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The influence of heat stress on auxin distribution in transgenic B. napus microspores and microspore-derived embryos.

Dubas E, Moravčíková J, Libantová J, Matušíková I, Benková E, Zur I, Krzewska M - Protoplasma (2014)

Bottom Line: Relative quantitative estimation of auxin, using time-lapse imaging, revealed that primordia possess up to 1.3-fold higher amounts than those found in the root apices of transgenic MDEs in the presence of exogenous auxin.Our results show, for the first time, which concentration of endogenous auxin coincides with the first cell division and how the high temperature interplays with auxin, by what affects delay early establishing microspore polarity.Moreover, we present how the local auxin accumulation demonstrates the apical-basal axis formation of the androgenic embryo and directs the axiality of the adult haploid plant.

View Article: PubMed Central - PubMed

Affiliation: The Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21, 30-239, Kraków, Poland, edubas@o2.pl.

ABSTRACT
Plant embryogenesis is regulated by differential distribution of the plant hormone auxin. However, the cells establishing these gradients during microspore embryogenesis remain to be identified. For the first time, we describe, using the DR5 or DR5rev reporter gene systems, the GFP- and GUS-based auxin biosensors to monitor auxin during Brassica napus androgenesis at cellular resolution in the initial stages. Our study provides evidence that the distribution of auxin changes during embryo development and depends on the temperature-inducible in vitro culture conditions. For this, microspores (mcs) were induced to embryogenesis by heat treatment and then subjected to genetic modification via Agrobacterium tumefaciens. The duration of high temperature treatment had a significant influence on auxin distribution in isolated and in vitro-cultured microspores and on microspore-derived embryo development. In the "mild" heat-treated (1 day at 32 °C) mcs, auxin localized in a polar way already at the uni-nucleate microspore, which was critical for the initiation of embryos with suspensor-like structure. Assuming a mean mcs radius of 20 μm, endogenous auxin content in a single cell corresponded to concentration of 1.01 μM. In mcs subjected to a prolonged heat (5 days at 32 °C), although auxin concentration increased dozen times, auxin polarization was set up at a few-celled pro-embryos without suspensor. Those embryos were enclosed in the outer wall called the exine. The exine rupture was accompanied by the auxin gradient polarization. Relative quantitative estimation of auxin, using time-lapse imaging, revealed that primordia possess up to 1.3-fold higher amounts than those found in the root apices of transgenic MDEs in the presence of exogenous auxin. Our results show, for the first time, which concentration of endogenous auxin coincides with the first cell division and how the high temperature interplays with auxin, by what affects delay early establishing microspore polarity. Moreover, we present how the local auxin accumulation demonstrates the apical-basal axis formation of the androgenic embryo and directs the axiality of the adult haploid plant.

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Local auxin distribution in the “mild” heat-treated microspores and MDEs of B. napus.a Uni-nucleated control microspores. Autofluorescence. b Uni-nucleated microspores 2 days co-cultured with A. tumefaciens. c -c1 Microspores with unequal auxin distribution on the one pole (arrows). d Two-celled pro-embryo with the basal (bc) and apical (ac) cells. e, f Linear file of three cells with the DR5 (e) or DR5rev (f) activities in all cells. g Linear file of four cells with higher DR5 activity in the basal cell (bc). h–l The pro-embryo proper with the suspensor-like structure. h Two-celled pro-embryo proper with a long suspensor-like filament. The tip cell of the suspensor-like structure was delineated to become the embryo proper after longitudinal division. i The embryo proper in the octant stage. j The globular embryo stage with the DR5 activity in the cells of the suspensor-like structure (j1). k The embryo proper in the dermatogen stage. Note the hypophysis region (h). l The globular embryo proper with maximum of the DR5 activity in the protoderm of the apical region of the embryo proper (square). The higher magnification of the region of protoderm with the DR5 activity on l (l1). m The DR5 activity in the cells of the suspensor-like structures at the globular stage embryo proper. n DR5rev activity in the apical part of the globular pro-embryo proper with suspensor. o Globular embryo proper with the suspensor. The DR5rev activity concentrated in the apical part of the pro-embryo, in the provasculature, and in the hypophysis (arrow). DIC image of the same representative as on o (o1). Blue and light green colors show the expression of the reporter gus and gfp genes driven by the DR5 or DR5rev promoters (respectively). Bar = 20 μm
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Fig1: Local auxin distribution in the “mild” heat-treated microspores and MDEs of B. napus.a Uni-nucleated control microspores. Autofluorescence. b Uni-nucleated microspores 2 days co-cultured with A. tumefaciens. c -c1 Microspores with unequal auxin distribution on the one pole (arrows). d Two-celled pro-embryo with the basal (bc) and apical (ac) cells. e, f Linear file of three cells with the DR5 (e) or DR5rev (f) activities in all cells. g Linear file of four cells with higher DR5 activity in the basal cell (bc). h–l The pro-embryo proper with the suspensor-like structure. h Two-celled pro-embryo proper with a long suspensor-like filament. The tip cell of the suspensor-like structure was delineated to become the embryo proper after longitudinal division. i The embryo proper in the octant stage. j The globular embryo stage with the DR5 activity in the cells of the suspensor-like structure (j1). k The embryo proper in the dermatogen stage. Note the hypophysis region (h). l The globular embryo proper with maximum of the DR5 activity in the protoderm of the apical region of the embryo proper (square). The higher magnification of the region of protoderm with the DR5 activity on l (l1). m The DR5 activity in the cells of the suspensor-like structures at the globular stage embryo proper. n DR5rev activity in the apical part of the globular pro-embryo proper with suspensor. o Globular embryo proper with the suspensor. The DR5rev activity concentrated in the apical part of the pro-embryo, in the provasculature, and in the hypophysis (arrow). DIC image of the same representative as on o (o1). Blue and light green colors show the expression of the reporter gus and gfp genes driven by the DR5 or DR5rev promoters (respectively). Bar = 20 μm

Mentions: In the presented study, mcs were firstly induced to embryogenesis by the “mild” or prolonged heat treatment and then, 7 or 21 days post heat stress, subjected to the genetic modification procedure for 2 days. The T-DNAs contained the gus or gfp reporter genes under control of the auxin-responsive DR5 or DR5rev promoters (respectively). The local auxin distribution was demonstrated by the imaging reporter genes expressions (Figs. 1, 2, 3 and 4).Fig. 1


The influence of heat stress on auxin distribution in transgenic B. napus microspores and microspore-derived embryos.

Dubas E, Moravčíková J, Libantová J, Matušíková I, Benková E, Zur I, Krzewska M - Protoplasma (2014)

Local auxin distribution in the “mild” heat-treated microspores and MDEs of B. napus.a Uni-nucleated control microspores. Autofluorescence. b Uni-nucleated microspores 2 days co-cultured with A. tumefaciens. c -c1 Microspores with unequal auxin distribution on the one pole (arrows). d Two-celled pro-embryo with the basal (bc) and apical (ac) cells. e, f Linear file of three cells with the DR5 (e) or DR5rev (f) activities in all cells. g Linear file of four cells with higher DR5 activity in the basal cell (bc). h–l The pro-embryo proper with the suspensor-like structure. h Two-celled pro-embryo proper with a long suspensor-like filament. The tip cell of the suspensor-like structure was delineated to become the embryo proper after longitudinal division. i The embryo proper in the octant stage. j The globular embryo stage with the DR5 activity in the cells of the suspensor-like structure (j1). k The embryo proper in the dermatogen stage. Note the hypophysis region (h). l The globular embryo proper with maximum of the DR5 activity in the protoderm of the apical region of the embryo proper (square). The higher magnification of the region of protoderm with the DR5 activity on l (l1). m The DR5 activity in the cells of the suspensor-like structures at the globular stage embryo proper. n DR5rev activity in the apical part of the globular pro-embryo proper with suspensor. o Globular embryo proper with the suspensor. The DR5rev activity concentrated in the apical part of the pro-embryo, in the provasculature, and in the hypophysis (arrow). DIC image of the same representative as on o (o1). Blue and light green colors show the expression of the reporter gus and gfp genes driven by the DR5 or DR5rev promoters (respectively). Bar = 20 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4125814&req=5

Fig1: Local auxin distribution in the “mild” heat-treated microspores and MDEs of B. napus.a Uni-nucleated control microspores. Autofluorescence. b Uni-nucleated microspores 2 days co-cultured with A. tumefaciens. c -c1 Microspores with unequal auxin distribution on the one pole (arrows). d Two-celled pro-embryo with the basal (bc) and apical (ac) cells. e, f Linear file of three cells with the DR5 (e) or DR5rev (f) activities in all cells. g Linear file of four cells with higher DR5 activity in the basal cell (bc). h–l The pro-embryo proper with the suspensor-like structure. h Two-celled pro-embryo proper with a long suspensor-like filament. The tip cell of the suspensor-like structure was delineated to become the embryo proper after longitudinal division. i The embryo proper in the octant stage. j The globular embryo stage with the DR5 activity in the cells of the suspensor-like structure (j1). k The embryo proper in the dermatogen stage. Note the hypophysis region (h). l The globular embryo proper with maximum of the DR5 activity in the protoderm of the apical region of the embryo proper (square). The higher magnification of the region of protoderm with the DR5 activity on l (l1). m The DR5 activity in the cells of the suspensor-like structures at the globular stage embryo proper. n DR5rev activity in the apical part of the globular pro-embryo proper with suspensor. o Globular embryo proper with the suspensor. The DR5rev activity concentrated in the apical part of the pro-embryo, in the provasculature, and in the hypophysis (arrow). DIC image of the same representative as on o (o1). Blue and light green colors show the expression of the reporter gus and gfp genes driven by the DR5 or DR5rev promoters (respectively). Bar = 20 μm
Mentions: In the presented study, mcs were firstly induced to embryogenesis by the “mild” or prolonged heat treatment and then, 7 or 21 days post heat stress, subjected to the genetic modification procedure for 2 days. The T-DNAs contained the gus or gfp reporter genes under control of the auxin-responsive DR5 or DR5rev promoters (respectively). The local auxin distribution was demonstrated by the imaging reporter genes expressions (Figs. 1, 2, 3 and 4).Fig. 1

Bottom Line: Relative quantitative estimation of auxin, using time-lapse imaging, revealed that primordia possess up to 1.3-fold higher amounts than those found in the root apices of transgenic MDEs in the presence of exogenous auxin.Our results show, for the first time, which concentration of endogenous auxin coincides with the first cell division and how the high temperature interplays with auxin, by what affects delay early establishing microspore polarity.Moreover, we present how the local auxin accumulation demonstrates the apical-basal axis formation of the androgenic embryo and directs the axiality of the adult haploid plant.

View Article: PubMed Central - PubMed

Affiliation: The Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21, 30-239, Kraków, Poland, edubas@o2.pl.

ABSTRACT
Plant embryogenesis is regulated by differential distribution of the plant hormone auxin. However, the cells establishing these gradients during microspore embryogenesis remain to be identified. For the first time, we describe, using the DR5 or DR5rev reporter gene systems, the GFP- and GUS-based auxin biosensors to monitor auxin during Brassica napus androgenesis at cellular resolution in the initial stages. Our study provides evidence that the distribution of auxin changes during embryo development and depends on the temperature-inducible in vitro culture conditions. For this, microspores (mcs) were induced to embryogenesis by heat treatment and then subjected to genetic modification via Agrobacterium tumefaciens. The duration of high temperature treatment had a significant influence on auxin distribution in isolated and in vitro-cultured microspores and on microspore-derived embryo development. In the "mild" heat-treated (1 day at 32 °C) mcs, auxin localized in a polar way already at the uni-nucleate microspore, which was critical for the initiation of embryos with suspensor-like structure. Assuming a mean mcs radius of 20 μm, endogenous auxin content in a single cell corresponded to concentration of 1.01 μM. In mcs subjected to a prolonged heat (5 days at 32 °C), although auxin concentration increased dozen times, auxin polarization was set up at a few-celled pro-embryos without suspensor. Those embryos were enclosed in the outer wall called the exine. The exine rupture was accompanied by the auxin gradient polarization. Relative quantitative estimation of auxin, using time-lapse imaging, revealed that primordia possess up to 1.3-fold higher amounts than those found in the root apices of transgenic MDEs in the presence of exogenous auxin. Our results show, for the first time, which concentration of endogenous auxin coincides with the first cell division and how the high temperature interplays with auxin, by what affects delay early establishing microspore polarity. Moreover, we present how the local auxin accumulation demonstrates the apical-basal axis formation of the androgenic embryo and directs the axiality of the adult haploid plant.

Show MeSH
Related in: MedlinePlus