Contrasting nitrogen fertilization treatments impact xylem gene expression and secondary cell wall lignification in Eucalyptus

Abstract

Background: 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.

Figure 1

Effects of limiting and luxuriant N fertilizations on the growth and phenotypes of Eucalyptus hybrids. (a) Representative young trees and corresponding leaves from the main stem submitted to limiting (N-) and luxuriant (N+) treatments for 30 days. Bar = 20 cm. (b) Plant height (cm; Anova: *, P < 0.01) (c) Plant basal diameter (cm; Anova: *, P < 0.01) (d) Closer view of the youngest leaves (upper part) and fully expanded leaves (lower part) from plants under N- and N + treatments. Bar = 2 cm.

Figure 2

Effects of N fertilization on Eucalyptus urophylla × E. grandis xylem secondary cell walls. (a) Phloroglucinol-HCl staining of the basal stem sections from samples grown under N fertilization treatments (N-, N and N+). Bar = 100 μm. (b) SEM images from xylem secondary cell walls under limiting (N-) and luxuriant (N+) treatments.

Figure 3

Distribution of all unigenes and differently expressed genes (DEG) in classes of transcript abundance. The classes were defined as high (RPKM ≥ 300), intermediate (300 > RPKM ≥ 25) and low (RPKM < 25) RPKM values. Axis “x” in Log₂ scale. All unigenes in grey, DEG in black.

Figure 4

Global analysis of the differently expressed genes between the contrasting N treatments. (a) PCA analysis of the DEG in each of the four treatment (b) Hierarchical clustering analysis of the DEG showing upregulated genes (red) and downregulated genes (green). (I) genes induced by nitrogen deprivation and repressed by nitrogen luxuriant supply, (II) genes repressed by nitrogen deprivation and induced by nitrogen luxuriant fertilization. All analyses were based on RPKM values for the 4 N treatments and p-value ≤ 0,01.

Figure 5

Functional analysis of the differently expressed genes between the contrasting N treatments. (a) Top ten categories of the genes induced by nitrogen deprivation and repressed by nitrogen luxuriant supply. (b) Top ten categories of the genes repressed by nitrogen deprivation and induced by nitrogen luxuriant fertilization. (c) Number of genes differently expressed in the five most represented categories of Eucalyptus DEG. Genes induced by nitrogen deprivation and repressed by nitrogen luxuriant supply are presented in dark green for Eucalyptus and light green for Populus [13]. Genes repressed by nitrogen deprivation and induced by nitrogen luxuriant fertilization in dark red for Eucalyptus and light red for Populus. FUNCAT software [35] categories: SL, subcellular localization; PS, protein synthesis; PWBF/CR, protein with binding function or cofactor requirement; M, metabolism; CTFR, cellular transport, transport facilitation and transport routes; IWE, interaction with the environment; CRDV, cell rescue, defense and virulence; UP, unclassified proteins; PF, protein fate; SIWE, systemic interaction with the environment; T, transcription. (a) and (b) values in %.

Figure 6

Expression of selected lignin biosynthesis genes by RT-qPCR.

Figure 7

Nitrogen and lignin metabolisms and expression profile from genes-related under contrasting N fertilization treatments. Nitrate (NO₃ ⁻) uptake is carried out by nitrate transporters (NRTs) and converted to nitrite (NO₂ ⁻) by the nitrate reductase (NR) and in the plastid reduced to ammonium (NH₄ ⁺). The NH₄ ⁺ is incorporated by glutamine synthethase/glutamate synthase (GS2/GOGAT) into the amino acids. Cells in N deficiency and undergoing active lignification use an efficient N recycling mechanism where the NH₄ ⁺ liberate by phenylalanine-ammonia lyase (PAL) and/or glutamate dehydrogenase (GDH) deamination by glutamate are incorporate by a cytosolic glutamine synthethase (GS1) and recycle into phenylalanine via Arogenate. Under N deficiency, high rates of lignification are maintained by the flux of N recycled. Expression profile by pairwise comparison between limiting (N-) and both luxuriant (N + and NO3) treatments; * = P < 0.01.