Secondary cell wall composition and candidate gene expression in developing willow (Salix purpurea) stems

Abstract

The properties of the secondary cell wall (SCW) in willow largely determine the suitability of willow biomass feedstock for potential bioenergy and biofuel applications. SCW development has been little studied in willow and it is not known how willow compares with model species, particularly the closely related genus Populus. To address this and relate SCW synthesis to candidate genes in willow, a tractable bud culture-derived system was developed in Salix purpurea, and cell wall composition and RNA-Seq transcriptome were followed in stems during early development. A large increase in SCW deposition in the period 0-2 weeks after transfer to soil was characterised by a big increase in xylan content, but no change in the frequency of substitution of xylan with glucuronic acid, and increased abundance of putative transcripts for synthesis of SCW cellulose, xylan and lignin. Histochemical staining and immunolabeling revealed that increased deposition of lignin and xylan was associated with xylem, xylem fibre cells and phloem fibre cells. Transcripts orthologous to those encoding xylan synthase components IRX9 and IRX10 and xylan glucuronyl transferase GUX1 in Arabidopsis were co-expressed, and showed the same spatial pattern of expression revealed by in situ hybridisation at four developmental stages, with abundant expression in proto-xylem, xylem fibre and ray parenchyma cells and some expression in phloem fibre cells. The results show a close similarity with SCW development in Populus species, but also give novel information on the relationship between spatial and temporal variation in xylan-related transcripts and xylan composition.

Fig. 1

Developmental stages of Salix purpurea. a Typical plants at the four developmental stages (0, 2, 4 and 6 weeks). Petri dish shows plantlets at the time of transfer to soil. Yellow lines and arrow in the inset image (0-week stage) indicate the position of internode 3. b Resin sections stained with Toluidine blue O showing the anatomical development of internode 3 at the same stages. px protoxylem, ca cambium, pf phloem fibres, sx secondary xylem. Bar 250 μm. Inset: dry weight of internode 3. Line is fitted exponential curve, y =  0.26.e^0.71x, R ² = 0.98

Fig. 2

Cell wall composition of internode 3 of developing stems. Averages from three replicate samples of pooled internodes. a HPAEC-PAD monosaccharide compositional analysis of willow AIR. b Xylan content. c Degree of substitution from PACE analysis of willow AIR following endo-xylanase digestion. The oligosaccharide products were quantified using PACE. Values are means of three biological replicates analysed four times ±SD. Asterisks indicate significant difference (Student’s t test, P < 0.01). d The amount of methylation of GlcA substitution of xylan was determined by endo-xylanase digestion of willow AIR. The oligosaccharides were quantified using DASH and the frequency of methylation was determined from the quantity of GlcAXyl₄ relative to the sum of GlcAXyl₄ and MeGlcAXyl₄. Values are means of three biological replicates analysed four times ±SD. The asterisk indicates significant difference (Student’s t test, P < 0.05)

Fig. 3

Abundance of transcripts associated with synthesis of cellulose (a, b), xylan (c, d) and lignin (e, f) in internode 3 of developing willow stem. The key shows the gene family with the closest gene homologue in Arabidopsis indicated in parentheses; some of these willow transcripts are referred to in the text using “Sp” followed by this Arabidopsis gene name. Some paralogues of transcripts shown in c and d have been omitted for clarity: a DUF579 IRX15, a GUX1-like and two DUF579 GXM transcripts; these all have profiles more similar in shape to the other xylan transcripts than the GXM transcript shown. A complete list of poplar gene ids to which the willow RNA-Seq reads mapped for the transcripts shown plus lower abundance ones associated with synthesis of cell wall polymers is given in Supplementary Table S1. Three xylan synthesis transcripts selected for in situ analysis are indicated (asterisk)

Fig. 4

Cell wall development and gene expression in stems at 0, 2, 4 and 6 weeks (from left to right). a Lignification shown by Phloroglucinol–HCl staining (pink colour). Staining is localised in the protoxylem at the 0-week stage and maturing secondary xylem and phloem fibres. b–m Expression of IRX10 (b–e), IRX9 (f–i) and GUX1 (j–m) transcripts shown by the purple staining occurs mainly in protoxylem (px), developing secondary xylem, especially in xylem fibres (xf) and in newly formed phloem fibres (pf). Expression is generally absent from mature phloem fibres (mpf) at the 6-week stage (arrows). Also indicated are cambium (ca) and ray parenchyma (rp). n–q Localisation of xylan in cell walls of 0- to 6-week-old stems detected with the LM10 antibody. Bars 250 μm (a), 100 μm (b–q)