Tissue and cell-specific transcriptomes in cotton reveal the subtleties of gene regulation underlying the diversity of plant secondary cell walls

Abstract

Background: Knowledge of plant secondary cell wall (SCW) regulation and deposition is mainly based on the Arabidopsis model of a 'typical' lignocellulosic SCW. However, SCWs in other plants can vary from this. The SCW of mature cotton seed fibres is highly cellulosic and lacks lignification whereas xylem SCWs are lignocellulosic. We used cotton as a model to study different SCWs and the expression of the genes involved in their formation via RNA deep sequencing and chemical analysis of stem and seed fibre.

Results: Transcriptome comparisons from cotton xylem and pith as well as from a developmental series of seed fibres revealed tissue-specific and developmentally regulated expression of several NAC transcription factors some of which are likely to be important as top tier regulators of SCW formation in xylem and/or seed fibre. A so far undescribed hierarchy was identified between the top tier NAC transcription factors SND1-like and NST1/2 in cotton. Key SCW MYB transcription factors, homologs of Arabidopsis MYB46/83, were practically absent in cotton stem xylem. Lack of expression of other lignin-specific MYBs in seed fibre relative to xylem could account for the lack of lignin deposition in seed fibre. Expression of a MYB103 homolog correlated with temporal expression of SCW CesAs and cellulose synthesis in seed fibres. FLAs were highly expressed and may be important structural components of seed fibre SCWs. Finally, we made the unexpected observation that cell walls in the pith of cotton stems contained lignin and had a higher S:G ratio than in xylem, despite that tissue's lacking many of the gene transcripts normally associated with lignin biosynthesis.

Conclusions: Our study in cotton confirmed some features of the currently accepted gene regulatory cascade for 'typical' plant SCWs, but also revealed substantial differences, especially with key downstream NACs and MYBs. The lignocellulosic SCW of cotton xylem appears to be achieved differently from that in Arabidopsis. Pith cell walls in cotton stems are compositionally very different from that reported for other plant species, including Arabidopsis. The current definition of a 'typical' primary or secondary cell wall might not be applicable to all cell types in all plant species.

Keywords: Cellulose synthase; Cotton; Gossypium hirsutum; Guaiacyl; Lignin; Primary cell wall; Secondary cell wall; Syringyl; Transcription factor; p-Hydroxyphenyl.

Fig. 1

2D–HSQC-NMR spectra of pith and xylem tissues. a, c Aliphatic/aldehyde and b, d aromatic regions of ¹H–¹³C (HSQC) spectra from cell wall gel samples in DMSO-d₆:pyridine-d₅ (4:1). a, b: Pith tissue. c, d: Xylem tissue. Levels of A, B and C in pith (A) were not quantified because of co-located polysaccharides with Aα, however the spectra allow the relative estimation. NMR lignin correlations are coloured to match the structures responsible. ABL refers to total lignin content measured as acetyl bromide lignin

Fig. 2

Summary of differentially expressed genes in cotton stem xylem, stem pith, and seed fibres. a Tissues sampled for transcriptome analysis. These included stem xylem (XYLM), stem pith (PITH), and seed fibre at 7 DPA (SF07), 14, 15, 16, DPA (SF15), and 25 DPA (SF25). DPA denotes Days Post Anthesis. b Typical cell walls found in each tissue type. c Hierarchical clustering of the differentially expressed genes across the stem and seed fibre samples. The biological replicate of each tissue is shown as a number (1–3). d Total overall number of differentially expressed genes in various comparisons between developmental or tissue types, at a p-value of 0.05. The processes that these differentially expressed genes might potentially be involved in is noted. SCW, secondary cell wall; PCW, primary cell wall

Fig. 3

NAC transcription factor expression during Gossypium hirsutum SCW and PCW formation. The heat map shows the RNA expression level as normalised FPKM of each differentially expressed NAC across the five tissues sampled. The differentially expressed NAC transcription factors were classified into ‘NAC TF groups’ based on phylogenetic similarity with Arabidopsis NACs (Additional file 7). XYLM, stem xylem; PITH, stem pith; SF07, seed fibre at 7 DPA; SF15, seed fibre at 14, 15, 16, DPA; SF25, seed fibre at 25 DPA; DPA, days post anthesis

Fig. 4

qPCR tissue profiling of select SND1 and NST1 NACs differentially expressed during cotton SCW development. qPCR based expression analysis was performed for key NACs that were differentially expressed between the different cotton SCW and PCW cell types. Comparative expression relative to ubiquitin is shown as relative abundance. These were SND1-like NACs (a) Gorai.003G077700 and (b) Gorai.008G259700, as well as NST1-related NACs (c) Gorai.007G112500 and (d) Gorai.008G130300. Expression profiling of the identified NACs in RNA from a range of tissues including a cotyledon, b hypocotyl, c young root, d petiole, e leaf blade, f petal, g stamen, h stigma, i boll coat, j upper stem, k middle stem, and a seed fibre developmental series from l 5 DPA, m 7DPA, n 10DPA, o 12 DPA, p 14 DPA, q 15 DPA, r 16 DPA, s 17 DPA, t 18 DPA, u 19 DPA, v 20 DPA, w 25 DPA Mean values ± SD are shown (n = 3), otherwise single replicates are shown. n.d., nil detected; DPA, days post anthesis

Fig. 5

MYB transcription factor expression during Gossypium hirsutum SCW and PCW formation. The heat map shows the RNA expression level as normalised FPKM of each differentially expressed MYB across the five tissues sampled. The groups are MYBs related to Arabidopsis SCW MYBs, or other MYBs/MYB-like genes. XYLM, stem xylem; PITH, stem pith; SF07, seed fibre at 7 DPA; SF15, seed fibre at 14, 15, 16, DPA; SF25, seed fibre at 25 DPA; DPA, days post anthesis

Fig. 6

WRKY transcription factor expression during Gossypium hirsutum SCW and PCW formation. The heat map shows the RNA expression level as normalised FPKM of each differentially expressed WRKY across the five tissues sampled. XYLM, stem xylem; PITH, stem pith; SF07, seed fibre at 7 DPA; SF15, seed fibre at 14, 15, 16, DPA; SF25, seed fibre at 25 DPA; DPA, days post anthesis

Fig. 7

CesA and CSL expression during Gossypium hirsutum SCW and PCW formation. The heat map shows the RNA expression level as normalised FPKM of each differentially expressed PCW CesA, SCW CesA, and CSL-like gene across the five tissues sampled. XYLM, stem xylem; PITH, stem pith; SF07, seed fibre at 7 DPA; SF15, seed fibre at 14, 15, 16, DPA; SF25, seed fibre at 25 DPA; DPA, days post anthesis

Fig. 8

FLA expression during Gossypium hirsutum SCW and PCW formation. The heat map shows the RNA expression level as normalised FPKM of each differentially expressed FLA gene. XYLM, stem xylem; PITH, stem pith; SF07, seed fibre at 7 DPA; SF15, seed fibre at 14, 15, 16, DPA; SF25, seed fibre at 25 DPA; DPA, days post anthesis

Fig. 9

Phenylpropanoid pathway gene expression during Gossypium hirsutum SCW and PCW formation. The heat map shows the RNA expression level as normalised FPKM of each differentially expressed phenylpropanoid pathway gene. These are grouped as general, lignin- specific, and flavonol-specific phenylpropanoid pathway genes. Spec, specific; XYLM, stem xylem; PITH, stem pith; SF07, seed fibre at 7 DPA; SF15, seed fibre at 14, 15, 16, DPA; SF25, seed fibre at 25 DPA; DPA, days post anthesis

Fig. 10

Concept map of cotton TF and cell wall genes for SCW synthesis. TF and cell wall gene expression in stem xylem (lignocellulosic cell walls), and in seed fibres (highly cellulosic cell walls) at transition to (SF15) and during (SF25) SCW synthesis. Bubble area is relative to transcript abundance of all the transcripts for each named gene/class. For visualisation, TF bubble size is at a larger scale (80×) than the biosynthetic and structural genes. Colour key: yellow - TF activators, orange – lignin-specific TF activator, black - TF repressors, blue - CesAs, grey - cellulose synthesis, structure and wall integrity, bronze - monolignol biosynthetic genes