Background Cellular components are controlled by genetic and physiological factors that define their shape and size. However, quantitively capturing the morphological characteristics and movement of cellular organelles from micrograph images is challenging, because the analysis deals with complexities of images that frequently lead to inaccuracy in the estimation of the features. Here we show a unique quantitative method to overcome biases and inaccuracy of biological samples from confocal micrographs. Results We generated 2D images of cell walls and spindle-shaped cellular organelles, namely ER bodies, with a maximum contrast projection of 3D confocal fluorescent microscope images. The projected images were further processed and segmented by adaptive thresholding of the fluorescent levels in the cell walls. Micrographs are composed of pixels, which have information on position and intensity. From the pixel information we calculated three types of features (spatial, intensity and Haralick) in ER bodies corresponding to segmented cells. The spatial features include basic information on shape, e.g., surface area and perimeter. The intensity features include information on mean, standard deviation and quantile of fluorescence intensities within an ER body. Haralick features describe the texture features, which can be calculated mathematically from the interrelationship between the pixel information. Together these parameters were subjected to multivariate analysis to estimate the morphological diversity. Additionally, we calculated the displacement of the ER bodies using the positional information in time-lapse images. We captured similar morphological diversity and movement within ER body phenotypes in several microscopy experiments performed in different settings and scanned under different objectives. We then described differences in morphology and movement of ER bodies between A. thaliana wild type and mutants deficient in ER body-related genes. Conclusions The findings unexpectedly revealed multiple genetic factors that are involved in the shape and size of ER bodies in A. thaliana. This is the first report showing morphological characteristics in addition to the movement of cellular components and it quantitatively summarises plant phenotypic differences even in plants that show similar cellular components. The estimation of morphological diversity was independent of the cell staining method and the objective lens used in the microscopy. Hence, our study enables a robust estimation of plant phenotypes by recognizing small differences in complex cell organelle shapes and their movement, which is beneficial in a comprehensive analysis of the molecular mechanism for cell organelle formation that is independent of technical variations.
Iron is an essential nutrient for plant photosynthesis and development, but excess iron leads to stress. After absorption from the soil, plants store iron in roots and distribute it to shoots via long-distance transport. Vacuole serves as the iron storage organ in root cells, maintaining cellular iron homeostasis, and vacuolar iron transporter (VIT) family proteins have been identified as plant vacuolar iron transporters. However, the contribution of vacuolar iron transporters to the overall iron homeostasis of plants is not fully understood. Here, we show that MEMBRANE PROTEIN OF ER BODY 3 (MEB3), a VIT family member, is a vacuolar iron transporter involved in root-shoot iron distribution in Arabidopsis thaliana. Heterologous expression of Arabidopsis MEB3 in yeast restored the iron resistance phenotype of the vacuolar iron transporter deficient mutant ccc1, indicating that MEB3 regulates iron transport. In Arabidopsis, MEB3 was expressed in almost all tissues, albeit to higher levels in roots and seedlings, and the MEB3 protein localized to the tonoplast. At low iron concentration, meb3 knockout mutants accumulated less iron in shoots, suggesting that MEB3 promotes iron accumulation in shoots. Consistently, meb3 mutants exhibited reduced growth compared with the wild type upon transfer to iron-deficient medium. However, at high iron concentration, meb3 mutants accumulated more iron in shoots and less iron in roots than the wild type, indicating the impairment of proper iron distribution in meb3 mutants. These findings demonstrate that MEB3 is a vacuolar iron transporter involved in root-to-shoot iron distribution.
Background: Cellular components are controlled by genetic and physiological factors that define their shape and size. However, quantitively capturing the morphological characteristics and movement of cellular organelles from micrograph images is challenging, because the analysis deals with complexities of images that frequently lead to inaccuracy in the estimation of the features. Here we show a unique quantitative method to overcome biases and inaccuracy of biological samples from confocal micrographs. Results: We generated 2D images of cell walls and spindle-shaped cellular organelles, namely ER bodies, with a maximum contrast projection of 3D confocal fluorescent microscope images. The projected images were further processed and segmented by adaptive thresholding of the fluorescent levels in the cell walls. Micrographs are composed of pixels, which have information on position and intensity. From the pixel information we calculated three types of features (spatial, intensity and Haralick) in ER bodies corresponding to segmented cells. The spatial features include basic information on shape, e.g., surface area and perimeter. The intensity features include information on mean, standard deviation and quantile of fluorescence intensities within an ER body. Haralick features describe the texture features, which can be calculated mathematically from the interrelationship between the pixel information. Together these parameters were subjected to multivariate analysis to estimate the morphological diversity. Additionally, we calculated the displacement of the ER bodies using the positional information in a time-lapse image. We captured similar morphological diversity and movement within ER body phenotypes on several microscopy experiments performed in different settings and scanned under different objectives. We then described differences in morphology and movement of ER bodies between A. thaliana wild type and mutants deficient in ER body-related genes. Conclusions: The findings unexpectedly revealed multiple genetic factors that are involved in the shape and size of ER bodies in A. thaliana. This is the first report showing morphological characteristics in addition to the movement of cellular components and quantitatively summarises plant phenotypic differences even in plants that show similar cellular components. The estimation of morphological diversity was independent of the cell staining method and the objective lens used in the microscopy. Hence, our study enables a robust estimation of plant phenotypes by recognizing small differences of complex cell organelle shapes and their movement, which is beneficial in a comprehensive analysis of the molecular mechanism for cell organelle formation that is independent of technical variations.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.