Determination of Fe3O4 and ZnO nanoparticles
The average hydrodynamic size and zeta potential for Fe3O4 NPs were 637.87 ± 42.35 nm and 2.71 ± 0.08 mV, respectively (Additional file 1: Fig. S1), while the average hydrodynamic size and zeta potential for ZnO NPs were 278.23 ± 12.72 nm and 3.09 ± 0.71 mV, respectively (Additional file 1: Fig. S1). We further analyzed the ions released from the NPs. The Fe or Zn contents of 50 mg·L−1 Fe3O4 NPs or ZnO NPs were 0.23 mg·L−1 and 0.87 mg·L−1, respectively (Additional file 1: Fig. S2), indicating a relatively low concentration of ions released from the NPs.
Fe3O4 and ZnO NPs affect plant growth and mineral element accumulation in tobacco
We first determined the effects of Cd on tobacco seedling growth. We found that Cd treatment markedly induced growth inhibition in plant height, shoot FW, root length and FW (Fig. 1). To confirm whether Fe3O4 and ZnO NPs played roles in mediating Cd response, we further applied foliar exposure to 50 mg·L−1 Fe3O4, ZnO NPs, and ion solutions to tobacco seedlings. The foliar applications of NPs and ions had no side effects on the plant height, shoot FW, root length and FW under untreated conditions (Fig. 1).
Compared with Cd-treated seedlings alone, Fe3O4, ZnO NPs, and ions significantly promoted root growth and FW. Both Fe3O4 and ZnO NPs enhanced plant growth, as indicated by higher plant height and shoot FW; however, FeSO4 elevated plant height but had no effects on the shoot FW. Similarly, ZnSO4 showed no noticeable promotion effects on the plant height and shoot FW (Fig. 1). These results suggested that moderate concentrations of NPs and ions played different roles in tobacco seedlings under Cd stress, and NPs were more efficient than ions.
Cd toxicity severely suppresses plant growth and ion balance, thus further disrupting plant metabolism and energy metabolism. We thus determined the elements in the leaves and roots. Cd treatment markedly reduced the contents of Fe, Mn and Zn but increased the Cu, Mg and K in the roots (Fig. 2a). Cd treatment also reduced the contents of Ca, Cu and Mn but increased Mg in the leaves (Fig. 2a). Under Cd stress, mineral element uptake and distribution were altered. Fe3O4 NPs or FeSO4 increased Mg but reduced Fe, Mn and Zn in the roots and decreased Cu, Zn and K in the leaves. In addition, ZnO NPs or ZnSO4 increased Cu, Mg and K but decreased Fe and Mn in the roots; however, they both increased Zn but reduced Ca, Cu and Mn in the leaves (Fig. 2a). In addition, FeSO4 or ZnSO4 decreased the Cd in the leaves, while ZnO NPs reduced it in the roots (Fig. 2b). These results showed that NPs and ions affected the balance of the element in tobacco seedlings.
Metabolomics profiling analysis
We examined differentially accumulated metabolites (DAMs) in the roots and leaves for the metabolomic profile. Compared to untreated conditions, metabolites in roots or leaves exposed to Fe3O4, ZnO NPs, FeSO4 ZnSO4, Cd toxicity, or their combinations presented a clear separation (Additional file 1: Figs. S3–5). This result indicated that Cd stress reprogrammed the metabolites, and NPs or ions altered the metabolites in the roots and leaves of untreated or Cd-exposed tobacco seedlings.
A total of 1013 and 890 metabolites were identified in tobacco roots and leaves, respectively. Then, a VIP ≥ 1 and P-value < 0.05 were set as the threshold of DAM characterization. As a result, 467 and 287 DAMs were identified in tobacco roots and leaves, respectively (Fig. 3a, Additional file 2: Table S1 and Additional file 3: Table S2). Among them, 131 DAMs were determined in both roots and leaves (Fig. 3, Additional file 2: Table S1 and Additional file 3: Table S2). Furthermore, several of the top 20 pathways, including arginine and proline metabolism, amino acid biosynthesis, nicotinate and nicotinamide metabolism, were significantly enriched in tobacco roots and leaves (Fig. 3d, e).
Under Cd stress, a total of 150 DAMs, including increases 132 and decreases 18 in tobacco roots, and 76 DAMs, including increases 54 and decreases 22 in tobacco leaves, were identified (Cd/control) (Fig. 3a, Additional file 2: Table S1 and Additional file 3: Table S2).
Fe3O4 NPs increased 16 DAMs and decreased 24 DAMs in tobacco roots, while increased 26 DAMs and decreased 19 DAMs in tobacco leaves (FeNP/control) (Fig. 3a, Additional file 2: Table S1 and Additional file 3: Table S2). FeSO4 increased 26 DAMs and decreased 14 DAMs in tobacco roots, while increased 26 DAMs and decreased 13 DAMs in tobacco leaves (Fe/control) (Fig. 3a, Additional file 2: Table S1 and Additional file 3: Table S2).
ZnO NPs increased 30 DAMs and decreased 22 DAMs in tobacco roots, while increased 12 DAMs and decreased 17 DAMs in tobacco leaves (ZnNP/control) (Fig. 3a, Additional file 2: Table S1 and Additional file 3: Table S2). On the other hand, ZnSO4 increased 34 DAMs and decreased 26 DAMs in tobacco roots, while increased 16 DAMs and decreased 17 DAMs in tobacco leaves (Zn/control) (Fig. 3a, Additional file 2: Table S1 and Additional file 3: Table S2).
Compared with Cd stress alone, Fe3O4 NPs resulted in 37 DAMs, including increased 10 and decreased 27, and 48 DAMs, including increased 30 and decreased 18, in Cd-treated tobacco roots and leaves (FeNP_Cd/Cd), respectively (Additional file 2: Table S1 and Additional file 3: Table S2). While FeSO4 resulted in 46 DAMs (37 showed increased levels and 9 showed decreased levels) and 72 DAMs (53 showed increased levels and 19 showed decreased levels) in the Cd-treated tobacco roots and leaves (Fe_Cd/Cd), respectively (Additional file 2: Table S1 and Additional file 3: Table S2).
ZnO NPs resulted in 50 DAMs, including increased 35 and decreased 15, and 47 DAMs, including increased 32 and decreased 15, in the Cd-treated tobacco roots and leaves (ZnNP_Cd/Cd), respectively (Additional file 2: Table S1 and Additional file 3: Table S2). On the other hand, ZnSO4 resulted in 68 DAMs, including increased 56 and decreased 12, and 64 DAMs, including increased 45 and decreased 19, in the Cd-treated tobacco roots and leaves (Zn_Cd/Cd), respectively (Additional file 2: Table S1 and Additional file 3: Table S2).
To explore whether NPs or ions alter the metabolic response to Cd, we further identified the common metabolites in the Cd-treated tobacco roots and leaves among treatments. KEGG pathway enrichment analysis indicated that several of the top 20 pathways, including arginine and proline metabolism, beta-alanine metabolism, and metabolic pathways, were markedly enriched in tobacco roots and leaves (Figs. 4, 5). Alkaloids, amino acids and their derivatives, and flavonoids were among the most altered metabolites in the Cd-treated seedlings. Specifically, 10 alkaloids, 17 amino acids and their derivatives, and 16 flavonoids showed increased accumulation, whereas 3 alkaloids, 1 amino acid and its derivative, and 3 flavonoids showed decreased accumulation in roots; While 2 alkaloids, 5 amino acids and their derivatives, and 13 flavonoids showed increased accumulation, whereas 2 alkaloids and 1 flavonoid showed decreased accumulation in the leaves of the Cd-treated seedlings (Figs. 4, 5, Additional file 4: Table S3 and Additional file 5: Table S4). Surprisingly, we found that NPs or ions recovered the Cd-responsive metabolites to normal levels in the Cd-treated tobacco roots and leaves (Figs. 4, 5, Additional file 4: Table S3 and Additional file 5: Table S4). For example, Fe3O4 NPs restored 75 DAMs (including 8 alkaloids, 7 amino acids and their derivatives, 11 flavonoids) in the roots and 53 DAMs (including 4 alkaloids, 1 amino acid and its derivative, 9 flavonoids) in leaves to normal levels. In comparison, FeSO4 restored 46 DAMs (including 9 alkaloids, 4 amino acids and their derivatives, 9 flavonoids) in the roots and 51 DAMs (including 3 alkaloids, 3 amino acids and their derivatives, 8 flavonoids) in the leaves of Cd-treated tobacco seedlings to normal levels (Figs. 4, 5). ZnO NPs restored 70 DAMs (including 8 alkaloids, 6 amino acids and their derivatives, 12 flavonoids) in the roots and 48 DAM (including 3 alkaloids, 3 amino acids and their derivatives, 6 flavonoids) in the leaves to normal levels. In comparison, ZnSO4 restored 57 DAMs (including 7 alkaloids, 3 amino acids and their derivatives, 9 flavonoids) in the roots and 45 DAMs (including 4 alkaloids, 3 amino acids and their derivatives, 5 flavonoids) in the leaves of Cd-treated tobacco seedlings to normal levels (Figs. 4, 5). These results collectively indicate that NP-reprogrammed DAMs may have great potential in alleviating Cd stress and further support the positive effects of NPs on growth under Cd stress.
Correlation analyses of DAMs and growth parameters of tobacco seedlings
The above results showed that NPs had greater efficiency in alleviating Cd stress, and DAMs profiling might correlate with plant growth. To address this question, we then conducted Pearson correlation coefficient analyses between the mean values of DAMs and growth parameters (including the plant height, shoot FW, root length, and root FW). In the roots, a diterpenoid (sterebin A), phytohormone (indole-3-acetic acid), and quinone (shikonin) were positively correlated with the root length, whereas 3 alkaloids (napellonine, piperidine, and vasicine), 10 amino acids and their derivatives (including L-theanine, L-threonine, proline), 4 terpenoids (including cinnzeylanol, retinoic acid), and 2 phenylpropanoids (cinnamyl cinnamate and ferulic acid) negatively correlated with the root length (Fig. 6a, Additional file 6: Table S5). A diterpenoid (sterebin A) and a flavonoid (hyperoside) were positively correlated with the root FW, whereas 5 amino acids and their derivatives (including proline, L-histidine, L-asparagine), 2 diterpenoids (cinnzeylanol and retinoic acid), a flavonoid (farrerol), and 2 phenylpropanoids (cinnamyl cinnamate and ferulic acid) negatively correlated with the root FW (Fig. 6a, Additional file 6: Table S5).
An organic acid (phosphoric acid) and an alkaloid (denudatine) were positively correlated with the plant height and shoot FW in the leaves. In addition, a sesquiterpenoid (catalpalactone) and miscellaneous compound (torachrysone 8-O-glucoside) showed a positive correlation with the plant height, while nicotinic acid showed a positive correlation with the shoot FW. However, 2 amino acids and their derivatives (N6-acetyl-l-lysine, l-theanine), 6 flavonoids (including quercetin, isorhamnetin, isoquercitrin), and 2 terpenoids (kaurenoic acid and limonin) showed negative correlations with both the plant height and shoot FW (Fig. 6b, Additional file 7: Table S6). In addition, 5-oxoproline, 5 flavonoids (including kaempferol-3-O-rutinoside, rutin, cynaroside), a diterpenoid (kaurenoic acid), and other metabolites showed negative correlations with the shoot FW (Fig. 6b, Additional file 7: Table S6).
Further analysis revealed that several DAMs in the roots and leaves correlated with plant growth. As shown in Fig. 6, 2 amino acids and their derivatives (5-oxoproline and l-theanine), a carbohydrate (l-gulose), cinnamic acid and derivative (ethyl trans-p-methoxycinnamate), carboxylic acid and derivative (NG, NG-dimethylarginine dihydrochloride), fatty acyl (kojibiose), and phenol (4,4′-methylenediphenol), showed negative correlations with plant growth parameters. In addition, phosphoric acid in the roots negatively correlated with the root length, whereas phosphoric acid in the leaves positively correlated with plant height and shoot FW (Fig. 6a, b, Additional file 8: Table S7). These results imply that changed metabolite profiling is indeed responsible for plant growth. The comparison of varied DAMs among different treatments is described in detail below.
Root and leaf metabolic profiling under cadmium stress
Cd toxicity resulted in more metabolites in the roots (88%), but it suppressed higher metabolites in the leaves (29%) of Cd-treated tobacco seedlings (Fig. 3 and Additional file 1: Fig. S6, Additional file 2: Table S1 and Additional file 3: Table S2). In the roots, Cd treatment increased metabolites, including10 alkaloids, 17 amino acids and their derivatives, 16 flavonoids, 4 phenylpropanoids, and 11 phenols (Fig. 3, Additional file 2: Table S1), whereas it reduced metabolites including 3 alkaloids, beta-alanine, 3 flavonoids, and sterebin A (Fig. 3, Additional file 2: Table S1). In addition, Cd stress increased a phytohormone, N-(-)-jasmonoyl)-S-isoleucine, and 5 carbohydrates, including d-glucose 6-phosphate, sucrose, and D-maltose (Fig. 3, Additional file 2: Table S1). By contrast, it decreased indole-3-acetic acid, a critical phytohormone that regulates plant growth and development, and maltotetraose (Fig. 3, Additional file 2: Table S1).
Cd treatment increased metabolites in the leaves, including 2 alkaloids, 5 amino acids and their derivatives, 13 flavonoids, and 2 phenylpropanoids (Fig. 3, Additional file 3: Table S2). By comparison, it decreased metabolites, including 2 alkaloids, 1 flavonoid, 2 phenylpropanoids, and 2 phenols (Fig. 3, Additional file 3: Table S2). Furthermore, Cd stress increased 2 carbohydrates, l-gulose and sucrose, and a phytohormone, (+)-abscisic acid (Fig. 3, Additional file 3: Table S2); By contrast, it decreased nicotinic acid accumulation (Fig. 3, Additional file 3: Table S2).
Venn diagram analysis showed that Cd stress increased 18 metabolites (8.7%), including 4 amino acids and their derivatives, 2 carbohydrates (l-gulose and sucrose), 3 flavonoids (cynaroside, naringenin chalcone, and quercetin), a phenylpropanoid (cinnamyl cinnamate), in both the roots and leaves of Cd-treated seedlings. Whereas it increased 2 metabolites (1%), a flavonoid, farrerol, and an organic acid, phosphoric acid in tobacco roots but decreased in tobacco leaves compared with the control (Additional file 1: Fig. S6, Additional file 2: Table S1 and Additional file 3: Table S2). These results suggest that Cd stress induces different metabolome profiles in the roots and leaves, indicating a distinct mechanistic response to Cd in tobacco roots and leaves.
Effects of Fe3O4 NPs on root and leaf metabolic profiling
Fe3O4 NPs increased 16 metabolites, including 1 alkaloid, 2 flavonoids, and 2 phenylpropanoids, whereas they reduced 24 metabolites, including 3 alkaloids, 3 flavonoids, and a phenylpropanoid, in the roots (Fig. 3, Additional file 2: Table S1). In the leaves, 26 metabolites, including 5 alkaloids, 7 amino acids and their derivatives, and 3 flavonoids, showed increased levels, whereas 19 metabolites, including 2 alkaloids, 2 flavonoids, and a phenylpropanoid, showed decreased levels compared with the control (Fig. 3, Additional file 3: Table S2). Venn diagram analysis showed that most of the DAMs were unique in the roots and leaves of Fe3O4 NP-exposed seedlings. Fe3O4 NPs increased 15 (17.9%) in roots and 26 DAMs (31%) in leaves, whereas they decreased 24 (28.6%) and 18 DAM (21.4%) accumulation levels in the roots and leaves, respectively. Furthermore, Fe3O4 NPs increased a flavonoid, farrerol in the roots but decreased in the leaves (Additional file 1: Fig. S7a).
FeSO4 increased 26 metabolites, including 3 alkaloids, 7 flavonoids, and 1 phenylpropanoid, whereas it decreased 14 metabolites, including 2 alkaloids, 2 amino acids and their derivatives, and 2 phytohormones, in the roots (Fig. 3, Additional file 2: Table S1). In addition, FeSO4 increased 26 metabolites, including 2 alkaloids, 2 amino acids and their derivatives, 4 flavonoids, whereas it reduced 13 metabolites, including a flavonoid, 2 phenols, a carbohydrate, in the leaves (Fig. 3, Additional file 3: Table S2). Venn diagram analysis showed FeSO4 increased 25 DAMs (32.1%) in the roots and 25 DAMs (32.1%) in the leaves, whereas it decreased 14 DAMs (17.9%) in the roots and 13 DAMs (16.7%) in the leaves. In addition, it increased only one metabolite, arachidonic acid, in both the roots and leaves (Additional file 1: Fig. S7b).
Effects of Fe3O4 NPs on the root and leaf metabolomes under Cd stress
Compared with Cd treatment alone, Fe3O4 NPs increased 10 metabolites, including 3 alkaloids, 2 amino acids and their derivatives, and a terpenoid, while they decreased 27 metabolites, including 6 alkaloids, an amino acid, and 3 flavonoids in the roots (Additional file 2: Table S1). In addition, Fe3O4 NPs increased 30 metabolites, including 3 alkaloids, 7 amino acids and their derivatives, and 3 flavonoids, whereas they decreased 18 metabolites, including an alkaloid, 4 flavonoids, and 2 phenylpropanoids, in the leaves of Cd-treated seedlings compared with Cd-treated seedlings (Additional file 3: Table S2). Venn diagram analysis showed that Fe3O4 NPs increased the levels of 10 (11.8%) metabolites in the roots (Additional file 1: Fig. S8a, Additional file 2: Table S1) and 30 (35.3%) metabolites in the leaves (Additional file 1: Fig. S8a, Additional file 3: Table S2) but decreased 27 (31.8%) metabolites in the roots (Additional file 1: Fig. S8a, Additional file 2: Table S1) and 18 (21.2%) metabolites in the leaves of Cd-treated tobacco seedlings (Additional file 1: Fig. S8a, Additional file 3: Table S2). These results indicated that Fe3O4 NPs reprogram different metabolic profiling responses under Cd stress in the roots and leaves.
FeSO4 increased 37 metabolites, including 5 alkaloids, 2 amino acids and their derivatives, and 6 flavonoids, whereas it decreased 9 metabolites, including 3 alkaloids, a phenylpropanoid, and a terpenoid, in tobacco roots (Additional file 2: Table S1). Additionally, FeSO4 increased 53 metabolites, including 5 alkaloids, 2 amino acids and their derivatives, and 8 flavonoids, whereas it reduced 19 metabolites, including an alkaloid, 2 flavonoids, and 3 phenylpropanoids, in the leaves (Additional file 3: Table S2). Venn diagram analysis showed that FeSO4 increased the accumulation levels of 37 (31.9%) in the roots and 53 DAMs (45.7%) in the leaves, while it decreased the accumulation levels of 9 (7.8%) and 19 DAMs (16.4%) in the roots and leaves, respectively (Additional file 1: Fig. S8b, Additional file 2: Table S1 and Additional file 3: Table S2). Furthermore, a flavonoid, homoferreirin, showed an increased accumulation level in the roots and leaves. By contrast, it reduced a phenylpropanoid, 3-methoxy-4,5-methylenedioxycinnamaldehyde, in both the roots and leaves (Additional file 1: Fig. S8b, Additional file 2: Table S1 and Additional file 3: Table S2).
Effects of ZnO NPs on root and leaf metabolic profiling
ZnO NPs increased 30 metabolites, including 3 alkaloids, 9 flavonoids, and 3 phenylpropanoids, whereas they reduced 22 metabolites, including an alkaloid, 5 amino acids and their derivatives, and 3 flavonoids, in tobacco roots (Fig. 3a, Additional file 2: Table S1). In addition, ZnO NPs increased 12 metabolites, including 2 alkaloids, 4 amino acids and their derivatives, and a flavonoid, whereas they reduced 17 metabolites, including 2 alkaloids, 5 flavonoids, and 2 nucleotides and their derivates, in tobacco leaves (Fig. 3a, Additional file 3: Table S2). Venn diagram analysis showed that ZnO NPs increased 30 (37%) in the roots and 12 DAM (14.8%) in the leaves, while they decreased 22 DAMs (27.2%) and 17 DAMs (21%) levels in the roots and leaves, respectively, compared with the control (Additional file 1: Fig. S9a, Additional file 2: Table S1 and Additional file 3: Table S2).
ZnSO4 increased 34 metabolites, including 3 alkaloids and 6 flavonoids, whereas it reduced 26 metabolites, including 7 alkaloids, 2 amino acids and their derivatives, and 4 flavonoids, in the roots (Fig. 3a, Additional file 2: Table S1). Meanwhile, ZnSO4 increased 16 metabolites, including an alkaloid, 4 amino acids and their derivatives, and a flavonoid, whereas it reduced 17 metabolites, including an alkaloid, 6 flavonoids, and a phytohormone (1-naphthylacetic acid), showed decreased levels in the leaves (Fig. 3a, Additional file 3: Table S2). Venn diagram analysis showed that ZnSO4 increased 34 (36.6%) in the roots and 16 DAMs (17.2%) in the leaves, while it decreased 26 (28%) in the roots and 17 DAMs (18.3%) in the leaves, compared with the control (Additional file 1: Fig. S9b).
Effects of ZnO NPs on root and leaf metabolomes under Cd stress
Compared with Cd treatment alone, ZnO NPs increased 35 metabolites, including 2 alkaloids, 3 amino acids and their derivatives, and 5 flavonoids, while it decreased 15 metabolites, including 2 alkaloids, 2 flavonoids, and a phenol, in the roots (Additional file 2: Table S1). In addition, ZnO NPs increased 32 metabolites, including 8 amino acids and their derivatives, 2 flavonoids, and 2 nucleotides and their derivatives. In contrast, they reduced 15 metabolites, including 3 alkaloids, 2 amino acids and their derivatives, and 2 flavonoids, in the leaves of Cd-treated tobacco (Additional file 3: Table S2). Venn diagram analysis showed that ZnO NPs increased 35 (36.4%) metabolites in tobacco roots (Additional file 1: Fig. S10a, Additional file 2: Table S1) and 32 (33.3%) metabolites in tobacco leaves (Additional file 1: Fig. S10a, Additional file 3: Table S2), whereas ZnO NPs decreased 15 (15.6%) metabolites in the roots (Additional file 1: Fig. S10a, Additional file 2: Table S1) and 15 (15.6%) metabolites in the leaves of Cd-treated tobacco (Additional file 1: Fig. S10a, Additional file 2: Table S1). Only one metabolite, an alkaloid [3-(carboxymethylamino) propanoic acid], was upregulated in the roots but reduced in the leaves of Cd-treated tobacco (Additional file 1: Fig. S10, Additional file 2: Table S1 and Additional file 3: Table S2).
ZnSO4 increased 56 metabolites, including 8 alkaloids, 8 amino acids and their derivatives, and 3 flavonoids, while it decreased 12 metabolites, including 2 alkaloids, 3 flavonoids, and a phytohormone, in the roots (Additional file 2: Table S1). In addition, ZnSO4 increased 45 metabolites, including 2 alkaloids, 5 amino acids and their derivatives, 5 flavonoids, and 5 phenols, whereas it reduced19 metabolites, including an alkaloid, 2 flavonoids, and a phenol, in the leaves of Cd-treated seedlings (Additional file 3: Table S2). Venn diagram analysis revealed that ZnSO4 increased 56 DAMs (43.7%) accumulation levels in the roots and 45 DAMs (35.1%) accumulation levels in the leaves. In comparison, it decreased 12 DAMs (9.4%) accumulation levels in the roots and 19 DAMs (14.9%) accumulation levels in the leaves (Additional file 1: Fig. S10b, Additional file 2: Table S1 and Additional file 3: Table S2). Furthermore, 3 metabolites, including beta-D-fructose 2-phosphate, l-acetylcarnitine, and l-citruline, showed an increased accumulation level in the roots and leaves. By contrast, cytidine 5′-monophosphate showed a decreased accumulation level in the roots and decreased in the leaves of Cd-treated seedlings (Additional file 1: Fig. S10b, Additional file 2: Table S1 and Additional file 3: Table S2).
Comparative analyses of DAMs between Fe3O4 and ZnO NP-treated seedlings under normal conditions and Cd stress
Under untreated control conditions, both Fe3O4 NPs and ZnO NPs increased 6 (4%) metabolites, including L-arginine, l-threonine, and quercetin, whereas they reduced 9 (6%) metabolites, including glycyrrhetinic acid, adenosine 3′-monophosphate, and uridine 5'-monophosphate, in the roots and/or leaves (Additional file 1: Fig. S11, Additional file 2: Table S1 and Additional file 3: Table S2).
Under Cd stress conditions, Fe3O4 NPs and ZnO NPs increased 13 (8.3%) metabolites, including 5 amino acids and their derivatives (e.g., isoleucine, l-leucine, l-phenylalanine), 2 flavonoids (7-hydroxyflavone and farrerol), 6-aminocaproic acid, and cytidine 5′-monophosphate; however, they decreased 9 (5.7%) metabolites, including 2 alkaloids [3-(carboxymethylamino) propanoic acid and jervine], and lupeol D-maltose, in the roots and/or leaves of Cd-treated tobacco seedlings (Additional file 1: Fig. S12, Additional file 2: Table S1 and Additional file 3: Table S2). In addition, Fe3O4 NPs increased the level, but ZnO NPs decreased the level of 2'-deoxyinosine-5'-monophosphate, while ZnO NPs increased the level, but Fe3O4 NPs decreased the level of (13E)-11a-hydroxy-9,15-dioxoprost-13-enoic acid in the Cd-treated tobacco roots and/or leaves (Additional file 1: Fig. S12, Additional file 2: Table S1 and Additional file 3: Table S2). The above results suggest that the two NPs mediate Cd tolerance through similar and distinct mechanisms.
Comparative analyses of DAMs between FeSO4- and ZnSO4-treated seedlings under normal conditions and Cd stress
Under untreated control conditions, FeSO4 and ZnSO4 increased 6 (3.7%) metabolites, including lupenone, nicotinic acid adenine dinucleotide, and synephrine, whereas they decreased 4 (2.5%) metabolites, including cantharidin, deethylatrazine, glycyrrhetinic acid, and L-glutamic acid, in the roots and/or leaves (Additional file 1: Fig. S13, Additional file 2: Table S1 and Additional file 3: Table S2).
Under Cd stress conditions, FeSO4 and ZnSO4 increased 21 (9.8%) metabolites, including 2 alkaloids (cinchonine, methylisopelletierine), 4 amino acids and their derivatives (e.g., l-citruline, l-lysine, and 4-aminobutyric acid), and 2 phenols (5-heneicosylresorcinol and oleocanthal). However, it decreased 4 (1.9%) metabolites, including dihydromyricetin, taraxasterone, and hypotaurine, in the Cd-treated tobacco roots and/or leaves (Additional file 1: Fig. S14, Additional file 2: Table S1 and Additional file 3: Table S2). However, FeSO4 increased the level, but ZnSO4 decreased 3 metabolites, including guggulsterone E&Z, cytidine 5′-monophosphate, and octadecyl p-coumarate, while ZnSO4 increased, but FeSO4 decreased the level of 2 metabolites, angelicin and picrasin B, in the Cd-treated tobacco roots and/or leaves (Additional file 1: Fig. S14, Additional file 2: Table S1 and Additional file 3: Table S2). These results indicate that the two ions might also modulate the Cd response through similar and distinct mechanisms.
Comparative analyses of DAMs between Fe3O4 NP- and FeSO4-treated seedlings under normal conditions and Cd stress
Under untreated control conditions, Fe3O4 NPs and FeSO4 increased 7 (4.8%) metabolites, including farrerol, isoalantolactone, and rosmarinine, whereas they decreased 8 (5.5%) metabolites, including 2 diterpenoids (picrasin B, kaurenoic acid), glycyrrhetinic acid, and adenosine 3′-monophosphate, in the roots and/or leaves. In addition, FeSO4 increased, but Fe3O4 NPs decreased the level of officinalisinin I (Additional file 1: Fig. S15, Additional file 2: Table S1 and Additional file 3: Table S2). Under Cd stress conditions, Fe3O4 NPs and FeSO4 increased 7 (3.9%) metabolites, including alpha-d-glucose, aconitine, and cytidine 5'-monophosphate. In contrast, they decreased the levels of 8 (4.4%) metabolites, including 3 alkaloids [3-(carboxymethylamino) propanoic acid, anacrotine, piperidine], dihydromyricetin, and erythritol, in the Cd-treated tobacco roots and/or leaves (Fig. 7 and Additional file 1: Fig. S16, Additional file 2: Table S1 and Additional file 3: Table S2). However, FeSO4 increased, but Fe3O4 NPs decreased 5 metabolites, including apiin, phyllalbine, and herniarin, in the Cd-treated tobacco roots and/or leaves (Fig. 7 and Additional file 1: Fig. S16, Additional file 2: Table S1 and Additional file 3: Table S2).
Comparative analyses of DAMs between ZnO NP- and ZnSO4-treated seedlings under normal conditions and Cd stress
ZnO NPs and ZnSO4 increased 8 (5.1%) metabolites, including l-homoserine, L-threonine, and dexamethasone. In contrast, they decreased 8 (5.1%) metabolites, including 3 flavonoids (kaempferol, methyl hesperidin, and hyperoside), 3-carbamyl-1-methylpyridinium 1-methylnicotinamide, and acarbose, in the roots and/or leaves under untreated control conditions (Additional file 1: Fig. S17, Additional file 2: Table S1 and Additional file 3: Table S2). Under Cd stress conditions, both ZnO NPs and ZnSO4 increased the levels of 16 (7.9%) metabolites, including 4 amino acids and their derivatives (e.g., DL-alanine, l-theanine, L-citruline), 3 nucleotides and their derivatives (NAD, beta-nicotinamide mononucleotide, and cytidine 5′-monophosphate), and astaxanthin. In contrast, they decreased the levels of 3 (1.5%) metabolites, tacrolimus, 13(S)-HPOT and cedeodarin, in the roots and/or leaves of Cd-treated tobacco seedlings (Fig. 7 and Additional file 1: Fig. S18, Additional file 2: Table S1 and Additional file 3: Table S2). However, ZnO NPs increased the level, but ZnSO4 decreased (13E)-11a-hydroxy-9,15-dioxoprost-13-enoic acid. Additionally, ZnSO4 increased, but ZnO NPs decreased 2 metabolites, d-proline and l-proline, in the Cd-treated tobacco roots and/or leaves (Fig. 7 and Additional file 1: Fig. S18, Additional file 2: Table S1 and Additional file 3: Table S2). The above results suggested that NPs and ions might largely modulate the Cd response through distinct mechanisms.
Fe3O4 or ZnO NPs altered critical metabolite pathway under Cd stress
The above results indicated that Fe3O4 and ZnO NPs showed more efficiency in facilitating tobacco growth than ions under Cd stress (Figs. 1, 3, 4, 5). Furthermore, the common Cd-induced DAMs were significantly enriched in the pathway of amino acids metabolism, flavone and flavonol biosynthesis, secondary metabolite biosynthesis, nicotinate and nicotinamide metabolism, indicating that these two NPs reprogram carbon/nitrogen metabolism and secondary metabolism (Figs. 3, 4, 5). Thus, we further analyzed these metabolites involved in amino acid, nicotinate and nicotinamide metabolism pathway in response to Cd stress with or without exposure to Fe3O4 or ZnO NPs as well as ions. Specifically, Cd stress increased 7 amino acids and their derivatives, including l-alanine, tryptophan, and 4-aminobutyric acid, but decreased beta-alanine in tobacco roots (Fig. 8a, Additional file 2: Table S1). Interestingly, Fe3O4 NPs, ZnO NPs, and ions increased 4 amino acids (proline, 4-aminobutyric acid, l-homoserine, l-threonine) and sinapyl alcohol in the Cd-treated tobacco roots compared with untreated control (Fig. 8a, Additional file 2: Table S1). However, only proline showed increased accumulation in the leaves, while the other amino acids were not affected after Cd treatment (Fig. 8b, Additional file 3: Table S2). Both Fe3O4 and ZnO NPs showed higher accumulation levels of 7 amino acids, including leucine, arginine, and threonine, in the Cd-treated tobacco leaves than untreated control (Fig. 8b, Additional file 2: Table S1 and Additional file 4: Table S3). Furthermore, Fe3O4 NPs increased phenylalanine, p-coumaryl alcohol, and quercetin levels, while ZnO NPs increased alanine and 4-aminobutyric acid levels in the Cd-treated tobacco leaves (Fig. 8b, Additional file 3: Table S2 and Additional file 5: Table S4). These results indicated that Fe3O4 or ZnO NPs improve seedling growth by reprogramming amino acid metabolism in the roots and leaves under Cd stress.
Nicotinate and nicotinamide metabolism play roles in plant response to stress [46]. We thus mapped the Cd-induced DAMs involved in the nicotinate and nicotinamide metabolism pathway (map00760) to the KEGG pathway database [47]. In the roots, Cd stress increased N1-methyl-2-pyridone-5-carboxamide (C05842), N1-methyl-4-pyridone-5-carboxamide (C05843), beta-nicotinamide mononucleotide (C00455) and 6-hydroxynicotinic acid (C01020), but decreased N1-methylnicotinamide (C02918) (Fig. 9, Additional file 2: Table S1). Similarly, NPs and ions increased the levels of N1-methyl-2-pyridone-5-carboxamide (C05842), N1-methyl-4-pyridone-5-carboxamide (C05843), and beta-nicotinamide mononucleotide (C00455) in the Cd-treated tobacco roots compared with the untreated control (Fig. 9, Additional file 2: Table S1). Furthermore, FeSO4 increased nicotinate (C00253), 6-hydroxynicotinic acid (C01020), and L-aspartic acid (C00049) levels, whereas Fe3O4 NPs decreased N1-methylnicotinamide (C02918) levels. ZnO NPs increased nicotinate (C00253) and 6-hydroxynicotinic acid (C01020) levels, while ZnSO4 increased nicotinate (C00253) and L-aspartic acid (C00049) levels in the Cd-treated tobacco roots compared with the untreated control (Fig. 9, Additional file 2: Table S1). In the leaves, FeSO4 and ZnO NPs increased N1-methyl-4-pyridone-5-carboxamide (C05843) levels under Cd stress, whereas Fe3O4 NPs or ZnSO4 did not affect its accumulation levels in the Cd-treated leaves compared with the untreated control (Fig. 9, Additional file 3: Table S2). In addition, the accumulation of nicotinate (C00253) showed a decrease in the Cd-treated leaves, whereas NPs did not affect its levels in the leaves of Cd-treated tobacco seedlings compared with the untreated control (Fig. 9, Additional file 3: Table S2). These results indicated that NPs or ions modulate nicotinate and nicotinamide metabolism to regulate plant response to Cd stress.