Abstract
BackgroundIn situ hybridization is a general molecular method typically used for the localization of mRNA transcripts in plants. The method provides a valuable tool to unravel the connection between gene expression and anatomy, especially in species such as pines which show large genome size and shortage of sequence information.ResultsIn the present study, expression of the catalase gene (CAT) related to the scavenging of reactive oxygen species (ROS) and the polyamine metabolism related genes, diamine oxidase (DAO) and arginine decarboxylase (ADC), were localized in developing Scots pine (Pinus sylvestris L.) seeds. In addition to specific signals from target mRNAs, the probes continually hybridized non-specifically in the embryo surrounding region (ESR) of the megagametophyte tissue, in the remnants of the degenerated suspensors as well as in the cells of the nucellar layers, i.e. tissues exposed to cell death processes and extensive nucleic acid fragmentation during Scots pine seed development.ConclusionsIn plants, cell death is an integral part of both development and defence, and hence it is a common phenomenon in all stages of the life cycle. Our results suggest that extensive nucleic acid fragmentation during cell death processes can be a considerable source of non-specific signals in traditional in situ mRNA hybridization. Thus, the visualization of potential nucleic acid fragmentation simultaneously with the in situ mRNA hybridization assay may be necessary to ensure the correct interpretation of the signals in the case of non-specific hybridization of probes in plant tissues.
Highlights
In situ hybridization is a general molecular method typically used for the localization of mRNA transcripts in plants
Our results suggest that extensive nucleic acid fragmentation during cell death processes can be a considerable source of non-specific signals in traditional in situ mRNA hybridization
We demonstrate that the traditional in situ mRNA hybridization technique may be inappropriate for the localization of mRNA transcripts in plant tissues that are exposed to extensive nucleic acid fragmentation during cell death
Summary
In situ hybridization is a general molecular method typically used for the localization of mRNA transcripts in plants. The method provides a valuable tool to unravel the connection between gene expression and anatomy, especially in species such as pines which show large genome size and shortage of sequence information. The in situ hybridization assay that is used for the localization of specific nucleic acid sequences in various organisms, species and specimens is a decades-old technology that is still continuously developed and remains applicable in many modern contexts, such as live-cell imaging [1] and medical diagnostics [2]. In pines, which show both shortage of sequence information and large genome size, in situ mRNA hybridization provides a molecular tool for a better understanding of the links between structural components and gene function. The main target of the PCD machinery is the nucleus, and the degradation processes include both chromatin and nuclear envelope [9]. In a cell death process with necrotic-like morphology, cells break down and fragmented nucleic acids may be detected in the surrounding extra-cellular space [6]
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