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Differential regulation of Knotted1-like genes during establishment of the shoot apical meristem in Norway spruce (Picea abies).

Larsson E, Sitbon F, von Arnold S - Plant Cell Rep. (2012)

Bottom Line: This was made both under normal embryo development and under conditions of reduced SAM formation by treatment with the polar auxin transport inhibitor NPA.Concomitantly with the formation of an embryonic SAM, the HBK2 and HBK4 genes displayed a significant up-regulation that was delayed by NPA treatment.Together, the results suggest that HBK2 and HBK4 exert similar functions related to the SAM differentiation and somatic embryo development in Norway spruce, while HBK1 and HBK3 have more general functions during embryo development.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences, P. O. Box 7080, 75007 Uppsala, Sweden. emma.larsson@slu.se

ABSTRACT
Establishment of the shoot apical meristem (SAM) in Arabidopsis embryos requires the KNOXI transcription factor SHOOT MERISTEMLESS. In Norway spruce (Picea abies), four KNOXI family members (HBK1, HBK2, HBK3 and HBK4) have been identified, but a corresponding role in SAM development has not been demonstrated. As a first step to differentiate between the functions of the four Norway spruce HBK genes, we have here analyzed their expression profiles during the process of somatic embryo development. This was made both under normal embryo development and under conditions of reduced SAM formation by treatment with the polar auxin transport inhibitor NPA. Concomitantly with the formation of an embryonic SAM, the HBK2 and HBK4 genes displayed a significant up-regulation that was delayed by NPA treatment. In contrast, HBK1 and HBK3 were up-regulated prior to SAM formation, and their temporal expression was not affected by NPA. Ectopic expression of the four HBK genes in transgenic Arabidopsis plants further supported similar functions of HBK2 and HBK4, distinct from those of HBK1 and HBK3. Together, the results suggest that HBK2 and HBK4 exert similar functions related to the SAM differentiation and somatic embryo development in Norway spruce, while HBK1 and HBK3 have more general functions during embryo development.

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Related in: MedlinePlus

Eight consecutive stages of control (a–d, h–k) and NPA-treated (e–g, l–o) somatic embryos of Norway spruce. The developmental stages of NPA-treated embryos were largely defined according to the sizes of phenotypically normal control embryos, and not according to age; however, the average exposure time to ABA for reaching a certain stage is presented. a Stage 1, proliferating proembryonic masses in the presence of the plant growth regulators (PGRs) auxin and cytokinin. NPA was added to the cultures simultaneously with the withdrawal of PGRs, and stage 1 cultures were hence not treated with NPA. b, e Stage 2, early embryos 1 week after withdrawal of PGRs. c, f Stage 3, beginning of late embryo development after 1–2 weeks of exposure to ABA. Note the cone shape of the embryo treated with NPA (f) compared to the more cylindrical shape of the control embryo (c). d, g Stage 4, late embryos after 2–3 weeks of exposure to ABA. Note that the suspensor is still present in the NPA-treated embryo (g), while it has been degraded in the control embryo (d). h, l Stage 5, early maturing embryos after 3–4 weeks of exposure to ABA. i, m Stage 6, maturing embryos after 4–5 weeks (i) or 5–6 weeks (m) of exposure to ABA. j, n Stage 7, almost fully matured control embryo after 5–6 weeks of exposure to ABA (j), almost fully matured embryo with doughnut-shaped apical part after 6–7 weeks of exposure to ABA and NPA (n). k, o Stage 8, fully matured cotyledonary embryo after 6–7 weeks of exposure to ABA (k), and fully matured embryo lacking separated cotyledons after 7–8 weeks of exposure to ABA and NPA (o). co Cotyledon, cp cotyledon primordia, dsc doughnut-shaped cotyledon, em embryonal mass, fc fused cotyledons, s suspensor, sp shoot apical meristem primordium, SAM shoot apical meristem, tc tube cells. Scale bars 100 µm (a–j, l–n) and 250 µm (k, o)
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Fig1: Eight consecutive stages of control (a–d, h–k) and NPA-treated (e–g, l–o) somatic embryos of Norway spruce. The developmental stages of NPA-treated embryos were largely defined according to the sizes of phenotypically normal control embryos, and not according to age; however, the average exposure time to ABA for reaching a certain stage is presented. a Stage 1, proliferating proembryonic masses in the presence of the plant growth regulators (PGRs) auxin and cytokinin. NPA was added to the cultures simultaneously with the withdrawal of PGRs, and stage 1 cultures were hence not treated with NPA. b, e Stage 2, early embryos 1 week after withdrawal of PGRs. c, f Stage 3, beginning of late embryo development after 1–2 weeks of exposure to ABA. Note the cone shape of the embryo treated with NPA (f) compared to the more cylindrical shape of the control embryo (c). d, g Stage 4, late embryos after 2–3 weeks of exposure to ABA. Note that the suspensor is still present in the NPA-treated embryo (g), while it has been degraded in the control embryo (d). h, l Stage 5, early maturing embryos after 3–4 weeks of exposure to ABA. i, m Stage 6, maturing embryos after 4–5 weeks (i) or 5–6 weeks (m) of exposure to ABA. j, n Stage 7, almost fully matured control embryo after 5–6 weeks of exposure to ABA (j), almost fully matured embryo with doughnut-shaped apical part after 6–7 weeks of exposure to ABA and NPA (n). k, o Stage 8, fully matured cotyledonary embryo after 6–7 weeks of exposure to ABA (k), and fully matured embryo lacking separated cotyledons after 7–8 weeks of exposure to ABA and NPA (o). co Cotyledon, cp cotyledon primordia, dsc doughnut-shaped cotyledon, em embryonal mass, fc fused cotyledons, s suspensor, sp shoot apical meristem primordium, SAM shoot apical meristem, tc tube cells. Scale bars 100 µm (a–j, l–n) and 250 µm (k, o)

Mentions: To study the effect of PAT on the expression of the HBK genes, embryogenic cultures were treated with 20 µM 1-N-naphthylphthalamic acid (NPA; Sigma-Aldrich, Schnelldorf, Germany) during both the pre-maturation and maturation phases, as described by Larsson et al. (2008). Samples for gene expression studies were collected from eight consecutive stages from both control and NPA-treated embryos (Fig. 1). The developmental stages of NPA-treated embryos were largely defined according to the sizes of phenotypically normal control embryos. Whole mount of tissue was sampled from proliferating PEMs (stage 1) and early embryos (stage 2). From stage 3 and onwards, the embryos were sampled individually and sorted according to their developmental stage to increase the specificity in subsequent expression analyses. Samples were stored at −80°C until use.Fig. 1


Differential regulation of Knotted1-like genes during establishment of the shoot apical meristem in Norway spruce (Picea abies).

Larsson E, Sitbon F, von Arnold S - Plant Cell Rep. (2012)

Eight consecutive stages of control (a–d, h–k) and NPA-treated (e–g, l–o) somatic embryos of Norway spruce. The developmental stages of NPA-treated embryos were largely defined according to the sizes of phenotypically normal control embryos, and not according to age; however, the average exposure time to ABA for reaching a certain stage is presented. a Stage 1, proliferating proembryonic masses in the presence of the plant growth regulators (PGRs) auxin and cytokinin. NPA was added to the cultures simultaneously with the withdrawal of PGRs, and stage 1 cultures were hence not treated with NPA. b, e Stage 2, early embryos 1 week after withdrawal of PGRs. c, f Stage 3, beginning of late embryo development after 1–2 weeks of exposure to ABA. Note the cone shape of the embryo treated with NPA (f) compared to the more cylindrical shape of the control embryo (c). d, g Stage 4, late embryos after 2–3 weeks of exposure to ABA. Note that the suspensor is still present in the NPA-treated embryo (g), while it has been degraded in the control embryo (d). h, l Stage 5, early maturing embryos after 3–4 weeks of exposure to ABA. i, m Stage 6, maturing embryos after 4–5 weeks (i) or 5–6 weeks (m) of exposure to ABA. j, n Stage 7, almost fully matured control embryo after 5–6 weeks of exposure to ABA (j), almost fully matured embryo with doughnut-shaped apical part after 6–7 weeks of exposure to ABA and NPA (n). k, o Stage 8, fully matured cotyledonary embryo after 6–7 weeks of exposure to ABA (k), and fully matured embryo lacking separated cotyledons after 7–8 weeks of exposure to ABA and NPA (o). co Cotyledon, cp cotyledon primordia, dsc doughnut-shaped cotyledon, em embryonal mass, fc fused cotyledons, s suspensor, sp shoot apical meristem primordium, SAM shoot apical meristem, tc tube cells. Scale bars 100 µm (a–j, l–n) and 250 µm (k, o)
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Related In: Results  -  Collection

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Fig1: Eight consecutive stages of control (a–d, h–k) and NPA-treated (e–g, l–o) somatic embryos of Norway spruce. The developmental stages of NPA-treated embryos were largely defined according to the sizes of phenotypically normal control embryos, and not according to age; however, the average exposure time to ABA for reaching a certain stage is presented. a Stage 1, proliferating proembryonic masses in the presence of the plant growth regulators (PGRs) auxin and cytokinin. NPA was added to the cultures simultaneously with the withdrawal of PGRs, and stage 1 cultures were hence not treated with NPA. b, e Stage 2, early embryos 1 week after withdrawal of PGRs. c, f Stage 3, beginning of late embryo development after 1–2 weeks of exposure to ABA. Note the cone shape of the embryo treated with NPA (f) compared to the more cylindrical shape of the control embryo (c). d, g Stage 4, late embryos after 2–3 weeks of exposure to ABA. Note that the suspensor is still present in the NPA-treated embryo (g), while it has been degraded in the control embryo (d). h, l Stage 5, early maturing embryos after 3–4 weeks of exposure to ABA. i, m Stage 6, maturing embryos after 4–5 weeks (i) or 5–6 weeks (m) of exposure to ABA. j, n Stage 7, almost fully matured control embryo after 5–6 weeks of exposure to ABA (j), almost fully matured embryo with doughnut-shaped apical part after 6–7 weeks of exposure to ABA and NPA (n). k, o Stage 8, fully matured cotyledonary embryo after 6–7 weeks of exposure to ABA (k), and fully matured embryo lacking separated cotyledons after 7–8 weeks of exposure to ABA and NPA (o). co Cotyledon, cp cotyledon primordia, dsc doughnut-shaped cotyledon, em embryonal mass, fc fused cotyledons, s suspensor, sp shoot apical meristem primordium, SAM shoot apical meristem, tc tube cells. Scale bars 100 µm (a–j, l–n) and 250 µm (k, o)
Mentions: To study the effect of PAT on the expression of the HBK genes, embryogenic cultures were treated with 20 µM 1-N-naphthylphthalamic acid (NPA; Sigma-Aldrich, Schnelldorf, Germany) during both the pre-maturation and maturation phases, as described by Larsson et al. (2008). Samples for gene expression studies were collected from eight consecutive stages from both control and NPA-treated embryos (Fig. 1). The developmental stages of NPA-treated embryos were largely defined according to the sizes of phenotypically normal control embryos. Whole mount of tissue was sampled from proliferating PEMs (stage 1) and early embryos (stage 2). From stage 3 and onwards, the embryos were sampled individually and sorted according to their developmental stage to increase the specificity in subsequent expression analyses. Samples were stored at −80°C until use.Fig. 1

Bottom Line: This was made both under normal embryo development and under conditions of reduced SAM formation by treatment with the polar auxin transport inhibitor NPA.Concomitantly with the formation of an embryonic SAM, the HBK2 and HBK4 genes displayed a significant up-regulation that was delayed by NPA treatment.Together, the results suggest that HBK2 and HBK4 exert similar functions related to the SAM differentiation and somatic embryo development in Norway spruce, while HBK1 and HBK3 have more general functions during embryo development.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences, P. O. Box 7080, 75007 Uppsala, Sweden. emma.larsson@slu.se

ABSTRACT
Establishment of the shoot apical meristem (SAM) in Arabidopsis embryos requires the KNOXI transcription factor SHOOT MERISTEMLESS. In Norway spruce (Picea abies), four KNOXI family members (HBK1, HBK2, HBK3 and HBK4) have been identified, but a corresponding role in SAM development has not been demonstrated. As a first step to differentiate between the functions of the four Norway spruce HBK genes, we have here analyzed their expression profiles during the process of somatic embryo development. This was made both under normal embryo development and under conditions of reduced SAM formation by treatment with the polar auxin transport inhibitor NPA. Concomitantly with the formation of an embryonic SAM, the HBK2 and HBK4 genes displayed a significant up-regulation that was delayed by NPA treatment. In contrast, HBK1 and HBK3 were up-regulated prior to SAM formation, and their temporal expression was not affected by NPA. Ectopic expression of the four HBK genes in transgenic Arabidopsis plants further supported similar functions of HBK2 and HBK4, distinct from those of HBK1 and HBK3. Together, the results suggest that HBK2 and HBK4 exert similar functions related to the SAM differentiation and somatic embryo development in Norway spruce, while HBK1 and HBK3 have more general functions during embryo development.

Show MeSH
Related in: MedlinePlus