Carbon Nanoparticle Exerts Positive Growth Effects with Increase in Productivity by Down-Regulating Phytochrome B and Enhancing Internal Temperature in Rice

doi:10.1016/j.rsci.2021.04.007

Abstract

Abstract:

The effects of carbon nanoparticle (CNP) on rice variety Swarna (MTU7029) were investigated. CNP induced effects similar to shade avoidance response (SAR) of Arabidopsis, with increase in shoot length, root length, root number, cotyledon area, chlorophyll content and total sugar content in rice seedlings. In mature plants, CNP treatment resulted increase in plant height, number of productive tillers per plant, normalized difference vegetation index, quantum yield and root growth. A total of 320 mg of CNP per plant administered in four doses resulted in improved grain traits such as filled grain rate, 100-grain weight, grain length/width ratio, hulling rate, milling rate and head rice recovery. Seeds from the CNP-treated plants showed increase in amylose, starch and soluble sugar contents compared to controls. Strikingly, CNP treatment showed an average of 17.5% increase in yield per plant. Upon investigation to the molecular mechanism behind CNP induction of SAR, a significant downregulation of phytochrome B transcript was found. Decrease in perception of red wavelengths led to responses similar to SAR. Increase in plant’s internal temperature by 0.5 ºC ± 0.1 ºC was recorded after CNP treatment. We suggest that the internalized CNP aggregates may serve to absorb extra photons thereby increasing the internal temperature of plants. Phytochrome B accounts the hike in internal temperature and initiates a feed-back reduction of its own transcription. We suggest that moderate SAR is beneficial for rice plants to improve agronomic traits and yield. It presents a potential non-transgenic method for improving rice yield by CNP treatment.

Key words: agronomic trait, carbon nanoparticle, flowering time, rice, grain quality, phytochrome B, yield, shade avoidance response, temperature sensing

Panigrahy Madhusmita, Das Subhashree, Poli Yugandhar, Kumar Sahoo Pratap, Kumari Khushbu, C. S. Panigrahi Kishore. Carbon Nanoparticle Exerts Positive Growth Effects with Increase in Productivity by Down-Regulating Phytochrome B and Enhancing Internal Temperature in Rice[J]. Rice Science, 2021, 28(3): 289-300.

Figures/Tables 13

Fig. 1. Effects of carbon nanoparticle (CNP) on seedling growth. Seedlings were grown either in darkness or irradiated with white light or different sources of monochromatic light [red (R), far-red (FR) and blue] or low R:FR on Murashige and Skoog (MS) medium with or without CNP (500 μg/mL) for 7 d. Low R:FR (R:FR = 0.06) was used to observe the effect of shade avoidance response. On the 8th day, seedling growth parameters such as shoot length (A), root length (B), rootlet number (C), total chlorophyll content (D), total carotenoid content (E), total sugar content (F) and cotyledon area (G) were measured. All data presented were from minimum 60 seedlings with 20 seedlings in each experiment. Total sugar content (F) was estimated from 50 mg shoot tissue without or with CNP treatment. Experiments were repeated for at least three independent experiments. Significances of values were obtained from one-way ANOVA using the Turkey’s multiple comparison in the Prism version 7.0 software, and were represented as *, P ≤ 0.05, **, P ≤ 0.01 and ***, P ≤ 0.001.

Fig. S1. Seedling phenotype after CNP treatment. Seedlings were grown for 7-day-old seedlings on MS medium with or without CNP (500 μg/mL) in darkness or under white light or under different monochromatic sources of light or low red:far-red. On the 8 d, seedlings were photographed. Scale Bars, 1 cm.

Fig. 2. Effects of carbon nanoparticle (CNP) treatment on rice plant phenotype and flowering.A, C, E, G, I and K represent the control plants, and B, D, F, H, J and L represent the CNP-treated plants. A and B, Plants on the 45 d after sowing (DAS) (1st CNP treatment).C and D, Plants on the 60 DAS (2nd CNP treatment).E and F, Plants on the 75 DAS (3rd CNP treatment).G and H, Plants on the 90 DAS (4th CNP treatment).I and J, Showing early flowering in CNP-treated plants on about 120 DAS (flowering indicated with red arrow, panicle showed in red circle, and panicle portion enlarged in M).K and L, Showing flowering in control plants on 127 DAS (flowering indicated with red arrows). Scale bars, 2.5 cm.

Fig. 3. Effects of carbon nanoparticle (CNP) treatment on plant growth. Plant growth phenotypes such as total tiller number (A), plant height (B), normalized difference vegetation index (NDVI) (C) and quantum yield (D) were recorded on the day of each CNP treatment. DAS, Days after sowing.Data were the mean of 60 plants (15?20 plants from each category in 3 seasons). Significant data points were marked with * at P ≤ 0.05. Test of significance was done using one-way ANOVA with the Turkey’s multiple comparison in the Prism version 7.0 software.

Table 1 Effects of carbon nanoparticle (CNP) treatment on yield and related attributes.

Trait	Control	CNP-treated
No. of productive tillers per plant	39.3 ± 11.4 a	49.5 ± 11.3 b
Days-to-flowering (d)	127.0 ± 2.0 a	122.5 ± 2.0 b
Panicle length (cm)	23.3 ± 1.1 a	24.3 ± 1.1 b
Flag leaf length (cm)	25.8 ± 2.1 a	29.6 ± 1.9 b
100-grain weight (g)	1.17 ± 0.02 a	1.25 ± 0.07 b
Filled grain rate (%)	8.21 ± 4.88 a	16.15 ± 2.33 b
Dry biomass matter (g)	139.50 ± 6.55 a	136.31 ± 6.73 b
Root dry weight (g)	36.97 ± 13.03 a	60.21 ± 0.27 b
Yield per plant (g)	42.11 ± 1.47 a	52.08 ± 3.93 b

Fig. S2. Effects of CNP treatment on root biomass. Roots of mature plants were recovered from pots after seed harvesting. The soil associated was gently removed by washing with plentiful of water followed by drying at 50 ºC for 3 d.. Roots of control plants without CNP treatment (A and B) or plants with CNP treatment (C and D). Scale Bars, 1 cm.

Fig. S3. Demonstration of carbon nanoparticle (CNP) and charcoal treatment. A and C, CNP-treated plants. B and D, Charcoal-treated plant.E?G, Control (E), CNP-treated (F) and charcoal treated plants (G) on 127 d after sowing. CNP- treated plants showing a greater number of panicles (red arrows) indicate early (3?5 d) flowering (F) than control (E) and charcoal- treated plants (G). Scale Bars, 1 cm. CNP or activated charcoal solutions prepared in water at 500 µg/mL were applied near the plant roots by pouring them on small holes made in the pot soil. A and B are photographs taken during the treatment, and C and D are plants just after treatment. Red arrows indicate the holes made in pot soil.

Fig. 4. Raman spectroscopic analysis of carbon nanoparticle (CNP) accumulation. Roots (1 g) of rice plants on 75 d after sowing were processed for Raman spectroscopy. CNP alone in solvent di-methyl formamide (DMF) showed a peak at 1 225 cm-1 (blue arrow). Roots from control plants showed no distinct peak, whereas roots from CNP-treated plants showed Raman spectra which had nearly 2-fold higher intensity with peak at the same wave number (red arrow) as that of the CNP alone. The observation of distinct peak in the root samples of CNP-treated plants was statistically significant at P ≤ 0.05.

Fig. 5. Visualization of carbon nanoparticle (CNP) aggregated inside plant tissue. A, D and G, Sections of leaf, root and sheath from the control plants after the 3rd CNP treatment, respectively.B, C, E, F, H and I, Sections of leaf (B and C), root (E and F) and sheath (H and I) from the CNP-treated plants after the 3rd CNP treatment, respectively.Red arrows indicate the CNP aggregates. Scale bars, 10 μm.

Fig. 6. Visualization of structure of rice plant parts treated with carbon nanoparticle (CNP) using a scanning electron microscopy. A?C, Extracts from rice leaves at 75 d after sowing with or without CNP treatment. Red arrows indicate projection like structures. Scale bars, 10 μm.D?F, Extracts from rice roots at 75 d after sowing with or without CNP treatment. Red circles indicate grooved impressions. Scale bars are 2 μm in D and E, and 10 μm in F.G?I, Extracts from rice seeds at 75 d after sowing with or without CNP treatment. Red arrows indicate more structured shapes. Scale bars, 2 μm.

Table 2 Effects of carbon nanoparticle (CNP) on grain quality.

Trait	Control	CNP-treated
Hulling rate (%)	80.60 ± 0.11 a	93.31 ± 0.12 b
Milling rate (%)	52.62 ± 0.11 a	63.95 ± 0.58 b
Head rice recovery (%)	44.05 ± 0.51 a	52.06 ± 0.43 b
Gelatinization temperature (ºC)	72.00 ± 2.00 a	72.00 ± 2.00 a
Gel consistency (mm)	75.33 ± 1.11 a	52.50 ± 0.83 b
Amylose content (%)	20.49 ± 2.19 a	29.69 ± 0.03 b
Total phosphate content (mg/g)	34.62 ± 0.77 a	45.07 ± 0.94 b
Total soluble sugar content (mg/g)	58.28 ± 0.34 a	65.32 ± 0.35 b
Starch content (mg/g)	75.66 ± 0.58 a	80.47 ± 0.58 b
Grain area (mm²)	0.10 ± 0.00 a	0.10 ± 0.01 a
Grain length/width	2.15 ± 0.06 a	2.28 ± 0.09 b

Fig. 7. Analysis of phytochrome B (PHYB) transcripts after carbon nanoparticle (CNP) treatment.A, RNAs isolated from the seedlings grown under white light and sampled of Zeitgeber at 8 h and 12 h after onset of light in the long-day cycle were named as Zt-8 and Zt-12, respectively. Relative transcript levels of PHYB were analyzed using semi-quantitative RT-PCR. Transcript levels of Actin at Zt-8 and Zt-12 were used for normalization and calculation of intensities of PHYB-Zt-8 and PHYB-Zt-12. MS-Zt-8 and MS-Zt-12 indicated control samples, whereas CNP-Zt-8 and CNP-Zt-12 indicated CNP-treated samples, respectively. Relative expression level was calculated using an Image lab software (Version 6.0.0, 2017, Bio-rad Laboratories Inc, USA). B, Real-time expression analysis of PHYB was done from flag leaf samples of CNP-treated or untreated plants. Data were representative images of experiments repeated three times. Statistical significances were confirmed using one-way ANOVA with the Turkey’s multiple comparison in the Prism version 7.0 software, and were represented as *, P ≤ 0.05 and **, P ≤ 0.01.

Fig. 8. Leaf internal temperature analysis using thermal imaging. Leaf temperature was studied from plants without carbon nanoparticle (CNP) treatment (A), with CNP treatment (B) or with charcoal treatment (C). The pictures were taken with the help of a FLUKE Infra-red Camera, and analyzed using the SMART VIEW software. Mostly leaf portions were selectively chosen to get the temperature data (D). Graph containing leaf temperature data from pictures of minimum of 15 plants from each CNP/charcoal treatment. Statistical significances were confirmed using one-way ANOVA with the Turkey’s multiple comparison in the Prism version 7.0 software, and were represented as * P ≤ 0.05.

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