Cryo-Treatment Enhances the Embryogenicity of Mature Somatic Embryos via the lncRNA-miRNA-mRNA Network in White Spruce.

In conifers, somatic embryogenesis is uniquely initiated from immature embryos in a narrow time window, which is considerably hindered by the difficulty to induce embryogenic tissue (ET) from other tissues, including mature somatic embryos. In this study, the embryogenic ability of newly induced ET and DNA methylation levels was detected, and whole-transcriptome sequencing analyses were carried out. The results showed that ultra-low temperature treatment significantly enhanced ET induction from mature somatic embryos, with the induction rate from 0.4% to 15.5%, but the underlying mechanisms remain unclear. The newly induced ET showed higher capability in generating mature embryos than the original ET. DNA methylation levels fluctuated during the ET induction process. Here, WGCNA analysis revealed that OPT4, TIP1-1, Chi I, GASA5, GST, LAX3, WRKY7, MYBS3, LRR-RLK, PBL7, and WIN1 genes are involved in stress response and auxin signal transduction. Through co-expression analysis, lncRNAs MSTRG.505746.1, MSTRG.1070680.1, and MSTRG.33602.1 might bind to pre-novel_miR_339 to promote the expression of WRKY7 genes for stress response; LAX3 could be protected by lncRNAs MSTRG.1070680.1 and MSTRG.33602.1 via serving as sponges for novel_miR_495 to initiate auxin signal transduction; lncRNAs MSTRG.505746.1, MSTRG.1070680.1, and MSTRG.33602.1 might serve as sponges for novel_miR_527 to enhance the expression of Chi I for early somatic embryo development. This study provides new insight into the area of stress-enhanced early somatic embryogenesis in conifers, which is also attributable to practical applications.

Different Roles of Auxins in Somatic Embryogenesis Efficiency in Two Picea Species.

The effects of auxins 2,4-D (2,4-dichlorophenoxyacetic acid), NAA (1-naphthaleneacetic acid) or picloram (4-amino-3,5,6-trichloropicolinic acid; 9 µM) and cytokinin BA (benzyloadenine; 4.5 µM) applied in the early stages of somatic embryogenesis (SE) on specific stages of SE in Picea abies and P. omorika were investigated. The highest SE initiation frequency was obtained after 2,4-D application in P. omorika (22.00%) and picloram application in P. abies (10.48%). NAA treatment significantly promoted embryogenic tissue (ET) proliferation in P. abies, while 2,4-D treatment reduced it. This reduction was related to the oxidative stress level, which was lower with the presence of NAA in the proliferation medium and higher with the presence of 2,4-D. The reduced oxidative stress level after NAA treatment suggests that hydrogen peroxide (H2O2) acts as a signalling molecule and promotes ET proliferation. NAA and picloram in the proliferation medium decreased the further production and maturation of P. omorika somatic embryos compared with that under 2,4-D. The quality of the germinated P. abies embryos and their development into plantlets depended on the auxin type and were the highest in NAA-originated embryos. These results show that different auxin types can generate different physiological responses in plant materials during SE in both spruce species.

Tissue-specific transposon-associated small RNAs in the gymnosperm tree, Norway spruce.

BACKGROUND: Small RNAs (sRNAs) are regulatory molecules impacting on gene expression and transposon activity. MicroRNAs (miRNAs) are responsible for tissue-specific and environmentally-induced gene repression. Short interfering RNAs (siRNA) are constitutively involved in transposon silencing across different type of tissues. The male gametophyte in angiosperms has a unique set of sRNAs compared to vegetative tissues, including phased siRNAs from intergenic or genic regions, or epigenetically activated siRNAs. This is contrasted by a lack of knowledge about the sRNA profile of the male gametophyte of gymnosperms. RESULTS: Here, we isolated mature pollen from male cones of Norway spruce and investigated its sRNA profiles. While 21-nt sRNAs is the major size class of sRNAs in needles, in pollen 21-nt and 24-nt sRNAs are the most abundant size classes. Although the 24-nt sRNAs were exclusively derived from TEs in pollen, both 21-nt and 24-nt sRNAs were associated with TEs. We also investigated sRNAs from somatic embryonic callus, which has been reported to contain 24-nt sRNAs. Our data show that the 24-nt sRNA profiles are tissue-specific and differ between pollen and cell culture. CONCLUSION: Our data reveal that gymnosperm pollen, like angiosperm pollen, has a unique sRNA profile, differing from vegetative leaf tissue. Thus, our results reveal that angiosperm and gymnosperm pollen produce new size classes not present in vegetative tissues; while in angiosperm pollen 21-nt sRNAs are generated, in the gymnosperm Norway spruce 24-nt sRNAs are generated. The tissue-specific production of distinct TE-derived sRNAs in angiosperms and gymnosperms provides insights into the diversification process of sRNAs in TE silencing pathways between the two groups of seed plants.

DNA methylome of the 20-gigabase Norway spruce genome.

DNA methylation plays important roles in many biological processes, such as silencing of transposable elements, imprinting, and regulating gene expression. Many studies of DNA methylation have shown its essential roles in angiosperms (flowering plants). However, few studies have examined the roles and patterns of DNA methylation in gymnosperms. Here, we present genome-wide high coverage single-base resolution methylation maps of Norway spruce (Picea abies) from both needles and somatic embryogenesis culture cells via whole genome bisulfite sequencing. On average, DNA methylation levels of CG and CHG of Norway spruce were higher than most other plants studied. CHH methylation was found at a relatively low level; however, at least one copy of most of the RNA-directed DNA methylation pathway genes was found in Norway spruce, and CHH methylation was correlated with levels of siRNAs. In comparison with needles, somatic embryogenesis culture cells that are used for clonally propagating spruce trees showed lower levels of CG and CHG methylation but higher level of CHH methylation, suggesting that like in other species, these culture cells show abnormal methylation patterns.

Overexpression of PaNAC03, a stress induced NAC gene family transcription factor in Norway spruce leads to reduced flavonol biosynthesis and aberrant embryo development.

BACKGROUND: The NAC family of transcription factors is one of the largest gene families of transcription factors in plants and the conifer NAC gene family is at least as large, or possibly larger, as in Arabidopsis. These transcription factors control both developmental and stress induced processes in plants. Yet, conifer NACs controlling stress induced processes has received relatively little attention. This study investigates NAC family transcription factors involved in the responses to the pathogen Heterobasidion annosum (Fr.) Bref. sensu lato. RESULTS: The phylogeny and domain structure in the NAC proteins can be used to organize functional specificities, several well characterized stress-related NAC proteins are found in III-3 in Arabidopsis (Jensen et al. Biochem J 426:183-196, 2010). The Norway spruce genome contain seven genes with similarity to subgroup III-3 NACs. Based on the expression pattern PaNAC03 was selected for detailed analyses. Norway spruce lines overexpressing PaNAC03 exhibited aberrant embryo development in response to maturation initiation and 482 misregulated genes were identified in proliferating cultures. Three key genes in the flavonoid biosynthesis pathway: a CHS, a F3'H and PaLAR3 were consistently down regulated in the overexpression lines. In accordance, the overexpression lines showed reduced levels of specific flavonoids, suggesting that PaNAC03 act as a repressor of this pathway, possibly by directly interacting with the promoter of the repressed genes. However, transactivation studies of PaNAC03 and PaLAR3 in Nicotiana benthamiana showed that PaNAC03 activated PaLAR3A, suggesting that PaNAC03 does not act as an independent negative regulator of flavan-3-ol production through direct interaction with the target flavonoid biosynthetic genes. CONCLUSIONS: PaNAC03 and its orthologs form a sister group to well characterized stress-related angiosperm NAC genes and at least PaNAC03 is responsive to biotic stress and appear to act in the control of defence associated secondary metabolite production.

Proteomic analysis of stress-related proteins and metabolic pathways in Picea asperata somatic embryos during partial desiccation.

Partial desiccation treatment (PDT) stimulates germination and enhances the conversion of conifer somatic embryos. To better understand the mechanisms underlying the responses of somatic embryos to PDT, we used proteomic and physiological analyses to investigate these responses during PDT in Picea asperata. Comparative proteomic analysis revealed that, during PDT, stress-related proteins were mainly involved in osmosis, endogenous hormones, antioxidative proteins, molecular chaperones and defence-related proteins. Compared with those in cotyledonary embryos before PDT, these stress-related proteins remained at high levels on days 7 (D7) and 14 (D14) of PDT. The proteins that differentially accumulated in the somatic embryos on D7 were mapped to stress and/or stimuli. They may also be involved in the glyoxylate cycle and the chitin metabolic process. The most significant difference in the differentially accumulated proteins occurred in the metabolic pathways of photosynthesis on D14. Furthermore, in accordance with the changes in stress-related proteins, analyses of changes in water content, abscisic acid, indoleacetic acid and H2 O2 levels in the embryos indicated that PDT is involved in water-deficit tolerance and affects endogenous hormones. Our results provide insight into the mechanisms responsible for the transition from morphologically mature to physiologically mature somatic embryos during the PDT process in P. asperata.

WUSCHEL-RELATED HOMEOBOX 2 is important for protoderm and suspensor development in the gymnosperm Norway spruce.

BACKGROUND: Distinct expression domains of WUSCHEL-RELATED HOMEOBOX (WOX) gene family members are involved in patterning and morphogenesis of the early embryo in Arabidopsis. However, the role of WOX genes in other taxa, including gymnosperms, remains elusive. Here, we use somatic embryos and reverse genetics for studying expression and function of PaWOX2, the corresponding homolog of AtWOX2 in the gymnosperm Picea abies (Pa; Norway spruce). RESULTS: The mRNA level of PaWOX2 was transiently up-regulated during early and late embryogeny. PaWOX2 mRNA in early and early late embryos was detected both in the embryonal mass and in the upper part of the suspensor. Down-regulation of PaWOX2 during development of early embryos resulted in aberrant early embryos, which failed to form a proper protoderm. Cells on the surface layer of the embryonal mass became vacuolated, and new embryogenic tissue differentiated from the embryonal mass. In addition, the aberrant early embryos lacked a distinct border between the embryonal mass, and the suspensor and the length of the suspensor cells was reduced. Down-regulation of PaWOX2 in the beginning of embryo development, before late embryos were formed, caused a significant decrease in the yield of mature embryos. On the contrary, down-regulation of PaWOX2 after late embryos were formed had no effect on further embryo development and maturation. CONCLUSIONS: Our data suggest an evolutionarily conserved function of WOX2 in protoderm formation early during embryo development among seed plants. In addition, PaWOX2 might exert a unique function in suspensor expansion in gymnosperms.

EXTRA SPINDLE POLES (Separase) controls anisotropic cell expansion in Norway spruce (Picea abies) embryos independently of its role in anaphase progression.

The caspase-related protease separase (EXTRA SPINDLE POLES, ESP) plays a major role in chromatid disjunction and cell expansion in Arabidopsis thaliana. Whether the expansion phenotypes are linked to defects in cell division in Arabidopsis ESP mutants remains elusive. Here we present the identification, cloning and characterization of the gymnosperm Norway spruce (Picea abies, Pa) ESP. We used the P. abies somatic embryo system and a combination of reverse genetics and microscopy to explore the roles of Pa ESP during embryogenesis. Pa ESP was expressed in the proliferating embryonal mass, while it was absent in the suspensor cells. Pa ESP associated with kinetochore microtubules in metaphase and then with anaphase spindle midzone. During cytokinesis, it localized on the phragmoplast microtubules and on the cell plate. Pa ESP deficiency perturbed anisotropic expansion and reduced mitotic divisions in cotyledonary embryos. Furthermore, whilst Pa ESP can rescue the chromatid nondisjunction phenotype of Arabidopsis ESP mutants, it cannot rescue anisotropic cell expansion. Our data demonstrate that the roles of ESP in daughter chromatid separation and cell expansion are conserved between gymnosperms and angiosperms. However, the mechanisms of ESP-mediated regulation of cell expansion seem to be lineage-specific.

Transcriptomic and proteomic analyses of embryogenic tissues in Picea balfouriana treated with 6-benzylaminopurine.

The cytokinin 6-benzylaminopurine (6-BAP) influences the embryogenic capacity of the tissues of Picea balfouriana during long subculture (after 3 months). Tissues that proliferate in 3.6 and 5 µM 6-BAP exhibit the highest and lowest embryogenic capacity, respectively, generating 113 ± 6 and 23 ± 3 mature embryos per 100 mg of tissue. In this study, a comparative transcriptomic and proteomic approach was applied to characterize the genes and proteins that are differentially expressed among tissues under the influence of different levels of 6-BAP. A total of 51 375 unigenes and 2617 proteins were obtained after quality filtering. There were 2770 transcripts for proteins found among these unigenes. Gene ontology (GO) analysis of the differentially expressed unigenes and proteins showed that they were involved in cell and binding activity and were enriched in ribosome and glutathione metabolism pathways. Ribosomal proteins, glutathione S-transferase proteins, germin-like proteins and calmodulin-independent protein kinases were up-regulated in the embryogenic tissues with the highest embryogenic ability (treated with 3.6 µM 6-BAP), which was validated via quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and these proteins might serve as molecular markers of embryogenic ability. Data are available via Sequence Read Archive (SRA) and ProteomeXchange with identifier SRP042246 and PXD001022, respectively.

Wuschel-related homeobox 8/9 is important for proper embryo patterning in the gymnosperm Norway spruce.

Proper embryo development is crucial as that is when the primary body axes are established. In Arabidopsis, AtWOX8 and AtWOX9, members of the Wuschel-related homeobox (WOX) gene family, are critical for embryo development. In Norway spruce, PaWOX8/9, which is expressed in embryos, is the homologue of AtWOX8 and AtWOX9. In this work, it is shown that the transcript abundance of PaWOX8/9 is high during early and late embryogeny and that it decreases when the maturation phase starts. To address the function of PaWOX8/9 during embryo development, RNAi lines were established to down-regulate the transcript level of PaWOX8/9, using both constitutive and inducible promoters. Embryos in the PaWOX8/9 RNAi lines show an aberrant morphology caused by disturbed orientation of the cell division plane at the basal part of the embryonal mass during early and late embryogeny. In addition, the transcript level of several key cell-cycle-regulating genes, for example, PaE2FAB-like and PaCYCLIN B-like, are affected in the PaWOX8/9 RNAi lines. Taken together, our results suggest that PaWOX8/9 may perform an evolutionarily conserved function as a regulator of the establishment of the apical-basal embryo pattern.

Potential link between biotic defense activation and recalcitrance to induction of somatic embryogenesis in shoot primordia from adult trees of white spruce (Picea glauca).

BACKGROUND: Among the many commercial opportunities afforded by somatic embryogenesis (SE), it is the ability to clonally propagate individual plants with rare or elite traits that has some of the most significant implications. This is particularly true for many long-lived species, such as conifers, but whose long generation times pose substantive challenges, including increased recalcitrance for SE as plants age. Identification of a clonal line of somatic embryo-derived trees whose shoot primordia have remained responsive to SE induction for over a decade, provided a unique opportunity to examine the molecular aspects underpinning SE within shoot tissues of adult white spruce trees. RESULTS: Microarray analysis was used to conduct transcriptome-wide expression profiling of shoot explants taken from this responsive genotype following one week of SE induction, which when compared with that of a nonresponsive genotype, led to the identification of four of the most differentially expressed genes within each genotype. Using absolute qPCR to expand the analysis to three weeks of induction revealed that differential expression of all eight candidate genes was maintained to the end of the induction treatment, albeit to differing degrees. Most striking was that both the magnitude and duration of candidate gene expression within the nonresponsive genotype was indicative of an intense physiological response. Examining their putative identities further revealed that all four encoded for proteins with similarity to angiosperm proteins known to play prominent roles in biotic defense, and that their high-level induction over an extended period is consistent with activation of a biotic defense response. In contrast, the more temperate response within the responsive genotype, including induction of a conifer-specific dehydrin, is more consistent with elicitation of an adaptive stress response. CONCLUSIONS: While additional evidence is required to definitively establish an association between SE responsiveness and a specific physiological response, these results suggest that biotic defense activation may be antagonistic, likely related to the massive transcriptional and metabolic reprogramming that it elicits. A major issue for future work will be to determine how and if suppressing biotic defense activation could be used to promote a physiological state more conducive to SE induction.

The effect of carbohydrates and osmoticum on storage reserve accumulation and germination of Norway spruce somatic embryos.

Somatic embryogenesis (SE) represents a useful experimental system for studying the regulatory mechanisms of embryo development. In this study, the effect of carbohydrates and osmoticum on storage reserve accumulation and germination of Norway spruce [Picea abies (L.) Karst] somatic embryos were investigated. Using time lapse photography, we monitored development from proliferation of proembryogenic masses (PEMs) to maturation of somatic embryos in two P. abies cell lines cultured on two maturation treatments. A combination of sugar assays, metabolic and proteomic analyses were used to quantify storage reserves in the mature somatic embryos. The maturation treatment containing a nonpermeating osmoticum, polyethylene glycol (PEG, 7.5%) and maltose (3%) as the carbohydrate gave significantly high maturation and low germination frequencies of somatic embryos compared to the treatment with only 3% sucrose. Somatic embryos treated with 3% sucrose contained high levels of sucrose, raffinose and late embryogenesis abundant (LEA) proteins. These compounds are known to be involved in the acquisition of desiccation tolerance during seed development and maturation. In addition the sucrose treatment significantly increased the content of starch in the somatic embryos while the maltose and PEG treatment resulted in somatic embryos with a high content of storage proteins. The high levels of sucrose, raffinose and LEA proteins in the embryos treated with 3% sucrose suggest that sucrose may improve the germination of somatic embryos by promoting the acquisition of desiccation tolerance.

A life cycle model to enable research of cryostorage recalcitrance in temperate woody species: the case of sitka spruce (Picea sitchensis).

Empirical testing of protocols and fundamental investigations are the approaches usually applied to study germplasm storage recalcitrance in temperate plants. However, they can fall short of practicable solutions, even after exhaustive experimentation, and the generation of negative survival data makes it difficult to plan further investigations. Picea sitchensis somatic embryos are amenable to cryopreservation whereas in vitro shoot meristems, although able to survive, are incapable of sustained recovery. Differential Scanning Calorimetry (DSC) revealed that these disparate responses could not be attributed to biophysical factors. A model is presented hypothesising that in some cases life cycle adaptations (cold hardening, dormancy) may have opposing influences on survival causing delayed-onset, cryogenically-induced loss of viability in temperate tree species.

Initiation of somatic embryos and regeneration of plants from primordial shoots of 10-year-old somatic white spruce and expression profiles of 11 genes followed during the tissue culture process.

Adult conifers are notoriously recalcitrant in vegetative propagation and micropropagation that would result in the regeneration of juvenile propagules through somatic embryogenesis (SE) has not been demonstrated to date. Because SE-derived material is more amenable in subsequent tissue culture experiments compared with seed-derived material, a multi-year study was conducted to investigate induction of SE from primordial shoot (PS) explants that were excised from shoot buds of somatic embryo-derived white spruce. The SE induction experiments were carried out first with greenhouse-grown and later with field-grown trees each year from 2002 (2-year-old) to 2010 (10-year-old). Of the four genotypes tested, 893-2 and 893-12 never responded, 893-1 responded up to year 4 and 893-6 consistently responded every year. In 2010, for the first time, three of the 17 893-6 clonal trees produced male strobili as well as SE from cultured PS explants. SE induction was associated with formation of a nodule on the surface of an elongated needle primordium or in callus. Early somatic embryos were detectable after about 3 weeks of culture. Of 11 genes whose expression profiles were followed during the PS cultures, CHAP3A, VP1, WOX2 and SAP2C were expressed exclusively in the early stages of SE, and could potentially be used as markers of embryogenecity. Mature somatic embryos and plants were produced from the explants of responding genotype. Implication of these results for future research on adult conifer recalcitrance in micropropagation is discussed.

Possible effect from shear stress on maturation of somatic embryos of Norway spruce (Picea abies).

Somatic embryogenesis is the only method with the potential for industrial scale clonal propagation of conifers. Implementation of the method has so far been hampered by the extensive manual labor required for development of the somatic embryos into plants. The utilization of bioreactors is limited since the somatic embryos will not mature and germinate under liquid culture conditions. The negative effect on mature embryo yields from liquid culture conditions has been previously described. We have described the negative effects of shear stress on the development of early stage somatic embryos (proembryogenic masses; PEMs) at shear stresses of 0.086 and 0.14 N/m(2). In the present study, additional flow rates were studied to determine the effects of shear stress at lower rates resembling shear stress in a suspension culture flask. The results showed that shear stress at 0.009, 0.014, and 0.029 N/m(2) inhibited the PEM expansions comparing with the control group without shear stress. This study also provides validation for the cross-correlation method previously developed to show the effect of shear stress on early stage embryo suspensor cell formation and polarization. Furthermore, shear stress was shown to positively affect the uptake of water into the cells. The results indicate that the plasmolyzing effect from macromolecules added to liquid culture medium to stimulate maturation of the embryos are affected by liquid culture conditions and thus can affect the conversion of PEMs into mature somatic embryos.

Somatic embryogenesis in mature zygotic embryos of Picea pungens.

This study developed somatic embryogenesis protocols for Picea pungens (Engelm), an important ornamental species, including initiation, proliferation, maturation, germination, and acclimation. Somatic embryogenic tissues were induced from mature zygotic embryos of five families, with a frequency of [Formula: see text] 22% for each. Embryogenic tissues (ET) from 13 clones of three families were proliferated for one week, achieving an average rate of 179.1%. The ET of 38 clones of three families were cultured in maturation medium for six weeks; 188 mature embryos on average were counted per gram ET cultured, of which [Formula: see text] 81.1% appeared normal, and each clone developed at least 28 normally matured embryos. A total of 69.9% or more of cotyledonary somatic embryos germinated normally and developed into normal emblings. The experiment of transplanting the emblings into a greenhouse had an average survival rate of 68.5%. Considerable variation among and within families during initiation and proliferation was observed, but this variation decreased in the maturation and germination. Changing the concentration of plant growth regulator of the initiation medium did not significantly change the initiation frequency. We recommend incorporating these protocols into the current Picea pungens practical programs, although further research is essential to increase efficiencies and reduce cost.

The role of actin isoforms in somatic embryogenesis in Norway spruce.

BACKGROUND: Somatic embryogenesis in spruce is a process of high importance for biotechnology, yet it comprises of orchestrated series of events whose cellular and molecular details are not well understood. In this study, we examined the role of actin cytoskeleton during somatic embryogenesis in Norway spruce line AFO 541 by means of anti-actin drugs. RESULTS: Application of low doses (50-100 nM) of latrunculin B (Lat B) during the maturation of somatic embryos predominantly killed suspensor cells while leaving the cells in meristematic centres alive, indicating differential sensitivity of actin in the two cell types. The treatment resulted in faster development of more advanced embryos into mature somatic embryos and elimination of insufficiently developed ones. In searching for the cause of the differential actin sensitivity of the two cell types, we analysed the composition of actin isoforms in the culture and isolated four spruce actin genes. Analysis of their expression during embryo maturation revealed that one actin isoform was expressed constitutively in both cell types, whereas three actin isoforms were expressed predominantly in suspensor cells and their expression declined during the maturation. The expression decline was greatly enhanced by Lat B treatment. Sequence analysis revealed amino-acid substitutions in the Lat B-binding site in one of the suspensor-specific actin isoforms, which may result in a different binding affinity for Lat B. CONCLUSIONS: We show that manipulating actin in specific cell types in somatic embryos using Lat B treatment accelerated and even synchronized the development of somatic embryos and may be of practical use in biotechnology.

Comparative expression pattern analysis of WUSCHEL-related homeobox 2 (WOX2) and WOX8/9 in developing seeds and somatic embryos of the gymnosperm Picea abies.

• In seed plants, current knowledge concerning embryonic pattern formation by polar auxin transport (PAT) and WUSCHEL-related homeobox (WOX) gene activity is primarily derived from studies on angiosperms, while less is known about these processes in gymnosperms. In view of the differences in their embryogeny, and the fact that somatic embryogenesis is used for mass propagation of conifers, a better understanding of embryo development is vital. • The expression patterns of PaWOX2 and PaWOX8/9 were followed with quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH) during seed and somatic embryo development in Norway spruce (Picea abies), and in somatic embryos treated with the PAT inhibitor N-1-naphthylphthalamic acid (NPA). • Both PaWOX2 and PaWOX8/9 were highly expressed at the early growth stages of zygotic and somatic embryos, and shared a similar expression pattern over the entire embryo. At later embryo stages, high expression of PaWOX8/9 became restricted to cotyledon primordia, epidermis, procambium and root apical meristem (RAM), which became most evident in NPA-treated somatic embryos, while expression of PaWOX2 was much lower. • Our results suggest an ancestral role of WOX in seed plant embryo development, and strengthen the proposed connection between PAT, PIN-FORMED (PIN) and WOX in the regulation of embryo patterning in seed plants.

Cryotolerance in Norway spruce and its association with growth rates, anatomical features and polyamines of embryogenic cultures.

Our study focused on the possible association between the cryotolerance of Norway spruce (Picea abies (L.) Karst.) embryogenic cultures and the anatomical structures of their embryogenic suspensor mass (ESM), their growth rate and their content of endogenous polyamines (PAs). The anatomical characteristics and PA content during cryopreservation and regrowth were studied in the ESMs of AFO 541 and C110 cultures, which have comparable ESM anatomy but diverse growth rates, PA content and regeneration abilities after cryopreservation. Different levels of tolerance to exogenous treatment were already apparent after transfer of the ESMs to liquid media. The endogenous free PAs were maintained at high levels, with spermidine being the predominant PA in the ESM of AFO 541, while in the ESM of C110 the content of putrescine and spermidine was almost identical and rather low, the content of spermidine being approximately one-third that in the ESM of AFO 541. Osmotic pretreatment, using a double application of sorbitol followed by an application of dimethyl sulfoxide (DMSO) resulted in the continual disintegration of polyembryogenic centers and suspensors in both cell lines. A continual decrease in the level of PAs was observed during the cell osmotic pretreatment. The cells that retained their viability and regrowth ability after cryopreservation were the meristematic cells inside the embryonal heads and the cells in the intermediate area between suspensor and meristems. Restoration of AFO 541 growth after cryopreservation was almost immediate; however, the C110 ESM culture regrew with difficulty, often exhibiting callogenesis. High levels of PA-soluble conjugates and an increase in the amount of PAs bound to high-molecular-mass substances was observed in cells of AFO 541 on Day 6 after thawing and also to some extent on Day 11. On Day 21 after thawing, the amount of free putrescine and spermidine in the AFO 541 cells reached the level observed in the suspension culture before the cryotreatment. The extremely low level of PAs determined in the ESM of C110 3 weeks after thawing agreed with the cell viability and rate of regrowth observed in this culture. The possible role of PAs in the process of cryopreservation of Norway spruce cultures is discussed.

Expression of a gymnosperm PIN homologous gene correlates with auxin immunolocalization pattern at cotyledon formation and in demarcation of the procambium during Picea abies somatic embryo development and in seedling tissues.

In seed plants, the body organization is established during embryogenesis and is uniform across gymnosperms and angiosperms, despite differences during early embryogeny. Evidence from angiosperms implicates the plant hormone auxin and its polar transport, mainly established by the PIN family of auxin efflux transporters, in the patterning of embryos. Here, PaPIN1 from Norway spruce (Picea abies [L.] Karst.), a gene widely expressed in conifer tissues and organs, was characterized and its expression and localization patterns were determined with reverse transcription polymerase chain reaction and in situ hybridization during somatic embryo development and in seedlings. PaPIN1 shares the predicted structure of other PIN proteins, but its central hydrophilic loop is longer than most PINs. In phylogenetic analyses, PaPIN1 clusters with Arabidopsis thaliana (L.) Heynh. PIN3, PIN4 and PIN7, but its expression pattern also suggests similarity to PIN1. The PaPIN1 expression signal was high in the protoderm of pre-cotyledonary embryos, but not if embryos were pre-treated with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). This, together with a high auxin immunolocalization signal in this cell layer, suggests a role of PaPIN1 during cotyledon formation. At later stages, high PaPIN1 expression was observed in differentiating procambium, running from the tip of incipient cotyledons down through the embryo axis and to the root apical meristem (RAM), although the mode of RAM specification in conifer embryos differs from that of most angiosperms. Also, the PaPIN1 in situ signal was high in seedling root tips including root cap columella cells. The results thus suggest that PaPIN1 provides an ancient function associated with auxin transport and embryo pattern formation prior to the separation of angiosperms and gymnosperms, in spite of some morphological differences.