Label-Free Comparative Proteomic Analysis Combined with Laser-Capture Microdissection Suggests Important Roles of Stress Responses in the Black Layer of Maize Kernels

Abstract

The black layer (BL) is traditionally used as an indicator for kernel harvesting in maize, as it turns visibly dark when the kernel reaches physiological maturity. However, the molecular roles of BL in kernel development have not been fully elucidated. In this work, microscopy images showed that BL began to appear at a growth stage earlier than 10 days after pollination (DAP), and its color gradually deepened to become dark as the development period progressed. Scanning electron microscopy observations revealed that BL is a tissue structure composed of several layers of cells that are gradually squeezed and compressed during kernel development. Laser-capture microdissection (LCM) was used to sample BL and its neighboring inner tissue, basal endosperm transfer layer (BETL), and outer tissue, inner epidermis (IEP), from 20 DAP of kernels. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling (MALDI-TOF MS profiling) detected 41, 104, and 120 proteins from LCM-sampled BL, BETL, and IEP, respectively. Gene ontology (GO) analysis indicated that the 41 BL proteins were primarily involved in the response to stress and stimuli. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis found that the BL proteins were enriched in several defense pathways, such as the ascorbate and aldarate metabolic pathways. Among the 41 BL proteins, six were BL-specific proteins that were only detected from BL. Annotations of five BL-specific proteins were related to stress responses. During kernel development, transcriptional expression of most BL proteins showed an increase, followed by a decrease, and reached a maximum zero to 20 DAP. These results suggest a role for BL in stress responses for protecting filial tissue against threats from maternal sides, which helps to elucidate the biological functions of BL.

Recent advances in proteomics and metabolomics in plants

“…Affinity binding is also commonly used as an enrichment method of PTMs like phosphorylation with immobilized metal affinity chromatography (Bontinck et al 2018 ). Laser capture microdissection (LCM) is a technique by which cells of a single-type can be harvested from tissue sections visualized under microscope (Chen et al 2020b ). Harvested cells can provide DNA, RNA, and protein for the profiling of genomic characteristics, gene expression, and protein abundance from single-type of cell.…”

Cell‐Type‐Specific Proteomics Analysis of a Small Number of Plant Cells by Integrating Laser Capture Microdissection with a Nanodroplet Sample Processing Platform

“…56 protein spots were picked and analyzed using mass spectrometry. In maize seed kernel, several cell types such as black layer, endosperm cells, and inner epidermal cells were dissected with LCM, which yielded 41, 104, and 120 cell‐type‐specific proteins (Chen et al., 2020). In all of the above LCM‐based proteomics studies, the protein identifications relied on many factors, including the number of dissected cells, protein loss during sample preparation, and the sensitivity of the mass spectrometry instrumentation.…”

Plant Proteomic Research 3.0: Challenges and Perspectives

“…In situ and single cell analysis remain some of the main challenges in proteomics studies. In this direction, Chen et al [ 14 ] used shotgun combined with laser-capture microdissection to analyze the black layer of maize kernels, a tissue rich in stress-responsive proteins. Lawrence et al [ 15 ] prepared stomatal guard cells from Arabidopsis thaliana using a Scotch-tape method to analyze changes in S-nitrosylation in guard cells during a pathogen challenge.…”

“…Proteomic techniques are used in the identification of stress-responsive mechanism in plants under conditions such as salt [ 14 ], cold [ 19 ], flood [ 20 ], and wounding [ 21 ]. Wang et al [ 19 ] provided insights into the cold stress responses of early imbibed castor seeds: proteins confer cold tolerance by promoting protein synthesis, protect the cell against damage caused by cold stress, and facilitate resistance or adaptation to cold stress by the increased content of unsaturated fatty acid.…”