Three-dimensional intracellular structure of a whole rice mesophyll cell observed with FIB-SEM

Oi, Takao; Enomoto, Sakiko; Nakao, Tomoyo; Arai, Shigeo; Yamane, Koji; Taniguchi, Masaru

doi:10.1093/aob/mcx036

Cited by 42 publications

(39 citation statements)

References 25 publications

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“…The sequential images were used for the reconstruction of 3D images (Figure ). In this study, the protocols to enhance signals for the backscatter electron imaging of resin‐embedded tissue (Ichimura et al, ) were not applied, because sufficient contrast was available for the observation of chloroplasts (Oi et al, ). The chloroplast structure of control was flat, and no protruded bodies were observed (Figure a,b).…”

Section: Resultsmentioning

confidence: 99%

“…Oi et al () reported a doughnut‐shaped chloroplast formed under a moderate growth condition, which is the structure related to surrounding other organelles. The doughnut shape would be formed by the curvature of the chloroplast itself along the cell lobe, and thus, the portion of the chloroplast body surrounding other organelles contains thylakoid membranes (Oi et al, ). On the other hand, a chloroplast pocket would be formed by a large sheet structure, which was a thin layer without thylakoid membranes in a cross section.…”

Section: Discussionmentioning

confidence: 99%

“…Although the 3D reconstruction method has been widely applied in biochemical research, only a few studies have investigated the ultrastructure of cells or organelles in higher plants (Lucas, Günthert, Gasser, Lucas, & Wepf, ). In our previous study, we applied the FIB‐SEM technique to rice leaves and reconstructed the 3D image of a rice mesophyll cell (Oi et al, ). Both traditional and new methods for 3D reconstruction are expected to be useful to reveal the entire structure of chloroplast pockets and identify the way in which they are formed.…”

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confidence: 99%

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Three‐dimensional ultrastructure of chloroplast pockets formed under salinity stress

Yamane

Enomoto

et al. 2018

Plant Cell & Environment

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We investigated the invagination structure of a chloroplast that surrounds organelles such as mitochondria and peroxisomes within a thin layer of chloroplast stroma, which is called a chloroplast pocket. In this study, chloroplast pockets were observed in rice plants subjected to salinity stress but not under moderate growth condition. They included cytosol, transparent structure, lipid bodies, mitochondria, and peroxisomes. We constructed the three-dimensional architecture of chloroplast pockets by using serial images obtained by transmission electron microscopy and focused ion beam-scanning electron microscopy. Three types of chloroplast pockets were observed by transmission electron microscopy: Organelles were completely enclosed in a chloroplast pocket (enclosed type), a chloroplast pocket with a small gap in the middle part (gap type), and a chloroplast pocket with one side open (open type). Of the 70 pockets observed by serial imaging, 35 were enclosed type, and 21 and 14 were gap and open types, respectively. Mitochondria and peroxisomes were often in contact with the chloroplast pockets. Focused ion beam-scanning electron microscopy revealed chloroplasts with a sheet structure partially surrounding peroxisomes. This fact suggests that chloroplasts might construct large sheet structures that would be related to the formation of chloroplast pockets.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Three‐dimensional ultrastructure of chloroplast pockets formed under salinity stress

Yamane

Enomoto

et al. 2018

Plant Cell & Environment

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“…Three‐dimensional leaf structures also have been explored using synchrotron radiation X‐ray laminography (Ho et al , ), microcomputed tomography imaging (Théroux‐Rancourt et al , ) and the incorporation of a focused ion beam (FIB) system within the SEM chamber which etches away atoms on the specimen surface (FIB‐SEM) (e.g. Jin et al , ; Oi et al , ; Reagan et al , ; Yamane et al , ). However, these 3D techniques have not been widely applied to date, which may limit our understanding of leaf anatomy (Théroux‐Rancourt et al , ).…”

Section: Introductionmentioning

confidence: 99%

Cell and chloroplast anatomical features are poorly estimated from 2D cross‐sections

et al. 2019

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Summary Leaf function is intimately related to the size, shape, abundance and position of cells and chloroplasts. Anatomy has long been assessed and quantified in two dimensions with 3D structure inferred from 2D micrographs. Serial block face scanning electron microscopy (SBF‐SEM) was used to reconstruct 95 cells and 1173 chloroplasts from three wheat and nine chickpea leaves (three samples each from three chickpea genotypes). Wheat chloroplast volume was underestimated by 61% in mesophyll cells and 45% in bundle sheath cells from 2D micrographs, whereas chickpea mesophyll chloroplast volume was underestimated by 60% using simple geometrical models. Models of chickpea spongy and palisade cells both under‐ and overestimated surface area and volume by varying degrees. These models did not adequately capture irregular shapes such as flattening of chloroplasts or lobed spongy mesophyll cells. It is concluded that simple geometrical models to estimate chloroplast and cell 3D volume and surface area from 2D micrographs are inadequate, and that SBF‐SEM has strong potential to contribute to improved understanding of leaf form and function.

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“…However, in mesophyll cells the vacuole is small or even absent. Most cells contain more chloroplasts [30] with smaller and denser intracellular spaces than those of hypocotyl cells, meaning they can withstand greater osmotic pressure. Therefore, it is speculated that the osmotic pressures tolerated by the two are also different due to differences in spatial composition between hypocotyl cells and mesophyll cells, resulting in a difference in the suitable mannitol concentration.…”

Section: Discussionmentioning

confidence: 99%

An Efficient and Economical Protocol for Isolating, Purifying and PEG-Mediated Transient Gene Expression of Chinese Kale Hypocotyl Protoplasts

Sun

Qiao

Zheng

et al. 2019

Plants

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In this study, we report the isolation and purification of protoplasts from Chinese kale (Brassica oleracea var. alboglabra) hypocotyls, and their transient gene expression transformation and subcellular localization of BaMYB75 (Bol042409). The upshot is that the vintage protocol included 5-d hypocotyls that were enzymatically hydrolyzed for 8 h in enzyme solution (3.0% cellulase, 0.5% pectolase, and 0.5 M mannitol), and the protoplasts were purified by precipitation. The total yield of protoplasts was 8 × 105 protoplast g−1 fresh weight, and the protoplasts’ viability was 90%. The maximum transformation efficiency obtained by using green fluorescent protein (GFP) as a detection gene was approximately 45% when the polyethylene glycol (PEG)4000 concentration was 40% and transformation time was 20 min. In addition, BaMYB75 was ultimately localized in the nucleus of Chinese kale hypocotyl protoplasts, verifying the validity and reliability of this transient transformation system. An effective and economical hypocotyl protoplast isolation, purification, and transformation system was established for Chinese kale in this study. This effectively avoided interference of chloroplast autofluorescence compared to using mesophyll cells, laying the foundation for future research in the molecular biology of Brassica vegetables.

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Three-dimensional intracellular structure of a whole rice mesophyll cell observed with FIB-SEM

Cited by 42 publications

References 25 publications

Three‐dimensional ultrastructure of chloroplast pockets formed under salinity stress

Three‐dimensional ultrastructure of chloroplast pockets formed under salinity stress

Cell and chloroplast anatomical features are poorly estimated from 2D cross‐sections

An Efficient and Economical Protocol for Isolating, Purifying and PEG-Mediated Transient Gene Expression of Chinese Kale Hypocotyl Protoplasts

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