Complex transmission of partiti-, ambi- and ourmiaviruses in the forest pathogen Heterobasidion parviporum.

Utilizing Heterobasidion partitivirus 13 strain an1 (HetPV13-an1) and 15 strain pa1 (HetPV15-pa1) in co-infection is considered a potential biocontrol approach against Heterobasidion root and butt rot. Both partitiviruses mediate debilitating effects in most Heterobasidion host isolates and are generally transmitted efficiently between host strains. In this investigation, we conducted transmission experiments in the laboratory (in vitro) using several H. parviporum isolates to test whether using dual partitivirus infections is a more efficient way of transmitting viruses to new hosts compared to using single partitivirus infections, and whether co-occurring single-stranded RNA (ssRNA) viruses are co-transmitted during the process. The results showed that H. parviporum donors carrying both partitiviruses, HetPV13-an1 and HetPV15-pa1, transmitted HetPV15-pa1 more efficiently to recipients than the same donors infected with only HetPV15-pa1. In contrast, the transmission of HetPV13-an1 did not differ significantly between donors infected with both or only one partitivirus. Altogether, the transmission rates of HetPV13-an1 and HetPV15-pa1 were high on artificial media. Moreover, the transmission of the ssRNA viruses Heterobasidion ourmia-like virus 1(HetOlV1-pa7) and 4 (HetOlV4-an1) as well as Heterobasidion ambi-like virus 3 (HetAlV3-pa4) across different recipients were found to be variable. This study demonstrated for the first time the transmission of ambi- and ourmiaviruses between H. parviporum isolates in dual cultures and showed that H. parviporum mycelia can be cured of these ssRNA viruses using heat treatment.

Virus transmission frequencies in the pine root rot pathogen Heterobasidion annosum.

The combined use of Heterobasidion partitiviruses 13 and 15 (HetPV13-an1 and HetPV15-pa1) is considered a promising biocontrol approach against Heterobasidion root and butt rot. In a previous study, the transmission frequency of HetPV15-pa1 was found to be higher from a double partitivirus-infected donor than from a single partitivirus-infected donor. In this study, we included a wider array of recipient isolates to assess whether the phenomenon is widespread across different host strains and conducted transmission experiments on artificial media (in vitro) using a total of 45 different H. annosum donor-recipient pairs. In addition to investigating whether double partitivirus infection improves the transmission of HetPV13-an1 and HetPV15-pa1, we examined for the first time how efficiently co-infecting ssRNA viruses are concomitantly transmitted with the partitiviruses, and whether pre-existing ssRNA viruses in the recipients affect virus transmission. Generally, the transmission rates of HetPV13-an1 and HetPV15-pa1 were high from both single partitivirus-infected and double partitivirus-infected donors to most of the H. annosum recipient strains, with few exceptions. However, in contrast to previous experiments, the transmission frequency was not higher from the double partitivirus-infected donors. Also, ourmiavirus was transmitted between H. annosum strains, but the presence of another ourmiavirus in the recipient might affect the efficacy.

Integrating viruses into soil food web biogeochemistry.

The soil microbiome is recognized as an essential component of healthy soils. Viruses are also diverse and abundant in soils, but their roles in soil systems remain unclear. Here we argue for the consideration of viruses in soil microbial food webs and describe the impact of viruses on soil biogeochemistry. The soil food web is an intricate series of trophic levels that span from autotrophic microorganisms to plants and animals. Each soil system encompasses contrasting and dynamic physicochemical conditions, with labyrinthine habitats composed of particles. Conditions are prone to shifts in space and time, and this variability can obstruct or facilitate interactions of microorganisms and viruses. Because viruses can infect all domains of life, they must be considered as key regulators of soil food web dynamics and biogeochemical cycling. We highlight future research avenues that will enable a more robust understanding of the roles of viruses in soil function and health.

Meeting report: The first soil viral workshop 2022.

Soil viral ecology is a growing research field; however, the state of knowledge still lags behind that of aquatic systems. Therefore, to facilitate progress, the first Soil Viral Workshop was held to encourage international scientific discussion and collaboration, suggest guidelines for future research, and establish soil viral research as a concrete research area. The workshop took place at Søminestationen, Denmark, between 15 and 17th of June 2022. The meeting was primarily held in person, but the sessions were also streamed online. The workshop was attended by 23 researchers from ten different countries and from a wide range of subfields and career stages. Eleven talks were presented, followed by discussions revolving around three major topics: viral genomics, virus-host interactions, and viruses in the soil food web. The main take-home messages and suggestions from the discussions are summarized in this report.

High Diversity of Novel Viruses in the Tree Pathogen Phytophthora castaneae Revealed by High-Throughput Sequencing of Total and Small RNA.

Phytophthora castaneae, an oomycete pathogen causing root and trunk rot of different tree species in Asia, was shown to harbor a rich diversity of novel viruses from different families. Four P. castaneae isolates collected from Chamaecyparis hodginsii in a semi-natural montane forest site in Vietnam were investigated for viral presence by traditional and next-generation sequencing (NGS) techniques, i.e., double-stranded RNA (dsRNA) extraction and high-throughput sequencing (HTS) of small RNAs (sRNAs) and total RNA. Genome organization, sequence similarity, and phylogenetic analyses indicated that the viruses were related to members of the order Bunyavirales and families Endornaviridae, Megabirnaviridae, Narnaviridae, Totiviridae, and the proposed family "Fusagraviridae." The study describes six novel viruses: Phytophthora castaneae RNA virus 1-5 (PcaRV1-5) and Phytophthora castaneae negative-stranded RNA virus 1 (PcaNSRV1). All six viruses were detected by sRNA sequencing, which demonstrates an active RNA interference (RNAi) system targeting viruses in P. castaneae. To our knowledge, this is the first report of viruses in P. castaneae and the whole Phytophthora major Clade 5, as well as of the activity of an RNAi mechanism targeting viral genomes among Clade 5 species. PcaRV1 is the first megabirnavirus described in oomycetes and the genus Phytophthora.

Discovery and Community Dynamics of Novel ssRNA Mycoviruses in the Conifer Pathogen Heterobasidion parviporum.

Heterobasidion species are highly destructive basidiomycetous conifer pathogens of the Boreal forest region. Earlier studies have revealed dsRNA virus infections of families Curvulaviridae and Partitiviridae in Heterobasidion strains, and small RNA deep sequencing has also identified infections of Mitoviridae members in these fungi. In this study, the virome of Heterobasidion parviporum was examined for the first time by RNA-Seq using total RNA depleted of rRNA. This method successfully revealed new viruses representing two established (+)ssRNA virus families not found earlier in Heterobasidion: Narnaviridae and Botourmiaviridae. In addition, we identified the presence of a recently described virus group tentatively named "ambiviruses" in H. parviporum. The H. parviporum isolates included in the study originated from experimental forest sites located within 0.7 km range from each other, and a population analysis including 43 isolates was conducted at one of the experimental plots to establish the prevalence of the newly identified viruses in clonally spreading H. parviporum individuals. Our results indicate that viral infections are considerably more diverse and common among Heterobasidion isolates than known earlier and include ssRNA viruses with high prevalence and interspecies variation.

Armillaria root rot fungi host single-stranded RNA viruses.

Species of Armillaria are distributed globally and include some of the most important pathogens of forest and ornamental trees. Some of them form large long-living clones that are considered as one of the largest organisms on earth and are capable of long-range spore-mediated transfer as well as vegetative spread by drought-resistant hyphal cords called rhizomorphs. However, the virus community infecting these species has remained unknown. In this study we used dsRNA screening and high-throughput sequencing to search for possible virus infections in a collection of Armillaria isolates representing three different species: Armillaria mellea from South Africa, A. borealis from Finland and Russia (Siberia) and A. cepistipes from Finland. Our analysis revealed the presence of both negative-sense RNA viruses and positive-sense RNA viruses, while no dsRNA viruses were detected. The viruses included putative new members of virus families Mymonaviridae, Botourmiaviridae and Virgaviridae and members of a recently discovered virus group tentatively named "ambiviruses" with ambisense bicistronic genomic organization. We demonstrated that Armillaria isolates can be cured of viruses by thermal treatment, which enables the examination of virus effects on host growth and phenotype using isogenic virus-infected and virus-free strains.

The virome from a collection of endomycorrhizal fungi reveals new viral taxa with unprecedented genome organization.

Mutualistic plant-associated fungi are recognized as important drivers in plant evolution, diversity, and health. The discovery that mycoviruses can take part and play important roles in symbiotic tripartite interactions has prompted us to study the viromes associated with a collection of ericoid and orchid mycorrhizal (ERM and ORM, respectively) fungi. Our study, based on high-throughput sequencing of transcriptomes (RNAseq) from fungal isolates grown in axenic cultures, revealed in both ERM and ORM fungi the presence of new mycoviruses closely related to already classified virus taxa, but also new viruses that expand the boundaries of characterized RNA virus diversity to previously undescribed evolutionary trajectories. In ERM fungi, we provide first evidence of a bipartite virus, distantly related to narnaviruses, that splits the RNA-dependent RNA polymerase (RdRP) palm domain into two distinct proteins, encoded by each of the two segments. Furthermore, in one isolate of the ORM fungus Tulasnella spp. we detected a 12 kb genomic fragment coding for an RdRP with features of bunyavirus-like RdRPs. However, this 12 kb genomic RNA has the unique features, for Bunyavirales members, of being tri-cistronic and carrying ORFs for the putative RdRP and putative nucleocapsid in ambisense orientation on the same genomic RNA. Finally, a number of ORM fungal isolates harbored a group of ambisense bicistronic viruses with a genomic size of around 5 kb, where we could identify a putative RdRP palm domain that has some features of plus strand RNA viruses; these new viruses may represent a new lineage in the Riboviria, as they could not be reliably assigned to any of the branches in the recently derived monophyletic tree that includes most viruses with an RNA genome.

Mixed infection by a partitivirus and a negative-sense RNA virus related to mymonaviruses in the polypore fungus Bondarzewia berkeleyi.

Virus communities of forest fungi remain poorly characterized. In this study, we detected two new viruses co-infecting an isolate of the polypore fungus Bondarzewia berkeleyi using high-throughput sequencing. One of them was a putative new partitivirus designated as Bondarzewia berkeleyi partitivirus 1 (BbPV1), with two linear dsRNA genome segments of 1928 and 1863 bp encoding a putative RNA-dependent RNA polymerase (RdRP) of 591 aa and a putative capsid protein of 538 aa. The other virus, designated as Bondarzewia berkeleyi negative-strand RNA virus 1 (BbNSRV1), had a non-segmented negative-sense RNA genome of 10,983 nt and was related to members of family Mymonaviridae. The BbNSRV1 genome includes six predicted open reading frames (ORFs) of 279, 425, 230, 174, 200 and 1970 aa. The longest ORF contained conserved regions corresponding to Mononegavirales RdRP and mRNA-capping enzyme region V constituting the mononegavirus Large protein. In addition, a low level of sequence identity was detected between the putative nucleocapsid protein-coding ORF2 of Lentinula edodes negative-strand RNA virus 1 and BbNSRV1. The viruses characterized in this study are the first ones described in Bondarzewia spp., and BbNSRV1 is the second mymona-like virus described in a basidiomycete host.

Virus population structure in the ectomycorrhizal fungi Lactarius rufus and L. tabidus at two forest sites in Southern Finland.

Lactarius fungi belong to the Russulaceae family and have an important ecological role as ectomycorrhizal symbionts of coniferous and deciduous trees. Two Lactarius species, L. tabidus and L. rufus have been shown to harbor bisegmented dsRNA viruses belonging to an unclassified virus group including the mutualistic Curvularia thermal tolerance virus (CThTV). In this study, we characterized the first complete genome sequences of these viruses designated as Lactarius tabidus RNA virus 1 (LtRV1) and Lactarius rufus RNA virus 1 (LrRV1), both of which included two genome segments of 2241 and 2049 bp. We also analyzed spatial distribution and sequence diversity of the viruses in sixty host strains at two forest sites, and showed that the viruses are species-specific at sites where both host species co-occur. We also found that single virus isolates inhabited several different conspecific host strains, and were involved in persistent infections during up to eight years.

Microscopical Detection of Cell Death Processes During Scots Pine Zygotic Embryogenesis.

Programmed cell death (PCD) processes are essential in the plant embryogenesis. To understand how PCD operates in a developing seed, the dying cells need to be identified in relation to their surviving neighbors. This can be accomplished by the means of in situ visualization of fragmented DNA-a well-known hallmark of PCD. In the developing Scots pine (Pinus sylvestris L.) seed, several tissues die via morphologically different PCD processes during the embryogenesis. Here, we describe the protocols for the characterization of Scots pine seeds at the early and late developmental stages and, further, the localization of nucleic acids and DNA fragmentation by the acridine orange staining and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling) assay in the dying seed tissues.

Viruses of fungi and oomycetes in the soil environment.

Soils support a myriad of organisms hosting highly diverse viromes. In this minireview, we focus on viruses hosted by true fungi and oomycetes (members of Stamenopila, Chromalveolata) inhabiting bulk soil, rhizosphere and litter layer, and representing different ecological guilds, including fungal saprotrophs, mycorrhizal fungi, mutualistic endophytes and pathogens. Viruses infecting fungi and oomycetes are characterized by persistent intracellular nonlytic lifestyles and transmission via spores and/or hyphal contacts. Almost all fungal and oomycete viruses have genomes composed of single-stranded or double-stranded RNA, and recent studies have revealed numerous novel viruses representing yet unclassified family-level groups. Depending on the virus-host combination, infections can be asymptomatic, beneficial or detrimental to the host. Thus, mycovirus infections may contribute to the multiplex interactions of hosts, therefore likely affecting the dynamics of fungal communities required for the functioning of soil ecosystems. However, the effects of fungal and oomycete viruses on soil ecological processes are still mostly unknown. Interestingly, new metagenomics data suggest an extensive level of horizontal virus transfer between plants, fungi and insects.

Expression of catalase and retinoblastoma-related protein genes associates with cell death processes in Scots pine zygotic embryogenesis.

BACKGROUND: The cell cycle and cellular oxidative stress responses are tightly controlled for proper growth and development of Scots pine (Pinus sylvestris L.) seed. Programmed cell death (PCD) is an integral part of the embryogenesis during which megagametophyte cells in the embryo surrounding region (ESR) and cells in the nucellar layers face death. In the present study, we show both the tissue and developmental stage specific expression of the genes encoding the autophagy related ATG5, catalase (CAT), and retinoblastoma related protein (RBR) as well as the connection between the gene expressions and cell death programs. RESULTS: We found strong CAT expression in the cells of the developing embryo throughout the embryogenesis as well as in the cells of the megagametophyte and the nucellar layers at the early embryogeny. The CAT expression was found to overlap with both the ATG5 expression and hydrogen peroxide localization. At the late embryogeny, CAT expression diminished in the dying cells of the nucellar layers as well as in megagametophyte cells, showing the first signs of incipient cell death. Accumulation of starch and minor RBR expression were characteristic of megagametophyte cells in the ESR, whereas strong RBR expression was found in the cells of the nucellar layers at the late embryogeny. CONCLUSIONS: Our results suggest that ATG5, CAT, and RBR are involved in the Scots pine embryogenesis and cell death processes. CAT seems to protect cells against hydrogen peroxide accumulation and oxidative stress related cell death especially during active metabolism. The opposite expression of RBR in the ESR and nucellar layers alongside morphological characteristics emphasizes the different type of the cell death processes in these tissues. Furthermore, the changes in ATG5 and RBR expressions specifically in the megagametophyte cells dying by necrotic cell death suggest the genetic regulation of developmental necrosis in Scots pine embryogenesis.

Phenolic compounds and expression of 4CL genes in silver birch clones and Pt4CL1a lines.

A small multigene family encodes 4-coumarate:CoA ligases (4CLs) catalyzing the CoA ligation of hydroxycinnamic acids, a branch point step directing metabolites to a flavonoid or monolignol pathway. In the present study, we examined the effect of antisense Populus tremuloides 4CL (Pt4CL1) to the lignin and soluble phenolic compound composition of silver birch (Betula pendula) Pt4CL1a lines in comparison with non-transgenic silver birch clones. The endogenous expression of silver birch 4CL genes was recorded in the stems and leaves and also in leaves that were mechanically injured. In one of the transgenic Pt4CL1a lines, the ratio of syringyl (S) and guaiacyl (G) lignin units was increased. Moreover, the transcript levels of putative silver birch 4CL gene (Bp4CL1) were reduced and contents of cinnamic acid derivatives altered. In the other two Pt4CL1a lines changes were detected in the level of individual phenolic compounds. However, considerable variation was found in the transcript levels of silver birch 4CLs as well as in the concentration of phenolic compounds among the transgenic lines and non-transgenic clones. Wounding induced the expression of Bp4CL1 and Bp4CL2 in leaves in all clones and transgenic lines, whereas the transcript levels of Bp4CL3 and Bp4CL4 remained unchanged. Moreover, minor changes were detected in the concentrations of phenolic compounds caused by wounding. As an overall trend the wounding decreased the flavonoid content in silver birches and increased the content of soluble condensed tannins. The results indicate that by reducing the Bp4CL1 transcript levels lignin composition could be modified. However, the alterations found among the Pt4CL1a lines and the non-transgenic clones were within the natural variation of silver birches, as shown in the present study by the clonal differences in the transcripts levels of 4CL genes, soluble phenolic compounds and condensed tannins.

Interaction with ectomycorrhizal fungi and endophytic Methylobacterium affects nutrient uptake and growth of pine seedlings in vitro.

Tissues of Scots pine (Pinus sylvestris L.) contain several endophytic microorganisms of which Methylobacterium extorquens DSM13060 is a dominant species throughout the year. Similar to other endophytic bacteria, M. extorquens is able to colonize host plant tissues without causing any symptoms of disease. In addition to endophytic bacteria, plants associate simultaneously with a diverse set of microorganisms. Furthermore, plant-colonizing microorganisms interact with each other in a species- or strain-specific manner. Several studies on beneficial microorganisms interacting with plants have been carried out, but few deal with interactions between different symbiotic organisms and specifically, how these interactions affect the growth and development of the host plant. Our aim was to study how the pine endophyte M. extorquens DSM13060 affects pine seedlings and how the co-inoculation with ectomycorrhizal (ECM) fungi [Suillus variegatus (SV) or Pisolithus tinctorius (PT)] alters the response of Scots pine. We determined the growth, polyamine and nutrient contents of inoculated and non-inoculated Scots pine seedlings in vitro. Our results show that M. extorquens is able to improve the growth of seedlings at the same level as the ECM fungi SV and PT do. The effect of co-inoculation using different symbiotic organisms was seen in terms of changes in growth and nutrient uptake. Inoculation using M. extorquens together with ECM fungi improved the growth of the host plant even more than single ECM inoculation. Symbiotic organisms also had a strong effect on the potassium content of the seedling. The results indicate that interaction between endophyte and ECM fungus is species dependent, leading to increased or decreased nutrient content and growth of pine seedlings.

Evolution, three-dimensional model and localization of truncated hemoglobin PttTrHb of hybrid aspen.

Thus far, research on plant hemoglobins (Hbs) has mainly concentrated on symbiotic and non-symbiotic Hbs, and information on truncated Hbs (TrHbs) is scarce. The aim of this study was to examine the origin, structure and localization of the truncated Hb (PttTrHb) of hybrid aspen (Populus tremula L. × tremuloides Michx.), the model system of tree biology. Additionally, we studied the PttTrHb expression in relation to non-symbiotic class1 Hb gene (PttHb1) using RNAi-silenced hybrid aspen lines. Both the phylogenetic analysis and the three-dimensional (3D) model of PttTrHb supported the view that plant TrHbs evolved vertically from a bacterial TrHb. The 3D model suggested that PttTrHb adopts a 2-on-2 sandwich of α-helices and has a Bacillus subtilis -like ligand-binding pocket in which E11Gln and B10Tyr form hydrogen bonds to a ligand. However, due to differences in tunnel cavity and gate residue (E7Ala), it might not show similar ligand-binding kinetics as in Bs-HbO (E7Thr). The immunolocalization showed that PttTrHb protein was present in roots, stems as well as leaves of in vitro -grown hybrid aspens. In mature organs, PttTrHb was predominantly found in the vascular bundles and specifically at the site of lateral root formation, overlapping consistently with areas of nitric oxide (NO) production in plants. Furthermore, the NO donor sodium nitroprusside treatment increased the amount of PttTrHb in stems. The observed PttTrHb localization suggests that PttTrHb plays a role in the NO metabolism.

The responses of Vitreoscilla hemoglobin-expressing hybrid aspen (Populus tremula × tremuloides) exposed to 24-h herbivory: expression of hemoglobin and stress-related genes in exposed and nonorthostichous leaves.

The responses of transcriptome and phenolic compounds were determined with Populus tremula L. × Populus tremuloides Michx. expressing the hemoglobin (Hb) of Vitreoscilla (VHb) and non-transformant (wt) line. After 24-h exposure of leaves to Conistra vaccinii L., the transcript levels of endogenous non-symbiotic class 1 Hb (PttHb1) and truncated Hb (PttTrHb) genes were modestly reduced and increased, respectively, in both wt and VHb-expressing line. Besides the herbivory exposed leaves showing the most significant transcriptome changes, alterations were also detected in the transcriptome of nonorthostichous leaves positioned directly above the exposed leaves. Both wt and VHb-expressing line displayed similar herbivory-induced effects on gene expression, although the extent of responses was more pronounced in the wt than in the VHb-expressing line. The contents of phenolic compounds were not altered due to herbivory and they were alike in the wt and VHb-expressing line. In addition, we determined the relative growth rates (RGRs) of Orthosia gothica L., Ectropis crepuscularia Denis & Schiff. and Orgyia antiqua L. larvae, and found no variation in the RGRs between the lines. Thus, VHb-expressing P. tremula × tremuloides lines showed to be comparable with wt in regards to the food quality of leaves.

Polyamine metabolism during exponential growth transition in Scots pine embryogenic cell culture.

Polyamine (PA) metabolism was studied in liquid cultures of Scots pine (Pinus sylvestris L.) embryogenic cells. The focus of the study was on the metabolic changes at the interphase between the initial lag phase and the exponential growth phase. PA concentrations fluctuated in the liquid cultures as follows. Putrescine (Put) concentrations increased, whereas spermidine (Spd) concentrations decreased in both free and soluble conjugated PA fractions. The concentrations of free and soluble conjugated spermine (Spm) remained low, and small amounts of excreted PAs were also found in the culture medium. The minor production of secondary metabolites reflected the undifferentiated stage of the embryogenic cell culture. Put was produced via the arginine decarboxylase (ADC) pathway. Futhermore, the gene expression data suggested that the accumulation of Put was caused neither by an increase in Put biosynthesis nor by a decrease in Put catabolism, but resulted mainly from the decrease in the biosynthesis of Spd and Spm. Put seemed to play an important role in cell proliferation in Scots pine embryogenic cells, but the low pH of the culture medium could also, at least partially, be the reason for the accumulation of endogenous Put. High Spd concentrations at the initiation of the culture, when cells were exposed to stress and cell death, suggested that Spd may act not only as a protector against stress but also as a growth suppressor, when proliferative growth is not promoted. All in all, Scots pine embryogenic cell culture was proved to be a favourable experimental platform to study PA metabolism and, furthermore, the developed system may also be beneficial in experiments where, e.g., the effect of specific stressors on PA metabolism is addressed.

Long-term cryopreservation of Greek fir embryogenic cell lines: recovery, maturation and genetic fidelity.

In coniferous species, including Greek fir (Abies cephalonica Loud), the involvement of somatic embryo plants in breeding and reforestation programs is dependent on the success of long-term cryostorage of embryogenic cultures during clonal field testing. In the present study on Greek fir, we assayed the recovery, morphological characteristics and genetic fidelity of embryogenic cell lines 6 and 8 during proliferation and maturation after long-term cryostorage. Our results indicate successful recovery of both cell lines after 6 years in cryostorage. In the maturation phase, both cell lines were capable of producing somatic embryos although some differences were detected among experiments. However, these changes were more dependent on the differences in the components of the maturation media or in the experimental set-up than on the long-term cryostorage. During both proliferation and maturation phases, the morphological fidelity of the embryogenic cultures as well as of the somatic embryos were alike before and after cryopreservation. The genetic fidelity of the cryopreserved cell line 6 that was assayed by random amplified polymorphic DNA (i.e. RAPD) markers demonstrated some changes in the RAPD profiles. The results indicate possible genetic aberrations caused by long-term cryopreservation or somaclonal variation during the proliferation stage. However, in spite of these changes the embryogenic cultures did not lose their proliferation or maturation abilities.

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

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.