Silicon via nutrient solution modulates deficient and sufficient manganese sugar and energy cane antioxidant systems

Manganese (Mn) is highly demanded by Poaceae, and its deficiency induces physiological and biochemical responses in plants. Silicon (Si), which is beneficial to plants under various stress conditions, may also play an important role in plants without stress. However, the physiological and nutritional mechanisms of Si to improve Mn nutrition in sugarcane and energy cane, in addition to mitigating deficiency stress, are still unclear. The objective of this study is to evaluate whether the mechanisms of action of Si are related to the nutrition of Mn by modulating the antioxidant defense system of sugarcane plants and energy cane plants cultivated in nutrient solution, favoring the physiological and growth factors of plants cultivated under Mn deficiency or sufficiency. Two experiments were carried out with pre-sprouted seedlings of Saccharum officinarum L. and Saccharum spontaneum L. grown in the nutrient solution. Treatments were arranged in a 2 × 2 factorial design. Plants were grown under Mn sufficiency (20.5 µmol L−1) and the deficiency (0.1 µmol L−1) associated with the absence and presence of Si (2.0 mmol L−1). Mn deficiency caused oxidative stress by increasing lipid peroxidation and decreasing GPOX activity, contents of phenols, pigments, and photosynthetic efficiency, and led to the growth of both studied species. Si improved the response of both species to Mn supply. The attenuation of the effects of Mn deficiency by Si depends on species, with a higher benefit for Saccharum spontaneum. Its performance is involved in reducing the degradation of cells by reactive oxygen species (21%), increasing the contents of phenols (18%), carotenoids (64%), proteins, modulating SOD activity, and improving photosynthetic and growth responses.


Results
Mn and Si accumulation and Mn use efficiency. The Si supplied via nutrient solution increased its accumulation in sugarcane plants (Fig. 1a) and energy cane plants (Fig. 1b). The change in the plant's Mn status (deficient or sufficient) does not affect the plant's ability to accumulate Si; however, this only occurred for sugarcane (Fig. 1a). For energy cane in a condition of Mn deficiency, it results in a low accumulation of Si compared to plants with sufficiency of Mn (Fig. 1b).
There was a decrease in Mn accumulation in sugarcane and energy cane under Mn deficiency in relation to Mn sufficiency in plants with the presence of Si and absence of Si (Fig. 1c,d). The presence of Si in the nutrient solution, in relation to the absence of Si, increased the accumulation of Mn by 23% in sugarcane cultivated under Mn sufficiency. There was no change in plants under Mn deficiency (Fig. 1c). For energy cane, the presence of Si, in relation to its absence in the nutrient solution, increased by 40% and 21% the accumulation of Mn in plants grown in nutrient solution deficient in Mn and in nutrient solution sufficient in Mn, respectively (Fig. 1d).
The presence of Si in the nutrient solution, in relation to the absence of Si, did not affect Mn use efficiency in sugarcane cultivated under Mn deficiency and sufficiency (Fig. 1e). However, for energy cane, the presence of Si in the nutrient solution, in relation to the absence of Si, increased the plant's Mn use efficiency by 10% and 18% when grown in nutrient solution with Mn sufficiency and deficiency, respectively (Fig. 1f).
Lipid peroxidation (MDA), SOD and GPOX activity, and phenol content. There (Fig. 2d). There was a decrease in GPOX activity in energy cane plants cultivated in nutrient solution   (Fig. 2f).
There was a decrease in the content of total phenols in sugarcane and energy cane plants cultivated under Mn deficiency in relation to plants sufficient in Mn and those that did not receive Si. There was an increase in the content of total phenols by 20% in sugarcane plants cultivated in nutrient solution with the presence of Si in the nutrient solution in relation to the absence of Si in plants sufficient in Mn (Fig. 2g). In energy cane plants, the presence of Si in the nutrient solution, in relation to the absence of Si, increased the contents of total phenols in Mn-deficient plants (18%) and in Mn-sufficient plants (16%) (Fig. 2h).
Pigments content and PSII quantum efficiency. There was a decrease in pigment content (Chl a, b, carotenoids) in sugarcane and energy cane plants (Fig. 3a-f) cultivated in nutrient solution under Mn deficiency in relation to sufficient Mn and those that did not receive Si in the nutrient solution. The presence of Si in the nutrient solution, in relation to its absence, increased the contents of Chl a, Chl b, and carotenoids (27%) in sugarcane plants under Mn sufficiency (Fig. 3a,c,e). In energy cane, the presence of Si in the nutrient solution, in relation to the absence of Si, increased the contents of Chl a (Fig. 3b), Chl b (Fig. 3d), and carotenoids by 64% and 51% (Fig. 3f) in Mn-deficient and Mn-sufficient plants, respectively.
There was a decrease in PSII quantum efficiency in sugarcane and energy cane plants cultivated in nutrient solution deficient in Mn in relation to a Mn-sufficient solution and those that did not receive Si in the nutrient solution (Fig. 3g,h). The presence of Si in the nutrient solution, in relation to the absence of Si, increased the PSII quantum efficiency in sugarcane plants under Mn sufficiency (Fig. 3g). In energy cane, the presence of Si in the nutrient solution, in relation to the absence of Si, increased the PSII quantum efficiency in Mn deficiency and in Mn sufficiency (Fig. 3h).
Protein content and growth. The sufficiency or deficiency conditions of Mn and the absence and presence of Si in the nutrient solution did not affect protein contents in sugarcane (Fig. 4a). There was a decrease in protein content in energy cane plants cultivated under Mn deficiency in relation to Mn-sufficient plants that did not receive Si in the nutrient solution. The presence of Si in the nutrient solution, in relation to the absence of Si, increased the protein content in energy cane under Mn deficiency and in Mn sufficiency (Fig. 4b).
There was a decrease in leaf area and dry mass of sugarcane plants and energy cane plants deficient in Mn in relation to plants sufficient in Mn and those that did not receive Si (Fig. 4c,d). The presence of Si, in relation to its absence in the nutrient solution, increased the sugarcane leaf area in plants grown in a sufficient nutrient solution of Mn (Fig. 4c). In energy cane, the presence of Si in the nutrient solution, in relation to the absence of Si, increased the leaf area in plants deficient in Mn and in plants sufficient in Mn (Fig. 4d).
There was an increase in the dry mass of sugarcane plants sufficient in Mn (11%) when cultivated in nutrient solution with Si in relation to the absence of Si (Fig. 4e). In energy cane, the presence of Si in relation to the absence of Si in the nutrient solution provided increases of 22 and 25% in plant dry mass in Mn sufficiency and Mn deficiency, respectively (Fig. 4f).

Discussion
Plants of both species grown in Mn-deficient nutrient solution with or without Si reduced Mn absorption, consequently causing biological damage to plants.
Manganese deficiency with no addition of Si to the nutrient solution promotes an increase in ROS, as observed in sorghum 5 , resulting in an increase in MDA content in both studied species. This plant response may be associated with cellular damage caused by the excess of ROS production in the metabolism and/or by a lower capacity of ROS elimination by the defense system, leading to formation of MDA, which is a secondary metabolite resulting from the degradation of hydroperoxides of polyunsaturated fatty acids 36 and an indicator of oxidative stress. This has not yet been reported for sugarcane, but it was observed in Mn-deficient sorghum 5

and corn plants 4 .
Our results also show that enzymatic responses under Mn deficiency may be different depending on species despite a clear occurrence of oxidative stress due to increased lipid peroxidation. SOD activity in Mn-deficient sugarcane plants was low in the absence of Si, a fact also reported in other studies 5,10 . Mn is a cofactor of the Mn-SOD enzyme in plants and a key enzyme in the O 2 •dismutation process in H 2 O 2 11,12 . The lack of this micronutrient may decrease this enzyme activity in the metabolism and consequently O 2 •accumulation in the cells, thus causing lipid degradation.
However, SOD activity in energy cane increased in Mn-deficient plants. Mn is one of the SOD cofactors, and therefore the total SOD activity may vary depending on species, reflecting the regulation of other isoforms of this enzyme, such as Cu/Zn-SOD and Fe-SOD, increasing in turn the total enzyme activity 8,37 . In addition, an increase in SOD activity in Mn-deficient plants suggests a higher ROS production 38 . Therefore, it is important to consider that the total SOD activity can be different depending on species even under the same type of stress.
Similar as SOD, peroxidases are part of the defense system components. They control the H 2 O 2 content produced in the metabolism by SOD catalysis and in a non-enzymatic manner 39 . Mn-deficient sugarcane and energy cane plants presented a decrease in the peroxidase activity studied (GPOX). The low activity of another peroxidase (APX) under Mn deficiency has already been reported and related to one of the main responses to the deprivation of this nutrient 10,38 . This low enzyme activity may have contributed to the lipid peroxidation observed in Mn-deficient energy cane plants due to excessive H 2 O 2 accumulation, which is a substrate of this enzyme and causes cell degradation 39 because SOD activity was high in this species.
In this scenario, Mn deficiency reduced the content of photosynthetic pigments in both studied species due to oxidative stress related to a direct ROS action, which causes thylakoid disorganization 13 40 . In addition, Mn deficiency decreased PSII quantum efficiency, as it causes a reduction in the complexes of this nutrient at the PSII core, inducing destabilization and disintegration of this photosystem 14 and increasing 1 O 2 formation 9 , which can in turn increase the degradation of chlorophylls. Also, reductions in the content of carotenoids and phenols increase the damage to chlorophylls, as they may cause accumulations of 1 O 2 , an active molecular oxygen, and H 2 O 2 , thus potentiating oxidative damage 39 .
On the other hand, Mn deficiency did not affect protein contents in sugarcane plants. However, energy cane plants showed an important decrease in protein contents. Mn activates RNA polymerase and the glycolytic enzymes of proteins 15,16 , with reflexes on protein synthesis. However, according to 12 , protein synthesis is not necessarily impaired and may even increase in Mn-deficient tissues, as 41 observed. The decrease in protein contents of Mn-deficient sugarcane plants may be the result of oxidative stress due to protein oxidation by ROS, suggesting a marked nutritional damage related to the lack of Mn in this species.
The damage Mn deficiency caused to plants, mainly in structures indispensable for photosynthesis, such as pigments and the PSII, with reflections on the oxidative metabolism, justifies the growth losses of both species given the decrease in leaf area and dry mass production. The effects that Mn deficiency causes on photosynthesis are mainly responsible for the impacts it exerts on plant growth 5,14,42 .
The physiological and biochemical damage of Mn deficiency, although not yet reported for sugarcane and energy cane, indicates the importance of this micronutrient for these species, thus confirming a need for strategies such as the use of Si to mitigate such effects. Our study presents unprecedented answers to these issues by evaluating possible mechanisms of action of Si and its relations with Mn.
Initially, the Si supply in the nutrient solution was efficient to increase Si accumulation in the sugarcane and energy cane grown in a nutrient solution deficient or not in Mn. The high amount of accumulated Si in these plants may be explained by the improved root absorption capacity of this element by this crop 26,43 , which is caused by the presence of specific carriers of this element, which controls its absorption 44 . The ability of S. spontaneum L. plants to accumulate Si, not yet reported, confirms that this species has a high Si absorption, similar as that of S. officinarum L.
The increase in Si absorption favored Mn absorption in both studied species regardless of deficiency or sufficiency in this micronutrient. This result is important because it contributes to understanding the role of Si in Mn absorption. It is one of the reasons that this element favors Mn nutrition whether in sufficiency or deficiency. The greater the absorption of Mn by the plant, the greater the expectation in increasing the plant's nutritional and physiological responses to this micronutrient. Therefore, this is a new finding for these species.
There are reports only for other grasses, such as sorghum under Mn deficiency 18 and corn and wheat without deficiency of this micronutrient 33 . This beneficial Si effect on Mn absorption could be related to the regulation of the H + -ATPase activity 28,45,46 , as well as to specific Mn transporters such as IRT1 and AtNramp 30 , which generate an electrochemical gradient for nutrient absorption, including Mn carriers in the membrane 29,31 . They were identified for sugarcane 32 .
The increase in Mn absorption caused by Si addition to Mn-sufficient sugarcane and Mn-sufficient and Mn-deficient energy cane could explain the increase in total phenol content. According to 6 , it occurs because Mn associates with the biosynthesis of phenolic compounds by activating enzymes involved in the formation of secondary metabolites that are precursors to phenols 23 . Some studies have reported that Si increases the amount of this antioxidant compound 34 . Also, the increased Mn due to Si provided increases of another important antioxidants (carotenoids), as Mn activates the enzyme phytoene synthase, which acts in the biosynthesis of isoprenoids, precursors of this pigment 47 . Therefore, Mn and Si may act together to increase the contents of these two non-enzymatic antioxidants in both studied species.
Thus, the increase in phenols provided by Si aids the defense system of plants subjected to different stresses 48,49 and plants under no stress 34 . Phenols act in balancing the antioxidant system, with a direct elimination of active molecular oxygen and H 2 O 2 , thus inhibiting lipid peroxidation 39 . Moreover, Mn-deficient and sufficient energy cane plants with Si presented reductions in SOD activity, but with a low lipid peroxidation. This may be related to a reduction in the O 2 •produced in the presence of the beneficial element in response to the best Fv/Fm ratio of these plants. Therefore, it suggests low losses of electrons in the transport chain in the photosystems II and I in chloroplasts because these electrons reduce molecular oxygen (O 2 ) and form O 2 •-50 , along with the action of other antioxidant compounds, and not with a direct action of SOD, in this case.
Other studies have reported reductions in SOD activity in the presence of Si 51-53 , including sugarcane 54 , and that they vary according to species. Despite this, there were reductions in oxidative stress promoted by Si associated with the action of other components linked to enzymatic and non-enzymatic antioxidant metabolism.
Carotenoids play an essential role in protecting the photosynthetic apparatus, contributing to decrease the formation of singlet oxygen ( 1 O 2 ). Singlet oxygen is formed by a deficiency in the dissipation of energy by the PSII 39 . Therefore, the action of Si, with mechanisms to reduce the production of ROS (O 2 ·− , H 2 O 2, 1 O 2 ) and to increase the antioxidant compounds that reduce the concentrations of H 2 O 2 (phenols) and 1 O 2 (carotenoids), may have contributed to reduce the lipid peroxidation observed. The regulation of antioxidant enzyme activity has been reported only in sorghum plants grown under Mn deficiency, modulating the activity of the enzymes SOD and ascorbate peroxidase (APX) 5 . However, the Si action in the enzyme and non-enzymatic antioxidant system occur in other stresses; recent studies have indicated Si benefits in reducing lipid peroxidation 55,56 .
Silicon did not benefit the enzymatic complexes SOD and GPOX in sugarcane. However, the reduction in lipid peroxidation in Mn-sufficient plants may have been the result of an action of the phenols and carotenoids studied in this species because they affect important ROS that degrades cells, thus improving metabolism and reducing natural damage to plant cells with no Mn deficiency. Also, Si may have aided other defense system complexes that have not been studied, but that may be the focus of further studies. The effects of Si described above have not yet been reported for S. officinarum L. and S. spontaneum L. This reveals the benefits of Si in reducing MDA in the leaves of the species evaluated as a result of the modulation of www.nature.com/scientificreports/ the antioxidant system. These actions may have delayed the natural degradation of chlorophyll, which resulted in an increase in pigments in leaves of sugarcane and energy cane under Mn sufficiency. However, the presence of Si in Mn-deficient sugarcane plants also decreased chlorophyll degradation. This occurs because Si promotes an increase in Mn accumulation in the plant. According to 13 , Mn decreases the damage to chloroplasts because it is involved with lipid synthesis. Also, ROS accumulation may have been suppressed in chloroplasts due to an improvement in the antioxidant system, which may in turn have reduced the oxidation of existing pigments, which explains the increase in this pigment in plants under deficiency stress. Gonzalo et al. 57 already described a low chlorophyll degradation by Si in soybeans and cucumber and 58 for cucumber. The application of silicon in energy cane plants promoted an increase in Mn use efficiency and protein content under Mn sufficiency and deficiency. In plants sufficient in Mn, the physiological benefits provided by this micronutrient, such as increased pigments and PSII efficiency, may have increased photosynthesis, reflecting on the increase in protein synthesis. In Mn-deficient plants, in addition to physiological benefits, the reduction of oxidative stress provided by Si reduced damage to cellular components, including proteins. This justifies increases in protein contents. In rice plants under cadmium stress, Si regulated six categories of proteins, including those involved with protein synthesis 59 , thus showing the benefits of Si for protein synthesis under stress conditions. Therefore, the nutritional, physiological, and biochemical improvements provided high conversions of photoassimilates into dry mass and into protein content 60,61 , as observed for other grasses with and without Mn deficiency stress 5,17 .
The presence of Si in sugarcane did not affect the Mn use efficiency and the protein content of plants. Use efficiency is partly modulated by the capacity and genetic characteristics that are intrinsic to the species or cultivar 62 . Similarly, the action of Si on soluble protein depends on the condition under which the plants grow and species metabolism. In our study, Si did not act on Mn deficiency in sugarcane. There are reports that the benefits of Si in increasing protein contents are observed in plants under higher stress levels 63,64 and that are also associated with reductions in ROS. Therefore, it occurred because of different species and metabolic variations.
The benefits promoted by the application of Si to the nutrient solution, especially for energy cane under Mn deficiency or not and sugarcane under Mn sufficiency, reflected, in a same proportion, in the growth of these species due to the increase in leaf area and dry mass. Si benefits plants mainly under stress conditions 25 , a fact that occurred in energy cane but not in sugarcane. In this case, the deficiency in this micronutrient was more severe in energy cane due to a more drastic decrease in the dry mass of this species (22%) compared to sugarcane (7%). Therefore, Si was not relevant to mitigate the slight Mn deficiency in sugarcane, as this element, under this condition, did not increase total phenols, carotenoids, and the activity of the antioxidant enzymes SOD and GPOX, which could act to maintain ROS homeostasis and improve the pigment content, the PSII quantum efficiency, and Mn use efficiency, and proteins, thus reflecting on plant growth.
However, there was a more severe Mn deficiency observed in energy cane. The Si increased the accumulation of Mn, which reflected in physiological gains shown by the increase in pigment contents and PSII quantum efficiency, in addition to biochemical gains by reducing lipid peroxidation (MDA) and increases in non-enzymatic antioxidants (phenols and carotenoids). This resulted in a greater use efficiency of Mn, protein contents, and production of dry matter. These are benefits of Si to dry matter contents of Mn-deficient plants, as verified for rice 65 and sorghum 5 .
The beneficial responses of Si application to a non-stressed plant were evident in this study for both species due to an increase in plant growth explained by increases in Mn accumulation, Mn use efficiency, and pigments. This result reinforces the indication that Si may enhance the response of plants without stress due to an increased Mn accumulation, as verified for corn and wheat 33 , total phenols in wheat 34 , and PSII quantum efficiency and chlorophyll content in rice 66 .
Therefore, in general, Si can mitigate the damage caused by Mn deficiency in energy cane plants and improve the responses of both species to the micronutrient supply by increasing Mn absorption and the production of antioxidant compounds, pigments, and PSII quantum efficiency. This is in line with the hypothesis of this study, which proposed such effects of Si on both species and that it could be more evident in plants under stress, depending on species. Thus, these facts are confirmed in our study, as the responses were different according to species.
Finally, the beneficial relationship between Si and Mn was unveiled, and the indication of this beneficial element was proposed for the cultivation of sugarcane without Mn deficiency and energy cane with or without Mn deficiency to increase the sustainability of the cultivation of these species through a better management of micronutrient supply.

Conclusion
The sugarcane and energy cane are sensitive to Mn deficiency as it causes important biological damage. At the same time, both species are responsive to the application of this micronutrient by presenting improvements in the antioxidant and physiological metabolism and an increase in total phenols, GPOX activity, pigments, and PSII quantum efficiency.
The attenuation of effects of manganese deficiency by silicon depends on species and stress level, with a greater benefit for Saccharum spontaneum L. The action of this beneficial element reflects on increases in the accumulation of Mn, which reduces oxidative stress by increasing antioxidant compounds, regulating the activity of SOD, and increasing the chlorophyll content, PSII quantum efficiency, use efficiency of Mn, and growth. It is thus a tool to improve the performance of this species in soils deficient in Mn.

Material and methods
Experimental conditions and plant growth. Two experiments were carried out using the sugarcane S. officinarum L., variety RB 966928, and the energy cane S. spontaneum L., type I, variety VX3, in a greenhouse at the São Paulo State University (UNESP), Jaboticabal Campus, Brazil, from January to July 2019. Pre-sprouted seedlings from mini-cuttings (5 cm long) with one bud were planted in a polypropylene tray filled with fine vermiculite. Si was applied via fertigation after the full emergence of shoots at intervals of four days for 50 days and via a complete Hoagland and Arnon 67 solution with a change in the Fe-EDTA source to Fe-EDDHA. Subsequently, the plants received the nutrient solution with low Mn content (0.1 µmol L −1 ) for 32 days to induce deficiency. The seedlings were then transplanted to 1.5-dm 3 polypropylene pots filled with washed sand.
Experimental design and treatments. The treatments were applied from the seedling transplant. They consisted of a 2 × 2 factorial design with five replications arranged in random blocks for each species. The treatments consisted of plants grown under Mn sufficiency (20.5 µmol L −1 ) and deficiency (0.1 µmol L −1 ) associated with the absence or presence of Si (2.0 mmol L −1 ). The Si source was sodium and potassium silicate stabilized with sorbitol (SiNaKE) at 107.9 g L −1 of Si and 16.44 g L −1 of K 2 O. The Hoagland and Arnon 67 nutrient solution, with a modification in Mn concentration, was used. The pH value of the nutrient solution was adjusted to 5.5 ± 0.5 using solutions of 1.0 mol L −1 HCl or 1.0 mol L −1 NaOH. The potassium concentration of the nutrient solution for the treatments without Si was adjusted with potassium chloride. The average relative air humidity and temperature were measured inside the greenhouse for both experiments during the growing period: 15 and 62% of minimum and maximum relative air humidity and 17 and 36 °C of minimum and maximum temperature.
Biochemical and physiological evaluations were carried out on plants at the end of the experiments, 58 days after the treatments were applied.

Analyses. Mn and Si accumulation and Mn use efficiency.
The Mn content was determined by digesting 0.1 g of dry and ground material from shoots and roots in 1.5 mL of nitro-perchloric solution 2:1 under heating blocks at an initial temperature of 80 °C, increased each 30 min until reaching 210 °C. After that, the reading was taken of the diluted extract in water in atomic absorption according to the method proposed in 68 .
Si content was determined in shoots and roots using 0.1 g of dry and ground material, which were added to 50-mL polyethylene screw-cap centrifuge tubes. The samples were moistened with 2 mL of hydrogen peroxide (H 2 O 2 ) after washing the sides of the tube to remove the sample. The tube was tightly capped and placed in an oven at 95 °C. After 30 min, the tubes were removed, and 4 mL of 50% sodium hydroxide (NaOH) were added to warm samples. The sample tubes were then gently vortexed, capped tightly, and returned to the oven (95 °C) for four hours according to the methodology described in 69 . The Si concentration was determined by colorimetry using 1 mL extract plus 19 mL of water, 1 mL of HCl (1:1), and 2 mL ammonium molybdate. After 5 min, 2 mL oxalic acid were added. The reading performed by a spectrophotometer at 410 nm as described in 70 . The total Mn and Si accumulation was obtained by the sum of the product of the dry mass and the content of elements in shoots and roots. Mn use efficiency in shoots was determined according to 71 based on Mn accumulation.
Lipid peroxidation (MDA). Lipid peroxidation was determined using 0.3 g of the first fully expanded leaf 58 days after transplanting (DAT) and treatment application, according to the methods described in 72 . The concentration of malondialdehyde (MDA) equivalents was determined by reading on a spectrophotometer between 535 and 600 nm and the data was calculated based on the extinction coefficient of 1.55 × 10 −5 mol −1 cm −1 , with MDA results expressed in nMol g −1 of fresh matter 73 .
Protein extraction. The total soluble protein was extracted using 1.0 g of the first fully developed leaf collected at 58 DAT and homogenized in a cooled mortar and pestle containing 100 mM potassium phosphate buffer (pH 7.5), 1 mM ethylenediaminetetraacetic acid (EDTA), 3 mM DL-dithiothreitol, and 5% (w/v) insoluble polyvinylpolypyrrolidone in a 3:1 volume/fresh mass ratio 74 . The material was centrifuged at 10,000×g for 30 min, and the supernatant was stored at − 80 °C for later determination of activities of the SOD and GPOX enzymes. Protein concentration was determined using the 75 method with bovine serum albumin as a standard, expressed as mg mL −1 protein.
Superoxide dismutase (SOD) activity. The SOD activity (SOD, EC 1.15.1.1) was determined in a spectrophotometer, as described in 76 , and the reaction was conducted in a reaction chamber (box) under the lighting of a 15 W fluorescent lamp at 25 °C. The reading was performed at 560 nm, and the SOD activity was expressed as U mg −1 protein.
Total phenol content. Total phenolic compounds were extracted using 0.1 g of fresh fully expanded leaves collected at 59 DAT and concentrated methanol in a water bath at 25 °C. After extraction, a colorimetric reaction of total phenols was induced with the 2 N Folin-Ciocalteu reagent, allowing reacting for three minutes, and 20% sodium carbonate, allowing reacting for two hours. In the end, the absorbance was read on a spectrophotometer Scientific Reports | (2021) 11:16900 | https://doi.org/10.1038/s41598-021-96427-z www.nature.com/scientificreports/ at a wavelength of 765 nm, while the content was determined using a standard curve with gallic acid, expressed as g gallic acid equivalent (GAE) 100 g −178 .
Chlorophyll a, Chlorophyll b, and carotenoids. The pigment content was determined using 0.027 g of leaf discs taken from the middle third of the leaf blade completely expanded at 59 DAT, according to the methodology proposed by 79 . Readings at 663 nm for chlorophyll a (Chl a), 647 nm for chlorophyll b (Chl b), and 470 nm for carotenoids were performed on a Beckman DU 640 spectrophotometer. The contents were defined based on fresh mass.
PSII quantum efficiency. The PSII quantum efficiency (Fv/Fm) was determined by measuring chlorophyll fluorescence using a fluorimeter (Opti-sciences-Os30P). For this, the sampled region was submitted to the dark for adaptation at least 30 min before the excitation of the red light pulse of 1 s. Measurements were performed between 7:30 and 8:30 a.m. on the middle third of leaf + 1 (first complete leaf with visible sheath) at 58 DAT.
Growth. The leaf area of sugarcane and energy cane was measured at 62 DAT using a leaf area measuring device (L-3100, Li-Cor, EUA). The value was given in cm 2 . The dry mass was determined by cutting the plants at the substrate level and separating shoots from roots to compose the total dry mass. The fractions were washed with detergent solution (0.1% v/v), acid solution with 1.0 mol L −1 HCl (0.3% v/v), and deionized water. Subsequently, the samples were stored in paper bags and dried in a forced air circulation oven at 65 ± 2 °C until constant mass. The dry mass of the whole plant was obtained in grams.
Statistical analysis. The data were checked for normality (Shapiro-Wilk test) and homogeneity of variances (Levene's test) in software libre R 4.0.3 and subjected to analysis of variance by F-test using the, the means were compared by Tukey test at 5% probability, and the SAS statistical program (Cary, NC, USA). The interactions were sliced even when not significant and the elaborated graphs in SigmaPlot 14.0 (Systat Software, Inc, San Jose, CA).