Characterization of graphene. The composition of graphene was analyzed by Fourier transform infrared (FT-IR) spectroscopy (Fig. 1A). FT-IR spectrum showed that graphene included different oxygen-containing functional groups, including the C-O (1093 cm-1), C-OH (1394 cm-1), C=C (1630 cm-1), -OH (3437 cm-1). Raman spectroscopy (Fig. 1B) shows that G peak appears near 1576 cm-1, which is generated by the stretching and far-moving of sp2 hybridized atoms in carbon rings or long chains, representing the ordered sp2 bond structure. Peak D appeared near 1348 cm-1, which was related to sp3 hybrid structure, representing defects and amorphous structure at the edge of graphene. A wide 2D peak appeared near 2707 cm-1, indicating that the number of graphene layers prepared was within 10 layers. High-resolution scanning electron microscopy (Fig. 1C) showed that the graphene presented a transparent sheet structure with slightly wrinkled and undulate surface.
Effects of exogenous graphene on the total root length of 48 plant species. In our previous study, we found that the concentration of 25 mg/L graphene could promote the total root length of maize 27. To explore whether the promotion effects on plant root lengths are universal, 25 mg/L graphene were used to treat the seedling roots of 48 plant species, including two gymnosperms and 46 angiosperms species. Among 46 angiosperms species, it contains six monocotyledon species, and 40 dicotyledonous species of plants (Supplementary Table 1). Two planting methods were utilized, including soil culture for 43 plant species and hydroponics for 5 plant species (Supplementary Table 1). After measuring the total root lengths of 48 plants between control and 25 mg/L graphene treatment, it was found that graphene increased the total root lengths of 30 plant species (Fig. 2 and Supplementary Fig. 1), showed no significant effect on the total root lengths of eight plant species (Supplementary Fig. 2), and inhibited the total root lengths of 10 plant species, including all five hydroponic species (Fig. 3 and Supplementary Fig. 3). These results proved that the total root length of the plants was decreased when cultured by hydroponics method with the concentration of 25 mg/L graphene. Whereas the total root length of plant species cultured by soil showed different growth effects, among which the ratio of promotion effect was 69.77%, the ratio of inhibition effect was 11.63% and the no-effect ratio was 18.60%. In addition, the effects of graphene on the total root lengths of these plants were not significantly correlated with plant classification (Supplementary Table 1).
Transcriptome data generation of 32 plant roots under graphene treatment. To gain insights into the molecular mechanisms by which graphene presents different growth effects on the root length in 48 plants, we performed RNA-seq for 32 plant roots treated with 25 mg/L graphene, including 30 soil-cultured plants and two hydroponic plants. Among them, 24 plant roots showed promoting effects, four showed inhibiting effects, and four showed no-effects (Supplementary Table 2). Three biological replicates, which each were pooled samples from at least three plants, were set up for all 32 plant roots, and totally, 5.17 billion high-quality reads were generated using the Illumina NovaSeq 6000 sequencing platform (Supplementary Table 3). The GC contents ranged from 42.44% in Petunia hybrida to 55.71% in Zea mays (Supplementary Table 3). Among the 32 plant species, RNA-seq data of 12 plants were mapped to their reference genomes, the number of unique mapped clean reads ranged from 59.53% in Paeonia suffruticosa to 92.40% in Solanum lycopersicum (Supplementary Table 4). Whereas for the other 20 plant species, as a lack of the reference genomes, we performed the de novo assembly of transcriptome data using Trinity 28. The ratio of mapped reads was from 66.67% in Hippophae rhamnoides to 82.02% in Lilium pumilum (Supplementary Table 5).
Before calculating the differentially expressed genes (DEGs) in 32 plant root samples in response to graphene treatment, we first calculated the Pearson correlation coefficient (PCC) for all genes in 32 plant species (as shown in Supplementary Fig. 4). The correlation coefficients of the three biological replicates in 32 plant species were all greater than 0.90. In addition, the transcriptional response observed in 32 plant roots exposed to 0 and 25 mg/L graphene treatments exhibited two levels of gene expression based on principal component analysis (as shown in Supplementary Fig. 5). These data indicated that the RNA-seq data were reliable for the subsequent analyses.
DEGs can be classified into six groups in response to graphene treatment by KEGG enrichment analysis. To identify the transcripts that were differentially expressed in 32 plant root samples in response to graphene treatment, the expression value of each gene was calculated using fragments per kilobase of transcript per million fragments mapped (FPKM). A two-fold change and a p-value of less than 0.05 were set as the cutoffs to define genes with significant differential expression. Transcriptome data showed that graphene treatment induced a large number of plant gene expression differences, in total, we identified 90,259 DEGs in 32 plant species, among which 55,537 were graphene-induced and 34,722 were graphene-repressed (Supplementary Table 2). For each plant species, the number of DEGs ranged from 81 in Chrysanthemum morifolium to 12,845 in Castanopsis hystrix in response to graphene treatment (Supplementary Table 2).
To investigate the biological functions of these DEGs in 32 plant species affected by 25 mg/L graphene, we performed KEGG pathway enrichment analysis for those DEGs. These DEGs were subjected to KEGG pathway analysis to identify the functional categorization, leading to the assignment of 127 KEGG pathways. Top 30 KEGG pathways of enriched differentially expressed genes were selected for subsequent analysis for each of the 32 plant species. In total, 43 pathways were assigned to these enriched differentially expressed genes in response to the graphene treatment in 32 plant species (Fig. 4). These 43 pathways were categorized on the following functional pathways: 1) cellular processes, including endocytosis, phagosome and peroxisome; 2) primary metabolisms, including carbon metabolism, starch and sucrose metabolism, glycolysis/gluconeogenesis, citrate cycle (TCA cycle), pyruvate metabolism, glyoxylate and dicarboxylate metabolism, pentose and glucuronate interconversions, biosynthesis of amino acids, amino sugar and nucleotide sugar metabolism, cysteine and methionine metabolism, alanine, aspartate and glutamate metabolism, valine, leucine and isoleucine degradation, fatty acid metabolism, alpha-Linolenic acid metabolism, 2-oxocarboxylic acid metabolism, biosynthesis of unsaturated fatty acids, carbon fixation in photosynthetic organisms as well as photosynthesis; 3) secondary metabolisms, including phenylpropanoid biosynthesis, glutathione metabolism, terpenoid backbone biosynthesis, steroid biosynthesis, stilbenoid, diarylheptanoid and gingerol biosynthesis, sulfur metabolism, sesquiterpenoid and triterpenoid biosynthesis, ubiquinone and other terpenoid-quinone biosynthesis; 4) organismal system of plant-pathogen interaction; 5) environmental information processing, including plant hormone signal transduction and phosphatidylinositol signaling system; 6) genetic information processing, including ribosome, protein processing in endoplasmic reticulum, spliceosome, RNA transport, RNA degradation, mRNA surveillance pathway, ubiquitin mediated proteolysis, ribosome biogenesis in eukaryotes, and proteasome.
Detoxification metabolism genes were induced after graphene treatment. Fig. 4 showed that DEGs from glutathione metabolism, phenylpropanoid biosynthesis and peroxisome were enriched in 27, 30 and 24 out of 32 plant species roots exposed to graphene, respectively. We choose the glutathione S-transferase (GST), peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) genes as a group of detoxification enzymes indicators 29. They could scavenge the toxic organic hydroperoxides and protect plant organs from oxidative damage or toxic reactive metabolites 30.
The glutathione S-transferase genes were up-regulated in 21 plant species, which showing promotion effect after graphene treatment. However, they were down-regulated in six plant species with negative or no effects, inchuding Aster subulatus, Petunia hybrida, Gossypium hirsutum, Astragalus propinquus, Toona sinensis and Pinus tabuliformis (Fig. 5A and Supplementary Table 1). The peroxidase (POD) genes were up-regulated in 24 (promotion effect) and down-regulated in six (negative or no effect) plant species (Fig. 5B). The superoxide dismutase (SOD) genes were up-regulated in 11 (promotion effect) and down-regulated in one (no effect) plant species (Fig. 5C). The catalase (CAT) genes were up-regulated in seven (promotion effect) and down-regulated in eight (seven promotion effect and one no effect) plant species (Fig. 5D). These results suggested that these four detoxification enzyme genes were mostly induced in plant species with promotion effect, but depressed in plant species with negative or no effects. The expressions change of detoxification enzyme genes indicated that graphene treatment would cause oxidative stress of plant roots.
As stated above, expressions of reactive oxygen species (ROS)-scavenging enzymes genes, including SOD, POD and CAT, were changed after graphene treatment. Therefore, we examined the antioxidants activities of SOD, POD, CAT in five plant species roots: Gossypium hirsutum (Fig. 6A), Castanopsis hystrix (Fig. 6B), Lycium chinense (Fig. 6C), Populus nigra (Fig. 6D) and Vicia faba (Fig. 6E). The results showed that SOD, POD and CAT activities was markedly elevated in response to 25 mg/L graphene treatment of four plant species with promotion effect (Fig. 6B-E), but decreased in G. hirsutum with negative effect (Fig. 6A). The enhancing activities of antioxidant enzymes in plant roots with promotion effect improved the plant's ability to survive from the oxidative stress exposed to graphene. For the plant roots with negative effect, the root cell might be severely damaged and not available to elevate the SOD, POD and CAT activities.
Graphene enters plant root cells by endocytosis. DEGs from endocytosis pathway were also enriched in 27 out of 32 plant species roots exposed to graphene (Fig. 4). Endocytosis requires the Hsp70 protein family genes 31, 32 and Rab (Ras-related protein) genes, which is a key regulator of cellular endocytosis 33, 34. These two endocytosis-related genes were selected to investigate their expression variation after graphene treatment. The Hsp70 protein genes were differentially expressed in 23 plant species, including up-regulated in 17 plant species and down-regulated in six plant species (Fig. 7A). The Rab protein genes were differentially expressed in 14 plant species, including up-regulated in eight plant species and down-regulated in six plant species (Fig. 7B). Then, we used the TEM method to observe the bio-distribution of graphene in the root cell of Populus nigra after 7 days exposure. The graphene was distributed on the outer and inner side of the plasma membrane in graphene-treated cells (Fig. 7C). In addition, an endocytosis-like structure was observed when the graphene was translocated across the plasma membrane (Fig. 7C). These results indicated that the graphene enters plant root cell by the endocytosis pathway.
Starch and sucrose metabolism. DEGs from starch and sucrose metabolism pathway were enriched in 32 plant species roots exposed to graphene (Fig. 4). DEGs from this pathway mainly included endoglucanases, glucosidase, amylase and invertase. Endoglucanases and glucosidase were involved in the conversion of plant cell-wall cellulose or starch into simple sugars 35. The roles of amylase and invertase in plants acted as functional enzymes to break down starches or catalyze hydrolysis of sucrose, and produces glucose or maltose 36, 37, 38, 39, 40.
We identified that endoglucanase genes were differentially expressed in 17 plant species, including up-regulated in 12 plant species and down-regulated in five plant species (Fig. 8A). The glucosidase genes were differentially expressed in 24 plant species, including up-regulated in 18 plant species and down-regulated in six plant species (Fig. 8B). The amylase genes were differentially expressed in 24 plant species, including up-regulated in 15 plant species and down-regulated in nine plant species (Fig. 8C). The invertase genes were up-regulated in three plant species (Fig. 8D). Most of these enzyme genes were induced in plant species with promotion effect, but depressed in plant species with negative or no effects. The up regulation of starch and sucrose metabolism enzyme genes suggested that graphene might facilitate the accumulation of simple sugars in plant roots increased length.
Graphene enhanced the expression of respiratory metabolism genes. The respiratory pathways of glycolysis, pyruvate metabolism, and the citrate cycle (TCA cycle)41, were also enriched in 32 plant species roots exposed to graphene (Fig. 4). Glycolysis process contains ten enzymes for dividing the glucose into two pyruvate molecules. We identified nine of ten enzyme genes were differentially expressed in these plant species except for phosphohexose isomerase after graphene treatment (Fig. 9A and Supplementary Fig. 6A-E, Supplementary Fig. 7A-E). Among them, the first enzyme in the glycolysis process, hexokinase, was up-regulated in 10 plant species and down-regulated in three plant species (Fig. 9A and Supplementary Fig. 6A). Whereas, pyruvate kinase, an enzyme for the last step of glycolysis pathway, was up-regulated in 12 plant species and down-regulated in four plant species (Fig. 9A and Supplementary Fig. 7D). Other enzymes showing differentially expression ranged from 10 (triosephosphate isomerase) to 24 (glyceraldehyde-3-phosphate dehydrogenase) plant species with more up-regulation than down-regulation. For the pyruvate metabolism, we observed that pyruvate dehydrogenase, which catalyze the pyruvate into acetyl-CoA, were up-regulated in eight plant species and down-regulated in eight plant species (Fig. 9A and Supplementary Fig. 7E).
Eight enzymes were involved in the process of the citrate cycle (TCA cycle) and we identified seven enzymes were differentially expressed in these plant species exposed to the graphene treatment (Fig. 9B and Supplementary Fig. 8A-G). Differentially expressed enzymes ranged from 6 to 17 plant species, for instance, citrate synthase was up-regulated in 11 plant species and down-regulated in five plant species (Fig. 9B and Supplementary Fig. 8A). Fumarase expressed highly in five plants, but expressed lowly in one plant species (Fig. 9B and Supplementary Fig. 8F), as well as malate dehydrogenase increased expression in nine plant species, but decreased in the eight plants (Fig. 9B and Supplementary Fig. 8G). In addition, for the respiratory metabolism enzymes that were down-regulated in plant species, most of plant roots resulted in negative effects after graphene treatment. These results indicated that graphene could enhance the expression of respiratory metabolism genes of plant roots with positive effect, further to promote the plant root growth.