Gellan gum and agar differently affect the change in root elongation by polyploidization
The measurement of root elongation rate in polyploid series grown on gellan gum and agar media (Fig. 2) confirmed our preliminary observation that the change in growth due to polyploidization differs between the two gelling agents, as the components of each medium were identical except for the gelling agent. This is the first study to demonstrate that the type of gelling agent influences the polyploidization effect on plant growth.
Physical properties of gelling agents are not key factors affecting the difference in change in growth due to polyploidization
Nakagawa et al. (2007) revealed that the primary root of the wild type of A. thaliana could penetrate the harder layer of double-layer agar medium, whereas mca1-null mutant could not. As MCA1 is considered to be a Ca2+-permeable stretch-activated (SA) channel (Yoshimura et al, 2021), the result of the experiment using double-layer agar medium suggests that the Ca2+-permeable SA channel could detect the gel hardness and affect the root elongation of A. thaliana. Therefore, we expected that each polyploid might detect the gel hardness of the gellan gum- and agar-solidified media differently, leading to differences in change in growth due to polyploidization between the two gelling agents (Fig. 1, 2).
However, the results suggest that the gel hardness was not a critical factor in determining the difference of change in growth by polyploidization (Fig. 2, 4). The root elongation rate in diploids, tetraploids, and hexaploids was promoted when they were grown on the softer agar-solidified medium (1.0% agar) with a gel hardness similar to that of the gellan gum-solidified medium; however, the relationship among the temporal profiles of root elongation rate in polyploids was mostly unchanged compared with those grown on the normal agar-solidified medium (1.5% agar) (Fig. 2b, 4). Schultz et al. (2016) analyzed the root growth grown on three kinds of gelling agents and revealed that differences in media type had more of an impact on root growth than hardness itself, which is consistent with the results of this study.
The control of water potential in media is related to the control of water availability, and the water availability is directly related to the plant growth. In the roots of A. thaliana, the lower the water potential of medium, the lower was the elongation rate (van der Weele et al. 2000). The results suggest that the water potential affects the root elongation rate and each polyploid may detect the water availability differently, leading to differences in change in growth due to polyploidization between gellan gum and agar.
However, the results of this study suggest that the water potential is not a critical factor in determining the difference in change in growth due to polyploidization (Fig. 2, 5). The temporal profiles of root elongation rate in polyploids in the normal 1.5% agar medium (0 mM mannitol) and in the 1.5% agar medium with the lower water potential (20 mM) are similar, which nearly corresponds to that of the 0.8% gellan gum medium. In a previous study, a difference of water potential between each pair of growth conditions was at least more than 0.1 Mpa (van der Weele et al. 2000); however, the difference of water potential between the normal 1.5% agar medium and the 1.5% agar medium with the lower water potential was 0.06 MPa in this study (Table 2), which may be too low to affect the change in growth due to polyploidization.
Aluminum in the gellan gum could partially explain the effects of agar and gellan gum on the root growth of polyploids
Quantitative analysis of trace elements in gellan gum and agar showed that the contents of some element differed significantly (Table 3). Agar contained significantly higher Na, Fe, and Cd than gellan gum. In contrast, gellan gum contains significantly higher Mg, Al, and Ca. The root elongation of A. thaliana was usually suppressed by more than 10 mM NaCl in previous studies (West et al. 2004; Jiang et al. 2016; Zhao et al. 2017; Fu et al. 2019), and the contents of Na in gellan gum and agar were significantly lower than 10 mM in this study (ca 7.8 mM in gellan gum and 1.8 mM, Table 3), which could not affect the root elongation because they are too low. The deficiency of Fe, Mg, and Ca in media severely suppressed the root elongation of A. thaliana (Gruber et al. 2013), but the 1/2 MS medium, which is the base medium used in this study, contains a large amount of Fe, Mg, and Ca (1500 µM Ca, 750 µM Mg and 45 µM Fe, Murashige & Skoog 1962). Therefore, the contents of Fe, Mg, and Ca in gellan gum and agar could not affect the root elongation (Table 3). Cd is well known to suppress root elongation in plants (Godbold and Hüttermann. 1985; Munzuroglu and Geckil 2004). However, less than 5 µM Cd did not severely suppressed the root elongation of A. thaliana (Wójcik and Tukiendorf 2004; Van Belleghem et al. 2007), which suggests that the contents of Cd in gellan gum and agar were too low to affect the root elongation (0.320 µM in agar and 0.028 µM in gellan gum, Table 3).
Table 3
Elemental analysis of gelling agents
Element
|
Agar
|
Gellan gum
|
p-value
|
|
B
|
154
|
±
|
66
|
9.88
|
±
|
9.80
|
0.094
|
|
Na
|
7802
|
±
|
184
|
1830
|
±
|
244
|
< 0.0001
|
*
|
Mg
|
139
|
±
|
2
|
260
|
±
|
33
|
0.021
|
*
|
Al
|
2.523
|
±
|
1.690
|
33.4
|
±
|
4.2
|
0.0009
|
*
|
Ca
|
166
|
±
|
3
|
439
|
±
|
55
|
0.0076
|
*
|
Ti
|
1.97
|
±
|
0.76
|
0.0552
|
±
|
0.0195
|
0.065
|
|
Cr
|
n. d.
|
n. d.
|
|
|
Mn
|
0.689
|
±
|
0.0357
|
0.776
|
±
|
0.099
|
0.44
|
|
Fe
|
18.07
|
±
|
1.74
|
7.40
|
±
|
0.92
|
0.0016
|
*
|
Cu
|
n. d.
|
n. d.
|
|
|
Cd
|
0.319
|
±
|
0.017
|
0.0281
|
±
|
0.00151
|
< 0.0001
|
*
|
Data shows mean (µM) ± SE (n = 5). *Indicates significant difference between agar and gellan gum at p < 0.05 (Welch's t test). n.d. indicates not detected |
On the other hand, Al significantly suppressed the root elongation of A. thaliana at 20 µM (Sun et al. 2010), 50 µM (Zhu et al. 2012), 6 µM (Yang et al. 2014). The content of Al in gellan gum was 33.401 µM (Table 3), which is within the range of content that could suppress the root elongation of A. thaliana. Therefore, we focused on the effect of Al in gellan gum and conducted Al addition experiment in agar media for polyploids.
The results of Al addition experiment showed that the root elongation rate of tetraploids and octoploids grown on the 1.5% agar medium with 30 µM Al was significantly suppressed, while that of diploids remained unchanged (Fig. 7a, b, d). Therefore, the root elongations of diploids and tetraploids were almost the same at 5–6, 6–7, and 7–8 DAS and the differences between the root elogations of diploids and tetraploids were relatively small at other measurement dates when grown on the 1.5% agar medium with 30 µM Al, whereas the root elongation rate of tetraploids was significantly higher than that of diploids at all measurement dates when grown on the 1.5% agar medium with no additional Al (0 µM Al) (Fig. 6a, b). In addition, the root elongation of octoploid grown on the 1.5% agar medium with 30 µM Al significantly decreased. These results suggest that the higher Al concentration in gellan gum could partially explain the differences in change in growth due to polyploidization between 0.8% gellan gum and 1.5% agar medium. As the root elongation rate of diploids was significantly higher than that of tetraploids grown on the 0.8% gellan gum medium, the addition of Al to the agar medium did not completely reproduce the relationship of temporal profiles in root elongation between diploids and tetraploids grown on the 0.8% gellan gum medium. The relationship between diploids and tetraploids grown on the agar medium with 30 µM Al, however, is clearly closer to that of those grown on the 0.8% gellan gum medium than that of those grown on the agar medium with 0 µM Al, which indicates that the decrease in root elongation of tetraploid in the 0.8% gellan gum medium should be partially attributed to the Al in gellan gum (Fig. 2, 6).
Al binds to cell wall components, especially hemicellulose, thereby reducing cell wall extensibility and inhibiting root elongation (Yang et al. 2011). Polyploids of A. thaliana have been shown to have an increased amount of hemicellulose compared with diploid plants (Corneillie et al., 2019), which may have contributed to the tetraploids and octoploids being more sensitive to Al than diploid in root elongation rate.
The root elongation rate of hexaploids grown on the 1.5% agar medium with 30 µM Al was similar to that of those grown on the 1.5% agar medium with 0 µM Al. However, further studies are needed to understand as to why the addition of Al did not have an effect on the root elongation of hexaploids, while it suppressed those of tetraploids and octoploids.