Effect of Nitrogen-doped Graphene Quantum Dots on Growth Direction of Primary Root in Arabidopsis thaliana

doi:10.11983/CBB22048

Abstract

Abstract: Graphene quantum dots (GQDs) have substantial application potentials in various fields such as electrochemical biosensor, bioimaging and biomedicine. Therefore, with the increasing exposure to the public and environment, their biosafety has aroused increasing concerns in recent years. So far, the influence of GQDs on the growth and development of plants is still poorly understood. In this study, we investigated the influence of nitrogen-doped GQDs (N-GQDs) treatment on the growth direction of primary root in Arabidopsis thaliana at cellular and molecular levels. We found that the N-GQDs were absorbed by roots and transported via vascular bundles. After the N-GQDs treatment at the concentration of 50-100 mg∙L^-1, the growth direction of primary roots was changed, curving towards the outside of the culture medium. Because of the N-GQDs treatment, the starch granule accumulation of columnar cells was reduced, the abundance of auxin efflux carrier PIN3 was repressed, and the PIN3 in columnar cells was relocated to outer lateral membrane distant from the culture medium (towards the air), which resulted in the asymmetric auxin distribution in the root tips and the curved growth of primary roots towards a direction distant from the medium in order to run away from the high-concentration N-GQDs environment. The study results provide a reference direction for exploring the possible coping mechanism of plants with N-GQDs treatment, and also provide corresponding data for biosafety evaluation of N-GQDs.

Key words: auxin, nitrogen-doped graphene quantum dots, PIN3, root growth, starch granule

Ye Qing, Yan Xiaoyan, Chen Huize, Feng Jinlin, Han Rong. Effect of Nitrogen-doped Graphene Quantum Dots on Growth Direction of Primary Root in Arabidopsis thaliana[J]. Chinese Bulletin of Botany, 2022, 57(5): 623-634.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB22048

https://www.chinbullbotany.com/EN/Y2022/V57/I5/623

Figures/Tables 9

Figure 1 Measurement method for bending angle of Arabidopsis primary root tip after nitrogen-doped graphene quantum dots (N-GQDs) treatment (A) Primary roots of 5-day-old Arabidopsis thaliana seedlings vertically cultured on 1/4MS medium; (B) The seedlings in (A) were transferred to 1/4MS medium containing 50 mg·L-1 N-GQDs, they were cultured vertically for 3 d, the roots were bent towards the direction distant from the medium. The bending angle α of primary roots between root growth direction and gravity direction was determined. The yellow part at the left side in figure meant the seedlings were perpendicularly placed at one side of the medium, and the white part at the right side denoted that the seedlings were placed at the air side distant from the medium. Bars=500 μm

Figure 2 Measurement method of GUS activity at two sides of Arabidopsis thaliana primary root tip after nitrogen-doped graphene quantum dots (N-GQDs) treatment (A) After 5-day-old Arabidopsis thaliana DR5-GUS plant was perpendicularly cultured in 1/4MS medium containing 50 mg·L-1 N-GQDs for 3 d, the root was bent towards a direction distant from the medium, and the GUS activity value at the left and right sides (close to the culture medium/distant from the medium) of primary root tips were determined, the red arrow at the left side represents the side close to the medium and that at the right side denotes the side distant from the medium (bar=200 μm); (B) Measurement method of GUS activity at the left and right sides of primary root tip of DR5-GUS plant, the red box in the figure was the determined left region (bar=50 μm).

Figure 3 The characterization of nitrogen-doped graphene quantum dots (N-GQDs) (A) Transmission electron microscope graph of N-GQDs, the red arrows represent N-GQDs (bar=20 nm); (B) Particle size distribution diagram of N-GQDs; (C) Photoluminescence (PL) spectrogram of N-GQDs

Figure 4 X-ray photoelectron spectroscopy (XPS) spectrum of nitrogen-doped graphene quantum dots (N-GQDs) (A) Full-scan XPS spectrum of N-GQDs; (B) High resolution C1s spectrum; (C) N1s spectrum; (D) O1s spectrum

Figure 5 Distribution of nitrogen-doped graphene quantum dots (N-GQDs) in primary roots of Arabidopsis thaliana seedlings (A), (C) Fluorescence micro images of meristem zone and elongation zone (A), and root hair zone (C) of primary roots in Arabidopsis thaliana seedlings untreated with N-GQDs (bars=200 μm); (B), (D) Fluorescence micro images of meristem zone and elongation zone (B), and root hair zone (D) of primary roots under N-GQDs treatment for 3 days (bars=200 μm), the N-GQDs presented red fluorescence under a fluorescence microscope; (E), (F) Confocal images of vascular bundles in the root hair zone under N-GQDs treatment for 3 days (bars=30 μm)

Figure 6 Effects of nitrogen-doped graphene quantum dots (N-GQDs) on growth direction of primary root of Arabidopsis thaliana (A) Primary root phenotypes of Arabidopsis thaliana under different concentrations of N-GQDs for 3 days (bar=1 cm); (B) The growth direction of primary roots under optical microscope (bars=500 μm); (C) Statistical analysis of primary roots length; (D) Statistical analysis of primary roots bending angles. The values are presented as means±SD of triplicate samples (n=30). Different lowercase letters represent significant differences (P<0.05).

Figure 7 Nitrogen-doped graphene quantum dots (N-GQDs) reduce the accumulation of starch grains in Arabidopsis thaliana primary root tips (A) Changes of starch grains content in A. thaliana primary root tips by staining with different concentrations of N-GQDs treatment for different time (bars=50 μm); (B) Statistical analysis of starch grains amount in primary root tips. The values are presented as means±SD of triplicate samples (n=15). Different lowercase letters represent significant differences (P<0.05).

Figure 8 Nitrogen-doped graphene quantum dots (N-GQDs) disrupt the auxin distribution in Arabidopsis thaliana root tips (A) The GUS activity on both sides of the primary roots were observed by staining after N-GQDs treatment for different time, the red arrow at the left side represents the side close to the culture medium (bars=50 μm); (B) Statistical analysis of GUS activity ratio at the left and right sides of primary roots; the left side corresponds to one side in (A) close to the culture medium while the right side corresponds to the side in (A) distant from the culture medium. The values are presented as means±SD of triplicate samples (n=15). Different lowercase letters represent significant differences (P<0.05).

Figure 9 Nitrogen-doped graphene quantum dots (N-GQDs) disrupt the abundance and distribution of PIN3 in Arabidopsis thaliana primary roots (A) GFP fluorescence of PIN3-GFP in the primary roots after N-GQDs treatment for different time (bars=50 μm); (B) Quantitative analysis of fluorescence intensity of PIN3-GFP in the primary roots; (C) Distribution of PIN3 in columnar cells (bars=20 μm). The red arrows represent the side close to the culture medium. The white arrows in the columnar cells indicate the polarization directions of PIN3. The values are presented as means±SD of triplicate samples (n=15). Different lowercase letters represent significant differences (P<0.05).

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