BACKGROUND
in vitro embryogenesis systems constitute important tools for investigating the regulating mechanisms of embryo formation, as well as for biotechnological applications in plant breeding, propagation and conservation strategies.
two different embryogenesis pathways, from microspores and immature zygotic embryos, have been developed in many species for rapid production of isogenic lines and clonal propagation of elite genotypes respectively.
tree breeding strategies have focused on ways to reduce cycle time and improve the efficiency of selection; here, propagation of selected trees by somatic embryogenesis and genetic engineering approaches applied to haploids and double-haploid plants produced in short-times by in vitro microspore embryogenesis have a high potential.
nevertheless, due to their recalcitrance, the efficiency of embryogenesis in vitro systems in many woody species is still very low.
quercus trees, oaks, are woody species of relevant economic and ecological interest; among them the cork oak, q. suber, is a species of high social and economic relevance in the mediterranean area as the natural source of cork.
different in vitro embryogenesis systems have been developed for quercus spps., with variable efficiency  <cit> .
despite the work performed in the last decades, factors responsible for embryogenesis initiation and embryo development in established cultures are still largely unknown.
the search for molecular and cellular markers during early stages of in vitro embryogenesis constitutes an important goal in the identification of cells committed to the embryogenesis developmental program as opposed to those cells which are non-responsive to the embryogenic pathway, as well as in the elucidation of the cellular mechanisms underlying in vitro embryo progression.
changes in various cell activities and in the structural organization of subcellular compartments have been reported as accompanying the microspore reprogramming process in some herbaceous and woody species  <cit> .
increasing evidences have indicated the relevance of some cell features like epigenetic marks  <cit> , cell wall components  <cit>  and hormones  <cit>  in the progression of in vitro organogenesis and embryogenesis in other systems, but no reports about the dynamics of these three cellular markers during in vitro early embryogenesis are available in two somatic embryogenesis pathways of the same species.
dynamic changes between chromatin states are relevant in the transcriptional regulation during microspore development and reprogramming to embryogenesis  <cit>  and epigenetic mechanisms play an essential role in the process of cellular differentiation allowing cells to be reprogrammed in order to generate new differentiation pathways  <cit> .
dna methylation constitutes a prominent epigenetic modification of the chromatin fiber, which becomes locked in a transcriptionally inactive conformation, thus leading to gene silencing.
stress-induced plant cell reprogramming involves changes in global genome organization, being the epigenetic modifications key factors of genome flexibility  <cit> .
previous studies have shown changes in dna methylation levels and distribution patterns during microspore embryogenesis of brassica napus and hordeum vulgare  <cit> , suggesting the existence of an epigenetic reprogramming after microspore induction to embryogenesis, but no information is available on dna methylation dynamics during in vitro embryogenesis in trees.
many of the molecular markers of in vitro embryogenesis and organogenesis have been found in cell walls  <cit> .
pectins are the major matrix components of dicotyledonous cell walls.
pectins are polymerized and methyl-esterified in the golgi, and secreted into the wall as highly methyl-esterified forms.
subsequently, they can be modified by pectin methylesterases, which catalyse the demethylesterification of homogalacturonans domains of pectins.
the relationship between the esterified and the non-esterified pectins, and their distribution in the plant cell walls is the result of different processes  <cit>  and their proportion and dynamics are involved in many plant developmental processes.
changes in the distribution of pectins have been reported in young embryos generated from microspores of quercus suber l.  <cit> , citrus clementina  <cit>  and olea europaea l.  <cit> .
abundant esterified pectins has been reported in the newly-formed walls of proliferating cells in different plant systems, like young microspore-derived embryos and root tip meristematic cells of herbaceous species  <cit> , suggesting that a high proportion of esterified pectins in walls is a marker for proliferation events while abundant low-esterified pectins correspond to low cell proliferation rates  <cit> .
the plant growth regulator  auxin, the predominant form of which is indole-3-acetic acid , is a major coordinating signal in the regulation of plant development, with key functions in many processes including embryo formation  <cit> .
despite it is widely known the stimulating effects of the addition of exogenous pgrs, including synthetic auxins like  <dig> -d, on in vitro embryogenesis induction  <cit> , little is known about endogenous levels of these regulators at the initial stages of embryogenesis.
in cork oak somatic embryos, the quantification of iaa showed the highest concentration during early embryo development and decreased afterwards  <cit> .
recent work has reported preliminary results showing that endogenous iaa localized in cells of early multicellular embryos developed in isolated microspore cultures of brassica napus  <cit> .
previous reports have indicated that microspore embryogenesis mimics zygotic embryogenesis in several species  <cit> .
proteomic analyses have approached the identification of protein expression patterns in q. suber embryos originated by different in vitro systems during advanced developmental stages and maturation  <cit> .
the phytohormones can act as signaling molecules for remodeling cell walls during cell elongation, as well as for modifying chromatin domains to activate gene expression programs.
in this sense, the information about the dynamics of epigenetic marks, pectin esterification changes in walls and auxin during early embryogenesis could inform on possible correlations among these processes, giving new insights into the complex regulatory mechanisms controlling cell reprogramming and embryogenesis induction.
in the present work, we have performed a comparative analysis of in vitro embryogenesis of microspores and immature zygotic embryos in cork oak in order to characterize early markers of embryo cells, in contrast with non-responsive cells, in both pathways and to gain new insights into the cellular mechanisms involved in the in vitro embryogenesis induction in woody species, in which there is scarce information, as well as to provide new evidences of the developmental pattern similarity between both embryogenesis pathways, from microspores and zygotic embryos.
we have analyzed the changes in dna methylation, esterification of pectins in cell walls and endogenous auxin distribution, during early embryogenesis of the two in vitro systems.
the results presented here revealed differential features in early multicellular embryo cells constituting early embryogenic markers which are similar in both pathways originated from microspores and zygotic embryos, providing additional evidences to the developmental pattern analogy of embryogenesis pathways.
RESULTS
characterization of early developmental stages of embryogenesis of both microspore and zygotic embryo origins in quercus suber
two different embryogenic in vitro systems have been compared in cork oak, embryogenesis of microspores  and immature zygotic embryos, to analyze the presence and distribution patterns of various cellular components at early embryogenesis stages and to identify common markers present in the two embryogenic pathways at the initial stages.
microspore embryogenesis was induced by heat treatment in anthers excised from catkins  at the developmental stage in which they contained microspores at the responsive stage of vacuolated microspores.
after 20– <dig> days in culture, white small multicellular and globular embryos emerged from inside the anther ; these embryos further developed to heart, torpedo and cotyledonary embryos.
mature cotyledonary embryos produced by microspore embryogenesis were subcultured monthly on medium without growth regulators producing embryogenic masses  from which new embryos emerged by secondary embryogenesis .figure  <dig>
main developmental stages of
in vitro
embryogenesis in cork oak.
a-g: microspore embryogenesis.
h-m: somatic embryogenesis of immature zygotic embryos.
a-d, h-j: explants and in vitro embryo development.
e-g, k-m: different stages of embryo development, resin sections stained by toluidine blue.
a: catkin selected for in vitro microspore embryogenesis.
b, c: multicellular and globular embryos  of different sizes emerging from inside anthers  after 20– <dig> days in culture.
d: secondary embryo  emerging from embryogenic masses .
e: early multicellular embryos  formed inside the anther and surrounded by the tissue of the anther wall .
f: cells of an early multicellular embryo at higher magnification.
g: large multicellular embryo formed by secondary embryogenesis attached to some cells of an embryogenic mass .
h: acorn selected for in vitro somatic embryogenesis of immature zygotic embryos.
i: embryos of different developmental stages and sizes emerging from the immature zygotic embryo explant after 30 days in culture, early multicellular embryo , globular, heart  and cotyledonary  embryos.
j: secondary embryo  at the torpedo stage originated from embryogenic masses .
k: early multicellular embryo  formed at the surface of an immature zygotic embryo .
l: cells of an early multicellular embryo at higher magnification.
m: early multicellular embryos  formed by secondary embryogenesis and emerging from inside an embryogenic mass .
bars: a, h: 100 μm; b, c, d, i, j: 1 mm; e, f, gk, l: 50 μm; m: 200 μm.
microscopic analysis of culture samples at early developmental stages showed small multicellular embryos  of different sizes inside the anthers and emerging out of the them.
the early multicellular embryos originated by either direct or secondary embryogenesis showed a typical structural organization consisting in a more or less rounded structure formed by small cells, with characteristic features of active proliferating cells.
the cells of these so-called early multicellular embryos were small, with medium-large nucleus, slightly dense cytoplasm and low vacuolation , very different from the cells of the anther wall tissues  and embryogenic masses  which were of larger size and highly vacuolated.
immature pollinated acorns  were the source of zygotic embryos which were cultured and used for somatic embryogenesis induction.
around one month after the culture initiation, early multicellular embryos of various sizes and globular embryos were observed on the surface of the immature zygotic embryos, they later developed to produce heart, torpedo and cotyledonary embryos .
embryos were transferred to proliferating medium where secondary embryogenesis was observed and after repetitive subcultures the existing embryos could proliferate by producing embryogenic masses  from which new embryos were originated .
the cells of early multicellular embryos of this somatic embryogenesis system showed a similar structural organization than those of the microspore embryogenesis pathway, early embryo cells exhibited small size, dense cytoplasm with small vacuoles and a mid-size nucleus , clearly distinguishable from the larger cells displayed by the immature zygotic embryo which exhibited a large vacuole .
secondary embryos of microspore and somatic origin emerged and developed from the embryogenic masses as small multicellular embryos  formed by cells which exhibited similar organization than described for cells of early multicellular embryos directly originated from microspores or immature zygotic embryos, very different than the larger and highly vacuolated cells of the embryogenic masses .
dna methylation changes during early embryogenesis of both microspore and zygotic embryo origins
the percentage of global dna methylation was analyzed at different developmental stages of embryogenesis as well as in microspores and zygotic embryos .
the results of the quantification showed similar temporal dna methylation profiles in both embryogenesis pathways.
dna methylation significantly decreased  after embryogenesis induction, in early multicellular embryos, in comparison with microspores in anthers, and zygotic embryos before embryogenesis initiation.
at later embryogenesis stages dna methylation levels significantly increased  in heart and torpedo embryos, and during late embryo development, reaching the highest level in cotyledonary embryos in the two in vitro systems .
in heart-torpedo embryos of the second pathway, derived from zygotic embryos, the increase in global dna methylation was higher than in the microspore embryogenesis pathway.figure  <dig>
global dna methylation during
in vitro
embryogenesis of microspore and immature zygotic embryo.
histograms representing the mean values of 5mdc percentage of total dna in different stages of microspore embryogenesis  and somatic embryogenesis of immature zygotic embryos , quantified by elisa-based immunoassay.
bars on columns represent the standard errors of the means.
different letters on columns indicate significant differences according to anova and tukey’s test at p ≤  <dig> .
to analyze the changes in the nuclear distribution pattern of methylated dna during early embryogenesis, immunofluorescence with anti-5mdc antibody followed by confocal analysis was performed at early stages of both primary and secondary embryogenesis, specifically at the stage of early multicellular embryos in the two pathways studied, microspores and immature zygotic embryos.
the 5mdc immunofluorescence signal was similar in early multicellular embryo cells of the two embryogenesis pathways  and in primary and secondary embryos.
the 5mdc immunofluorescence signal was very different in early embryo cells than inf microspores of anthers , immature zygotic embryos  and embryogenic masses .
early multicellular embryos directly originated from in vitro microspore or zygotic embryo cultures by primary embryogenesis showed the same 5mdc pattern of localization than multicellular embryos  produced from embryogenic masses  by secondary embryogenesis in the two pathways.
early embryo cells displayed a distribution pattern of 5mdc immunofluorescence in small bright spots over the nucleus, which were clearly identified by dapi staining , these 5mdc fluorescence spots probably correspond to small heterochromatin masses.
on the contrary, nuclei of microspores , immature zygotic embryos  and embryogenic masses  showed an intense 5mdc immunofluorescence signal which covered almost the entire nuclear region.
the exine, the special pollen wall, showed unspecific autofluorescence  which did not interfere with the specific immunofluorescence signals detected throughout the microspore embryogenesis analysis.
control experiments avoiding the first antibody or the denaturation step, required for 5mdc recognition by the antibody, did not show labeling in any sample and developmental stage, assessing the specificity of the antibody.figure  <dig>
methylated dna nuclear patterns at early
in vitro
embryogenesis of microspore and immature zygotic embryo.
a-d: microspore embryogenesis.
e-i: somatic embryogenesis of immature zygotic embryos.
a, a’, e, e’: confocal images of 5mdc immunofluorescence in early multicellular embryos  and embryogenic masses .
differential interference contrast, dic, images , and merged images of 5mdc immunofluorescence  and dapi staining  for the nuclei  of the same sections.
b-b”, c-c”, f-f”, g-g”: high magnification images of representative nuclei of early multicellular embryos, “emb” , embryogenic masses, “ms” , microspores  and immature zygotic embryos, “zye”  showing 5mdc immunofluorescence in green , dapi staining of nuclei in blue  and merged images of both green and blue channels .
bars: a, a´, e, e´: 50 μm; b, b´,b´´,c, c´, c´´, f, f´, f´´, g, g’, g”, i, i’: 5 μm; d, d’: 10 μm.
changes in pectin esterification of cell walls during early embryogenesis of both microspore and zygotic embryo origins
the changes in the proportion of pectin esterification in the cell walls during early stages of embryogenesis of microspore and zygotic embryos were analyzed by using jim <dig> and jim <dig> specific antibodies, which recognize non-esterified and highly-esterified pectins respectively, in immunodot-blot and immunofluorescence assays.
dot-blot analysis was performed in microspores and zygotic embryos before embryogenesis induction as well as in embryos during consecutive developmental stages of both embryogenic pathways.
the differences in the signal intensities of dot-blots, which were performed over equal amounts of protein extracts, as illustrated by the ponceau red staining, revealed changes in the proportion of highly-esterified and non-esterified pectins of cell walls during embryogenesis .
the quantification of the signal intensities showed similar temporal profiles in the two embryogenesis pathways for each antibody .
jim <dig> signal, corresponding to non-esterified pectins, was high in microspores and zygotic embryos before induction whereas it progressively diminished with embryogenesis progression in heart, torpedo and cotyledonary embryos ; in the microspore embryogenesis pathway, the lowering of jim <dig> signal was significant also at earlier stages, in multicellular embryos .
on the contrary, jim <dig> antibody which recognizes highly-esterified pectins, showed a significant signal increase  in early multicellular embryos in comparison with microspores and immature zygotic embryos .
the intensities of jim <dig> signals at advanced embryogenesis stages progressively decreased, in heart, torpedo and cotyledonary embryos .
control experiments avoiding the first antibodies did not show labeling in any case.figure  <dig>
distribution patterns of non-esterified  and highly-esterified  pectins during
in vitro
embryogenesis of microspore and zygotic embryo origins.
a, b: representative immuno dot-blot assays of jim <dig>  and jim <dig>  at different developmental stages of microspore embryogenesis.
ponceau red staining for total proteins and immuno-dot-blot of the same strip is shown for each antibody.
c-f: histograms representing the mean values of dot color intensities in arbitrary units of jim <dig>  and jim <dig>  during microspore embryogenesis  and somatic embryogenesis of immature zygotic embryos .
bars on columns indicate standard errors of the means.
different letters on columns indicate significant differences according to anova and tukey’s test at p ≤  <dig> .
immunofluorescence assays revealed analogous localization patterns in both embryogenesis pathways from microspore and immature zygotic embryos for each pectin epitope, non-esterified  and highly-esterified  pectins.
jim <dig> labelling was intense on peripheral and inner cell walls of early multicellular embryos .
in contrast, early multicellular embryos showed very low or no labeling with jim <dig> antibody on inner cell walls, only the peripheral wall layer surrounding the multicellular embryos exhibited jim <dig> labelling .
at more advanced developmental stages, globular embryos developed by direct and secondary embryogenesis showed very low or no labeling with jim <dig> or jim <dig> antibodies, whereas cell walls of embryogenic masses appeared labeled with both antibodies .
the fact that the results of the dot-blot assays showed faint positive jim <dig> and jim <dig> signals on advanced embryo stages indicated a higher sensitivity of this assay in comparison with immunofluorescence on resin sections where only epitopes exposed on the section surface can be detected; on the other hand, the possibility that some cells of the embryogenic masses would remain attached to embryos in the samples used for dot-blot assays and slightly contribute to dot-blot signals, cannot be completely excluded.figure  <dig>
immunolocalization of non-esterified  and highly-esterified  pectins at early
in vitro
embryogenesis of microspore and immature zygotic embryos.
a, a’, c, c’, e, e’, g, g’: microspore embryogenesis.
b, b’, d, d’, f, f’, h, h’: somatic embryogenesis of immature zygotic embryos.
a’-h’: confocal merged images of immunofluorescence signal  of jim <dig>  or jim <dig>  combined with dapi staining of nuclei .
a-h: differential interference contrast, dic, images of the same sections.
a, a’, b, b’, e, e’, f, f’: early multicellular embryos.
c, c’, d, d’, g, g’, h, h’: large multicellular and globular embryos  emerging from embryogenic masses  by secondary embryogenesis.
bars: 100 μm.
localization of endogenous auxin during early embryogenesis of both microspore and zygotic embryo origins
the localization of the endogenous auxin indol acetic acid, iaa, was approached by immunofluorescence assays using specific anti-iaa antibodies.
results showed similar patterns of iaa localization in the two embryogenesis pathways, from microspores and from zygotic embryos.
after embryogenesis induction, very low or no iaa labeling was observed in microspores  and immature zygotic embryos  in culture.
in microspores, the special pollen wall, the exine, showed unspecific autofluorescence .
however, an intense iaa labeling was found in the cytoplasm of cells of early multicellular embryos produced in the two pathways by primary embryogenesis  b, f).
during early secondary embryogenesis, at the surface of the embryogenic masses  the dense cells which originate new multicellular embryos, also showed intense iaa labeling , whereas the subjacent embryogenic mass cells  did not show any labeling.
controls by immunodepletion of the iaa antibody did not show labeling in any cellular structure and developmental stage, supporting the specificity of the immunofluorescence results.figure  <dig>
immunolocalization of auxin  at early
in vitro
embryogenesis of microspore and immature zygotic embryos.
a-f: microspore embryogenesis.
g-l: somatic embryogenesis of immature zygotic embryos.
a, b, c, g, h, i: resin sections stained by toluidine blue.
d, e, f, j, k, l: confocal images of iaa immunofluorescence signal ; in some images, dapi staining of nuclei  is also shown merged with the green iaa signal .a, d: microspores .
b, e, h, k: early multicellular embryos .
c, f, i, l: dense cells  from which early embryos originate at the surface of embryogenic masses .
bars a, d: 50 μm; b, c, e-l: 100 μm.
to assess the involvement of endogenous auxin accumulations in the early stages of embryogenesis of the two pathways, treatments with the inhibitor of the polar auxin transport 1-n-naphthylphthalamic acid  were performed to in vitro cultures with embryogenic masses originated from the two systems.
results were similar in embryogenic cultures of both origins microspore and immature zygotic embryos; they showed that npa treatment affected embryo development in comparison with control cultures .
after 20 days of npa treatment, neither new embryos nor development of the embryogenic masses were observed on treated cultures, whereas in parallel embryogenic cultures without npa embryogenesis normally initiated and progressed.
new embryos emerging from the embryogenic masses and development of previous embryos were observed at the same time period .figure  <dig>
effects of inhibiting auxin transport by npa on
in vitro
embryogenesis initiation and progression.
embryogenic masses originated by secondary embryogenesis from immature zygotic embryos.
a, b: control cultures, without npa, at the beginning of the treatment  and 20 days afterwards , when embryo development and new embryos  were observed.
c, d: culture containing 40 μm npa at the beginning of the treatment  and 20 days afterwards , no further growth or new embryos were observed.
bars 5 mm.
distribution patterns of the cellular markers used  in other developmental systems with proliferating and differentiated cells
in order to evaluate the patterns of the selected cellular features  as early markers of embryogenesis, their distribution was analyzed in other two plant developmental systems with proliferating and differentiated cells, taken as positive and negative controls.
one system was the proliferating cells of developing zygotic embryos in which the markers should be found, and in a different plant species, brassica napus, to additionally evaluate whether those features were also present in other species.
the other system was a non-embryogenic system with differentiated cells, the epidermis and subjacent cell layers of mature anthers, differentiated tissues where the markers should not be present.
the results of the comparative analysis between the two systems, proliferating embryo cells and non-embryogenic differentiated cells are shown in figure  <dig>  in proliferating cells of the zygotic torpedo embryos , the patterns of immunolocalization observed with all antibodies were similar than the patterns found in early multicellular embryos developed in vitro.
methylated dna, localized by anti-5mdc immunofluorescence, was scarce and distributed in small spots over nuclei .
jim <dig> and jim <dig> antibodies revealing non-esterified and highly-esterified pectins respectively, exhibited localization patterns in which the walls of proliferating embryo cells appeared higher labeled with jim <dig> than with jim <dig>  indicating a higher proportion of esterified pectins in these cells.
auxine immunofluorescence signal was intense in the cytoplasm of the proliferating embryo cells  localized at the developing cotyledon tips of the torpedo embryo.
on the contrary, the patterns of localization in the differentiated tissue of the anther wall  were opposite to those found in embryo cells.
5mdc signal was intense and covered the whole nuclear area , corresponding to abundant hypermethylated dna.
the cell walls of the anther tissues showed intense jim <dig> signal and very low jim <dig> signal which indicated a higher proportion of non-esterified pectins in these cell walls.
very low or no iaa signal was observed in differentiated cells of the anther wall layers .figure  <dig>
immunolocalization of 5mdc, esterified  and non-esterified  pectins, and iaa in other systems.
a-e: zygotic torpedo embryo of brassica napus.
f-k: mature anther of quercus suber.
a, f: panoramic views of zygotic torpedo embryo and mature anther, resin sections stained by toluidine blue; squares in a and f indicate representative regions of proliferating embryo cells  and differentiated cells of anther wall  where the analysis is focused.
b-e, g-k: higher magnifications of immunofluorescence signals  for 5mdc , jim <dig> , jim <dig>  and iaa  antibodies.
in b, dapi staining for nuclei  is merged with 5mdc immunofluorescence .
insets in b and g show representative nuclei of each cell types, at high magnification.
bars: a, f: 50 μm, b-e, g-k: 25 μm, insets: 5 μm.
discussion
global dna methylation decrease accompanies nuclear remodeling in early embryo cells
dna methylation plays an essential role in plant growth as a mechanism to epigenetically maintain developmental fates in proliferating and differentiating cells  <cit> .
in the present work, dna methylation level and distribution pattern, in relation to chromatin changes, have been analyzed during early embryogenesis of the two pathways, of microspores and zygotic embryos, results suggesting that the initiation of both embryogenic processes is epigenetically regulated.
differences in global dna methylation levels and changes in the 5-methyl-deoxy-cytidine  distribution patterns were found in the early embryo cells of the two in vitro embryogenesis pathways analyzed, suggesting changes of nuclear activity.
a significant decrease in global dna methylation was observed in early multicellular embryos in comparison with microspores, immature zygotic embryos or embryogenic mass cells from both embryogenesis in vitro systems.
reports in tree species showed that the morphogenic capacity was associated with low levels of dna methylation  <cit>  and a transient dna demethylation of the ovules occurred after pollination  <cit> .
in brassica napus, a decrease of dna methylation has been also reported at early stages of microspore embryogenesis  <cit> .
the immunolocalization essays showed not only a decrease on the 5mdc labelling in the cells of early embryos derived from microspores and zygotic embryos, but also a change in the distribution pattern of 5mdc in small nuclear spots, in contrast with the 5mdc distribution pattern over large nuclear regions corresponding with highly condensed chromatin masses of non-embryo cells.
this 5mdc distribution pattern over small heterochromatin masses correlates with the pattern found in active proliferating cells of root meristems  <cit> , and early microspore-derived embryos of rapeseed, brassica napus, with high transcriptional activity and decondensed chromatin patterns  <cit> .
in contrast, 5mdc distribution over large heterochromatin masses has been associated with decreased cellular activity stages and cell differentiation, as in the generative/sperm cells of pollen grains, in the differentiating cells of advanced embryos  <cit>  or in the tapetal cells during the high chromatin condensation occurring during their developmental programmed cell death  <cit> .
the data presented here revealed an epigenetic change associated with the switch towards the embryogenic developmental program and with the activation of cell proliferation occurring at the beginning of embryogenesis in the two pathways studied, microspore embryogenesis through anther culture and somatic embryogenesis from immature zygotic embryos, for the first time in a woody species, cork oak.
cell walls of early embryo cells exhibit higher levels of esterified pectins
increasing evidence has linked the cell wall remodeling with many processes involved in plant growth and development, including somatic embryogenesis  <cit> .
several monoclonal antibodies have been used to unravel the involvement of specific cell wall epitopes in controlling cell growth and morphogenesis; specifically, there are different studies on antigen distribution detected by the antibodies jim <dig> and jim <dig> recognizing non-esterified and highly-esterified pectins respectively, in several plant tissues and organs  <cit> .
in the present work, we have used both jim <dig> and jim <dig> antibodies to analyze changes in cell wall associated with in vitro embryogenesis initiation in quercus suber l.
in very young microspore-derived embryos of herbaceous species , a high level of esterified pectins was observed in the cell walls  <cit> .
using an in situ localization approach at both light and electron microscopy levels, it has been reported that in walls of proliferating cells, the levels of esterified pectins were higher than in the differentiating cells, which showed cell walls rich in non-esterified pectins  <cit> .
differences in the proportion of esterified pectins between microspores and microspore-derived embryos were reported in tree species, such as quercus suber, citrus clementina, and olea europaea  <cit> .
the results presented here showed differences in the distribution pattern of highly-esterified pectins in early embryo cells in comparison with non-embryogenic cells, in the two pathways  in cork oak.
highly-esterified pectins were more abundant in early embryo cell walls than in microspores, zygotic embryo and embryogenic mass cells, while they diminished with embryo development and differentiation.
this evidence was consistent with the abundance of esterified pectins in the newly-formed walls of proliferating cells of young microspore-derived embryos and root tip meristematic cells of some herbaceous species  <cit> , thus providing experimental support indicating that a high proportion of esterified pectins in walls is not only a marker for proliferation events, but also an early marker of both reprogramming pathways to embryogenesis, of microspores and immature zygotic embryos origins.
our results showed that, in contrast with the inner cell walls, the peripheral wall layer that surrounds the early multicellular embryos was labeled by jim <dig>  previous reports have described the presence of a thick wall underneath the exine, the special microspore wall, in embryogenic microspores and young embryos of various species, like capsicum annuum and olea europaea  <cit> .
the development of those peripheral thick wall in early stages of somatic embryogenesis and organogenesis, and also microspore embryogenesis, has been reported as a specific feature of these processes  <cit> , though the nature of this wall has not been elucidated yet.
it has been proposed that this layer could constitute an organized network with differential wall components which may create a specific cellular environment with altered permeability and receptors, providing conditions to express morphogenic competence  <cit> .
the present study reveals that at early stages of the two quercus suber in vitro embryogenesis pathways, from microspores and immature zygotic embryos, there is also a peripheral wall in early multicellular embryos with a differentially higher proportion of non-esterified pectins than the inner cell walls, providing additional evidences of the existence of special wall structures surrounding groups of cells committed for entering the cell cycle and initiate the embryogenic program.
high endogenous auxin level is present in early embryo cells
many aspects of auxin action depend on its local biosynthesis and differential distribution within plant tissues, mainly regulated by its directional transport between cells  <cit> .
we have analyzed the endogenous auxin iaa distribution and level during early stages in the two embryogenesis pathways, from microspore and immature zigotic embryos, in cork oak, in vitro systems in which the embryo development occurs in a medium free of exogenous iaa  <cit> .
interestingly, the iaa immunolocalization assays revealed a differential and significant increase of the iaa endogenous levels in the early multicellular embryo cells, in comparison with the microspores, immature zygotic embryos and embryogenic mass cells which showed very low iaa signal.
in arabidopsis, auxin has been localized by the expression of the auxin-responsive promoter dr <dig> in cells of the embryo proper at very early stages  <cit> .
iaa biosynthesis is associated with rapidly dividing and rapidly growing tissues, especially in shoots and roots, free auxin concentrations in the living plant are in the apical meristems of shoots, young leaves, root meristems and lateral root initiation sites  <cit> .
in a new brassica napus microspore embryogenesis in vitro system at low temperature, our group reported preliminary results showing that iaa also localized in cells of early multicellular embryos  <cit> , similarly to early zygotic embryogenesis.
the results presented here indicate the increase of endogenous levels of iaa in the formation of early multicellular embryos of both microspore and zygotic embryo origins, in quercus suber.
this result, together with the fact that the inhibition of auxin transport by npa negatively affected embryogenesis initiation and development suggest that endogenous iaa biosynthesis and transport would be involved in the activation of proliferation and the switch of the embryogenic program of the reprogrammed microspores, zygotic embryo and embryogenic mass cells.
early embryogenesis from microspores and zygotic embryos exhibits similar early markers: epigenetic changes by dna hypomethylation, cell wall remodelling by high pectin esterification and high endogenous auxin levels
in vitro embryogenesis initiation and progression is conditioned by many factors, still largely unknown.
the comparative analysis performed here during the early stages of embryogenesis in two different in vitro systems, microspore embryogenesis through anther cultures and somatic embryogenesis from immature zygotic embryo cultures, in a woody species, quercus suber, has permitted the identification of differential cell features, summarized in table  <dig>  specific to the reprogramming process towards embryogenesis, as early markers which are similar in both embryogenesis pathways from microspore and immature zygotic embryos in cork oak.
these differential features are the decrease of dna methylation, the remodeling of cell walls by increasing the proportion of esterified pectins, and the accumulation of endogenous auxin in the cells that initiate the embryogenic program.
the present work provides new evidences of two important questions for the understanding of the in vitro embryogenesis process.
on one hand, the reported data reveal that several basic cellular features undergo similar changes in the two different embryogenesis pathways studied at early stages, indicating that basic cellular processes underlying early in vitro embryogenesis are common to various systems and suggesting that general mechanisms could regulate the process independently of the cell type origin and culture conditions .
moreover, the results illustrate the relevance of three cellular processes which specifically occur in early embryo cells just after induction, while the same cellular changes are not observed neither before embryogenesis initiation neither at advanced developmental stages, fact that permits to consider these features  as early markers of the process, opening the possibility to design strategies for efficient manipulation of in vitro protocols to improve yields.table  <dig>
early markers present in
in vitro
embryogenesis of both microspores and immature zygotic embryos origins
early markers
before embryogenesis induction
after embryogenesis induction
cells from which embryos originate
early embryos
a previous cytochemical and immunocytochemical study compared the structural organization of advanced globular embryos of both origins, microspores and immature zygotic embryos, finding similar anatomy and cellular organization patterns in both of them; the common features found concerned high vacuolization, small nuclear size and structure, abundant ribosomal population, and presence of starch granules  <cit> .
studies in stress-induced microspore embryogenesis and somatic embryogenesis of other species have indicated the involvement of epigenetic marks  <cit> , cell wall components  <cit>  and endogenous phytohormones  <cit>  in the initiation and progression of organogenesis and embryogenesis.
some of the markers described here in cork oak have been also found in other plant species, for example, dna hypomethylation has been observed in microspore embryos of brassica napus  <cit>  and hordeum vulgare  <cit> , a high proportion of esterified pectins in cell walls was described in microspore embryos of capsicum annum  <cit> , and production of endogenous auxin in early embryos was found in microspore embryogenesis of rapeseed  <cit> .
t he present work is the first report on microspore and somatic embryogenesis in a forest tree, that shows all these cellular processes during the initiation of two different embryogenesis pathways which are induced by different in vitro protocols.
interestingly, the reported cell modifications were observed not only in early embryos originated directly from microspore and zygotic embryos, but also in secondary embryos produced from embryogenic masses, at early stages.
in contrast, these features were not found in the cells that originated the embryos  before embryogenesis induction.
moreover, they were also found in proliferating cells of zygotic embryos of other plant species, b. napus, whereas differentiated cells of mature organs, as revealed in mature anthers here, showed opposite features than early embryo cells regarding dna methylation, pectin esterification and endogenous auxin content.
the fact that the same differential cell features appear in the two early embryo cells of both origins, from microspores and immature zygotic embryos, produced by primary or secondary embryogenesis indicate that they can be considered as early markers of in vitro embryogenesis.
as stated before, some of them have been reported individually in embryos of other plants  <cit>  and, in the present work, the three features were found on proliferating cells of zygotic embryos in a different species, brassica napus, these data support the idea that these early markers reflect a general behavior that could be exploited for other species.
increasing evidences have documented the effect of different phytohormones, including auxin, in signaling processes of cell wall remodeling, necessary for cell elongation and growth, and in chromatin remodeling for the activation of specific gene expression developmental programs.
the changes reported here affecting epigenetic marks and cell wall polymers are associated with the accumulation of auxin in early embryo cells, this data could reflect different processes that are interconnected in complex regulatory mechanisms that control cell reprogramming and embryogenesis initiation.
CONCLUSIONS
the identification of early markers of embryogenesis can help to monitor the metabolic processes involved in the induction providing a wider understanding of the physiology and mechanisms controlling plant cell reprogramming and embryogenic competence acquisition.
the present work is the first report that shows, by a comparative analysis during the early stages of embryogenesis, that various cellular processes, dna hypomethylation, cell wall remodeling by increasing pectin esterification, and endogenous auxin increase, occur concomitantly at early stages of two embryogenesis pathways, microspore embryogenesis and embryogenesis from immature zygotic embryos, which are induced by different in vitro protocols, identifying similar early embryogenic markers in quercus suber.
the data reported here, in a woody species, which have shown to be extremely recalcitrant to in vitro embryogenesis, is of particular relevance.
the information gained will be useful to increase our knowledge on the regulatory mechanisms and involved factors in the in vitro induction of embryogenesis, opening the door to identify novel strategies and selective targets for improving the efficiency of the process in biotechnology, agronomic and forestry breeding programs.

 in vitro embryogenesis systems constitute important tools for investigating the regulating mechanisms of embryo formation, as well as for biotechnological applications in plant breeding, propagation and conservation strategies. two different embryogenesis pathways, from microspores and immature zygotic embryos, have been developed in many species for rapid production of isogenic lines and clonal propagation of elite genotypes respectively. tree breeding strategies have focused on ways to reduce cycle time and improve the efficiency of selection; here, propagation of selected trees by somatic embryogenesis and genetic engineering approaches applied to haploids and double-haploid plants produced in short-times by in vitro microspore embryogenesis have a high potential. nevertheless, due to their recalcitrance, the efficiency of embryogenesis in vitro systems in many woody species is still very low. quercus trees, oaks, are woody species of relevant economic and ecological interest; among them the cork oak, q. suber, is a species of high social and economic relevance in the mediterranean area as the natural source of cork. different in vitro embryogenesis systems have been developed for quercus spps., with variable efficiency. despite the work performed in the last decades, factors responsible for embryogenesis initiation and embryo development in established cultures are still largely unknown. the search for molecular and cellular markers during early stages of in vitro embryogenesis constitutes an important goal in the identification of cells committed to the embryogenesis developmental program as opposed to those cells which are non-responsive to the embryogenic pathway, as well as in the elucidation of the cellular mechanisms underlying in vitro embryo progression. changes in various cell activities and in the structural organization of subcellular compartments have been reported as accompanying the microspore reprogramming process in some herbaceous and woody species. increasing evidences have indicated the relevance of some cell features like epigenetic marks, cell wall components and hormones in the progression of in vitro organogenesis and embryogenesis in other systems, but no reports about the dynamics of these three cellular markers during in vitro early embryogenesis are available in two somatic embryogenesis pathways of the same species. dynamic changes between chromatin states are relevant in the transcriptional regulation during microspore development and reprogramming to embryogenesis and epigenetic mechanisms play an essential role in the process of cellular differentiation allowing cells to be reprogrammed in order to generate new differentiation pathways. dna methylation constitutes a prominent epigenetic modification of the chromatin fiber, which becomes locked in a transcriptionally inactive conformation, thus leading to gene silencing. stress-induced plant cell reprogramming involves changes in global genome organization, being the epigenetic modifications key factors of genome flexibility. previous studies have shown changes in dna methylation levels and distribution patterns during microspore embryogenesis of brassica napus and hordeum vulgare, suggesting the existence of an epigenetic reprogramming after microspore induction to embryogenesis, but no information is available on dna methylation dynamics during in vitro embryogenesis in trees. many of the molecular markers of in vitro embryogenesis and organogenesis have been found in cell walls. pectins are the major matrix components of dicotyledonous cell walls. pectins are polymerized and methyl-esterified in the golgi, and secreted into the wall as highly methyl-esterified forms. subsequently, they can be modified by pectin methylesterases, which catalyse the demethylesterification of homogalacturonans domains of pectins. the relationship between the esterified and the non-esterified pectins, and their distribution in the plant cell walls is the result of different processes and their proportion and dynamics are involved in many plant developmental processes. changes in the distribution of pectins have been reported in young embryos generated from microspores of quercus suber l., citrus clementina and olea europaea l.. abundant esterified pectins has been reported in the newly-formed walls of proliferating cells in different plant systems, like young microspore-derived embryos and root tip meristematic cells of herbaceous species, suggesting that a high proportion of esterified pectins in walls is a marker for proliferation events while abundant low-esterified pectins correspond to low cell proliferation rates. the plant growth regulator auxin, the predominant form of which is indole-3-acetic acid, is a major coordinating signal in the regulation of plant development, with key functions in many processes including embryo formation. despite it is widely known the stimulating effects of the addition of exogenous pgrs, including synthetic auxins like -d, on in vitro embryogenesis induction, little is known about endogenous levels of these regulators at the initial stages of embryogenesis. in cork oak somatic embryos, the quantification of iaa showed the highest concentration during early embryo development and decreased afterwards. recent work has reported preliminary results showing that endogenous iaa localized in cells of early multicellular embryos developed in isolated microspore cultures of brassica napus. previous reports have indicated that microspore embryogenesis mimics zygotic embryogenesis in several species. proteomic analyses have approached the identification of protein expression patterns in q. suber embryos originated by different in vitro systems during advanced developmental stages and maturation. the phytohormones can act as signaling molecules for remodeling cell walls during cell elongation, as well as for modifying chromatin domains to activate gene expression programs. in this sense, the information about the dynamics of epigenetic marks, pectin esterification changes in walls and auxin during early embryogenesis could inform on possible correlations among these processes, giving new insights into the complex regulatory mechanisms controlling cell reprogramming and embryogenesis induction. in the present work, we have performed a comparative analysis of in vitro embryogenesis of microspores and immature zygotic embryos in cork oak in order to characterize early markers of embryo cells, in contrast with non-responsive cells, in both pathways and to gain new insights into the cellular mechanisms involved in the in vitro embryogenesis induction in woody species, in which there is scarce information, as well as to provide new evidences of the developmental pattern similarity between both embryogenesis pathways, from microspores and zygotic embryos. we have analyzed the changes in dna methylation, esterification of pectins in cell walls and endogenous auxin distribution, during early embryogenesis of the two in vitro systems. the results presented here revealed differential features in early multicellular embryo cells constituting early embryogenic markers which are similar in both pathways originated from microspores and zygotic embryos, providing additional evidences to the developmental pattern analogy of embryogenesis pathways.  characterization of early developmental stages of embryogenesis of both microspore and zygotic embryo origins in quercus suber two different embryogenic in vitro systems have been compared in cork oak, embryogenesis of microspores and immature zygotic embryos, to analyze the presence and distribution patterns of various cellular components at early embryogenesis stages and to identify common markers present in the two embryogenic pathways at the initial stages. microspore embryogenesis was induced by heat treatment in anthers excised from catkins at the developmental stage in which they contained microspores at the responsive stage of vacuolated microspores. after 20– days in culture, white small multicellular and globular embryos emerged from inside the anther; these embryos further developed to heart, torpedo and cotyledonary embryos. mature cotyledonary embryos produced by microspore embryogenesis were subcultured monthly on medium without growth regulators producing embryogenic masses from which new embryos emerged by secondary embryogenesis.figure main developmental stages of in vitro embryogenesis in cork oak. a-g: microspore embryogenesis. h-m: somatic embryogenesis of immature zygotic embryos. a-d, h-j: explants and in vitro embryo development. e-g, k-m: different stages of embryo development, resin sections stained by toluidine blue. a: catkin selected for in vitro microspore embryogenesis. b, c: multicellular and globular embryos of different sizes emerging from inside anthers after 20– days in culture. d: secondary embryo emerging from embryogenic masses. e: early multicellular embryos formed inside the anther and surrounded by the tissue of the anther wall. f: cells of an early multicellular embryo at higher magnification. g: large multicellular embryo formed by secondary embryogenesis attached to some cells of an embryogenic mass. h: acorn selected for in vitro somatic embryogenesis of immature zygotic embryos. i: embryos of different developmental stages and sizes emerging from the immature zygotic embryo explant after 30 days in culture, early multicellular embryo, globular, heart and cotyledonary embryos. j: secondary embryo at the torpedo stage originated from embryogenic masses. k: early multicellular embryo formed at the surface of an immature zygotic embryo. l: cells of an early multicellular embryo at higher magnification. m: early multicellular embryos formed by secondary embryogenesis and emerging from inside an embryogenic mass. bars: a, h: 100 μm; b, c, d, i, j: 1 mm; e, f, gk, l: 50 μm; m: 200 μm. microscopic analysis of culture samples at early developmental stages showed small multicellular embryos of different sizes inside the anthers and emerging out of the them. the early multicellular embryos originated by either direct or secondary embryogenesis showed a typical structural organization consisting in a more or less rounded structure formed by small cells, with characteristic features of active proliferating cells. the cells of these so-called early multicellular embryos were small, with medium-large nucleus, slightly dense cytoplasm and low vacuolation, very different from the cells of the anther wall tissues and embryogenic masses which were of larger size and highly vacuolated. immature pollinated acorns were the source of zygotic embryos which were cultured and used for somatic embryogenesis induction. around one month after the culture initiation, early multicellular embryos of various sizes and globular embryos were observed on the surface of the immature zygotic embryos, they later developed to produce heart, torpedo and cotyledonary embryos. embryos were transferred to proliferating medium where secondary embryogenesis was observed and after repetitive subcultures the existing embryos could proliferate by producing embryogenic masses from which new embryos were originated. the cells of early multicellular embryos of this somatic embryogenesis system showed a similar structural organization than those of the microspore embryogenesis pathway, early embryo cells exhibited small size, dense cytoplasm with small vacuoles and a mid-size nucleus, clearly distinguishable from the larger cells displayed by the immature zygotic embryo which exhibited a large vacuole. secondary embryos of microspore and somatic origin emerged and developed from the embryogenic masses as small multicellular embryos formed by cells which exhibited similar organization than described for cells of early multicellular embryos directly originated from microspores or immature zygotic embryos, very different than the larger and highly vacuolated cells of the embryogenic masses. dna methylation changes during early embryogenesis of both microspore and zygotic embryo origins the percentage of global dna methylation was analyzed at different developmental stages of embryogenesis as well as in microspores and zygotic embryos. the results of the quantification showed similar temporal dna methylation profiles in both embryogenesis pathways. dna methylation significantly decreased after embryogenesis induction, in early multicellular embryos, in comparison with microspores in anthers, and zygotic embryos before embryogenesis initiation. at later embryogenesis stages dna methylation levels significantly increased in heart and torpedo embryos, and during late embryo development, reaching the highest level in cotyledonary embryos in the two in vitro systems. in heart-torpedo embryos of the second pathway, derived from zygotic embryos, the increase in global dna methylation was higher than in the microspore embryogenesis pathway.figure global dna methylation during in vitro embryogenesis of microspore and immature zygotic embryo. histograms representing the mean values of 5mdc percentage of total dna in different stages of microspore embryogenesis and somatic embryogenesis of immature zygotic embryos, quantified by elisa-based immunoassay. bars on columns represent the standard errors of the means. different letters on columns indicate significant differences according to anova and tukey’s test at p ≤ . to analyze the changes in the nuclear distribution pattern of methylated dna during early embryogenesis, immunofluorescence with anti-5mdc antibody followed by confocal analysis was performed at early stages of both primary and secondary embryogenesis, specifically at the stage of early multicellular embryos in the two pathways studied, microspores and immature zygotic embryos. the 5mdc immunofluorescence signal was similar in early multicellular embryo cells of the two embryogenesis pathways and in primary and secondary embryos. the 5mdc immunofluorescence signal was very different in early embryo cells than inf microspores of anthers, immature zygotic embryos and embryogenic masses. early multicellular embryos directly originated from in vitro microspore or zygotic embryo cultures by primary embryogenesis showed the same 5mdc pattern of localization than multicellular embryos produced from embryogenic masses by secondary embryogenesis in the two pathways. early embryo cells displayed a distribution pattern of 5mdc immunofluorescence in small bright spots over the nucleus, which were clearly identified by dapi staining, these 5mdc fluorescence spots probably correspond to small heterochromatin masses. on the contrary, nuclei of microspores, immature zygotic embryos and embryogenic masses showed an intense 5mdc immunofluorescence signal which covered almost the entire nuclear region. the exine, the special pollen wall, showed unspecific autofluorescence which did not interfere with the specific immunofluorescence signals detected throughout the microspore embryogenesis analysis. control experiments avoiding the first antibody or the denaturation step, required for 5mdc recognition by the antibody, did not show labeling in any sample and developmental stage, assessing the specificity of the antibody.figure methylated dna nuclear patterns at early in vitro embryogenesis of microspore and immature zygotic embryo. a-d: microspore embryogenesis. e-i: somatic embryogenesis of immature zygotic embryos. a, a’, e, e’: confocal images of 5mdc immunofluorescence in early multicellular embryos and embryogenic masses. differential interference contrast, dic, images, and merged images of 5mdc immunofluorescence and dapi staining for the nuclei of the same sections. b-b”, c-c”, f-f”, g-g”: high magnification images of representative nuclei of early multicellular embryos, “emb”, embryogenic masses, “ms”, microspores and immature zygotic embryos, “zye” showing 5mdc immunofluorescence in green, dapi staining of nuclei in blue and merged images of both green and blue channels. bars: a, a´, e, e´: 50 μm; b, b´,b´´,c, c´, c´´, f, f´, f´´, g, g’, g”, i, i’: 5 μm; d, d’: 10 μm. changes in pectin esterification of cell walls during early embryogenesis of both microspore and zygotic embryo origins the changes in the proportion of pectin esterification in the cell walls during early stages of embryogenesis of microspore and zygotic embryos were analyzed by using jim and jim specific antibodies, which recognize non-esterified and highly-esterified pectins respectively, in immunodot-blot and immunofluorescence assays. dot-blot analysis was performed in microspores and zygotic embryos before embryogenesis induction as well as in embryos during consecutive developmental stages of both embryogenic pathways. the differences in the signal intensities of dot-blots, which were performed over equal amounts of protein extracts, as illustrated by the ponceau red staining, revealed changes in the proportion of highly-esterified and non-esterified pectins of cell walls during embryogenesis. the quantification of the signal intensities showed similar temporal profiles in the two embryogenesis pathways for each antibody. jim signal, corresponding to non-esterified pectins, was high in microspores and zygotic embryos before induction whereas it progressively diminished with embryogenesis progression in heart, torpedo and cotyledonary embryos; in the microspore embryogenesis pathway, the lowering of jim signal was significant also at earlier stages, in multicellular embryos. on the contrary, jim antibody which recognizes highly-esterified pectins, showed a significant signal increase in early multicellular embryos in comparison with microspores and immature zygotic embryos. the intensities of jim signals at advanced embryogenesis stages progressively decreased, in heart, torpedo and cotyledonary embryos. control experiments avoiding the first antibodies did not show labeling in any case.figure distribution patterns of non-esterified and highly-esterified pectins during in vitro embryogenesis of microspore and zygotic embryo origins. a, b: representative immuno dot-blot assays of jim and jim at different developmental stages of microspore embryogenesis. ponceau red staining for total proteins and immuno-dot-blot of the same strip is shown for each antibody. c-f: histograms representing the mean values of dot color intensities in arbitrary units of jim and jim during microspore embryogenesis and somatic embryogenesis of immature zygotic embryos. bars on columns indicate standard errors of the means. different letters on columns indicate significant differences according to anova and tukey’s test at p ≤ . immunofluorescence assays revealed analogous localization patterns in both embryogenesis pathways from microspore and immature zygotic embryos for each pectin epitope, non-esterified and highly-esterified pectins. jim labelling was intense on peripheral and inner cell walls of early multicellular embryos. in contrast, early multicellular embryos showed very low or no labeling with jim antibody on inner cell walls, only the peripheral wall layer surrounding the multicellular embryos exhibited jim labelling. at more advanced developmental stages, globular embryos developed by direct and secondary embryogenesis showed very low or no labeling with jim or jim antibodies, whereas cell walls of embryogenic masses appeared labeled with both antibodies. the fact that the results of the dot-blot assays showed faint positive jim and jim signals on advanced embryo stages indicated a higher sensitivity of this assay in comparison with immunofluorescence on resin sections where only epitopes exposed on the section surface can be detected; on the other hand, the possibility that some cells of the embryogenic masses would remain attached to embryos in the samples used for dot-blot assays and slightly contribute to dot-blot signals, cannot be completely excluded.figure immunolocalization of non-esterified and highly-esterified pectins at early in vitro embryogenesis of microspore and immature zygotic embryos. a, a’, c, c’, e, e’, g, g’: microspore embryogenesis. b, b’, d, d’, f, f’, h, h’: somatic embryogenesis of immature zygotic embryos. a’-h’: confocal merged images of immunofluorescence signal of jim or jim combined with dapi staining of nuclei. a-h: differential interference contrast, dic, images of the same sections. a, a’, b, b’, e, e’, f, f’: early multicellular embryos. c, c’, d, d’, g, g’, h, h’: large multicellular and globular embryos emerging from embryogenic masses by secondary embryogenesis. bars: 100 μm. localization of endogenous auxin during early embryogenesis of both microspore and zygotic embryo origins the localization of the endogenous auxin indol acetic acid, iaa, was approached by immunofluorescence assays using specific anti-iaa antibodies. results showed similar patterns of iaa localization in the two embryogenesis pathways, from microspores and from zygotic embryos. after embryogenesis induction, very low or no iaa labeling was observed in microspores and immature zygotic embryos in culture. in microspores, the special pollen wall, the exine, showed unspecific autofluorescence. however, an intense iaa labeling was found in the cytoplasm of cells of early multicellular embryos produced in the two pathways by primary embryogenesis b, f). during early secondary embryogenesis, at the surface of the embryogenic masses the dense cells which originate new multicellular embryos, also showed intense iaa labeling, whereas the subjacent embryogenic mass cells did not show any labeling. controls by immunodepletion of the iaa antibody did not show labeling in any cellular structure and developmental stage, supporting the specificity of the immunofluorescence results.figure immunolocalization of auxin at early in vitro embryogenesis of microspore and immature zygotic embryos. a-f: microspore embryogenesis. g-l: somatic embryogenesis of immature zygotic embryos. a, b, c, g, h, i: resin sections stained by toluidine blue. d, e, f, j, k, l: confocal images of iaa immunofluorescence signal; in some images, dapi staining of nuclei is also shown merged with the green iaa signal.a, d: microspores. b, e, h, k: early multicellular embryos. c, f, i, l: dense cells from which early embryos originate at the surface of embryogenic masses. bars a, d: 50 μm; b, c, e-l: 100 μm. to assess the involvement of endogenous auxin accumulations in the early stages of embryogenesis of the two pathways, treatments with the inhibitor of the polar auxin transport 1-n-naphthylphthalamic acid were performed to in vitro cultures with embryogenic masses originated from the two systems. results were similar in embryogenic cultures of both origins microspore and immature zygotic embryos; they showed that npa treatment affected embryo development in comparison with control cultures. after 20 days of npa treatment, neither new embryos nor development of the embryogenic masses were observed on treated cultures, whereas in parallel embryogenic cultures without npa embryogenesis normally initiated and progressed. new embryos emerging from the embryogenic masses and development of previous embryos were observed at the same time period.figure effects of inhibiting auxin transport by npa on in vitro embryogenesis initiation and progression. embryogenic masses originated by secondary embryogenesis from immature zygotic embryos. a, b: control cultures, without npa, at the beginning of the treatment and 20 days afterwards, when embryo development and new embryos were observed. c, d: culture containing 40 μm npa at the beginning of the treatment and 20 days afterwards, no further growth or new embryos were observed. bars 5 mm. distribution patterns of the cellular markers used in other developmental systems with proliferating and differentiated cells in order to evaluate the patterns of the selected cellular features as early markers of embryogenesis, their distribution was analyzed in other two plant developmental systems with proliferating and differentiated cells, taken as positive and negative controls. one system was the proliferating cells of developing zygotic embryos in which the markers should be found, and in a different plant species, brassica napus, to additionally evaluate whether those features were also present in other species. the other system was a non-embryogenic system with differentiated cells, the epidermis and subjacent cell layers of mature anthers, differentiated tissues where the markers should not be present. the results of the comparative analysis between the two systems, proliferating embryo cells and non-embryogenic differentiated cells are shown in figure  in proliferating cells of the zygotic torpedo embryos, the patterns of immunolocalization observed with all antibodies were similar than the patterns found in early multicellular embryos developed in vitro. methylated dna, localized by anti-5mdc immunofluorescence, was scarce and distributed in small spots over nuclei. jim and jim antibodies revealing non-esterified and highly-esterified pectins respectively, exhibited localization patterns in which the walls of proliferating embryo cells appeared higher labeled with jim than with jim indicating a higher proportion of esterified pectins in these cells. auxine immunofluorescence signal was intense in the cytoplasm of the proliferating embryo cells localized at the developing cotyledon tips of the torpedo embryo. on the contrary, the patterns of localization in the differentiated tissue of the anther wall were opposite to those found in embryo cells. 5mdc signal was intense and covered the whole nuclear area, corresponding to abundant hypermethylated dna. the cell walls of the anther tissues showed intense jim signal and very low jim signal which indicated a higher proportion of non-esterified pectins in these cell walls. very low or no iaa signal was observed in differentiated cells of the anther wall layers.figure immunolocalization of 5mdc, esterified and non-esterified pectins, and iaa in other systems. a-e: zygotic torpedo embryo of brassica napus. f-k: mature anther of quercus suber. a, f: panoramic views of zygotic torpedo embryo and mature anther, resin sections stained by toluidine blue; squares in a and f indicate representative regions of proliferating embryo cells and differentiated cells of anther wall where the analysis is focused. b-e, g-k: higher magnifications of immunofluorescence signals for 5mdc, jim, jim and iaa antibodies. in b, dapi staining for nuclei is merged with 5mdc immunofluorescence. insets in b and g show representative nuclei of each cell types, at high magnification. bars: a, f: 50 μm, b-e, g-k: 25 μm, insets: 5 μm. discussion global dna methylation decrease accompanies nuclear remodeling in early embryo cells dna methylation plays an essential role in plant growth as a mechanism to epigenetically maintain developmental fates in proliferating and differentiating cells. in the present work, dna methylation level and distribution pattern, in relation to chromatin changes, have been analyzed during early embryogenesis of the two pathways, of microspores and zygotic embryos, results suggesting that the initiation of both embryogenic processes is epigenetically regulated. differences in global dna methylation levels and changes in the 5-methyl-deoxy-cytidine distribution patterns were found in the early embryo cells of the two in vitro embryogenesis pathways analyzed, suggesting changes of nuclear activity. a significant decrease in global dna methylation was observed in early multicellular embryos in comparison with microspores, immature zygotic embryos or embryogenic mass cells from both embryogenesis in vitro systems. reports in tree species showed that the morphogenic capacity was associated with low levels of dna methylation and a transient dna demethylation of the ovules occurred after pollination. in brassica napus, a decrease of dna methylation has been also reported at early stages of microspore embryogenesis. the immunolocalization essays showed not only a decrease on the 5mdc labelling in the cells of early embryos derived from microspores and zygotic embryos, but also a change in the distribution pattern of 5mdc in small nuclear spots, in contrast with the 5mdc distribution pattern over large nuclear regions corresponding with highly condensed chromatin masses of non-embryo cells. this 5mdc distribution pattern over small heterochromatin masses correlates with the pattern found in active proliferating cells of root meristems, and early microspore-derived embryos of rapeseed, brassica napus, with high transcriptional activity and decondensed chromatin patterns. in contrast, 5mdc distribution over large heterochromatin masses has been associated with decreased cellular activity stages and cell differentiation, as in the generative/sperm cells of pollen grains, in the differentiating cells of advanced embryos or in the tapetal cells during the high chromatin condensation occurring during their developmental programmed cell death. the data presented here revealed an epigenetic change associated with the switch towards the embryogenic developmental program and with the activation of cell proliferation occurring at the beginning of embryogenesis in the two pathways studied, microspore embryogenesis through anther culture and somatic embryogenesis from immature zygotic embryos, for the first time in a woody species, cork oak. cell walls of early embryo cells exhibit higher levels of esterified pectins increasing evidence has linked the cell wall remodeling with many processes involved in plant growth and development, including somatic embryogenesis. several monoclonal antibodies have been used to unravel the involvement of specific cell wall epitopes in controlling cell growth and morphogenesis; specifically, there are different studies on antigen distribution detected by the antibodies jim and jim recognizing non-esterified and highly-esterified pectins respectively, in several plant tissues and organs. in the present work, we have used both jim and jim antibodies to analyze changes in cell wall associated with in vitro embryogenesis initiation in quercus suber l. in very young microspore-derived embryos of herbaceous species, a high level of esterified pectins was observed in the cell walls. using an in situ localization approach at both light and electron microscopy levels, it has been reported that in walls of proliferating cells, the levels of esterified pectins were higher than in the differentiating cells, which showed cell walls rich in non-esterified pectins. differences in the proportion of esterified pectins between microspores and microspore-derived embryos were reported in tree species, such as quercus suber, citrus clementina, and olea europaea. the results presented here showed differences in the distribution pattern of highly-esterified pectins in early embryo cells in comparison with non-embryogenic cells, in the two pathways in cork oak. highly-esterified pectins were more abundant in early embryo cell walls than in microspores, zygotic embryo and embryogenic mass cells, while they diminished with embryo development and differentiation. this evidence was consistent with the abundance of esterified pectins in the newly-formed walls of proliferating cells of young microspore-derived embryos and root tip meristematic cells of some herbaceous species, thus providing experimental support indicating that a high proportion of esterified pectins in walls is not only a marker for proliferation events, but also an early marker of both reprogramming pathways to embryogenesis, of microspores and immature zygotic embryos origins. our results showed that, in contrast with the inner cell walls, the peripheral wall layer that surrounds the early multicellular embryos was labeled by jim previous reports have described the presence of a thick wall underneath the exine, the special microspore wall, in embryogenic microspores and young embryos of various species, like capsicum annuum and olea europaea. the development of those peripheral thick wall in early stages of somatic embryogenesis and organogenesis, and also microspore embryogenesis, has been reported as a specific feature of these processes, though the nature of this wall has not been elucidated yet. it has been proposed that this layer could constitute an organized network with differential wall components which may create a specific cellular environment with altered permeability and receptors, providing conditions to express morphogenic competence. the present study reveals that at early stages of the two quercus suber in vitro embryogenesis pathways, from microspores and immature zygotic embryos, there is also a peripheral wall in early multicellular embryos with a differentially higher proportion of non-esterified pectins than the inner cell walls, providing additional evidences of the existence of special wall structures surrounding groups of cells committed for entering the cell cycle and initiate the embryogenic program. high endogenous auxin level is present in early embryo cells many aspects of auxin action depend on its local biosynthesis and differential distribution within plant tissues, mainly regulated by its directional transport between cells. we have analyzed the endogenous auxin iaa distribution and level during early stages in the two embryogenesis pathways, from microspore and immature zigotic embryos, in cork oak, in vitro systems in which the embryo development occurs in a medium free of exogenous iaa. interestingly, the iaa immunolocalization assays revealed a differential and significant increase of the iaa endogenous levels in the early multicellular embryo cells, in comparison with the microspores, immature zygotic embryos and embryogenic mass cells which showed very low iaa signal. in arabidopsis, auxin has been localized by the expression of the auxin-responsive promoter dr in cells of the embryo proper at very early stages. iaa biosynthesis is associated with rapidly dividing and rapidly growing tissues, especially in shoots and roots, free auxin concentrations in the living plant are in the apical meristems of shoots, young leaves, root meristems and lateral root initiation sites. in a new brassica napus microspore embryogenesis in vitro system at low temperature, our group reported preliminary results showing that iaa also localized in cells of early multicellular embryos, similarly to early zygotic embryogenesis. the results presented here indicate the increase of endogenous levels of iaa in the formation of early multicellular embryos of both microspore and zygotic embryo origins, in quercus suber. this result, together with the fact that the inhibition of auxin transport by npa negatively affected embryogenesis initiation and development suggest that endogenous iaa biosynthesis and transport would be involved in the activation of proliferation and the switch of the embryogenic program of the reprogrammed microspores, zygotic embryo and embryogenic mass cells. early embryogenesis from microspores and zygotic embryos exhibits similar early markers: epigenetic changes by dna hypomethylation, cell wall remodelling by high pectin esterification and high endogenous auxin levels in vitro embryogenesis initiation and progression is conditioned by many factors, still largely unknown. the comparative analysis performed here during the early stages of embryogenesis in two different in vitro systems, microspore embryogenesis through anther cultures and somatic embryogenesis from immature zygotic embryo cultures, in a woody species, quercus suber, has permitted the identification of differential cell features, summarized in table  specific to the reprogramming process towards embryogenesis, as early markers which are similar in both embryogenesis pathways from microspore and immature zygotic embryos in cork oak. these differential features are the decrease of dna methylation, the remodeling of cell walls by increasing the proportion of esterified pectins, and the accumulation of endogenous auxin in the cells that initiate the embryogenic program. the present work provides new evidences of two important questions for the understanding of the in vitro embryogenesis process. on one hand, the reported data reveal that several basic cellular features undergo similar changes in the two different embryogenesis pathways studied at early stages, indicating that basic cellular processes underlying early in vitro embryogenesis are common to various systems and suggesting that general mechanisms could regulate the process independently of the cell type origin and culture conditions. moreover, the results illustrate the relevance of three cellular processes which specifically occur in early embryo cells just after induction, while the same cellular changes are not observed neither before embryogenesis initiation neither at advanced developmental stages, fact that permits to consider these features as early markers of the process, opening the possibility to design strategies for efficient manipulation of in vitro protocols to improve yields.table early markers present in in vitro embryogenesis of both microspores and immature zygotic embryos origins early markers before embryogenesis induction after embryogenesis induction cells from which embryos originate early embryos a previous cytochemical and immunocytochemical study compared the structural organization of advanced globular embryos of both origins, microspores and immature zygotic embryos, finding similar anatomy and cellular organization patterns in both of them; the common features found concerned high vacuolization, small nuclear size and structure, abundant ribosomal population, and presence of starch granules. studies in stress-induced microspore embryogenesis and somatic embryogenesis of other species have indicated the involvement of epigenetic marks, cell wall components and endogenous phytohormones in the initiation and progression of organogenesis and embryogenesis. some of the markers described here in cork oak have been also found in other plant species, for example, dna hypomethylation has been observed in microspore embryos of brassica napus and hordeum vulgare, a high proportion of esterified pectins in cell walls was described in microspore embryos of capsicum annum, and production of endogenous auxin in early embryos was found in microspore embryogenesis of rapeseed. t he present work is the first report on microspore and somatic embryogenesis in a forest tree, that shows all these cellular processes during the initiation of two different embryogenesis pathways which are induced by different in vitro protocols. interestingly, the reported cell modifications were observed not only in early embryos originated directly from microspore and zygotic embryos, but also in secondary embryos produced from embryogenic masses, at early stages. in contrast, these features were not found in the cells that originated the embryos before embryogenesis induction. moreover, they were also found in proliferating cells of zygotic embryos of other plant species, b. napus, whereas differentiated cells of mature organs, as revealed in mature anthers here, showed opposite features than early embryo cells regarding dna methylation, pectin esterification and endogenous auxin content. the fact that the same differential cell features appear in the two early embryo cells of both origins, from microspores and immature zygotic embryos, produced by primary or secondary embryogenesis indicate that they can be considered as early markers of in vitro embryogenesis. as stated before, some of them have been reported individually in embryos of other plants and, in the present work, the three features were found on proliferating cells of zygotic embryos in a different species, brassica napus, these data support the idea that these early markers reflect a general behavior that could be exploited for other species. increasing evidences have documented the effect of different phytohormones, including auxin, in signaling processes of cell wall remodeling, necessary for cell elongation and growth, and in chromatin remodeling for the activation of specific gene expression developmental programs. the changes reported here affecting epigenetic marks and cell wall polymers are associated with the accumulation of auxin in early embryo cells, this data could reflect different processes that are interconnected in complex regulatory mechanisms that control cell reprogramming and embryogenesis initiation.  the identification of early markers of embryogenesis can help to monitor the metabolic processes involved in the induction providing a wider understanding of the physiology and mechanisms controlling plant cell reprogramming and embryogenic competence acquisition. the present work is the first report that shows, by a comparative analysis during the early stages of embryogenesis, that various cellular processes, dna hypomethylation, cell wall remodeling by increasing pectin esterification, and endogenous auxin increase, occur concomitantly at early stages of two embryogenesis pathways, microspore embryogenesis and embryogenesis from immature zygotic embryos, which are induced by different in vitro protocols, identifying similar early embryogenic markers in quercus suber. the data reported here, in a woody species, which have shown to be extremely recalcitrant to in vitro embryogenesis, is of particular relevance. the information gained will be useful to increase our knowledge on the regulatory mechanisms and involved factors in the in vitro induction of embryogenesis, opening the door to identify novel strategies and selective targets for improving the efficiency of the process in biotechnology, agronomic and forestry breeding programs. 
despite its high potential in tree breeding strategies, due to their recalcitrancy, the efficiency of embryogenesis in vitro systems in many woody species is still very low since factors responsible for embryogenesis initiation and embryo development are still largely unknown. rearrangements of the cell structural organization, changes in epigenetic marks, cell wall polymers modifications and endogenous auxin changes were analyzed at early embryogenesis stages of the two in vitro systems by a multidisciplinary approach. in quercus suber, cork oak, a mediterranean forest tree of economic and social interest, rapid production of isogenic lines and clonal propagation of elite genotypes have been achieved by developing in vitro embryogenesis from microspores and zygotic embryos respectively. these findings suggest that dna hypomethylation, cell wall remodeling by pectin esterification and auxin increase are involved in early in vitro embryogenesis in woody species, providing new evidences of the developmental pattern similarity between both embryogenesis pathways, from microspores and immature zygotic embryos, in woody species. in this work, we have performed a comparative analysis of two embryogenesis pathways derived from microspores and immature zygotic embryos in cork oak in order to characterize early markers of reprogrammed cells in both pathways. results showed that early embryo cells exhibited defined changes of cell components which were similar in both embryogenesis in vitro systems, cellular features that were not found in non-embryogenic cells. the search for molecular and cellular markers during early stages of in vitro embryogenesis constitutes an important goal to distinguish, after induction, responsive from non-responsive cells, and to elucidate the mechanisms involved in embryogenesis initiation for their efficient manipulation. somatic embryogenesismicrospore embryogenesisanther culturedna methylation5-methyl-deoxy-cytidinepectin esterificationcell wallauxiniaaissue-copyright-statement© the author 2014 dna methylation level and nuclear pattern, proportion of esterified pectins in cell walls, and endogenous auxin levels were different in embryo cells in comparison with microspores and immature zygotic embryo cells from which embryos originated, constituting early embryogenesis markers.
 in quercus suber, cork oak, a mediterranean forest tree of economic and social interest, rapid production of isogenic lines and clonal propagation of elite genotypes have been achieved by developing in vitro embryogenesis from microspores and zygotic embryos respectively. despite its high potential in tree breeding strategies, due to their recalcitrancy, the efficiency of embryogenesis in vitro systems in many woody species is still very low since factors responsible for embryogenesis initiation and embryo development are still largely unknown. the search for molecular and cellular markers during early stages of in vitro embryogenesis constitutes an important goal to distinguish, after induction, responsive from non-responsive cells, and to elucidate the mechanisms involved in embryogenesis initiation for their efficient manipulation. in this work, we have performed a comparative analysis of two embryogenesis pathways derived from microspores and immature zygotic embryos in cork oak in order to characterize early markers of reprogrammed cells in both pathways. rearrangements of the cell structural organization, changes in epigenetic marks, cell wall polymers modifications and endogenous auxin changes were analyzed at early embryogenesis stages of the two in vitro systems by a multidisciplinary approach.  results showed that early embryo cells exhibited defined changes of cell components which were similar in both embryogenesis in vitro systems, cellular features that were not found in non-embryogenic cells. dna methylation level and nuclear pattern, proportion of esterified pectins in cell walls, and endogenous auxin levels were different in embryo cells in comparison with microspores and immature zygotic embryo cells from which embryos originated, constituting early embryogenesis markers.  these findings suggest that dna hypomethylation, cell wall remodeling by pectin esterification and auxin increase are involved in early in vitro embryogenesis in woody species, providing new evidences of the developmental pattern similarity between both embryogenesis pathways, from microspores and immature zygotic embryos, in woody species. keywords somatic embryogenesismicrospore embryogenesisanther culturedna methylation5-methyl-deoxy-cytidinepectin esterificationcell wallauxiniaaissue-copyright-statement© the author 2014 