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. 2019 Dec;181(4):1552-1572.
doi: 10.1104/pp.19.00743. Epub 2019 Sep 26.

Ray Parenchymal Cells Contribute to Lignification of Tracheids in Developing Xylem of Norway Spruce

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Ray Parenchymal Cells Contribute to Lignification of Tracheids in Developing Xylem of Norway Spruce

Olga Blokhina et al. Plant Physiol. .
Free PMC article

Abstract

A comparative transcriptomic study and a single-cell metabolome analysis were combined to determine whether parenchymal ray cells contribute to the biosynthesis of monolignols in the lignifying xylem of Norway spruce (Picea abies). Ray parenchymal cells may function in the lignification of upright tracheids by supplying monolignols. To test this hypothesis, parenchymal ray cells and upright tracheids were dissected with laser-capture microdissection from tangential cryosections of developing xylem of spruce trees. The transcriptome analysis revealed that among the genes involved in processes typical for vascular tissues, genes encoding cell wall biogenesis-related enzymes were highly expressed in both developing tracheids and ray cells. Interestingly, most of the shikimate and monolignol biosynthesis pathway-related genes were equally expressed in both cell types. Nonetheless, 1,073 differentially expressed genes were detected between developing ray cells and tracheids, among which a set of genes expressed only in ray cells was identified. In situ single cell metabolomics of semi-intact plants by picoliter pressure probe-electrospray ionization-mass spectrometry detected monolignols and their glycoconjugates in both cell types, indicating that the biosynthetic route for monolignols is active in both upright tracheids and parenchymal ray cells. The data strongly support the hypothesis that in developing xylem, ray cells produce monolignols that contribute to lignification of tracheid cell walls.

Figures

Figure 1.
Figure 1.
Photomicrographs of tangential cryosections of 4-year-old and 40-year-old Norway spruce stems stained with safranin and Alcian Blue. Note the smaller cell size in the young stem (A) in comparison with the old stem (B). Developing xylem (blue) and mature xylem (red) are stained with Alcian Blue and safranin, for cellulosic and lignified cell walls, respectively. In B, only the developing xylem is shown. Tracheid (T), resin duct (RD), and rays (R) are indicated. Bars = 200 μm.
Figure 2.
Figure 2.
REVIGO Treemaps of 500 most highly expressed genes in developing xylem. The GO enrichment was realized using the Web-based tool agriGO (http://bioinfo.cau.edu.cn/agriGO/index.php; Du et al., 2010). Developing tracheids (A) and developing ray parenchymal cells (B) are shown. See Supplemental Table S1 for gene identifiers.
Figure 3.
Figure 3.
REVIGO Treemaps of differentially expressed genes. Genes more highly expressed in developing tracheids are shown in A, and those more highly expressed in developing ray parenchymal cells are shown in B (Padj < 0.01). The GO enrichment was realized using the Web-based tool agriGO (http://bioinfo.cau.edu.cn/agriGO/index.php; Du et al., 2010). See Supplemental Table S2 for gene identifiers.
Figure 4.
Figure 4.
Average-normalized expression of genes important in lignification. NAC and MYB transcription factors (A), shikimate, Phe, and Tyr pathway genes leading to Phe and Tyr (B), and phenylpropanoid and monolignol biosynthesis pathway genes leading to monolignols (C) are shown in ray parenchymal cells, upright tracheids, and whole sections of developing xylem with expression levels higher than 5 (variance-stabilizing transformation [VST] > 5) at least in one of the sample types. Red stars count for significantly higher and blue for significantly lower levels of expression in ray cells than in tracheids (Padj ≤ 0.05). Shikimate, Phe, and Tyr pathway genes are according to Maeda and Dudareva (2012): ADH, arogenate dehydrogenase; ADT, arogenate dehydratase; CM, chorismate mutase; CS, chorismate synthase; DAHP synthase, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase; DHD-SDH, 3-dehydroquinate dehydratase-shikimate dehydrogenase; DHQS, 3-dehydroquinate synthase; EPSP synthase, 5-enolpyruvylshikimate 3-phosphate synthase; PDT, prephenate dehydratase; PPA-AT, prephenate aminotransferase; SK, shikimate kinase. General phenylpropanoid pathway genes are as follows: CCoAOMT, caffeoyl-CoA 3-O-methyltransferase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate-CoA ligase; HCT, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase; PAL, phenylalanine ammonia lyase. Monolignol pathway genes are as follows: CAD, cinnamyl alcohol dehydrogenase; CCR, cinnamoyl-CoA reductase; COMT, caffeate/5-hydroxyconiferaldehyde O-methyltransferase. p-Coumaroyl shikimate 3-hydroxylase is missing from the figure due to annotation difficulties related to cytochrome P450 genes, as enzyme activity cannot be inferred from the gene sequence. See Supplemental Table S3 for gene identifiers.
Figure 5.
Figure 5.
Coexpression subnetworks of transcription factors differentially expressed in developing ray cells and tracheids. Red color indicates transcription factors more highly expressed in ray cells, and blue indicates those more highly expressed in tracheids (Padj < 0.01). Solid lines indicate positive correlation, and dashed lines indicate negative correlation. Note that MA_95898g0010, which is more highly expressed in tracheids than in ray cells, is a sequence homolog of PgNAC8.
Figure 6.
Figure 6.
PicoPPESI-MS negative ion mode mass spectra directly obtained from ray cells (RC) and tracheids (TR) in semi-intact Norway spruce plants. Data are representative of similar experiments with eight to nine cells in each cell type.
Figure 7.
Figure 7.
Putative lignin biosynthesis route in Norway spruce developing xylem based on single-cell metabolomics. Red arrows indicate transport of monolignol glucosides through the membrane via, for example, secondary active transport based on a proton gradient. Purple arrows indicate transport of monolignol alcohols through the membrane via, for example, ABC transporters (Miao and Liu, 2010; Alejandro et al., 2012). Yellow arrows indicate transport of monolignol glucosides to a neighboring cell via plasmodesmata. Green arrows indicate liberation of monolignol glucosides into the apoplast after vacuolar collapse. Monolignol names in gray were not detected. Bar = 50 μm.

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