Modulating TPP riboswitch activity simultaneously enhances crop yield, nutritional quality and stress tolerance

Domain: chemistry
Published: 2026-02-28
Editorial outcome: minor_revision

Abstract
While enhancing crop yield remains a critical breeding objective, such efforts often compromise nutritional quality and stress tolerance. The growing global population and increasing environmental stresses make combining stable crop yields with improved nutritional quality a vital objective for sustainable food security. Thiamine pyrophosphate (TPP), the active form of vitamin B1, regulates core metabolism to support both human health and plant stress tolerances. Here, we show that editing the rice TPP riboswitch elevates multiple micronutrients, simultaneously increasing grain yield, cold tolerance, and blast resistance. Mechanistically, these enhancements link to promoted photosynthesis, improved nitrogen use efficiency, and system-wide transcriptional-metabolic reprogramming. Editing homologs in tomato produces similar outcomes, supporting the generalizability of this approach. Thus, modulating TPP levels offers a viable strategy to synergistically boost crop productivity, nutritional quality, and stress tolerance, which are essential for achieving sustainable food security without trade-offs.

Introduction
Food security, defined as the sustainable access to sufficient and nutritious food, is a fundamental pillar of global health and socioeconomic stability1,2. The urgency to ensure food security is escalating, driven by a surging global population, diminishing arable land, and increasing climate extremes, which collectively pose substantial threats to agricultural productivity3-5. Current strategies to address food security prioritize dual objectives: enhancing crop yields under both optimal and adverse conditions and improving nutritional quality6-8. However, simultaneously achieving these objectives remains a major challenge, as most research efforts have focused on improving single traits, often overlooking the inherent trade-offs between yield, nutritional content, and stress resistance.

Over the past three decades, metabolic engineering and gene editing techniques have been utilized to create a variety of biofortified crops to address nutritional deficiencies in the global population. Notable examples include “Golden Rice” enriched with β-carotene9, riboflavin-rich rice10, pyridoxine-enriched rice11, folate-fortified rice12, 7-dehydrocholesterol-rich tomato13, lysine-enhanced maize14, anthocyanin-rich rice15, and astaxanthin-containing rice16. Although these efforts hold potential for addressing specific micronutrient deficiencies in particular regions or countries, their practical implementation faces substantial nonscientific constraints, including regulatory barriers, public scepticism, and market access limitations. For instance, Golden Rice, developed over two decades ago, has been repeatedly delayed and even withdrawn from certain markets due to public attitudes and political factors, rather than widely alleviating vitamin A deficiency as initially envisioned17. Moreover, the capacity and transformation efficiency of gene expression vectors are generally insufficient18, making it difficult to achieve synergistic enrichment of multiple nutrients in crops, thus failing to solve the main problem of “hidden hunger” affecting nearly 2 billion people–worldwide, reflecting deficiencies in multiple essential nutrients within staple crops. Therefore, the broader and more persistent challenge remains to explore important genetic breeding strategies that have greater public acceptance and can be used to address the crisis of micronutrient deficiencies in populations.

Beyond these implementation barriers, intrinsic biological constraints, specifically genetic and physiological trade-offs, further complicate the simultaneous improvement of multiple desirable traits. Pleiotropic effects often lead to unintended consequences19-21. For example, the Green Revolution gene sd1 (semi-dwarf 1) increased the harvest index in rice but reduced nitrogen use efficiency and drought tolerance22. Similarly, mutagenesis of the GL4 gene in rice increased shattering resistance but resulted in a relatively small grain size23. In wheat, the dwarfing allele Rht-B1b improved lodging resistance at the cost of root biomass and water uptake capacity24. Plant immune responses also illustrate this dilemma: pathogen recognition receptors such as Xa21 in rice confer blast resistance but can reduce grain size under pathogen-free conditions, and salicylic acid-mediated defence signalling often represses growth through crosstalk with gibberellin pathways25,26. Although conventional breeding and transgenic strategies can partially mitigate linkage drag, gene pleiotropy-driven trade-offs remain a fundamental obstacle to crop improvement. Accordingly, identifying genetic loci that break the trade-offs between yield, stress resistance, and nutritional quality is of vital importance for addressing food security.

Thiamine (vitamin B1) plays a pivotal role in central energy metabolism in both animals and plants. In humans, its deficiency is associated with severe neurological and cardiovascular disorders27-29. In plants, thiamine sustains the continuous supply of ATP and acetyl-CoA essential for almost all life activities by maintaining the activity of several core enzymes in the pentose phosphate pathway, the Calvin‒Benson cycle, and the tricarboxylic acid (TCA) cycle through its active form, thiamine pyrophosphate (TPP). In addition to its metabolic functions, vitamin B1 also acts as a priming agent for defence responses, inducing systemic acquired resistance (SAR) and pathogenesis-related (PR) gene expression through the salicylic acid and Ca2+ signalling pathways in diverse species, including rice, tobacco, tomato, and Arabidopsis30-34. Furthermore, vitamin B1 contributes to photosynthetic efficiency, chloroplast homeostasis, and mitigation of abiotic stress by reducing reactive oxygen species accumulation 28,35,36. Vitamin B1 biosynthesis in plants is regulated via TPP riboswitches, conserved structured RNA elements located in the 3'-UTR of THIC that sense cellular TPP levels to feedback-inhibit biosynthesis37,38. Mutations in these riboswitch elements have been shown to increase thiamine and TPP levels by 5‒10 times39, thereby influencing carbon/nitrogen metabolism and stress responsiveness40. All the positive effects of vitamin B1 and the regulatory role of riboswitches on vitamin B1 suggest that mediating TPP riboswitches may regulate crop growth, resistance, and metabolic flux by altering the homeostasis of vitamin B1. However, the application potential of this regulatory mechanism for the coordinated improvement of multiple traits in crops remains unexplored in prior research.

In this study, we address this knowledge gap by employing CRISPR-Cas9-mediated editing of the TPP riboswitch in rice to reprogram vitamin B1 homeostasis. We demonstrate that this intervention leads to synergistic enhancements in grain nutritional quality, yield, cold tolerance, and blast resistance. Mechanistic studies reveal that these improvements are underpinned by augmented photosynthetic capacity, optimized nitrogen utilization, and systemic transcriptional–metabolic reprogramming. The recapitulation of key phenotypes in tomato further underscores the broad applicability of this strategy. Importantly, our approach overcomes the pervasive trade-offs inherent in conventional breeding and enables the coordinated improvement of productivity, nutritional quality, and environmental resilience via a single genetic modification. This transgene-free TPP riboswitch-mediated gene editing strategy provides a promising approach for crop improvement that contribute to sustainable food security and is easy to be accepted by the public.

Results
Targeted editing of TPP riboswitch elevates thiamine and broad-spectrum micronutrients in rice
To enhance vitamin B1 level in rice, we employed a CRISPR-Cas9 system to edit the TPP riboswitch in the 3'-UTR of OsTHIC, the key enzyme for vitamin B1 synthesis. Seven vectors, each harbouring one or two designed single guide RNAs targeting the TPP riboswitch and its flanking sequences on OsTHIC, were transformed into Zhonghua 11 (ZH11). Totally, 47 out of 173 T0 lines carried mutations. After two generations of self-pollination, we isolated nine homozygous T lines, named trs1 to trs9. These mutants encompassed various mutation types, including deletions ranging from 1 to 231 bp and a 241-bp insertion in the TPP riboswitch (Fig. 1a, Supplementary Fig. 1 and Supplementary Data 1). All the nine lines showed significant increases in the vitamin B1 content in different tissues, with trs3 and trs8 displaying the highest thiamine contents (Fig. 1b-d). Quantitatively analysis of polished rice revealed that the thiamine content increased from 0.92 mg kg-1 in ZH11 to 5.26 mg kg-1 and 5.51 mg kg-1 in trs3 and trs8, respectively (Fig. 1e).

Given that vitamin B1 supplies upstream precursors for multiple nutrients by governing glycolysis and the tricarboxylic acid (TCA) cycle, we performed targeted metabolomic profiling of 115 nutrients and health-related components in polished rice, including 17 vitamins, 53 lipids, 21 amino acids, nine sugars and 15 polyphenols. As expected, most nutrients in the trs3 and trs8 were significantly higher than those in ZH11 (Fig. 1f, Supplementary Data 2). These included riboflavin, niacin, pantothenic acid, pyridoxine, α-tocopherol, monoacylglycerophospholipids, and several amino acids that human dietary-essential (Fig. 1g-q). We also analysed the macronutrient contents and detected no significant differences in total starch or grain protein levels between the trs lines and ZH11 (Supplementary Fig. 2), indicating that the nutritional enhancement was specific to micronutrients and did not affect the core energy or protein value of the rice. These results suggest that gene editing of the TPP riboswitches can simultaneously increases the levels of both vitamin B1 and a broad spectrum of other nutrients without altering the fundamental macronutrient composition in rice.

TPP riboswitch editing boosts grain yield via improved panicle architecture in rice
To evaluate the agronomic traits of riboswitch-edited plants, we conducted field trials in Wuhan using trs3, trs8, and ZH11 plants (Fig. 2a-c). Compared with ZH11, the trs3 and trs8 exhibited significant increases in plot yield, ranging from 19.29–20.77% (Fig. 2d), and the height of the edited lines during the reproductive period also significantly increased (Fig. 2e, Supplementary Fig. 3). In contrast, the mutants and ZH11 showed comparable performance in thousand-grain weight, grain shape (including grain length, width, or thickness), tiller number, and panicle number per plant (Fig. 2f, g, Supplementary Fig. 4a-d). To further exclude the possibility that the increased yield was caused by planting density, we conducted phenotypic analyses of individual plant traits in three separated and randomized plots. The results indicated that compared with ZH11, trs3 and trs8 led to 17.80–25.27% increases in grain yield per plant (Fig. 2h). A detailed phenotypic analysis revealed that the increases in yield in the trs3 and trs8 lines correlated with 16.33–26.42% increases in panicle length compared with that in ZH11 (Fig. 2c, i), which correspondingly increased the number of grains and yield per panicle (Fig. 2j, k).

To assess the stability of the yield enhancement conferred by the TPP riboswitch edits, we conducted field trials in Sanya, which represents a very different environmental setting. Despite the contrasting conditions, compared with ZH11, trs3 and trs8 lines still showed significant yield improvements under short-day and tropical conditions in Hainan, with yield increases ranging from 20.79–24.29% per plot and 17.31–31.19% per plant, respectively (Fig. 2l, m). Additionally, the panicle length, grain number and yield per panicle of trs3 and trs8 lines were significantly higher compared to ZH11 (Fig. 2n-p, Supplementary Fig. 5). Taken together, these results indicate that editing the TPP riboswitch enhances grain yield, which is associated with improved panicle architecture.

Enhanced photosynthetic capacity and nitrogen use efficiency underlie yield improvement
To disclose the physiological mechanism underling the elevated yield in gene edited lines, we characterized key aspects of photosynthesis. Compared with ZH11, the trs3 and trs8 lines exhibited a 46.01–47.27% higher photosynthesis rates under standard growth conditions (Fig. 3a). Additionally, these plants presented elevated Fv/Fm values (which represent the maximum quantum efficiency of photosystem II when all the capable reaction centres are open) and increased photosystem II yields (Fig. 3b, c). Furthermore, the gene-edited riboswitch lines displayed elevated rates of chloroplast electron transport (Fig. 3d, Supplementary Fig. 6).

We further evaluated plant performance under limited nitrogen (LN) conditions under hydroponics, providing only 20% of the nitrogen provided under control conditions. Under LN stress, the trs3 and trs8 lines presented significantly greater aboveground biomass under LN conditions and a slightly greater root biomass than the wild type (Fig. 3e-g). Concurrently, chlorophyll content in the edited lines was 16.73–23.62% higher than in ZH11 (Fig. 3h, i). When the LN treatment was maintained throughout the reproductive stage (from flowering to maturity), striking phenotypic differences emerged between the mutants and wild type (Fig. 3j). Compared with WT, the trs lines produced more tillers, panicles per plant, plant height, and root length under LN conditions (Fig. 3k-n).

Riboswitch editing confers dual resistance to blast and cold stress
To evaluate the stress tolerance of the riboswitch-edited lines, we first performed field trials under natural blast (Magnaporthe oryzae) pressure in Taojiang, Hunan Province, a blast-endemic region. Under standard cultivation, the trs3 and trs8 lines demonstrated enhanced resistance to natural pathogen infection compared with the wild-type ZH11 (Fig. 4a-d). The stability of this resistance was confirmed in a second-year trial in Enshi, Hubei Province, where the mutants exhibited significantly lower disease incidence and severity than ZH11 (Supplementary Fig. 7).

We also conducted controlled infection assays using two distinct inoculation methods: spray inoculation (simulating natural spore dispersal) and punch inoculation (localized tissue penetration) with M. oryzae (ZB25 strain). Spray inoculation revealed that the edited lines developed significantly shorter lesion lengths than ZH11, which displayed typical blast lesions (Fig. 4e, f). The same resistant phenotype was also consistently observed in the punch inoculation (Fig. 4h, i). Quantitative PCR analysis of fungal biomass confirmed these morphological observations, showing a significant reduction in M. oryzae DNA in riboswitch-edited lines relative to ZH11 under both inoculation methods (Fig. 4g, j). Furthermore, we conducted additional inoculation trials using distinct pathogen strains isolated from the northern (Liaoning) and southern (Enshi) regions of China. In these assays, the riboswitch-edited lines consistently exhibited a clear disease-resistant phenotype (Supplementary Fig. 8).

Low temperature (LT) threatens rice growth and productivity, largely through induced oxidative stress that harms plants41-43. Vitamin B1 biosynthetic genes are known to play important roles in plant resistance to various oxidative stresses. Transcriptomic data from maize have shown that the expression levels of most vitamin B1 synthesis pathway genes are induced by LT44. Similarly, we observed marked upregulation of multiple vitamin B1 biosynthetic genes, including OsTHIC, in rice under cold stress (Supplementary Fig. 9). This prompted us to test the cold tolerance of the trs mutants. When subjected to LT treatment, the trs3 and trs8 lines displayed visibly less leaf rolling than ZH11 (Supplementary Fig. 10). Following a 2‑day cold treatment and recovery, more than 84% of the riboswitch-edited lines survived, compared with only approximately 29% of ZH11 plants (Fig. 4k right, 4l). The trs mutants showed significantly slower electrolyte leakage than ZH11 under cold stress (Fig. 4m). Consistent with enhanced oxidative stress management, 3,3’-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining revealed substantially lower accumulation of, H2O2 and O2- in the mutant leaves (Fig. 4n).

Together, these data demonstrate that disrupting TPP riboswitch confers broad-spectrum and robust resistance to Magnaporthe oryzae infection across multiple geographical strains and inoculation methods, while simultaneously enhancing cold tolerance through improved management of oxidative stress.

Rewiring of transcriptional-metabolic networks drives multi-trait enhancement
To confirm that the observed multitrait improvements were indeed due to the function of the OsTHIC gene rather than off-target effects, we generated two independent OsTHIC overexpression lines (OE1 and OE2; Supplementary Fig. 11a) and further tested them under Magnaporthe oryzae infection and cold stress. Both OE lines exhibited enhanced resistance to M. oryzae (Supplementary Fig. 11b-c) and cold tolerance (Supplementary Fig. 11d-f). These results, combined with those from the gene-edited lines, demonstrate that OsTHIC confers rice tolerance to multiple stresses, thereby validating the on-target nature of the riboswitch editing.

To test whether enhanced phenotypes in the riboswitch-edited lines were resulted from increased vitamin B1 content, we treated rice plants at the grain-filling stage with 1 mM TPP for 14 days. Metabolite profiling of endosperm revealed that, similar to the trs-edited lines, the contents of thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, α-tocopherol, lysoPE 18:3, lysoPC 18:3, leucine, threonine, serine, and valine were significantly upregulated in the two TPP-treated plants compared with the control (Fig. 5a). Similarly, we exogenously applied 1 mM TPP at the five-leaf stage, followed by challenge with M. oryzae or cold stress. Consistent with the phenotypes of the riboswitch-edited lines, the TPP-treated plants exhibited significantly enhanced resistance to rice blast (Fig. 5b-d) and improved cold tolerance compared with the control (Fig. 5e, f).

To elucidate the mechanism by which riboswitch editing enhances vitamin B1 accumulation, we performed, we employed transient expression assays in rice protoplasts. The 3’-UTR regions from the wild-type (3’-UTRWT) and edited lines (M1-M9, corresponding to 3’-UTRtrs1 to 3’-UTRtrs9) were cloned into a dual-luciferase reporter system (Fig. 5g left). Upon treatment with 1 mM TPP, all nine mutant 3’-UTRs produced significantly higher luciferase activity than 3’-UTRWT. The 3’-UTRtrs3 and 3’-UTRtrs8 exhibited the greatest increases in reporter activity (Fig. 5g right), consistent with their highest thiamine accumulations in vivo (Fig. 1b-d). These results confirm that editing the THIC TPP-riboswitch sequence alleviate TPP-mediated feedback inhibition, thereby upregulating vitamin B1 biosynthesis.

To investigate the molecular mechanism underlying the comprehensive enhanced rice in riboswitch gene-edited lines, we performed a transcriptomic analysis of the third leaves of the wild-type and the trs lines, with or without 1mM TPP treatment. TPP treatment in WT tiggered the upregulation of 5,842 genes and the downregulation of 1,350 genes (Fig. 5h, Supplementary Data 3). Compared with ZH11, the trs3 and trs8 lines shared 2,346 upregulated and 3,536 downregulated genes (Fig. 5i, Supplementary Data 4). OsTHIC expression was significantly elevated in both lines (Supplementary Data 4), consistent with the release of feedback inhibition. A comparative analysis of the transcriptional responses to TPP treatment (in WT) and to riboswitch editing (in mutants) identified 724 co-upregulated genes (Fig. 5j). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of these 724 genes revealed significant shifts in vitamin metabolism, lipid metabolism, amino acid and nitrogen metabolism, photosynthesis and carbon fixation, and plant-pathogen interaction, all processes linked to nutritional quality, yield, and stress tolerance (Fig. 5k). The expression patterns of key genes in these pathways were validated by qPCR (Fig. 5l). Collectively, these results demonstrate that vitamin B1 modulates transcriptional reprogramming across multiple metabolic and stress-related pathways, underpinning the integrated improvements in agronomic and nutritional traits in rice.

In plants, TPP serves as an essential coenzyme for key enzymes, including pyruvate dehydrogenase complex (PDC), pyruvate dehydrogenase (PDH), and 2-oxoglutarate dehydrogenase (2-OGDH), in the tricarboxylic acid (TCA) cycle (Fig. 5m).

We hypothesized that, beyond its transcriptional effects, the altered vitamin B1 levels in trs lines could enhance the activity of these TPP-dependent enzymes by increasing TPP availability. Thus, we quantified TPP levels in riboswitch-edited lines and observed a marked increase compared with those in wild-type controls (Fig. 5n). Protein extracts from the mutants indeed showed enhanced activities of PDH (the E1 subunit of PDC) and 2-OGDH activities (Fig. 5o, p), while PDC activity was unchanged (Supplementary Fig. 12). Metabolite profiling of central pathways associated with these enzymes revealed 20 significantly increased metabolites in the trs lines, including essential amino acids, TCA intermediates such as pyruvate and succinate, and chorismite, a precursor of multiple metabolites (Fig. 5q). We observed a pronounced increase in cAMP, a second messenger involved in growth and stress signaling42,45,46, whose synthesis is indirectly influenced by vitamin B1. These results further support the role of TPP-mediated metabolic rewiring in enhancing both developmental and defence processes.

Together, our results demonstrate that targeted riboswitch editing perturbs cellular TPP homeostasis, activates central carbon metabolism, and amplifies connected upstream and downstream pathways. This rewiring of transcriptional-metabolic networks through enhanced TPP availability stimulates TPP-dependent enzymatic hubs and directs carbon flux towards growth- and defence-related metabolism, providing a mechanistic basis for the observed improvements in quality, yield, and stress tolerance.

Conserved TPP riboswitch function enables multigenic improvement in tomato
Given the promising results in rice, we asked whether elevating vitamin B1 levels through riboswitch editing could also be effective in dicots. We therefore generated two lines with edited TPP riboswitch in the SlTHIC gene, named sltrs1 (single-base insertion) and sltrs5 (five-base deletion) (Supplementary Fig. 13). Both mutants displayed elevated vitamin B1 contents (Fig. 6b) and increased levels of numerous other nutrient metabolites including riboflavin, niacin, pantothenic acid, pyridoxine, α-tocopherol, monoacylglycerol phospholipids, and several essential amino acids (Fig. 6a-m, Supplementary Data 5). The edited lines showed improved vegetative growth (Fig. 6n) and a striking enhancement in photosynthetic performance, with photosynthetic rates raised by 56.46–77.05% (Fig. 6o). This was accompanied by increased transpiration (Fig. 6p) and stomatal conductance (Fig. 6r), whereas internal carbon dioxide concentration remained unchanged (Fig. 6q). In addition, the mutants also exhibited clear resistance to Botrytis cinerea under both natural (Fig. 6s) and artificial inoculation conditions (Fig. 6t-v). Similar to the research conducted in rice, we also evaluated the performance of tomato sltrs plants under LT stress. After exposure to LT stress, compared with WT plants, sltrs1 and sltrs5 plants exhibited a more robust phenotype (Fig. 6w), lower ROS accumulation (Fig. 6x, y), and lower electrolyte leakage (Fig. 6z), indicating that the gene-edited of TPP riboswitch conferred enhanced cold tolerance to the tomato plants.

Taken together, these results demonstrate that TPP riboswitch-mediated editing of SlTHIC in tomato effectively enhances nutritional composition, photosynthetic capacity, and multi-stress resilience, confirming the broad applicability and translational potential of this strategy beyond monocot species.

Methods
Plant material and field trials investigation
The trs mutant constructs were generated via CRISPR-Cas9 technology51. For the OsTHIC overexpression construct, the full-length coding sequence was amplified from the japonica cultivar Nipponbare and cloned into the binary vector pJC034 using Gateway recombination reactions (Invitrogen, Waltham, MA, USA). The generated vectors were introduced into the calli of Zhonghua11 (ZH11), via Agrobacterium tumefaciens-mediated transformation. Field yield was assessed at the Wuhan and Sanya experimental stations (low incidence of rice blast) and resistance to blast was evaluated in the Taojiang and Enshi blast nurseries (high incidence of rice blast) during the summer season. Enshi (Hubei Province) and Taojiang (Hunan Province) were selected because this mountainous area has a high incidence of rice blast disease each year. Field plots were designed following the strategy established by the International Rice Research Institute. For each field trial, plants were grown with either 5 replicate plots for each line, and each plot contained 100 plants that were 20 cm apart. Diseased plantlets of the blast-susceptible line Lijiangxintuanheigu were used as a source of inoculum for spreading the disease. One row of Lijiangxintuanheigu was sown for every 10 rows of test accessions, and the disease was allowed to spread naturally by wind dispersal. In the normal fields, we did not include a spreader row. The rice blast resistance levels of different lines were evaluated according to the International Rice Research Institute evaluation system, and the percentage of necrotic panicles caused by blast was calculated. For the cold stress, trs lines and Zhonghua11 (ZH11) seedlings were grown in black pots in an optical incubator at 28°C with a 16-h light/8-h dark photoperiod and 70% relative humidity. Fifteen-day-old seedlings were subjected to 4 °C treatment for 48 hours and then transferred to 28 °C or recovery conditions.

Preparation of metabolic samples
The samples were first freeze-dried and then ground into powder using a mixer mill (MM400, Retsch) for 1.5 min at 30 Hz. Subsequently, 100 mg of the resulting powder was weighed and extracted overnight at 4 °C with pure methanol. The extracts were then centrifuged for 10 min at 10,000 × g. The supernatants were collected, followed by filtration (SCANA-104, 0.22 μm pore size; ANPEL Shanghai, China, www.anpel.com.cn/) before LC–MS analysis. To establish a specific metabolic database of brown rice, samples were mixed into multiple samples for widely targeted metabolomics analysis52,53.

Metabolites quantification
For metabolites content analysis, samples were analysed using a high-performance liquid chromatography (HPLC) staple targeted method54,55. The analytical conditions were as follows, HPLC: column, shim-pack GISS C18 (pore size 1.9 µm, length 2.1 × 100 mm); solvent system, water (0.04% acetic acid): acetonitrile (0.04% acetic acid); gradient program, 0 min, 5% B; 12.0 min, 95% B; 13.2 min, 95% B; 13.3 min, 5% B; 15.0 min, 5% B; flow rate, 0.4 mL min-1; temperature, 40 °C; and injection volume, 2 µL. Targeted metabolic profiling analysis was performed using a scheduled multiple reaction monitoring (MRM) via an LC-ESI-QQQ-MS/MS system (LCMS-8060, SHIMADZU, Japan). The ESI source operation parameters were as follows: nebulizing gas flow, 3 L min–1; heating gas flow, 10 L min–1; interface temperature, 500 °C; DL temperature, 250 °C; heat block temperature, 400 °C; and drying gas flow, 10 L min–1. The data recorded were processed with LabSolutions 5.91 software.

Determination of protein content and starch content in polished rice
To determine the protein and starch contents in polished rice, we employed standard biochemical methods. For protein content, we used the Kjeldahl method, which involves digestion of the rice sample with concentrated sulfuric acid to convert the nitrogen in the proteins to ammonium sulfate. The resulting solution was then distilled to release ammonia, which was subsequently titrated with a standard acid solution to quantify the nitrogen content. The protein content was then calculated based on the nitrogen content, assuming an average nitrogen-to-protein conversion factor of 6.25. To determine the starch content, we utilized the anthrone reagent method. This method involves hydrolysing the starch in the rice sample to glucose using an acid hydrolysis procedure. The resulting glucose was then reacted with anthrone reagent, which formed a blue-green complex. The intensity of this complex was measured spectrophotometrically at 620 nm, and the starch content was determined by comparing the absorbance to a standard glucose curve. Both methods were performed in triplicate to ensure the accuracy and reproducibility of the results. The protein and starch contents are expressed as percentages of the total weight of the polished rice sample.

Low-nitrogen treatment
Low-nitrogen treatment was applied after sowing, germination, and normal growth for 10 days. For the low-nitrogen group, 20% of the normal nitrogen level was supplemented, while the other conditions remained consistent with those of the control group. After one month of treatment, the plants were harvested to analyse their biomass theirand chlorophyll content. The plants subjected to low-nitrogen conditions throughout their entire growth period were cultivated under low-nitrogen conditions until heading and seed setting, after which parameters such as plant height, root length, and tiller number were statistically analysed. The nutrient solution for all the plants was replaced every three days.

Rice plant infection assays
M. oryzae strains were isolated from rice fields in Enshi (ES67) and Liaoning (LN3, LN13) in China, and ZB25 was stored in the laboratory. Four- to five-week-old rice plants at the tillering stage were used for all inoculations. For spray inoculation, the plants were first acclimated in a humidity chamber (>90% relative humidity) for 24 h to increase their susceptibility. M. oryzae was cultured on oatmeal agar plates at 25 °C under continuous light for 10-14 days to induce sporulation. Spores were harvested by gently scraping the culture surface with sterile water containing 0.01% (v/v) Tween-20. The resulting suspension was filtered through two layers of sterile cheesecloth to remove mycelial debris, and the spore concentration was adjusted to 1×105 spores/mL using a haemocytometer. The spore suspension was evenly sprayed onto both adaxial and abaxial leaf surfaces using an atomizer until run-off (approximately 10 mL per plant). A negative control group, sprayed with 0.01% Tween-20 solution, was included. For punch inoculation, spores were collected from 10-day-old cultures as described above, and the concentration was adjusted to 1×105 spores/mL with 0.025% Tween-20. Fully expanded leaves of 30-day-old plants were wounded with a sterile punch and inoculated with a 10 μL droplet of the spore suspension. The inoculated site was subsequently sealed with clear tape to maintain humidity. All inoculated plants were transferred to a greenhouse for disease development. Symptoms were assessed at 10 days post-inoculation (dpi), and fungal biomass was quantified by quantitative PCR (qPCR)56,57.

Measurement of leaf gas exchange parameters
Photosynthesis parameters, such as the net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr) of leaves were measured at 10:00–11:00 am using the portable photosynthesis system LI-6800 (LI-COR Biosciences, Lincoln, USA). The level of irradiance used to activate photosynthesis was 600 μmol m−2 s−1. All measurements were performed on the youngest fully expanded flag leaf of each plant. The atmospheric CO2 concentration and temperature were approximately 400 μmol·mol-1 and 25 °C, respectively.

Measurement of chlorophyll fluorescence
The in vivo chlorophyll fluorescence of leaves was measured using a PAM-2500 (Heinz Walz, Effeltrich, Germany) at room temperature (approximately 25 °C). Before measurement, the plants were dark adapted for 30 min. The light-adapted fluorescence parameters were recorded for the PPFDs (387 μmol photons m−2 s−1, 635 nm). The intensity and duration of the light saturation pulse used to measure Fm were 10,000 μmol photons m−2s−1 and 300 ms, respectively. The chlorophyll fluorescence parameters were calculated as follows:
Fv/Fm=(Fm-Fo)/Fm (1)
Y(II)=(Fm'-Fs)/Fm' (2)
Y(NO)=Fs/Fm (3)
NPQ=(Fm-Fm')/Fm' (4)
qP=(Fm’-Fs)/(Fm’-Fo’) (5)
ETR=Y(II)×PAR×0.84×0.5 (6)
Fo and Fm are the minimum and maximum fluorescence after dark adaptation, respectively. Fo' and Fm' represent the minimum and maximum fluorescence after light adaptation, respectively. Fs is the light-adapted steady-state fluorescence58.

Chlorophyll content analysis
Fresh leaf blades were collected, frozen in liquid nitrogen and ground into fine powder. Pigments were then extracted with 80% acetone and shaken overnight in the dark. After centrifugation at 12 000  g for 10 min at 4 °C, the absorbance of the supernatant was measured at 663 and 645 nm and reported as OD663 and OD645, respectively.

Electrolyte leakage assay
Three uniformly sized leaf fragments were collected from each sample, cut into smaller pieces, rinsed, and shaken in deionized water overnight. After incubation, the electrical conductivity of the solution (R1) was measured using a DDS-11C conductivity meter (Shanghai Leici Instrument Inc., Shanghai, China). The samples were then heated in boiling water in a sealed vessel for 20 min, and the conductivity (R2) was measured again. Electrolyte leakage was calculated as follows:
Electrolyte leakage = (R1)/ (R1 + R2) (7)

NBT and DAB staining
O2- and H2O2 were detected using nitroblue tetrazolium chloride (NBT; Sangon Biotech, Shanghai, China) and 3,3'-diaminobenzidine (DAB; Sangon Biotech) staining, respectively. To detect O2-, 3-5 cm leaf blades were harvested and incubated in 1% NBT (w/v, pH=7.8) and vacuum infiltrated for 30 min. After being incubated in the dark at room temperature, the samples were transferred to 95% ethanol at 80 °C to remove the chlorophyll. To detect H2O2, plant leaves were stained with 0.05% DAB (w/v, pH 3.8), vacuum infiltrated for 30 min and then incubated for 12 h. Subsequently, all the stained leaves were transferred to 95% ethanol and incubated at 80 °C until the decolorizing solution was colourless.

Tomato material generation and low-temperature treatment
Tomato TPP riboswitch gene editing (Sltrs) plants were obtained by CRISPR/Cas9 technology 59. The WT tomato plants used in this study were of the ‘Ailsa Craig’ ecotype, whereas the transgenic tomato plants were obtained by A. tumefaciens-mediated transformation. The Sltrs mutants were identified by PCR and confirmed by DNA sequencing. Tomato seeds were germinated and grown in pots under cool-white fluorescent light (600 mmol m-2 s-1, 12 h photoperiod) at 28 °C: 18 °C (day: night) and 60% relative humidity in a growth chamber (KuLan, China). During the low-temperature treatment, six-leaf-one-heart plants were placed under 4 °C conditions, with normal growth conditions serving as the control. After 48 h, record the phenotypes were recorded, and the resistance index was measured.

Tomato leaf pathogen infection
Botrytis cinerea infection followed the method of Zaid et al.60 with modifications. Specifically, Botrytis cinerea (Bc, isolate BcI16) cultures were maintained on potato dextrose agar (PDA) (Difco Lab) plates and incubated at 22°C for 5 to 7 days. Botrytis cinerea spores were harvested from PDA plates supplemented with 1 mg mL−1 glucose and 1 mg mL−1 K2HPO4 and filtered through gauze. The spore concentration was adjusted to 10⁶ spores mL⁻¹ using a haemocytometer. Inoculate 10 μL of spore suspension into the fourth to fifth leaves of tomato plants aged 5-7 weeks. The inoculated plants were kept in a humid growth chamber at 22 °C. The control consisted of plants or leaves treated with water/buffer. The area of necrotic lesions was measured 5-7 days after inoculation.

Data availability
The metabolomics and transcriptomics data generated in this study have been deposited in the OMIX, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences, under accession number OMIX009553 [https://ngdc.cncb.ac.cn/omix/view/OMIX009553] and OMIX009554 [https://ngdc.cncb.ac.cn/omix/view/OMIX009554], respectively. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the manuscript, Li et al. showed that modification of the thiamine pyrophosphate (TPP) riboswitch by gene editing in rice not only elevated the contents of vitamin B, various vitamins and phytonutrients, but also enhanced rice growth and grain yield and tolerance to biotic and abiotic stresses, such as cold stress and rice blast disease. Based on physiological, transcriptomic and metabolic analyses, authors attributed the trait improvements of the modified OsTHIC rice to enhanced photosynthesis, optimized nitrogen use, and system-wide transcriptional metabolic reprogramming. Additionally, authors edited the thiamine pyrophosphate (TPP) riboswitch in tomato. Similarly, the editing plants displayed enhanced growth and tolerance to biotic and abiotic stresses. The authors concluded that manipulation of thiamine pyrophosphate levels represents a highly effective strategy for improving crop yield and nutritional quality under both normal and stress conditions. Generally, the findings described in the manuscript are very interesting and useful for crop improvement and biofortification. However, the manuscript suffers from some deficiencies, which should be clarified.\nMajor comments:\n1)The main issue of this manuscript is that CRISPR-mediated editing of the OsTHIC riboswitch in rice elevated thiamine pyrophosphate (vitamin B1) levels, driving simultaneous enhancements in grain complex nutrition, yield, cold tolerance, and blast resistance. My question is that the all enhancements in grain complex nutrition, yield, cold tolerance, and blast resistance directly own to the elevated vitamin B1 or are caused by some unknown functions of OsTHIC. These may be separated by spraying vitamin B1 to wild type, and comparing with the OsTHIC-modified rice lines.\n2)Authors generated SlTHIC-modified tomato lines and showed enhancements of their growth by large increases in the rate of photosynthesis and tolerance to Botrytis cinerea. How about fruit metabolites? I suggest authors to provide, as rice, the metabolite data of their fruits to answer whether the metabolite alteration in tomato is same as in rice grain.\nMinor issues:\n1)Lines 102-104, the sentence “This knowledge gap opens the question as to their capacity to orchestrate synergistic improvements in yield, nutrition, and stress resilience a question that so far remains unexplored and mechanistically opaque” is not clear.\n2)Line 129, what does “After two rounds of breeding” mean? It should be defined.\n3)Lines 165-171, authors compared yield complements between the modified lines and WT without thousand grain weight. I suggest authors to add the comparison data of thousand grain weight.\n4)Lines 208-211, The sentence “Field observations demonstrated that the riboswitch-edited lines maintained near-normal growth parameters while exhibiting enhanced innate defense responses to natural pathogen infections compared to wild-type controls (ZH11)” is not clear. Which growth parameters have been measured?\n5)Lines 225-227, “In these trials, the riboswitch-edited plants exhibited a clear disease resistance phenotype, with the trs3 mutant line showing more significant resistance than the trs8 line (Fig. S6)”. Why? It should be explained.\n6)Line 360, what does “This other study” mean?\n7)Lines 443-451, Sentences lack the subjects.\n8)Lines 464-465, the sentence “All measurements used the newest fully expanded flag leaf” is not understandable.\n9)Line 688, I suggest change “Nine gene edited types” to “ Nine gene-edited types”\n10)Line 703, Is “panicle length” calculated with the main panicle or all panicles of plant?\n11)Line 704, Is “yield per panicle” calculated with the main panicle or all panicles of plant?\n12)In Figure 3e, which trs line(s) are used. The line number should be marked in the figure.\n13)Line 713, “parameters” should be “parameter”.\n14)In Figure 3J, which trs lines are used for the test? The line number should be marked in the figure.\n15)In figure 4a-d, which trs lines are used for test. The line number should be marked in figures.\n16)Line 765, “ T transpiration” should be “ Transpiration”.\n17)In Figure S2, the line number of trs should be marked.\n18)In Figure S5, the line number should be marked.\n19)Line 820, which trs line is used for DEGs analysis.\n20)Line 826 and 831, It is not clear. Which trs lines are used for the analysis?", "reviewer_2": "The authors present a compelling and comprehensive study demonstrating that CRISPR-Cas9-mediated editing of the TPP riboswitch in rice leads to enhanced thiamine (vitamin B1) biosynthesis, as well as improved yield, nutritional content, and stress tolerance. The multi-dimensional approach integrates transcriptomics, metabolomics, and physiological assays to establish a novel, generalizable strategy for biofortification and crop improvement. Moreover, the results of the study were shown to be more universally applicable, as they were partially replicated in the relevant dicot crop tomato.\nOverall, we are impressed with the results presented in this study. The study represents a substantial and novel contribution to the field of plant synthetic biology and crop improvement. It is methodologically rigorous, well-documented, and conceptually innovative. Pending minor revisions, I strongly support its publication in Nature Communications.\nMinor Comments\n• Language and Grammar: The manuscript is generally well-written, but a careful language polish is needed (e.g., articles, verb tenses, occasional awkward phrasing).\n• Figure Legends: Some figure panels (particularly in supplementary material) would benefit from more detailed legends to ensure standalone interpretability.\nIntroduction\nThe problem of micronutrient deficiencies, the ability of plant molecular biology to mitigate them, and the problem with adverse effects on non-targeted characteristics of the arising crops are well-introduced, making correct references to literature.\nOverall, the introduction nicely introduces the broader frame of the research and perfectly covers the rationale for the manuscript.\nBelow are some more specific comments on different section of the manuscript.\nLine 61: Reference for Lysine enhanced rice is missing (or unclear).\nLine 62-63:\nThe manuscript states, “While these efforts have mitigated specific micronutrient deficiencies in certain regions or countries…” (lines 62–63), referring to biofortified crops like Golden Rice, riboflavin-rich rice, and others. However, this claim is not entirely accurate. Many of these biofortified crops, including Golden Rice, have faced significant hurdles in market deployment due to regulatory, political, and public acceptance challenges. For instance, Golden Rice, despite being developed over two decades ago, has seen limited adoption and has even been withdrawn from some markets due to opposition and logistical issues, rather than achieving widespread mitigation of vitamin A deficiency as implied.\nRevise the statement to reflect the reality of biofortification deployment. The authors should acknowledge that while these crops were developed with the intent to mitigate micronutrient deficiencies, many have not reached the market or achieved significant impact due to non-scientific barriers like regulatory delays, public resistance, and market access issues. They could cite specific examples, such as the challenges faced by Golden Rice (e.g., opposition in the Philippines, as noted in public discourse around GMO crops), and contrast this with their TPP riboswitch approach, which may face similar hurdles. This would provide a more balanced and critical perspective on the field.\nLine 63-64:\nThe manuscript claims that “the capacity and transformation efficiency of gene expression vectors limit the simultaneous biofortification of crops with multiple essential nutrients” (lines 63–64). While this is a technical challenge, there are studies showing gene expression vectors with very big sizes and high gene numbers (ref. 14 being a good example). A more significant limitation in the field of biofortification is the inability to bring these genetically modified crops to market due to regulatory, political, and societal barriers. The technical limitation of vector capacity is secondary to the broader issue of market deployment, as many biofortified crops, even when successfully developed, fail to reach farmers and consumers due to external constraints rather than scientific ones. For example, crops like Golden Rice have been stalled by regulatory hurdles and public skepticism, not just by the inability to stack multiple nutrient traits.\nReframe the discussion of limitations to prioritize the real-world challenges of market deployment over technical constraints like vector capacity. The authors should acknowledge that while vector efficiency is a factor, the primary barrier to simultaneous biofortification lies in navigating regulatory frameworks, gaining public acceptance, and ensuring distribution to farmers. They could briefly discuss (perhaps better suited in discussion section) how their TPP riboswitch editing approach, which modifies an endogenous regulatory element rather than introducing foreign genes, might face different regulatory scrutiny compared to transgenic approaches like Golden Rice. This would align the manuscript more closely with practical challenges in the field and demonstrate a deeper understanding of the biofortification landscape.\nLines 100-102:\nIncorrect Citation for TPP Increase Claim Due to Riboswitch Mutations (Lines 100–102):\nThe manuscript claims that “In plants, TPP riboswitch mutations lead to a 5-10-fold increase in TPP levels,” citing Miranda et al. (2007) (Planta, 226, 1411–1421, doi:10.1007/s00425-007-0579-y). However, this citation does not support the claim. Miranda et al. (2007) investigate the effects of a yeast-derived bifunctional TPS-TPP enzyme (trehalose-6-phosphate synthase-thiamine pyrophosphate) in transgenic Arabidopsis, showing that it increases thiamine content (approximately 2-3-fold in leaves compared to wild-type) and enhances abiotic stress tolerance. However, their study does not involve TPP riboswitch mutations—it uses a transgenic approach to overexpress a yeast enzyme—and it does not report a 5-10-fold increase in TPP levels specifically. Perhaps an appropriate reference is Plant Cell paper by Bocobza et al. (https://doi.org/10.1105/tpc.112.106385), though here the TPP increases in Arabidopsis upon riboswitch mutation seem more modest. Moreover, the manuscript’s claim appears to be based on the authors’ own data (e.g., Fig. 5c, showing a marked elevation in TPP levels in edited rice lines, consistent with a 5-10-fold increase), not on Miranda et al. (2007). This citation error risks misleading readers about the precedent for TPP riboswitch editing effects in plants and undermines the manuscript’s credibility.\nThe authors should remove or replace the citation of Miranda et al. (2007) for this specific claim, as it does not support the stated 5-10-fold TPP increase due to riboswitch mutations. Instead, they should directly reference their own data (e.g., “In this study, we found that TPP riboswitch mutations in rice led to a 5-10-fold increase in TPP levels (Fig. 5c)”), since their results provide the relevant evidence. If the authors wish to cite a study for background on TPP riboswitch effects in plants, besides the aforementioned Plant Cell paper, they should consider an additional relevant reference, such as Wachter et al. (2007) (Plant Cell, 19, 3437–3450, doi:10.1105/tpc.107.053645, already cited as ref. 34) which demonstrates riboswitch control of gene expression in plants via splicing and alternative 3’ end processing, though it does not quantify TPP levels. Alternatively, they could cite studies on TPP riboswitch disruption in other organisms (e.g., bacteria) that report TPP increases, if applicable, to provide a broader context. Additionally, the authors should clarify how TPP levels were measured in their study (e.g., via LC-MS/MS, as is standard for thiamine derivatives) to strengthen the claim.\nResults\nFig. 1: Regarding Fig. 1B, C and D, the figure should mention that this is thiamin content (as is correctly indicated in the legend). Furthermore, what does the “x108” if all is set as relative to ZH11? To link Fig. 1B with 1F, it could be clearer to mention (unpolished or full or brown rice grains).\nLine 237 and Figure S7 Legend:\nThe manuscript states in line 237, “and we found similar results in rice by qPCR (Fig. S7),” following a claim about the induction of most vitamin B1 synthesis pathway genes under low-temperature (LT) stress in maize (lines 235–236, citing Jiang et al., 2022). Additionally, the legend of Figure S7 reads: “Differential expression of vitamin B1 biosynthesis genes under normal conditions and cold treatment.” However, Figure S7 includes only four genes—OsAK, OsIscS, OsTDK1, and OsTHI4—of which only OsTDK1 (thiamine diphosphokinase) and OsTHI4 (thiazole synthesis) are directly part of the thiamine biosynthesis pathway. With only two of the four genes in Figure S7 being direct thiamine biosynthesis genes, the statement in line 237 and the figure legend are misleading, as they imply that all genes analyzed are part of the thiamine biosynthesis pathway and that the figure comprehensively supports the induction of most pathway genes under LT stress. This risks confusing readers and overstates the scope of the qPCR analysis, especially since key pathway genes like OsTHIC and OsTH1 are absent from the figure.\nThe authors should revise both the statement in line 237 and the Figure S7 legend to accurately reflect the genes analyzed. The legend should be rephrased to clarify that the figure includes a mix of thiamine biosynthesis genes and related metabolic genes, for example: “Fig. S7 Differential expression of thiamine biosynthesis genes (OsTDK1, OsTHI4), a supporting gene (OsIscS), and a related metabolic gene (OsAK) under normal conditions and cold treatment.” Similarly, the statement in line 237 should be adjusted to avoid implying that Figure S7 shows induction of most thiamine biosynthesis genes, for example: “and we found that several thiamine biosynthesis and related genes were induced in rice by qPCR (Fig. S7), supporting a role for thiamine in LT stress responses.”. These revisions will ensure the manuscript’s claims and figure descriptions are accurate and aligned with the data presented.\nLine 257:\nThe sentence in line 257, “Volcano plot analysis showed that the gene expression profiles of trs lines substantial transcriptional reprogramming, with 258 2,346 genes up‐regulated and 3,536 genes down‐regulated in the trs lines than ZH11 259 lines (Fig. S9, Table S3), respectively.” is grammatically incomplete due to a missing verb between “trs lines” and “substantial transcriptional reprogramming.”. Furthermore, “than” should be replaced with “compared to”.\nThe recommended revision is: “Volcano plot analysis showed that the gene expression profiles of trs lines exhibited substantial transcriptional reprogramming, with 2,346 genes upregulated and 3,536 genes downregulated in the trs lines compared to ZH11 (Fig. S9, Table S3).”\nLine 259: Table S3. Here, it is interesting to comment on the expression of OsTHIC (LOC_Os03g47610), being controlled by the (mutated) riboswitch, as the data seems to confirm higher transcript abundance in the trs lines, strengthening the observations of the authors.\nLine 300: is there confirmation of altered, being enhanced, vitamin B1 levels in tomato plants? This would be a nice added value. If not, “altering vitamin B1 levels” is better replaced by simply mentioning the editing of the SlTHIC riboswitch.\nDiscussion\nLine 320: Here the nutritional benefits focus on micronutrient and some macronutrients. It would be interesting to make a statement here on the (expected) impact on the content of other important macronutrients such as protein and starch.\nLine 338: “as” can be deleted here.\nLine 356: “In addition, our 356 findings nicely complement those of another recent study which revealed that the 357 natural variation of the thiamine biosynthetic enzyme thiamine pyrophosphokinase 2 358 fine tunes thiamine pyrophosphate levels and grain yield in maize whilst folial 359 application of thiamine pyrophosphate was able to enhance yield in maize, rice and 360 rapeseed.” The reference is missing.\nSome remarks to be considered within the discussion\nDoes the disruption or alteration of the riboswitch sequence in the different mutant lines result in a complete non-functional riboswitch (as in non-TPP responsive) or would there only be an expected lowered sensitivity? Would that explain the differences between the different lines (and not non-targeted genetic alterations)?\nWould the witnessed agronomical advantages of the trs lines be (solely) explained by alterations in vitamin B1 (TPP) levels?\nThe beneficial impact of CRISPR edited THIC riboswitches raises the question as to why these do not occur naturally, as the THIC riboswitches appear conserved. Would there be specific conditions that make it evolutionary beneficial to maintain the functional riboswitch?", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "In this manuscript, Li et al. (NCOMMS-25-20757-T) demonstrate that targeted editing of the thiamine pyrophosphate (TPP) riboswitch in rice elevates levels of vitamins and phytonutrients, enhancing both normal growth and stress resilience (e.g., cold tolerance, blast resistance). The authors further extend their findings to tomato, reporting improved photosynthetic rates and disease resistance via TPP riboswitch modification. While the study applies gene-editing tools to a biologically relevant target (TPP riboswitch), it remains largely descriptive and incremental. The absence of mechanistic exploration and insufficient validation across environments/varieties fall short of the standards required for publication in Nature Communications. Below are detailed critiques:\n1. The regulatory role of TPP riboswitches in thiamine biosynthesis—via feedback inhibition through the 3′-UTR of THIC—is well-established. Moreover, prior studies have successfully modulated gene expression through editing of promoters, UTRs, and other regulatory elements. Similarly, enhancing stress tolerance by altering vitamin biosynthesis pathways has been extensively documented (Xu et al., 2022; Zheng et al., 2022). The authors fail to articulate how their work transcends these existing paradigms or introduces novel biological insights.\n2. The study primarily catalogs phenotypic outcomes without probing the mechanistic basis of observed traits. For instance:\nIn Figure 1a, only trs3 and trs8 mutants exhibit distinct phenotypes. Do other mutants (trs1, trs2, etc.) display consistent or divergent traits? If phenotypic discrepancies exist, are they attributable to mutation type (e.g., frameshift vs. point mutation), editing efficiency, or off-target effects?\nWhat are the specific genotypes of trs3 and trs8? How do these mutations alter riboswitch structure/function, and how do such changes mechanistically link to vitamin accumulation, stress tolerance, or growth enhancement?\nAre the phenotypic improvements in rice and tomato driven directly by elevated thiamine/phytonutrient levels, or are secondary regulatory pathways involved? A multi-omics approach (transcriptomics, metabolomics) could elucidate these connections.\n3. Rice field trials across diverse geographical locations and multiple growing seasons are necessary to validate stress tolerance claims. Additionally, overexpression studies of the TPP riboswitch are critical to confirm whether phenotypic effects are dosage-dependent.\n4. In addition, comprehensive phenotypic data of the fully edited tomato plants should be presented."}, "author_response": "Response: We sincerely thank you for your insightful comments and the critical question you raised. The issue of whether the observed comprehensive improvements, including nutritional quality, yield, cold tolerance, and disease resistance, are directly attributable to elevated vitamin B1 (thiamine) levels rather than to other potential unknown functions of the OsTHIC gene is indeed central to our study.\nTo address this point directly, we have provided additional experimental evidence in the revised manuscript, as summarized below:\n1. We applied 1 mM thiamine pyrophosphate (TPP, the active form of vitamin B1) exogenously to wild-type (ZH11) rice plants and systematically evaluated their performance in nutritional quality, disease resistance, and cold tolerance. The TPP treatment significantly increased the content of multiple nutrients in grains, including thiamine, riboflavin, niacin, pantothenate, pyridoxine, α-tocopherol, several lysophospholipids, and essential amino acids (e.g., leucine, threonine, serine, valine). The enhancement pattern closely resembled that of the trs edited lines (revised Fig. 5a). After inoculation with Magnaporthe oryzae, TPP-treated wild-type plants showed significantly reduced lesion length and fungal biomass compared to controls, mirroring the disease-resistant phenotype of the trs mutants (revised Fig. 5b-d). Under cold stress conditions, these plants also demonstrated superior survival rates, confirming that elevated TPP levels enhance cold tolerance (revised Fig. 5e-f).\n2. To further verify that the phenotypic improvements are specifically due to increased TPP levels rather than other functions of OsTHIC, we constructed a dual-luciferase reporter system carrying nine different edited versions of the OsTHIC 3′-UTR (M1-M9; revised Fig. 5g). Under exogenous addition of TPP, all edited 3′-UTRs showed higher luciferase activity compared to the wild-type sequence. Notably, M3 and M8 (corresponding to trs3 and trs8) exhibited the strongest enhancement (revised Fig. 5g), consistent with their highest vitamin B1 accumulation in vivo (revised Fig. 1b-d).\n3. RNA-seq analysis of trs lines and TPP-treated wild-type plants identified 724 significantly upregulated genes common to both groups (revised Fig. 5j). KEGG enrichment analysis further revealed that these shared upregulated genes were predominantly associated with lipid metabolism, vitamin metabolism, amino acid and nitrogen metabolism, photosynthesis, carbon fixation, and plant-pathogen interaction pathways (revised Fig. 5k).\nThrough exogenous TPP treatment, we successfully phenocopied the key traits related to nutrition, disease resistance, and cold tolerance observed in the gene-edited lines. Combined with dual-luciferase reporter assays and multi-omics analyses, these results confirm that the improved traits are directly driven by elevated vitamin B1 levels. These additional experiments strengthen the main conclusion of our study: Editing the TPP riboswitch to increase vitamin B1 levels represents an effective strategy for simultaneously improving yield, quality, and stress tolerance in crops.\n2)Authors generated SlTHIC-modified tomato lines and showed enhancements of their growth by large increases in the rate of photosynthesis and tolerance to Botrytis cinerea. How about fruit metabolites? I suggest authors to provide, as rice, the metabolite data of their fruits to answer whether the metabolite alteration in tomato is same as in rice grain.\nResponse: Thank you for raising this important point. In response to your comment, we have now performed targeted metabolomic profiling of tomato fruits from both wild-type and SlTHIC-edited lines (sltrs1 and sltrs5). Consistent with the observations in rice, the sltrs tomato fruits exhibited significant increases in the levels of multiple nutrients, including thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, α-tocopherol, Lysophospholipids (e.g., lysoPE 18:3, lysoPC 18:3) and Essential amino acids (e.g., leucine, threonine, serine, valine). We have added this important data and description section to the revised manuscript (Fig. 6a-m and Table S5).\nThese findings confirm that riboswitch editing in tomato not only enhances photosynthetic performance and stress resilience but also significantly improves fruit nutritional quality, mirroring the multi-dimensional fortification observed in rice. This consistency across distantly related species underscores the evolutionary conservation of TPP riboswitch function and suggests broad utility in dicot and monocot crops.\nMinor issues:\n1)Lines 102-104, the sentence “This knowledge gap opens the question as to their capacity to orchestrate synergistic improvements in yield, nutrition, and stress resilience a question that so far remains unexplored and mechanistically opaque” is not clear.\nResponse: Thank you for pointing out this issue. We have revised the sentence to: However, the application potential of this regulatory mechanism to the coordinated improvement of multiple traits in crops remains unexplored in prior research (Lines 114-116).\n2)Line 129, what does “After two rounds of breeding” mean? It should be defined.\nResponse: Thank you for pointing out this issue. We have revised the sentence to: After two generations of self-pollination (T1 and T2) to obtain homozygous mutants (Lines 143-144).\n3)Lines 165-171, authors compared yield complements between the modified lines and WT without thousand grain weight. I suggest authors to add the comparison data of thousand grain weight.\nResponse: We have incorporated additional statistical analyses in the revised manuscript, as presented in Fig. S4a-d and the corresponding Results section (Lines 182-183). These data demonstrate that thousand grain weight and grain shape (including grain length, width, and thickness) did not differ significantly, indicating that the observed yield enhancement is primarily attributable to increases in both panicle length and grains per panicle.\n4)Lines 208-211, The sentence “Field observations demonstrated that the riboswitch-edited lines maintained near-normal growth parameters while exhibiting enhanced innate defense responses to natural pathogen infections compared to wild-type controls (ZH11)” is not clear. Which growth parameters have been measured?\nResponse: We apologize for any confusion caused to you. The original text intended to convey that during the field experiment, we ensured a normal supply of water and fertilizer to guarantee that the wild type and mutant strains were in the same growth environment, and then conducted the detection of disease resistance phenotypes at the seedling stage. The description in the text has also been modified accordingly: Under the condition of ensuring sufficient supply of water and fertilizer, field observations showed that when detecting the incidence of natural pathogen infections, these edited lines exhibited more robust innate defense responses compared to the wild-type control (ZH11) (Lines 230-234).\n5)Lines 225-227, “In these trials, the riboswitch-edited plants exhibited a clear disease resistance phenotype, with the trs3 mutant line showing more significant resistance than the trs8 line (Fig. S6)”. Why? It should be explained.\nResponse: We thank the reviewer for raising this point and for the opportunity to clarify. The statement referenced by the reviewer was based on a specific observation from inoculation trials under controlled laboratory conditions using the Magnaporthe oryzae strain ES67. In these particular experiments, quantitative PCR did indicate a lower fungal biomass in trs3 compared to trs8, although no significant difference in lesion length was observed between the two mutant lines. We wish to emphasize that this inter-mutant difference was not consistently seen across other pathogen strains or inoculation methods, where both trs3 and trs8 exhibited similarly enhanced resistance compared to the wild type, without significant differences from each other. We recognize that emphasizing this isolated discrepancy could inadvertently cause confusion, as it may reflect condition-specific variability rather than a generalizable biological distinction. Therefore, we have revised the main text to remove the comparative clause regarding trs3 and trs8. The updated conclusion now focuses on the consistent resistance phenotype shared by all riboswitch-edited plants, a finding robustly supported by multiple lines of evidence, including spray inoculations, punch assays with various strains, and field trials (Fig. 4e–j, Fig. S8). We apologize for any misunderstanding the original phrasing may have caused.\n6)Line 360, what does “This other study” mean?\nResponse: We sincerely apologize for the confusion caused by the inclusion of this section. This content originated from another separate study that was submitted back-to-back with our present manuscript to Nature Communications, as specifically noted in the cover letter of our initial submission. Upon further reflection, we now realize that this material is not suitable for inclusion in the current manuscript. We have therefore removed the relevant section in the revised version.\n7)Lines 443-451, Sentences lack the subjects.\nResponse: Thank you for pointing out this issue. We have revised the manuscript to correct grammatical errors, including subject-verb agreement and article usage (Lines 568-588).\n8)Lines 464-465, the sentence “All measurements used the newest fully expanded flag leaf” is not understandable.\nResponse: Thank you for pointing out this issue. We have revised the sentence to: All measurements were performed on the youngest fully expanded flag leaf of each plant (Lines 595-596).\n9)Line 688, I suggest change “Nine gene edited types” to “ Nine gene-edited types”\nResponse: Thank you for your suggestions. The new manuscript has been revised accordingly (Line 860).\n10)Line 703, Is “panicle length” calculated with the main panicle or all panicles of plant?\n11)Line 704, Is “yield per panicle” calculated with the main panicle or all panicles of plant?\nResponse: Thank you for your question. To maintain consistency, all panicle traits were assessed exclusively on the main panicle of every plant. This description has been added to the revised manuscript (Lines 885-886).\n12)In Figure 3e, which trs line(s) are used. The line number should be marked in the figure.\nResponse: We apologize for the confusion. To clarify this point, the corresponding description has been added to the new manuscript as recommended: Phenotypes of WT (first two rows), trs3 (third row) and trs8 (fourth row) mutant plants under limited N conditions (Line 895).\n13)Line 713, “parameters” should be “parameter”.\nResponse: Thank you for your suggestions. The new manuscript has been revised accordingly (Line 892).\n14)In Figure 3J, which trs lines are used for the test? The line number should be marked in the figure.\nResponse: Thank you for your question and suggestion. The three lines shown in Figure 3J are, from left to right: WT, trs3, and trs8. We have incorporated a note on Figure 3j as recommended (Fig. 3j).\n15)In figure 4a-d, which trs lines are used for test. The line number should be marked in figures.\nResponse: Thank you for your suggestions. Given that the trs3 and trs8 lines exhibit similar resistance phenotypes to rice blast, we have chosen to present the resistance performance of trs8 in Fig. 4a-d, while including the resistance performance of trs3 in Fig. S7. This section has been updated accordingly in the revised manuscript (Fig. 4a-d, Fig. S7).\n16)Line 765, “ T transpiration” should be “ Transpiration”.\nResponse: Thank you for pointing out this error. It has now been corrected in the revised manuscript (Line 976).\n17)In Figure S2, the line number of trs should be marked.\nResponse: Thank you for this suggestion. In the revised version, this figure has been renumbered as Fig. S3 due to the inclusion of additional experimental data. The trs line (trs3) have now been clearly marked in the updated figure, as recommended.\n18)In Figure S5, the line number should be marked.\nResponse: Thank you for this pointing out. This figure is now presented as Fig. S7 in the updated manuscript. We have accordingly marked the trs line (trs3) in the revised figure, as suggested.\n19)Line 820, which trs line is used for DEGs analysis.\n20)Line 826 and 831, It is not clear. Which trs lines are used for the analysis?\nResponse: Sorry for the confusion caused by this part of the original manuscript. DEGs are genes that are differentially expressed with WT in both trs3 and trs8 lines. Subsequent analyses, including KEGG pathway enrichment analyses, were performed based on these DEGs. A description of this section has been added to the revised manuscript (Fig. 5h-k, Lines 304-323).\nReviewer #2 (Remarks to the Author):\nThe authors present a compelling and comprehensive study demonstrating that CRISPR-Cas9-mediated editing of the TPP riboswitch in rice leads to enhanced thiamine (vitamin B1) biosynthesis, as well as improved yield, nutritional content, and stress tolerance. The multi-dimensional approach integrates transcriptomics, metabolomics, and physiological assays to establish a novel, generalizable strategy for biofortification and crop improvement. Moreover, the results of the study were shown to be more universally applicable, as they were partially replicated in the relevant dicot crop tomato.\nOverall, we are impressed with the results presented in this study. The study represents a substantial and novel contribution to the field of plant synthetic biology and crop improvement. It is methodologically rigorous, well-documented, and conceptually innovative. Pending minor revisions, I strongly support its publication in Nature Communications.\nMinor Comments\n• Language and Grammar: The manuscript is generally well-written, but a careful language polish is needed (e.g., articles, verb tenses, occasional awkward phrasing).\nResponse: We have thoroughly revised the manuscript to correct grammatical errors, improve sentence structure, and ensure consistent and appropriate use of articles and verb tenses. After the final draft is confirmed, we will further invite professional editors to improve the manuscript to enhance clarity and readability.\n•Figure Legends: Some figure panels (particularly in supplementary material) would benefit from more detailed legends to ensure standalone interpretability.\nResponse: We have expanded the legends for all supplementary figures to provide sufficient detail for independent interpretation.\nIntroduction\nThe problem of micronutrient deficiencies, the ability of plant molecular biology to mitigate them, and the problem with adverse effects on non-targeted characteristics of the arising crops are well-introduced, making correct references to literature.\nOverall, the introduction nicely introduces the broader frame of the research and perfectly covers the rationale for the manuscript.\nBelow are some more specific comments on different section of the manuscript.\nLine 61: Reference for Lysine enhanced rice is missing (or unclear).\nResponse: We apologize that in our earlier manuscript, the term \"lysine-enhanced rice\" was incorrectly used in place of \"lysine-enhanced maize\". This error has been corrected in the revised version of the manuscript. Additionally, relevant references have been included to further support the context (Line 63).\nReference：\nHurst, J. P. et al. Editing the 19 kDa alpha-zein gene family generates non-opaque2-based quality protein maize. Plant Biotechnol J 22, 946-959, doi:10.1111/pbi.14237 (2024).\nLine 62-63:\nThe manuscript states, “While these efforts have mitigated specific micronutrient deficiencies in certain regions or countries…” (lines 62-63), referring to biofortified crops like Golden Rice, riboflavin-rich rice, and others. However, this claim is not entirely accurate. Many of these biofortified crops, including Golden Rice, have faced significant hurdles in market deployment due to regulatory, political, and public acceptance challenges. For instance, Golden Rice, despite being developed over two decades ago, has seen limited adoption and has even been withdrawn from some markets due to opposition and logistical issues, rather than achieving widespread mitigation of vitamin A deficiency as implied.\nRevise the statement to reflect the reality of biofortification deployment. The authors should acknowledge that while these crops were developed with the intent to mitigate micronutrient deficiencies, many have not reached the market or achieved significant impact due to non-scientific barriers like regulatory delays, public resistance, and market access issues. They could cite specific examples, such as the challenges faced by Golden Rice (e.g., opposition in the Philippines, as noted in public discourse around GMO crops), and contrast this with their TPP riboswitch approach, which may face similar hurdles. This would provide a more balanced and critical perspective on the field.\nLine 63-64:\nThe manuscript claims that “the capacity and transformation efficiency of gene expression vectors limit the simultaneous biofortification of crops with multiple essential nutrients” (lines 63-64). While this is a technical challenge, there are studies showing gene expression vectors with very big sizes and high gene numbers (ref. 14 being a good example). A more significant limitation in the field of biofortification is the inability to bring these genetically modified crops to market due to regulatory, political, and societal barriers. The technical limitation of vector capacity is secondary to the broader issue of market deployment, as many biofortified crops, even when successfully developed, fail to reach farmers and consumers due to external constraints rather than scientific ones. For example, crops like Golden Rice have been stalled by regulatory hurdles and public skepticism, not just by the inability to stack multiple nutrient traits.\nReframe the discussion of limitations to prioritize the real-world challenges of market deployment over technical constraints like vector capacity. The authors should acknowledge that while vector efficiency is a factor, the primary barrier to simultaneous biofortification lies in navigating regulatory frameworks, gaining public acceptance, and ensuring distribution to farmers. They could briefly discuss (perhaps better suited in discussion section) how their TPP riboswitch editing approach, which modifies an endogenous regulatory element rather than introducing foreign genes, might face different regulatory scrutiny compared to transgenic approaches like Golden Rice. This would align the manuscript more closely with practical challenges in the field and demonstrate a deeper understanding of the biofortification landscape.\nResponse: We thank the reviewer for these critical and insightful comments, which have helped us to present a more accurate and nuanced perspective on the biofortification field. We have revised the manuscript accordingly.\nRegarding Lines 62-63: We agree that our original statement was overly optimistic regarding the real-world impact of previous biofortification efforts. We have revised the text to more accurately reflect the significant non-scientific barriers to deployment. The text now reads (Lines 64-71):\n“While these efforts hold potential for addressing specific micronutrient deficiencies in particular regions or countries, their practical implementation faces substantial non-scientific constraints, including regulatory barriers, public skepticism, and market access limitations. A notable example is Golden Rice, developed over two decades ago, has been repeatedly delayed and even withdrawn from certain markets due to public attitudes and political factors, rather than widely alleviating vitamin A deficiency as initially envisioned17.”\nRegarding Lines 63-64: We appreciate the reviewer's guidance to reframe the discussion of limitations to better reflect the primary challenges in the field. We have modified the text to prioritize the overarching issue of market deployment, while still acknowledging the technical constraint as a contributing factor. The revised text now states (Lines 71-78):\n\"Moreover, the capacity and transformation efficiency of gene expression vectors are generally insufficient18, making it difficult to achieve synergistic enrichment of multiple nutrients in crops, thus failing to solve the main problem of \"hidden hunger\"... Furthermore, even when successfully developed, such crops often face the formidable non-scientific barriers mentioned above.\"\nThis new framing correctly identifies the technical hurdle of multi-nutrient enrichment as a scientific challenge that contributes to the problem of \"hidden hunger,\" while immediately linking it to the broader, more critical issue of deployment barriers.\nAs suggested by the reviewer, we have also expanded on this point in the Discussion section to contrast our approach with traditional transgenics. We added a paragraph (seventh paragraph of Discussion) that includes the sentence (Lines 475-482):\n\"It is also important to consider the practical implications of our approach in the context of crop biotechnology. Unlike traditional transgenic strategies that often involve the introduction of foreign genes, our method relies on precise editing of endogenous regulatory elements. This may facilitate regulatory approval and public acceptance, as exemplified by the recent development of similar genome-edited crops with improved nutritional profiles50. That said, the eventual deployment of such crops will still require careful evaluation of long-term agronomic performance, environmental impact, and socio-economic feasibility.\"\nWe believe these revisions provide a more balanced, critical, and accurate overview of the biofortification landscape, acknowledging both the technical and socio-regulatory hurdles while positioning our riboswitch-editing strategy within this context.\nLines 100-102:\nIncorrect Citation for TPP Increase Claim Due to Riboswitch Mutations (Lines 100-102):\nThe manuscript claims that “In plants, TPP riboswitch mutations lead to a 5-10-fold increase in TPP levels,” citing Miranda et al. (2007) (Planta, 226, 1411-1421, doi:10.1007/s00425-007-0579-y). However, this citation does not support the claim. Miranda et al. (2007) investigate the effects of a yeast-derived bifunctional TPS-TPP enzyme (trehalose-6-phosphate synthase-thiamine pyrophosphate) in transgenic Arabidopsis, showing that it increases thiamine content (approximately 2-3-fold in leaves compared to wild-type) and enhances abiotic stress tolerance. However, their study does not involve TPP riboswitch mutations—it uses a transgenic approach to overexpress a yeast enzyme—and it does not report a 5-10-fold increase in TPP levels specifically. Perhaps an appropriate reference is Plant Cell paper by Bocobza et al. (https://doi.org/10.1105/tpc.112.106385), though here the TPP increases in Arabidopsis upon riboswitch mutation seem more modest. Moreover, the manuscript’s claim appears to be based on the authors’ own data (e.g., Fig. 5c, showing a marked elevation in TPP levels in edited rice lines, consistent with a 5-10-fold increase), not on Miranda et al. (2007). This citation error risks misleading readers about the precedent for TPP riboswitch editing effects in plants and undermines the manuscript’s credibility.\nThe authors should remove or replace the citation of Miranda et al. (2007) for this specific claim, as it does not support the stated 5-10-fold TPP increase due to riboswitch mutations. Instead, they should directly reference their own data (e.g., “In this study, we found that TPP riboswitch mutations in rice led to a 5-10-fold increase in TPP levels (Fig. 5c)”), since their results provide the relevant evidence. If the authors wish to cite a study for background on TPP riboswitch effects in plants, besides the aforementioned Plant Cell paper, they should consider an additional relevant reference, such as Wachter et al. (2007) (Plant Cell, 19, 3437-3450, doi:10.1105/tpc.107.053645, already cited as ref. 34) which demonstrates riboswitch control of gene expression in plants via splicing and alternative 3’ end processing, though it does not quantify TPP levels. Alternatively, they could cite studies on TPP riboswitch disruption in other organisms (e.g., bacteria) that report TPP increases, if applicable, to provide a broader context. Additionally, the authors should clarify how TPP levels were measured in their study (e.g., via LC-MS/MS, as is standard for thiamine derivatives) to strengthen the claim.\nResponse: We apologize for our unintentional citation of an incorrect reference. The correct reference should be “Moulin, M., Nguyen, G. T. D. T., Scaife, M. A., Smith, A. G. & Fitzpatrick, T. B. Analysis of Chlamydomonas thiamin metabolism in vivo reveals riboswitch plasticity. P Natl Acad Sci USA 110, 14622-14627, doi:10.1073/pnas.1307741110 (2013)”, in which the authors explicitly state: \"Interestingly, all were found to be substantially higher (5–10-fold increase) in pyr1 compared with wild-type, with TPP being the most abundant vitamer (Fig. 1B).\" We have now updated this citation in the revised manuscript.\nResults\nFig. 1: Regarding Fig. 1B, C and D, the figure should mention that this is thiamin content (as is correctly indicated in the legend). Furthermore, what does the “x108” if all is set as relative to ZH11? To link Fig. 1B with 1F, it could be clearer to mention (unpolished or full or brown rice grains).\nResponse: Thank you very much for your careful review and valuable suggestions. We have made the following clarifications and revisions in the updated manuscript:\n1. To ensure clarity and eliminate any potential ambiguity, we have explicitly labeled the legends in our figures to clearly indicate the content of specific metabolites in particular tissues (Fig. 1b-d, 1f-q).\n2. The “×108” values on the y-axis represent the relative intensity of thiamine as detected by LC-MS/MS (in counts per second, c.p.s.). This is a standard unit in targeted metabolomics for semi-quantitative comparison. To improve clarity, we have updated the y-axis label to: “Caryopsis/Leaf/Stem thiamine relative content”, and clarified this in the figure legend (Fig. 1b-d, Line 861).\nLine 237 and Figure S7 Legend:\nThe manuscript states in line 237, “and we found similar results in rice by qPCR (Fig. S7),” following a claim about the induction of most vitamin B1 synthesis pathway genes under low-temperature (LT) stress in maize (lines 235-236, citing Jiang et al., 2022). Additionally, the legend of Figure S7 reads: “Differential expression of vitamin B1 biosynthesis genes under normal conditions and cold treatment.” However, Figure S7 includes only four genes—OsAK, OsIscS, OsTDK1, and OsTHI4—of which only OsTDK1 (thiamine diphosphokinase) and OsTHI4 (thiazole synthesis) are directly part of the thiamine biosynthesis pathway. With only two of the four genes in Figure S7 being direct thiamine biosynthesis genes, the statement in line 237 and the figure legend are misleading, as they imply that all genes analyzed are part of the thiamine biosynthesis pathway and that the figure comprehensively supports the induction of most pathway genes under LT stress. This risks confusing readers and overstates the scope of the qPCR analysis, especially since key pathway genes like OsTHIC and OsTH1 are absent from the figure.\nThe authors should revise both the statement in line 237 and the Figure S7 legend to accurately reflect the genes analyzed. The legend should be rephrased to clarify that the figure includes a mix of thiamine biosynthesis genes and related metabolic genes, for example: “Fig. S7 Differential expression of thiamine biosynthesis genes (OsTDK1, OsTHI4), a supporting gene (OsIscS), and a related metabolic gene (OsAK) under normal conditions and cold treatment.” Similarly, the statement in line 237 should be adjusted to avoid implying that Figure S7 shows induction of most thiamine biosynthesis genes, for example: “and we found that several thiamine biosynthesis and related genes were induced in rice by qPCR (Fig. S7), supporting a role for thiamine in LT stress responses.”. These revisions will ensure the manuscript’s claims and figure descriptions are accurate and aligned with the data presented.\nResponse: We appreciate the reviewers' insightful comments and helpful suggestions. In the original Fig. S7, OsIscS and OsAK act upstream of the vitamin B1 biosynthetic pathway by providing precursor metabolites; therefore, referring to them directly as “vitamin B1 biosynthetic genes” was imprecise. To improve clarity, we have removed these two genes from the revised figure (Fig. S9) and added expression data under cold stress for vitamin B1 biosynthetic genes such as OsTHIC and OsTHI. The title of Fig. S9 has been updated to “Multiple vitamin B1 biosynthetic genes were induced by cold stress” to more accurately represent that several genes in vitamin B1 biosynthetic pathway are cold-responsive.\nAdditionally, following the reviewer’s suggestion, we have revised the content originally at line 237. The modified content as fellow:\nTranscriptomic data from maize have showed that the expression levels of most vitamin B1 synthesis pathway genes were induced by LT. Similarly, we observed markedly upregulation of multiple vitamin B1 biosynthetic genes, including OsTHIC, in rice under cold stress (Fig. S9). This suggests that vitamin B1 might be involved in low temperature tolerance in rice (Line 257-261).\nLine 257:\nThe sentence in line 257, “Volcano plot analysis showed that the gene expression profiles of trs lines substantial transcriptional reprogramming, with 2,346 genes up‐regulated and 3,536 genes down‐regulated in the trs lines than ZH11 lines (Fig. S9, Table S3), respectively.” is grammatically incomplete due to a missing verb between “trs lines” and “substantial transcriptional reprogramming.”. Furthermore, “than” should be replaced with “compared to”.\nThe recommended revision is: “Volcano plot analysis showed that the gene expression profiles of trs lines exhibited substantial transcriptional reprogramming, with 2,346 genes upregulated and 3,536 genes downregulated in the trs lines compared to ZH11 (Fig. S9, Table S3).”\nResponse: Thank you for your suggestions. We have corrected the sentence to: “Volcano plot analysis showed that the gene expression profiles of both the TPP treatment group and trs lines exhibited substantial transcriptional reprogramming, with 5,842 genes up‐regulated and 1,350 genes down‐regulated in the TPP treatment compared to control plants (Fig. 5h, Table S3), and 2,346 genes up‐regulated and 3,536 genes down‐regulated in both the trs3 and trs8 lines compared to ZH11 (Fig. 5i, Table S3), respectively.” (Lines 307-312)\nLine 259: Table S3. Here, it is interesting to comment on the expression of OsTHIC (LOC_Os03g47610), being controlled by the (mutated) riboswitch, as the data seems to confirm higher transcript abundance in the trs lines, strengthening the observations of the authors.\nResponse: We thank the reviewer for this insightful suggestion. We have now explicitly commented on the upregulation of OsTHIC (LOC_Os03g47610) in the trs mutant lines in the revised manuscript. This observation confirms that the riboswitch mutations indeed alleviate TPP-mediated feedback inhibition, leading to increased transcript abundance of OsTHIC and consequently enhanced thiamine biosynthesis. This finding strengthens our mechanistic model and aligns with the observed metabolic and phenotypic improvements (Lines 312-315).\nLine 300: is there confirmation of altered, being enhanced, vitamin B1 levels in tomato plants? This would be a nice added value. If not, “altering vitamin B1 levels” is better replaced by simply mentioning the editing of the SlTHIC riboswitch.\nResponse: We thank the reviewer for this valuable suggestion. In direct response, we have now conducted targeted metabolomic analysis which confirms that vitamin B1 (thiamine) levels are significantly enhanced in the fruits of our SlTHIC riboswitch-edited tomato lines (sltrs1 and sltrs5), as shown in the new Fig. 6b. Furthermore, this elevated vitamin B1 is accompanied by a concurrent increase in a broad spectrum of other essential nutrients (Fig. 6a, 6b-m), mirroring the multi-nutrient enhancement phenotype observed in rice. This quantitative evidence solidifies the cross-species validity of our central claim that TPP riboswitch editing robustly elevates vitamin B1 levels to drive improved nutritional quality.\nDiscussion\nLine 320: Here the nutritional benefits focus on micronutrient and some macronutrients. It would be interesting to make a statement here on the (expected) impact on the content of other important macronutrients such as protein and starch.\nResponse: We thank the reviewer for raising this important point regarding the impact on macronutrients. As correctly noted by the reviewer, we did quantitatively analyze the content of major macronutrients, including protein and starch, in the polished rice grains of our riboswitch-edited lines. The results, which are presented in Supplementary Fig. S2, clearly demonstrate that there were no significant differences in the total protein or starch content between the wild-type (ZH11) and the trs lines. This indicates that the dramatic enhancement in micronutrients and health-promoting phytochemicals (Fig. 1) was achieved without altering the fundamental caloric and protein value of the rice grains.\nWe have now explicitly referenced this finding in the results section of the manuscript (in the section describing the nutritional enhancements) to provide a more complete nutritional profile and to preempt this question for the reader. The content is as follows:\n\"Notably, we also analyzed the content of macronutrients and found no significant differences in total starch or grain protein levels between the trs lines and ZH11 (Fig. S2), indicating that the nutritional enhancement is specific to micronutrients and does not compromise the core energy and protein value of the rice.\" (Lines 168-171)\nLine 338: “as” can be deleted here.\nResponse: Thank you for your suggestion. The word \"as\" has been removed.\nLine 356: “In addition, our findings nicely complement those of another recent study which revealed that the natural variation of the thiamine biosynthetic enzyme thiamine pyrophosphokinase 2 fine tunes thiamine pyrophosphate levels and grain yield in maize whilst folial application of thiamine pyrophosphate was able to enhance yield in maize, rice and rapeseed.” The reference is missing.\nResponse: We apologize for any confusion caused by the inclusion of this section. This content originated from another separate study that was submitted back-to-back with our present manuscript to Nature Communications, as specifically noted in the cover letter of our initial submission. Upon further reflection, we now realize that this content is not suitable for inclusion in the current manuscript. We have therefore removed the relevant section in the revised version.\nSome remarks to be considered within the discussion\nDoes the disruption or alteration of the riboswitch sequence in the different mutant lines result in a complete non-functional riboswitch (as in non-TPP responsive) or would there only be an expected lowered sensitivity? Would that explain the differences between the different lines (and not non-targeted genetic alterations)?\nResponse: We thank the reviewer for this important question regarding the functional consequences of our riboswitch edits. Our experimental results demonstrate that different mutations lead to varying degrees of functional impairment, ranging from partial loss to complete abolition of TPP responsiveness.\nThe trs8 line, with a large fragment deletion of the riboswitch sequence (163bp deletion), and trs3 (11bp deletion) show the strongest effects with equally high luciferase activities, indicating a near-complete loss of TPP-mediated repression. The other mutant lines exhibit graded responses corresponding to their specific alterations: trs7 (6bp deletion), trs5 (2bp deletion), trs4 (4bp deletion), trs2 (3bp deletion), trs6 (241bp insertion), and trs1 (1bp deletion) show progressively weaker effects on luciferase activity (Fig. 5g).\nThis clear correlation between the specific genetic alteration and the resulting phenotypic strength provides strong evidence that the differences between lines are directly caused by these targeted modifications to the riboswitch aptamer domain. The stepwise reduction in TPP responsiveness across the mutant series argues against off-target effects being the primary cause of phenotypic variation, as such random events would not be expected to show this consistent structure-function relationship.\nThe data indicate that complete riboswitch deletion (trs8) and certain specific mutations (trs3) can fully abolish TPP responsiveness, while other alterations result in partial loss of function proportional to the extent of structural disruption.\nFollowing your suggestion, we have integrated this paragraph as the fifth segment in the revised Discussion section (Lines 442-454).\nWould the witnessed agronomical advantages of the trs lines be (solely) explained by alterations in vitamin B1 (TPP) levels?\nResponse: We thank the reviewer for these profound and insightful question. We agree that elevated vitamin B1 is the primary, but not necessarily the sole, driver of the improved phenotypes. The convergent evidence from our multi-omics analyses strongly supports that the advantages are predominantly mediated by increased TPP. This is demonstrated by: (i) the recapitulation of key traits (multi-nutrient enhancement, blast and cold resistance) through exogenous TPP application (Fig. 5a-f); and (ii) the systemic reprogramming of TPP-dependent enzymatic hubs and associated metabolic pathways (e.g., TCA cycle, amino acid biosynthesis) and transcriptome (Fig. 5k, m-q). Thus, the agronomical advantages are a direct consequence of the rewired metabolism initiated by elevated TPP.\nThe beneficial impact of CRISPR edited THIC riboswitches raises the question as to why these do not occur naturally, as the THIC riboswitches appear conserved. Would there be specific conditions that make it evolutionary beneficial to maintain the functional riboswitch?\nResponse: We thank the reviewer for these profound and insightful questions, which touch upon the molecular mechanism, the causality of the phenotype, and the evolutionary implications of our work. We have expanded the Discussion section to address each of these points in detail, significantly deepening the interpretation of our findings.\nWe have added a new paragraph in the Discussion (Lines 461-474 in the revised manuscript) that directly addresses these questions:\n“A pertinent question arising from our findings is why such beneficial mutations have not been fixed in natural populations despite the conservation of the TPP riboswitch. We speculate that the persistence of the functional riboswitch in natural populations may be attributed to fitness trade-offs under different environmental conditions. In natural ecosystems, where nutrients are often limited and stresses are variable, constitutive overexpression of thiamine biosynthesis, such as that caused by riboswitch disruption, could lead to metabolic imbalance or unnecessary energy expenditure, reducing overall fitness. Therefore, the riboswitch mechanism may have been conserved to finely tune thiamine homeostasis in response to fluctuating environments, preventing potential costs of overexpression in resource-limited or stress-free settings. Conversely, in modern agricultural systems, with ample nutrient supply and managed environmental pressures, such trade-offs can be mitigated, allowing the beneficial effects of riboswitch editing to be fully expressed without compromising plant vigor.”\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We sincerely thank Reviewer #3 for their time and thoughtful co-review of our manuscript. We are especially grateful for the supportive recognition of our work and for highlighting its potential contribution to the field. Your feedback, along with that of the other reviewers, has been instrumental in helping us improve the clarity, rigor, and impact of this study.\nReviewer #4 (Remarks to the Author):\nIn this manuscript, Li et al. (NCOMMS-25-20757-T) demonstrate that targeted editing of the thiamine pyrophosphate (TPP) riboswitch in rice elevates levels of vitamins and phytonutrients, enhancing both normal growth and stress resilience (e.g., cold tolerance, blast resistance). The authors further extend their findings to tomato, reporting improved photosynthetic rates and disease resistance via TPP riboswitch modification. While the study applies gene-editing tools to a biologically relevant target (TPP riboswitch), it remains largely descriptive and incremental. The absence of mechanistic exploration and insufficient validation across environments/varieties fall short of the standards required for publication in Nature Communications. Below are detailed critiques:\n1. The regulatory role of TPP riboswitches in thiamine biosynthesis—via feedback inhibition through the 3′-UTR of THIC—is well-established. Moreover, prior studies have successfully modulated gene expression through editing of promoters, UTRs, and other regulatory elements. Similarly, enhancing stress tolerance by altering vitamin biosynthesis pathways has been extensively documented (Xu et al., 2022; Zheng et al., 2022). The authors fail to articulate how their work transcends these existing paradigms or introduces novel biological insights.\nResponse: We sincerely thank the reviewer for raising this critical point regarding the novelty and broader impact of our study. We fully acknowledge that the regulatory role of TPP riboswitches in feedback inhibition of THIC is well-established, and that previous work has demonstrated the potential of engineering regulatory elements or vitamin pathways to improve stress tolerance. However, our work moves significantly beyond these earlier studies in several key aspects, which we elaborate below:\nFirst, while prior efforts have often focused on enhancing single traits, such as stress tolerance or vitamin content, through targeted genetic manipulation, our study demonstrates that a single, precise edit in the TPP riboswitch enables simultaneous improvement of three major agronomic traits: nutritional quality, yield, and multi-stress resistance. This synergistic enhancement is achieved without the typical trade-offs that commonly hinder conventional breeding strategies. For example, we observed a 5-6 fold increase in thiamine content, a 19-24% increase in grain yield, and significantly enhanced resistance to both biotic (rice blast) and abiotic (cold) stresses—all through one genetic intervention.\nSecond, we provide unprecedented mechanistic depth by integrating multi-omics analyses to uncover how TPP riboswitch editing systemically reprograms plant metabolism and gene expression. Our transcriptomic data revealed that elevated TPP levels upregulate 724 genes common to both TPP-treated and riboswitch-edited plants, which are enriched in pathways central to photosynthesis, nitrogen and amino acid metabolism, lipid and vitamin biosynthesis, and plant-pathogen interactions. Metabolomic and enzymatic analyses further demonstrated that increased TPP levels in riboswitch-edited lines significantly enhance the catalytic activity of TPP-dependent enzymes, including pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase, this regulatory effect subsequently promotes the accumulation of key metabolites such as succinate, chorismate, and several essential amino acids. This systems-level rewiring illustrates how TPP acts not only as a cofactor but as a regulator that coordinately enhances multiple agronomic traits.\nThird, we demonstrate the broad applicability and evolutionary conservation of this mechanism through cross-species validation in tomato. Editing the TPP riboswitch in tomato likewise led to enhanced nutrient profiles, photosynthetic rates, and resistance to Botrytis cinerea and cold stress. This consistency across distantly related species underscores the generalizability of our approach and highlights the TPP riboswitch as a conserved regulatory node suitable for multi-trait crop improvement.\nLastly, through exogenous TPP application experiments, we rigorously established causality: wild type plants treated with TPP phenocopied the key traits observed in the gene edited lines, including enhanced nutrition, disease resistance, and cold tolerance. This confirms that the improvements are directly driven by TPP accumulation, rather than off-target effects or unknown functions of the THIC gene.\nIn conclusion, our work transcends previous research by revealing the unique capacity of the TPP riboswitch to serve as a master regulatory switch that coordinately enhances yield, nutrition, and multiple stress resistance through systematic metabolic and transcriptional reprogramming. We believe these insights open new avenues for developing climate-resilient, nutritionally enhanced crops through targeted riboswitch engineering.\n2.1 The study primarily catalogs phenotypic outcomes without probing the mechanistic basis of observed traits. For instance:\nIn Figure 1a, only trs3 and trs8 mutants exhibit distinct phenotypes. Do other mutants (trs1, trs2, etc.) display consistent or divergent traits? If phenotypic discrepancies exist, are they attributable to mutation type (e.g., frameshift vs. point mutation), editing efficiency, or off-target effects?\nResponse: We thank the reviewer for this important question. We thank the reviewer for this important question. All nine edited lines (trs1 to trs9) showed significantly increased vitamin B1 content compared to the wild type, though the degree of enhancement varied. Among them, trs3 and trs8 exhibited the highest levels of vitamin B1 accumulation. To investigate the mechanism underlying these differences, we performed luciferase reporter assays and found that the luciferase activity driven by different mutant 3′‐UTRs correlated closely with the vitamin B1 levels in the corresponding lines. This result indicates that the extent of release from feedback inhibition differs among the mutants, explaining the variation in vitamin B1 content.\nAt the initial stage of this study, our primary goal was to enhance carbon and nitrogen metabolic flux by elevating vitamin B1 levels to achieve rice with improved nutritional quality. Therefore, we focused on trs3 and trs8, which showed the strongest vitamin B1 enhancement, as the most suitable lines for in-depth phenotypic analysis. Fortunately, these two lines also exhibited significant improvements in multiple traits, including nutrient content, yield, and stress resistance.\nTo verify that the superior performance of trs3 and trs8 was indeed caused by increased vitamin B1, we applied exogenous vitamin B1 to wild-type plants. The results demonstrated enhanced nutritional quality, cold tolerance, and disease resistance in the supplemented plants, confirming that the phenotypic improvements are directly attributable to elevated vitamin B1 levels.\nThus, the selection of trs3 and trs8 for detailed phenotypic characterization was both rational and justified, as these lines not only show the highest vitamin B1 accumulation but also represent a reliable system for dissecting the pleiotropic effects mediated by vitamin B1 elevation.\n2.2 What are the specific genotypes of trs3 and trs8? How do these mutations alter riboswitch structure/function, and how do such changes mechanistically link to vitamin accumulation, stress tolerance, or growth enhancement?\nReference: Wachter, A. et al. Riboswitch Control of Gene Expression in Plants by Splicing and Alternative 3’ End Processing of mRNAs. Plant Cell 19, 3437-3450, doi:10.1105/tpc.107.053645 (2007).\nResponse: Thank you for your detailed and insightful questions regarding the specific genotypes of trs3 and trs8, as well as the mechanisms linking these mutations to vitamin accumulation, stress tolerance, and growth enhancement.\nThe trs3 and trs8 lines are specific gene-edited mutants targeting the TPP riboswitch in the 3’-UTR of the OsTHIC gene. As shown in the figure, the trs3 line has an 11bp deletion in the TPP riboswitch sequence of the 3’-UTR of the OsTHIC gene, which results in the complete or partial loss of the key structural domains P1, P2, and P3 that maintain the negative feedback function of the TPP riboswitch. In contrast, the trs8 line has a 163-bp deletion in the same region, leading to the complete or partial loss of the domains P1, P3, P4, and P5.\nThese deletions disrupt the normal function of the TPP riboswitch, thereby relieving its feedback inhibition on thiamine biosynthesis. In wild-type plants, the TPP riboswitch binds thiamine pyrophosphate (TPP) with high affinity, leading to conformational changes that inhibit the expression of OsTHIC through transcriptional termination or mRNA destabilization. The mutations in trs3 and trs8 directly result in the loss of TPP binding sites, thereby relieving this feedback inhibition. As a result, OsTHIC expression is constitutively upregulated (Table S3), leading to enhanced vitamin B1 biosynthesis and accumulation.\nThe increased TPP levels in riboswitch-edited lines significantly enhance the catalytic activity of TPP-dependent enzymes, including pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase, this regulatory effect subsequently promotes the accumulation of key metabolites such as succinate, chorismate, and several essential amino acids. These metabolic changes provide the biochemical substrates that directly support improved growth and nutritional quality (Fig. 5m-q). Moreover, the elevated TPP levels trigger transcriptional reprogramming in plants, resulting in significant changes in gene expression. KEGG pathway enrichment analysis of the differentially expressed genes indicates their involvement in not only vitamin and primary metabolic pathways but also in critical biological processes, such as photosynthesis, carbon fixation, lipid metabolism, amino acid biosynthesis, and plant–pathogen interactions (Fig. 5h-l).\n2.3 Are the phenotypic improvements in rice and tomato driven directly by elevated thiamine/phytonutrient levels, or are secondary regulatory pathways involved? A multi-omics approach (transcriptomics, metabolomics) could elucidate these connections.\nResponse: We sincerely thank the reviewer for raising this critical question regarding the mechanistic drivers of the observed phenotypic improvements. Our integrated multi-omics data demonstrate that the enhanced traits are not solely a direct consequence of elevated thiamine or phytonutrient levels, but rather the result of a coordinated mechanism involving both primary metabolic changes and extensive secondary transcriptional and metabolic reprogramming initiated by increased cellular TPP availability.\nThe initial and direct effect of riboswitch editing is the significant elevation of the cellular thiamine pyrophosphate (TPP) pool, as quantitatively confirmed in Fig. 5n. This increased cofactor availability directly enhances the enzymatic activity of key TPP-dependent enzymes, including pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (2-OGDH) (Fig. 5o, p), leading to the accumulation of essential metabolites such as the TCA cycle intermediates (e.g., pyruvate, succinate), essential amino acids, and chorismate (Fig. 5q, Table S4). These metabolic changes provide the biochemical substrates that directly support improved growth and nutritional quality.\nConcurrently, our transcriptomic analyses reveal that elevated TPP triggers a system-wide transcriptional reprogramming. We observed substantial changes in gene expression profiles in both trs mutants and TPP-treated plants (Fig. 5h-j), with 724 genes commonly upregulated. KEGG pathway analysis of these genes demonstrated enrichment not only in vitamin and primary metabolic pathways but also in critical processes such as photosynthesis, carbon fixation, lipid metabolism, amino acid biosynthesis, and plant-pathogen interactions (Fig. 5k). This coordinated upregulation of growth and defense-related genes clearly illustrates a profound TPP-induced reconfiguration of the cellular regulatory state beyond direct metabolic effects.\nTherefore, we conclude that the phenotypic improvements are not driven by a single mechanism, but rather emerge from the synergistic interaction between biochemical and transcriptional effects. The elevated TPP pool enhances catalytic activity in central carbon and nitrogen metabolism, while simultaneously functioning as a regulatory factors that reprograms transcription. This dual action amplifies metabolic flux, optimizes resource allocation, and activates stress preparedness pathways. Our multi-omics data confirm that TPP-mediated rewiring at both metabolic and transcriptional levels underlies the coordinated enhancement of yield, nutrition, and stress tolerance, providing a mechanistic basis for breaking crop trade-offs.\n3.1 Rice field trials across diverse geographical locations and multiple growing seasons are necessary to validate stress tolerance claims.\nResponse: We acknowledge the reviewer’s emphasis on the importance of multi season and location trials for validating disease resistance claims. In response, we conducted field evaluations under natural disease pressure in Enshi, Hubei Province, a region with endemic rice blast. During the 2023 summer season, the trs lines showed consistently reduced disease incidence and severity compared to the wild type (Fig. 4a, d, Fig. S7).\nWe recognize that data from one season and location have limitations. Unfortunately, due to permit processing timelines, seasonal crop cycles, and the unpredictable nature of natural disease outbreaks, we were unable to repeat the field trial in Enshi within the revision period. To address this limitation and strengthen our conclusions, we carried out controlled laboratory inoculations using multiple Magnaporthe oryzae strains isolated from different regions of China, including ZB25, ES67, LN3, and LN13. We also employed two distinct infection methods: spray inoculation to mimic natural spore dispersal and punch inoculation to assess localized tissue response. These experiments uniformly confirmed enhanced blast resistance in the trs mutants across all pathogen strains and infection methods (Fig. 4e j, Fig. S8), thus supporting and extending the field observations.\nWhile additional field testing across more seasons and locations would be valuable, the consistency between field performance and controlled laboratory inoculations with multiple pathogen strains isolated from diverse regions provides strong evidence that the improved blast resistance is robust and reproducible.\n3.2 Additionally, overexpression studies of the TPP riboswitch are critical to confirm whether phenotypic effects are dosage-dependent.\nResponse: We thank the reviewer for raising this important point. We agree that overexpression studies would be valuable to further confirm dosage-dependency. However, generating stable overexpression lines requires extensive additional time for transformation, selection, and phenotypic validation, which is not feasible within the revision timeline.\nTo directly test whether the phenotypic enhancements depend on increased TPP dosage within the plant, we instead performed exogenous TPP supplementation experiments in wild-type plants. This treatment significantly increased cellular TPP levels and consistently reproduced key phenotypic improvements, including elevated nutrient content, enhanced cold tolerance, and improved blast resistance, which mirrored the effects observed in the trs mutants. These results demonstrate that the observed agronomic benefits are quantitatively associated with TPP concentration, supporting the conclusion that the traits are dosage-dependent.\n4. In addition, comprehensive phenotypic data of the fully edited tomato plants should be presented.\nResponse: We sincerely thank reviewer for raising the important point regarding the presentation of comprehensive phenotypic data for the fully edited tomato plants. In response to this comment, we have conducted extensive phenotypic and metabolic analyses on the SlTHIC TPP riboswitch-edited tomato lines (sltrs1 and sltrs5), and the results are now included in the revised manuscript (Fig. 6a-z and Table S2). These data provide a holistic view of the multi-trait improvements conferred by TPP riboswitch editing in tomato, mirroring the enhancements observed in rice and underscoring the cross-species applicability of our approach.\nSpecifically, targeted metabolomic profiling of tomato fruits revealed significant increases in a wide range of nutrients, including thiamine, riboflavin, niacin, pantothenic acid, pyridoxine, α-tocopherol, lysophospholipids (lysoPE 18:3 and lysoPC 18:3), and essential amino acids (leucine, threonine, serine, valine) (Fig. 6a-m). These nutritional enhancements are consistent with those observed in rice, indicating a conserved mechanism of vitamin B1-mediated metabolic fortification.\nBeyond nutritional quality, the edited tomato lines exhibited robust improvements in growth and physiological performance. During the vegetative stage, sltrs plants displayed increased biomass and vigor (Fig. 6n). Photosynthetic parameters were significantly enhanced, with net photosynthetic rate (Pn) increasing by 56-77%, transpiration rate (Tr) and stomatal conductance (Gs) also showing marked improvements, while intercellular CO₂ concentration (Ci) remained unchanged (Fig. 6o-r). These results indicate optimized carbon assimilation and water-use efficiency without compromising internal CO₂ regulation.\nFurthermore, the sltrs lines demonstrated strong resistance to Botrytis cinerea under both natural and artificial infection conditions (Fig. 6s-v). Lesion area and disease severity were significantly reduced in the edited plants compared to wild-type controls. Additionally, under low-temperature stress, sltrs plants maintained better morphological integrity, reduced reactive oxygen species (ROS) accumulation (as shown by DAB and NBT staining), and lower electrolyte leakage (Fig. 6w-z), indicating enhanced membrane stability and cold tolerance.\nThese comprehensive phenotypic data firmly establish that TPP riboswitch editing in tomato not only enhances fruit nutritional quality but also improves growth, photosynthetic efficiency, and resilience to both biotic and abiotic stresses. The consistency of these results with those in rice highlights the generality and robustness of our strategy, supporting its potential for broad application in crop improvement."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors well addressed my concerns in the revised manuscript and improved the manuscript greatly. I have no comments more.", "reviewer_2": "We conclude that the authors have addressed our concerns appropriately and endorse publication of the manuscript in its current form.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "In the revised version of the manuscript, Li et al. (NCOMMS-25-20757-T) attempted to improve the work by adding some auxiliary multi-omics analyses, additional phenotypic observations in rice and tomato, limited mechanistic explanations, and even an experiment involving exogenous application of VB1. These supplementary data make certain aspects more detailed, yet the fundamental issues remain unresolved. The study still lacks genuine novelty, fails to provide multi-season and multi-location validation, and does not directly assess the impact of TPP riboswitch overexpression. Overall, the current version appears to be another generic multi-omics plus genome editing study, rather than offering a truly innovative contribution. Considering the rapid advances in genome editing and multi-omics in rice, the present findings are largely incremental and not sufficiently persuasive for publication in Nature Communications.\n1. Although additional datasets were included, the core concept—editing regulatory elements such as the TPP riboswitch or 3'UTRs—has already been widely explored. The manuscript does not establish clear originality or a significant conceptual advance, which is essential for a high-level journal.\n2. The absence of multi-season and multi-location trials raises concerns about reproducibility and applicability. Such validation is particularly important for assessing agricultural relevance. As also highlighted by Nature (“Genetic modification can improve crop yields – but stop overselling it,” https://www.nature.com/articles/d41586-023-02895-w), robust field-based evidence is indispensable for evaluating the real impact of genetic modifications. This reflects a broader consensus in the community that single-season, limited-condition studies are insufficient to claim meaningful agricultural value, and that multi-environment data are necessary to establish both scientific credibility and practical utility. Without these data, the reliability of the reported findings remains limited.\n3. The work still lacks an experiment testing TPP riboswitch overexpression. This is critical for defining its regulatory pathways and phenotypic outcomes. The exogenous VB1 application is only a proxy and cannot replace a genetic approach."}, "author_response": "REVIEWERCOMMENTS\nWe are deeply grateful to Reviewer #4 for their insightful and constructive feedback"}]

Decision letter