Development of a laser capture microscope-based single-cell-type proteomics tool for studying proteomes of individual cell layers of plant roots.

Yingde Zhu,Sarabjit Bhatti,Yong Yang,Hui Li,Tara Fish,Theodore W Thannhauser,Suping Zhou

doi:10.1038/hortres.2016.26

Yingde Zhu, Sarabjit Bhatti + Show 5 more

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https://doi.org/10.1038/hortres.2016.26

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Abstract

Single-cell-type proteomics provides the capability to revealing the genomic and proteomics information at cell-level resolution. However, the methodology for this type of research has not been well-developed. This paper reports developing a workflow of laser capture microdissection (LCM) followed by gel-liquid chromatography-tandem mass spectrometry (GeLC-MS/MS)-based proteomics analysis for the identification of proteomes contained in individual cell layers of tomato roots. Thin-sections (~10-μm thick, 10 sections per root tip) were prepared for root tips of tomato germinating seedlings. Epidermal and cortical cells (5000–7000 cells per tissue type) were isolated under a LCM microscope. Proteins were isolated and then separated by SDS–polyacrylamide gel electrophoresis followed by in-gel-tryptic digestion. The MS and MS/MS spectra generated using nanoLC-MS/MS analysis of the tryptic peptides were searched against ITAG2.4 tomato protein database to identify proteins contained in each single-cell-type sample. Based on the biological functions, proteins with proven functions in root hair development were identified in epidermal cells but not in the cortical cells. Several of these proteins were found in Al-treated roots only. The results demonstrated that the cell-type-specific proteome is relevant for tissue-specific functions in tomato roots. Increasing the coverage of proteomes and reducing the inevitable cross-contamination from adjacent cell layers, in both vertical and cross directions when cells are isolated from slides prepared using intact root tips, are the major challenges using the technology in proteomics analysis of plant roots.

Highlights

The structure of a typical root is organized from the outermost to the innermost rings as: epidermis, cortex, endodermis, pericycle and the stele tissues
The process of single-cell-type proteomics consists of four steps (Figure 1): (1) Preparation of thin-sectioning of root tips; (2) laser capture microdissection (LCM) and single-cell-type containing 75% (v/v) ethanol and 25% (v/v) acetic acid, at a 1:10 volume ratio of tissue to fixative on ice
The anatomical structures of the cross sections were covered by OCT solution (Figures 2a1–b2), but the epidermal and cortical cell layers were still clearly defined which allowed for collection of the two distinct types of tissues

Summary

Introduction

The structure of a typical root is organized from the outermost to the innermost rings as: epidermis, cortex, endodermis, pericycle and the stele tissues. The root epidermis, endodermis and pericyle are each formed of a single layer of cells, whereas the cortex comprises one to several cellular layers. In Arabidopsis thaliana, roots contain a single-layer cortex and tomato (Solanum lycoperisicum) roots have three layers.[1] Each of the root tissue layers is comprised of a unique cell population, demonstrating different degrees of morphological, as well as functional specialization.[2]. Due to the spatial distribution of the layers, suboptimal soil conditions impart varying degrees of effects on root cells. These cellular layers play different roles in affecting the root architectural system and functions therein. Similar phenomena of positional effects of root cellular layers are observed under osmotic, drought and salt stress treatments.[5,6] a single-cell-type analysis approach is preferred to effectively reveal the underlying molecular mechanisms regulating root developmental processes and plasticity when grown under suboptimal conditions

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