sitl1 mutant decreases root growth and leaf chlorophyll content
Previously, we have developed 15,000 fertile M3 TILLING rice mutant lines (Oryza sativa , cv Donganbyeo) via the application of gamma-ray irradiation with 200 and 300 Gy (Hwang, Hwang, Kim, & Jang, 2014; L. Li et al., 2001). From this TILLING population, we selected 100 mutant lines based on evaluation on abiotic stresses, and subsequently developed M10 homozygous lines. This core collection was used to screen germplasm collections for seedling-stage salinity tolerance. Of these, one mutant line (300Gy-1011) showed significantly increase in both shoot and root growth compared to wild-type (WT) plants after salt treatment (Figure S2). This mutant line was named salt-insensitive TILLING line 1(sitl1 ) and was selected for further phenotypic analysis.
In order to evaluate phenotype differences between sitl1 and WT, plants were grown in hydroponic solution and root and shoot growth was monitored for 3 weeks. Although plants showed no significant difference in germination rate (Figure S3), the sitl1 showed significantly decreased root growth as measured by either length or fresh weight of root tissues in seedlings (Figure 1a). The sitl1 showed a 0.79-fold decrease in root length, and 0.84-fold decrease in root fresh weight, relative to WT (Figure 1b, c). However, no significant differences were observed in lengths and fresh weights of coleoptile, leaf sheath, or leaf blade between the sitl1 and WT plants. We also noticed that leaves of the sitl1 were slightly paler green compared to WT in seedlings (Figure 1d). In addition, relatively old leaves (2nd and 3rd leaf) of thesitl1 displayed an accelerated leaf senescence phenotype compared to WT at the same development stage (Figure 1d). Consistent with the observed leaf color, leaf chlorophyll content of the sitl1 were significantly less than that of WT (Figure 1e). The observed decreases in root growth and leaf chlorophyll content of the sitl1 did not appear when the sitl1 was grown in DW (Figure 1f-i).
Next, we tested whether the observed phenotypes in the sitl1 is potentially linked to Mg2+ deficient symptoms. Seedlings were grown in nutrient solution containing various levels of Mg2+ ranged from 0 to 500 µM. Although, thesitl1 exhibited root growth defects in the solution containing 0, 10, 100 µM of Mg2+, the root growth inhibition was rescued in the solution containing 500 µM of Mg2+(Figure S4a,b). Fresh weights of aerial shoot between the sitl1and WT were not significantly different under low Mg2+conditions, but the sitl1 had significantly higher shoot weight in the solution containing 500 µM of Mg2+ (Figure S4c). We also observed reduced total chlorophyll content in the leaves of sitl1 which was rescued when the solution Mg2+ levels reached to 500 µM (Figure S4d,e). These results suggest that the sitl1 might have less ability to uptake Mg2+ ions, which affects root growth, leaf senescence, and chlorophyll synthesis.