Dealing with the problem of non-specific in situ mRNA hybridization signals associated with plant tissues undergoing programmed cell death

Abstract

Background: 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.

Results: In 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.

Conclusions: In 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.

Figure 1

Nucleic acid fragmentation in immature Scots pine seed. Nucleic acid fragmentation in the embryo surrounding region (ESR), cells of the megagametophyte tissue and the degenerated suspensor tissue in a developing Scots pine seed. (A) In the acridine orange (AO) -stained section, cell wall remnants and degraded nucleic acid formed a zone between the ESR and the developing embryo (arrows). (B) TUNEL-positive nuclei of the megagametophyte cells in the ESR (arrows). (C) TUNEL-positive signal in fragmented DNA among the cell wall remnants in the corrosion cavity (arrow). (D) AO-stained section with subordinate embryos and fragmented nucleic acids in the corrosion cavity close to the remnants of the degenerated suspensor tissue (arrows). CC = corrosion cavity, E = embryo, M = megagametophyte.

Figure 2

Catalase (CAT) localization by in situ mRNA hybridization in immature Scots pine seed. Localization of catalase (CAT) mRNA transcripts by in situ mRNA hybridization in a developing Scots pine seed. (A and B) In addition to the specific in situ hybridization signal (blue colour) in the embryo, unspecific signals (arrows) were found in the broken megagametophyte cells in the embryo surrounding region (ESR) (A) and in the degenerated suspensor tissue in the corrosion cavity (B) in the section hybridized with the CAT antisense probe. (C and D) With the CAT sense probe, no signal was found in the embryo but unspecific signals (arrows) were detected in the ESR cells of the megagametophyte and in the arrow-shaped region in front of the expanding corrosion cavity (C) as well as in the degenerated suspensor tissue in the corrosion cavity (D). CC = corrosion cavity, E = embryo, M = megagametophyte.

Figure 3

Nuclear DNA degradation and unspecific in situ hybridization signal in immature Scots pine seed. Nuclear DNA degradation and an unspecific in situ hybridization signal in the nucellar layers of a developing Scots pine seed. (A) Fragmented DNA (arrows) in the AO-stained section. (B) TUNEL-positive nuclei (arrows) and autofluorescence of granulous phenols (arrow heads) in the nucellar layers. Localization of diamine oxidase (DAO) mRNA transcripts by in situ mRNA hybridization (arrows) with antisense (C) and sense (D) probes resulted in equal hybridization signals in the cells of the nucellar layers. M = megagametophyte, N = nucellar layers.