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Conifer embryology: a study of polar auxin transport and WOX transcription factors
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences. (Inger Hakman)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Plants, like animals, use endogenous signaling molecules to coordinate their own physiology and development. One such molecule in plants is the hormone auxin and recent research has implicated auxin and its polar transport, together with the WOX transcription factors, in regulating embryo patterning and development in angiosperms (flowering plants), the most diverse group of land plants. No extensive investigation of this has been carried out on the more evolutionary distant gymnosperms, the other major taxa of seed plants. Thus, in this thesis I, together with my co-authors, have analyzed the regulation of embryonic pattern formation in the coniferous gymnosperm Picea abies (Norway spruce) using seed and somatic embryos. Conifers are important forestry species and knowledge of their embryology is vital for reforestration programs, where the method of somatic embryogenesis is employed for mass propagation as well as it is used as a model system for embryo development.

Mature spruce embryos display a basic body plan and contain a shoot apical meristem (SAM), cotyledons, hypocotyl, embryonic root and a root apical meristem (RAM), with the meristems generating differentiated cells for organ formation. Treatment of embryos with a polar auxin transport (PAT) inhibitor produce embryos with poor SAM and, in some cases, fused cotyledons. Thus, PAT is essential for the correct patterning of conifer embryos. In angiosperms, PAT is mainly established and maintained by members of the auxin efflux facilitator PIN-FORMED (PIN) family. I isolated a PIN homologue (PaPIN1), of high abundance in conifer tissues, that is localized, together with auxin, to the epidermis of precotyledonary spruce embryos and upregulated as well as delocalized from the epidermis in early PAT inhibited embryos. Since also auxin concentration seems to decrease in the epidermis of these embryos, my data indicate that local auxin accumulation in the epidermis is mediated by PIN-dependent auxin transport.

Further, I investigated WOX genes in conifers and isolated two homologues, PaWOX2 and PaWOX8/9, that are expressed throughout spruce embryo development. Their expression profile suggests involvement in cell proliferation and specification, and, indeed, they both serve as markers for conifer somatic embryogenesis. The expression of PaWOX2 and PaWOX8/9 overlap with PaPIN1 in differentiating vascular tissue (procambium) of spruce embryos and is also upregulated in early PAT inhibited embryos. Thus, PaWOX2, PaWOX8/9, and PaPIN1 may all act together in procambium differentiation. With this, I conclude that a common mechanism, involving PAT and WOX, regulate embryo pattern formation in seed plants. Results in post-embryonic tissue imply a common mechanism regulating pattern formation also here.

Place, publisher, year, edition, pages
Kalmar/Växjö: Linnaeus University Press , 2010.
Series
Linnaeus University Dissertations ; 10/2010
National Category
Botany
Research subject
Natural Science, Cell and Organism Biology
Identifiers
URN: urn:nbn:se:hik:diva-2932ISBN: 978-91-86491-12-3 (print)OAI: oai:DiVA.org:hik-2932DiVA, id: diva2:300836
Public defence
2010-04-16, Hörsalen Fullrigaren, Landgången 4, Kalmar, 09:30 (English)
Opponent
Supervisors
Available from: 2010-04-09 Created: 2010-03-01 Last updated: 2014-05-12Bibliographically approved
List of papers
1. Expression of a gymnosperm PIN homologous gene correlates with auxin immunolocalization pattern at cotyledon formation and in demarcation of the procambium during Picea abies somatic embryo development and in seedling tissues
Open this publication in new window or tab >>Expression of a gymnosperm PIN homologous gene correlates with auxin immunolocalization pattern at cotyledon formation and in demarcation of the procambium during Picea abies somatic embryo development and in seedling tissues
Show others...
2010 (English)In: Tree Physiology, ISSN 0829-318X, E-ISSN 1758-4469, Vol. 30, no 4, p. 479-489Article in journal (Refereed) Published
Abstract [en]

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

Place, publisher, year, edition, pages
Oxford University Press, 2010
Keywords
conifer, embryo patterning, in situ hybridization, meristem, NPA, polar auxin transport (PAT), protoderm, RT-PCR
National Category
Botany
Research subject
Natural Science, Cell and Organism Biology
Identifiers
urn:nbn:se:hik:diva-2928 (URN)10.1093/treephys/tpp126 (DOI)000276305100005 ()2-s2.0-77950538656 (Scopus ID)
Available from: 2010-03-01 Created: 2010-03-01 Last updated: 2017-12-12Bibliographically approved
2. Comparative expression pattern analysis of WUSCHEL-related homeobox 2 (WOX2) and WOX8⁄9 in developing seeds and somatic embryos of the gymnosperm Picea abies
Open this publication in new window or tab >>Comparative expression pattern analysis of WUSCHEL-related homeobox 2 (WOX2) and WOX8⁄9 in developing seeds and somatic embryos of the gymnosperm Picea abies
2010 (English)In: New Phytologist, ISSN 0028-646X, E-ISSN 1469-8137, Vol. 188, no 1, p. 122-135Article in journal (Refereed) Published
Abstract [en]

In seed plants, current knowledge concerning embryonic pattern formation by polar auxin transport (PAT) and WUSCHEL-related homeobox (WOX) gene activity is primarily derived from studies on angiosperms, while less is known about these processes in gymnosperms. In view of the differences in their embryogeny, and the fact that somatic embryogenesis is used for mass propagation of conifers, a better understanding of embryo development is vital.

The expression patterns of PaWOX2 and PaWOX8/9 were followed with quantitative reverse transcription–polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH) during seed and somatic embryo development in Norway spruce (Picea abies), and in somatic embryos treated with the PAT inhibitor N-1-naphthylphthalamic acid (NPA).

Both PaWOX2 and PaWOX8/9 were highly expressed at the early growth stages of zygotic and somatic embryos, and shared a similar expression pattern over the entire embryo. At later embryo stages, high expression of PaWOX8/9 became restricted to cotyledon primordia, epidermis, procambium and root apical meristem (RAM), which became most evident in NPA-treated somatic embryos, while expression of PaWOX2 was much lower.

Our results suggest an ancestral role of WOX in seed plant embryo development, and strengthen the proposed connection between PAT, PIN-FORMED (PIN) and WOX in the regulation of embryo patterning in seed plants.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Keywords
archegonia, conifer, in situ hybridization, N-1-naphthylphthalamic acid (NPA), PCR, polar auxin transport (PAT), rocambium, transcription factor
National Category
Botany
Research subject
Natural Science, Cell and Organism Biology
Identifiers
urn:nbn:se:hik:diva-2929 (URN)10.1111/j.1469-8137.2010.03336.x (DOI)2-s2.0-77956295063 (Scopus ID)
Available from: 2010-03-01 Created: 2010-03-01 Last updated: 2017-12-12Bibliographically approved
3. Conifer WOX-related homedomain transcription factors, developmental considerations and expression dynamic of WOX2 during Picea abies somatic embryogenesis
Open this publication in new window or tab >>Conifer WOX-related homedomain transcription factors, developmental considerations and expression dynamic of WOX2 during Picea abies somatic embryogenesis
2008 (English)In: Plant Molecular Biology, ISSN 0167-4412, E-ISSN 1573-5028, Vol. 66, no 5, p. 533-549Article in journal (Refereed) Published
Abstract [en]

In angiosperms, the WOX family of transcription factors has important functions in meristem regulation and in control of the partitioning of developing embryos into functional domains. In this study, a putative WOX2 homologous gene was isolated from Picea abies, and its expression pattern during somatic embryo development was followed using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). We used strategies of both absolute and relative quantification of gene expression, and benefits and disadvantages of the two methods are presented and discussed. During embryogenesis, PaWOX2 expression was highest at the earliest stages of development, but low levels were also detected in seedling tissues. No PaWOX2expression was detected in a non-embryogenic cell culture, indicating thatPaWOX2 plays a fundamental role during early somatic embryo development, and can be used as a possible marker for embryogenic potential. Additional results show that conifers, like angiosperms, contain a large number of WOX-related genes, many of them expressed during embryo development. In phylogenetic analysis based on the deduced homeodomain of retrieved pine and spruce EST sequences, no conifer WUS homolog was found. Neither did we find any homeodomain to cluster with WOX5. Interestingly, a clade including only conifer sequences derived from various tissues was resolved as sister to a PhyscomitrellaWOX-like gene, suggestive of the early origin of this gene family. Our results thus provide basic information for further studies of the evolution of this gene family and of their function in relation to meristem dynamics and specification of stem cells in gymnosperms.

Place, publisher, year, edition, pages
Springer, 2008
Keywords
Conifer, Picea abies, qRT-PCR, Somatic embryogenesis, transcription factor, WOX
National Category
Botany
Research subject
Computer and Information Sciences Computer Science, Computer Science
Identifiers
urn:nbn:se:hik:diva-2924 (URN)10.1007/s11103-008-9289-5 (DOI)
Available from: 2010-03-01 Created: 2010-03-01 Last updated: 2017-12-12Bibliographically approved
4. WOX2 and polar auxin transport during spruce embryo pattern formation
Open this publication in new window or tab >>WOX2 and polar auxin transport during spruce embryo pattern formation
2009 (English)In: Plant Signalling & Behavior, ISSN 1559-2316, E-ISSN 1559-2324, Vol. 4, no 2, p. 153-155Article in journal (Other academic) Published
Abstract [en]

The WOX family of transcription factors and polar auxin transport (PAT) are both essential for embryonic patterning and thus normal embryo development in angiosperms. Recent analysis by us of WOX-related genes in Picea and Pinus suggests that they play fundamental roles during embryo development also in conifers.1 It has been proposed that there is a connection between the spatial separation of WOX2 and WOX8, and PAT in the formation of the apical-basal axis in Arabidopsis embryos and that both are involved in the regulation of the auxin efflux carrier PIN1. Auxin also seems to play a crucial role in apical-basal axis formation in conifer embryos based on studies using the polar auxin inhibitor NPA. We recently analyzed the expression of a PIN1-like gene in NPA-treated and untreated precotyledonary somatic spruce embryos and could see a significant upregulation of the PIN1-like gene in the NPA-treated embryos.2 Here we show that PaWOX2 is also significantly upregulated in the same embryos. Taken together, this suggests that PAT is involved in regulating both PIN1 and WOX2 expression in conifers and strengthens the evidence for the proposed connection between WOX and PIN genes in seed plants.

Place, publisher, year, edition, pages
Landes Biosciences, 2009
Keywords
conifer, Picea abies, embryo development, polar auxin transport, WOX, PIN, NPA, somatic embryogenesis, qRT-PCR
National Category
Botany
Research subject
Natural Science, Cell and Organism Biology
Identifiers
urn:nbn:se:hik:diva-2927 (URN)10.4161/psb.4.2.7684 (DOI)
Available from: 2010-03-01 Created: 2010-03-01 Last updated: 2017-12-12Bibliographically approved
5. The polar auxin transport inhibitor NPA impairs embryo morphology and increases the expression of an auxin efflux facilitator protein PIN during Picea abies somatic embryo development
Open this publication in new window or tab >>The polar auxin transport inhibitor NPA impairs embryo morphology and increases the expression of an auxin efflux facilitator protein PIN during Picea abies somatic embryo development
2009 (English)In: Tree Physiology, ISSN 0829-318X, E-ISSN 1758-4469, Vol. 29, no 4, p. 483-496Article in journal (Refereed) Published
Abstract [en]

Auxin and polar auxin transport have been implicated in controlling embryo patterning and development in angiosperms but less is known from the gymnosperms. The aims of this study were to determine at what stages of conifer embryo development auxin and polar auxin transport are the most important for normal development and to analyze the changes in embryos after treatment with the polar auxin inhibitor N-1-naphthylphthalamic acid (NPA). For these studies, somatic embryos of Norway spruce (Picea abies L. Karst) were used. Growth on medium containing NPA leads to the formation of embryos with poor shoot apical meristem (SAM) and fused cotyledons, and to a pin-formed phenotype of the regenerated plantlets. The effect of NPA on embryo morphology was most severe if embryos were transferred to NPA-containing medium immediately before cotyledon initiation and SAM specification. Indole-3-acetic acid (IAA) was identified by immunolocalization in developing embryos. The highest staining intensity was seen in early staged embryos and then decreased as the embryos matured. No clear IAA-maxima was seen, although the apical parts of embryos, particularly the protoderm, and the suspensor cells appear to accumulate more IAA, as reflected by the staining pattern. The NPA treatment also caused expanded procambium and a broader root apical meristem in embryos, and a significant increase in the expression of a PIN1-like gene. Taken together, our results show that, for proper cotyledon initiation, correct auxin transport is needed only during a short period at the transition stage of embryo development, probably involving PIN efflux proteins and that a common mechanism is behind proper cotyledon formation within the species of angiosperms and conifers, despite their cotyledon number which normally differs.

Place, publisher, year, edition, pages
Oxford University Press, 2009
Keywords
conifer, meristem, qRT-PCR
National Category
Biological Sciences
Research subject
Natural Science, Cell and Organism Biology
Identifiers
urn:nbn:se:hik:diva-2925 (URN)10.1093/treephys/tpn048 (DOI)
Available from: 2010-03-01 Created: 2010-03-01 Last updated: 2017-12-12Bibliographically approved

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