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Molecular evolution and transcriptional regulation of the oilseed rape proline dehydrogenase genes suggest distinct roles of proline catabolism during development

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Abstract

Main conclusion

Six BnaProDH1 and two BnaProDH2 genes were identified in Brassica napus genome. The BnaProDH1 genes are mainly expressed in pollen and roots’ organs while BnaProDH2 gene expression is associated with leaf vascular tissues at senescence.

Abstract

Proline dehydrogenase (ProDH) catalyzes the first step in the catabolism of proline. The ProDH gene family in oilseed rape (Brassica napus) was characterized and compared to other Brassicaceae ProDH sequences to establish the phylogenetic relationships between genes. Six BnaProDH1 genes and two BnaProDH2 genes were identified in the B. napus genome. Expression of the three paralogous pairs of BnaProDH1 genes and the two homoeologous BnaProDH2 genes was measured by real-time quantitative RT-PCR in plants at vegetative and reproductive stages. The BnaProDH2 genes are specifically expressed in vasculature in an age-dependent manner, while BnaProDH1 genes are strongly expressed in pollen grains and roots. Compared to the abundant expression of BnaProDH1, the overall expression of BnaProDH2 is low except in roots and senescent leaves. The BnaProDH1 paralogs showed different levels of expression with BnaA&C.ProDH1.a the most strongly expressed and BnaA&C.ProDH1.c the least. The promoters of two BnaProDH1 and two BnaProDH2 genes were fused with uidA reporter gene (GUS) to characterize organ and tissue expression profiles in transformed B. napus plants. The transformants with promoters from different genes showed contrasting patterns of GUS activity, which corresponded to the spatial expression of their respective transcripts. ProDHs probably have non-redundant functions in different organs and at different phenological stages. In terms of molecular evolution, all BnaProDH sequences appear to have undergone strong purifying selection and some copies are becoming subfunctionalized. This detailed description of oilseed rape ProDH genes provides new elements to investigate the function of proline metabolism in plant development.

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Abbreviations

CDS:

Coding domain sequence

DAS:

Days after sowing

ProDH:

Proline dehydrogenase

P5C:

Pyrroline-5-carboxylate

qRT-PCR:

Quantitative reverse transcription PCR

TFAA:

Total free amino acids

WGD:

Whole genome duplication

WGT:

Whole genome triplication

References

  • Adams KL, Percifield R, Wendel JF (2004) Organ-Specific silencing of duplicated genes in a newly synthesized cotton allotetraploid. Genetics 168:2217–2226

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Albert B, Le Caherec F, Niogret MF, Faes P, Avice JC, Leport L, Bouchereau A (2012) Nitrogen availability impacts oilseed rape (Brassica napus L.) plant water status and proline production efficiency under water-limited conditions. Planta 236:659–676

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  PubMed  Google Scholar 

  • Auger B, Baron C, Lucas M-O, Vautrin S, Bergès H, Chalhoub B, Fautrel A, Renard M, Nesi N (2009) Brassica orthologs from BANYULS belong to a small multigene family, which is involved in procyanidin accumulation in the seed. Planta 230:1167–1183

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Avice J-C, Etienne P (2014) Leaf senescence and nitrogen remobilization efficiency in oilseed rape (Brassica napus L.). J Exp Bot 65:3813–3824

    Article  PubMed  Google Scholar 

  • Avila-Ospina L, Moison M, Yoshimoto K, Masclaux-Daubresse C (2014) Autophagy, plant senescence, and nutrient recycling. J Exp Bot 65:3799–3811

    Article  PubMed  Google Scholar 

  • Bekaert M, Edger PP, Pires JC, Conant GC (2011) Two-phase resolution of polyploidy in the Arabidopsis metabolic network gives rise to relative and absolute dosage constraints. Plant Cell 23:1719–1728

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi I, Lipman DJ, Ostell J, Sayers EW (2013) GenBank. Nucleic Acids Res 41:D36–D42

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Buchanan-Wollaston V, Page T, Harrison E, Breeze E, Lim PO, Nam HG, Lin J-F, Wu S-H, Swidzinski J, Ishizaki K, Leaver CJ (2005) Comparative transcriptome analysis reveals significant differences in gene expression and signalling pathways between developmental and dark/starvation-induced senescence in Arabidopsis. Plant J 42:567–585

    Article  CAS  PubMed  Google Scholar 

  • Carter C, Shafir S, Yehonatan L, Palmer R, Thornburg R (2006) A novel role for proline in plant floral nectars. Naturwissenschaften 93:72–79

    Article  CAS  PubMed  Google Scholar 

  • Chalhoub B, Denoeud F, Liu S et al (2014) Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome. Science 345:950–953

    Article  CAS  PubMed  Google Scholar 

  • Chen ZJ, Pikaard CS (1997) Transcriptional analysis of nucleolar dominance in polyploid plants: biased expression/silencing of progenitor rRNA genes is developmentally regulated in Brassica. Proc Natl Acad Sci 94:3442–3447

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Cheng F, Liu S, Wu J, Fang L, Sun S, Liu B, Li P, Hua W, Wang X (2011) BRAD, the genetics and genomics database for Brassica plants. BMC Plant Biol 11:136

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Chiang HH, Dandekar AM (1995) Regulation of proline accumulation in Arabidopsis thaliana (L.) Heynh during development and in response to desiccation. Plant, Cell Environ 18:1280–1290

    Article  CAS  Google Scholar 

  • Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791

    Article  Google Scholar 

  • Franzke A, Lysak MA, Al-Shehbaz IA, Koch MA, Mummenhoff K (2011) Cabbage family affairs: the evolutionary history of Brassicaceae. Trends Plant Sci 16:108–116

    Article  CAS  PubMed  Google Scholar 

  • Funck D, Eckard S, Muller G (2010) Non-redundant functions of two proline dehydrogenase isoforms in Arabidopsis. BMC Plant Biol 10:70

    Article  PubMed Central  PubMed  Google Scholar 

  • Funck D, Winter G, Baumgarten L, Forlani G (2012) Requirement of proline synthesis during Arabidopsis reproductive development. BMC Plant Biol 12:191

    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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Graur D, Li W (1999) Fundamentals of molecular evolution. Sinauer, Sunderland

    Google Scholar 

  • Horton P, Park K-J, Obayashi T, Fujita N, Harada H, Adams-Collier CJ, Nakai K (2007) WoLF PSORT: protein localization predictor. Nucleic Acids Res 35:W585–W587

    Article  PubMed Central  PubMed  Google Scholar 

  • Hruz T, Laule O, Szabo G, Wessendorp F, Bleuler S, Oertle L, Widmayer P, Gruissem W, Zimmermann P (2008) Genevestigator v3: a reference expression database for the meta-analysis of transcriptomes. Adv Bioinform 2008:420747

    Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    CAS  PubMed Central  PubMed  Google Scholar 

  • Jenczewski E, Chèvre AM, Alix K (2013) Chromosomal and gene expression changes in Brassica Allopolyploids. Polyploid and hybrid genomics. Wiley, UK, pp 171–186

    Book  Google Scholar 

  • Kavi Kishor PB, Sreenivasulu N (2014) Is proline accumulation per se correlated with stress tolerance or is proline homeostasis a more critical issue? Plant Cell Environ 37:300–311

    Article  CAS  PubMed  Google Scholar 

  • Kishor P, Hong Z, Miao GH, Hu C, Verma D (1995) Overexpression of [delta]-Pyrroline-5-Carboxylate Synthetase increases proline production and confers osmotolerance in transgenic plants. Plant Physiol 108:1387–1394

    CAS  PubMed Central  PubMed  Google Scholar 

  • Kiyosue T, Yoshiba Y, Yamaguchi-Shinozaki K, Shinozaki K (1996) A nuclear gene encoding mitochondrial proline dehydrogenase, an enzyme involved in proline metabolism, is upregulated by proline but downregulated by dehydration in Arabidopsis. Plant Cell 8:1323–1335

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Koncz C, Kreuzaler F, Kalman Z, Schell J (1984) A simple method to transfer, integrate and study expression of foreign genes, such as chicken ovalbumin and alpha-actin in plant tumors. EMBO J 3:1029–1037

    CAS  PubMed Central  PubMed  Google Scholar 

  • Lancashire PD, Bleiholder H, Boom TVD, Langelüddeke P, Stauss R, Weber E, Witzenberger A (1991) A uniform decimal code for growth stages of crops and weeds. Ann Appl Biol 119:561–601

    Article  Google Scholar 

  • Larher FR, Aziz A, Gibon Y, Trotel-Aziz P, Sulpice R, Bouchereau A (2003) An assessment of the physiological properties of the so-called compatible solutes using in vitro experiments with leaf discs. Plant Physiol Biochem 41:657–666

    Article  CAS  Google Scholar 

  • Larher FR, Gagneul D, Niogret M-F, Emery N, Monnier C, Maerskalck V, Quillien G, Lefort C, Plasman M, Bouchard F, Bouchereau A (2013) Potentiation of proline accumulation in oilseed rape leaf discs exogenously supplied with combinations of PEG and cryoprotective agents is associated with overproduction of ABA. Environ Exp Bot 87:167–178

    Article  CAS  Google Scholar 

  • Leport L, Larher F (1988) Free proline levels in the flowers and other aerial organs of several higher plants at flowering. Comptes rendus de l’Académie des sciences 3:299–304

    Google Scholar 

  • Liang X, Zhang L, Natarajan SK, Becker DF (2013) Proline mechanisms of stress survival. Antioxid Redox Signal 19:998–1011

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Lysak MA, Cheung K, Kitschke M, Bureš P (2007) Ancestral chromosomal blocks are triplicated in Brassiceae species with varying chromosome number and genome size. Plant Physiol 145:402–410

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Magné C, Larher F (1992) High sugar content of extracts interferes with colorimetric determination of amino acids and free proline. Anal Biochem 200:115–118

    Article  PubMed  Google Scholar 

  • Mattioli R, Costantino P, Trovato M (2009) Proline accumulation in plants: not only stress. Plant Signal Behav 4:1016–1018

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Mondal WA, Dey BB, Choudhuri MA (1985) Proline accumulation as a reliable indicator of monocarpic senescence in rice cultivars. Experientia 41:346–348

    Article  CAS  Google Scholar 

  • Nakashima K, Satoh R, Kiyosue T, Yamaguchi-Shinozaki K, Shinozaki K (1998) A gene encoding proline dehydrogenase is not only induced by proline and hypoosmolarity, but is also developmentally regulated in the reproductive organs of Arabidopsis. Plant Physiol 118:1233–1241

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Nanjo T, Fujita M, Seki M, Kato T, Tabata S, Shinozaki K (2003) Toxicity of free proline revealed in an Arabidopsis T-DNA-tagged mutant deficient in proline dehydrogenase. Plant Cell Physiol 44:541–548

    Article  CAS  PubMed  Google Scholar 

  • Orsel M, Moison M, Clouet V, Thomas J, Leprince F, Canoy A-S, Just J, Chalhoub B, Masclaux-Daubresse C (2014) Sixteen cytosolic glutamine synthetase genes identified in the Brassica napus L. genome are differentially regulated depending on nitrogen regimes and leaf senescence. J Exp Bot 65:3927–3947

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ostergaard L, King G (2008) Standardized gene nomenclature for the Brassica genus. Plant methods 4:10

    Article  PubMed Central  PubMed  Google Scholar 

  • Parkin IA, Gulden SM, Sharpe AG, Lukens L, Trick M, Osborn TC, Lydiate DJ (2005) Segmental structure of the Brassica napus genome based on comparative analysis with Arabidopsis thaliana. Genetics 171:765–781

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Pfaffl MW, Hageleit M (2001) Validities of mRNA quantification using recombinant RNA and recombinant DNA external calibration curves in real-time RT-PCR. Biotechnol Lett 23:275–282

    Article  CAS  Google Scholar 

  • Posada D (2003) Using MODELTEST and PAUP* to select a model of nucleotide substitution. Current protocols in bioinformatics/editorial board, Andreas D. Baxevanis et al. Chapter 6: Unit 6 5

  • Saxena SN, Kaushik N, Sharma R (2008) Effect of abscisic acid and proline on in vitro flowering in Vigna aconitifolia. Biol Plant 52:181–183

    Article  CAS  Google Scholar 

  • Schertl P, Cabassa C, Saadallah K, Bordenave M, Savouré A, Braun H-P (2014) Biochemical characterization of proline dehydrogenase in Arabidopsis mitochondria. FEBS J 281:2794–2804

    Article  CAS  PubMed  Google Scholar 

  • Schwacke R, Grallath S, Breitkreuz KE, Stransky E, Stransky H, Frommer WB, Rentsch D (1999) LeProT1, a transporter for proline, glycine betaine, and gamma-amino butyric acid in tomato pollen. Plant Cell 11:377–392

    CAS  PubMed Central  PubMed  Google Scholar 

  • Sémon M, Wolfe KH (2007) Consequences of genome duplication. Curr Opin Genet Dev 17:505–512

    Article  PubMed  Google Scholar 

  • Szabados L, Savoure A (2010) Proline: a multifunctional amino acid. Trends Plant Sci 15:89–97

    Article  CAS  PubMed  Google Scholar 

  • Tajima F (1993) Simple methods for testing the molecular evolutionary clock hypothesis. Genetics 135:599–607

    CAS  PubMed Central  PubMed  Google Scholar 

  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30:2725–2729

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Team RC (2012) R: A language and environment for statistical computing

  • Tilsner J, Kassner N, Struck C, Lohaus G (2005) Amino acid contents and transport in oilseed rape (Brassica napus L.) under different nitrogen conditions. Planta 221:328–338

    Article  CAS  PubMed  Google Scholar 

  • Town CD, Cheung F, Maiti R, Crabtree J, Haas BJ, Wortman JR, Hine EE, Althoff R, Arbogast TS, Tallon LJ, Vigouroux M, Trick M, Bancroft I (2006) Comparative genomics of Brassica oleracea and Arabidopsis thaliana reveal gene loss, fragmentation, and dispersal after polyploidy. Plant Cell 18:1348–1359

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Trotel-Aziz P, Niogret M-F, Deleu C, Bouchereau A, Aziz A, Larher FR (2003) The control of proline consumption by abscisic acid during osmotic stress recovery of canola leaf discs. Physiol Plant 117:213–221

    Article  CAS  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: research0034.0031–0034.0011

  • Verbruggen N, Hermans C (2008) Proline accumulation in plants: a review. Amino Acids 35:753–759

    Article  CAS  PubMed  Google Scholar 

  • Verslues PE, Sharp RE (1999) Proline accumulation in maize (Zea mays L.) primary roots at low water potentials. II. Metabolic source of increased proline deposition in the elongation zone. Plant Physiol 119:1349–1360

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Verwoerd TC, Dekker BMM, Hoekema A (1989) A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res 17:2362

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wan P-J, Lü D, Guo W-C, Ahmat T, Yang L, Mu L-L, Li G-Q (2014) Molecular cloning and characterization of a putative proline dehydrogenase gene in the Colorado potato beetle Leptinotarsa decemlineata. Insect Sci 21:147–158

    Article  CAS  PubMed  Google Scholar 

  • Wang X, Wang H, Wang J et al (2011) The genome of the mesopolyploid crop species Brassica rapa. Nat Genet 43:1035–1039

    Article  CAS  PubMed  Google Scholar 

  • Widodo Patterson JH, Newbigin E, Tester M, Bacic A, Roessner U (2009) Metabolic responses to salt stress of barley (Hordeum vulgare L.) cultivars, Sahara and Clipper, which differ in salinity tolerance. J Exp Bot 60:4089–4103

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Xue X, Liu A, Hua X (2009) Proline accumulation and transcriptional regulation of proline biosynthesis and degradation in Brassica napus. BMB Rep 42:28–34

    Article  CAS  PubMed  Google Scholar 

  • Yu J, Zhao M, Wang X, Tong C, Huang S, Tehrim S, Liu Y, Hua W, Liu S (2013) Bolbase: a comprehensive genomics database for Brassica oleracea. BMC Genom 14:664

    Article  CAS  Google Scholar 

  • Zhang Z, Li J, Zhao X-Q, Wang J, Wong GK-S, Yu J (2006) KaKs_calculator: calculating Ka and Ks through model selection and model averaging. Genomics, Proteomics Bioinform 4:259–263

    Article  CAS  Google Scholar 

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Acknowledgments

We gratefully acknowledge the Genetic Resource Center (BrACySol BRC, UMR IGEPP, INRA Ploudaniel, France) for providing the seeds of the Tenor cultivar, and Fanchon Divol, Bertrand Dubreucq and Jean-Christophe Palauqui (INRA Versailles) for the kind gift of the pBI101-R1R2-GUS vector. We thank Laurent Charlon, Patrick Rolland, Patrick Leconte, Françoise Leprince and Sophie Rolland for technical assistance in plant culture, sampling and biochemical measurements. For this work Pascal Faës was supported by a PhD grant from the French Ministry of Higher Education and Research (Ministère de l’Enseignement Supérieur et de la Recherche).

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. UMR 1349 Institut de Génétique, Environnement et Protection des Plantes, INRA, Agrocampus Ouest, Université de Rennes 1, 35653, Le Rheu, France

    Pascal Faës, Carole Deleu, Françoise Le Cahérec, Emilie Montes, Vanessa Clouet, Anne-Marie Gouraud, Benjamin Albert, Gilles Lassalle, Laurent Leport, Alain Bouchereau & Marie-Françoise Niogret

  2. UMR 6553 Ecobio, CNRS Université de Rennes 1, Campus scientifique de Beaulieu, Bât 14A, 35042, Rennes, France

    Abdelkader Aïnouche

  3. UMR 1345 Institut de Recherche en Horticulture et Semences, INRA Agrocampus Ouest, Université d’Angers, 49071, Beaucouzé, France

    Mathilde Orsel

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  1. Pascal Faës
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  2. Carole Deleu
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Correspondence to Marie-Françoise Niogret.

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Faës, P., Deleu, C., Aïnouche, A. et al. Molecular evolution and transcriptional regulation of the oilseed rape proline dehydrogenase genes suggest distinct roles of proline catabolism during development. Planta 241, 403–419 (2015). https://doi.org/10.1007/s00425-014-2189-9

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