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Reproductive phase change is well characterized in angiosperm model species, but less studied in gymnosperms. We utilize the early cone-setting acrocona mutant to study reproductive phase change in the conifer Picea abies (Norway spruce), a gymnosperm. The acrocona mutant frequently initiates cone-like structures, called transition shoots, in positions where wild-type P. abies always produces vegetative shoots. We collect acrocona and wild-type samples, and RNA-sequence their messenger RNA (mRNA) and microRNA (miRNA) fractions. We establish gene expression patterns and then use allele-specific transcript assembly to identify mutations in acrocona. We genotype a segregating population of inbred acrocona trees. A member of the SQUAMOSA BINDING PROTEIN-LIKE (SPL) gene family, PaSPL1, is active in reproductive meristems, whereas two putative negative regulators of PaSPL1, miRNA156 and the conifer specific miRNA529, are upregulated in vegetative and transition shoot meristems. We identify a mutation in a putative miRNA156/529 binding site of the acrocona PaSPL1 allele and show that the mutation renders the acrocona allele tolerant to these miRNAs. We show co-segregation between the early cone-setting phenotype and trees homozygous for the acrocona mutation. In conclusion, we demonstrate evolutionary conservation of the age-dependent flowering pathway and involvement of this pathway in regulating reproductive phase change in the conifer P. abies.

Sugar beet root rot, caused by the oomycete Aphanomyces cochlioides, is a serious and economically important disease of sugar beets world-wide. Today, disease risk assessment consists of a time-consuming greenhouse bioassay using bait plants. In the present study, a real-time quantitative PCR (qPCR) assay for determination of A. cochlioides DNA in field-infested soil samples was developed and validated using the standard bioassay. The qPCR assay proved to be species-specific and was optimized to give high amplification efficiency suitable for target copy quantification. A high correlation (R2 > 0.98, p

Recent efforts to sequence the genomes and transcriptomes of several gymnosperm species have revealed an increased complexity in certain gene families in gymnosperms as compared to angiosperms. One example of this is the gymnosperm sister clade to angiosperm TM3-like MADS-box genes, which at least in the conifer lineage has expanded in number of genes. We have previously identified a member of this sub-clade, the conifer gene DEFICIENS AGAMOUS LIKE 19 (DAL19), as being specifically upregulated in cone-setting shoots. Here, we show through Sanger sequencing of mRNA-derived cDNA and mapping to assembled conifer genomic sequences that DAL19 produces six mature mRNA splice variants in Picea abies. These splice variants use alternate first and last exons, while their four central exons constitute a core region present in all six transcripts. Thus, they are likely to be transcript isoforms. Quantitative Real-Time PCR revealed that two mutually exclusive first DAL19 exons are differentially expressed across meristems that will form either male or female cones, or vegetative shoots. Furthermore, mRNA in situ hybridization revealed that two mutually exclusive last DAL19 exons were expressed in a cell-specific pattern within bud meristems. Based on these findings in DAL19, we developed a sensitive approach to transcript isoform assembly from short-read sequencing of mRNA. We applied this method to 42 putative MADS-box core regions in P. abies, from which we assembled 1084 putative transcripts. We manually curated these transcripts to arrive at 933 assembled transcript isoforms of 38 putative MADS-box genes. 152 of these isoforms, which we assign to 28 putative MADS-box genes, were differentially expressed across eight female, male, and vegetative buds. We further provide evidence of the expression of 16 out of the 38 putative MADS-box genes by mapping PacBio Iso-Seq circular consensus reads derived from pooled sample sequencing to assembled transcripts. In summary, our analyses reveal the use of mutually exclusive exons of MADS-box gene isoforms during early bud development in P. abies, and we find that the large number of identified MADS-box transcripts in P. abies results not only from expansion of the gene family through gene duplication events but also from the generation of numerous splice variants.

Understanding complex biological systems requires functional characterization of specialized tissue domains. However, existing strategies for generating and analysing high-throughput spatial expression profiles were developed for a limited range of organisms, primarily mammals. Here we present the first available approach to generate and study high-resolution, spatially resolved functional profiles in a broad range of model plant systems. Our process includes high-throughput spatial transcriptome profiling followed by spatial gene and pathway analyses. We first demonstrate the feasibility of the technique by generating spatial transcriptome profiles from model angiosperms and gymnosperms microsections. In Arabidopsis thaliana we use the spatial data to identify differences in expression levels of 141 genes and 189 pathways in eight inflorescence tissue domains. Our combined approach of spatial transcriptomics and functional profiling offers a powerful new strategy that can be applied to a broad range of plant species, and is an approach that will be pivotal to answering fundamental questions in developmental and evolutionary biology.

Reproductive organs in seed plants are morphologically divergent and their evolutionary history is often unclear. The mechanisms controlling their development have been extensively studied in angiosperms but are poorly understood in conifers and other gymnosperms. Here, we address the molecular control of seed cone development in Norway spruce, Picea abies. We present expression analyses of five novel MADS-box genes in comparison with previously identified MADS and LEAFY genes at distinct developmental stages. In addition, we have characterized the homeotic transformation from vegetative shoot to female cone and associated changes in regulatory gene expression patterns occurring in the acrocona mutant. The analyses identified genes active at the onset of ovuliferous and ovule development and identified expression patterns marking distinct domains of the ovuliferous scale. The reproductive transformation in acrocona involves the activation of all tested genes normally active in early cone development, except for an AGAMOUS-LIKE6/SEPALLATA (AGL6/SEP) homologue. This absence may be functionally associated with the nondeterminate development of the acrocona ovule-bearing scales. Our morphological and gene expression analyses give support to the hypothesis that the modern cone is a complex structure, and the ovuliferous scale the result of reductions and compactions of an ovule-bearing axillary short shoot in cones of Paleozoic conifers.

Conifers normally go through a long juvenile period, for Norway spruce (Picea abies) around 20 to 25 years, before developing male and female cones. We have grown plants from inbred crosses of a naturally occurring spruce mutant (acrocona). One-fourth of the segregating acrocona plants initiate cones already in their second growth cycle, suggesting control by a single locus. The early cone-setting properties of the acrocona mutant were utilized to identify candidate genes involved in vegetative-to-reproductive phase change in Norway spruce. Poly(A+) RNA samples from apical and basal shoots of cone-setting and non-cone-setting plants were subjected to high-throughput sequencing (RNA-seq). We assembled and investigated 33,383 expressed putative protein-coding acrocona transcripts. Eight transcripts were differentially expressed between selected sample pairs. One of these (Acr42124_1) was significantly up-regulated in apical shoot samples from cone-setting acrocona plants, and the encoded protein belongs to the MADS box gene family of transcription factors. Using quantitative real-time polymerase chain reaction with independently derived plant material, we confirmed that the MADS box gene is up-regulated in both needles and buds of cone-inducing shoots when reproductive identity is determined. Our results constitute important steps for the development of a rapid cycling model system that can be used to study gene function in conifers. In addition, our data suggest the involvement of a MADS box transcription factor in the vegetative-to-reproductive phase change in Norway spruce.

Background and Aims During embryo development in most gymnosperms, the establishment of the shoot apical meristem (SAM) occurs concomitantly with the formation of a crown of cotyledons surrounding the SAM. It has previously been shown that the differentiation of cotyledons in somatic embryos of Picea abies is dependent on polar auxin transport (PAT). In the angiosperm model plant, Arabidopsis thaliana , the establishment of cotyledonary boundaries and the embryonal SAM is dependent on PAT and the expression of the CUP-SHAPED COTYLEDON (CUC) genes, which belong to the large NAC gene family. The aim of this study was to characterize CUC -like genes in a gymnosperm, and to elucidate their expression during SAM and cotyledon differentiation, and in response to PAT. Methods Sixteen Picea glauca NAC sequences were identified in GenBank and deployed to different clades within the NAC gene family using maximum parsimony analysis and Bayesian inference. Motifs conserved between angiosperms and gymnosperms were analysed using the motif discovery tool MEME. Expression profiles during embryo development were produced using quantitative real-time PCR. Protein conservation was analysed by introducing a P. abies CUC orthologue into the A. thaliana cuc1cuc2 double mutant. Key Results Two full-length CUC -like cDNAs denoted PaNAC01 and PaNAC02 were cloned from P. abies. PaNAC01, but not PaNAC02, harbours previously characterized functional motifs in CUC1 and CUC2. The expression profile of PaNAC01 showed that the gene is PAT regulated and associated with SAM differentiation and cotyledon formation. Furthermore, PaNAC01 could functionally substitute for CUC2 in the A. thaliana cuc1cuc2 double mutant. Conclusions The results show that CUC -like genes with distinct signature motifs existed before the separation of angiosperms and gymnosperms approx. 300 million years ago, and suggest a conserved function between PaNAC01 and CUC1/CUC2.

Opponents of genetically modified crops continue to raise concerns about risk, despite 20 years of research disproving their claims. Science should close the book on risk research and turn to studying the economic and environmental benefits of agricultural biotechnology

SHORT-INTERNODES/STYLISH (SHI/STY)-family proteins redundantly regulate development of lateral organs in Arabidopsis thaliana. We have previously shown that STY1 interacts with the promoter of the auxin biosynthesis gene YUCCA (YUC)4 and activates transcription of the genes YUC4, YUC8 and OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF (ORA)59 independently of protein translation. STY1 also affects auxin levels and auxin biosynthesis rates. Here we show that STY1 induces the transcription of 16 additional genes independently of protein translation. Several of these genes are tightly co-expressed with SHI/STY-family genes and/or down-regulated in SHI/STY-family multiple mutant lines, suggesting them to be regulated by SHI/STY proteins during plant development. The majority of the identified genes encode transcription factors or cell expansion-related enzymes and functional studies suggest their involvement in stamen and leaf development or flowering time regulation.

The phosphatidyl ethanolamine-binding protein (PEBP) gene family is present in all eukaryote kingdoms, with three subfamilies identified in angiosperms (FLOWERING LOCUS T [FT], MOTHER OF FT AND TFL1 [MFT], and TERMINAL FLOWER1 [TFL1] like). In angiosperms, PEBP genes have been shown to function both as promoters and suppressors of flowering and to control plant architecture. In this study, we focus on previously uncharacterized PEBP genes from gymnosperms. Extensive database searches suggest that gymnosperms possess only two types of PEBP genes, MFT-like and a group that occupies an intermediate phylogenetic position between the FT-like and TFL1-like (FT/TFL1-like). Overexpression of Picea abies PEBP genes in Arabidopsis (Arabidopsis thaliana) suggests that the FT/TFL1-like genes (PaFTL1 and PaFTL2) code for proteins with a TFL1-like function. However, PaFTL1 and PaFTL2 also show highly divergent expression patterns. While the expression of PaFTL2 is correlated with annual growth rhythm and mainly confined to needles and vegetative and reproductive buds, the expression of PaFTL1 is largely restricted to microsporophylls of male cones. The P. abies MFT-like genes (PaMFT1 and PaMFT2) show a predominant expression during embryo development, a pattern that is also found for many MFT-like genes from angiosperms. P. abies PEBP gene expression is primarily detected in tissues undergoing physiological changes related to growth arrest and dormancy. A first duplication event resulting in two families of plant PEBP genes (MFT-like and FT/TFL1-like) seems to coincide with the evolution of seed plants, in which independent control of bud and seed dormancy was required, and the second duplication resulting in the FT-like and TFL1-like clades probably coincided with the evolution of angiosperms.

Genetically modified (GM) plants aimed at producing food/feed are part of regular agriculture in many areas of the World. Commodity plants have also found application as bioreactors, designated non-food/non-feed GM (NFGM) plants, thereby making raw material for further refinement to industrial, diagnostic or pharmaceutical preparations. Many among them may pose health challenge to consumers or livestock animals, if occurring in food/feed. NFGM plants are typically released into the environment, but are grown under special oversight and any among several containment practices, none of which provide full protection against accidental dispersal. Adventitious admixture with food or feed can occur either through distributional mismanagement or as a consequence of gene flow to plant relatives. To facilitate NFGM surveillance we propose a new mandatory tagging of essentially all such plants, prior to cultivation or marketing in the European Union. The suggested tag--Plant-Made Industrial or Pharmaceutical Products Tag (PMIP-T)--is envisaged to occur as a transgenic silent DNA identifier in host plants and designed to enable technically simple identification and characterisation of any NFGM. Implementation of PMIP-T would permit inexpensive, reliable and high-throughput screening for NFGM specifically. The paper outlines key NFGM prospects and challenges as well as the PMIP-T concept.

In orthodox seeds, the transcriptional activator ABI3 regulates two major stages in embryo maturation: a mid-maturation (MAT) stage leading to accumulation of storage compounds, and a late maturation (LEA) stage leading to quiescence and desiccation tolerance. Our aim was to elucidate mechanisms for transcriptional shutdown of MAT genes during late maturation, to better understand phase transition between MAT and LEA stages. Using transgenic and transient approaches in Nicotiana, we examined activities of two ABI3-dependent reporter genes driven by multimeric RY and abscisic acid response elements (ABREs) from a Brassica napus napin gene, termed RY and ABRE, where the RY reporter requires ABI3 DNA binding. Expression of RY peaks during mid-maturation and drops during late maturation, mimicking the MAT gene program, and in Arabidopsis thaliana RY elements are over-represented in MAT, but not in LEA, genes. The ABI3 transactivation of RY is inhibited by staurosporine, by a PP2C phosphatase, and by a repressor of maturation genes, VAL1/HSI2. The RY element mediates repression of MAT genes, and we propose that transcriptional shutdown of the MAT program during late maturation involves inhibition of ABI3 DNA binding by dephosphorylation. Later, during seedling growth, VAL1/HSI2 family repressors silence MAT genes by binding RY elements.

Flower development in plants depends not only on a set of nuclear genes but also on the coordinate action of the mitochondrion. Certain mitochondrial genomes in combination with certain nuclear genomes lead to the expression of cytoplasmic male-sterility (CMS). Both mitochondrial genes that determine male-sterility and nuclear Restorer-of-fertility genes that suppress the male-sterile phenotype have been cloned. Lately, the interactions between mitochondrial and nuclear genes through retrograde signalling in CMS-systems have been dissected. Of special interest are the altered expression patterns of floral homeotic genes in certain CMS-systems. Here, we review the mitochondrial influence on flower development and give examples from CMS-systems developed in Brassica, Daucus carota, Nicotiana tabacum and Triticum aestivum.

To gain new insights into the mechanism underlying cytoplasmic male sterility (CMS), we compared the nuclear gene expression profiles of flowers of a Brassica napus CMS line with that of the fertile B. napus maintainer line using Arabidopsis thaliana flower-specific cDNA microarrays. The CMS line used has a B. napus nuclear genome, but has a rearranged mitochondrial (mt) genome consisting of both B. napus and A. thaliana DNA. Gene expression profiling revealed that a large number of genes differed in expression between the two lines. For example, nuclear genes coding for proteins that are involved in protein import into organelles, genes expressed in stamens and pollen, as well as genes implicated in either cell-wall remodeling or architecture, were repressed in the CMS line compared with B. napus. These results show that the mt genome of the CMS line strongly influences nuclear gene expression, and thus reveal the importance of retrograde signalling between the mitochondria and the nucleus. Furthermore, flowers of the CMS line are characterized by a replacement of stamens with carpelloid organs, and thus partially resemble the APETALA3 (AP3) and PISTILLATA (PI) mutants. In accordance with this phenotype, AP3 expression was downregulated in the stamens, shortly before these organs developed carpelloid characteristics, even though it was initiated correctly. Repression of PI succeeded that of AP3 and might be a consequence of a loss of AP3 activity. These results suggest that AP3 expression in stamens depends on proper mt function and a correct nuclear–mt interaction, and that mt alterations cause the male sterility phenotype of the CMS line.

The plant nucleoside diphosphate kinase (NDPK, EC 2.7.4.6) gene family consists of three groups whose gene products are found in different subcellular locations. In this study we discuss the evolutionary history, localization and expression of the NDPK genes, addressing the question of functional specialization of the different NDPKs. A phylogenetic analysis revealed that the three NDPK isoforms were present already in the last common ancestor of vascular plants and mosses. Our data also imply that the NDPK3 genes possess a higher degree of conservation than the NDPK1 and NDPK2 genes. The expression levels of the different NDPKs in Arabidopsis thaliana inflorescences, leaves and roots were evaluated using quantitative PCR as well as in silico methods. This analysis showed that NDPK1 is the most highly expressed NDPK gene in all the studied tissues. NDPK3a has the second highest NDPK expression, while NDPK3b is expressed to a very low extent. However, expression of NDPK3b is elevated in inflorescence tissue. In situ hybridization experiments performed on inflorescences showed NDPK3a expression in actively dividing cells. NDPK3b expression was observed during later stages of flower development, specifically in the tapetum, ovules and petals. Additionally, we show that an NDPK3 protein is able to direct the green fluorescent protein to both mitochondria and chloroplasts using transient expression in leaf protoplasts. The dual localisation of NDPK3 was confirmed by Western blot, which also demonstrated that the majority of the NDPK3 protein is found in the mitochondria.

Antisense oligodeoxynucleotide (asODN) inhibition was developed in the 1970s, and since then has been widely used in animal research. However, in plant biology, the method has had limited application because plant cell walls significantly block efficient uptake of asODN to plant cells. Recently, we have found that asODN uptake is enhanced in a sugar solution. The method has promise for many applications, such as a rapid alternative to time-consuming transgenic studies, and high potential for studying gene functionality in intact plants and multiple plant species, with particular advantages in evaluating the roles of multiple gene family members. Generation of transgenic plants relies on the ability to select transformed cells. This screening process is based on co-introduction of marker genes into the plant cell together with a gene of interest. Currently, the most common marker genes are those that confer antibiotic or herbicide resistance. The possibility that traits introduced by selectable marker genes in transgenic field crops may be transferred horizontally is of major public concern. Marker genes that increase use of antibiotics and herbicides may increase development of antibiotic-resistant bacterial strains or contribute to weed resistance. Here, we describe a method for selection of transformed plant cells based on asODN inhibition. The method enables selective and high-throughput screening for transformed cells without conferring new traits or functions to the transgenic plants. Due to their high binding specificity, asODNs may also find applications as plant-specific DNA herbicides.

Reproductive organs in seed plants are morphologically divergent and their evolutionary history is often unclear. The mechanisms controlling their development have been extensively studied in angiosperms but are poorly understood in conifers and other gymnosperms. Here, we address the molecular control of seed cone development in Norway spruce, Picea abies. We present expression analyses of five novel MADS-box genes in comparison with previously identified MADS and LEAFY genes at distinct developmental stages. In addition, we have characterized the homeotic transformation from vegetative shoot to female cone and associated changes in regulatory gene expression patterns occurring in the acrocona mutant. The analyses identified genes active at the onset of ovuliferous and ovule development and identified expression patterns marking distinct domains of the ovuliferous scale. The reproductive transformation in acrocona involves the activation of all tested genes normally active in early cone development, except for an AGAMOUS-LIKE6/SEPALLATA (AGL6/SEP) homologue. This absence may be functionally associated with the nondeterminate development of the acrocona ovule-bearing scales. Our morphological and gene expression analyses give support to the hypothesis that the modern cone is a complex structure, and the ovuliferous scale the result of reductions and compactions of an ovule-bearing axillary short shoot in cones of Paleozoic conifers.

The establishment and maintenance of auxin maxima in vascular plants is regulated by auxin biosynthesis and polar intercellular auxin flow. The disruption of normal auxin biosynthesis in mouse-ear cress (Arabidopsis thaliana) leads to severe abnormalities, suggesting that spatiotemporal regulation of auxin biosynthesis is fundamental for normal growth and development. We have shown previously that the induction of the SHORT-INTERNODES/STYLISH (SHI/STY) family member STY1 results in increased transcript levels of the YUCCA (YUC) family member YUC4 and also higher auxin levels and auxin biosynthesis rates in Arabidopsis seedlings. We have also shown previously that SHI/STY family members redundantly affect development of flowers and leaves. Here, we further examine the function of STY1 by analyzing its DNA and protein binding properties. Our results suggest that STY1, and most likely other SHI/STY members, are DNA binding transcriptional activators that target genes encoding proteins mediating auxin biosynthesis. This suggests that the SHI/STY family members are essential regulators of auxin-mediated leaf and flower development. Furthermore, the lack of a shoot apical meristem in seedlings carrying a fusion construct between STY1 and a repressor domain, SRDX, suggests that STY1, and other SHI/STY members, has a role in the formation and/or maintenance of the shoot apical meristem, possibly by regulating auxin levels in the embryo.

Programmed cell death (PCD) is executed by proteases, which cleave diverse proteins thus modulating their biochemical and cellular functions. Proteases of the caspase family and hundreds of caspase substrates constitute a major part of the PCD degradome in animals. Plants lack close homologues of caspases, but instead possess an ancestral family of cysteine proteases, metacaspases. Although metacaspases are essential for PCD, their natural substrates remain unknown. Here we show that metacaspase mcII-Pa cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease), during both developmental and stress-induced PCD. TSN knockdown leads to activation of ectopic cell death during reproduction, impairing plant fertility. Surprisingly, human TSN (also known as p100 or SND1), a multifunctional regulator of gene expression, is cleaved by caspase-3 during apoptosis. This cleavage impairs the ability of TSN to activate mRNA splicing, inhibits its ribonuclease activity and is important for the execution of apoptosis. Our results establish TSN as the first biological substrate of metacaspase and demonstrate that despite the divergence of plants and animals from a common ancestor about one billion years ago and their use of distinct PCD pathways, both have retained a common mechanism to compromise cell viability through the cleavage of the same substrate, TSN.

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film. Anna Karlgren and Jenny Carlsson contributed equally to this study. Corresponding authors: Anna Karlgren at Anna.Karlgren@ebc.uu.se and Jens F. Sundström at Jens.Sundstrom@vbsg.slu.se

The high-throughput expression analysis technologies available today give scientists an overflow of expression profiles but their resolution in terms of tissue specific expression is limited because of problems in dissecting individual tissues. Expression data needs to be confirmed and complemented with expression patterns using e.g. in situ hybridization, a technique used to localize cell specific mRNA expression. The in situ hybridization method is laborious, time-consuming and often requires extensive optimization depending on species and tissue. In situ experiments are relatively more difficult to perform in woody species such as the conifer Norway spruce (Picea abies). Here we present a modified DIG in situ hybridization protocol, which is fast and applicable on a wide range of plant species including P. abies. With just a few adjustments, including altered RNase treatment and proteinase K concentration, we could use the protocol to study tissue specific expression of homologous genes in male reproductive organs of one gymnosperm and two angiosperm species; P. abies, Arabidopsis thaliana and Brassica napus. The protocol worked equally well for the species and genes studied. AtAP3 and BnAP3 were observed in second and third whorl floral organs in A. thaliana and B. napus and DAL13 in microsporophylls of male cones from P. abies. For P. abies the proteinase K concentration, used to permeablize the tissues, had to be increased to 3 g/ml instead of 1 g/ml, possibly due to more compact tissues and higher levels of phenolics and polysaccharides. For all species the RNase treatment was removed due to reduced signal strength without a corresponding increase in specificity. By comparing tissue specific expression patterns of homologous genes from both flowering plants and a coniferous tree we demonstrate that the DIG in situ protocol presented here, with only minute adjustments, can be applied to a wide range of plant species. Hence, the protocol avoids both extensive species specific optimization and the laborious use of radioactively labeled probes in favor of DIG labeled probes. We have chosen to illustrate the technically demanding steps of the protocol in our film.

Background In most gymnosperms the cotyledons develop as a crown surrounding the incipient shoot apical meristem (SAM), which maintains the radial symmetry of the plant throughout embryogenesis. This is in contrast to the Arabidopsis embryo in which a symmetry-breaking event (from radial to bilateral symmetry) is associated with the emergence of the SAM and the two cotyledons [1]. We have previously shown that the differentiation of cotyledons and the establishment of the SAM and the root apical meristem (RAM) in somatic embryos of Norway spruce (Picea abies) is dependent on polar auxin transport (PAT) [2]. In Arabidopsis PAT restricts the expression of several genes belonging to the large plant specific NAC family of transcription factors [3-5], which in turn induce developmental processes involved in booth shoot and root formation. Members of the NAC family have a conserved DNA-binding domain in the Nterminal region known as the NAC domain, and a more diverse domain in the C-terminal region, proposed to be responsible for the trans-activation capacity of the protein [6]. Despite the diversity of the NAC gene family, and the importance of NAC proteins in fundamental processes, no NAC genes have to our knowledge been characterized in conifers.

Somatic embryogenesis is used for vegetative propagation of conifers. Embryogenic cultures can be established from zygotic embryos; however, the embryogenic potential decreases during germination. In Arabidopsis, LEAFY COTYLEDON (LEC) genes are expressed during the embryonic stage, and must be repressed to allow germination. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) causes de-repression of LEC genes. ABSCISICACID3 (ABI3) and its Zeamays ortholog VIVIPAROUS1 (VP1) act together with the LEC genes to promote embryo maturation. In this study, we have asked the question whether TSA treatment in a conifer affects the embryogenic potential and the expression of embryogenesis-related genes. We isolated two conifer LEC1-type HAP3 genes, HAP3A and HAP3B, from Picea abies and Pinus sylvestris. A comparative phylogenetic analysis of plant HAP3 genes suggests that HAP3A and HAP3B are paralogous genes originating from a duplication event in the conifer lineage. The expression of HAP3A is high, in both somatic and zygotic embryos, during early embryo development, but decreases during late embryogeny. In contrast, the expression of VP1 is initially low but increases during late embryogeny. After exposure to TSA, germinating somatic embryos of P. abies maintain the competence to differentiate embryogenic tissue, and simultaneously the germination progression is partially inhibited. Furthermore, when embryogenic cultures of P. abies are exposed to TSA during embryo maturation, the maturation process is arrested and the expression levels of PaHAP3A and PaVP1 are maintained, suggesting a possible link between chromatin structure and expression of embryogenesis-related genes in conifers. Electronic supplementary material The online version of this article (doi:10.1007/s00425-011-1418-8) contains supplementary material, which is available to authorized users.