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

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.

Hunting monolignol transporters: membrane proteomics and biochemical transport assays with membrane vesicles of Norway spruce

“…In the datasets analyzed, ~5% of the genes correlated with the baits with a correlation coefficient of >0.8. Altogether, 8494 genes had a correlation coefficient >0.8 with the bait genes in at least one of the datasets ( Supplementary Table S1B ), of which 765 were present in the Norwood data ( Jokipii-Lukkari et al , 2017 ), 3630 in the tissue culture data ( Laitinen et al , 2017 ), 3236 in the ray-tracheid data ( Blokhina et al , 2019 ), and 2587 in the ConGenIE data ( Nystedt et al , 2013 ). The gene lists showed some overlap ( Fig.…”

“… Venn diagram representing genes whose expression correlated with the bait genes in four different datasets: Tissue culture ( Laitinen et al , 2017 ), ConGenIE ( Nystedt et al , 2013 ), Norwood ( Jokipii-Lukkari et al , 2017 ), and Ray-tracheid ( Blokhina et al , 2019 ). The number of ABC, MATE, and MFS transporters is given in parentheses.…”

“…Data from pre-existing gene expression studies in Norway spruce were used to search for suitable monolignol transporter candidates. The datasets were: (i) ConGenIE ( http://congenie.org/; Nystedt et al , 2013 ), 22 libraries from different organs and different time points of the whole aerial part of a tree; (ii) Norwood ( Jokipii-Lukkari et al , 2017 ), 51 libraries of thin layers cut from phloem and cambium over the developing xylem into the mature xylem; (iii) Ray-tracheid comparison ( Blokhina et al , 2019 ), nine libraries comprising developing ray parenchymal cells and tracheids separately, and whole sections of developing xylem; (iv) Tissue culture ( Laitinen et al , 2017 ), 18 libraries of lignin-forming cultured cells under lignin-forming and non-lignin-forming conditions. In a recent study, gene expression in xylem throughout the year was examined and a highly correlating set of 12 monolignol biosynthesis genes with a peak of expression during xylem development was identified ( Jokipii-Lukkari et al , 2018 ).…”

“…Partially enriched PM vesicles (upper phase fraction; UP) ( Kärkönen et al , 2014 ) were prepared from MF material and their proteomes were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). To take a closer look at membrane-localized, putatively lignification-related proteins, protein identifications were compared with several published gene expression studies ( Nystedt et al , 2013 ; Jokipii-Lukkari et al , 2017 ; Laitinen et al , 2017 ; Blokhina et al , 2019 ), and candidates for monolignol transport and other lignification-related processes were identified. Biochemical monolignol transport assays with multiple candidate transport forms showed that MF prepared from developing xylem, but not those from developing phloem, had a H + -gradient-dependent, secondary active, tonoplast-localized transport of coniferin and p -coumaryl alcohol glucoside.…”

On-site single pollen metabolomics reveals varietal differences in phosphatidylinositol synthesis under heat stress conditions in rice

“…This analytical method consists of single cell metabolomics, called 'picolitre pressure-probe-electrospray-ionization mass spectrometry (picoPPESI-MS)' 19 and accurate environmental control. The picoPPESI-MS technique has been successfully applied to several cell-specific research in different plant tissues, such as tomato trichomes 19 , rice endosperms 18 , and developing xylems in Norway spruce 20 . In this study, we have directly inserted the tip of a finely tapered quartz capillary tip into the developing pollen grains in intact plants growing under controlled high temperature environments to identify the internal metabolites.…”