Yi Wang, Heng Xu, Jiaojiao Kou, Lei Shi, Chunyu Zhang & Fangsen Xu

Authors
  1. Yi Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Heng Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jiaojiao Kou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lei Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chunyu Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Fangsen Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fangsen Xu.

Additional information

Responsible Editor: Jian Feng Ma.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplemental Fig. 1

Sequence analysis of 431 bp fragment of BnMATE. Alignment of the nucleotides sequences of BnMATE and other known plant citrate transporter genes (AtMATE, HvAACT1, SbMATE) was carried by CLUSTAL 2.1 software (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The result showed that BnMATE shared 90%, 76% and 73% similarities with AtMATE, HvAACT1 and SbMATE, respectively. (DOC 80 kb)

Supplemental Fig. 2

Nucleotide sequence and deduced amino acids alignment of a candidate Al-activated citrate transporter (BnMATE) in Brassica napus. a, 1554 bp coding sequence of BnMATE. b, Alignment of the MATE protein from Arabidopsis (AtMATE), barley (HvAACT1), sorghum (SbMATE) and the hypothetical protein from B. napus (BnMATE). Alignments were carried out using ClustalW software (http://www.ch.embnet.org/software/ClustalW.html). Identical amino acids and similar amino acids were indicated by dark shading and light shading, respectively, using BOXSHADE software (http://www.ch.embnet.org/software/BOX_form.html). The four black lines above the sequence indicate the position of degenerate primers. (DOC 547 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, Y., Xu, H., Kou, J. et al. Dual effects of transgenic Brassica napus overexpressing CS gene on tolerances to aluminum toxicity and phosphorus deficiency. Plant Soil 362, 231–246 (2013). https://doi.org/10.1007/s11104-012-1289-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11104-012-1289-1

Keywords

Search

Navigation