Calcium ion significance on the maintenance of barley (Hordeum vulgare) chromosome compaction
Introduction
The integrity of chromosome structure is very important to facilitate the proper segregation of the sister chromatid during cell division. Improper segregation of the chromatid resulted in numerous abnormalities, such as producing aneuploid or polyploid cells, fragments of whole chromosomes, change in the relative dosage of products from genes, perturbations in gene expression, and even detrimental to the health and survival of the organism (Potapova and Gorbsky, 2017). Thus, it is important to evaluate factors responsible for the maintenance of chromosome structure. Among the major factors responsible for chromosome condensation, cations have been studied intensively. Cations contained within the cell and play various roles such as in nucleic acid structure and functional regulation, cell cycle, apoptotic (Ohyama, 2019), and chromosome compaction (Engelhardt, 2004). The significant role of divalent cations in the plant was reported by Gerbeau et al. (2002). They showed the importance of cations on cell signaling and metabolism in Arabidopsis by regulating water transport through the membrane plasma. Furthermore, the chelation of cations by ethylenediaminetetraacetic acid resulted in the mitotic and meiotic abnormalities in Allium cepa and Tradescantia paludosa as shown by Davidson (1958). Other roles of Ca2+ on the plant are reviewed by White and Broadley (2003).
Monovalent cations such as Na+ and K+ have been confirmed to be important in the primary level of chromatin compaction especially for histone binding, while the condensation of chromatin into the higher-order structure of chromosome requires divalent cations such as Ca2+ and Mg2+ (Strick et al., 2001). Compared to Mg2+ and the other divalent cations, Ca2+ is the most abundant one inside the cells. The concentration of Ca2+ in the entire cells, nuclei, and metaphase chromosomes during mitosis were reported to be 4−8 mM, 12−24 mM, and 20−32 mM respectively (Strick et al., 2001), suggesting that this ion plays a critical role in chromosome condensation. Phengchat et al. (2016) and Dwiranti et al. (2016) had reported that the HeLa chromosome structure was decondensed by the lack of Ca2+ depleted by a calcium chelator, BAPTA (1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) or BAPTA-AM (acetoxymethyl ester). Nevertheless, to date, reports about the role of cations on chromosome structure have been limited to the human chromosome only. Plant genome and chromosome have somehow different characteristics compared to human and animal chromosome structures such as genome size, polyploidization, chromosome stabilization, and plasticity as reported by Murat et al. (2012). The effects of Ca2+ on plant chromosome has not been revealed so far. It is thus necessary to evaluate the effects of Ca2+ on plant chromosomes.
Barley (Hordeum vulgare L.) is one of the plant species which is often used as a model organism in cytogenetics and the karyotype is quantitatively reported (Fukui and Kakeda, 1990). The haploid genome size and the chromosome number of Barley (Hordeum vulgare) are ∼5.3 Gb and 2n = 14 chromosomes, respectively. The ultrastructure of the barley chromosome observed by Scanning Electron Microscope (SEM) and Focused Ion Beam/Scanning Electron Microscope (FIB/SEM) has been reported (Iwano et al., 1997; Hamano et al., 2014). However, the visualization of barley chromosome ultrastructure affected by a calcium chelator like BAPTA has not been revealed yet. Thus, in this study, we aimed to investigate the effects of Ca2+ on barley chromosome structure by using a scanning electron microscope (SEM). This study presents the first report of Ca2+ roles on the chromosome structure of plant species. The results give deeper insights into the comprehensive understanding of Ca2+ on DNA compaction to universal organisms.
Section snippets
Barley chromosomes preparation
Chromosomes were prepared from the roots following Lysák et al. (1999) and Hayashihara et al. (2008) with minor modification. The chromosomes used in this study were obtained from barley roots. At first, barley seeds (Hordeum vulgare 2n = 14) were germinated on moist filter papers at 25 °C in dark for 48 h until the root tips reached 0.5–1 cm. After that, the samples were fixed in paraformaldehyde for 1 h, washed for 30 min, and subjected to enzymatic treatment consisting of 2.5 % Cellulase
Mitotic index and percentage of metaphase cells
In order to facilitate a proper chromosome structure analysis under the different Ca2+ concentrations, chromosomes in metaphase are required. Thus, at first, we calculated the mitotic index and the percentage of metaphase chromosomes (Fig. 1).
According to the data obtained, it is shown that the mitotic index of the cells was relatively high 87 % (Fig. 1.A), with 32 % of metaphase cells. In this study, the sample was not subjected to any pretreatment of cell synchronization to get more natural
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The SEM observations were carried out at the Research Center for Ultrahigh Voltage Electron Microscopy and The Institute of Scientific and Industrial Research, Osaka University. We are grateful to Dr. Kanako Inoue at the Ultra-High Voltage Electron Microscope Center for her help on the SEM observation. This study was supported by the International Research Collaboration Grant Universitas Indonesia, 2019 No. NKB-1945/UN2.R3.1/HKP.05.00/2019 to A.D., The Japan Science and Technology Agency (JST),
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