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The two major taxa of extant seed plants, gymnosperms and angiosperms, are believed to have diverged from a common ancestor > 300 Myr ago (Stuessy, 2004), although their evolutionary relationship is still a matter of controversy (Chaw et al., 2000; Burleigh & Mathews, 2004). Many aspects of growth and development differ between gymnosperms and angiosperms, including early embryogeny (Raghavan & Sharma, 1995). For example, in gymnosperms, the multicellular haploid female gametophyte both holds egg cells within several archegonia and serves as a nutritive tissue for the growing embryo, similar to the triploid endosperm of angiosperms. Unlike those of most angiosperms, conifer proembryos have a brief period of free nuclear divisions before cellularization of the embryo (Singh, 1978), and embryos do not contain a cell corresponding to the hypophysis, which in the model plant Arabidopsis thaliana has been shown to be important for radicle and root cap formation (Berleth & Chatfield, 2002).
In seed plants, the mature embryo is a structure that displays basic body polarities and contains a shoot apical meristem (SAM), cotyledons, a hypocotyl, an embryonic root and a root apical meristem (RAM). This primary body plan is established through a process called embryonic pattern formation, and current knowledge concerning its genetic regulation is primarily derived from studies on angiosperm models and is mostly focused on A. thaliana (reviewed e.g. by Jürgens, 2001; Laux et al., 2004; Weijers & Jürgens, 2005; Chandler et al., 2008). Studies show that the polar transport of the plant hormone auxin (indole-3-acetic acid (IAA)), which is referred to as ‘polar auxin transport’ (PAT) and is mainly established and maintained by members of the auxin efflux facilitator PIN-FORMED (PIN) family (Friml et al., 2003; Petrásek et al., 2006; Forestan et al., 2010), is important for apical–basal patterning in angiosperm embryos (Schiavone, 1988; Liu et al., 1993; reviewed e.g. by Jenik et al., 2007; Petrásek & Friml, 2009). Work on somatic embryos of the conifer Picea abies suggests that PAT is of similar importance in gymnosperms (Hakman et al., 2009; Palovaara et al., 2010).
There seems to be a connection between PAT and the spatial separation of WUSCHEL-related homeobox (WOX) transcription factors in the formation of the main body axis in A. thaliana embryos. Haecker et al. (2004) recently identified two members of the WOX gene family, WOX2 and WOX8, as being initially co-expressed in the A. thaliana egg cell and the zygote, and then specifically expressed in the apical and basal cell lineages, respectively, after zygotic division. STIMPY/WOX9, a close homologue of WOX8, is expressed after the first division of the zygote and is required to maintain cell division in the embryo and the suspensor (Wu et al., 2007), suggesting that WOX genes are intrinsic determinants in early asymmetric divisions during A. thaliana embryogenesis. WOX2 and WOX8 act redundantly with MONOPTEROS (MP), an auxin response factor (ARF) involved in the TRANSPORT INHIBITOR RESPONSE 1 (TIR1)-AUXIN (AUX)/INDOLE-3-ACETIC ACID (IAA)-ARF pathway, in PIN1 regulation (Breuninger et al., 2008), while the expression of STIMPY/WOX9 is altered in mp mutants (Haecker et al., 2004). Auxin is thought to modulate the transcription of multiple PIN genes through the TIR1-Aux/IAA-ARF pathway (Sauer et al., 2006; Wenzel et al., 2007). After the establishment of the apical–basal pattern, other members of the gene family, WUSCHEL (WUS) and WOX5, can be detected in the SAM and RAM, respectively, with both genes promoting stem cell identity (Mayer et al., 1998; Sarkar et al., 2007). Other angiosperms show similar WOX expression patterns, including the monocot Zea mays, indicating that the WOX gene family is ancient in the angiosperm lineage (Nardmann et al., 2007; reviewed e.g. by Dodsworth, 2009; Van der Graaff et al., 2009).
Previous WOX gene phylogenies for basal land plants, gymnosperms, and angiosperms support a distribution of genes into three evolutionary lineages with a modern clade (WUS/WOX1-7) restricted to seed plants (Deveaux et al., 2008; Palovaara & Hakman, 2008; Nardmann et al., 2009; Van der Graaff et al., 2009). Recently, Nardmann et al. (2009) identified a single WUS/WOX5 homologue in two nonconiferous gymnosperm genomes, suggesting that distinct WUS and WOX5 genes are restricted to angiosperms and that the genome of the last common gymnosperm/angiosperm ancestor contained a single WUS/WOX5 precursor. Given the importance and proposed connection of PAT and WOX transcription factors during embryo development in angiosperms, and the evolutionary considerations and developmental differences mentioned above, we set out to analyse the expression patterns of a WOX2 (PaWOX2) and a WOX8/9 (PaWOX8/9) homologue, genes that had earlier been identified as being expressed in conifer embryos (Palovaara & Hakman, 2008), during P. abies seed and somatic embryo development, and in somatic embryos treated with the PAT inhibitor N-1-napthylphthalamic acid (NPA).
The results of the present study suggest that PaWOX2 and PaWOX8/9 are involved in P. abies embryo formation and differentiation, and, together with PaPIN1 and PAT (Hakman et al., 2009; Palovaara et al., 2010), act in embryo patterning. Thus, despite changes during seed plant evolution, our results strengthen the proposed connection between PAT, PIN and WOX in regulating seed embryo patterning. Also, the comparison of expression in somatic and zygotic embryos reveals somatic embryogenesis as a useful model system for conifer embryo development.
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In angiosperms, the establishment of distinct transcriptional domains during early embryo development leads to correct apical–basal axis formation, which is essential for optimal post-embryonic growth. In gymnosperms, a better understanding of this process is essential, not only from an evolutionary perspective, but also because of the potential of somatic embryogenesis as a propagation method in forestry and as a model system for basic studies of embryo development (Hakman, 1985; Hakman et al., 1985). A fascinating aspect of somatic embryogenesis is the similarity between somatic and zygotic embryo developmental patterns, even though the physical and chemical environments differ. Conifer somatic embryos develop into polar structures with a well-defined embryo proper and suspensor, similar to zygotic embryos, without the influence of the surrounding tissue of the archegonium and the female gametophyte. The early stages of zygotic proembryo development, however, are not easily recognized in somatic embryo development. In this study, we show that similar expression patterns of PaWOX2 and PaWOX8/9 occur in P. abies embryos, regardless of their origin.
WOX genes are expressed in both zygotic and somatic embryos during early development
PaWOX2 and PaWOX8/9 were both expressed in the egg cell/zygote (Fig. 5a,f), similar to WOX2 and WOX8 in A. thaliana (Haecker et al., 2004) and WOX2A in Z. mays (Nardmann et al., 2007), and at high levels in proembryos (Fig. 5b,g). High transcript levels of both genes were also seen in the small rapidly dividing cells, present as single cells or in cell aggregates, in the calli (Fig. 3a,e) which presumably gave rise to the somatic embryos seen in the cultures (Hakman et al., 1987). At early embryo stage, which starts when primary suspensor cells elongate and ends at meristem differentiation, high PaWOX2 and PaWOX8/9 expression was seen in both the embryo proper but also in the suspensor cells of both somatic and zygotic embryos (Figs 3b,f, 5c,h), which may reflect the importance of both genes during early P. abies embryo development. This is further suggested by the up-regulation of PaWOX8/9 during early somatic embryo development (Fig. 2). The expression of PaWOX8/9 throughout the early embryo is similar to that seen for STIMPY/WOX9 in the proembryo of A. thaliana (Wu et al., 2007).
In A. thaliana, the wox2 mutation confers division defects in the apical cell lineage of the embryo, while embryo arrest is seen in wox8 stimpy/wox9 double mutants, with aberrant divisions in both embryo and suspensor cell lineages (Breuninger et al., 2008). As WOX2 restores apical cell fate in wox8 stimpy/wox9 mutant embryos, its expression is a downstream function of WOX8 and STIMPY/WOX9.
The gene activity of both PaWOX2 and PaWOX8/9 in P. abies suspensor cells (Fig. 3b,c,f,g) could perhaps reflect their higher mitotic activity compared with that of A. thaliana, in which the suspensor only consists of a few cells that rarely divide. Also, the archegonial jacket cells (Fig. 5a,f), which are implicated in the nutrition of the egg/proembryo (Hakman & Oliviusson, 2002), expressed both WOX genes. The jacket cells are derived from cells surrounding the archegonial initials (Singh, 1978) and mitotic divisions are common also here. Thus, high WOX expression could be linked to cell proliferation as well as to cell specification. Indeed, PaWOX2 is highly expressed in a somatic cell line with a very high proliferation rate, but with poor embryo maturation capacity, suggesting its involvement in regulating cell proliferation and/or differentiation (Palovaara & Hakman, 2008).
WOX2 and WOX8/9 may act together in conifer embryo patterning
In later stages of P. abies embryo development, the transcription pattern of PaWOX8/9 share many similarities with that of STIMPY/WOX9 in A. thaliana embryos (Figs 3g–l, 5i,j) (Wu et al., 2007). After being expressed throughout the A. thaliana proembryo, STIMPY/WOX9 becomes restricted to the cotyledon primordia and the outer cell layers, including the epidermis, in the basal half of the embryo, much like what was seen for PaWOX8/9 in P. abies embryos (Figs 3g–j, 5i). As STIMPY/WOX9 seems to promote, partially redundantly with WOX8, cell division during A. thaliana embryo development (Wu et al., 2007), expression patterns here indicate a similar role for PaWOX8/9 in P. abies embryos.
We recently showed that PaPIN1 is transcribed in the differentiating procambium of P. abies somatic embryos (Palovaara et al., 2010), and that correct auxin transport is needed for proper cotyledon and SAM and RAM initiation and development (Hakman et al., 2009). Here, high levels of PaWOX8/9 transcripts overlapped with a weaker PaWOX2 transcription pattern in the differentiating procambium of embryos, which was most clearly seen after NPA treatment of embryos (Fig. 4b–d,f). The up-regulation of both genes in NPA-treated precotyledonary embryos (Fig. 2) indicates that PaWOX8/9 and PaWOX2 may act together in procambium initiation and/or maintenance together with PaPIN1, which is also up-regulated in such embryos (Hakman et al., 2009; Palovaara et al., 2010).
Although WOX2, WOX8 and STIMPY/WOX9 are not expressed in the procambium of A. thaliana embryos (Haecker et al., 2004; Wu et al., 2007), it was recently shown that WOX2 and WOX8 act redundantly with MP to promote PIN1 expression in the cotyledonary vasculature, while also both being involved in cotyledon separation (Wu et al., 2007; Breuninger et al., 2008). Presumably, a PIN1-mediated auxin maximum is required to regulate cotyledon initiation and separation, partially through the MP-mediated control of CUP-SHAPED COTYLEDON (CUC) gene expression (Aida et al., 2002; Furutani et al., 2004). Furthermore, evidence suggests that auxin and WOX4 are both involved in procambium differentiation and/or maintenance in A. thaliana by controlling the HD-Zip class III gene HOMEOBOX GENE 8 (ATHB8), which specifies procambial cells, possibly through MP action (Donner et al., 2009; Ji et al., 2010). Together with the recent discovery of a CUC2-like and MP-like gene in EST libraries from Pinus taeda embryos (Cairney et al., 2006), our results thus imply that there are conserved embryo pattern events regulating procambium initiation/maintenance and, indirectly, cotyledon separation in gymnosperms and angiosperms.
WOX genes may contribute to shoot apex function in conifers
As in embryos, PaWOX8/9 and STIMPY/WOX9 share similar transcription patterns in the shoot apex of P. abies seedlings (Fig. 6b,c) and the SAM of A. thaliana, respectively (Wu et al., 2005). Recently, Skylar et al. (2010) showed that cytokinin signalling is involved in activation of STIMPY/WOX9 expression in meristematic tissues where it promotes undifferentiated growth by maintaining cell division and WUS expression (Wu et al., 2005). Loss of STIMPY/WOX9 leads, for example, to a failure in leaf-primordia initiation. High PaWOX8/9 expression in P. abies needle primordia could thus be linked to cytokinin signalling in needle formation, but also to cell cycle control, as some single cells of the needle primordia stained more intensely than others. However, whether these cells really are cycling and also express genes concerned with cell cycle control needs to be determined. When the PaWOX2 homeobox probe was used, a faint expression pattern was seen in the P. abies SAM (Fig. 6a), which might indicate transcription of WOX genes other than PaWOX2 and PaWOX8/9. Several WOX family members are expressed in the SAM of A. thaliana, O. sativa, Z. mays and Petunia × hybrida, with different roles in organ development, including leaf formation (Matsumoto & Okada, 2001; Nardmann et al., 2004, 2007; Park et al., 2005; Dai et al., 2007; Deveaux et al., 2008; Rebocho et al., 2008; Shimizu et al., 2009; Vandenbussche et al., 2009). Thus, our results indicate that PaWOX8/9 and possibly other WOX genes may contribute to shoot apex function, including needle initiation, also in P. abies.
STIMPY/WOX9 is expressed in the roots of A. thaliana seedlings and has a similar role as in the aerial part of the plant; that is, to maintain cell division and growth (Wu et al., 2005). As no expression of PaWOX8/9 could be found in P. abies seedling root after ISH, this suggests that WOX9 has acquired new functions in angiosperms or that other WOX8/9-like genes function in this tissue in conifers, which needs to be investigated further. Also, the PaWOX2 homeobox probe, which should have broader and less specific hybridization binding, did not detect any WOX transcripts in the seedling root or in the developing SAM of embryos. However, as genes homologous to CLAVATA1, 2 and 3, which are involved in WUS regulation in A. thaliana (Schoof et al., 2000), have been discovered in EST libraries from loblolly pine embryos (Cairney & Pullman, 2007), this implies that a WUS/WOX-CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (ESR)-related (CLE) protein signalling regulation mechanism also functions in conifers, and so this needs to be investigated further.
Taken together, our results indicate that WOX2 and WOX8/9 are involved in conifer embryo formation and differentiation, and, together with PIN1 and PAT, in embryo patterning, similar to the situation in angiosperms. Thus, despite changes during seed plant evolution, a conserved mechanism, involving PAT and WOX transcription factors, may regulate seed embryo patterning. The comparison of expression in somatic and zygotic embryos also reveals somatic embryogenesis as a useful model system for conifer embryo development. Finally, similar mechanisms, including WOX gene activities, seem to function in both cotyledon and needle formation.