Contrasting Nitrogen Fertilization Treatments Impact Xylem Gene Expression And Secondary Cell Wall Lignification In Eucalyptus

Nitrogen (N) is a main nutrient required for tree growth and biomass accumulation. In this study, we analyzed the effects of contrasting nitrogen fertilization treatments on the phenotypes of fast growing Eucalyptus hybrids (E. urophylla x E. grandis) with a special focus on xylem secondary cell walls and global gene expression patterns. Results: Histological observations of the xylem secondary cell walls further confirmed by chemical analyses showed that lignin was reduced by luxuriant fertilization, whereas a consistent lignin deposition was observed in trees grown in N-limiting conditions. Also, the syringyl/guaiacyl (S/G) ratio was significantly lower in luxuriant nitrogen samples. Deep sequencing RNAseq analyses allowed us to identify a high number of differentially expressed genes (1,469) between contrasting N treatments. This number is dramatically higher than those obtained in similar studies performed in poplar but using microarrays. Remarkably, all the genes involved the general phenylpropanoid metabolism and lignin pathway were found to be down-regulated in response to high N availability. These findings further confirmed by RT-qPCR are in agreement with the reduced amount of lignin in xylem secondary cell walls of these plants. Conclusions: This work enabled us to identify, at the whole genome level, xylem genes differentially regulated by N availability, some of which are involved in the environmental control of xylogenesis. It further illustrates that N fertilization can be used to alter the quantity and quality of lignocellulosic biomass in Eucalyptus, offering exciting prospects for the pulp and paper industry and for the use of short coppices plantations to produce second generation biofuels.141Rennenberg, H., Schmidt, S., Perennial lifestyle - an adaptation to nutrient limitation? (2010) Tree Physiol, 30, pp. 1047-1049. , 20696885Carpita, N.C., Progress in the biological synthesis of the plant cell wall: new ideas for improving biomass for bioenergy (2011) Curr Opin Biotechnol, 23, pp. 1-8Séguin, A., How could forest trees play an important role as feedstock for bioenergy production? (2011) Curr Opin Environ Sustain, 3, pp. 90-94Mansfield, S.D., Kang, K.Y., Chapple, C., Designed for deconstruction - poplar trees altered in cell wall lignification improve the efficacy of bioethanol production (2012) New Phytol, 194, pp. 91-101. , 22239166Mizrachi, E., Mansfield, S.D., Myburg, A.A., Cellulose factories: advancing bioenergy production from forest trees (2012) New Phytol, 194, pp. 54-62. , 22474687Demura, T., Ye, Z.H., Regulation of plant biomass production (2010) Curr Opin Plant Biol, 13, pp. 299-304. , 20381410Xu, G., Fan, X., Miller, A.J., Plant nitrogen assimilation and use efficiency (2012) Annu Rev Plant Biol, 63, pp. 153-182. , 22224450Pitre, F.E., Cooke, J.E.K., Mackay, J.J., Short-term effects of nitrogen availability on wood formation and fibre properties in hybrid poplar (2007) Trees, 21, pp. 249-259Pitre, F.E., Pollet, B., Lafarguette, F., Cooke, J.E.K., Mackay, J.J., Lapierre, C., Effects of increased nitrogen supply on the lignification of poplar wood (2007) J Agric Food Chem, 55, pp. 10306-10314. , 17988087Pitre, F.E., Lafarguette, F., Boyle, B., Pavy, N., Caron, S., Dallaire, N., Poulin, P.L., Mackay, J.J., High nitrogen fertilization and stem leaning have overlapping effects on wood formation in poplar but invoke largely distinct molecular pathways (2010) Tree Physiol, 30, pp. 1273-1289. , 20739427Euring, D., Löfke, C., Teichmann, T., Polle, A., Nitrogen fertilization has differential effects on N allocation and lignin in two Populus species with contrasting ecology (2012) Trees, 26, pp. 1933-1942Li, H., Li, M., Luo, J., Cao, X., Qu, L., Gai, Y., Jiang, X., Luo, Z.B., N-fertilization has different effects on the growth, carbon and nitrogen physiology, and wood properties of slow- and fast-growing Populus species (2012) J Exp Bot, 63, pp. 6173-6185. , 23028021Plavcová, L., Hacke, U.G., Almeida-Rodriguez, A.M., Li, E., Douglas, C.J., Gene expression patterns underlying changes in xylem structure and function in response to increased nitrogen availability in hybrid poplar (2012) Plant Cell Environ, 36, pp. 186-199. , 22734437Novaes, E., Kirst, M., Chiang, V., Winter-Sederoff, H., Sederoff, R., Lignin and biomass: a negative correlation for wood formation and lignin content in trees (2010) Plant Physiol, 154, pp. 555-561. , 20921184Eldridge, K., Davidson, J., Harwood, C., Van Wyk, G., (1993) Eucalypt Domestication and Breeding, , Clarendon Press, Oxford, UKPotts, B.M., Genetic Improvement of Eucalypt (2004) Encyclopedia of Forest Science, pp. 1480-1490. , Elsevier Science, Oxford, UK, Burley J, Evans J, Youngquist JA, 10.1016/B0-12-145160-7/00084-3Grattapaglia, D., Kirst, M., Eucalyptus applied genomics: from gene sequences to breeding tools (2008) New Phytol, 179, pp. 911-929. , 18537893Mizrachi, E., Hefer, C.A., Ranik, M., Joubert, F., Myburg, A.A., De novo assembled expressed gene catalog of a fast-growing Eucalyptus tree produced by Illumina mRNA-Seq (2010) BMC Genomics, 11, p. 681. , 21122097Zhong, R., Ye, Z.H., Transcriptional regulation of lignin biosynthesis (2009) Plant Signal Behav, 4 (11), pp. 1028-1034. , 19838072Baucher, M., Halpin, C., Petit-Conil, M., Boerjan, W., Lignin: genetic engineering and impact on pulping (2003) Crit Rev Biochem Mol Biol, 38, pp. 305-350. , 14551235Vanholme, R., Brecht Demedts, B., Morreel, K., Ralph, J., Boerjan, W., Lignin biosynthesis and structure (2010) Plant Physiol, 153 (3), pp. 895-905. , 20472751Canam, T., Campbell, M.M., Genes and nitrogen fuel wood formation (2009) New Phytol, 182, pp. 783-785. , 19646065Novaes, E., Osorio, L., Drost, D.R., Miles, B.L., Boaventura-Novaes, C.R., Benedict, C., Dervinis, C., Kirst, M., Quantitative genetic analysis of biomass and wood chemistry of Populus under different nitrogen levels (2009) New Phytol, 182, pp. 878-890. , 19291008Grima-Pettenati, J., Soler, M., Camargo, E.L.O., Wang, H., Transcriptional Regulation of the Lignin Biosynthetic Pathway Revisited: New Players and Insights (2012) Advances in Botanical Research 61, pp. 173-218. , Academic Press, Burlington, Jouanin L, Lapierre CPeng, M., Hudson, D., Schofield, A., Tsao, R., Yang, R., Gu, H., Bi, Y.M., Rothstein, S.J., Adaptation of Arabidopsis to nitrogen limitation involves induction of anthocyanin synthesis which is controlled by the NLA gene (2008) J Exp Bot, 59 (11), pp. 2933-2944. , 18552353Lepikson-Neto, J., Alves, A., Simões, R., Deckmann, A.C., Camargo, E.L.O., Salazar, M.M., Scatollin Rio, M.C., Rodrigues, J.C., Flavonoid supplementation reduces the extractive content and increases the Syringyl/Guaiacyl ratio in eucalyptus grandis x eucalyptus urophylla hybrid trees (2013) BioResources, 8 (2), pp. 1747-1757Voelker, L.S., Lachenbruch, B., Meinzer, F.C., Strauss, S.H., Reduced wood stiffness and strength, and altered stem form, in young antisense 4CL transgenic poplars with reduced lignin contents (2011) New Phytol, 189, pp. 1096-1109. , 21158867Plomion, C., Leprovost, G., Stokes, A., Wood formation in trees (2001) Plant Physiol, 127 (4), pp. 1513-1523. , 11743096Pilate, G., Déjardin, A., Laurans, F., Leplé, J.C., Tension wood as a model for functional genomics of wood formation (2004) New Phytol, 164, pp. 63-72Pilate, G., Chabbert, B., Cathala, B., Yoshinaga, A., Leplé, J.C., Laurans, F., Lapierre, C., Ruel, K., Lignification and tension wood (2004) C R Biol, 327, pp. 889-901. , 15587080Paux, E., Carocha, V., Marques, C., Mendes de Sousa, A., Borralho, N., Sivadon, P., Grima-Pettenati, J., Transcript profiling of Eucalyptus xylem genes during tension wood formation (2005) New Phytol, 167, pp. 89-100. , 15948833Luo, Z.-B., Langenfeld-Heyser, R., Calfapietra, C., Polle, A., Influence of free air CO2 enrichment (EUROFACE) and nitrogen fertilisation on the anatomy of juvenile wood of three poplar species after coppicing (2005) Trees, 19, pp. 109-118Cassan-Wang, H., Soler, M., Yu, H., Camargo, E.L.O., Carocha, V., Ladouce, N., Savelli, B., Grima-Pettenati, J., Reference genes for high-throughput quantitative RT-PCR analysis of gene expression in organs and tissues of eucalyptus grown in various environmental conditions (2012) Plant Cell Physiol, 53 (12), pp. 2101-2116. , 23161857Salazar, M.M., Nascimento, L.C., Camargo, E.L., Gonçalves, D.C., Lepikson Neto, J., Marques, W.L., Teixeira, P.J., Pereira, G.A., Xylem transcription profiles indicate potential metabolic responses for economically relevant characteristics of Eucalyptus species (2013) BMC Genomics, 14, p. 201. , 23521840Ruepp, A., Zollner, A., Maier, D., Albermann, K., Hani, J., Mokrejs, M., Tetko, I., Mewes, H.W., The FunCat, a functional annotation scheme for systematic classification of proteins from whole genomes (2004) Nucleic Acids Res, 32, pp. 5539-5545. , 15486203Åkerfelt, M., Morimoto, R.I., Sistonen, L., Heat shock factors: integrators of cell stress, development and lifespan (2010) Nat Rev Mol Cell Biol, 11, pp. 545-555. , 20628411Xu, C., Jiang, Z., Huang, B., Nitrogen deficiency-induced protein changes in immature and mature leaves of creeping bentgrass (2011) J Am Soc Hortic Sci, 136, pp. 399-407Scheller, H.V., Ulvskov, P., Hemicelluloses (2010) Annu Rev Plant Biol, 61, pp. 263-289. , 20192742Doering, A., Lathe, R., Persson, S., An update on xylan synthesis (2012) Mol Plant, 5 (4), pp. 769-771. , 22628543Berthet, S., Demont-Caulet, N., Pollet, B., Bidzinski, P., Cézard, L., Le Bris, P., Borrega, N., Jouanin, L., Disruption of LACCASE4 and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems (2011) Plant Cell, 23 (3), pp. 1124-1137. , 21447792Corea, O.R.A., Bedgar, D.L., Davin, L.B., Lewis, N.G., The arogenate dehydratase gene family: towards understanding differential regulation of carbon flux through phenylalanine into primary versus secondary metabolic pathways (2012) Phytochemistry, 82, pp. 22-37. , 22818526Cantón, F.R., Suárez, M.F., Cánovas, F.M., Molecular aspects of nitrogen mobilization and recycling in trees (2005) Photosynth Res, 83, pp. 265-278. , 16143856Watanabe, M., Yumi, O., Itoh, Y., Yasuda, K., Kamachi, K., Ratcliffe, R.G., Deamination role of inducible glutamate dehydrogenase isoenzyme 7 in Brassica napus leaf protoplasts (2011) Phytochemistry, 72 (7), pp. 587-593. , 21353684Hanson, A.D., Roje, S., One-carbon metabolism in higher plants (2001) Annu Rev Plant Physiol Plant Mol Biol, 52, pp. 119-137. , 11337394van den Broeck, H.C., Maliepaard, C., Ebskamp, M.J.M., Toonen, M.A.J., Koops, A.J., Differential expression of genes involved in C1 metabolism and lignin biosynthesis in wooden core and bast tissues of fibre hemp (Cannabis sativa L.) (2008) Plant Sci, 174, pp. 205-220Demura, T., Fukuda, H., Transcriptional regulation in wood formation (2007) Trends Plant Sci, 12 (2), pp. 64-70. , 17224301Du, J., Groover, A., Transcriptional regulation of secondary growth and wood formation (2010) J Integr Plant Biol, 52 (1), pp. 17-27. , 20074137Déjardin, A., Laurans, F., Arnaud, D., Breton, C., Pilate, G., Leplé, J.C., Wood formation in angiosperms (2010) C R Biol, 333, pp. 325-334. , 20371107Goicoechea, M., Lacombe, E., Legay, S., Mihaljevic, S., Rech, P., Jauneau, A., Lapierre, C., Grima-Pettenati, J., EgMYB2, a new transcriptional activator from Eucalyptus xylem, regulates secondary cell wall formation and lignin biosynthesis (2005) Plant J, 43, pp. 553-567. , 16098109Legay, S., Lacombe, E., Goicoechea, M., Brière, C., Séguin, A., Mackay, J., Grima-Pettenati, J., Molecular characterization of EgMYB1, a putative transcriptional repressor of the lignin biosynthetic pathway (2007) Plant Sci, 173, pp. 542-549Legay, S., Sivadon, P., Blervacq, A.S., Pavy, N., Baghdady, A., Tremblay, L., Levasseur, C., Grima-Pettenati, J., EgMYB1, an R2R3 MYB transcription factor from eucalyptus negatively regulates secondary cell wall formation in Arabidopsis and poplar (2010) New Phytol, 188, pp. 774-786. , 20955415Wang, H., Avci, U., Nakashima, J., Hahn, M.G., Chen, F., Dixon, R.A., Mutation of WRKY transcription factors initiates pith secondary wall formation and increases stem biomass in dicotyledonous plants (2010) PNAS, 107 (51), pp. 22338-22343. , 21135241Zhong, R., Lee, C., Zhou, J., McCarthy, R.L., Ye, Z.H., A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis (2008) Plant Cell, 20, pp. 2763-2782. , 18952777Pratelli, R., Voll, L.M., Horst, R.J., Frommer, W.B., Pilot, G., Stimulation of nonselective amino acid export by glutamine dumper proteins (2010) Plant Physiol, 152 (2), pp. 762-773. , 20018597Alekseeva, A.A., Savin, S.S., Tishkov, V.I., NAD(+)-dependent formate dehydrogenase from plants (2011) Acta Nat, 3 (4), pp. 38-54Lin, S.-H., Kuo, H.-F., Canivenc, G., Lin, C.-S., Lepetit, M., Hsu, P.-K., Tillard, P., Tsay, Y.-F., Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport (2008) Plant Cell, 20 (9), pp. 2514-2528. , 18780802Wang, R., Guegler, K., LaBrie, S.T., Crawford, N.M., Genomic analysis of a nutrient response in Arabidopsis reveals diverse expression patterns and novel metabolic and potential regulatory genes induced by nitrate (2000) Plant Cell, 12, pp. 1491-1509. , 10948265Trevisan, S., Manoli, A., Begheldo, M., Nonis, A., Enna, M., Vaccaro, S., Caporale, G., Quaggiotti, S., Transcriptome analysis reveals coordinated spatiotemporal regulation of hemoglobin and nitrate reductase in response to nitrate in maize roots (2011) New Phytol, 192, pp. 338-352. , 21762167Perazzoli, M., Dominici, P., Romero-Puertas, M.C., Zago, E., Zeier, A., Sonoda, M., Lamb, C., Delledonne, M., Arabidopsis nonsymbiotic hemoglobin AHb1 modulates nitric oxide bioactivity (2004) Plant Cell, 16, pp. 2785-2794Rubin, G., Tohge, T., Matsuda, F., Saito, K., Scheible, W.R., Members of the LBD family of transcription factors repress anthocyanin synthesis and affect additional nitrogen responses in Arabidopsis (2009) Plant Cell, 21 (11), pp. 3567-3584. , 19933203Bernard, S.M., Habash, D.Z., The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling (2009) New Phytol, 182, pp. 608-620. , 19422547Cánovas, F.M., Avila, C., Cantón, F.R., Cañas, R.A., de la Torre, F., Ammonium assimilation and amino acid metabolism in conifers (2007) J Exp Bot, 58 (9), pp. 2307-2318. , 17490997Suárez, M.F., Avila, C., Gallardo, F., Cantón, F.R., García-Gutiérrez, A., Claros, M.G., Cánovas, F.M., Molecular and enzymatic analysis of ammonium assimilation in woody plants (2002) J Exp Bot, 53, pp. 891-904. , 11912232Graindorge, M., Giustini, C., Jacomin, A.C., Kraut, A., Curien, G., Matringe, M., Identification of a plant gene encoding glutamate/aspartate- prephenate aminotransferase: the last homeless enzyme of aromatic amino acids biosynthesis (2010) FEBS Lett, 584, pp. 4357-4360. , 20883697Maeda, H., Yoo, H., Dudareva, N., Prephenate aminotransferase directs plant phenylalanine biosynthesis via arogenate (2011) Nat Chem Biol, 7, pp. 19-21. , 21102469Sarruge, J.R., Soluções nutritivas (1975) Summa Phytopatologica, 1, pp. 231-233Fritz, C., Palacios-Rojas, N., Feil, R., Stitt, M., Regulation of secondary metabolism by the carbon-nitrogen status in tobacco: nitrate inhibits large sectors of phenylpropanoid metabolism (2006) Plant J, 46, pp. 533-548. , 16640592Alves, A., Schwanninger, M., Pereira, H., Rodrigues, J., Analytical pyrolysis as a direct method to determine the lignin content in wood - Part 1: Comparison of pyrolysis lignin with Klason lignin" (2006) J Anal Appl Pyrolysis, 76 (1-2), pp. 209-213Alves, A., Simões, R., Stackpole, D.J., Vaillancourt, R.E., Potts, B.M., Schwanninger, M., Rodrigues, J., Determination of the syringyl/guaiacyl ratio of Eucalyptus globulus wood lignin by near infrared-based partial least squares regression models using analytical pyrolysis as the reference method (2011) J Near Infrared Spectrosc, 19 (5), pp. 343-348Alves, A., Simões, R., Santos, C., Potts, B., Rodrigues, J., Schwanninger, M., Determination of Eucalyptus globulus wood extractives content by NIR-based PLS-R models: comparison between extraction procedures (2012) J Near Infrared Spectrosc, 20, pp. 275-285Rodrigues, J.C., Meier, D., Faix, O., Pereira, H., Determination of tree to tree variation in syringyl/guaiacyl ratio of Eucalyptus globulus wood lignin by analytical pyrolysis (1999) J Anal Appl Pyrolysis, 48 (2), pp. 121-128Le Provost, G., Herrera, R., Paiva, J.A.P., Chaumeil, P., Salin, F., Plomion, C., A micromethod for high throughput RNA extraction in forest trees (2007) Biol Res, 40, pp. 291-297. , 18449457Quail, M.A., Kozarewa, I., Smith, F., Scally, A., Stephens, P.J., Durbin, R., Swerdlow, H., Turner, D.J., A large genome center's improvements to the Illumina sequencing system (2008) Nat Methods, 5, pp. 1005-1010. , 19034268Rengel, D., San Clemente, H., Servant, F., Ladouce, N., Paux, E., Wincker, P., Couloux, A., Grima-Pettenati, J., A new genomic resource dedicated to wood formation in Eucalyptus (2009) BMC Plant Biol, 9, p. 36. , 19327132Keller, G., Marchal, T., San Clemente, H., Navarro, M., Ladouce, N., Wincker, P., Coloux, A., Marque, C., Development and functional annotation of an 11,303-EST collection from Eucalyptus for studies of cold tolerance (2009) Tree Genet Genomes, 5, pp. 317-327Li, R., Yu, C., Li, Y., Lam, T.W., Yiu, S.M., Kristiansen, K., Wang, J., SOAP2: an improved ultrafast tool for short read alignment (2009) Bioinformatics, 25 (15), pp. 1966-1967. , 19497933Grabherr, M.G., Haas, B.J., Yassour, M., Levin, J.Z., Thompson, D.A., Amit, I., Adiconis, X., Regev, A., Full-length transcriptome assembly from RNA-Seq data without a reference genome (2011) Nat Biotechnol, 29, pp. 644-652. , 21572440Koski, L.B., Gray, L.W., Lang, B.F., Burger, G., AutoFACT: an automatic functional annotation and classification tool (2005) BMC Bioinformatics, 6, p. 151. , 15960857Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs (1997) Nucleic Acids Res, 25 (17), pp. 3389-3402. , 9254694Suzek, B.E., Huang, H., McGarvey, P., Mazumber, R., Wu, C.H., Uniref: comprehensive and non-redundant UniProt reference clusters (2007) Bioinformatics, 23 (10), pp. 1282-1288. , 17379688Bateman, A., Birney, E., Cerruti, L., Durbin, R., Etwiller, L., Griffiths-Jones, S., Howe, K.L., Sonnhammer, E.L.L., The Pfam protein families database (2002) Nucleic Acids Res, 30 (1), pp. 276-280. , 11752314Kanehisa, M., Goto, S., KEGG: Kyoto encyclopedia of genes and genomes (2000) Nucleic Acids Res, 28 (1), pp. 27-30. , 10592173Conesa, A., Gotz, S., García-Gómez, J.M., Terol, J., Talón, M., Robles, M., Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research (2005) Bioinformatics, 21 (18), pp. 3674-3676. , 16081474Mortazavi, A., Williams, B.A., McCue, K., Schaeffer, L., Wold, B., Mapping and quantifying mammalian transcriptomes by RNA-Seq (2008) Nat Methods, 5, pp. 621-628. , 18516045Ulitsky, I., Maron-Katz, A., Shavit, S., Sagir, D., Linhart, C., Elkon, R., Tanay, A., Shamir, R., Expander: from expression microarrays to networks and functions (2010) Nat Protoc, 5 (2), pp. 303-322. , 20134430Pfaffl, M.W., A new mathematical model for relative quantification in real-time RT-PCR (2001) Nucleic Acids Res, 29 (900), pp. 2002-200