Laser Capture Microdissection Protocol for Xylem Tissues of Woody Plants

Abstract

Laser capture microdissection (LCM) enables precise dissection and collection of individual cell types from complex tissues. When applied to plant cells, and especially to woody tissues, LCM requires extensive optimization to overcome such factors as rigid cell walls, large central vacuoles, intercellular spaces, and technical issues with thickness and flatness of the sections. Here we present an optimized protocol for the laser-assisted microdissection of developing xylem from mature trees: a gymnosperm (Norway spruce, Picea abies) and an angiosperm (aspen, Populus tremula) tree. Different cell types of spruce and aspen wood (i.e., ray cells, tracheary elements, and fibers) were successfully microdissected from tangential, cross and radial cryosections of the current year’s growth ring. Two approaches were applied to achieve satisfactory flatness and anatomical integrity of the spruce and aspen specimens. The commonly used membrane slides were ineffective as a mounting surface for the wood cryosections. Instead, in the present protocol we use glass slides, and introduce a glass slide sandwich assembly for the preparation of aspen sections. To ascertain that not only the anatomical integrity of the plant tissue, but also the molecular features were not compromised during the whole LCM procedure, good quality total RNA could be extracted from the microdissected cells. This showed the efficiency of the protocol and established that our methodology can be integrated in transcriptome analyses to elucidate cell-specific molecular events regulating wood formation in trees.

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

“…S1). Whole cryosections of developing xylem were also analyzed to address RNA quality issues during LCM (Blokhina et al, 2017) and to serve as a reference transcriptome. RNA of adequate to good quality was isolated from the tracheids and ray cells, with RNA integrity numbers in the range 6.1 to 9.2.…”

“…Trunk discs were immediately frozen in liquid nitrogen and stored at 280°C. Cryosectioning was performed as described previously (Blokhina et al, 2017). Briefly, frozen stem discs were quickly (in approximately 1 min) sawn at room temperature into approximately 0.9-3 0.9-3 8-to 10-cm strips.…”

“…Earlier, LCM was used successfully with other plant tissues (Ruel et al, 2009;Abbott et al, 2010;Hogekamp et al, 2011;Cañas et al, 2014;Nagy et al, 2014). To resolve the possible contribution of ray cells in lignification of tracheids in Norway spruce developing xylem, the LCM protocol for cryosections of developing xylem was first optimized, then ray cells and tracheids were collected separately with LCM (Blokhina et al, 2017). The isolated cells were then used for RNA extraction and transcriptome analysis.…”

Tissue-specific transcriptome profiling of theArabidopsis thalianainflorescence stem reveals local cellular signatures

“…Laser capture microdissection (LCM) is an alternative tool for a precise isolation of cell material (Schad et al, 2005;Chandran et al, 2010;Agusti et al, 2011;Blokhina et al, 2016). Spatial specificity of the profiling process is lower in this case, because genetically encoded markers for cell identity can hardly be used.…”

Fixation and Laser Capture Microdissection of Plant Tissue for RNA Extraction and RNASeq Library Preparation

“…Plant tissue is usually fixed and embedded before sectioning (Weigel & Glazebrook, 2008), though there are some tissues, such as woody plant xylem tissues, for which cryosectioning is preferred (Blokhina et al, 2017;Knapp et al, 2012). Fixatives used in the preparation of tissue for LCM with the intent of downstream RNA analysis depend on the resin to be used for embedding.…”