Natural lignin modulators improve bagasse saccharification of sugarcane and energy cane in field trials

The burgeoning cellulosic ethanol industry necessitates advancements in enzymatic saccharification, effective pretreatments for lignin removal, and the cultivation of crops more amenable to saccharification. Studies have demonstrated that natural inhibitors of lignin biosynthesis can enhance the saccharification of lignocellulose, even in tissues generated several months post‐treatment. In this study, we applied daidzin (a competitive inhibitor of coniferaldehyde dehydrogenase), piperonylic acid (a quasi‐irreversible inhibitor of cinnamate 4‐hydroxylase), and methylenedioxy cinnamic acid (a competitive inhibitor of 4‐coenzyme A ligase) to 60‐day‐old crops of two conventional Brazilian sugarcane cultivars and two energy cane clones, bred specifically for enhanced biomass production. The resultant biomasses were evaluated for lignin content and enzymatic saccharification efficiency without additional lignin‐removal pretreatments. The treatments amplified the production of fermentable sugars in both the sugarcane cultivars and energy cane clones. The most successful results softened the most recalcitrant lignocellulose to the level of the least recalcitrant of the biomasses tested. Interestingly, the softest material became even more susceptible to saccharification.


| INTRODUCTION
Biofuels are recognized as a clean, economically viable energy alternative to combat the greenhouse effect and global warming, phenomena that are now evident worldwide.They can be used as an energy source for vehicular movement, replacing fossil fuels either partially or entirely, and can be derived from energy-rich plants such as sugarcane (Saccharum spp.;GranBio, 2020).Ethanol biofuel (bioethanol) from sugarcane can be directly produced from sucrose and starch (first-generation ethanol) or from lignocellulosic biomass (second-generation ethanol, 2G ethanol).Lignocellulosic biomass consists of cellulose fibers embedded in an amorphous matrix of polysaccharides and lignin.In addition to supporting plant growth, lignin serves as a natural defense against microbial and enzymatic attacks.Biofuels derived from lignocellulosic biomass represent a potential renewable, nonpolluting, and sustainable energy source.Consequently, the cultivation of sugarcane clones with increased lignocellulosic biomass has become a focus of breeding programs.
The Inter-University Network for the Development of the Sugar-Energy Sector (RIDESA: Rede Interuniversitária para o Desenvolvimento do Setor Sucroenergético) initiated a hybridization program involving S. spontaneum, S. robustum, and commercial cultivars (Saccharum ssp.) to develop clones with fiber content exceeding 17% while preserving the existing 13% sucrose content (Ming et al., 2006;Ramos, Brasileiro, Kist, et al., 2017;Ramos, Brasileiro, Silveira, et al., 2017;Silveira et al., 2015).The species S. spontaneum and S. robustum exhibit high resistance to pests and diseases, robust vigor, elevated fiber content, and a high tillering capacity (Carvalho-Netto et al., 2014;Matsuoka et al., 2014).The breeding program yielded a more robust cane characterized by increased fiber content, enhanced productivity potential, and a greater number of cutting and regrowth cycles.This product, referred to as "supercane" or "energy cane" (Bonomi, 2017;Grassi & Pereira, 2019), is deemed ideal for biofuel production.The robust supercanes developed by entities such as GranBio, Vignis, the Instituto Agronômico de Campinas (IAC), and RIDESA are recognized as a means to augment the yield of the nation's sugarcane fields, thereby facilitating the production of 2G ethanol and enhancing energy cogeneration.Besides high fiber yield per unit of cultivated area, energy cane exhibits high cold tolerance, reduced fertilizer and water requirements, and a lower replanting rate, sustaining high productivity through at least eight cutting stages (Bischoff et al., 2008).Energy cane is identified as an optimal crop for the sustainable production of biofuels (Kim & Day, 2011;Qiu et al., 2012).
The primary obstacle in large-scale ethanol production from lignocellulosic biomass is the technological challenge of deconstructing plant biomass (specifically, lignin in the cell walls) to release sugars for fermentation into ethanol.Lignocellulose, with its complex and resistant structure, necessitates a pretreatment phase before enzymatic hydrolysis and subsequent fermentation for ethanol can occur.The primary goal of pretreatment is the delignification of plant biomass, as discussed in Mohapatra et al. (2017).This process also enhances cell wall porosity, increases the surface area of cellulose for enzymatic hydrolysis, and removes more intricate polysaccharides such as hemicelluloses and pectins for separate processing to boost efficiency.Lignin, a polymer composed of various monomeric units connected by chemically diverse and low-reactivity bonds, obstructs the access of exogenous enzymes needed to hydrolyze cellulose (Weng et al., 2008).Conversely, the by-products of lignin degradation through chemical treatments inhibit enzymatic action during fermentation, reducing biofuel production yield (Simmons et al., 2010).Therefore, the conversion of lignocellulosic biomass into biofuel is a relatively intricate process that necessitates investment in alternative strategies to reduce cell wall rigidity and facilitate enzyme access for cellulose breakdown into hexoses for subsequent fermentation (Vanholme et al., 2010).
Efforts have been invested to enhance the accessibility of cell wall cellulose in various grass species (Marriott et al., 2014;Mohapatra et al., 2017), as well as in sugarcane (Bottcher et al., 2013;Figueiredo et al., 2019;Isaac et al., 2018;Martarello et al., 2021;Mishra & Ghosh, 2019;Miyamoto et al., 2018;Mota et al., 2021;Zhang et al., 2018).The goal is to improve lignocellulose digestibility.However, several factors influence lignocellulose digestibility, including the content of lignin and suberin, cross-linking with hemicellulose, porosity, surface area, the ratio of lignin monomers, crystallinity, and the degree of polymerization.These factors complicate the access of cellulolytic enzymes to the polysaccharides in the cell wall (Pu et al., 2013).
Lignin significantly hinders cellulose saccharification, making it a primary focus of research in the past decade.Studies have aimed to modify the regulation and structure of lignin to boost saccharification efficiency without compromising yield (Martarello et al., 2021;Mota et al., 2021).Saccharification yield, which is indicative of the quantity of fermentable sugars produced through enzymatic hydrolysis of cell wall polysaccharides within a specific timeframe, is a critical measure in these studies.
The compound's activation with coenzyme A readies it for ester linkage to lignin and polysaccharides, and for reduction to form p-coumaraldehyde, ultimately leading to alcohols (monolignols).A crucial regulatory step involves the transesterification of p-coumaroyl-CoA with shikimate from the shikimate pathway to yield p-coumaroyl-shikimate (Marchiosi et al., 2020), which is hydroxylated at carbon 3, forming caffeoyl-shikimate.Subsequently, another transesterification releases caffeoyl-CoA.Caffeoylshikimate can alternatively be released free as caffeate.The production of the catechol caffeoyl ester through a shikimate-dependent linkage is believed to regulate the carbon flux through the phenylpropanoid pathway, but only when shikimate pathway products are available.This typically occurs when the primary metabolism (aromatic amino acids) is functioning optimally (Marchiosi et al., 2020).Caffeoyl-CoA is then methoxylated at carbon 3 to produce feruloyl-CoA.In turn, free caffeate also can be converted into ferulic acid and subsequently activated to form feruloyl-CoA by 4-CL.Feruloyl-CoA can either be ester-linked to cell wall components or converted into coniferaldehyde by the enzyme coniferaldehyde dehydrogenase (CALDH).Coniferaldehyde is then hydroxylated to 5-hydroxyconiferaldehyde and further methoxylated at hydroxyl in carbon 5 to render sinapaldehyde.Finally, p-coumaraldehyde, coniferaldehyde, and sinapaldehyde are reduced to produce their respective alcohols: p-coumaryl, sinapyl, and coniferyl alcohol.
In previous reports, we have shown that several enzyme inhibitors of the phenylpropanoid pathway such as nitecapone (Parizotto et al., 2021), entacapone (Parizotto et al., 2020), and naringenin (Ferro, Parizotto, et al., 2020) applied in maize plants in an appropriate range of concentrations are capable to increase saccharification of maize lignocellulose without impairing growth or biomass production.
Benzohydrazide (BHZ) was selected from a set of phenolic derivatives screened in bench-scale for their ability to induce lignocellulose saccharification without affecting productivity.We then tested it in field-scale unrevealing that young maize crops (30 days old) sprayed with BHZ induce a long-term change in lignocellulose that turn mature plants lignocellulose substantially prone for saccharification with or without pretreatment for removal of lignin (HPAC) with any impact on maize productivity (Martarello et al., 2023).
We also had reported that PIP, MDCA, and DZN sprayed in young crops of sugarcane, Brachiaria, and soybean induced long-term improvement on saccharification of lignocellulosic residues of these mature harvested crops, with no prejudice to plant health, productivity, or average lignin content (dos Santos et al., 2023).We conducted an anatomical investigation that indicated that PIP induced a tissue-specific change, which increases lignin content in fiber and xylem, and decreases it in parenchyma cell walls.
Here, we examine the effect of PIP, MDCA, and DZN on bagasse saccharification of two commercial sugarcane cultivars and two energy cane clones aiming to assess the potential and limits of these compounds as active principles for formulating agrochemicals able to contribute with the nascent industry of cellulosic ethanol.

| MATERIALS AND METHODS
2.1 | Planting of Saccharum spp.

cultivars and clones of energy cane
Commercial cultivars RB867515 and RB966928 of Saccharum spp., along with clones PRBIO 130 and PRBIO 172 of energy cane, were procured from RIDESA.These sugarcane stalks, each with 3-cm axillary buds, were planted in trays filled with a mixture of commercial substrate MCPlant and filter cake from a sugar mill in a 3:1 ratio.The stalks were harvested from plants that had been cultivated for 10 months and were at the same maturity level.PRBIO 172, a "Type I" energy cane, contains 18% fiber, while PRBIO 130, a "Type II" clone, contains 23% fiber.
Twenty days of postbud germination, seedlings underwent evaluation for leaf count and color, tillering, and root quality.Those exhibiting favorable conditions were subsequently transplanted to the experimental field of the Technical Center for Irrigation (CTI; 23°25′57" S, 51°57′08" W, and 542 m).The planting process was executed in three blocks, each comprising eight prefertilized and prepared furrows.Within each furrow, five seedlings from each genotype were individually planted, maintaining a distance of 1.5 m between each.Each block was planted with 40 seedlings from cultivars RB966928, RB867515, and clones PRBIO 172 and PRBIO 130.

| Spray of enzyme inhibitors of the phenylpropanoid pathway
Sixty days of postplanting, the leaves of the plants were sprayed with inhibitors of the phenylpropanoid pathway: methylenedioxy(cinnamic) acid (MDCA; at concentrations of 1.0 and 2.0 mmol L −1 ), piperonylic acid (PIP; at concentrations of 0.25 and 0.50 μmol L −1 ), and daidzin (DZN; at concentrations of 1.0 and 2.0 mmol L −1 ).The application was performed using a hand sprayer, with a canvas curtain placed between the furrows to prevent the inhibitors from contaminating the environment or affecting other plants.A set of five plants was left untreated to serve as a control group.
Following the application of the inhibitor, five stalks measuring 50 cm each were collected from five clumps of each genotype (RB867515, RB966928, Type I-PRBIO 172, and Type II-PRBIO 130) 180 days later.This resulted in a total of 25 stalks for each sugarcane genotype.A manual machine was used to extract the broth from these 25 stems to determine the Brix degree.The average biometry of the two sugarcane cultivars (RB966928 and RB867515) and of the two energy cane clones (PRBIO 172 and PRBIO 130) is shown in Table 1.
The bagasse underwent drying in a forced circulation oven at 55°C until it was completely dry.Once the material achieved a constant mass, it was milled again using a knife mill to ensure homogeneity.Subsequently, the material was subjected to further milling in a ball mill to reduce it into smaller particles.

| Extraction of soluble sugars
The dried ground biomass was encased in small TNT bags (2.5 × 2.5 cm) and subjected to a minimum of 8 h of reflux in a Soxhlet apparatus with 80% ethanol at 80°C to eliminate soluble sugars.The phenol-sulfuric method (Dubois et al., 1956) was employed to assess the efficacy of sugar extraction.A portion of the supernatant was isolated and allowed to react for 15 min.Following incubation, absorbance was measured using a spectrophotometer at 490 nm.Complete extraction of soluble sugars was confirmed when the absorbance reached zero.The TNT bags with the samples were then dried in an oven at 70°C for a minimum of 24 h, after which the biomass was classified as alcohol-insoluble residue (AIR).

| Enzymatic saccharification
The commercial enzyme blend, NovoZyme (NS22086), which is abundant in cellulase, was employed for enzymatic saccharification.This enzyme was diluted twofold with a sodium acetate buffer (pH 5.0).The assessment of the liberation of reducing sugars from the cell wall was conducted using 10 mg of AIR.This was placed in 2-mL microtubes, each containing 50 mM sodium acetate buffer (pH 5.0), 2% sodium azide, and 20 U of the enzyme.
The samples underwent incubation in a water bath at 50°C for durations of 4 and 24 h.Aliquots of 50 μL were extracted at both the 4-h and 24-h incubation marks.The enzymatic action's resultant reducing sugars were quantified through a reaction with 1% DNS (3.5 dinitrosalicylic acid), as per Bernfeld (1955).The readings were conducted at 540 nm using a spectrophotometer (Spectrum Meter SP-2000 UV).The saccharification was expressed in mg of reducing sugars g −1 bagasse.

| Lignin quantification and monomeric composition
The quantification of lignin was determined using the acetyl bromide method.The alcohol-insoluble residue (AIR) from each sample, weighing 0.3 g, was subjected to wall protein extraction.This process involved thoroughly washing the AIR with a phosphate buffer (50 mM, pH 7.0), 1% Triton® (v/v) prepared in a phosphate buffer (pH 7.0), 1.0 M NaCl in a phosphate buffer (pH 7.0), distilled water, and acetone.The resulting precipitate was flocculated under vacuum conditions for 24 h.The product of this process is referred to as the protein-free cell wall fraction, as outlined by Ferrarese et al. (2002).
The protein-free wall fraction, weighing 0.002 g, was subjected to lignin quantification through acid hydrolysis, facilitated by 25% acetyl bromide in ice-cold acetic acid.The reaction involved the use of 50 μL of 25% acetyl bromide solution in acetic acid, followed by incubation in a dry bath for 30 min at 70°C.Subsequently, the samples were cooled on ice for 2 min and neutralized with 90 μL of 2 M NaOH.The addition of 10 μL of hydroxylamine ensued, and the lignin was solubilized with 450 μL of icecold acetic acid.After centrifugation, the supernatant was diluted fivefold in ice-cold acetic acid.Spectrophotometric readings were then conducted at a wavelength of 280 nm, and the Lambert-Beer law was applied for calculations.Monolignols were extracted via alkaline nitrobenzene oxidation (Dean, 1997) and subjected to reverse-phase chromatography analysis.The content of lignin monomer was expressed in μg monomer mg −1 of the protein-free cell wall.

| Data analysis
Lignin quantification and enzymatic saccharification analysis were conducted using the GraphPad Prism® (Version 6.0) and Statistica® software, employing the Dunnett test with a significance level of p ≤ 0.05.The reported values represent the mean of five repetitions for each treatment within each genotype.

| RESULTS
The sugarcane bagasse contains a high and variable content of free, reducing sugars (see Tab. 1, Brix%).To prevent these free sugars from interfering with the determination of released sugars, they were extensively removed with 80% ethanol prior to saccharification.The application of varying concentrations of MDCA, PIP, and DZN to plant leaves enhanced the biomass saccharification of cultivar RB966928 when using the commercial Novozymes enzyme pool (20 U) for a 4-h period (Figure 1).Notably, DZN 2.0 mmol L −1 , PIP 0.25 and 0.5 μmol L −1 , and MDCA 1.0 and 2.0 mmol L −1 increased the saccharification of the lignocellulosic biomass of clone PRBIO 172.The lignocellulosic biomass of cultivar RB867515 exhibited a significantly higher release of reducing sugars following treatments with DZN 1.0 mmol L −1 and MDCA 1.0 mmol L −1 .However, the saccharification of the lignocellulosic biomass from clone PRBIO 130 did not improve after the application of MDCA, PIP, or DZN during the initial 4 h of incubation.
An increase in enzymatic saccharification was observed in the lignocellulosic biomass of clone PRBIO 130 treated with PIP 0.5 μmol L −1 and MDCA 1.0 mmol L −1 , following 24 h of bagasse saccharification (Figure 1b).Treatment with DZN 1.0 mmol•L −1 also increased saccharification by 42.40% in cultivar RB867515 when incubated for 24 h.Saccharification over 24 h also enhanced enzymatic saccharification in the lignocellulosic biomass of sugarcane cultivar RB966928 treated with DZN 1.0 mmol L −1 , PIP 0.5 μmol L −1 , and MDCA 1.0 and 2.0 mmol L −1 .The highest enzymatic saccharification was achieved when cultivar RB867515 was exposed to DZN 1.0 mmol L −1 and incubated for 24 h.However, 24-h saccharification did not alter the saccharification in clone PRBIO 130's lignocellulosic biomass.In cultivar RB966928, saccharification increased 47.7% in plants treated with DZN 1.0 mmol L −1 ; 109.93% with PIP 0.50 μmol L −1 ; 106.57% with MDCA 1.0 mmol L −1 ; and 77.20% with MDCA 2.0 mmol L −1 .Saccharification was promoted in clone PRBIO 130 treated with PIP 0.50 μmol L −1 and MDCA 1.0 mmol L −1 , with increases of 68.00% and 65.20%, respectively.For PRBIO 172, there was no significant increase in the saccharification under different treatments.Despite the varying effects of the three inhibitors on different plants, the release of reducing sugars was generally higher in lignocellulosic plants treated with inhibitors, when incubated for 4 h.
Figure 2 illustrates the lignin content in sugarcane bagasse derived from the culms of cultivars RB867515 and RB966928, as well as energy cane clones PRBIO 130 and PRBIO 172, 180 days of postleaf-spray with phenylpropanoids pathway inhibitors.Piperonylic acid did not significantly alter the lignin content in any of the genotypes.However, MDCA notably decreased lignin levels in both RB cultivars and clone PRBIO 172.Daidzin, on the other hand, only reduced the lignin content in cultivar RB966928.
The PRBIO 130 clones exhibited no reduction in lignin content.Instead, lignin increased with treatment with MDCA 1,0 μmol L −1 .The application of PIP at concentrations of 0.25 and 0.5 μmol L −1 resulted in a decrease in lignin content in the bagasse of cultivar RB867515 and energy cane clone PRBIO 172.Additionally, PIP at 0.25 μmol L −1 significantly reduced the lignin content in the lignocellulosic biomass of cultivar RB966928.Although the traditional nitrobenzene method used to measure S/G ratio cannot distinguish between guaiacyl (G) and feruloyl (FA) residues linked to cell wall, which are particularly significant in sugarcane, nitrobenzene analysis indicated an increased S/G ratio in energy cane clone PRBIO 130 (Figure 3).However, it had no impact on the S/G ratio of energy cane clone PRBIO 172.The analysis also suggested that nearly all treatments reduced the S/G ratio in cultivar RB966928, but none affected the S/G ratio in sugarcane cultivar RB867515.In PRBIO 130, the significant increase in the S/G ratio resulted from a slight tendency for all treatments to reduce G (or FA) and increase S. In contrast, the reduction in the S/G ratio observed in cultivar RB966928 was due to a slight tendency to increase G (or FA) and decrease S. In RB867515 and PROBIO 172, where the S/G ratio was unaffected by neither treatment, both G and S monomers remained unaltered across all treatments.

| DISCUSSION
The three inhibitors of lignin synthesis, MDCA, PIP, and DZN, were able to enhance the saccharification of the lignocellulosic biomass.However, this effect was contingent upon the genetic makeup of the plant.The greatest saccharification of lignocellulosic biomass did not correspond with the reduction in lignin content observed.Despite no detectable decrease in lignin content, DZN 1.0 mmol L −1 and MDCA 1.0 mmol L −1 facilitated an increase in the saccharification of lignocellulosic biomass from the RB867515 cultivar.Similarly, PIP 0.5 μmol L −1 and MDCA at concentrations of 1.0 and 2.0 mmol L −1 also led to an increase in saccharification of lignocellulosic biomass from the RB966928 cultivar, again without a corresponding reduction in lignin content.Furthermore, DZN 2.0 mmol L −1 and MDCA at concentrations of 1.0 and 2.0 mmol L −1 enhanced the digestibility of lignocellulosic biomass from the energy cane clone PRBIO 172, even in the absence of lignin content reduction.
Preliminary assays were conducted to select inhibitors and determine their optimal dose/effect on saccharification at a bench scale (e.g., Ferro et al., 2020;Martarello et al., 2021).These assays revealed that at higher (toxic) concentrations, the inhibitors significantly reduced lignin content (dos Santos et al., 2008).However, at doses compatible with normal growth-those selected for field assays-the effect on lignin content was minimal.A field test on sugarcane cultivar SP803280 indicated that at concentrations conducive to normal growth and development, the inhibitors might decrease lignification in parenchymatic cells while increasing lignin in fibers.This could result in a negligible impact on total lignin, potentially leading to a net increase in lignin.This trend was observed in energy cane clones treated with daidzin.Similar tissue-specific effects of the inhibitors were also noted in core (S, G, and H) and noncore (e.g., ferulic and p-coumaric acids) lignin monolignols that are ester-linked to cell wall polymers (dos Santos et al., 2023).
The observed differences in saccharification among cultivars, the nature of inhibitors, and doses at identical time intervals of enzymatic saccharification (4 or 24 h) underscore the complex and inadequately understood interaction between inhibitors and plant phenotypes.We believe that plant phenotypes possess significant adaptability, enabling them to withstand environmental stresses throughout their lifecycle.The disruption of the phenylpropanoid pathway appears to initiate one of these enduring responses to environmental stimuli, as evidenced by the increase in saccharification in tissues synthesized long after the plant's exposure to the compounds.Generally, we noted that in the more recalcitrant plants, such as RB966928, the gains in saccharification following treatment can be more substantial.
This study did not subject plant biomasses to ex vivo pretreatments for lignin removal.However, evidence suggests that applying a chemical pretreatment to biomass from plants treated in vivo results in higher conversion than biomass untreated in vivo.Biomass from plants treated both in vivo and ex vivo can achieve complete saccharification in less time (Martarello et al., 2021).A comparison of the saccharification of control plants revealed that PRBIO 172 exhibited a lignocellulose more susceptible to saccharification (Figure 1).Treatment with lignin inhibitors enhanced this characteristic, enabling the production of reducing sugars to reach a saccharification threshold of approximately 250 mg g −1 in just 4 h.This value did not increase with an additional 20 h of saccharification, except in the case of control plants, which achieved the saccharification level of the treated plants (Figure 2).These findings suggest that in vivo treatments with lignin inhibitors could be employed to decrease the enzyme load or time required for complete saccharification of biomass in the bioethanol industry.
Among the cultivars evaluated, cultivar RB867515 exhibited the most recalcitrant biomass, as determined by comparing the saccharification of untreated plants.All inhibitors amplified the production of reducing sugars after a 4-h saccharification period.However, the saccharification rate was inferior to that observed in PRBIO 172, resulting in a continuous increase in the amount of reducing sugar over the 24-h saccharification period.Despite this, the reducing sugar levels observed in PRBIO 172 were achieved through the saccharification of biomass from plants treated with PIP 0.5 μmol L −1 and MDCA.Consequently, it can be inferred that in vivo treatments with PIP 0.5 μmol L −1 and MDCA transformed the most recalcitrant lignocellulose among the tested plants into a material with recalcitrance comparable to the most susceptible material evaluated.However, the saccharification of the most susceptible lignocellulose examined showed further improvement.
The behavior of PRBIO 130 exhibited significant idiosyncrasies.The majority of treatments involving lignin inhibitors resulted in a decrease in the release of reducing sugars following a 4-h digestion period.However, after 24 h, the production of reducing sugars in the treated material, on average, exceeded the control values, achieving the highest recorded values of reducing sugars in the entire assay (PIP 0.5 μmol L −1 and MDCA 1.0 mmol L −1 ; Figure 1b).The underlying causes of this phenomenon remain unclear, but it may be associated with the fiber content, which was higher in PRBIO 130 (23%) compared with PRBIO 172 (18%).Thus, PRBIO 130 may necessitate extended incubation periods for enzymes to effectively release sugars from lignocellulose.
The cultivar RB867515, which was the second most resistant material tested, generally showed insensitivity to the treatments.However, a significant deviation was noted in the treatments involving 1.0 mmol L −1 daidzin.This treatment notably stimulated the release of reducing sugars by 43.80% after 4 h and 26.80% after 24 h of saccharification.
We have been testing several lignin modulators that are unique in their nature and targets.They commonly inhibit enzymes of the phenylpropanoid pathway and are rapidly metabolized in both plants and soil.Despite this, they persistently enhance saccharification (up to 10 months of postapplication) in a diverse range of plant species, including soybean, Brachiaria decumbens, sugarcane (dos Santos et al., 2023), and maize (Martarello et al., 2023).Consequently, these inhibitors could play a significant role in the burgeoning cellulosic ethanol industry.
Several mechanisms impact lignocellulose saccharification such as lignin size; cross-linkage with hemicellulose through ferulic acid; the content of tricin, ferulic, p-coumaric acids linked to lignin and to hemicellulose; the content suberin; ratio of lignin monomers; microfibril crystallinity and so on.Saccharification is an emergent property of many factors and one or more of them may be altered in tissue-specific manner after the contact of the plants with active principles that improve saccharification.Ongoing research is being conducted to enhance our understanding of the mechanisms through which lignin inhibitors improve the saccharification of lignocellulose as well as to assess the putative tissue-specific modifications observed in previous study.

| CONCLUSION
The use of enzyme inhibitors, specifically MDCA, PIP, and DZN, in the lignin biosynthesis process has been found to improve lignocellulose saccharification in commercial cultivars of Saccharum spp.(RB867515 and RB966928) and the energy cane clone PRBIO 172.The agroindustrial application of these compounds in the near future could potentially reduce both the quantity of enzymes required and the associated costs for producing cellulosic ethanol and other biorefinery products.

AUTHOR CONTRIBUTIONS
CAM, WDS, and VFO conceptualized the whole study.VFO, CAM, HZN, and WDS selected and collected plant material for the present study.HZN supervised the planting and field experiments.VFO, LF, and GOC cultivated, treated, and harvested the.VFO, LF, and GOC carried out the extraction of soluble sugars, enzymatic saccharification, and lignin quantification.VFO, CAM, and WDS assisted in the data analysis.CAM, WDS, HZN, and MFPSM supervised the project and data analysis, discussed results, and contributed toward the drafting of the manuscript.All authors read and approved the final manuscript.

total of 25 stalks of each sugarcane genotype)
Biometry of the two sugarcane cultivars (RB 966928 and RB 867515) and of the two energy cane clones (PRBIO 172 and PRBIO 130).
T A B L E 1