Abstract
The tangentially oriented polyphenolic parenchyma (PP) and radially organized ray parenchyma in the phloem are central in the defense of conifer stems against insects and pathogens. Laser micro-dissection enables examination of cell-specific defense responses. To examine induced defense responses in Norway spruce stems inoculated with the necrotrophic blue-stain fungus Ceratocystis polonica, RNA extracted from laser micro-dissected phloem parenchyma and vascular cambium was analyzed using real-time RT-PCR (qRT-PCR) to profile transcript levels of selected resistance marker genes. The monitored transcripts included three pathogenesis-related proteins (class IV chitinase (CHI4), defensin (SPI1), peroxidase (PX3), two terpene synthesis related proteins (DXPS and LAS), one ethylene biosynthesis related protein (ACS), and a phenylalanine ammonia-lyase (PAL). Three days following inoculation, four genes (CHI4, PAL, PX3, SPI1) were differentially induced in individual cell and tissue types, both close to the inoculation site (5 mm above) and, to a lesser degree, further away (10 mm above). These resistance marker genes were all highly induced in ray parenchyma, supporting the important role of the rays in spruce defense propagation. CHI4 and PAL were also induced in PP cells and in conducting secondary phloem tissues. Our data suggests that different cell types in the secondary phloem of Norway spruce have overlapping but not fully redundant roles in active host defense. Furthermore, the study demonstrates the usefulness of laser micro-dissection coupled with qRT-PCR to characterize gene expression in different cell types of conifer bark.
Highlights
Norway spruce (Picea abies (L.) Karst.), a dominant species in Europe’s boreal forests, is susceptible to the blue-stain fungus Ceratocystis polonica that is vectored into the stem by the spruce bark beetle Ips typographus L
In phloem cross-sections polyphenolic parenchyma (PP) cells can be identified by their rounded shape, their occurrence in multiple axial rows separated by 5–7 layers of empty sieve cells, and the presence of polyphenolic globules and starch grains in their cell lumen
The ray parenchyma cells have a characteristic radially elongated shape, form chain-like rows radiating through the bark and extending into the xylem, and contain polyphenolic aggregates and starch grains that appear irregularly in small amounts (Figs. 2B and 2C)
Summary
Norway spruce (Picea abies (L.) Karst.), a dominant species in Europe’s boreal forests, is susceptible to the blue-stain fungus Ceratocystis polonica that is vectored into the stem by the spruce bark beetle Ips typographus L. The beetle-fungus complex causes extensive tree mortality over large areas (Christiansen & Bakke, 1988; How to cite this article Nagy et al (2014), Using laser micro-dissection and qRT-PCR to analyze cell type-specific gene expression in Norway spruce phloem. Among the cell types thought to be pivotal in bark defense are polyphenolic parenchyma (PP) cells and radial ray cells, which make up the major proportion of living cells of the phloem. The rays consist of parenchyma cells forming radial plates in the stem of conifers (Franceschi et al, 2005). Ray parenchyma store starch and form a living connection between the vascular cambium and the cork cambium, serving as a radial transport route for materials and signals in the bark
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