Our bright field imaging and FTIR spectroscopy analysis clearly indicated the absorption and translocation of fullerols in the plant organs (roots, stems, petioles, leaves, flowers, and fruits), and their generational transmission, consistent with an earlier study on the uptake of fullerene C70 (suspended in natural organic matter) in rice . Most of the stem and fruit samples (excluding C0 and C1) exhibited distinct FTIR features common to fullerols across the 1500–1700 cm-1 spectral region (see Figure 3b), suggesting the presence of fullerols in the samples. Importantly, fullerol-like IR features were absent in sample C0, obviously reflecting the absence of the nanomaterial. As seen in Figure 3c, only the fruits from C3 and C5 samples exhibited intense FTIR signal for fullerols. This result is expected since the C5 seeds were treated at the highest fullerol concentration. The major mechanism for the uptake of fullerol in our study is believed to be transpiration resulting from the water evaporation from the shoot organs, concentration gradient of the nanoparticles within the plant continuum, as well as hydrophobic interaction between the nanoparticles and the waxy layers between the plant cells (see Figure 1, panels for C3- and C5-petiole and C2-leaf).
The results revealed that seed treatment with fullerol at different concentrations led to varying effects on biomass, fruit characters and phytomedicine content. The extent of these effects also varied significantly. Among the five different fullerol concentrations, C2 promoted the highest fruit yield and its component characters, whereas C3 produced the highest biomass yield. C2, C4, and C5 led to increased contents of charantin, cucurbitacin-B, insulin, and lycopene, respectively. In all cases, the remaining concentrations either superseded or were on par with the control. Moreover, the same individual concentrations produced effects of different directions and degrees on different variables. Therefore, selection of proper concentration of nanoparticle is important for realizing higher benefits for a target agroeconomic trait. Two exhaustive lists of positive or non-consequential effects and negative effects of nanoparticles on different food crops presented in a recent review  substantiate our findings. It exemplified that the nanoparticles which were of same sizes and treated by similar methods could produce three types of effects on the same seedling trait in the same crop species. Besides, the effects were different in different seedling parameters such as germination, root length, shoot length and their ratios. While fullerols show no effect on mammalian cell viability [22, 23], at 70 mg/l, they induced 5% cell damage in onion after 9 h of incubation as a result of their accumulation between the rigid cell walls and the fluidic plasma membranes. In contrast, the more hydrophobic fullerene C70 nanoparticles were largely retained by the cell-walls and elicited no toxicity . Other previous works deliberated in two recent reviews [1, 2] also report similar variability in effects of nanopartciles on plant growth and development. It is evident then that independent genetic regulation exists for the biosynetheic and physiological pathways for production of biomass, fruits, and phytomedicines in fruits.
Exploratory research on the positive impacts of nanoparticles on plant growth and development and the underlying physiological and genetic factors have been conducted mostly at seedling stages [1, 2]. To the best of our knowledge, improvement of any agronomic yield was reported only in one instance in soybean , wherein increased leaf and pod dry weight resulting in a 48% increase in grain yield by nano-iron oxide treatment was reported. However, this report does not decipher the causal factors for such increases. We also observed strikingly high enhancement in biomass yield, fruit yield, and phytomedicine content by fullerol treatment at different concentrations. However, with the available data, it is not possible to precisely decipher the causal physiological and genetic factors underlying such genetic improvements. However, a previous study in tomato  indicated that seeds exposed to MWCNTs had higher level of moisture as compared to the untreated seeds. The authors hypothesized that their observed enhanced germination parameters, including germination rate, length of stem and fresh vegetative biomass, were based on the role of the carbon nanotubes in the process of water uptake inside the seed embryo. Therefore, we verified the plausible association of plant water content with the effects on biomass yield, fruit yield and its component characters, and phytomedicine content. However, we observed no significant correlation of plant water content with the agro-economic traits including biomass yield, fruit yield and phytomedicine contents in fruits. On the other hand, we observed that plant water content had a non-significant, but highly positive, association with biomass yield.
Reviews on previous research provide evidence for enhancement of various physiological factors related to photosynthesis and nitrogen metabolism [1, 2, 34]. Earlier, nitrate reductase activity was reported to increase the absorption and utilization of water/fertilizer and enhanced antioxidant system using a mixture of nano-SiO2 and TiO2 in soybean . These might be the physiological mechanisms underlying the increased germination and shoot growth in their experiment. Exposure to nano-TiO2 in spinach resulted in increased chlorophyll formation, ribulosebiphosphate carboxylase/oxygenase activity and acceleration of the rate of evolution of oxygen in the chloroplasts that could have promoted photosynthesis leading to increased germination, germination and vigor indices, and ultimately plant dry weight [4, 5]. From the follow-up studies, the authors reported enhanced activity of rubisco activase, rubisco carboxylation, rate of photosynthetic carbon reaction and chlorophyll content that could have resulted in increased plant dry weight [6, 7]. From a later study in spinach, nano-TiO2 treatment was found to improve light absorbance, transformation from light energy to electron energy and chemical energy, and promoted carbon dioxide assimilation . Magnetic nanopartciles coated with tetramethylammonium hydroxide also led to an increase in chlorophyll-a level in maize . Recently, use of iron-oxide was claimed as facilitators for iron and photosynthate transfer to the leaves of peanut . Use of iron-oxide in pumpkin was also observed to increase root elongation that was attributed to the Fe-dissolution .
There are few, but highly suggestive, reports on genetic implication for changes in plant growth and development due to nanoparticle-treatment. Germinating maize seeds in presence of magnetic fluid followed by exposure to electromagnetic field was observed to cause a pronounced increase in nucleic acid level due to the regeneration reactions of plant metabolism processes . Nano-TiO2 treatment led to a highly enhanced mRNA expressions and protein level in spinach . Expression of several water channel genes including important prolactin-induced protein (PIP) genes was characterized during rice seed germination . Recently, it has been deciphered that MWCNTs induce novel changes in gene expression in tomato leaves and roots, particularly up-regulation of the stress-related genes including those induced by pathogens and the water channel LeAqp2 gene employing microarray analysis of transcripts . In a later extensive study in tobacco, these authors have detected a correlation between activation of growth of cells exposed to MWCNTs and up-regulation of genes underlying cell division and cell wall formation, and water transport . They also observed expression of tobacco aquaporin gene (NtPIP1) along with production of the NtPIP1 protein, significantly increased in cells exposed to MWCNTs compared to the control cells. They also detected up-regulation of expression of marker genes for cell division (CycB) and cell wall extension (NtLRX1) in the exposed cells.