Bioherbicides for sustainable barnyard grass management in paddy field: an in-silico perspective

Abstract Paddy (Oryza sativa) yield is greatly influenced by the insidious presence of rice-mimicking weed, widely known as barnyard grass. This study explores the promising natural ACCase inhibitors that could enhance paddy yield by controlling weeds. A total of 2828 natural compounds were examined using diverse computational techniques. The results of this study depict that CNP0390839 (xanthoangelol) exhibited a better XP Gscore (−7.328 kcal/mol) and MM/GBSA score (−84.24 kcal/mol) than other investigated compounds. Importantly, ACCase-xanthoangelol complexes was thermodynamically stable with an RMSD value of ∼1.2 nm. Of note, 72% xanthoangelol resides in the Angelica keiskei plant root which exhibits 55% weed-inhibitory action. The A. keiskei plant mainly inhibits the hypocotyl (71.8 ± 5.4%) and root region (55.3 ± 4.7%) of weeds. Moreover, the existence of dihydroxyphenyl scaffold in xanthoangelol was also witnessed in literatures for weed inhibitory action. Overall, xanthoangelol might prove to be an effective ACCase herbicide in paddy weed management. Graphical Abstract


Introduction
Paddy (Oryza sativa L.) is the major food source for more than half of the world's population.Globally, rice harvesting covers 150 million hectares, of which Asia accounts for about 90%.India is the world's leading paddy cultivator; however, its average rice production is 3.76 t/ha and ranking in 42nd position (Fahad et al. 2019).Barnyard grass (Echinochloa crus-galli) is the most troublesome post-emergence weed that often evades from manual or mechanical weeding can cause up to 57% of yield loss in paddy field (Bajwa et al. 2015).Target-specific herbicides play an inevitable role in post-emergence weed management.Precisely, monocot (grass) weeds can be controlled by targeting the CT domain of homomeric plastidic Acetyl-CoA Carboxylase (ACCase) enzyme.It is crucial for the production of fatty acids in plants, thus disrupting this enzyme leads to plant death.Though paddy resembles grass, natural detoxifiers including Cytochrome P450 and Glutathione-S-transferase (GST) in paddy rapidly metabolize herbicides into inactive compounds.Considering this benefit, ACCase herbicides can differentiate paddy from weeds (Dimaano and Iwakami 2021).Fenoxaprop-P-ethyl (FPPE) is one of the grass-selective ACCase inhibitors of rice field.However, consistent use of FPPE elicits weed resistance, residual effects, poor solubility and phytotoxic effects in rice plants.Herbicides from natural agrochemicals are devoid of persistent properties and also not harmful to non-target organisms (Shen et al. 2017).Moreover, the short environmental half-life of bioherbicides reduces environmental toxicology, conserves soil biodiversity and leads to sustainable weed management (Hasan et al. 2021).Thus, the present study intended to determine toxic-free ACCase inhibitors from natural sources using computational techniques.

Results and discussion
The 3-dimensional structure of ACCase protein was modelled using available yeast structure (PDB: 3TV5) with the aid of Schr€ odinger software (Figure S1).The predicted model was evaluated with the Ramachandran plot which reveals that 91.5% of amino acids were found in the favoured region (Figure S2a).The overall quality factor (OQF) of the ERRAT plot yielded a score of 89.936 confirms the model quality (Figure S2b).Further, the negative value of the Z-score (À7.95) implies a good quality protein model (Figure S2c) (Panda et al. 2019).It is to note that the template structure is found to exhibit higher similarity (54%) with HQ395759.1 sequence than the template structure used in the recent literature (Fang et al. 2020).This highlights that the modelled structure is much more reliable to work in the modelling environment.Further, Tanimoto coefficients (Tc) technique was implemented for ligand-based screening.A similarity between reference compound (FPPE) and 2828 natural compounds was observed using BulkTanimoto similarity from the RDKit package.The Tc values between 0.2 and 0.5 provides an ideal range for filtering out similar molecules.Considering this, the threshold value of 0.3 was set and yielded 1826 compounds.
Note that the Pearson coefficient was calculated between the XP Gscores and experimental IC 50 values of commercial ACCase herbicides was found to be 0.68 (Table S1).This signifies the accuracy of glide algorithm in predicting the herbicidal activity of the ACCase compounds.The Tc filtered compounds was subjected to hierarchical virtual screening (HTVS, SP and XP).The XP Gscore (À5.977 kcal/mol) of FPPE was used as a threshold which yielded 364 compounds (Table S1).Further, the XP resultant compounds were subjected to MM/GBSA analysis for calculating the binding free energy (DG).It is note that seven lead compounds exhibit the higher negative DG values (À90.08 to À80.26 kcal/mol) than the FPPE (À77.03 kcal/mol) (Table S3).
On the basis of the Tice rules, the seven hit molecules were compared for herbicidal likeness, and thus the five lead molecules have an optimal range of molecular descriptors (Table S4).Amongst, the compound xanthoangelol CNP0390839 (xanthoangelol) and its scaffold dihydroxyphenyl was widely reported for its herbicidal activity in literatures.Xanthoangelol is found in Angelica keiskei plant and 72% of the compound resides in its plant roots (Aulifa et al. 2020).Moreover, 55% of the A. keiskei plant root inhibits the hypocotyl (71.8 ± 5.4%) and root (55.3 ± 4.7%) regions of weeds (Sunohara et al. 2015).In essence, the existence of dihydroxyphenyl scaffold in xanthoangelol was also reported for their herbicidal action (Javaid et al. 2010).Further, hydrogen bond (H-bond) interaction in both complexes indicates protein-ligand stability (Figure S3&S4).Notably, ASN373 residue forms H-bond in both complexes.In addition, THR320 and ALA413 form H-bond with hit and THR318 form H-bond with reference (Table S5).Moreover, solvent-exposed ligands stabilize protein-ligand complexes by interacting with water molecules (Trisciuzzi et al. 2019).Here, the solventexposed scaffold of xanthoangelol form H-bond with THR320 residue of ACCase signifying the solvent-friendly binder (Figure S3).
Finally, molecular dynamics was performed to illustrate the atomic movement of atoms in a protein-hit complex (Panda et al. 2019).The RMSD plot of the hit (xanthoangelol) complex was stable throughout the simulation (RMSD $1.18 nm).In contrast, the FPPE complex greatly fluctuated until $85ns (RMSD $1.35 nm), at the end, the line subsided quickly around $90ns (RMSD $1.18 nm) (Figure 1).In RMSF, 61.5% of active site residues of hit complex were exhibiting minimum deviation (<0.1 nm) (Figure S5).Notably, ASN373 residue in the hit complex (0.0279 nm) was remarkably stable than the native complex (0.0364 nm).Moreover, the covariance matrix of the hit complex signifies the positive correlation (red colour) of proteins while in the native complex it is vice versa (Figure S6a, b).Similarly, the 2D projection plot of the hit complex occupied minimal space than FPPE (Figure S6C).In FEL analysis, both complexes showed similar kinds of global energy minima conformation (Figure S7).Thus, the dynamics findings revealed that the xanthoangelol was thermodynamically stable and rigid.A study by Ye et al. (2018) synthesized and docked series of cyclohexanedione derivatives and found 3-(2,4-Dichlorophenoxyacetyloxy)-2-Phenyl-3-Cyclohexen-1-One compound has the best inhibitory activity against barnyard grass (CDocker energy ¼ À9.87 kcal/mol).Yan et al. (2019) reported that 2-(4-aryloxyphenoxy) propionate derivatives had better lipid solubility and herbicidal activity against barnyard grass in biological assays and docking studies (À7.60 kcal/mol).Despite the higher efficacy of chemical herbicides against barnyard grass, its long-term usage poses a serious threat to the environment (Dayan and Duke 2014).Therefore, ligand-based screening strategy led to the identification of xanthoangelol as an effective FPPE herbicide with minimal effects on the environment.Overall, this study highlights that xanthoangelol as a bioherbicide has a potential allelopathic activity against ACCase barnyard yard grass.

Conclusion
The ongoing toxicological effects of commercial herbicides emphasize the importance of designing the bioherbicides for effective paddy-weed management.An intensive computational approach was used to identify promising ACCase herbicides from 2828 natural compounds against E. crus-galli weed.Priorly, tanimoto coefficient removed non-identical compounds and molecular docking filters 394 compounds with better affinity than FPPE.Further, MM/GBSA technique resulted seven lead molecules by reducing false positives.To ensure the herbicidal property of lead molecules, physiochemical properties were analysed and ultimately five lead molecules were agreeable with the Tice rules.Interestingly, the literature evidence divulged that the compound xanthoangelol has a remarkable herbicidal activity against weeds.Moreover, the Hbond formation and solvent exposure of xanthoangelol was consistent with FPEE-ACCase complex.In essence, a dynamic study unveiled that hit complex was stabled throughout the simulation.Altogether, xanthoangelol, a natural compound, can lay the promising foothold for a sustainable paddy-weed management in the near future.