Flavonoid-mediated bacterial spermidine biosynthesis enhances vitamin accumulation in tomato fruits

Domain: biology
Published: 2026-01-12
Editorial outcome: major_revision

Abstract
Rhizosphere microbes benefit plant growth and health. How plant-microbe interactions regulate fruit quality remains poorly understood. Here, we elucidate the multi-level modulation of vitamin accumulation in tomato by flavonoid-mediated crosstalk between host plants and rhizosphere microbes. SlMYB12-overexpressing plants with up-regulated flavonoid biosynthesis accumulate higher levels of vitamins C and B6 in fruits compared to wild-type plants grown in natural soil. Flavonoid-mediated improvement of fruit quality depends on the presence of soil microbiomes and relates to rhizosphere enrichment of key taxa (e.g. Lysobacter). Multi-omics analyses reveal that flavonoids attract Lysobacter soli by stimulating its twitching motility and spermidine biosynthesis, which in turn boosts vitamin accumulation in fruits across tomato cultivars and soil types. RpoN acts as a dual regulator in L. soli that is responsive to flavonoids, controlling bacterial motility and spermidine production. Our study provides insight into flavonoid-mediated rhizosphere signalling and underscores plant-microbiome orchestration for improved tomato fruit quality.

Introduction
Tomato (Solanum lycopersicum Mill.) is one of the most popular horticultural crops worldwide1, offering a unique flavour and substantial nutritional value2. The tomato fruit is a favourable source of bioactive compounds, especially vitamins involved in various metabolic processes3. Owing to their antioxidant capacity, vitamins can scavenge free radicals in the human body, lowering the risk of cardiovascular diseases and cancers4–6. Therefore, substantial effort has been made to develop cost-effective and environmentally friendly methods (e.g., microbial agents) for enhancing vitamin accumulation in tomato fruits.

Plant growth-promoting rhizobacteria (PGPR) are a diverse group of beneficial microbes that can modify plant traits. Numerous PGPR are able to stimulate plant growth or strengthen stress resistance in crops at the seedling stage by releasing auxins, siderophores and 1-aminocyclopropane-1-carboxylate (ACC) deaminases7–9. There are insufficient studies on the potential role of PGPR in fruit quality regulation of crops at the later growth stages8,9. Among common PGPR, Lysobacter species exhibit strong antagonistic effects against a broad range of pathogenic bacteria, fungi, oomycetes and nematodes10,11. Previous studies have demonstrated the role of Lysobacter in plant disease control10,11. An interesting but unsolved question is whether Lysobacter species contribute to fruit quality in crops, such as tomato.

Root exudates, as mediators of plant-microbe communication, regulate plant growth, development and health12. Many root exudates play a role in improving the rhizosphere micro-environment by enhancing nutrient availability and recruiting plant-beneficial microbes13–15. Flavonoids, one of the major groups of root exudates14, are active molecules mediating plant-PGPR interactions16. Flavonoids can alleviate nutrient deficiencies17,18 and drought stress19,20 to maintain normal plant growth. These effects are achieved by mediating rhizosphere microbiome restructuring and beneficial taxa enrichment. However, the feedback mechanism of flavonoid-responsive microbes to regulate fruit quality in tomato has not been deciphered. It remains unclear whether flavonoids modulate secondary metabolism in key rhizosphere taxa, leading to fruit metabolic reprogramming and consequently promoting bioactive constituent accumulation.

In this work, we demonstrate that tomato plants recruit specific rhizosphere microbes (e.g., Lysobacter) and modulate microbial metabolic patterns through root exudates (e.g., flavonoids), thereby enhancing fruit vitamin accumulation. We show that flavonoids reshape the rhizosphere microbiome and stimulate bacterial spermidine biosynthesis via the regulator RpoN. This microbial response subsequently increases vitamin C and B6 levels in tomato fruits across different cultivars and soil types. Our study identifies a flavonoid-mediated signalling pathway that underpins plant-microbiome coordination for improved fruit quality.

Results
Flavonoids mediate fruit vitamin accumulation driven by the soil microbiome

To determine whether flavonoid-mediated variations in the rhizosphere microbiome contribute to vitamin accumulation in tomato fruits, we conducted independent pot experiments under different soil conditions (Fig. 1A–C). First, wild-type plants of Micro-Tom tomato and SlMYB12-overexpressing plants with up-regulated flavonoid biosynthesis21 were grown in natural soil from Yangling, China. Flavonoid-targeted metabolomic analysis revealed that 19 root metabolites and three rhizosphere soil metabolites were differentially accumulated (DAMs; fold-change ≥ 10, P<0.01) in SlMYB12-overexpressing plants compared to wild-type plants. Among them, rutin, naringenin chalcone (NC) and kaempferol-3-O-rutinoside (K3OR) showed the most prominent differences in their relative abundance between groups (Supplementary Fig. 1). Based on widely targeted metabolomic analysis, we compared the metabolite profiles of tomato fruits between wild-type and SlMYB12-overexpressing plants. A total of 3,092 metabolites were identified in all fruit samples, representing 13 chemical classes. The most predominant classes were “flavonoids” (14.46%), “amino acids and its derivatives” (13.69%) and “alkaloids” (13.53%; Supplementary Fig. 2A). There were 679 DAMs (fold-change ≥ 2, P<0.05), with 409 up-regulated and 270 down-regulated (Supplementary Fig. 2B). To identify the physiological processes responsible for the DAMs, we performed KEGG pathway analysis. “Vitamin B6 metabolism” and “ascorbate and aldarate metabolism” were predominantly enriched for the DAMs (P<0.01; Supplementary Fig. 2C). The relative abundances of vitamin C (ascorbic acid) and vitamin B6 (pyridoxine, pyridoxal and pyridoxamine) in fruit samples were significantly higher in SlMYB12-overexpressing plants compared to wild-type plants (≥7-fold, P<0.001; Supplementary Fig. 2D). This pattern was confirmed by hydrophilic interaction chromatography (HILIC) and high-performance liquid chromatography (HPLC) of vitamins C and B6 in fruit samples (P<0.05; Supplementary Fig. 3A–D), but not in sterilised soil (Supplementary Fig. 3E–H). These results allowed us to posit that flavonoids promote vitamin accumulation in tomato fruits depending on the presence of soil microbiomes.

To verify the involvement of soil microbiomes in fruit vitamin accumulation across different tomato cultivars, we devised a flavonoid addition experiment with wild-type Micro-Tom and Sheng Nv-Guo plants under natural and sterilised soil conditions. The experiment included a negative control (wild-type plants with no exogenous flavonoids, group Con) and a positive control (SlMYB12-overexpressing plants with up-regulated flavonoid biosynthesis, group O). In natural soil, exogenous addition of flavonoids, either in combination or individually, increased the vitamin contents in fruit samples of Micro-Tom plants compared to those of group Con. Equal mass combinations of rutin, NC and K3OR at different concentrations (A: 10 µmol g−1, B: 3 µmol g−1, C: 1 µmol g−1, D: 0.1 µmol g−1) showed greater effects on fruit vitamin accumulation than individual flavonoids (R: rutin, N: NC, K: K3OR; 1 µmol g−1 each). The highest vitamin contents in fruit samples were observed for Micro-Tom plants of groups C and O (P<0.05; Supplementary Fig. 4). Comparable results were obtained from Sheng Nv-Guo plants in natural soil, where the combined and individual flavonoid treatments increased the vitamin contents in fruit samples compared to group Con, and the highest values were observed in groups C (P<0.05; Supplementary Fig. 4). In sterilised soil, exogenous addition of flavonoids did not affect fruit vitamin contents in Micro-Tom and Sheng Nv-Guo plants (Supplementary Fig. 5). Moreover, we analysed soil chemical properties and found no significant differences in key soil variables (pH and nutrient contents) between the control and flavonoid-treated groups (Supplementary Table 1). These results supported that flavonoids facilitate fruit vitamin accumulation across tomato genotypes in a soil microbiome-dependent manner.

Flavonoids broadly contribute to rhizosphere bacterial diversity

To explore flavonoid-induced variations in rhizosphere microbial diversity, we performed 16S rRNA gene amplicon sequencing. Exogenous addition of flavonoids prominently enhanced bacterial alpha-diversity in the rhizosphere of wild-type Micro-Tom and Sheng Nv-Guo plants under natural soil conditions. The bacterial richness (Chao1) and diversity (Shannon) in rhizosphere soils of Micro-Tom plants were greater in groups B, C, D and N than group Con (P<0.05), with peak values in group C (Fig. 2A, B). Soil samples from SlMYB12-overexpressing plants (group O) also showed greater bacterial richness and diversity compared to group Con (P<0.05), and no significant differences were observed between groups O and C (Supplementary Fig. 6A, B). The fungal alpha-diversity in rhizosphere soils of Micro-Tom plants did not respond to exogenous flavonoids (Fig. 2D, E). Similar patterns were observed in alpha-diversity of bacteria and fungi when flavonoids were added to natural soil with Sheng Nv-Guo plants (Supplementary Figs. 6D, E and 7A–D). Furthermore, we added exogenous flavonoids to unplanted natural soil and observed microbial alpha-diversity patterns comparable to those seen in planted natural soil (Supplementary Fig. 8A–D).

Beta-diversity analysis by principal coordinate analysis (PCoA) revealed distinct variations in rhizosphere bacterial communities among the flavonoid treatment, Con and O groups (P<0.05; Fig. 2C and Supplementary Fig. 6C, F). However, there was no significant difference in bacterial community composition between groups O and C (Fig. 2C and Supplementary Fig. 6C). The bacterial community composition in unplanted natural soil also shifted following exogenous addition of flavonoids (Supplementary Fig. 8E). Irrespective of the presence or absence of plants, fungal beta-diversity did not respond to any exogenous flavonoid treatments (Fig. 2F, Supplementary Fig. 7F and Supplementary Fig. 8F). The results provided robust evidence for flavonoid-mediated enhancement of soil bacterial diversity, which is not interfered by other metabolites from root exudates.

Flavonoids induce rhizosphere bacterial community restructuring

To disentangle the effect of flavonoids on rhizosphere microbial community structure, we focused on the composition and relative abundance of bacterial taxa at the phylum and genus levels. Exogenous addition of flavonoids altered the structure of rhizosphere bacterial communities associated with tomato plants, causing changes in the composition and relative abundance of major taxa (Supplementary Fig. 9). At the phylum level, Proteobacteria, Acidobacteria, Bacteroidetes, Chloroflexi, Planctomycetes, Gemmantimonadetes and Actinobacteria showed noticeable responses to flavonoid treatment. At the genus level, linear discriminant analysis (LDA) effect size (LEfSe) analysis identified 26 flavonoid-responsive biomarkers in rhizosphere soils of wild-type Micro-Tom plants (LDA>3). Among them, six genera were enriched in group O, with eight genera enriched in group C. In both groups O and C, Lysobacter and Bacillus were two common genera that increased in relative abundance under flavonoid treatment (Fig. 2G). Furthermore, we identified 32 flavonoid-responsive genera in rhizosphere soils of Sheng Nv-Guo plants (LDA>3), including Lysobacter and Bacillus that were also enriched in group C (Supplementary Fig. 7G).

Exogenous addition of flavonoids led to shifts in the microbial community structure in unplanted natural soil (Supplementary Fig. 10). LEfSe analysis uncovered the enrichment of both Lysobacter and Bacillus in soil samples of group C after flavonoid treatment (LDA>3; Fig. 2H). Moreover, we performed quantitative PCR (qPCR) assays targeting the 16S rRNA genes to quantify taxa-specific abundances (Supplementary Method 1, Supplementary Table 2 and 3). Both Lysobacter and Bacillus showed significantly higher absolute abundances in soil samples of groups C and O compared to the control samples (P<0.05; Supplementary Fig. 11). The results indicated that flavonoids preferentially attract Lysobacter and Bacillus in the rhizosphere microbiome.

Mantel’s test unravelled the relationship between differentially abundant taxa in rhizosphere soils and bioactive compounds in tomato fruits. Fruit vitamin contents were significantly positively correlated with the relative abundances of both Bacillus (P<0.05) and Lysobacter (P<0.01; Supplementary Fig. 12). These results provided important clues that flavonoids likely mediate rhizosphere enrichment of key taxa to boost fruit vitamin accumulation.

Flavonoids increase fruit vitamin content through Lysobacter enrichment

To ascertain the potential role of Bacillus and Lysobacter in regulating tomato fruit quality, we obtained a total of 104 bacterial isolates from rhizosphere soils treated with flavonoids. Using 16S rRNA gene sequencing, 54 species representing 27 genera were identified (Fig. 3A). Among them, three dominant Lysobacter strains (designated P18, P25 and P58) and three dominant Bacillus strains (designated P54, P64 and P66) were purified (Fig. 3A). Based on high-throughput sequencing data of operational taxonomic units (OTUs), Lysobacter OTU69 and Bacillus OTU341 showed the highest relative abundance in rhizosphere soils, with significant enrichment in groups O and C (Supplementary Fig. 13). We aligned these two representative OTU sequences to the full-length 16S rRNA gene sequences of the dominant bacterial strains. Lysobacter OTU69 shared high similarity to L. soli P18 (95.8%), L. fragariae P25 (94.1%) and L. niastensis P58 (92.8%). Bacillus OTU341 matched to B. subtilis P66, B. amyloliquefaciens P64 and B. cereus P54 (91.7–94.4% similarity). Therefore, these six strains were selected as key bacteria for plant growth assay, and all of them showed growth-promoting effect on wild-type Micro-Tom tomato seedlings (P<0.05; Supplementary Fig. 14, Supplementary Table 4).

To validate the function of key bacteria in fruit vitamin accumulation, we conducted a pot experiment in sterile soil with wild-type Micro-Tom and Sheng Nv-Guo plants (Fig. 3B). Compared to the control group, the vitamin C, pyridoxine, pyridoxal and pyridoxamine contents in fruit samples of Micro-Tom plants increased across the six experimental groups treated with different bacterial cell suspensions (P<0.05; Fig. 3C–F). The highest vitamin contents in fruit samples were observed for the treatment group of L. soli P18 (2.13–5.00-fold). Consistently, the treatment with L. soli P18 increased vitamin contents in fruit samples of Sheng Nv-Guo plants (P<0.05; Supplementary Fig. 15), confirming that the positive effects of L. soli P18 on fruit vitamin levels are robust across plant genotypes. Moreover, L. soli P18 treatment significantly elevated vitamin contents in fruit samples of Micro-Tom plants in sterilised soils from Kashgar, Ordos, Chengdu and Nanning. There was no significant difference in fruit vitamin contents between L. soli P18-treated plants and SlMYB12-overexpressing plants (P<0.05; Fig. 3G). These results indicated that the observed effects of L. soli P18 on fruit vitamin levels are consistent across geographically distinct soils. We therefore selected L. soli P18 as a flavonoid-enriched key bacterial strain for subsequent assays.

Flavonoids enhance twitching motility of Lysobacter soli P18

To decipher the mechanism of flavonoid-mediated Lysobacter enrichment in the rhizosphere, we performed metagenomic sequencing. A total of 22,759 and 22,355 unigenes of Lysobacter were identified in rhizosphere soils of groups C/Con and O/Con, respectively. There were 1,374 and 1,297 differentially expressed genes (DEGs, P<0.05) in groups C/Con and O/Con, respectively (Supplementary Fig. 16A, B). Based on Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis, 190 DEGs of group C/Con and 185 DEGs of group O/Con were both enriched in the two-component system (Supplementary Fig. 16C, D). Among them, 34 and 32 up-regulated DEGs were identified as related to bacterial twitching motility (P<0.05; Supplementary Fig. 17). These DEGs comprised genes that encode bacterial type IV pilus assembly protein, twitching motility protein, twitching motility response regulator and chemosensory pili system protein (Supplementary Fig. 17).

We subsequently delved into the transcriptomic changes of L. soli P18 induced by flavonoids (treatment C: 1 µmol g−1). Principal component analysis (PCA) showed a clear separation of flavonoid-treated and control samples along the first principal component (PC1, 75.05 %; Supplementary Fig. 18A). A total of 509 DEGs (P<0.05) were identified between the two groups (Supplementary Fig. 18B). Categorisation of the up- and down-regulated DEGs indicated that flavonoids broadly influenced a plethora of cellular processes in L. soli P18 (Supplementary Fig. 18C). Importantly, 27 up-regulated DEGs were identified as related to bacterial twitching motility (P<0.05; Fig. 4A). These DEGs included genes that encode pilus assembly protein, pilus biogenesis protein, twitching motility response regulator, type IV pilus twitching motility protein, type IV pilus modification protein and chemosensory pili system protein.

We next verified the mechanism of rhizosphere colonisation by L. soli P18 in response to flavonoids (treatment C: 1 µmol g−1). Heightened bacterial colonisation was observed in rhizosphere soils of SlMYB12-overexpressing plants and flavonoid-treated wild-type Micro-Tom plants compared to the control group (Fig. 4B). Furthermore, bacterial motility assays unveiled that flavonoids enhanced the twitching motility, rather than the swarming or swimming motility, in L. soli P18 on agar plates (Fig. 4C–E). Cell growth assay demonstrated minimal effects of flavonoids on the growth rate of L. soli P18 (Fig. 4F). Among the up-regulated genes related to bacterial twitching motility, RpoN emerged as the hub node in the gene co-expression network. There was a significant positive correlation between the expression levels of RpoN and other genes related to twitching motility (P<0.01, weight > 0.9; Fig. 4G).

To verify the role of RpoN as a hub gene in response to flavonoid signalling and a regulator of bacterial twitching motility, we constructed a RpoN knockout mutant (ΔRpoN) and a complemented strain (ΔRpoN-C). ΔRpoN showed significantly lower twitching motility and colonisation ability under flavonoid treatment compared to its wild-type strain (P<0.01), and this phenotype was restored in ΔRpoN-C (Fig. 4H, I). In summary, these results indicated that flavonoids stimulate the twitching motility of L. soli P18 in a RpoN-dependent manner, which enables bacterial enrichment in the rhizosphere (P<0.05; Fig. 4J).

Flavonoids stimulate spermidine biosynthesis in Lysobacter soli P18

To unravel whether flavonoids regulate bacterial secondary metabolism in the rhizosphere, we carried out widely targeted metabolomic profiling of L. soli P18. Based on PCA, the culture samples treated with flavonoids (treatment C: 1 µmol g−1) were clearly distinguished from the control samples by PC1 (43.05%; Supplementary Fig. 19A). A total of 1,708 metabolites were identified in all culture samples. The most predominant classes were “amino acids”, “alkaloids”, “aldehyde” and “lipids”. A total of 584 metabolites were differentially accumulated between groups (P<0.05). Specifically, 354 metabolites were up-regulated and 230 metabolites were down-regulated by flavonoids (Supplementary Fig. 19B).

We then looked at the relationship between flavonoid-induced metabolites in L. soli P18 and vitamins in tomato fruits. Mantel’s test revealed that the relative abundance of bacterial alkaloids was significantly correlated with fruit vitamin contents (P<0.01; Supplementary Fig. 20). Among alkaloids, spermidine emerged as the greatest contributor to fruit vitamin accumulation based on random forest analysis. Based on the cross-validation results, the random forest model achieved optimal stability and accuracy when selecting the top 20 metabolites (Supplementary Fig. 21). Widely targeted metabolomic profiling unveiled the up-regulation of spermidine (1.89-fold) and its precursors (cysteine: 1.67-fold; methionine: 1.81-fold) in the metabolome of flavonoid-treated L. soli P18 (P<0.05; Fig. 5A). Therefore, we focused on spermidine as a DAM of L. soli P18 in response to flavonoids.

Spermidine biosynthesis is controlled by a set of genes that encode S-adenosylmethionine decarboxylase (SpeD) and spermidine synthase (SpeE)22 (Fig. 5B). Metagenomic sequencing uncovered that the relative abundances of SpeD and SpeE genes were 1.60–1.95-fold up-regulated in soil samples of groups C and O (Fig. 5C and D). Consistently, prokaryotic transcriptomic profiling revealed 1.50–1.65-fold up-regulation of SpeD and SpeE in L. soli P18 under flavonoid treatment (Fig. 5E). To validate the effect of flavonoids on spermidine biosynthesis in L. soli P18, we performed chemical extraction and quantitative analysis. The spermidine content in culture samples added with combined flavonoids (treatment C: 1 µmol g−1) was 2.8-fold higher than that of the control group (P<0.05). Combined flavonoids exhibited a greater effect on spermidine production in L. soli P18 compared to individual flavonoids (treatments R, K, N; P<0.05; Fig. 5F). The results indicated that flavonoids induce up-regulation of genes related to spermidine biosynthetic enzymes in L. soli P18, stimulating spermidine production. Interestingly, the gene co-expression network revealed a positive correlation between the expression levels of RpoN and spermidine biosynthesis related genes, SpeD and SpeE (P<0.01, weight > 0.9; Fig. 4G).

Further, the function of RpoN was validated by gene knockout in L. soli P18. We found that fruit vitamin contents were significantly higher in Micro-Tom plants treated with cell suspensions of the wild-type strain and the complemented strain ΔRpoN-C compared to the control group (P<0.01). Cell suspension of the mutant strain ΔRpoN had no significant effect on fruit vitamin contents (Fig. 5G). Under flavonoid treatment, ΔRpoN showed a significantly lower ability to produce spermidine than its wild-type strain (P<0.01), and this phenotype was restored in ΔRpoN-C; Fig. 5H). In summary, these results indicated that flavonoids enhance spermidine biosynthesis of L. soli P18 under the control of RpoN.

Bacterially produced spermidine boosts fruit vitamin accumulation

To elucidate the role of spermidine in fruit vitamin accumulation, we added exogenous spermidine (1 μmol g−1) to sterilised soil with wild-type Micro-Tom plants. The spermidine treatment significantly increased vitamin C, pyridoxine, pyridoxal and pyridoxamine contents in tomato fruits (P<0.05; Fig. 5I–L). We then profiled the metabolome of tomato fruits after spermidine addition (Supplementary Fig. 22). A total of 2,476 metabolites were identified in all fruit samples, representing 13 chemical classes. The most predominant classes were “amino acids and its derivatives” (15.11 %), “alkaloids” (12.0 %) and “lipids” (12.4 %; Supplementary Fig. 23A). PCA uncovered that the treatment and control groups were well separated along PC1 (44.49%; Supplementary Fig. 23B), suggestive of a remarkable difference in the fruit metabolome between groups. The heatmap shows that some metabolites were differentially accumulated in response to spermidine treatment. A total of 500 DAMs were identified (fold-change ≥ 1.6, P<0.05), with 267 up-regulated and 233 down-regulated (Supplementary Fig. 23C). To identify the physiological processes responsible for the DAMs, we performed KEGG pathway analysis. “Vitamin B6 metabolism” and “ascorbate and aldarate metabolism” were predominantly enriched for the DAMs (P<0.05; Supplementary Fig. 23D).

Transcriptomic analysis yielded 34,688 annotated unigenes. Among them, 4,976 genes were differentially expressed (fold-change ≥ 1.6, P<0.05), with 3,065 up-regulated and 1,911 down-regulated in spermidine treatment (Supplementary Fig. 24A). PCA showed a distinct separation between the treatment and control groups along PC1 (60.84%; Supplementary Fig. 24B), indicating a notable difference in the fruit transcriptome between groups. To identify the putative functions of potential transcripts, we performed KEGG enrichment analysis of DEGs. The DEGs were mainly enriched in “ascorbate and aldarate metabolism” and “vitamin B6 metabolism” (P<0.05; Supplementary Fig. 24C).

By integrating transcriptomics and metabolomics, we selected DAMs and DEGs enriched in the pathways of “ascorbate and aldarate metabolism” and “vitamin B6 metabolism”. The results indicated that gene-regulated changes in the metabolites and associated metabolic pathways could alter vitamin accumulation in spermidine-treated fruits (Supplementary Figs. 23D, 24C). Specifically, the relative abundances of pyridoxamine, pyridoxal, pyridoxine, 4-pyridoxic acid, pyridoxal-5P and L-ascorbate were 1.80–8.97-fold up-regulated by spermidine (P<0.05; Fig. 5M and Supplementary Fig. 25). Consistently, transcriptomic data showed 1.61–3.27-fold up-regulation of genes modulating EC1.1.3.8 (L-gulonolactone oxidase), EC1.2.3.1 (aldehyde oxidase), EC3.1.3.74 (pyridoxal phosphatase) and EC4.3.3.6 (pyridoxal 5′-phosphate synthase) in spermidine treatment (P<0.05; Fig. 5M). The collective results indicated that spermidine produced by L. soli P18 promotes vitamin accumulation in tomato fruits, conferring benefits to human health.

Methods
Soil and plants
Different types of natural soil were collected from cropland fields in Yangling, Nanning, Chengdu, Ordos, and Kashgar, China. After removing plant debris, worms and stones, the soil was homogenised manually using a 2.5-mm sieve. The chemical properties of the soils are provided in Supplementary Table 5.

Two wild-type tomato cultivars (Micro-Tom, Sheng Nv-Guo) and a SlMYB12-overexpressing transgenic line that over-accumulates flavonoids (Micro-Tom)21 were used as plant materials. Surface sterilisation of seeds was conducted by soaking in 75% (v/v) ethanol for 30 s followed by sodium hypochlorite solution (10% active chlorine) for 1 min. After three washes with sterile water, clean seeds were transferred onto Petri dishes and incubated at 4°C in the dark for 2 d of germination. Then, the germinated seeds were transferred into a humidity chamber and incubated at 22°C and 60–70% relative humidity for 3 d until the length of roots reached ~1 cm. Seedlings were transferred to a 45-hole tray for 30 d and watered once a week. After that, 35-d-old seedlings were transplanted to plastic pots (90mm × 130mm × 100mm) containing 5 kg of natural soil from Yangling with an initial moisture content of 100% (v/w). The pots (one plant per pot) were kept in a humidity chamber with a day/night temperature of 26°C/18°C, a relative humidity of 60–70%, a light intensity of 12,000 Lux and a photoperiod of 18h light/6h dark. In the seedling, flowering and fruiting stages, 10 mL of Hoagland’s nutrient solution was supplied to each pot without chemical fertiliser application.

Flavonoid-targeted metabolomic analysis
Samples were collected from wild-type and SlMYB12-overexpressing plants at 90 d after transplanting (DAT). A 20-g sample of rhizosphere soil was obtained from each pot. The soil samples from two pots were pooled as one biological replicate, with three biological replicates per treatment. Each soil sample was extracted with 50 mL of sterile water shaken at 120 rpm for 60 min. The soil extract was centrifuged at 4°C and 12,000×g for 10 min, and the supernatant was collected. Additionally, tomato roots were washed with running water and then rinsed with sterile water five times. The root samples from two pots were pooled as one biological replicate, and three biological replicates were included for each treatment. All samples (soil extracts and tomato roots) were stored at -80°C until use for targeted metabolomic analysis.

After thawing and vortexing, each root sample was vacuum-lyophilised and ground to powder using a grinding mill at 30 Hz for 1.5 min. Then, 2 mL of soil extract or 20 mg of root powder was transferred to a 10-mL centrifuge tube containing liquid nitrogen. The sample was completely freeze-dried in a lyophiliser and then added with 200 µL of 70% methanol internal standard extract. The mixture was oscillated for 15 min followed by centrifugation at 4°C and 12,000×g for 3 min. The supernatant was filtered through a 0.22-μm microporous filter membrane before use. The sample extracts were analysed by targeted metabolomics (Supplementary Method 2) based on ultra-performance liquid chromatography (UPLC, Nexera X2; Shimadzu, Kyoto, Japan) coupled to tandem mass spectrometry (MS/MS, Applied Biosystems 6500 Q-TRAP; Applied Biosystems, Framingham, MA, USA). Declustering potential and collision energy were optimised for individual MRM transitions. A specific set of MRM transitions were monitored for each period based on the eluted metabolites. Identification of metabolites was performed by comparison of the m / z values, retention time, and searching an internal custom database of MetWare Biotechnology Co., Ltd (Wuhan, China). DAMs were identified by based on the variable importance in projection (VIP≥1), P<0.05 (Two-sided Student’s t-test) and fold-change ≥ 1.6.

Flavonoid addition experiment
An exogenous flavonoid addition assay was carried out with tomato plants grown in natural soil from Yangling. Flavonoids (Solarbio, Beijing, China) were dissolved in 85% (v/v) methanol with a dilution factor of 50 and passed through a 0.22-μm sterile microporous filter membrane. Seven flavonoid treatments were established for wild-type plants based on the design of previous studies60,61 and our targeted metabolomics data of flavonoids (Supplementary Fig. 1). Four treatment groups received a mixture of rutin, NC and K3OR at different concentrations (1:1:1, m/m/m; A: 10 µmol g−1, B: 3 µmol g−1, C: 1 µmol g−1 and D: 0.1 µmol g−1). The other three treatment groups received individual flavonoids at the same concentration (R: rutin, N: NC and K: K3OR; 1 µmol g−1 each). At 15 DAT, a sterile bamboo stick was used to puncture holes into the soil around the plants at a distance of 1 cm. The prepared flavonoid solutions were repeatedly injected into the holes with a sterile disposable syringe (10 mL per pot, 90mm×130 mm×100mm) at 22 and 29 DAT. Two controls (wild-type Miro-Tom and SlMYB12-overexpressing plants) received an equal volume of sterile 50-fold diluted 85% methanol. In the seedling, flowering and fruiting stages, 10 mL of Hoagland’s nutrient solution was supplied to each pot. All other management practices were consistent for the treatment and control groups. At 90 DAT, rhizosphere soil samples (50 g each) were collected by gently shaking the roots and kept in a -80°C freezer until use for DNA extraction. Another set of rhizosphere soil samples (50 g each) were obtained and stored in a 4°C refrigerator before bacterial isolation. Fruit samples were harvested to measure vitamin contents. Samples from two pots (one plant per pot) were collected as one biological replicate, with three biological replicates per treatment.

The same procedures were followed to conduct a second flavonoid addition assay with tomato plants grown in soil from Yangling that had been sterilised by γ-irradiation (20 kGy, 10 MeV γ-ray). Only one combined flavonoid treatment (1 µmol g−1) was applied to wild-type Micro-Tom and Sheng Nv-Guo plants at 15, 22 and 29 DAT, with two controls (wild-type Micro-Tom and SlMYB12-overexpressing plants) receiving an equal volume of sterile 50-fold diluted 85% methanol. Pots were managed as described for the natural soil experiment. At 90 DAT, rhizosphere soil and fruit samples from three pots (one plant per pot) were collected as one biological replicate, and three biological replicates per treatment were prepared for vitamin C and B6 analysis.

A third flavonoid addition assay was performed in plastic pots (50mm×60mm×60mm) containing 1 kg of natural soil from Yangling without plants. Exogenous flavonoids were applied at 15, 22 and 29 days for seven treatments and one control as performed in the natural soil experiment, and pots were managed as described for the tomato growth test. At 90 days, soil samples (5 g each) from three pots were collected as one biological replicate, and three biological replicates were included for each treatment. The samples were frozen at -80°C until DNA extraction for 16S rRNA gene sequencing and metagenomic profiling.

Soil 16S rRNA gene sequencing
The PowerLyzer PowerSoil DNA Isolation Kit (Qiagen GmbH, Hilden, Germany) was used to isolate total genomic DNA from natural soil samples (0.5 g each) as per the manufacturer’s protocol. DNA quality and quantity were respectively assessed using 1% agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). For taxonomic profiling, the V3-V4 region of the bacterial 16S rRNA genes was amplified by PCR with the universal primers 515F (5’-GTGYCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACNVGGGTWTCTAAT-3’)62, yielding 292-bp amplicons. PCR reactions, product purification, and library construction are described in Supplementary Method 3. Paired-end libraries (2×250bp) were sequenced on an Illumina NovaSeq 6000 platform (Illumina Inc., San Diego, CA, USA).

After raw data processing and taxonomic assignment (Supplementary Method 4), bioinformatic analysis was carried out using the Magigene Cloud platform (http://cloud.magigene.com/). Based on OTU data, rarefaction curves were created and alpha-diversity metrics (Chao1 and Shannon) were estimated using Mothur v1.30.163. Community similarities among samples were determined by PCoA based on unweighted UniFrac distances using Vegan v2.5-3 (https://CRAN.R-project.org/package=vegan/). Permutational multivariate analysis of variance was performed to assess the percentage of variation explained by the treatment and its statistical significance using Vegan v2.5-3 (https://CRAN.R-project.org/package=vegan/). LEfSe64 was used to identify differentially abundant bacterial taxa (phyla to genera) among the different treatment groups (LDA>3). LDA scores were calculated using R v4.2.065 to maximise between-class variance while minimising within-class variance. Data were standardised prior to analysis (mean-centred and scaled to unit variance). The discriminant axes were ranked by their associated eigenvalues, and scores were projected onto the first two axes for visualisation.

Soil metagenomic sequencing
The E.Z.N.A.® Soil DNA Kit (Omega Bio-tek) was used for genomic DNA isolation from natural rhizosphere soil samples (5 g each) following the manufacturer’s instructions. DNA concentration was measured using TBS-380 (Turner BioSystems, Sunnyvale, CA, USA). DNA purity was evaluated with NanoDrop 2000 (NanoDrop Technologies Inc., Wilmington, DE, USA), and DNA quality was checked by 1% agarose gel electrophoresis. Then, DNA extracts were fragmented to an average size of ~400 bp using Covaris M220 (Gene Company Limited, China), and paired-end libraries were generated using NEXTFLEX® Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA). Adaptors containing the full complement of sequencing primer hybridisation sites were ligated to the blunt-end of DNA fragments. Paired-end sequencing was accomplished on Illumina NovaSeq 6000 (Illumina Inc.) at Meige Gene Technology Co., Ltd. (Guangzhou, Guangdong Province, China). Sequencing data were analysed on the Magigene Cloud platform (http://cloud.magigene.com/). Detailed data analyses are provided in Supplementary Method 5. All unigenes of Lysobacter species were selected from the sequenced genes for DEG identification and KEGG enrichment analysis. Genes with fold-change ≥ 1.3 and P<0.05 (two-sided Student’s t-test) were identified as DEGs. KEGG annotation was conducted against the KEGG database with an e-value cutoff of 10-5 using Diamond v0.8.3566.

Bacterial isolation and identification
Culturable bacteria were isolated from fresh natural rhizosphere soil samples by serial dilution and spread plate techniques67. Briefly, a 10-g soil sample was added to a flask containing 90 mL of sterile water with glass beads and shaken at 120 rpm for 30 min. The soil suspension was serially diluted to 10-5–10-7, and 10-μL aliquots of each dilution were evenly spread on beef extract peptone (BEP) agar plates. The plates were incubated at 28°C for 3 d, with five replicates for each dilution. Representative colonies of the dominant bacteria were purified and preserved on BEP agar slants at low temperature. Identification of purified bacteria was based on PCR amplification and 16S rRNA gene sequencing68. Details of DNA extraction, PCR amplification and sequencing have been provided in Supplementary Method 6. The obtained sequences were blasted in the GenBank database (http://blast.ncbi.nlm.nih.gov/). Phylogenetic trees were constructed using the neighbour-joining method with MEGA v1169.

Tomato growth test
A 72-h broth culture of key bacteria in BEP medium was centrifuged at 12,000×g (4°C, 15 min) and passed through a 0.22-μm sterile microporous filter membrane. The cell-free filtrate was diluted 50-, 100-, 200- and 400-fold with sterile water before use. Spermidine aqueous solutions were formulated at gradient concentrations of 0.1, 1.0 and 10.0 μmol g−1. Surface-sterilised seeds of wild-type Micro-Tom were evenly placed in Petri dishes with double layers of sterilised filter paper, followed by the addition of 10 mL of cell-free filtrate or spermidine solution. During the incubation period, each dish received 5 mL of cell-free filtrate or spermidine solution everyday. An equal volume of sterile BEP medium at corresponding dilutions and sterile water served as the control for bacterial and spermidine treatments, respectively, with five biological replicates per treatment. All dishes were incubated in a light incubator for 15 d under the following conditions: day/night temperature, 26°C/18°C; photoperiod, 14h/10h light/dark; relative humidity, 65%; and light intensity, 12,000 Lux.

Single colonies of key bacteria were transferred from 24-h cultures to fresh BEP medium and incubated at 36°C shaken at 160 rpm for 24 h. Bacterial cultures were harvested by centrifugation at 12,000×g (4°C, 10 min). The cell pellet was resuspended in 1× phosphate-buffered saline (PBS), with an effective viable count of 1×108 colony-forming units (CFU)·mL−1. In the treatment group, pot sterile soil was mixed with 5 mL of cell suspension using a pipette before transplanting. At 15, 30 and 50 DAT, cell suspension (5 mL per pot) was added to the soil around roots. The control group received an equal volume of 1× PBS. Fruit samples from three pots (one plant per pot) were collected as one biological replicate, and three biological replicates of each treatment were included for determination of vitamin contents.

Construction of L. soli RpoN mutant
A RpoN knockout mutant of L. soli P18 was constructed by homologous recombination using the sucrose lethal factor70. First, upstream and downstream homology arms were obtained using the primer pair RpoN-F1/R1 and RpoN-F2/R2 (Supplementary Table 3 and 6). All oligonucleotide primers were designed based on the genome sequence of L. soli P18 (GenBank accession PRJNA1177803) using Primer Premier 6.0 software. The primers were synthesised by Sangon Biotech

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript describes the effects of flavonoids on the root microbiome and the accumulation of vitamins in tomato fruit.\nThe use of the Micro-tom tomato in these experiments is concerning. Micro-tom is a brassinosteroid mutant (\nhttps://doi.org/10.1093/jxb/erj154 ) and brassinosteroids have been implicated in root microbiome signaling\n(http://dx.doi.org/10.1016/j.molp.2015.12.003) , so the results for other tomato varieties may be different. Wang et al., 2018\nshowed that overexpression of SlMYB12 affect flavonol biosynthesis in three tomato varieties. A full size tomato plant\nwithout the brassinosteroid mutations would be more appropriate for this work.\nThe title is very wordy. Could this be simplified? Figure 1 is far too complex. Is spermidine the only factor involved?\nThe amount of nutrients in the soil may affect the interaction of the plants and the microbiome. How was the soil used\nselected? Was any fertilizer added to the soil?\nLines 40-41: What about everything in between the seedling stage and ripening? Are there studies on the effects of PGPR\non plant growth, development or flowering?\nFigure 1. Could it be replaced with a more generalized workflow? The graphs are too small to see. Even they are not the\nactual results, there is no need for them. It is confusing for the reader.\nThe statistical analysis section of the methods is lacking information. How were LDA scores calculated? How were the\nPCAs done?\nFigure 1D. Which metabolites are significantly different between the two genotypes? All of them? Why do you only focus on\nascorbic acid and vitamin B6, if there are differences in many compounds?\nFigure 2. A list of metabolite abbreviations within the figure would be helpful for the reader.\nFigure 2. A definition of Chao1 and Shannon in the figure legend would be nice.\nFigure 2. What is the difference between figures 2C and 2F? Is one fungal and the other bacterial communities? Why are\nbacterial communities p< 0.05 and fungal p>0.05?\nFigure 2E. The metabolite order is different from the other graphs. Please change.\nFigure 3. Explain what the key bacteria in the figure are. Are they the bacteria listed in the tree?\nFigure 5A. Cysteine is spelled wrong.\nFigure 6. The numbers in the legend do not match the numbers in the figure.\nFigure S1A. What are the other compounds that are significant in tomato roots? Only three are labeled.\nFigure S5-S9. Add the abbreviation meanings to the figure or figure legend so the reader does not have to refer back to the\nmain manuscript.\nLine 362: Per cent humidity in moisture chamber?\nLine 382: What is the internal standard? Is it methanol or was the internal standard diluted in methanol?\nThroughout the methods section L. soli is misspelled as L. soil.", "reviewer_2": "This study explores a hot topic in the plant-microbe interaction, revealing how flavonoids secreted by tomato roots recruit\nspecific soil microbes, such as Lysobacter soli, to the rhizosphere.\nThe recruited microbes produce spermidine, which the authors propose plays a pivotal role in enhancing vitamin C and B6\naccumulation in tomato fruits. By combining advanced microbial profiling with metabolomic and plant biochemical analyses,\nthe authors present a compelling data to prove the hypothesis that: 1) flavonoid-driven mechanism shapes the rhizosphere\nmicrobiome, contribute to the strain-level recruitment and eventually influences plant nutritional quality.\nThese study benefit from broad analysis, and multi layer omic findings to provide mechanistic insights into the flavonoid\nmediated plant-microbiome interaction and its effects on tomato nutritional traits.\nThe research is well-designed, employs an adequate level of analysis, and benefits from a clear and effective writing style.\nHowever, I have some concerns regarding the conceptual aspects of this study, which I invite the authors to clarify.\nSpecific-level selection VS overall diversity effect\n1) The manuscript hypothesis turns around the fact “that tomato plants recruit specific rhizosphere microbes and modulate\nmicrobial metabolic patterns through root exudates (e.g. flavonoids), thereby enhancing fruit vitamin accumulation”. The\nauthors used 16S rRNA gene amplicon and metagenomic sequencing, Culture-based experiments are conducted to\nascertain the role of flavonoid-responsive microbial taxa represented by Lysobacter in fruit vitamin accumulation.\nCan the flavonoid effect specifically increase the abundance of the taxa described by the authors, such as Bacillus and L.\nsoli, or does it enhance overall microbial diversity, with these taxa increasing as a result of the general rise in bacterial\nabundance? If the latter is true, the 'strain-level recruitment' hypothesis would not be supported. How does the presented\ndata effectively disprove the second hypothesis, which suggests a general increase in all taxa, including the target taxa?\n2. Co-founding factor to clarified or acknowledge\nThe study establishes a strong correlation between flavonoid-mediated changes in the microbiome and vitamin\naccumulation in fruits but may not conclusively demonstrate causation. The enrichment of Lysobacter soli and increased\nvitamin levels might be due to other indirect factors influenced by flavonoids, such as soil pH, nutrient availability, or plant\nroot exudates. Has the study thoroughly ruled out other variables that might confound the flavonoid-microbiome-vitamin\npathway?\n3. Generalization of Findings\nThe flavonoid positive effects are shown on the other context in the tomato Micrtom, for example disease resistant or overall\ngrowth. See please this study by Yang et al, 2023: For example see this paper by : RIN enhances plant disease resistance\nvia root exudate-mediated assembly of diseasesuppressive rhizosphere microbiota\nThe study focuses on the specific interaction between flavonoids, Lysobacter soli, and tomato plants. It is unclear if these\nfindings are broadly applicable across different plant species, soil types, or environmental conditions.\n4. Vitamin Accumulation Pathway\nThe study attributes vitamin C and B6 accumulation to microbial spermidine production but does not explore whether\nflavonoids directly influence the plant’s own vitamin biosynthesis pathways? Was there an attempt to decouple the plant’s\nintrinsic biosynthetic response from the microbial influence?", "reviewer_3": "This study examines how flavonoids influence interactions between plants and rhizosphere microbes, contributing to\nchanges in vitamin levels in Micro-Tom tomato fruits. Overexpression of the SlMYB12 gene, which enhances flavonoid\nbiosynthesis, was associated with increased levels of vitamins C and B6 in fruits grown in natural soil, highlighting the\ninvolvement of the soil microbiome. Metagenomic and transcriptomic analyses indicated that flavonoids promote the\nenrichment of rhizosphere microbes such as Lysobacter by enhancing bacterial twitching motility. Metabolomic and\ntranscriptomic profiling further suggested that flavonoids stimulate spermidine biosynthesis in Lysobacter soli P18, which\nmay influence vitamin metabolic pathways in the fruits. These findings outline a possible mechanism for flavonoid-mediated\nrhizosphere signaling and its impact on fruit quality. While I appreciate the findings' potential influence on the nutritional\nquality of horticultural crops, the manuscript falls short of fully addressing its stated objectives, exhibiting notable gaps in the\ncomprehensiveness of the research and weaknesses in the methodological approach. While I like the potential of these\nfindings to contribute to the understanding of plant-microbe interactions and their impact on enhancing the nutritional value\nof horticultural crops, I feel that the manuscript fails to meet its objectives in several aspects, including the workload, depth of\nresearch, and construction of methodological frameworks.\nMajor\n1. In L208-213, the study employs a random forest algorithm for predictive analysis, however, the manuscript lacks an\nexplanation for the rationale behind the algorithm selection and does not compare it with other algorithms, which limits the\ngeneralizability and robustness of the results. The discussion on the performance metrics of the chosen algorithm is\ninsufficient, and there is a high dependency on the predictive outcomes without adequate empirical evidence to support\nthem. To enhance the transparency, reliability and validity of the research, it is recommended to supplement the rationale for\nalgorithm selection, introduce comparative analysis with other machine learning algorithms, and provide a detailed\ndiscussion on performance metrics. Additionally, presenting more experimental empirical data would strengthen the\ncredibility of the predictive results.\n2. In the experimental design, the authors employed different concentrations and combinations of flavonoid treatments to\ninvestigate their compatibility with SlMYB12-overexpressing plants. Notably, this study found that the addition of three\nflavonoids at a concentration of 1 µmol g-¹ in a 1:1:1 ratio could simulate the conditions of the SlMYB12-overexpressing\ngroup, providing new insights into the role of flavonoids in gene regulation. However, we also acknowledge a significant\nlimitation of this study: subsequent experiments did not include SlMYB12-overexpressing treatment group. While the\naddition of flavonoids may mimic the effects of gene over-expression, it is not sufficient to fully substitute for actual gene\nover-expression experiments. Experiments involving the gene over-expression group are critical for verifying whether\nflavonoid treatments can truly replace the effects, as they provide direct evidence of gene expression regulation and\npotential molecular mechanisms. Therefore, I recommend that the authors incorporate a gene over-expression treatment\ngroup in future studies to compare the differences and connections between flavonoid treatments and gene over-expression.\nSuch an experimental design would contribute to a more comprehensive understanding of the mechanisms underlying the\neffects of flavonoids and enhance the persuasiveness and scientific value of this study’s findings.\n3. In this article, the authors did not explain why the simultaneous addition of three flavonoids in a 1:1:1 ratio yielded better\neffects. This indirectly reflects the lack of clarity regarding the specific pathways influencing spermidine biosynthesis in\nLysobacter soli P18 in this study. The study primarily relied on analyses of microbial metagenomics, transcriptomics, and\nmetabolomics to calculate correlations between microbial enrichment and compound variation using statistical methods,\nwithout further validation through molecular experiments. The specific molecular mechanisms by which flavonoids promote\nspermidine biosynthesis in Lysobacter soli P18 were not elucidated in this research.\n4. In this article, the pathway by which plant-secreted flavonoid metabolites mediate microbial spermidine synthesis and\nsubsequently up-regulate plant vitamin levels is presented as an innovative finding. However, the evidence is insufficient,\nparticularly regarding the lack of direct evidence for the molecular mechanisms involved in this pathway. In L294-296, it is\nnoted that research on Lysobacter spp. has primarily focused on their role in the biocontrol of soilborne diseases. This study\nhighlights the regulatory effects of these bacteria on fruit quality. Nonetheless, the role of spermidine in promoting vitamin\naccumulation has already been extensively studied and is not a core innovative point. This study does not evaluate whether\nthe described vitamin synthesis pathway is a major determinant of plant vitamin production. If it is not a primary factor\ninfluencing vitamin synthesis, the practical application value of this finding may be limited.\n5. In Figure S8, the direct application of flavonoids exhibited a noticeable effect on microbiome shaping from day 7 to day 15.\nHowever, this effect gradually diminished between day 15 and day 30, accompanied by a decrease in the population of\nLysobacter soli P18. The duration of the effect induced by direct flavonoid application was relatively short, limiting its\npractical applicability. This study did not fully evaluate the genetically modified plants at the gene level. To obtain results of\npractical significance, further analysis is needed to assess the vitamin-accumulating potential of SlMYB12-overexpressing\nplants.\n6. In this study, metagenomic, transcriptomic, and metabolomic analyses revealed that the increased production of\nflavonoids can promote the enrichment of Lysobacter soli P18 and the biosynthesis of spermidine. In Figure 4F, growth\ncurve analysis demonstrated that flavonoids did not significantly promote the reproductive growth of Lysobacter soli P18.\nHowever, metagenomic and transcriptomic analyses predicted that flavonoids could up-regulate the expression of genes\nrelated to twitching motility in Lysobacter soli P18. This prediction was validated by bacterial motility experiments shown in\nFigures 4C-E, which confirmed that flavonoids enhance the twitching motility of Lysobacter soli P18. Additionally, Figure 4B\nprovided evidence that flavonoids indeed promote the enrichment of Lysobacter soli. However, the molecular mechanisms\nunderlying the attraction of Lysobacter soli P18 enrichment have not been validated. Authors need to conduct further\nresearch on the specific molecular mechanisms that attract Lysobacter soli P18 to accumulate in the rhizosphere in order to\nprovide full evidence.\n7. In this study, although the reasons for not further investigating the specific regulatory sites of spermidine in plant vitamin\nbiosynthesis were described in L337-342—mainly due to the potential lethality caused by gene deletions in this pathway—\nthe evidence supporting spermidine's role in promoting plant vitamin synthesis seems insufficient. The evidence primarily\nrelies on metabolomic and transcriptomic data analysis, which may not be adequate to substantiate the newly discovered\nvitamin synthesis pathway reported in this study. In L251-259, specifically in the last paragraph of the results section, the\nauthors presented the transcriptomic data results. However, in L225, in section Bacterially produced spermidine promotes\nfruit vitamin accumulation, insufficient emphasis was placed on detailing the up-regulation of upstream gene expression of\nenzymes involved in vitamin biosynthesis upon spermidine treatment. Authors should conduct further molecular-level\nanalyses to provide more direct evidence. Additionally, the results section should reduce the descriptive focus on data\noutcomes and instead prioritize discussing the underlying mechanisms and specific pathways.\nMinor points\n1. In L17-18, the experimental subject analyzed is the rhizosphere microbe, whereas in the experimental methods described\nin L445-449, sequencing is conducted using natural soil or sterilized soil. This indicates a conceptual inconsistency in the\nstudy. Rigorous sampling is essential in research focusing on rhizosphere microbe-plant interactions.\n2. The random forest mentioned in L209 is not explained in the experimental methods section. It is essential to provide\ndetails of the experimental methods and data used in the study, as they serve as critical references for verifying the validity\nand reproducibility of the experiment.\n3. The verb \"enhance\" in the title is inconsistent with the singular subject \"biosynthesis\"; it should be revised to \"enhances\"\nto match the subject.\n4. In lines 209-211, aside from spermidine being up-regulated by 1.89-fold, cysteine and methionine also showed significant\nup-regulation. However, the study did not explain why these markedly up-regulated metabolites were not further\ninvestigated.\n5. In the KEGG enrichment plot analyzed in L245-246 (Figure S23D), the DEGs appear to be less significantly enriched in\n\"ascorbate and aldarate metabolism\" than described by the authors.\n6. In Figure S1, only three flavonoid compounds are up-regulated. However, these three flavonoids play significant roles in\npromoting the synthesis of vitamins in plants, which raises doubts about the accuracy of the result.\n7. In Figure S20, the results predicted using random forests show that, in addition to spermidine, other alkaloids also exhibit\nspecific up-regulation effects on different types of vitamins, which were not studied in this manuscript.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_2": "Point-by-point responses to the Reviewers’ comments\nReviewer #1 (Remarks to the Author):\n1. The manuscript describes the effects of flavonoids on the root microbiome and\nthe accumulation of vitamins in tomato fruit. The use of the Micro-tom tomato in\nthese experiments is concerning. Micro-tom is a brassinosteroid mutant\n(https://doi.org/10.1093/jxb/erj154) and brassinosteroids have been implicated in\nroot microbiome signaling (http://dx.doi.org/10.1016/j.molp.2015.12.003) , so the\nresults for other tomato varieties may be different. Wang et al., 2018 showed that\noverexpression of SlMYB12 affect flavonol biosynthesis in three tomato varieties.\nA full size tomato plant without the brassinosteroid mutations would be more\nappropriate for this work.\nANSWER: DONE. We appreciate your insightful comment regarding the potential\ninfluence of the Micro-tom tomato cultivar on our study’s outcomes. Indeed, Micro-\ntom is a brassinosteroid (BR) mutant and the role of BRs in root microbiome signalling\nhas been documented, which may affect the generalizability of our results to other\ntomato cultivars. Below, we address these points and provide additional analyses to\nstrengthen our manuscript.\nFirst, we selected Micro-tom because of its compact size, rapid life cycle, and\nwell-annotated genome, which are advantageous for high-throughput profiling of the\nroot microbiome and metabolome. Wang et al. (2018) reported that SlMYB12\noverexpression (OE) in three different Cherry tomato cultivars (Csl09-03, Micro-Tom,\nand Sheng Nv-Guo) led to an average increase of 5.07, 11.80, and 10.19-fold in\nflavonoid content compared with wild-type tomato, respectively. The average flavonoid\ncontent was highest in SlMYB12-overexpressing Micro-Tom tomato, which also\nrenders this cultivar more suitable for our study.\nSecond, we acknowledge that Micro-tom tomato carries a mutation in the\nbrassinosteroid biosynthesis pathway, which may influence root-microbe interactions.\nOur primary goal was to establish a proof-of-concept for flavonoid-mediated\nmodulation of the root microbiome and fruit vitamin accumulation. Going a step further,\nvalidating our findings in a non-BR-mutant tomato cultivar would demonstrate broader\napplicability and scientificity of our results. To address this issue, we conducted an\nadditional experiment to compare different wild-type tomato cultivars (Micro-tom vs.\nSheng Nv-Guo). This allowed us to confirm whether the observed effects of flavonoids\non root microbiome profiles and fruit vitamin levels are consistent across different\ngenotypes. After adding flavonoids to Sheng Nv-Guo tomato, rhizosphere microbial\n1\ncommunity structure changed in a similar pattern to that observed with Micro-Tom\ntomato. Meanwhile, enrichment of Lysobacter was observed in the flavonoid treatment,\nwhich contributed to increase vitamin content in tomato fruits.\nLine 94 to 129, revised & added, “To verify the involvement of soil microbiomes\nin fruit vitamin accumulation across different tomato cultivars, we devised a flavonoid\naddition experiment with wild-type Micro-Tom and Sheng Nv-Guo plants under natural\nand sterilised soil conditions. The experiment included a negative control (wild-type\nplants with no exogenous flavonoids, group Con) and a positive control (SlMYB12-\noverexpressing plants with up-regulated flavonoid biosynthesis, group O). In natural\nsoil, exogenous addition of flavonoids, either in combination or individually, increased\nthe vitamin contents in fruit samples of Micro-Tom plants compared to those of group\nCon. Equal mass combinations of rutin, NC and K3OR at different concentrations (A:\n10 µmol g-1"}, "author_response": "Point-by-point responses to the Reviewers’ comments\nReviewer #1 (Remarks to the Author):\n1. The manuscript describes the effects of flavonoids on the root microbiome and\nthe accumulation of vitamins in tomato fruit. The use of the Micro-tom tomato in\nthese experiments is concerning. Micro-tom is a brassinosteroid mutant\n(https://doi.org/10.1093/jxb/erj154) and brassinosteroids have been implicated in\nroot microbiome signaling (http://dx.doi.org/10.1016/j.molp.2015.12.003) , so the\nresults for other tomato varieties may be different. Wang et al., 2018 showed that\noverexpression of SlMYB12 affect flavonol biosynthesis in three tomato varieties.\nA full size tomato plant without the brassinosteroid mutations would be more\nappropriate for this work.\nANSWER: DONE. We appreciate your insightful comment regarding the potential\ninfluence of the Micro-tom tomato cultivar on our study’s outcomes. Indeed, Micro-\ntom is a brassinosteroid (BR) mutant and the role of BRs in root microbiome signalling\nhas been documented, which may affect the generalizability of our results to other\ntomato cultivars. Below, we address these points and provide additional analyses to\nstrengthen our manuscript.\nFirst, we selected Micro-tom because of its compact size, rapid life cycle, and\nwell-annotated genome, which are advantageous for high-throughput profiling of the\nroot microbiome and metabolome. Wang et al. (2018) reported that SlMYB12\noverexpression (OE) in three different Cherry tomato cultivars (Csl09-03, Micro-Tom,\nand Sheng Nv-Guo) led to an average increase of 5.07, 11.80, and 10.19-fold in\nflavon fruit vitamin accumulation than individual flavonoids (R: rutin, N: NC and K: K3OR;\n1 µmol g-1 each). The highest vitamin contents in fruit samples were observed for\nMicro-Tom plants of groups C and O (P < 0.05; Supplementary Fig. 4). Comparable\nresults were obtained from Sheng Nv-Guo plants in natural soil, where the combined\nand individual flavonoid treatments increased the vitamin contents in fruit samples\ncompared to group Con, and the highest values were observed in groups C (P < 0.05;\nSupplementary Fig. 4). In sterilised soil, exogenous addition of flavonoids did not affect\nfruit vitamin contents in Micro-Tom and Sheng Nv-Guo plants (Supplementary Fig. 5).\nMoreover, we analysed soil chemical properties and found no significant differences in\nkey soil variables (pH and nutrient contents) between the control and flavonoid-treat\ngroups (Supplementary Table 1). These results supported that flavonoids facilitate fruit\nvitamin accumulation across tomato genotypes in a soil microbiome-dependent manner.\nFlavonoids broadly contribute to rhizosphere bacterial diversity. To explore\nflavonoid-induced variations in rhizosphere microbial diversity, we performed 16S\nrRNA gene amplicon sequencing. Exogenous addition of flavonoids prominently\nenhanced bacterial alpha-diversity in the rhizosphere of wild-type Micro-Tom and\nSheng Nv-Guo plants under natural soil conditions. The bacterial richness (Chao1) and\ndiversity (Shannon) in rhizosphere soils of Micro-Tom plants were greater in groups B,\nC, D and N than group Con (P < 0.05), with peak values in group C (Fig. 2A, B). Soil\nsamples from SlMYB12-overexpressing plants (group O) also showed greater bacterial\nrichness and diversity compared to group Con (P < 0.05), and no significant differences\nwere observed between groups O and C (Supplementary Fig. 6A, B). The fungal alpha-\ndiversity in rhizosphere soils of Micro-Tom plants did not respond to exogenous\nflavonoids (Fig. 2D, E). Similar patterns were observed in alpha-diversity of bacteria\nand fungi when flavonoids were added to natural soil with Sheng Nv-Guo plants\n(Supplementary Figs. 6D, E and 7A–D). Furthermore, we added exogenous flavonoids\n2\nto unplanted natural soil and observed microbial alpha-diversity patterns comparable to\nthose seen in planted natural soil (Supplementary Fig. 8 A–D).”\nFig. 2 Flavonoids mediate variations in rhizosphere microbial diversity and community composition\nof wild-type Micro-Tom tomato in natural soil. A, B Bacterial richness (Chao1) and diversity\n(Shannon) under combined and individual flavonoid treatments. D, E Fungal richness (Chao1) and\ndiversity (Shannon) under different flavonoid treatments. Flavonoids were applied in combination\n(rutin: naringenin chalcone: kaempferol-3-O-rutinoside = 1:1:1, m/m/m; A: 10 µmol g-1, B: 3 µmol\ng-1, C: 1 µmol g-1 and D: 0.1 µmol g-1) and individually (N: naringenin chalcone, 1 µmol g-1; R:\nrutin, 1 µmol g-1; and K: kaempferol-3-O-rutinoside; 1 µmol g-1). Wild-type (Con) and SlMYB12-\noverexpressing (O) plants served as controls. Alpha-diversity data are presented as means ± standard\nerror, and different letters above the error bars indicate significant differences between treatments\n(n = 3, Tukey’s HSD test, P < 0.05).\nSheng Nv-Guo tomato plants in natural soil. A Vitamin C content. B Pyridoxine content. C\nPyridoxal content. D Pyridoxamine content. For detailed legend, see caption of Fig. 2.\nSheng Nv-Guo tomato plants in sterilised soil. A Vitamin C content. B Pyridoxine content. C\nPyridoxal content. D Pyridoxamine content. SC: rutin : naringenin chalcone : kaempferol-3-O-\nrutinoside = 1:1:1, m/m/m, 1 µmol g-1, wild-type (SCon) and SlMYB12-overexpressing (SO) plants\nserved as controls. Error bars represent standard deviation of the means (n = 3, Tukey’s HSD test).\nSignificant differences among treatments are indicated by different lowercase letters above the error\nbars (P < 0.05).\nFig. S6 Flavonoid-induced variations in rhizosphere bacterial diversity of wild-type Micro-Tom and\nSheng Nv-Guo tomato plants in natural soil. Bacterial alpha diversity in Micro-Tom (A, B) and\nSheng Nv-Guo (D, E) plants. Significant differences among treatments are indicated by different\nlowercase letters above the error bars (n = 3, Tukey’s HSD test, P < 0.05). Bacterial beta diversity\nin Micro-Tom (C) and Sheng Nv-Guo (F) plants revealed by principal coordinate analysis (PCoA)\nbased on unweighted UniFrac distance (P < 0.05). C: rutin : naringenin chalcone : kaempferol-3-O-\nrutinoside = 1:1:1, m/m/m, 1 µmol g-1, wild-type (Con) and SlMYB12-overexpressing (O) plants\nserved as controls\ncomposition of wild-type Sheng Nv-Guo tomato plants in natural soil. A, B Bacterial richness\n(Chao1) and diversity (Shannon) under combined and individual flavonoid treatments. C, D Fungal\nrichness (Chao1) and diversity (Shannon) under different flavonoid treatments. For detailed legend,\nsee caption of Fig. 2.\nFig. S8 Effects of exogenous flavonoids on microbial community diversity in unplanted natural soil.\nA, B Bacterial richness (Chao1) and diversity (Shannon) under combined and individual flavonoid\ntreatments. C, D Fungal richness (Chao1) and diversity (Shannon) under different flavonoid\ntreatments. For detailed legend, see caption of Fig. 2.\n2. The title is very wordy. Could this be simplified?\nANSWER: DONE. The title is simplified as suggested. “Flavonoid-mediated bacterial\n6\nspermidine biosynthesis enhances vitamin accumulation in tomato fruits”\n3. Figure 1 is far too complex. Is spermidine the only factor involved?\nANSWER: DONE. We have simplified Figure 1 for better readability. Detailed data\ncharts were replaced with simple text box or schematic diagrams.\nYou also raised an important point on spermidine, which is not the only factor\ncontributing to fruit vitamin accumulation, but the only metabolite of Lysobacter soli\nP18 responsive to flavonoids. Our Mantel’s test revealed a highly significant correlation\nbetween the relative abundance of alkaloids in the metabolites of P18 and the contents\nof vitamins C and B6 in tomato fruits (Line 246 to 248). Random forest analysis\nidentified a notably strong positive correlation between the contents of spermidine in\nbacterial culture and four vitamins in fruits (Line 248 to 251). Furthermore, we added\nexogenous spermidine (1 μmol g-1) to sterilised soil and observed increased vitamin\ncontents in wild-type Micro-Tom tomato (Line 279 to 281). Modified, “we added\nexogenous spermidine (1 μmol g-1) to sterilised soil with wild-type Micro-Tom plants.\nThis treatment significantly increased vitamin C, pyridoxine, pyridoxal and\npyridoxamine contents in tomato fruits (P < 0.05; Fig. 5I–L).”\nWe designated spermidine as a distinctive metabolite of P18 that is responsive to\nflavonoids. In follow-up studies, we also found a significant positive correlation\nbetween the expression levels of RpoN and spermidine biosynthesis-related genes.\nWe found that inoculation with the wild-type strain P18 and the complementary strain\nP18ΔRpoN-C increased the vitamin content compared to the control. However,\ninoculation with the mutant strain P18ΔRpoN had no significant effect on fruit vitamin\ncontent. Line 270 to 277, added, “Further, the function of RpoN was validated by gene\nknockout in L. soli P18. We found that fruit vitamin contents were significantly higher\nin Micro-Tom plants treated with cell suspensions of the wild-type strain and the\ncomplemented strain ΔRpoN-C compared to the control group (P < 0.01). Cell\nsuspension of the mutant strain ΔRpoN had no significant effect on fruit vitamin\ncontents (Fig. 5G). Under flavonoid treatment, ΔRpoN showed a significantly lower\nability to produce spermidine than its wild-type strain (P < 0.01), and this phenotype\nwas restored in ΔRpoN-C; Fig. 5H). In summary, these results indicated that flavonoids\nenhance spermidine biosynthesis of L. soli P18 under the control of RpoN.”\nfile.\nFig. 5 Flavonoids promote vitamin accumulation fruits of in Micro-Tom tomato by up-regulating\nspermidine production of Lysobacter soli P18. G Vitamin contents in fruits treated with cell\nsuspensions of L. soli strains (n = 5). P18 represents the wild-type strain, ΔRpoN is a RpoN mutant\nstrain, and ΔRpoN-C is a complemented strain. H Spermidine content in broth cultures of L. soli\nstrains treated without and with flavonoids (C: 1 µmol g-1, n = 10). I–L Vitamin contents in fruits\nof tomato plants treated with spermidine or not (n = 5, Tukey’s HSD test).\n8\n4. The amount of nutrients in the soil may affect the interaction of the plants and\nthe microbiome. How was the soil used selected? Was any fertilizer added to the\nsoil?\nANSWER: DONE. Different types of natural soil were collected from cropland fields\nin Yangling, Nanning, Chengdu, Ordos, and Kashgar, China. After removing plant\ndebris, worms and stones, the soil was homogenized manually using a 2.5-mm sieve.\nThe chemical properties of the soilsare provided in Supplementary Table 3. (Line 424\nto 427).\nIn the seedling, flowering and fruiting stages, 10 mL of Hoagland’s nutrient\nsolution was supplied to each pot without chemical fertilizer application. (Line 440 to\n441).\n5. Lines 40-41: What about everything in between the seedling stage and ripening?\nAre there studies on the effects of PGPR on plant growth, development or\nflowering?\nANSWER: Regarding plant developmental stages, we agree that investigating the\neffects of PGPR across the entire plant growth cycle would be valuable. However, our\nstudy specifically focused on establishing the novel mechanism whereby flavonoids\nmediate Lysobacter recruitment to enhance vitamin accumulation in tomato fruits. Our\ndata indicate certain plant growth-promoting effects of bacterial strains that responded\nto flavonoid enrichment in the rhizosphere of tomato (Fig. 2B, Supplementary table 2).\nWhile this part of work is not considered in the present study, we will further explore\nthe molecular mechanisms of tomato growth promotion by flavonoid-responsive PGPR\nin the future.\n6. Figure 1. Could it be replaced with a more generalized workflow? The graphs\nare too small to see. Even they are not the actual results, there is no need for them.\nIt is confusing for the reader.\nANSWER: DONE. Figure 1 has been replaced with a more generalized workflow as\nsuggested. Please also see our response to your Comment 3.\n7. The statistical analysis section of the methods is lacking information. How were\nLDA scores calculated? How were the PCAs done?\nANSWER: DONE. We have explained the calculation of LDA scores in the Methods.\nLine 518 to 522, added, “LDA scores were calculated using R v4.2.065 to maximize\nbetween-class variance while minimizing within-class variance. Data were\nstandardized prior to analysis (mean-centered and scaled to unit variance). The\ndiscriminant axes were ranked by their associated eigenvalues, and scores were\n9\nprojected onto the first two axes for visualization.”\nWe additionally provided more details for PCA. Line 659 to 660, added,\n“Multivariate analysis of gene expression profiles was conducted using PCA\nimplemented in R v4.3.265.” Line 687 to 688, added, “PCA was performed to analyse\nthe differences in metabolite composition using R v4.3.265.”\n8. Figure 1D. Which metabolites are significantly different between the two\ngenotypes? All of them? Why do you only focus on ascorbic acid and vitamin B6,\nif there are differences in many compounds?\nANSWER: DONE. Based on widely targeted metabolomic analysis, we found 679\ndifferential metabolites between the two genotypes in natural soil. KEGG enrichment\nanalysis revealed predominant enrichment of \"Vitamin B metabolism\" and \"aspartate\n6\nand alginate metabolism\" for the differential metabolites. Notably, ascorbic acid\n(vitamin C) and vitamin B (pyridoxine, pyridoxal and pyridoxamine) were the\n6\nmetabolites with the largest fold change in relative abundance, whereas these vitamins\nhad no significant difference in sterilised soil. Therefore, we focused on ascorbic acid\nand vitamin B in this part.\nand SlMYB12-overexpressing plants with up-regulated flavonoid biosynthesis21 were\ngrown in natural soil from Yangling, China. Flavonoid-targeted metabolomic analysis\nrevealed that 19 root metabolites and three rhizosphere soil metabolites were\ndifferentially accumulated (DAMs; fold-change ≥ 10, P < 0.01) in SlMYB12-\noverexpressing plants compared to wild-type plants. Among them, rutin, naringenin\nchalcone (NC) and kaempferol-3-O-rutinoside (K3OR) showed the most prominent\ndifferences in their relative abundance between groups (Supplementary Fig. 1). Based\non widely targeted metabolomic analysis, we compared the metabolite profiles of\ntomato fruits between wild-type and SlMYB12-overexpressing plants. A total of 3,092\nmetabolites were identified in all fruit samples, representing 13 chemical classes. The\nmost predominant classes were “flavonoids” (14.46 %), “amino acids and its\nderivatives” (13.69 %) and “alkaloids” (13.53 %; Supplementary Fig. 2A). There were\n(Supplementary Fig. 2B). To identify the physiological processes responsible for the\nDAMs, we performed KEGG pathway analysis. “Vitamin B metabolism” and\n6\n“ascorbate and aldarate metabolism” were predominantly enriched for the DAMs (P <\n0.01; Supplementary Fig. 2C). The relative abundances of vitamin C (ascorbic acid)\nand vitamin B (pyridoxine, pyridoxal and pyridoxamine) in fruit samples were\n6\nsignificantly higher in SlMYB12-overexpressing plants compared to wild-type plants (≥\n7-fold, P < 0.001; Supplementary Fig. 2D). This pattern was confirmed by hydrophilic\ninteraction chromatography (HILIC) and high-performance liquid chromatography\n10\n(HPLC) of vitamins C and B in fruit samples (P < 0.05; Supplementary Fig. 3A–D),\n6\nbut not in sterilised soil (Supplementary Fig. 3E–H). These results allowed us to posit\nthat flavonoids promote vitamin accumulation in tomato fruits depending on the\npresence of soil microbiomes.”\nFig. S1 Comparison of root and soil flavonoids between wild-type Micro-Tom and SlMYB12-\noverexpressing plants based on targeted metabolomics. A Volcano plot of differentially abundant\nflavonoids in tomato roots. B Volcano plot of differentially abundant flavonoids in rhizosphere soils.\noverexpressing fruits in natural soil. A Classification of metabolites in fruits. B Volcano plot of\ndifferentially abundant metabolites (P < 0.05). C KEGG pathway enrichment of differentially\nabundant metabolites. D Relative abundance (fold change, FC) of differentially abundant\nmetabolites (P < 0.001).\nSlMYB12-overexpressing plants under different soil conditions. A–D Vitamin C content, pyridoxine\ncontent, pyridoxal content, and pyridoxamine content in natural soil. E–H Vitamin C content,\npyridoxine content, pyridoxal content, and pyridoxamine content in sterilised soil. Error bars\nrepresent standard deviation of the means (n = 3, Tukey’s HSD test). Significant differences among\ntreatments are indicated by different lowercase letters above the error bars (P < 0.05).\n9. Figure 2. A list of metabolite abbreviations within the figure would be helpful\nfor the reader.\nANSWER: DONE. We added a list of metabolite abbreviations in the legend of Figure\n2. “Flavonoids were applied in combination (rutin: naringenin chalcone: kaempferol-3-\nO-rutinoside = 1:1:1, m/m/m; A: 10 µmol g-1, B: 3 µmol g-1, C: 1 µmol g-1 and D: 0.1\nµmol g-1) and individually (N: naringenin chalcone, 1 µmol g-1; R: rutin, 1 µmol g-1;\nand K: kaempferol-3-O-rutinoside; 1 µmol g-1). Wild-type (Con) and SlMYB12-\noverexpressing (O) plants served as controls.”\n10. Figure 2. A definition of Chao1 and Shannon in the figure legend would be nice.\nANSWER: DONE. We have added a definition of alpha diversity indices to the legend\nof Figure 2. “A, B Bacterial richness (Chao1) and diversity (Shannon) under combined\nand individual flavonoid treatments. D, E Fungal richness (Chao1) and diversity\n(Shannon) under different flavonoid treatments.”\n11. Figure 2. What is the difference between figures 2C and 2F? Is one fungal and\n13\nthe other bacterial communities? Why are bacterial communities p< 0.05 and\nfungal p>0.05?\nANSWER: DONE. Figure 2C shows bacterial beta diversity and Figure 2F shows\nfungal beta diversity. We have modified figure legend for clarify.\nBacterial beta diversity in rhizosphere soils indicated significant compositional\nshifts (p < 0.05) in response to flavonoid treatment (Figure 2C). This aligns with our\nfinding that bacterial communities were highly responsive to exogenous flavonoids. In\ncontrast, fungal beta diversity in rhizosphere soils did not significantly change (p > 0.05)\nafter flavonoid treatment (Figure 2F). This suggests fungal communities were less\nsensitive to flavonoids under our experimental conditions. Moreover, it has been found\nthat flavonoids can alleviate nutrient deficiencies and drought stress to maintain normal\nplant growth by mediating rhizosphere bacterial microbiome reshaping and beneficial\ntaxa enrichment (Yu, P. et al. 2021; He, P. et al. 2021; Wu, P. et al. 2025; Xu, P. et al.\n2025). This provides additional evidence supporting that bacterial communities in the\nrhizosphere soil are more sensitive to flavonoids.\n12. Figure 2E. The metabolite order is different from the other graphs. Please\nchange.\nANSWER: DONE. We have unified the metabolic order in Figure 2E for consistency\nwith Figure 2D.\nFig. 2 Flavonoids mediate variations in rhizosphere microbial diversity and community composition\nof wild-type Micro-Tom tomato in natural soil. D, E Fungal richness (Chao1) and diversity\n(Shannon) under different flavonoid treatments.\n13. Figure 3. Explain what the key bacteria in the figure are. Are they the bacteria\nlisted in the tree?\nANSWER: DONE. We have marked the key bacteria in the phylogenetic tree with\nasterisks (Fig. 3A) and explained strain names (Fig. 3B).\nenrichment. A Phylogenetic tree of culturable bacterial isolates from flavonoid-treated rhizosphere\nsoils based on the neighbour-joining method. Six key bacteria are marked with an asterisk (*). B\nGrowth of tomato plants treated with cell suspensions of key bacteria.\n14. Figure 5A. Cysteine is spelled wrong.\nANSWER: DONE. Corrected “Cystenine” to “Cysteine”.\nspermidine production of Lysobacter soli P18. A Relative abundance of spermidine, cysteine and\nmethionine in fruits of flavonoid treatment (C: 1 µmol g-1) vs. control groups based on widely\ntargeted metabolomic profiling.\n15. Figure 6. The numbers in the legend do not match the numbers in the figure.\nANSWER: DONE. We have modified the numbers in the legend in the same order as\nshown in the figure and explained more detail for the mechanism of action of flavonoids.\nFig. 6 Schematic presentation of the mechanism that enables root-derived flavonoids\nto promote vitamin accumulation in tomato fruits. (1) Plant roots release flavonoid\nsignals into the rhizosphere, where they interact with soil microbes. (2) Flavonoids\nrecruit Lysobacter to the rhizosphere by enhancing bacterial twitching motility, which\nreshapes the rhizosphere microbiome. (3) Lysobacter responds to flavonoids in a RpoN-\ndependent manner and produces more spermidine. (4) Spermidine up-regulates fruit\nvitamin metabolism through the L-galactose pathway and thereby boosts the\naccumulation of vitamins B and C in fruits.\n6\n16. Figure S1A. What are the other compounds that are significant in tomato roots?\nOnly three are labeled.\nANSWER: DONE. Micro-Tom tomato plants overexpress the SlMYB12 gene, which\nup-regulates flavonoid biosynthesis including rutin, naringenin chalcone (NC), and\nkaempferol-3-O-rutinoside (K3OR; Wang, S. et al, 2018). As stated in Line 71 to 78,\n“First, wild-type plants of Micro-Tom tomato and SlMYB12-overexpressing plants with\nup-regulated flavonoid biosynthesis21 were grown in natural soil from Yangling, China.\nFlavonoid-targeted metabolomic analysis revealed that 19 root metabolites and three\nrhizosphere soil metabolites were differentially accumulated (DAMs; fold-change ≥ 10,\nP < 0.01) in SlMYB12-overexpressing plants compared to wild-type plants.\nAmong them, rutin, naringenin chalcone (NC) and kaempferol-3-O-rutinoside (K3OR)\nshowed the most prominent differences in their relative abundance between groups\n(Supplementary Fig. 1).”\noverexpressing plants based on targeted metabolomics. A Volcano plot of differentially abundant\nflavonoids in tomato roots. B Volcano plot of differentially abundant flavonoids in rhizosphere soils.\n17. Figure S5-S9. Add the abbreviation meanings to the figure or figure legend so\nthe reader does not have to refer back to the main manuscript.\nANSWER: DONE. All abbreviations used in Figures S5-S10 have been defined in the\nlegend.\nFig. S4, added, Flavonoids were applied in combination (rutin : naringenin chalcone :\nkaempferol-3-O-rutinoside = 1:1:1, m/m/m; A: 10 µmol g-1, B: 3 µmol g-1, C: 1 µmol\ng-1 and D: 0.1 µmol g-1) and individually (N: naringenin chalcone, 1 µmol g-1; R: rutin,\n1 µmol g-1; and K: kaempferol-3-O-rutinoside; 1 µmol g-1). Wild-type (Con) and\nSlMYB12-overexpressing (O) plants served as controls….\nFig. S5, added, SC: rutin : naringenin chalcone : kaempferol-3-O-rutinoside = 1:1:1,\nm/m/m, 1 µmol g-1; wild-type (SCon) and SlMYB12-overexpressing (SO) plants served\nas controls….\nFig. S6, added, C: rutin : naringenin chalcone : kaempferol-3-O-rutinoside = 1:1:1,\nm/m/m, 1 µmol g-1; wild-type (Con) and SlMYB12-overexpressing (O) plants served as\ncontrols.\nFigs. S7–S10, added, Flavonoids were applied in combination (rutin: naringenin\nchalcone: kaempferol-3-O-rutinoside = 1:1:1, m/m/m; A: 10 µmol g-1, B: 3 µmol g-1, C:\n1 µmol g-1 and D: 0.1 µmol g-1) and individually (N: naringenin chalcone, 1 µmol g-1;\nR: rutin, 1 µmol g-1; and K: kaempferol-3-O-rutinoside; 1 µmol g-1).\n17\n18. Line 362: Percent humidity in moisture chamber?\nANSWER: DONE. The pots (one plant per pot) were kept in a humidity chamber with\na day/night temperature of 26°C/18°C, a relative humidity of 60–70% ...... (Line 438 to\n439).\n19. Line 382: What is the internal standard? Is it methanol or was the internal\nstandard diluted in methanol?\nANSWER: DONE. The sample was completely freeze-dried in a lyophiliser and then\nadded with 200 µL of 70% methanol internal standard extract. (Line 454 to 456).\n20. Throughout the methods section L. soli is misspelled as L. soil.\nANSWER: DONE. We appreciate your close attention and have corrected the spelling\nto \"L. soli\" (e.g., Line 541, 574, 579, 590, 599, 612).\nThis study explores a hot topic in the plant-microbe interaction, revealing how\nflavonoids secreted by tomato roots recruit specific soil microbes, such as\nLysobacter soli, to the rhizosphere.\nThe recruited microbes produce spermidine, which the authors propose plays a\npivotal role in enhancing vitamin C and B accumulation in tomato fruits. By\n6\ncombining advanced microbial profiling with metabolomic and plant biochemical\nanalyses, the authors present a compelling data to prove the hypothesis that: 1)\nflavonoid-driven mechanism shapes the rhizosphere microbiome, contribute to the\nstrain-level recruitment and eventually influences plant nutritional quality.\nThese study benefit from broad analysis, and multi layer omic findings to provide\nmechanistic insights into the flavonoid mediated plant-microbiome interaction\nand its effects on tomato nutritional traits.\nThe research is well-designed, employs an adequate level of analysis, and benefits\nfrom a clear and effective writing style. However, I have some concerns regarding\nthe conceptual aspects of this study, which I invite the authors to clarify.\nANSWER: Thank you very much for your suggestions, which are helpful and\nconstructive for improving the quality of our MS. We have addressed your concerns as\nfollows.\nSpecific-level selection VS overall diversity effect\n1. The manuscript hypothesis turns around the fact “that tomato plants recruit\nspecific rhizosphere microbes and modulate microbial metabolic patterns through\nroot exudates (e.g. flavonoids), thereby enhancing fruit vitamin accumulation”.\nThe authors used 16S rRNA gene amplicon and metagenomic sequencing,\nCulture-based experiments are conducted to ascertain the role of flavonoid-\nresponsive microbial taxa represented by Lysobacter in fruit vitamin\naccumulation.\nCan the flavonoid effect specifically increase the abundance of the taxa described\nby the authors, such as Bacillus and L. soli, or does it enhance overall microbial\ndiversity, with these taxa increasing as a result of the general rise in bacterial\nabundance? If the latter is true, the 'strain-level recruitment' hypothesis would\nnot be supported. How does the presented data effectively disprove the second\nhypothesis, which suggests a general increase in all taxa, including the target taxa?\nANSWER: Our data robustly support flavonoid-mediated strain-specific recruitment,\nrather than general microbial enrichment in the rhizosphere of tomato. Differential taxa\nabundance analysis based on 16S sequencing data revealed that flavonoids exclusively\nincreased specific taxa (e.g., Lysobacter, Bacillus), while other rhizosphere microbial\n19\ntaxa barely changed. Previously, specific recruitment of microorganisms by root\nexudates has been reported. For example, Yang et al. (2023) found that microbial alpha\ndiversity was enhanced in wild-type Micro-Tom (WT) tomato compared to RIN mutant\n(rin) tomato. This was accompanied by increased proportions of Streptomyces,\nBacillus, and Pseudomonas in the microbiota of WT plants. In particular, the role of\nStreptomyces in the bacterial community was enhanced in WT plants than in rin plants.\nFurthermore, rin plants showed defects in the production of ethylene and root exudates,\nwhich weakened the inhibitory effect of rhizosphere microbiota on bacterial wilt. In contrast,\nWT plants produced ethylene and secreted specific root exudates to attract disease-\ninhibiting Streptomyces species and protect the plants from bacterial wilt.\nMany microorganisms in rhizosphere soil respond differentially to root exudates,\nconferring benefits to host plants. For example, root-derived flavonoids mediate the\nreshaping of rhizosphere microbiomes and enrichment of beneficial taxa (e.g.,\nOxalobacteraceae, Aeromonas sp., Pseudomonas), thereby alleviate plant nutrient\ndeficiencies and drought stress (Xu et al. 2021, He et al. 2021, Wu et al. 2025, Xu et al.\n2025). Soybean can secrete xanthine to recruit Pseudomonas to the rhizosphere soil,\nenhancing salt tolerance in the plants (Zhang et al. 2021). Methyl-p-hydroxyvanillate\nfrom root exudates inhibits plant pathogens and promotes rhizospheric enrichment of\nBacillus as regulated by caffeic acid O-methyltransferase NtCOMT10, thus bolstering\nplant disease resistance (Ma et al. 2021).\nTaken together, these findings allow us to posit that flavonoids induce an increase\nin the relative abundance of specific taxa, rather than the total abundance of microbial\ncommunity. Lysobacter and Bacillus emerge as the most responsive genera to\nflavonoids and are most sensitive to flavonoid signalling molecules.\n2. Co-founding factor to clarified or acknowledge\nThe study establishes a strong correlation between flavonoid-mediated changes in\nthe microbiome and vitamin accumulation in fruits but may not conclusively\ndemonstrate causation. The enrichment of Lysobacter soli and increased\nvitamin levels might be due to other indirect factors influenced by flavonoids, such as\nsoil pH, nutrient availability, or plant root exudates. Has the study thoroughly\nruled out other variables that might confound the flavonoid-microbiome-vitamin\npathway?\nANSWER: we analysed soil chemical properties and found no significant differences\nin key soil variables (pH and nutrient contents) between the control and flavonoid-treat\ngroups (Supplementary Table 1) (Line 110 to 112). This rules out the influence of pH-\ndriven microbiome changes and nutrient availability on the flavonoid-microbiome-\nvitamin pathway.\ntype Micro-Tom\nTreat pH Organic matter Total N content Total P content\nment content (g/kg) (g/kg)\n(g/kg)\nCon 7.23±0.06a 12.16±0.95a 0.96±0.09a 0.81±0.05a\nC 7.25±0.08a 11.98±0.89a 0.90±0.07a 0.79±0.07a\nO 7.29±0.07a 11.58±0.71a 0.93±0.06a 0.79±0.06a\nTotal K content Alkali-hydrolysable N Available P content Available K\n(g/kg) content (mg/kg) (mg/kg) content\n(mg/kg)\nCon 14.46±1.25a 47.08±2.69a 14.69±1.11a 121.62±9.54a\nC 14.66±1.56a 45.30±2.87a 13.56±1.04a 126.18±8.28a\nO 14.11±1.47a 45.30±2.18a 13.93±0.97a 125.88±7.12a\nNote: The soil was collected from farmland in Yangling, China. Wild-type Micro-Tom (Con) and\nSlMYB12-overexpressing (O) plants served as controls. C: rutin : naringenin chalcone : kaempferol-\n3-O-rutinoside = 1:1:1, m/m/m, 1 µmol g-1. Data are presented as means ± standard error, and\ndifferent lowercase letters indicate significant differences between treatments (n = 3, Tukey’s HSD\ntest, P < 0.05).\n3. Generalization of Findings\nThe flavonoid positive effects are shown on the other context in the tomato\nMicrotom, for example disease resistant or overall growth. See please this study\nby Yang et al, 2023: For example see this paper by: RIN enhances plant disease\nresistance via root exudate-mediated assembly of disease suppressive rhizosphere\nmicrobiota.\nThe study focuses on the specific interaction between flavonoids, Lysobacter soli,\nand tomato plants. It is unclear if these findings are broadly applicable across\ndifferent plant species, soil types, or environmental conditions.\nANSWER: DONE. Cross-species and cross-environment validation is critical for\ntranslating mechanistic insights into broader applications. We supplemented an\nexperiment with Micro-Tom tomato grown in different soil types from geographically\ndistant regions. Line 188 to 193, added, “Moreover, L. soli P18 treatment significantly\nelevated vitamin contents in fruit samples of Micro-Tom plants in sterilised soils from\nKashgar, Ordos, Chengdu and Nanning. There was no significant difference in fruit\nvitamin contents between L. soli P18-treated plants and SlMYB12-overexpressing\nplants (P < 0.05; Fig. 3G). These results indicated that the observed effects of L. soli\nP18 on fruit vitamin levels are consistent across geographically distinct soils.”\ncultivars (Micro-Tom vs. Sheng Nv-Guo) in the same soil from Yangling. Line 186 to\n188, added, “Consistently, the treatment with L. soli P18 increased vitamin contents in\nfruit samples of Sheng Nv-Guo plants (P < 0.05; Supplementary Fig. 14), confirming\nthat the positive effects of L. soli P18 on fruit vitamin levels are robust across plant\ngenotypes.”\nWhile our findings require further testing in other crops systems, the consistency\nof observations across soil types and tomato cultivars supports their potential relevance\nbeyond the specific conditions examined here.\nFig. 3 Flavonoids promote vitamin accumulation in fruits of Micro-Tom tomato through Lysobacter\nenrichment. G Vitamin contents in fruit samples of tomato plants grown in geographically distant\nsoils that had been sterilised and treated with L. soli P18 cell suspensions (n = 5). SO represents\nSlMYB12 overexpression, SCon represents sterile 1× phosphate-buffered saline (control) and SP18–\nSP66 represent cell suspensions of key bacterial strains diluted with 1× phosphate-buffered saline.\nFig. S14 Vitamin contents in fruits of wild-type Sheng Nv-Guo tomato treated with bacterial cell\nsuspensions of Lysobacter soli P18. A Vitamin C content. B Pyridoxine content. C Pyridoxal content.\nD Pyridoxamine content. Error bars represent standard deviation of the means (n = 5, Tukey’s HSD\ntest). Significant differences among treatments are indicated by different lowercase letters above the\nerror bars (P < 0.05). SCon represents sterile 1× phosphate-buffered saline (control) and SP18\n22\nrepresent cell suspensions diluted with 1× phosphate-buffered saline.\n4. Vitamin Accumulation Pathway\nThe study attributes vitamin C and B6 accumulation to microbial spermidine\nproduction but does not explore whether flavonoids directly influence the plant’s\nown vitamin biosynthesis pathways? Was there an attempt to decouple the plant’s\nintrinsic biosynthetic response from the microbial influence?\nANSWER: DONE. Decoupling plant-intrinsic pathways from microbial influences is\nessential for mechanistic clarity. Our results (including new data) explicitly addressed\nyour concern through the evidence from sterile and natural soil experiments.\n(1) Sterile soil experiments rule out plant's intrinsic responses\nWe compared fruit vitamin levels in SlMYB12-overexpressing tomato plants (with\nupregulated flavonoid biosynthesis) vs. wild-type Micro-Tom plants grown in sterilised\nsoil. When soil microbiota was eliminated, endogenous flavonoids failed to stimulate\nvitamin accumulation in fruits. Line 89 to 93, modified, “This pattern was confirmed\nby hydrophilic interaction chromatography (HILIC) and high-performance liquid\nchromatography (HPLC) of vitamins C and B in fruit samples (P < 0.05;\nresults allowed us to posit that flavonoids promote vitamin accumulation in tomato\nfruits depending on the presence of soil microbiomes.”\nWe found no significant changes in vitamin content in fruits treated with exogenous\nflavonoids in sterilised soil. Line 108 to 110, modified, “In sterilised soil, exogenous\naddition of flavonoids did not affect fruit vitamin contents in Micro-Tom and Sheng\nNv-Guo plants (Supplementary Fig. 5).”\nIn summary, fruit vitamin content did not change in response to exogenous or\nendogenous flavonoids in the absence of soil microbiomes.\nSlMYB12-overexpressing plants under different soil conditions. A–D Vitamin C content, pyridoxine\ncontent, pyridoxal content, and pyridoxamine content in natural soil. E–H Vitamin C content,\npyridoxine content, pyridoxal content, and pyridoxamine content in sterilised soil. Error bars\nrepresent standard deviation of the means (n = 3, Tukey’s HSD test). Significant differences among\ntreatments are indicated by different lowercase letters above the error bars (P < 0.05).\nFig. S5 Effect of exogenous flavonoids on fruit vitamin accumulation of wild-type Micro-Tom and\nSheng Nv-Guo tomato plants in sterilised soil. A Vitamin C content. B Pyridoxine content. C\nPyridoxal content. D Pyridoxamine content. SC: rutin : naringenin chalcone : kaempferol-3-O-\nrutinoside = 1:1:1, m/m/m, 1 µmol g-1, wild-type (SCon) and SlMYB12-overexpressing (SO) plants\nserved as controls. Error bars represent standard deviation of the means (n = 3, Tukey’s HSD test).\nSignificant differences among treatments are indicated by different lowercase letters above the error\n24\nbars (P < 0.05).\n(2) Natural soil experiments confirm microbial dependence\nComparing SlMYB12-overexpressing plants to wild-type plants grown in natural\nsoil revealed: 1) Rhizosphere enrichment of Lysobacter (Fig. 2G, Supplementary Fig\n7G). Line 147 to 154, revised & added, “At the genus level, linear discriminant\nanalysis (LDA) effect size (LEfSe) analysis identified 26 flavonoid-responsive\nbiomarkers in rhizosphere soils of wild-type Micro-Tom plants (LDA > 3). Among\nthem, six genera were enriched in group O, with eight genera enriched in group C.\nIn both groups O and C, Lysobacter and Bacillus were two common genera that increased\nin relative abundance under flavonoid treatment (Fig. 2G). Furthermore, we identified\n32 flavonoid-responsive genera in rhizosphere soils of Sheng Nv-Guo plants (LDA >\n3), including Lysobacter and Bacillus that were also enriched in group C\n(Supplementary Fig. 7G).”\n2) Elevation of fruit vitamin C/B levels (Supplementary Fig. 3A-D,\ninteraction chromatography (HILIC) and high-performance liquid chromatography\n(HPLC) of vitamins C and B in fruit samples (P < 0.05; Supplementary Fig. 3A–D),\n6\nbut not in sterilised soil (Supplementary Fig. 3E–H). These results allowed us to posit\nthat flavonoids promote vitamin accumulation in tomato fruits depending on the\npresence of soil microbiomes (Line 89 to 97).” “Comparable results were obtained from\nSheng Nv-Guo plants in natural soil, where the combined and individual flavonoid\ntreatments increased the vitamin contents in fruit samples compared to group Con, and\nthe highest values were observed in groups C (P < 0.05; Supplementary Fig. 4) (Line\n105 to 108).”\nThese results demonstrate that fruit vitamin accumulation requires both microbial\nflavonoid secretion and a functional soil microbiome containing Lysobacter and\nBacillus.\nFig. 2 Flavonoids mediate variations in rhizosphere microbial diversity and community composition\nof wild-type Micro-Tom tomato in natural soil. G List of rhizosphere biomarkers in planted soil\nidentified by linear discriminant analysis (LDA) effect size. H List of rhizosphere biomarkers in\n25\nunplanted soil (n = 3). Flavonoids were applied in combination (rutin: naringenin chalcone:\nkaempferol-3-O-rutinoside = 1:1:1, m/m/m; A: 10 µmol g-1, B: 3 µmol g-1, C: 1 µmol g-1\nand D: 0.1 µmol g-1) and individually (N: naringenin chalcone, 1 µmol g-1; R: rutin, 1 µmol g-1; and K:\nkaempferol-3-O-rutinoside; 1 µmol g-1). Wild-type (Con) and SlMYB12-overexpressing (O) plants\nserved as controls.\nFig. S7 Flavonoid-mediated variations in rhizosphere microbial diversity and community\ncomposition of wild-type Sheng Nv-Guo tomato plants in natural soil. G List of rhizosphere\nbiomarkers identified by linear discriminant analysis (LDA) effect size. For detailed legend, see\ncaption of Fig. 2.\nSheng Nv-Guo tomato plants in natural soil. A Vitamin C content. B Pyridoxine content. C\nPyridoxal content. D Pyridoxamine content. For detailed legend, see caption of Fig. 2.\n(3) Multilevel analyses identify microbial spermidine as a direct causal agent\nOur analyses validated the complete pathway by which root-derived flavonoids\nenhance fruit vitamin accumulation: Plant roots release flavonoid signals →\nFlavonoids recruit L. soli → L. soli produces spermidine → Spermidine enhances\nfruit vitamin accumulation (Fig. 6).\nvitamin accumulation in tomato fruits. (1) Plant roots release flavonoid signals into the rhizosphere,\nwhere they interact with soil microbes. (2) Flavonoids recruit Lysobacter to the rhizosphere by\nenhancing bacterial twitching motility, which reshapes the rhizosphere microbiome. (3) Lysobacter\nresponds to flavonoids in a RpoN-dependent manner and produces more spermidine. (4) Spermidine\nup-regulates fruit vitamin metabolism through the L-galactose pathway and thereby boosts the\naccumulation of vitamins B and C in fruits.\n6\nThis study examines how flavonoids influence interactions between plants and\nrhizosphere microbes, contributing to changes in vitamin levels in Micro-Tom\ntomato fruits. Overexpression of the SlMYB12 gene, which enhances flavonoid\nbiosynthesis, was associated with increased levels of vitamins C and B6 in fruits\ngrown in natural soil, highlighting the involvement of the soil microbiome.\nMetagenomic and transcriptomic analyses indicated that flavonoids promote the\nenrichment of rhizosphere microbes such as Lysobacter by enhancing bacterial\ntwitching motility. Metabolomic and transcriptomic profiling further suggested\nthat flavonoids stimulate spermidine biosynthesis in Lysobacter soli P18, which\nmay influence vitamin metabolic pathways in the fruits. These findings outline a\npossible mechanism for flavonoid-mediated rhizosphere signaling and its impact\non fruit quality. While I appreciate the findings' potential influence on the\nnutritional quality of horticultural crops, the manuscript falls short of fully\naddressing its stated objectives, exhibiting notable gaps in the\ncomprehensiveness of the research and weaknesses in the methodological approach.\nWhile I like the potential of these\nfindings to contribute to the understanding of plant-microbe interactions and their impact on enhancing the nutritional value\nof horticultural crops, I feel that the manuscript fails to meet its objectives in several\naspects, including the workload, depth of research, and construction of methodological\nframeworks.\nANSWER: We sincerely thank you for thoughtful consideration of our work and\nacknowledge your concerns regarding the study’s scope and methodological depth. The\nMS has been substantially improved to address these issues. Please see our response in\nthe following sections.\nMajor\n1. In L208-213, the study employs a random forest algorithm for predictive\nanalysis, however, the manuscript lacks an explanation for the rationale behind\nthe algorithm selection and does not compare it with other algorithms, which\nlimits the generalizability and robustness of the results. The discussion on the\nperformance metrics of the chosen algorithm is insufficient, and there is a high\ndependency on the predictive outcomes without adequate empirical evidence to\nsupport them. To enhance the transparency, reliability and validity of the research,\nit is recommended to supplement the rationale for algorithm selection, introduce\ncomparative analysis with other machine learning algorithms, and provide a\ndetailed discussion on performance metrics. Additionally, presenting more\nexperimental empirical data would strengthen the credibility of the predictive\n29\nresults.\nANSWER: DONE. We have explained the rationale of algorithm selection to\nstrengthen the generalizability and robustness of predictive modeling. Random forest\nwas selected for its ability to: (i) handle high-dimensional metabolomics data with\npotential multicollinearity; (ii) capture non-linear relationships between bacterial\nmetabolites and fruit vitamins without pre-specified parametric assumptions; (iii) rank\nintrinsic feature importance (mean decrease in Gini impurity) for identifying key\nmetabolic drivers; and (iv) resist overfitting via ensemble bootstrapping and feature\nsubsampling. These advantages make random forest analysis an ideal tool to predict\ncomplex biological interactions.\nModel performance was quantified using 10-fold cross-validation to ensure\nrobustness of the results. Line 248 to 251, added, “Among alkaloids, spermidine\nemerged as the greatest contributor to fruit vitamin accumulation based on random\nforest analysis. Based on the cross-validation results, the random forest model achieved\noptimal stability and accuracy when selecting the top 20 metabolites (Supplementary\nFig. 20).”\nFig. S20 The top 20 marker metabolites of Lysobacter soli P18 identified by random forest\n30\nclassification. A Importance to vitamin C content in tomato fruits. B Importance to pyridoxine\ncontent in tomato fruits. C Importance to pyridoxal content in tomato fruits. D Importance to\npyridoxamine content in tomato fruits. The marker metabolites are ranked in descending order of\nimportance concerning the model’s accuracy. The inset represents 10-fold cross-validation error as\na function of the number of input metabolites in order of variable importance.\n2. In the experimental design, the authors employed different concentrations and\ncombinations of flavonoid treatments to investigate their compatibility with\nSlMYB12-overexpressing plants. Notably, this study found that the addition of\nthree flavonoids at a concentration of 1 µmol g-¹ in a 1:1:1 ratio could simulate the\nconditions of the SlMYB12-overexpressing group, providing new insights into the\nrole of flavonoids in gene regulation. However, we also acknowledge a significant\nlimitation of this study: subsequent experiments did not include SlMYB12-\noverexpressing treatment group. While the addition of flavonoids may mimic the\neffects of gene over-expression, it is not sufficient to fully substitute for actual gene\nover-expression experiments. Experiments involving the gene over-expression\ngroup are critical for verifying whether flavonoid treatments can truly replace the\neffects, as they provide direct evidence of gene expression regulation and\npotential molecular mechanisms. Therefore, I recommend that the authors incorporate a\ngene over-expression treatment group in future studies to compare the differences\nand connections between flavonoid treatments and gene over-expression. Such an\nexperimental design would contribute to a more comprehensive understanding of\nthe mechanisms underlying the effects of flavonoids and enhance the\npersuasiveness and scientific value of this study’s findings.\nANSWER: DONE. In the revision, we conducted a new experiment with SlMYB12-\noverexpressing plants as control to explore whether Lysobacter soli P18 exhibits\nuniversal effects on vitamin accumulation in the fruits of tomato plants grown in soils\nfrom different geographical regions. Line 188 to 192, added, “Moreover, L. soli P18\ntreatment significantly elevated vitamin contents in fruit samples of Micro-Tom plants\nin sterilised soils from Kashgar, Ordos, Chengdu and Nanning. There was no significant\ndifference in fruit vitamin contents between L. soli P18-treated plants and SlMYB12-\noverexpressing plants (P < 0.05; Fig. 3G).”\nenrichment. G Vitamin contents in fruit samples of tomato plants grown in geographically distant\nsoils that had been sterilised and treated with L. soli P18 cell suspensions (n = 5). SO represents\nSlMYB12 overexpression, SCon represents sterile 1× phosphate-buffered saline (control) and SP18–\nSP66 represent cell suspensions of key bacterial strains diluted with 1× phosphate-buffered saline.\n3. In this article, the authors did not explain why the simultaneous addition of\nthree flavonoids in a 1:1:1 ratio yielded better effects. This indirectly reflects the\nlack of clarity regarding the specific pathways influencing spermidine biosynthesis\nin Lysobacter soli P18 in this study. The study primarily relied on analyses of\nmicrobial metagenomics, transcriptomics, and metabolomics to calculate\ncorrelations between microbial enrichment and compound variation using\nstatistical methods, without further validation through molecular experiments.\nThe specific molecular mechanisms by which flavonoids promote spermidine\nbiosynthesis in Lysobacter soli P18 were not elucidated in this research.\nANSWER: DONE. In the original study, treatment C (rutin : naringenin chalcone :\nkaempferol-3-O-rutinoside = 1:1:1, m/m/m, 1 µmol g-1) showed the greatest effect on\nLysobacter recruitment. In the revision, we explored whether flavonoids, in\ncombination or separately, could promote spermidine production in Lysobacter soli P18\nunder treatment C. Our new data indicated that treatment C had a greater ability to\npromote spermidine production in Lysobacter soli P18 than individual flavonoid\ntreatments, indicating synergy of the three flavonoids. Line 260 to 265, added, “To\nvalidate the effect of flavonoids on spermidine biosynthesis in L. soli P18, we\nperformed chemical extraction and quantitative analysis. The spermidine content in\nculture samples added with combined flavonoids (treatment C: 1 µmol g-1) was 2.8-\nfold higher than that of the control group (P < 0.05). Combined flavonoids exhibited a\ngreater effect on spermidine production in L. soli P18 compared to individual\nflavonoids (treatments R, K, N; P < 0.05; Fig. 5F).”\nflavonoids significantly upregulated RpoN expression—a sigma factor regulating\nspermidine synthase genes. The gene co-expression network revealed a positive\ncorrelation of RpoN to SpeD and SpeE. We validated this cascade using RpoN knockout\nmutants, where flavonoids failed to induce spermidine biosynthesis. This confirms that\nflavonoids activate the RpoN-spermidine regulatory axis and RpoN is indispensable for\nflavonoid-driven spermidine production in Lysobacter soli P18. Line 267 to 277, added, “Interestingly, the gene co-expression network revealed a positive correlation between\nthe expression levels of RpoN and spermidine biosynthesis related genes, SpeD and\nSpeE (P < 0.01, weight > 0.9; Fig. 4G).\nFurther, the function of RpoN was validated by gene knockout in L. soli P18. We\nfound that fruit vitamin contents were significantly higher in Micro-Tom plants treated\nwith cell suspensions of the wild-type strain and the complemented strain ΔRpoN-C\ncompared to the control group (P < 0.01). Cell suspension of the mutant strain ΔRpoN\nhad no significant effect on fruit vitamin contents (Fig. 5G). Under flavonoid treatment,\nΔRpoN showed a significantly lower ability to produce spermidine than its wild-type\nstrain (P < 0.01), and this phenotype was restored in ΔRpoN-C; Fig. 5H). In summary,\nthese results indicated that flavonoids enhance spermidine biosynthesis of L. soli P18\nunder the control of RpoN.”\nFig. 4 Flavonoids mediate rhizosphere enrichment of Lysobacter soli P18 by enhancing bacterial\nmotility and colonisation. G A hub gene identified in the co-expression network with edges carrying\nweights > 0.9. The hub gene is shown in red colour, with twitching motility-related genes in purple\ncolour and spermidine biosynthetic-related genes in green colour. Positive correlations between\nRpoN and other genes are indicated by grey lines.\nspermidine production of Lysobacter soli P18. F Spermidine content in broth culture of L. soli P18\ntreated without and with flavonoids (C: 1 µmol g-1, n = 5). G Vitamin contents in fruits treated with\ncell suspensions of L. soli strains (n = 5). P18 represents the wild-type strain, ΔRpoN is a RpoN\nmutant strain, and ΔRpoN-C is a complemented strain. H Spermidine content in broth cultures of L.\nsoli strains treated without and with flavonoids (C: 1 µmol g-1, n = 10).\n4. In this article, the pathway by which plant-secreted flavonoid metabolites\nmediate microbial spermidine synthesis and subsequently up-regulate plant\nvitamin levels is presented as an innovative finding. However, the evidence is\ninsufficient, particularly regarding the lack of direct evidence for the molecular\nmechanisms involved in this pathway. In L294-296, it is noted that research on\nLysobacter spp. has primarily focused on their role in the biocontrol of soilborne\ndiseases. This study highlights the regulatory effects of these bacteria on fruit\nquality. Nonetheless, the role of spermidine in promoting vitamin accumulation\nhas already been extensively studied and is not a core innovative point. This study\ndoes not evaluate whether the described vitamin synthesis pathway is a major\ndeterminant of plant vitamin production. If it is not a primary factor influencing\nvitamin synthesis, the practical application value of this finding may be limited.\nANSWER: Molecular validation would strengthen the proposed flavonoid-microbe-\nspermidine-vitamin pathway. While our data support a causal chain, we acknowledge\nthat direct evidence of molecular signalling between flavonoids and bacterial\nspermidine synthesis is limited. To address this issue, we propose the following three\npoints.\n1. Direct evidence for molecular mechanisms\n1) Available evidence includes: ① Transcriptome data show that flavonoids\nsignificantly upregulated twitching motility genes (e.g., PilA, PilE, PilH; Fig. 4A) and\nspermidine biosynthesis genes (SpeD/SpeE; Fig. 5C-E) in L. soli P18; and ②\nMetabolic data show that the relative abundances of spermidine and its precursors\n(cysteine, methionine) increased 1.8–2.8-fold in flavonoid-treated L. soli (Fig. 5A, F).\n2) Further molecular validation, we included new data from Lysobacter gene\nknockout experiments targeting spermidine regulating genes (e.g., RpoN) in the\nrevision. This demonstrate whether flavonoid-induced vitamin accumulation is\nabolished in knockout strains. Please also see our response to your major point 3.\nmotility and colonisation. A Flavonoid-induced changes in twitching motility-related genes\nrevealed by prokaryotic transcriptomic sequencing.\nFig. 5 Flavonoids promote vitamin accumulation fruits of in Micro-Tom tomato by up-regulating\nspermidine production of Lysobacter soli P18. A Relative abundance of spermidine, cysteine and\nmethionine in fruits of flavonoid treatment (C: 1 µmol g-1) vs. control groups based on widely\ntargeted metabolomic profiling. B Spermidine biosynthetic pathway. C, D Expression levels (fold\nchange, FC) of spermidine biosynthetic enzyme genes in fruits of flavonoid-treated plants (C: rutin :\nnaringenin chalcone : kaempferol-3-O-rutinoside = 1:1:1, m/m/m, 1 µmol g-1; R: rutin, 1 µmol g-1;\nN: naringenin chalcone, 1 µmol g-1; K: kaempferol-3-O-rutinoside, 1 µmol g-1; C: 1 µmol g-1) and\nSlMYB12-overexpressing plants vs. wild-type Micro-Tom plants based on metagenomic sequencing.\nE Expression levels of spermidine biosynthesis-related genes in fruits of flavonoid treatment (C: 1\nµmol g-1) vs. control groups based on prokaryotic transcriptomic sequencing. F Spermidine content\nin broth culture of L. soli P18 treated without and with flavonoids (C: 1 µmol g-1, n = 5).\n2. Innovative role of spermidine\nWhile spermidine’s role in plant physiology is well documented, our work adds\nnovel insights in four aspects. 1) Presenting the first evidence for regulation of fruit\nquality by microbial-derived spermidine: L. soli-derived spermidine (induced by root\n35\nflavonoids) is the primary driver of vitamin accumulation in fruits. 2) Proposing an\ninnovative mechanism of plant-microbial signalling mediated by flavonoids:\nFlavonoids act as signalling molecules to recruit Lysobacter via enhancing twitching\nmotility and stimulate spermidine biosynthesis. 3) Emphasizing the central role of\nrhizosphere microbiomes in fruit vitamin accumulation: This pathway is microbiome-\ndependent, as the enhancement of fruit vitamin accumulation disappears in sterilised\nsoil. 4) Uncovering a new function of Lysobacter in regulating fruit vitamin\naccumulation: Lysobacter is indeed well-known for biocontrol, but our work expands\nits functional repertoire beyond pathogen suppression. To sum up, the innovation of this\nstudy lies in the discovery of a new plant-microbiome cross-border regulatory pathway\n(root flavonoids → microbial spermidine → fruit vitamins),” rather than a single\nmolecular function of spermidine.\n3. Importance & practical value of vitamin synthesis pathway\nOur data suggest that the proposed pathway is significant but not exclusive for\nvitamin synthesis in tomato fruits. Flavonoid supplementation led to increase in fruit\nvitamin contents (Supplementary Fig. 4), and this effect vanished in sterilised soil\n(Supplementary Fig. 5), confirming microbial dependence. We acknowledge that other\nfactors (e.g., light, nutrients) may also contribute to fruit vitamin accumulation. Future\nwork will be conducted to quantify the relative contributions of different pathways\nusing specific inhibitors or multi-omics approaches. The practical value lies in\nleveraging the proposed pathway for prebiotic strategies (e.g., flavonoid amendments)\nto enhance fruit nutrition without genetic modification. Our next step is to test the\ndominance of flavonoid in the field.\nSheng Nv-Guo tomato plants in natural soil. A Vitamin C content. B Pyridoxine content. C\nPyridoxal content. D Pyridoxamine content. Flavonoids were applied in combination (rutin :\nnaringenin chalcone : kaempferol-3-O-rutinoside = 1:1:1, m/m/m; A: 10 µmol g-1, B: 3 µmol g-1, C:\n1 µmol g-1 and D: 0.1 µmol g-1) and individually (N: naringenin chalcone, 1 µmol g-1; R: rutin,\n1 µmol g-1; and K: kaempferol-3-O-rutinoside; 1 µmol g-1). Wild-type (Con) and SlMYB12-\noverexpressing (O) plants served as controls. Error bars represent standard deviation of the means\n(n = 3, Tukey’s HSD test). Significant differences among treatments are indicated by different\nlowercase letters above the error bars (P < 0.05).\nSheng Nv-Guo tomato plants in sterilised soil. A Vitamin C content. B Pyridoxine content. C\nPyridoxal content. D Pyridoxamine content. SC: rutin : naringenin chalcone : kaempferol-3-O-\nrutinoside = 1:1:1, m/m/m, 1 µmol g-1, wild-type (SCon) and SlMYB12-overexpressing (SO) plants\nserved as controls. Error bars represent standard deviation of the means (n = 3, Tukey’s HSD test).\nSignificant differences among treatments are indicated by different lowercase letters above the error\nbars (P < 0.05).\n5. In Figure S8, the direct application of flavonoids exhibited a noticeable effect on\nmicrobiome shaping from day 7 to day 15. However, this effect gradually\ndiminished between day 15 and day 30, accompanied by a decrease in the\npopulation of Lysobacter soli P18. The duration of the effect induced by direct\nflavonoid application was relatively short, limiting its practical applicability. This\nstudy did not fully evaluate the genetically modified plants at the gene level. To\nobtain results of practical significance, further analysis is needed to assess the\nvitamin-accumulating potential of SlMYB12-overexpressing plants.\nANSWER: DONE. First, the flavonoid addition experiments were designed to test the\neffects of flavonoids on rhizosphere microbiome reshaping (especially Lysobacter and\nBacillus). We found that the effect of flavonoids diminished over time, with concurrent\ndecrease in Lysobacter populations (original Assay 3). This is because in the original\nAssay 3, we profiled rhizosphere microbiomes on days 7, 15 and 30 after one addition\nof flavonoids. In Assay 2 with three additions at 15, 22 and 29 DAT, the effect of\nflavonoids persisted. In future applications, if gene editing is used to modify tomato\nplants for continuous secretion of flavonoids, this effect reduction will not occur.\nSecond, we added a new experiment (Assay 3) by incubating natural soil in pots\nfor 90 days and adding flavonoids as described for Assay 2. We found that different\n38\nflavonoid treatments led to enrichment of Lysobacter and Bacillus, which aligns with\nthe results of 125-day pot experiment (Assay 2). Line 127 to 129, added, “Furthermore,\nwe added exogenous flavonoids to unplanted natural soil and observed microbial alpha-\ndiversity patterns comparable to those seen in planted natural soil (Supplementary Fig.\nnatural soil also shifted following exogenous addition of flavonoids (Supplementary\nFig. 8E). Irrespective of the presence or absence of plants, fungal beta-diversity did not\nrespond to any exogenous flavonoid treatments (Fig. 2F, Supplementary Fig. 7F and\nSupplementary Fig. 8F).” Line 156 to 158, added, “LEfSe analysis uncovered the\nenrichment of both Lysobacter and Bacillus in soil samples of group C after flavonoid\ntreatment (LDA > 3; Fig. 2H).”\nThird, the vitamin accumulation potential of SlMYB12-overexpressing plants, we\nfound that their fruit vitamin contents in natural soil exceeded those of wild-type plants;\nhowever, there were no significant differences in fruit vitamin content between wild-\ntype and SlMYB12-overexpressing plants in sterilised soil (Assays 1, 4). We concluded\nthat fruit vitamin accumulation in SlMYB12-overexpressing plants was upregulated due\nto the presence of soil microbiome. In this regard, the vitamin accumulation potential\nof SlMYB12-overexpressing plants is attributed to root-derived flavonoid signalling\nmolecules, but not directly controlled by the SlMYB12 gene.\nIn the subsequent analyses, we found that flavonoids stimulated the synthesis of\nspermidine in L. soli P18; then, spermidine promoted vitamin accumulation in tomato\nfruits. This unlocks new possibilities to improve tomato fruit quality by using\nflavonoids, L. soli P18 or spermidine.\nFig. 2 Flavonoids mediate variations in rhizosphere microbial diversity and community composition\nof wild-type Micro-Tom tomato in natural soil. F Fungal beta-diversity revealed by principal\ncoordinate analysis (PCoA) based on unweighted UniFrac distances. H List of rhizosphere\nbiomarkers in unplanted soil (n = 3). Flavonoids were applied in combination (rutin: naringenin\nchalcone: kaempferol-3-O-rutinoside = 1:1:1, m/m/m; A: 10 µmol g-1, B: 3 µmol g-1, C: 1 µmol g-1\nand D: 0.1 µmol g-1) and individually (R: rutin, 1 µmol g-1; N: naringenin chalcone, 1 µmol g-1; and\nK: kaempferol-3-O-rutinoside; 1 µmol g-1). Wild-type (Con) and SlMYB12-overexpressing (O)\nplants served as controls.\nA, B Bacterial richness (Chao1) and diversity (Shannon) under combined and individual flavonoid\ntreatments. C, D Fungal richness (Chao1) and diversity (Shannon) under different flavonoid\ntreatments. E, F Bacterial and fungal beta-diversity revealed by principal coordinate analysis (PCoA)\nbased on unweighted UniFrac distances. For detailed legend, see caption of Fig. 2.\n6. In this study, metagenomic, transcriptomic, and metabolomic analyses revealed\nthat the increased production of flavonoids can promote the enrichment of\nLysobacter soli P18 and the biosynthesis of spermidine. In Figure 4F, growth\ncurve analysis demonstrated that flavonoids did not significantly promote the\nreproductive growth of Lysobacter soli P18. However, metagenomic and\ntranscriptomic analyses predicted that flavonoids could up-regulate the\nexpression of genes related to twitching motility in Lysobacter soli P18. This\nprediction was validated by bacterial motility experiments shown in Figures 4C-\nE, which confirmed that flavonoids enhance the twitching motility of Lysobacter\nsoli P18. Additionally, Figure 4B provided evidence that flavonoids indeed\npromote the enrichment of Lysobacter soli. However, the molecular mechanisms\nunderlying the attraction of Lysobacter soli P18 enrichment have not been\nvalidated. Authors need to conduct further research on the specific molecular\nmechanisms that attract Lysobacter soli P18 to accumulate in the rhizosphere in\norder to provide full evidence.\nANSWER: DONE. We have conducted additional experiments to elucidate the\nmolecular mechanisms underlying flavonoid-mediated rhizosphere recruitment of L.\nsoli P18. Our new results revealed that the alternative sigma factor RpoN serves as a\nkey regulator in this process. The experimental evidence is detailed below and has been\n40\nincorporated into the revised MS.\n1) Identification of RpoN as a central regulator: Co-expression network analysis\nrevealed that the RpoN gene acts as a hub node exhibiting significant positive\ncorrelations with twitching motility genes. This strongly suggests that RpoN\norchestrates the transcriptional response of motility genes to flavonoid signals.\n2) Functional validation of RpoN via genetic mutagenesis: To determine the role of\nRpoN in flavonoid sensing and response, we constructed an isogenic RpoN knockout\nmutant (ΔRpoN) and its complemented strain (ΔRpoN-C). Key findings under\nflavonoid treatment include: twitching motility and rhizosphere colonization.\nThese results establish that RpoN is the molecular switch through which flavonoids\nactivate twitching motility and drive rhizosphere enrichment of L. soli P18. We propose\nthe following mechanistic pathway: root flavonoid secretion – bacterial RpoN\nactivation – upregulation of bacterial twitching motility genes – enhanced bacterial\nmigration – rhizosphere bacterial enrichment. This work provides the first genetic\nevidence linking flavonoid perception to RpoN-dependent transcriptional control of\nbacterial motility and colonization in L. soli P18.\nLine 224 to 235, added, “Among the up-regulated genes related to bacterial\ntwitching motility, the RpoN gene emerged as the hub node in the gene co-expression\nnetwork. There was a significant positive correlation between the expression levels of\nRpoN and other genes related to twitching motility (P < 0.01, weight > 0.9; Fig. 4G).\nTo verify the role of RpoN as a hub gene in response to flavonoid signalling and a\nregulator of bacterial twitching motility, we constructed a RpoN knockout mutant\n(ΔRpoN) and a complemented strain (ΔRpoN-C). ΔRpoN showed significantly lower\ntwitching motility and colonisation ability under flavonoid treatment compared to its\nwild-type strain (P < 0.01), and this phenotype was restored in ΔRpoN-C (Fig. 4H–I).\nIn summary, these results indicated that flavonoids stimulate the twitching motility of\nL. soli P18 in a RpoN-dependent manner, which enables bacterial enrichment in the\nrhizosphere (P < 0.05; Fig. 4J).”\nmotility and colonisation. G A hub gene identified in the co-expression network with edges carrying\nweights > 0.9. The hub gene is shown in red colour, with twitching motility-related genes in purple\ncolour and spermidine biosynthetic-related genes in green colour. Positive correlations between\nRpoN and other genes are indicated by grey lines. H, I Effects of flavonoids on twitching motility\nand colonisation of L. soli strains (n = 10). P18 represents the wild-type strain, ΔRpoN is a RpoN\nmutant strain, and ΔRpoN-C is a complemented strain. J Bacterial twitching motility-related\npathways in the two-component system (P < 0.05).\n7. In this study, although the reasons for not further investigating the specific\nregulatory sites of spermidine in plant vitamin biosynthesis were described in\nL337-342—mainly due to the potential lethality caused by gene deletions in this\npathway—the evidence supporting spermidine's role in promoting plant vitamin\nsynthesis seems insufficient. The evidence primarily relies on metabolomic and\ntranscriptomic data analysis, which may not be adequate to substantiate the\nnewly discovered vitamin synthesis pathway reported in this study. In L251-259,\nspecifically in the last paragraph of the results section, the authors presented the\ntranscriptomic data results. However, in L225, in section Bacterially produced\nspermidine promotes fruit vitamin accumulation, insufficient emphasis was\nplaced on detailing the up-regulation of upstream gene expression of enzymes\ninvolved in vitamin biosynthesis upon spermidine treatment. Authors should\nconduct further molecular-level analyses to provide more direct evidence.\nAdditionally, the results section should reduce the descriptive focus on data\noutcomes and instead prioritize discussing the underlying mechanisms and\nspecific pathways.\nANSWER: Partially DONE. When treating tomato plants, we added spermidine to\n42\nsterilised soil, rather than foliar spraying (by which spermidine could directly act on\nfruits). Following soil addition, spermidine was taken up by roots and transported to\nfruits, where it affects vitamin accumulation. Validation of this complete process\nnecessitates a series of experiments that require extensive effort.\nInstead, we analysed metabolomics and transcriptomics data to explore the content\nof other metabolites and the expression of related synthetic enzymes in the vitamin\nsynthesis pathway after spermidine treatment (Line 278 to 313). This lays the\ngroundwork for further exploring the molecular mechanisms of spermidine on fruit\nvitamin accumulation. For this part of the work, virtual screening and gene editing\ntechnologies can be used to address the remaining issue.\nWe supplemented the molecular mechanisms of flavonoid-mediated Lysobacter\nenrichment and spermidine biosynthesis. We identified a dual regulator, RpoN, which\nis responsive to flavonoids and controls both cell motility and spermidine production\nin L. soli P18. Please also see our response to your major points 3 and 6.\nMinor points\n1. In L17-18, the experimental subject analyzed is the rhizosphere microbe,\nwhereas in the experimental methods described in L445-449, sequencing is\nconducted using natural soil or sterilized soil. This indicates a conceptual\ninconsistency in the study. Rigorous sampling is essential in research focusing on\nrhizosphere microbe-plant interactions.\nANSWER: DONE. The “natural soil” subjected to sequencing refers specifically to the\nrhizosphere (or bulk) soil collected from the rhizosphere of tomato plants (or unplanted\npots) in Assays 2 and 3. The “sterilised soil” mentioned in Line 483 to 491 pertains\nexclusively to Assay 4, and the purpose was fundamentally different from that of\nmicrobial sequencing in Assays 2 and 3. Assay 4 was designed as a microbiome-\ndepletion assay to test the dependence of flavonoid-mediated improvement in tomato\nfruit vitamin accumulation on the presence of soil microbiomes. Soil sterilisation was\nused to eliminate microbial components. Crucially, microbial sequencing was not\nperformed on soil samples from Assay 4, as this assay involved measuring plant\nphenotype (vitamin accumulation) under microbiome-present (natural soil) vs.\nmicrobiome-absent (sterilised soil) conditions. Therefore, there is no conceptual\ninconsistency in our study. In the revision, we make sure the methodology explicitly\ndescribes the sequenced samples as “natural soil”.\n2. The random forest mentioned in L209 is not explained in the experimental\nmethods section. It is essential to provide details of the experimental methods and\ndata used in the study, as they serve as critical references for verifying the validity\nand reproducibility of the experiment.\nStatistical analysis section as suggested. Line 695 to 698, added, “Random forest\nanalysis was performed using the R ‘RandomForest’ package to identify the most\nimportant metabolite predictors for fruit vitamin accumulation91.”\n3. The verb \"enhance\" in the title is inconsistent with the singular subject\n\"biosynthesis\"; it should be revised to \"enhances\" to match the subject.\nANSWER: DONE. We have revised the title as suggested.\n4. In lines 209-211, aside from spermidine being up-regulated by 1.89-fold, cysteine\nand methionine also showed significant up-regulation. However, the study did not\nexplain why these markedly up-regulated metabolites were not further\ninvestigated.\nANSWER: DONE. Based on widely targeted metabolomic profiling, we looked at the\nrelationship between flavonoid-induced metabolites in L. soli P18 and vitamins in\ntomato fruits. Line 248to 251, modified, “Among alkaloids, spermidine emerged as the\ngreatest contributor to fruit vitamin accumulation based on random forest analysis.\nBased on the cross-validation results, the random forest model achieved optimal\nstability and accuracy when selecting the top 20 metabolites (Supplementary Fig. 20).”\nThe relative abundances of cystine and methionine (precursors of spermidine) also\nincreased significantly in L. soli P18 after flavonoid treatment, which was beneficial to\nspermidine synthesis. Therefore, we identified spermidine as a differential metabolite\nin L. soli P18 that is responsive to flavonoids during fruit vitamin accumulation.\nMoreover, our new data confirm the importance of spermidine in vitamin C and B\n6\naccumulation in tomato fruits. Line 270 to 274, added, “Further, the function of RpoN\nwas validated by gene knockout in L. soli P18. We found that fruit vitamin contents\nwere significantly higher in Micro-Tom plants treated with cell suspensions of the wild-\ntype strain and the complemented strain ΔRpoN-C compared to the control group (P <\n0.01). Cell suspension of the mutant strain ΔRpoN had no significant effect on fruit\nvitamin contents (Fig. 5G).”\nand vitamins in wild-type Micro-Tom tomato fruits. A colour gradient denotes Pearson’s correlation\ncoefficients. Edge width corresponds to the Mantel’s r statistic, and edge colour denotes the\nstatistical significance based on 9,999 permutations. *, P < 0.05; **, P < 0.01; and ***, P < 0.001.\nFig. S20, Fig. 5G Please see our response to your major point 1, 3.\n5. In the KEGG enrichment plot analyzed in L245-246 (Figure S23D), the DEGs\nappear to be less significantly enriched in \"ascorbate and aldarate metabolism\"\nthan described by the authors.\nANSWER: DONE. After checking the raw data, we found that the P value for the\n\"sorbate and alginate metabolism\" pathway was 0.04786. This P value is presented as-\nLog10(pvalue) = 1.32 after transformation in the KEGG pathway, consistent with our\ndescription (P < 0.05). The reason for your misunderstanding is the colour difference\nin the figure.\n6. In Figure S1, only three flavonoid compounds are up-regulated. However, these\nthree flavonoids play significant roles in promoting the synthesis of vitamins in\nplants, which raises doubts about the accuracy of the result.\nANSWER: DONE. Micro-Tom tomato mutant plants overexpress the SlMYB12 gene,\nwhich up-regulates the biosynthesis of flavonoids including rutin, naringenin chalcone\n(NC), and kaempferol-3-O-rutinoside (K3OR; Wang, S. et al, 2018). As stated in Line\n73 to 78, “Flavonoid-targeted metabolomic analysis revealed that 19 root metabolites\nand three rhizosphere soil metabolites were differentially accumulated (DAMs; fold-\nchange ≥ 10, P < 0.01) in SlMYB12-overexpressing plants compared to wild-type plants.\nAmong them, rutin, naringenin chalcone (NC) and kaempferol-3-O-rutinoside (K3OR)\nshowed the most prominent differences in their relative abundance between groups\n(Supplementary Fig. 1).\"\noverexpressing plants based on targeted metabolomics. A Volcano plot of differentially abundant\nflavonoids in tomato roots. B Volcano plot of differentially abundant flavonoids in rhizosphere soils.\n7. In Figure S20, the results predicted using random forests show that, in addition\nto spermidine, other alkaloids also exhibit specific up-regulation effects on\ndifferent types of vitamins, which were not studied in this manuscript.\nANSWER: The random forest analysis indeed showed that additional alkaloids beyond\nspermidine might induce up-regulation of specific vitamin accumulation. We focused\non spermidine because this metabolite emerged as the greatest contributor to fruit\naccumulation of four vitamins (Supplementary Fig. 20). Our new data also confirm the\nimportant role of spermidine in vitamin C and B accumulation in tomato fruits (Fig.\n6\n5H). Please also see our response to you Minor Point 4.\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training\nin peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.\nANSWER: Many thanks for your kind consideration. We have thoroughly revised the\nMS as per comments from other reviewers. Inclusion of these comments is a\ntremendous help to our work.\nReviewer #1 (Remarks to the Author):\nThe authors have responded to the reviewers' comments on the first version of the\nmanuscript. This manuscript is much improved over the original, and in my\nopinion can be accepted.\nANSWER: Thank you very much for your positive feedback of our manuscript.\nReviewer #2 (Remarks to the Author):\nDear Authors,\nThank you for your detailed and constructive revisions. Most issues have been well\naddressed. However, two aspects still require clarification or acknowledgment to\nmake the conclusions more robust and balanced.\nANSWER: We are pleased that our previous revision is well-received. We agree that\nclarifying and acknowledging the two remaining issues is crucial for enhancing the\nrobustness and balance of our conclusions. We have addressed these points by\nproviding new evidence and adding brief discussion.\n1) Strain-specific recruitment: need for absolute abundance evidence\nThe manuscript argues that flavonoids specifically recruit Lysobacter and\nBacillus in the tomato rhizosphere. However, the supporting evidence relies\nprimarily on 16S rRNA amplicon differential abundance and LEfSe biomarker\nanalysis, both of which are based on relative abundance data. Such data are\ncompositional — an apparent increase in relative abundance can occur because\nother taxa decline, sequencing depth varies, or total microbial biomass changes,\nnot necessarily because the focal taxa truly increased in cell numbers.\nWhat is missing are direct measures of absolute abundance or total bacterial\nload (e.g., 16S qPCR, spike-in standards during sequencing, flow cytometry, or\nCFU counts) showing that Lysobacter and Bacillus actually increased in cell\nnumber or gene copy number, and that total bacterial biomass did not simply rise\nacross all taxa. Without such quantitative evidence, the “strain-specific\nrecruitment” claim remains somewhat inferential. If absolute quantification\ncannot be performed at this stage, please explicitly acknowledge this limitation in\nthe Discussion and rephrase conclusions more cautiously (e.g., “flavonoids\nselectively increase the relative abundance of specific taxa” rather than “recruit\nspecific strains”).\nANSWER: Your critical and constructive comment is very much appreciated, which\nhas prompted us to strengthen the rigor of our conclusion. We performed 16S rRNA\ngene qPCR assays and obtained new quantitative evidence for strain-specific\nrecruitment.\nResults: Lines 158–162, added, “Moreover, we performed quantitative PCR (qPCR)\nassays targeting the 16S rRNA genes to quantify taxa-specific abundances. The absolute\nabundances of Lysobacter and Bacillus were significantly higher in soil samples of\ngroups C and O compared to the control samples (P < 0.05; Supplementary Fig. 11).\nThe results indicated that flavonoids preferentially attract Lysobacter and Bacillus in\nthe rhizosphere microbiome.”\nFig. S11 Absolute quantification of Bacillus and Lysobacter in the rhizosphere of\nMicro-Tom tomato in natural soil after flavonoid treatment. Flavonoids (C: 1 µmol g-1)\nwere applied as a mixture of rutin, naringenin chalcone, and kaempferol-3-O-rutinoside\n(1:1:1, m/m/m, 1 µmol g-1). Wild-type (Con) and SlMYB12-overexpressing (O) plants\nserved as controls. Data are presented as means ± standard error (n = 3), and significant\ndifferences among treatments are indicated by different lowercase letters above the\nerror bars (Student’s t-test, P < 0.05).\nDiscussion: Lines 350 to 358, added, “Microbiomic analysis and qPCR-based absolute\nquantification underscored the uniqueness of Lysobacter, which was not only enriched\nin the rhizosphere of SlMYB12-overexpressing tomato plants. The enrichment of\nLysobacter was also detected in the rhizosphere of wild-type tomato plants and\nunplanted soil treated with flavonoids. The specific recruitment of Lysobacter by\nflavonoids is supported by both differential relative abundance and absolute abundance.\nThese results confirm that the increased relative abundance of Lysobacter in flavonoid-\ntreated samples reflects a true growth in their absolute cell numbers, rather than\ncompositional artifacts from sequencing or quantitative changes in other taxa.”\nMethods: Text S8. Absolute quantification of target taxa. DNA isolation, quality\nassessment, and quantification followed the protocol for Soil 16S rRNA gene\nsequencing. Quantitative PCR (qPCR) reactions were prepared using the UltraSYBR\nMixture (Takara, Japan). The 20 μL reaction contained: 10 μL ArtiCanCEO SYBR\nqPCR Mix, 0.8 μL forward primer (10 μM), 0.8 μL reverse primer (10 μM), 1 μL\ntemplate DNA, and 7 μL double-distilled H₂O. Each sample was prepared with three\ntechnical replicates, alongside a blank control (ddH2O instead of template DNA) and a\nstandard curve gradient. The following reaction conditions were used: initial\ndenaturation at 95°C for 20 s, followed by 40 cycles of denaturation at 95°C for 3 s and\nannealing at 60°C for 30 s. After that, a melting curve analysis was carried out by\nheating from 60°C to 95°C at a rate of 0.5°C s-1 to verify the amplification specificity.\nFor more details, including the primer sequences, see Supplementary Table 6. The\nabsolute abundances (A, 16S rRNA gene copies g-1 soil) of target taxa were calculated\nas follows: A = Total abundance of bacterial 16S rRNA genes (copies g-1 soil) ×\nBacillus\nRelative abundance of Bacillus (%), and A = Total abundance of bacterial 16S\nLysobacter\nrRNA genes (copies g-1 soil) × Relative abundance of Lysobacter (%).\nTable S6 Melting curve analysis\nStage Temperature Time Cycles\nPre denaturation 95℃ 5 min 1cycles\nCycle stage 95℃ 15s 40cycles\n60℃ 20s\n72℃ 20s\nMelting stage 95℃ 15s 1cycles\n65℃ 1min 1cycles\n95℃ 0.1℃/s 1cycles\n95℃ 15s 1cycles\n2) Possible indirect effects of flavonoids\nWhile the sterile-soil and microbial inoculation experiments strongly support a\nmicrobiome-dependent mechanism, flavonoids may also influence plant\nphysiology or soil chemistry in ways that were not directly assessed. For instance,\nflavonoids could affect root architecture, redox balance, or secretion of other\nmetabolites that indirectly alter vitamin biosynthesis. The authors have already\nreported soil parameters such as organic matter and nitrogen, but such\nmeasurements cannot fully exclude indirect effects. I acknowledge that new\ntranscriptomic analyses could help clarify this, but these are likely beyond the\nscope of the current study. Therefore, please include a short acknowledgment in\nthe Discussion noting that flavonoid–microbiome interactions are likely a\ndominant but not necessarily exclusive—pathway influencing fruit vitamin\naccumulation (to recognize, potential indirect flavonoid effects, smooth causal\ninterpretation, and leave room for future research.\nANSWER: We take your thoughtful suggestion into consideration. While our data\nstrongly support a microbiome-dependent mechanism, we cannot rule out the additional,\nindirect effects of flavonoids on plant physiology or soil chemistry. To address this issue,\nwe have included a brief acknowledgment in the Discussion section as suggested. Line\n432–441, added, “Our experiments strongly support a microbiome-dependent\nmechanism as the dominant pathway driving fruit vitamin accumulation. However, we\nacknowledge that flavonoids may also exert indirect effects that were not directly\nassessed in this study. For instance, flavonoids could alter vitamin biosynthesis\npathways in tomato fruits potentially by modulating root architecture, redox balance,\nor secretion of other plant metabolites. Thus, flavonoid–microbiome interactions\nrepresent a dominant but not necessarily exclusive pathway influencing fruit vitamin\naccumulation. This recognition highlights an important direction for future research—\nassessing the relative contributions of direct microbiome effects versus indirect\nflavonoid-driven plant/soil responses to vitamin biosynthesis in tomato fruits.”\nReviewer #3 (Remarks to the Author):\nAll of the concerns have been addressed. I have no further comments.\nANSWER: We appreciate very much your acceptance of our revised manuscript.\nPoint-by-point Responses to the Reviewers’ Comments\nREVIEWERS' COMMENTS\nEditor: Previous Reviewer #2 states in Remark to Editor section that they are\nsatisfied with the revision.\nANSWER: Thank you very much for your positive feedback of our manuscript."}]

Decision letter