Imaging early stages of the female reproductive structure of arabidopsis by confocal laser scanning microscopy

Reyes-Olalde, J. Irepan; Marsch-Martínez, Nayelli; Folter, Stefan de

doi:10.1002/dvdy.24301

Cited by 9 publications

(9 citation statements)

References 30 publications

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“…Tissue preparation and confocal microscopy analysis To observe fluorescence signal, gynoecia were dissected and observed as previously described [116]. In summary, gynoecia were observed longitudinally or cut transversely using a scalpel and mounted in glycerol.…”

Section: Methodsmentioning

confidence: 99%

The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium

et al. 2017

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Fruits and seeds are the major food source on earth. Both derive from the gynoecium and, therefore, it is crucial to understand the mechanisms that guide the development of this organ of angiosperm species. In Arabidopsis, the gynoecium is composed of two congenitally fused carpels, where two domains: medial and lateral, can be distinguished. The medial domain includes the carpel margin meristem (CMM) that is key for the production of the internal tissues involved in fertilization, such as septum, ovules, and transmitting tract. Interestingly, the medial domain shows a high cytokinin signaling output, in contrast to the lateral domain, where it is hardly detected. While it is known that cytokinin provides meristematic properties, understanding on the mechanisms that underlie the cytokinin signaling pattern in the young gynoecium is lacking. Moreover, in other tissues, the cytokinin pathway is often connected to the auxin pathway, but we also lack knowledge about these connections in the young gynoecium. Our results reveal that cytokinin signaling, that can provide meristematic properties required for CMM activity and growth, is enabled by the transcription factor SPATULA (SPT) in the medial domain. Meanwhile, cytokinin signaling is confined to the medial domain by the cytokinin response repressor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFERASE 6 (AHP6), and perhaps by ARR16 (a type-A ARR) as well, both present in the lateral domains (presumptive valves) of the developing gynoecia. Moreover, SPT and cytokinin, probably together, promote the expression of the auxin biosynthetic gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and the gene encoding the auxin efflux transporter PIN-FORMED 3 (PIN3), likely creating auxin drainage important for gynoecium growth. This study provides novel insights in the spatiotemporal determination of the cytokinin signaling pattern and its connection to the auxin pathway in the young gynoecium.

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Section: Methodsmentioning

confidence: 99%

The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium

et al. 2017

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“…In the case of propidium iodide (PI) staining, 1 μL of stock solution (Sigma-Aldrich; 5 mg mL -1 dissolved in water) was added to 1 mL of water, sections were stained in this solution for 30 s, washed with water, and mounted on a glass slide. Samples were observed with a confocal laser scanning-inverted microscope LSM 510 META (Carl Zeiss, Germany), the fluorophore was excited with a 514 nm laser line, and emission was filtered with a LP 575 nm filter ( Reyes-Olalde et al, 2015 ). The following objectives were used: EC Plan-Neofluar 20×/0.5 and EC Plan-Neofluar 40×/0.75 ( Reyes-Olalde et al, 2015 ).…”

Section: Methodsmentioning

confidence: 99%

“…Samples were observed with a confocal laser scanning-inverted microscope LSM 510 META (Carl Zeiss, Germany), the fluorophore was excited with a 514 nm laser line, and emission was filtered with a LP 575 nm filter ( Reyes-Olalde et al, 2015 ). The following objectives were used: EC Plan-Neofluar 20×/0.5 and EC Plan-Neofluar 40×/0.75 ( Reyes-Olalde et al, 2015 ).…”

Section: Methodsmentioning

confidence: 99%

Exploring Cell Wall Composition and Modifications During the Development of the Gynoecium Medial Domain in Arabidopsis

Herrera-Ubaldo

Folter

2018

Front. Plant Sci.

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In Arabidopsis, the gynoecium, the inner whorl of the flower, is the female reproductive part. Many tissues important for fertilization such as the stigma, style, transmitting tract, placenta, ovules, and septum, comprising the medial domain, arise from the carpel margin meristem. During gynoecium development, septum fusion occurs and tissues form continuously to prepare for a successful pollination and fertilization. During gynoecium development, cell wall modifications take place and one of the most important is the formation of the transmitting tract, having a great impact on reproductive competence because it facilitates pollen tube growth and movement through the ovary. In this study, using a combination of classical staining methods, fluorescent dyes, and indirect immunolocalization, we analyzed cell wall composition and modifications accompanying medial domain formation during gynoecium development. We detected coordinated changes in polysaccharide distribution through time, cell wall modifications preceding the formation of the transmitting tract, mucosubstances increase during transmitting tract formation, and a decrease of mannan distribution. Furthermore, we also detected changes in lipid distribution during septum fusion. Proper cell wall composition and modifications are important for postgenital fusion of the carpel (septum fusion) and transmitting tract formation, because these tissues affect plant reproductive competence.

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“…Another type of microscope often used to characterize plant phenotypes with detail, is the confocal laser scanning microscope (CLSM), which allows the observation of fluorescent signals and produces high-resolution images ( Reddy et al, 2007 ; Reyes-Olalde et al, 2015 ). It also produces three-dimensional (3D) reconstructions, such as Z-stacks (tridimensional Z-stack images are obtained by merging several two-dimensional images obtained at different focal planes along the volume of a sample).…”

Section: Introductionmentioning

confidence: 99%

Non-destructive Plant Morphometric and Color Analyses Using an Optoelectronic 3D Color Microscope

et al. 2018

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Gene function discovery in plants, as other plant science quests, is aided by tools that image, document, and measure plant phenotypes. Tools that acquire images of plant organs and tissues at the microscopic level have evolved from qualitative documentation tools, to advanced tools where software-assisted analysis of images extracts quantitative information that allows statistical analyses. They are useful to perform morphometric studies that describe plant physical characteristics and quantify phenotypes, aiding gene function discovery. In parallel, non-destructive, versatile, robust, and user friendly technologies have also been developed for surface topography analysis and quality control in the industrial manufacture sector, such as optoelectronic three-dimensional (3D) color microscopes. These microscopes combine optical lenses, electronic image sensors, motorized stages, graphics engines, and user friendly software to allow the visualization and inspection of objects of diverse sizes and shapes from different angles. This allow the integration of different automatically obtained images along the Z axis of an object, into a single image with a large depth-of-field, or a 3D model in color. In this work, we explored the performance of an optoelectronic microscope to study plant morphological phenotypes and plant surfaces in different model species. Furthermore, as a “proof-of-concept,” we included the phenotypic characterization (morphometric analyses at the organ level, color, and cell size measurements) of Arabidopsis mutant leaves. We found that the microscope tested is a suitable, practical, and fast tool to routinely and precisely analyze different plant organs and tissues, producing both high-quality, sharp color images and morphometric and color data in real time. It is fully compatible with live plant tissues (no sample preparation is required) and does not require special conditions, high maintenance, nor complex training. Therefore, though barely reported in plant scientific studies, optoelectronic microscopes should emerge as convenient and useful tools for phenotypic characterization in plant sciences.

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Imaging early stages of the female reproductive structure of arabidopsis by confocal laser scanning microscopy

Cited by 9 publications

References 30 publications

The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium

The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium

Exploring Cell Wall Composition and Modifications During the Development of the Gynoecium Medial Domain in Arabidopsis

Non-destructive Plant Morphometric and Color Analyses Using an Optoelectronic 3D Color Microscope

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