Abstract
The chromosome compaction of chromatin fibers results in the formation of the nucleosome, which consists of a DNA unit coiled around a core of histone molecules associated with linker histone. The compaction of chromatin fibers has been a topic of controversy since the discovery of chromosomes in the 19th century. Although chromatin fibers were first identified using electron microscopy, the chromatin fibers on the surface of chromosome structures in plants remain unclear due to shrinking and breaking caused by prior chromosome isolation or preparation with alcohol and acid fixation, and critical point drying occurred into dehydration and denatured chromosomal proteins. This study aimed to develop a high-quality procedure for the isolation and preparation of plant chromosomes, maintaining the native chromosome structure, to elucidate the organization of chromatin fibers on the surface of plant chromosomes by electron microscopy. A simple technique to isolate intact barley (Hordeum vulgare) chromosomes with a high yield was developed, allowing chromosomes to be observed with a high-resolution scanning ion microscopy and helium ion microscopy (HIM) imaging technology, based on a scanning helium ion beam. HIM images from the surface chromatin fibers were analyzed to determine the size and alignment of the chromatin fibers. The unit size of the chromatin fibers was 11.6 ± 3.5 nm and was closely aligned to the chromatin network model. Our findings indicate that compacting the surface structure of barley via a chromatin network and observation via HIM are powerful tools for investigating the structure of chromatin.
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Data Availability
All data analyzed during this study are included in this published article.
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Abbreviations
- APM:
-
Amiprophos methyl
- CPD:
-
Critical point drying
- EM:
-
Electron microscopy
- FIB/SEM:
-
Focused ion beam-SEM
- HIM:
-
Helium ion microscopy
- HU:
-
Hydroxyurea
- IL:
-
Ionic liquid
- PMSF:
-
Phenylmethylsulfonyl fluoride
- PI:
-
Propidium iodide
- SEM:
-
Scanning electron microscopy
- SHIM:
-
Scanning helium ion microscopy
- SEs:
-
Secondary electrons (SEs)
- TEM:
-
Transmission microscopy (TEM)
- DTT:
-
1,4-Dithiothreitol (DTT)
- DAPI:
-
4’,6-Diamidino-2-phenylindole (DAPI)
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Acknowledgements
We would like to thank Dr. Kimihiro Norizawa, the Nanotechnology Open Facilities Center in Osaka University for all his guidance regarding the HIM technique. We also gratefully acknowledge scholarship support provided by the Japanese Ministry of Education, Culture, Sports, Science, and Technology (to CS). We would like to thank Editage (www.editage.com) for English language editing.
Funding
This work was supported by “e-ASIA Joint Research Program,” “Development of nanovisualization for structural analyses of genetic materials and early infection process for further innovation of functional bio-nanotechnology” from SICORP and the Mobility Plus Project (grant no. JSPS-18-07) (to NO). This work was also supported by “The 51th Research Grants in the Natural Sciences” from The Mitsubishi Foundation (to NO) and Nanotechnology Platform of MEXT, Grant Number JPMXP09F20OS0004 (to NO).
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NO and KF conceived and designed research. CS and RP conducted experiments. TW contributed the development of image analysis tools. CS analyzed data. CS and NO wrote the manuscript. All authors read and approved the manuscript.
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Sartsanga, C., Phengchat, R., Fukui, K. et al. Surface structures consisting of chromatin fibers in isolated barley (Hordeum vulgare) chromosomes revealed by helium ion microscopy. Chromosome Res 29, 81–94 (2021). https://doi.org/10.1007/s10577-021-09649-2
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DOI: https://doi.org/10.1007/s10577-021-09649-2