Plant and Soil

, Volume 359, Issue 1–2, pp 297–319 | Cite as

Microarray analysis of humic acid effects on Brassica napus growth: Involvement of N, C and S metabolisms

  • Laëtitia JanninEmail author
  • Mustapha Arkoun
  • Alain Ourry
  • Philippe Laîné
  • Didier Goux
  • Maria Garnica
  • Marta Fuentes
  • Sara San Francisco
  • Roberto Baigorri
  • Florence Cruz
  • Fabrice Houdusse
  • José-Maria Garcia-Mina
  • Jean-Claude Yvin
  • Philippe Etienne
Regular Article


Background & aims

Winter rapeseed (Brassica napus) is characterized by a low N recovery in seeds and requires high rates of fertilization to maintain yield. Its nutrient use efficiency could be improved by addition of a biostimulant such as humic acids whose physiological effects have been described previously in some plant species. However, to our knowledge, no study has focused on transcriptomic analyses to determine metabolic targets of this extract.


A preliminary screening of ten humic acids revealed a significant effect of one of them (HA7) on rapeseed root growth. Microarray analysis was then used on HA7-treated or non-treated plants to characterize changes in gene expression that were further supported by physiological evidence.


Stimulation of nitrogen uptake (+15% in shoots and +108% in roots) and assimilation was found to be increased in a similar manner to growth while sulfate content (+76% in shoots and +137% in roots) was more strongly stimulated leading to higher sulfate accumulation. In parallel, microscopic analysis showed an enhancement of chloroplast number per cell.


It is therefore suggested that HA7, which promotes plant growth and nutrient uptake, could be used as a supplementary tool to improve rapeseed nitrogen use efficiency.


Brassica napus Humic acid Microarray Growth promotion Nutrient uptake Chloroplast 



This study was a part of AZOSTIMER project selected and supported by the Pôle de compétitivité Mer-Bretagne and funded by the French FUI (Fond Unique Interministériel), Brittany Region and Saint-Malo Agglomeration. We thank Marie-Paule Bataillé and Raphaël Ségura for IRMS analyses. We acknowledge Patrick Beauclair for LICOR measurements, Julie Levallois for technical assistance in RNA extractions and q-PCR analyses, Xavier Sarda and Anne-Françoise Ameline for helping with plant culture and harvest and finally Nicolas Elie from GRECAN (Groupe Régional d’Etude sur le CANcer, Histo-imagerie quantitative, Caen, France) for microscopic analysis. We thank Laurence Cantrill for improving the English of the manuscript.

Supplementary material

11104_2012_1191_MOESM1_ESM.xls (296 kb)
Supplemental Table S1 List of the differentially expressed genes in shoots and roots of rapeseed after 3 days of HA7 supply to the roots. (XLS 295 kb)
11104_2012_1191_MOESM2_ESM.xls (35 kb)
Supplemental Table S2 List of the differentially expressed genes in shoots and roots of rapeseed after 30 days of HA7 supply to the roots. (XLS 35 kb)


  1. Abdallah M, Etienne P, Ourry A, Meuriot F (2011) Do initial S reserve and mineral S availability alter leaf S-N mobilization and leaf senescence in oilseed rape? Plant Sci 180:511–520PubMedCrossRefGoogle Scholar
  2. Abiven S, Menasseri S, Chenu C (2009) The effect of organic input over time on soil aggregate stability—a literature analysis. Soil Biol Biochem 41:1–12CrossRefGoogle Scholar
  3. Aguirre E, Leménager D, Bacaicoa E, Fuentes M, Baigorri R, Zamarreño AM, García-Mina JM (2009) The root application of a purified leonardite humic acid modifies the transcriptional regulation of the main physiological root responses to Fe deficiency in Fe-sufficient cucumber plants. Plant Physiol Biochem 47:215–223PubMedCrossRefGoogle Scholar
  4. Andersson S, Nilsson SI, Saetre P (2000) Leaching of dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) in mor humus as affected by temperature and pH. Soil Biol Biochem 32:1–10CrossRefGoogle Scholar
  5. Arancon NQ, Edwards CA, Bierman P, Metzger JD, Lucht C (2005) Effects of vermicomposts produced from cattle manure, food waste and paper waste on the growth and yield of peppers field. Pedobiologia 49:297–306CrossRefGoogle Scholar
  6. Arancon NQ, Adwards CA, Bierman P (2006) Influence of vermicomposts on field strawberries: Part 2. Effects on soil microbiological and chemical properties. Bioresource Technology 97:831–840PubMedCrossRefGoogle Scholar
  7. Atiyeh RM, Lee S, Edwards CA, Arancon NQ, Metzger JD (2002) The influence of humic acids derived from earthworm-processed organic wastes on plant growth. Bioresource Technology 84:7–14PubMedCrossRefGoogle Scholar
  8. Ayuso M, Hernandez T, Garcia C, Pascual JA (1996) Stimulation of barley growth and nutrient absorption by humic substances originating from various organic materials. Bioresource Technology 57:251–257CrossRefGoogle Scholar
  9. Bossuyt H, Six J, Hendrix PF (2007) Protection of soil carbon by microaggregates within earthworm casts. Soil Biol Biochem 37:251–258CrossRefGoogle Scholar
  10. Bungard R, Wingler A, Morton J, Andrews M, Press M, Scholes J (1999) Ammonium can stimulate nitrate and nitrite reductase in the absence of nitrate in Clematis vitalba. Plant Cell Environ 22:859–866CrossRefGoogle Scholar
  11. Canellas LP, Olivares FL, Okorokova-Façanha AL, Façanha AR (2002) Humic acids isolated from earthworm compost enhance root elongation, lateral root emergence, and plasma membrane H+-ATPase activity in maize roots. Plant Physiol 130:1951–1957PubMedCrossRefGoogle Scholar
  12. Canellas LP, Piccolo A, Dobbss LB, Spaccini R, Olivares FL, Zandonadi DB, Façanha AR (2010) Chemical composition and bioactivity properties of size-fractions separated from a vermicompost humic acid. Chemosphere 78:457–466PubMedCrossRefGoogle Scholar
  13. Carletti P, Masi A, Spolaore B, Polverino De Laureto P, De Zorzi M, Turetta L, Ferretti M, Nardi S (2008) protein expression changes in maize roots in response to humic substances. J Chem Ecol 34:804–818PubMedCrossRefGoogle Scholar
  14. Castaings L, Marchive C, Meyer C, Krapp A (2010) Nitrogen signaling in Arabidopsis: how to obtain insights into a complex signaling network. J Exp Bot 62:1391–1347PubMedCrossRefGoogle Scholar
  15. Cesco S, Nikolic M, Römheld V, Varanini Z, Pinton R (2002) Uptake of 59Fe from 59Fe-humate complexes by cucumber and barley plants. Plant Soil 241:121–128CrossRefGoogle Scholar
  16. Christl I, Kretzschmar R (2001) Relating ion binding by fulvic and humic acids to chemical composition and molecular size. 1. Proton binding. Environ Sci Technol 35:2505–2511PubMedCrossRefGoogle Scholar
  17. Conte P, Piccolo A (1999) Conformational arrangement of dissolved humic substances. Influence of solution composition on association of humic molecules. Environ Sci Technol 33:1682–1690CrossRefGoogle Scholar
  18. Cookson WR, Abaye DA, Marschner P, Murphy DV, Stockdale EA, Goulding KWT (2005) The contribution of soil organic matter fractions to carbon and nitrogen mineralization and microbial community size and structure. Soil Biol Biochem 37:1726–1737CrossRefGoogle Scholar
  19. Daniel-Vedele F, Filleur S, Caboche M (1998) Nitrate transport: a key step in nitrate assimilation. Curr Opin Plant Biol 1:235–239PubMedCrossRefGoogle Scholar
  20. Dejoux JF, Recous S, Meynard JM, Trinsoutrot I, Leterme P (2000) The fate of nitrogen from winter-frozen rapeseed leaves: mineralization, fluxes to the environment and uptake by rapeseed crop in spring. Plant Soil 218:257–272CrossRefGoogle Scholar
  21. Dell’Agnola G, Nardi S (1987) Hormone-like effect and enhanced nitrate uptake induced by depolycondensed humic fractions obtained from Allolobophora rosea and A. caliginosa faeces. Biol Fertil Soils 4:115–118Google Scholar
  22. Desclos M, Dubousset L, Etienne P, Bonnefoy J, Lecahérec F, Satoh H, Ourry A, Avice JC (2008) A proteomic profiling approach to reveal a novel role of BnD22 (Brassica napus drought 22)/water soluble chlorophyll binding protein in young leaves during nitrogen remobilization induced by stressful condition. Plant Physiol 147:1830–1844PubMedCrossRefGoogle Scholar
  23. Dobrev P, Kamiek M (2002) Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction. J Chromatogr A 950:21–29PubMedCrossRefGoogle Scholar
  24. Dreccer MF, Schapendonk AHM, Slafer GA, Rabbinge R (2000) Comparative response of wheat and oilseed rape to nitrogen supply: absorption and utilisation efficiency of radiation and nitrogen during reproductive stage determining yield. Plant Soil 220:189–205CrossRefGoogle Scholar
  25. Esparza I, Salinas I, Santamaria C, Garcia-Mina JM, Fernandez JM (2005) Electrochemical and theoretical complexation studies for Zn and Cu with individual polyphenols. Analytica Chimica Acta 543:267–274CrossRefGoogle Scholar
  26. Etienne P, Desclos M, Le Gou L, Gombert J, Bonnefoy J, Maurel K, Le Dily F, Ourry A, Avice JC (2007) N-protein mobilization associated with the leaf senescence process in oilseed rape in concomitant with the disappearance of trypsin inhibitor activity. Funct Plant Biol 34:895–906CrossRefGoogle Scholar
  27. Eyheraguibel B, Silvestre J, Morard P (2008) Effects of humic substances derived from organic waste enhancement on the growth and mineral nutrition of maize. Bioresource Technology 99:4206–4212PubMedCrossRefGoogle Scholar
  28. Garcia-Mina JM, Antolin MC, Sanchez-Diaz M (2004) Metal-humic complexes and plant micronutrient uptake: a study based on different plant species cultivated in diverse soil types. Plant Soil 258:57–68CrossRefGoogle Scholar
  29. Garcia-Mina JM (2006) Stability, solubility and maximum metal-binding capacity in metal-humic complexes involving humic substances extracted from peat and organic compost. Org Geochem 37:1960–1972CrossRefGoogle Scholar
  30. Imbufe AU, Patti AF, Burrow D, Surapaneni A, Jackson WR, Milner AD (2005) effects of potassium humate on aggregate stability of two soils from Victoria, Australia. Geoderma 125:321–330CrossRefGoogle Scholar
  31. Itoh R, Fujiwara M, Nagata N, Yoshida S (2001) A chloroplast protein homologous to the eubacterial topotogical specificity factor MinE plays a role in chloroplast division. Plant Physiol 127:1644–1655PubMedCrossRefGoogle Scholar
  32. Keeling AA, McCallum KR, Beckwith CP (2003) Mature green waste compost enhances growth and nitrogen uptake in wheat (Triticum aestivum L.) and oilseed rape (Brassica napus L.) through the action of water-extractable factors. Bioresource Technology 90:127–132PubMedCrossRefGoogle Scholar
  33. Kojo KH, Fujiwara MT, Itoh RD (2009) Involvement of AtMinE in plastid morphogenesis in various tissues of Arabidopsis thaliana. Biosci Biotechnol Biochem 73:2632–2639PubMedCrossRefGoogle Scholar
  34. Kozuch J, Pempkowiak J (1996) Molecular weight of humic acids as a major property of the substances influencing the accumulation rate of cadmium by a blue mussel (Mytilus edulis). Environ Int 22:585–589CrossRefGoogle Scholar
  35. Krouk G, Lacombe B, Bielach A, Perrine-Walker F, Malinska K, Mounier E, Hoyerova K, Tillard P, Leon S, Ljung K, Zazimalova E, Benkova E, Nacry P, Gojon A (2010) Nitrate-regulated auxin transport by NRT1.1 defines a mechanism for nutrient sensing in plants. Dev Cell 18:927–937PubMedCrossRefGoogle Scholar
  36. Livak KJ, Schmittgen TD (2001) Analysis of gene expression data using real-time quantitative PCR and the 2(-DDc(t)) method. Methods 25:402–408PubMedCrossRefGoogle Scholar
  37. Malagoli P, Laine P, Rossato L, Ourry A (2005) Dynamics of nitrogen uptake and mobilization in field-grown winter oilseed rape (Brassica napus L.) from stem extension to harvest. II A 15N-labelling based simulation model of N partitioning between vegetative and reproductive tissues. Ann of Bot 95:1187–1198CrossRefGoogle Scholar
  38. Mora V, Bacaicoa E, Zamarreno AM, Aguirre E, Garnica M, Fuentes M, Garcia-Mina JM (2010) Action of humic acid on promotion of cucumber shoot growth involves nitrate-related changes associated with the root-to-shoot distribution of cytokinins, polyamines and mineral nutrients. J Plant Physiol 167:633–642PubMedCrossRefGoogle Scholar
  39. Moroney JV, Bartlett SG, Samuelsson G (2001) Carbonic anhydrase in plants and algae. Plant Cell Envir 24:141–153CrossRefGoogle Scholar
  40. Muscolo A, Panuccio MR, Abenavoli MR, Concheri G, Nardi S (1996) Effect of molecular complexity and acidity of earthworm faeces humic fractions on glutamate dehydrogenase, glutamine synthetase, and phosphoenolpyruvate carboxylase in Daucus carota άII cells. Biol Fertil Soils 22:83–88CrossRefGoogle Scholar
  41. Muscolo A, Cutrupi S, Nardi S (1998) IAA detection in humic substances. Soil Biol Biochem 30:1199–1201CrossRefGoogle Scholar
  42. Muscolo A, Bovalo F, Gionfriddo F, Nardi S (1999) Earthworm humic matter produces auxin-like effects on Daucus carota cell growth and nitrate metabolism. Soil Biol Biochem 31:1303–1311CrossRefGoogle Scholar
  43. Muscolo A, Sidari M, Attina M, Francioso O, Tugnoli V, Nardi S (2007a) Biological activity of humic substances is related to their chemical structure. Soil Sci Soc Am J 71:75–85CrossRefGoogle Scholar
  44. Muscolo A, Sidari M, Francioso O, Tugnoli V, Nardi S (2007b) The auxin-like activity of humic substances is related to membrane interactions in carrot cell culture. J Chem Ecol 33:115–129PubMedCrossRefGoogle Scholar
  45. Nardi S, Concheri G, Pizzeghello D, Sturaro A, Rella R, Parvoli G (2000a) Soil organic matter mobilization by root exudates. Chemosphere 41:653–658PubMedCrossRefGoogle Scholar
  46. Nardi S, Pizzeghello D, Gessa C, Ferrarese L, Trainotti L, Casadoro G (2000b) A low molecular weight humic fraction on nitrate uptake and protein synthesis in maize seedlings. Soil Biol Biochem 32:415–419CrossRefGoogle Scholar
  47. Nardi S, Pizzeghello D, Reniero F, Rascio N (2000c) Chemical and biochemical properties of humic substances isolated from forest soils and plant growth. Soil Sci Soc Am J 64:639–645CrossRefGoogle Scholar
  48. Nardi S, Pizzeghello D, Muscolo A, Vianello A (2002a) Physiological effects of humic substances on higher plants. Soil Biol Biochem 34:1527–1536CrossRefGoogle Scholar
  49. Nardi S, Sessi E, Pizzeghello D, Sturaro A, Rella R, Parvoli G (2002b) Biological activity of soil organic matter mobilized by root exudates. Chemosphere 46:1075–1081PubMedCrossRefGoogle Scholar
  50. Nardi S, Tosoni M, Pizzeghello D, Provenzano MR, Cilenti A, Sturaro A, Rella R, Vianello A (2005) Chemical characteristics and biological activity of organic substances extracted from soil by root exudates. Soil Sci Soc Am J 69:2012–2019CrossRefGoogle Scholar
  51. Nardi S, Muscolo A, Vaccaro S, Baiano S, Spaccini R, Piccolo A (2007) Relationship between molecular characteristics of soil humic fractions and glycolytic pathway and krebs cycle in maize seedlings. Soil Biol Biochem 39:3138–3146CrossRefGoogle Scholar
  52. Okazaki K, Kabeya Y, Suzuki K, Mori T, Ichikawa T, Matsui M, Nakanishi H, Miyagishima S (2009) The PLASTID DIVISION1 and 2 component of the chloroplast division machinery determine the rate of chloroplast division in land plant cell differentiation. Plant Cell 21:1769–1780PubMedCrossRefGoogle Scholar
  53. Park SY, Yu JW, Li J, Yoo SC, Lee NY, Lee SK, Jeong SW, Seo HS, Koh HJ (2007) The senescence-induced staygreen protein regulates chlorophyll degradation. Plant Cell 19:1649–1664PubMedCrossRefGoogle Scholar
  54. Piccolo A, Pietramellara G, Mbagwu JSC (1997a) Reduction in soil loss from erosion-susceptible soils amended with humic substances from oxidized coal. Soil Technology 10:235–245CrossRefGoogle Scholar
  55. Piccolo A, Pietramellara G, Mbagwu JSC (1997b) Use of humic substances as soil conditioners to increase aggregate stability. Geoderma 75:267–277CrossRefGoogle Scholar
  56. Pinton R, Cesco S, Iacolettig G, Astolfi S, Varanini Z (1999) Modulation of NO3- uptake by water-extractable humic substances: involvement of root plasma membrane H+ATPase. Plant Soil 215:155–161CrossRefGoogle Scholar
  57. Puglisi E, Fragoulis G, Ricciuti P, Cappa F, Spaccini R, Piccolo A, Trevisan M, Grecchio C (2009) Effects of humic acid and its size-fractions on the bacterial community of soil rhizosphere under maize (Zea mays L.). Chemosphere 77:829–837PubMedCrossRefGoogle Scholar
  58. Quaggiotti S, Ruperti B, Pizzeghello D, Francioso O, Tugnoli V, Nardi S (2004) Effect of low molecular size humic substances on nitrate uptake and expression of genes involved in nitrate transport in maize (Zea mays L.). J Exp Bot 55:803–813PubMedCrossRefGoogle Scholar
  59. Ramanan R, Kannan K, Vinayagamoorthy N, Ramkumar KM, Sivanesan SD, Chakrabarti T (2009) Purification and characterization of a novel plant-type carbonic anhydrase from Bacillus subtilis. Biotechnol Bioproc Eng 14:32–37CrossRefGoogle Scholar
  60. Schmidt W, Santi S, Pinton R, Varanini Z (2007) Water-extractable humic substances alter root development and epidermal cell pattern in Arabidopsis. Plant Soil 300:259–267CrossRefGoogle Scholar
  61. Smejkalova D, Piccolo A (2008) Aggregation and disaggregation of humic supramolecular assemblies by NMR diffusion ordered spectrometry (DOSY-NMR). Environ Sci Technol 42:699–706PubMedCrossRefGoogle Scholar
  62. Sutton R, Sposito G (2005) Molecular structure in soil humic substances: the new view. Environ Sci Technol 39:9009–9015PubMedCrossRefGoogle Scholar
  63. Swift RS (1996) Organic matter characterization. In: Sparks DL (ed) Methods of soil analysis Part 3 Chemical methods SSSA Book Ser5. SSSA, Madison, pp 1011–1069Google Scholar
  64. Tejada M, Gonzalez JM (2004) Effects of foliar application of a byproduct of the two-step olive oil mill process on rice yield. Eur J Agron 21:31–40CrossRefGoogle Scholar
  65. Trevisan S, Botton A, Vaccaro S, Vezzaro A, Quaggiotti S, Nardi S (2011) Humic substances affect Arabidopsis thaliana physiology by altering the expression of genes involved in primary metabolism, growth and development. Environ. Exp Bot doi: 10.1016/j.envexpbot.2011.04.017
  66. Varga B, Kiss G, Galambos I, Gelencser A, Hlavay J, Krivacsy Z (2000) Secondary structure of humic acids. Can micelle-like conformation be proved by aqueous size exclusion chromatography? Environ Sci Technol 34:3303–3306CrossRefGoogle Scholar
  67. Vaughan DA, Balazs E, Heslop-Harrison JS (2007) From crop domestication to super-domestication. Annals of Botany 100:893–901PubMedCrossRefGoogle Scholar
  68. Visser SA (1985) Viscometric studies on molecular weight fractions of fulvic and humic acids of aquatic, terrestrial and microbial origin. Plant Soil 87:209–221CrossRefGoogle Scholar
  69. Zandonadi DB, Canellas LP, Façanha AR (2007) Indolacetic and humic acids induce lateral root development through a concerted plasmalemma and tonoplast H+ pumps activation. Planta 225:1583–1595PubMedCrossRefGoogle Scholar
  70. Zhang X, Schmidt RE (1997) The impact of growth regulators on the α-tocopherol status in water-stressed Poa pratensis. Int Turfgrass Soc Res J 8:1364–1371Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Laëtitia Jannin
    • 1
    • 2
    Email author
  • Mustapha Arkoun
    • 1
    • 2
  • Alain Ourry
    • 1
    • 2
  • Philippe Laîné
    • 1
    • 2
  • Didier Goux
    • 3
  • Maria Garnica
    • 4
  • Marta Fuentes
    • 4
  • Sara San Francisco
    • 4
  • Roberto Baigorri
    • 4
  • Florence Cruz
    • 5
  • Fabrice Houdusse
    • 5
  • José-Maria Garcia-Mina
    • 4
  • Jean-Claude Yvin
    • 5
  • Philippe Etienne
    • 1
    • 2
  1. 1.Université de Caen Basse-Normandie, UMR 950 Ecophysiologie VégétaleCaen CedexFrance
  2. 2.INRA, UMR 950 Ecophysiologie VégétaleCaen CedexFrance
  3. 3.Université de Caen Basse-Normandie, Centre de Microscopie Appliquée à la Biologie (CMABio)Caen CedexFrance
  4. 4.TIMAC Agro SpainOrcoyenSpain
  5. 5.Centre de Recherche International en Agroscience, CRIAS-TAI, Groupe RoullierDinardFrance

Personalised recommendations