Comprehensive and quantitative analysis of intracellular structure polarization at the apical-basal axis in elongating Arabidopsis zygotes.

A comprehensive and quantitative evaluation of multiple intracellular structures or proteins is a promising approach to provide a deeper understanding of and new insights into cellular polarity. In this study, we developed an image analysis pipeline to obtain intensity profiles of fluorescent probes along the apical-basal axis in elongating Arabidopsis thaliana zygotes based on two-photon live-cell imaging data. This technique showed the intracellular distribution of actin filaments, mitochondria, microtubules, and vacuolar membranes along the apical-basal axis in elongating zygotes from the onset of cell elongation to just before asymmetric cell division. Hierarchical cluster analysis of the quantitative data on intracellular distribution revealed that the zygote may be compartmentalized into two parts, with a boundary located 43.6% from the cell tip, immediately after fertilization. To explore the biological significance of this compartmentalization, we examined the positions of the asymmetric cell divisions from the dataset used in this distribution analysis. We found that the cell division plane was reproducibly inserted 20.5% from the cell tip. This position corresponded well with the midpoint of the compartmentalized apical region, suggesting a potential relationship between the zygote compartmentalization, which begins with cell elongation, and the position of the asymmetric cell division.

Different effects of gellan gum and agar on change in root elongation in Arabidopsis thaliana by polyploidization: the key role of aluminum.

Agar and gellan gum have been considered to have different effects on polyploidy-dependent growth in plants. We aim to demonstrate that agar and gellan gum differently affect the change in root elongation in Arabidopsis thaliana by polyploidization and examined the physico-chemical parameters in each gelling agent to elucidate key factors that caused the differences. Each polyploid strain was cultured vertically on agar and gellan gum solidified medium under fixed conditions. Root elongation rate was measured during 4-10 days after sowing. As a result, agar promoted root elongation of polyploids more than the gellan gum. Then water potential, gel hardness, and trace elements of each medium were quantified in each medium. Water potential and gel hardness of agar medium were significantly higher than those of gellan gum medium. The decrease in water potential and gel hardness in agar medium, however, did not affect the change in polyploidy-dependent growth. Elemental analysis showed that gellan gum contained more aluminum than agar. Subsequently, the polyploids were grown on agar media with additional aluminum, on which the root elongation in tetraploids and octoploids was significantly suppressed. These results revealed that agar and gellan gum affect the change in growth of root elongation in A. thaliana by polyploidization in different ways and the different effects on change in polyploidy-dependent growth is partially caused by aluminum in the gellan gum, which may be due to cell wall composition of polyploids.

Novel whole-mount FISH analysis for intact root of Arabidopsis thaliana with spatial reference to 3D visualization.

Whole-mount fluorescent in situ hybridization (WM-FISH) is an effective tool to observe chromosome behavior in tissues or organs. However, it is difficult to obtain a precise spatial profile of fluorescent signals in roots using conventional WM-FISH mainly because of the severe damage caused during the processing. To address this problem, we established a novel WM-FISH analysis for intact roots of Arabidopsis thaliana and successfully obtained a precise spatial profile of nuclear size and centromere signals. The two main improvements in the novel WM-FISH analysis are: (i) hybridization was performed directly on MAS-coated glass slides covered with silicon wells and (ii) conditions for enzyme treatment were optimized (37 °C, 45 s). After the WM-FISH using a centromere probe, we analyzed the results by 3D data processing to quantify the nuclear volume and number of centromere signals of the obtained cortical cell files and determined the position of each nucleus in intact roots. Then we plotted the nuclear volume and number of centromere signals versus distance from the quiescent center to evaluate the precise spatial profile of each parameter.