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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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The relative fluorescence intensity (RFI) of DR5rev::GFP in MDEs exposed to the exogenous auxin in darkness. Application of IBA decreases the DR5rev::GFP fluorescence. DR5rev-GFP expression patterns in the leaf primordia and root tips at 10–30 min intervals over 220 min. Interval 0–30 min is dedicated for control (culture without the presence of exogenous auxin)
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Fig5: The relative fluorescence intensity (RFI) of DR5rev::GFP in MDEs exposed to the exogenous auxin in darkness. Application of IBA decreases the DR5rev::GFP fluorescence. DR5rev-GFP expression patterns in the leaf primordia and root tips at 10–30 min intervals over 220 min. Interval 0–30 min is dedicated for control (culture without the presence of exogenous auxin)

Mentions: The shoot- and root-derived auxin pools participate in the control of plant development (Davies 2004). In order to determine the physiological response of the individual MDEs to applied exogenous auxin, the expression of GFP fluorescence was quantified on the basis of time-lapse imaging analysis. The relative fluorescence was fluctuating in a period of 220 min and changed in the range of 34.85–27.77 (max–min) in leaf primordia and 33.26–21.91 (max–min) in the root tips. Indole-3-butiric acid that induced decrease in the DR5:GFPrev fluorescence shows that IBA is probably able to influence the transcription of auxin-induced genes in embryonic leaf and root primordia. However, this effect was different from those usually observed in the case of IAA (Lewis et al. 2013). Unfortunately, there is no information available describing the mechanism of regulation of IBA response gene expression. Expected of an active auxin, the RFI intensity of primordia exposed to auxin was 1.31× higher of that measured in the root tips (Fig. 5). The data presented are in agreement with results received in classical auxin location experiments (Davies 2004). Quantitative estimation (spectrophotometric analysis) of auxin in the segments of plant seedlings revealed that the stem apex possesses 1.5- to 2.0-fold higher amounts than that found in the root apex. Although shoot apices synthesize more auxin than the root apices and both places of auxin production are critical for primordia, shoot, and root apical meristems formation and subsequent growth, we have noticed that the leaf primordia were more influenced by exogenous auxin than the roots in MDEs at the early stages.Fig. 5


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)

The relative fluorescence intensity (RFI) of DR5rev::GFP in MDEs exposed to the exogenous auxin in darkness. Application of IBA decreases the DR5rev::GFP fluorescence. DR5rev-GFP expression patterns in the leaf primordia and root tips at 10–30 min intervals over 220 min. Interval 0–30 min is dedicated for control (culture without the presence of exogenous auxin)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: The relative fluorescence intensity (RFI) of DR5rev::GFP in MDEs exposed to the exogenous auxin in darkness. Application of IBA decreases the DR5rev::GFP fluorescence. DR5rev-GFP expression patterns in the leaf primordia and root tips at 10–30 min intervals over 220 min. Interval 0–30 min is dedicated for control (culture without the presence of exogenous auxin)
Mentions: The shoot- and root-derived auxin pools participate in the control of plant development (Davies 2004). In order to determine the physiological response of the individual MDEs to applied exogenous auxin, the expression of GFP fluorescence was quantified on the basis of time-lapse imaging analysis. The relative fluorescence was fluctuating in a period of 220 min and changed in the range of 34.85–27.77 (max–min) in leaf primordia and 33.26–21.91 (max–min) in the root tips. Indole-3-butiric acid that induced decrease in the DR5:GFPrev fluorescence shows that IBA is probably able to influence the transcription of auxin-induced genes in embryonic leaf and root primordia. However, this effect was different from those usually observed in the case of IAA (Lewis et al. 2013). Unfortunately, there is no information available describing the mechanism of regulation of IBA response gene expression. Expected of an active auxin, the RFI intensity of primordia exposed to auxin was 1.31× higher of that measured in the root tips (Fig. 5). The data presented are in agreement with results received in classical auxin location experiments (Davies 2004). Quantitative estimation (spectrophotometric analysis) of auxin in the segments of plant seedlings revealed that the stem apex possesses 1.5- to 2.0-fold higher amounts than that found in the root apex. Although shoot apices synthesize more auxin than the root apices and both places of auxin production are critical for primordia, shoot, and root apical meristems formation and subsequent growth, we have noticed that the leaf primordia were more influenced by exogenous auxin than the roots in MDEs at the early stages.Fig. 5

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