Metabolomic profiling of C. canadensis at various timepoints post caprylic acid (CAP) treatment versus the untreated control.

At 4 h after foliar application of CAP treatment (625 μM) and untreated control, a total of 2 g of C. canadensis leaves were collected for analysis by GC-MS. (A) The heatmap analysis of significantly different metabolites The complete list of perturbed metabolites is in Supplemental Table 2S, Student’s t-test p valure<0.05; (B) The top ten pathways affected KEGG Metabolic pathway by CAP treatment after proteomic and metabolomic co-analysis. C: metabolomics and proteomics data correlation pathway analysis. (D)The occurrence frequencies of the top nine genes involved in the top eight KEGG pathways were key indicators of the differences between CAP-treated samples and untreated controls. The occurrence frequencies of the genes increased by 20% and appeared at least once in the KEGG pathways, according to the standard calculating module.

CcSHMT1 is a typical PLP enzyme

(A) Multiple sequence alignment of SHMTs. The sequences in the boxes are conserved amino acid residues. Source species of SHMT proteins and their GenBank accession numbers: BdSHM1, Brachypodium distachyon, XP_003559966.1; CcSHM1, Conyza canadensis, MN586851.1; HaSHM1, Helianthus annuus, XM_022184227.1; NtSHM1, Nicotiana tabacum, XP_016446471.1; HvSHM1, Hordeum vulgare, KAE8805790.1; AtSHM1, Arabidopsis thaliana, NC_003075.7; OsSHM1, Oryza sativa. (B) A molecular phylogenetic tree of SHMT gene based on amino acid sequences with homologous proteins from other species (neighbor-joining method, 1, 000 replicates). The sequences were obtained from GenBank. Source species of SHMT proteins and their GenBank accession numbers. (C) The subcellular Localization of CcSHMT1, the bar=10 μm. (D) The homology modeling structure of CcSHMT1.

CcSHMT1 gene expression, protein abundance, and enzyme assays of CAP treatment versus the untreated control

(A) Relative expression of the top five genes in C. canadensis following CAP treatment. Primer-Q were designed by DOMAN 7 according to the conserved sequences from NBCI BLAST; SHMT1, FBA5, PGK3, PGK2, PGM1 and GLO1 were had homologous genes from Arabidopsis; the mRNA was extracted from C. canadensis 30 min after CAP treatment (625 μM). (B) Relative expression of CcSHMT1 in C. canadensis under CAP and Vanillic acid (VA) treatments. The mRNA was extracted from C. canadensis 0, 0.5, 1, 2, 4, and 8 h after CAP and VA treatment (625 μM). (C) Effect of CcSHMT1 concentration in vivo under CAP and VA stress. The CcSHMT1 protein was extracted from C. canadensis leaves 0, 0.5, 1, 2, 4, and 8 h after CAP and VA treatment (625 μM). The protein levels were measured with a SHMT ELISA Kit according to 5 the manufacturer’s instructions. (D) Effect of CcSHMT1 activity in vivo under CAP and VA stress. The CcSHMT1 protein was extracted from the C. canadensis leaves after CAP and VA treatment (0, 78.125, 156.25, 312.5, 625, 1250 μM). The SHMT activity was assayed based on the product rate of benzaldehyde. (E) The effect of CcSHMT1 activity in vitro under CAP and VA stress. Recombinant CcSHMT1 was purified using Ni2+-chelating columns. SHMT activity was assayed by the product rate of benzaldehyde 0.5 h after CAP and VA treatment (0.63, 0.95, 1.26, 1.58, 1.90, 2.53, 3.16, 6.32, 9.48, 12.64, 15.80, 18.96, 22.12, 25.28, 28.44, 31.60, 34.76, 37.92, 41.08, 44.24 μM). (F) Kinetic analysis of CcSHMT1 in vitro under CAP and VA stress. Recombinant CcSHMT1 proteins were purified with Ni2+-chelating columns, and kinetic analyses for DL-β-phenylserine were performed (1 mM, 1.25 mM, 2.00 mM, 2.5 mM, 5 mM, 10 mM of DL-β-phenylserine). CAP: caprylic acid, VA: Vanillic acid. Each independent measurement was repeated at least three times. Data are the mean±standard deviation (s.d.). Statistically significance was at p-value < 0.05 according to the Student’s t-test, *P<0.05, **P<0.01, ***P<0.001 and NS, not significant (P>0.05).

The CcSHMT1-overexpressing and WT in Arabidopsis and rice under CAP treatment versus the untreated control

(A) The two lines of CcSHMT1-overexpressing Arabidopsis plants (AtCcT1OE1 and AtCcT1OE2) and WT 7 days after 316 μM CAP treatment. (B) The inhibition rate of CcSHMT1-overexpressing and WT seedlings treated with CAP (189.6, 316, 505.6, 632 and 1264 μM). The inhibition rate was calculated after 7 days relative to the untreated control plants (Inhibition rate % = (Fresh Weight of untreated control plants-Fresh Weight of treatment plants)/ Fresh Weight of untreated control plants * 100%). The bioactivity assays comprised three biological replicates, each with three technical replicates. (C) The SHMT ac in CcSHMT1-overexpressing and WT Arabidopsis seedlings under CAP treatment (189.6, 316, 505.6, 632 and 1264 μM). (D) The two lines of CcSHMT1-overexpressing Nipponbare plants (OsCcT1OE20 and OsCcT1OE26) and WT 7 days after 2.78 mM CAP treatment. (E) The inhibition rate of CcSHMT1-overexpressing and WT seedlings treated with CAP (0.69, 1.39, 2.78, 4.17, 8.34 and 11.12 mM). The inhibition rate was calculated after 3 days relative to the untreated control plants. Data are the mean±standard deviation (s.d.). Statistically significance was at p-value < 0.05 according to Student’s t-test, *P<0.05, **P<0.01, ***P<0.001 and NS, not significant (P>0.05).

The SHMT1-overexpressing Arabidopsis and rice under CAP treatment versus the untreated control

(A) The two lines of AtSHMT1-overexpressing Arabidopsis plants (AtT1-OE1 and AtT1-OE2) and WT 7 days after 150 μM CAP treatment. (B) The inhibition rate of AtSHMT1-overexpressing and WT seedlings treated with CAP (62.5, 93.75, 115.62, 125, 150, 250, 316, 505.6, 632, and 1264 μM). The inhibition rate was calculated after 7 days relative to the untreated control plants (Inhibition rate % = (Fresh Weight of untreated control plants-Fresh Weight of treatment plants)/Fresh Weight of untreated control plants * 100%). The bioactivity assays comprised three biological replicates, each with three technical replicates. (C) The typical phenotype OsSHMT1 knock-out mutant lines (osshmt1-qc-9, osshmt1-qc-10, osshmt1-qc-12). Bar= 2cm (D) The two lines of OsSHMT1-overexpressing Nipponbare plants (OsT1-OE13 and OsT1-OE9) and WT 7 days after 2.78 mM CAP treatment. Bar= 5cm (E) The inhibition rate of CcSHMT1-overexpressing and WT seedlings treated with CAP (0.69, 1.39, 2.78, 4.17, 8.34 and 11.12 mM). The inhibition rate was calculated after 3 days relative to the untreated control plants. (F) The SHMT activity of CcSHMT1-overexpressing and WT seedlings treated with CAP (2.78 mM). Data are the mean±standard deviation (s.d.). Statistically significance was at p-value < 0.05 according to Student’s t-test, *P<0.05, **P<0.01, ***P<0.001 and NS, not significant (P>0.05).

Target evolution of OsSHMT1 for CAP-resistant mutations by base editor

(A) Architectures of evoCDA1-BE4max, evoAPOBEC1-BE4max, ABE8e, and ACEs. Linker, a 32-aa linker; bpNLS, bipartite nuclear localization signal; TadA8e contains a V106W amino acid substitution. (B) Sanger sequencing chromatograms of four resistant mutants evolved by ACE5. Lower sequences are corresponding target sgRNAs for the mutants. The PAM motif is marked in bold and the boxes indicate the mutated sequences corresponding to the amino acid changes. (C) The typical phenotype OsSHMT1 mutant lines (Y197C, L198S, T203I, R198G). Bar= 2cm (D)The five lines of mutant plants (D181N, L199S, T194A, T194A/M195V and D209N) and WT 3 days after 2.78 mM CAP treatment. Bar= 5cm (E) The inhibition rate of rice mutant and WT seedlings treated with CAP (0.69, 1.39, 2.78, 4.17, 8.34 and 11.12 mM). The inhibition rate was calculated after 3 days relative to the untreated control plants. (F)The SHMT activity of CcSHMT1-overexpressing and WT seedlings treated with CAP (2.78 mM). (G) The influence of CAP (2.78 mM) on SHMT1 content in rice mutants and the wild type by western bolt. Data are the mean±standard deviation (s.d.). Statistically significance was at p-value < 0.05 according to Student’s t-test, *P<0.05, **P<0.01, ***P<0.001 and NS, not significant (P>0.05).

Interaction between CcSHMT1 and CAP

The docking results revealed hydrogen bonding between CAP and homology modeling structure of CcSHMT1 amino acid residues Ala191, Ser190, and Val192. The key residues surrounding the active site are shown as green sticks, and CAP is shown in yellow. Hydrogen atoms of Ser190 were within hydrogen bonding distance of the carboxyl group of CAP (2.5 Å). The hydrogen atom of Ala191 exhibited hydrogen bonds with the carboxyl group at a distance of 2.9 Å. The hydrogen atom of Val192 also exhibited hydrogen bonds with the carboxyl group at a distance of 2.4 Å. The results were produced with PYMOL (V.1.7.0, DeLano Scientific LLC). (B) MST titration profile of the interaction between CAP and CcSHMT1 amino acids. The error bars represent the standard deviation (s.d.) of each data point calculated from three independent thermophoresis measurements. (C) Effect of mutations to CcSHMT1 activity under CAP. CcSHMT1S190A: Ser190 was mutated to Ala; CcSHMTA191T: Ala191 was mutated to Thr; CcSHMTV192L: Val192 were mutated to Leu; CcSHMT1 S190L/A191E/V192G: Ser190, Ala191 and Val192 was mutant to Leu, Gly, and Glu. SHMT activity was assayed for purified recombinant CcSHMT1 proteins by the product rate of benzaldehyde 0.5 h after CAP treatment (3.16, 6.32, 9.48, 12.64 μM). (D) The inhibition rate of two lines of site-directed mutagenesis lines (atshmt1-5 and −8), and WT seedlings treated with CAP (62.5, 93.75, 115.62, 125, 150, 250, 316, and 505.6 μM). The inhibition rate was calculated after 7 days relative to the untreated control plants (Inhibition rate % = (Freash weight of untreated control plants-Freash Weight of treatment plants)/Freash Weight of untreated control plants * 100%). (E) The two lines of site-directed mutagenesis lines (atshmt1-5, −8, Ser190, Ala191, and Val192 were mutated to Leu, Gly, and Glu) and the WT 7 days after 115.62 μM CAP treatment. Bar=1 cm. The MST analysis and activity assay comprised three biological replicates, each with three technical replicates. Data are reported as mean ± s.d. Results with a p-value < 0.05 according to Student’s t-test were considered statistically significant.

Model of Caprylic acid inhibition of SHMT1 to subdue weeds and tolerant crop.

Under normal conditions, SHMT1 catalyzes the reversible interconversion of serine and glycine. The non-mutation CcSHMT1 showed good binding affinities with caprylic acid. In normal CcSHMT1 plants, caprylic acid competitively binds to the key domains (Ser190, Ala191, and Vla192) of CcSHMT1, thus disturbing SHMT function and resulting in the inhibition of C. canadensis growth. In the overexpression CcSHMT1 plants, caprylic acid competitively binds to the key site of CcSHMT1. However, serine can still be converted to glycine in surplus CcSHMT1, resulting in higher crop survival rates. In the mutant OsSHMT1 plants, we obtained the T194A, T194A/ M195V, and D209N site mutant lines. Caprylic acid cannot competitively bind to the key site (Ser190, Ala191, and Vla192) of CcSHMT1. Serine can be converted into glycine, resulting in higher crop survival rates. SHMT1: Serine hydroxymethyltransferase 1.