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

Li, Yufei; Li, Kang; Lu, Jiazhi; Bulut, Mustafa; Hou, Huanteng; Zaman, Qamar U.; Zhang, Ran; Yang, Zhuang; Yang, Chenkun; Zhan, Chuansong; Wang, Guan; Chen, Tong; Liu, Xianqing; Zhao, Qiao; Shen, Shuangqian; Fernie, Alisdair R.; Luo, Jie

doi:10.1038/s41467-026-69730-4

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Published: 28 February 2026

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

Yufei Li^1,2^na1,
Kang Li^2,3^na1,
Jiazhi Lu¹^na1,
Mustafa Bulut ORCID: orcid.org/0000-0002-6038-518X^4,5^na1,
Huanteng Hou^1,2,
Qamar U. Zaman ORCID: orcid.org/0000-0002-2665-7436¹,
Ran Zhang^1,2,
Zhuang Yang²,
Chenkun Yang¹,
Chuansong Zhan¹,
Guan Wang¹,
Tong Chen^1,2,
Xianqing Liu²,
Qiao Zhao ORCID: orcid.org/0000-0002-0958-4300⁶,
Shuangqian Shen¹,
Alisdair R. Fernie ORCID: orcid.org/0000-0001-9000-335X⁴ &
…
Jie Luo ORCID: orcid.org/0000-0002-4931-7469^1,2

Nature Communications volume 17, Article number: 3328 (2026) Cite this article

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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.

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Introduction

Food security, defined as the sustainable access to sufficient and nutritious food, is a fundamental pillar of global health and socioeconomic stability^1,2. 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 productivity^3,4,5. Current strategies to address food security prioritize dual objectives: enhancing crop yields under both optimal and adverse conditions and improving nutritional quality^6,7,8. 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 consequences^19,20,21. 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 pathways^25,26. 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 disorders^27,28,29. 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 Arabidopsis^{30,31,32,33,34}. 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 biosynthesis^37,38. 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 T₂ 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).

**Fig. 1: Increased contents of multiple nutrients in the TPP riboswitch gene edited lines.**

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).

Fig. 2: trs rice lines showed increased yield related parameters. — **Fig. 2: *trs* rice lines showed increased yield related parameters.**

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).

**Fig. 3: The gene-edited TPP riboswitch improves photosynthetic capacity and nitrogen utilization.**

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).

**Fig. 4: The gene-edited TPP riboswitch increased rice blast disease and cold resistance.**

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 plants^41,42,43. 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, 4 l). 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).

**Fig. 5: Multidimensional regulation of TPP riboswitch editing drives crop enhancement.**

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’-UTR^WT) and edited lines (M1-M9, corresponding to 3’-UTR^trs1 to 3’-UTR^trs9) 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’-UTR^WT. The 3’-UTR^trs3 and 3’-UTR^trs8 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 1 mM TPP treatment. TPP treatment in WT tiggered the upregulation of 5842 genes and the downregulation of 1350 genes (Fig. 5h, Supplementary Data 3). Compared with ZH11, the trs3 and trs8 lines shared 2346 upregulated and 3536 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 signaling^42,45,46, 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).

**Fig. 6: The gene-edited TPP riboswitch improves quality, photosynthesis and stress tolerance in tomato.**

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.

Discussion

Our study demonstrated that targeted editing of the TPP riboswitch in the 3’-UTR of THIC represents a highly effective strategy for simultaneously improving nutritional quality, yield, and stress tolerance in crops. We observed that CRISPR-Cas9-induced mutations in the riboswitch region led to a significant increase in vitamin B1 content in rice grains-up to 5.5 mg kg^-1, which is substantially higher than the typical concentration of 1 mg kg^-1 found in conventional polished rice. This elevation implies that approximately 300 g of rice from the edited lines would suffice to meet the daily recommended intake of vitamin B1 for adults, offering a practical solution to mitigate deficiencies that affect nearly two billion people worldwide^47,48. In addition to thiamine, we documented broad-spectrum enhancements in other essential nutrients, including B vitamins (riboflavin, niacin, pantothenic acid, pyridoxine), α-tocopherol (vitamin E), phospholipids, and multiple essential amino acids. All of these compounds are highly important for human health⁴⁹. This multinutrient fortification effectively addresses the complex nature of “hidden hunger,” which often involves concurrent deficiencies in several micronutrients.

The agronomic benefits of the riboswitch edits were equally remarkable. Field trials conducted across diverse environments (Wuhan and Sanya) have consistently shown yield increases of 19.29–20.77% in edited lines, primarily attributable to improved panicle architecture—longer panicles with more grains—rather than changes in tiller number or grain weight. Importantly, these gains were achieved without compromising nitrogen use efficiency; under low-nitrogen conditions, the edited lines maintained superior growth, chlorophyll content, and biomass accumulation, indicating a more efficient metabolic platform. These phenotypic improvements are underpinned by significant enhancements in photosynthetic parameters, including higher net photosynthetic rates (Pn), maximal photochemical efficiency (Fv/Fm), and electron transport rates (ETRs). We propose that increased TPP availability enhances the activity of key enzymes in the Calvin–Benson and TCA cycles, thereby promoting carbon fixation and energy metabolism.

A key finding of this study is the concurrent increases in of biotic and abiotic stress tolerance in riboswitch-edited lines. We observed strong resistance to rice blast (Magnaporthe oryzae) under both field and laboratory conditions, with reduced lesion formation and fungal biomass accumulation. This finding is consistent with the established role of thiamine as a priming agent for systemic acquired resistance^30,34. Similarly, the edited lines exhibited pronounced cold tolerance, which was likely mediated by reduced membrane damage (as indicated by lower electrolyte leakage) and decreased reactive oxygen species (ROS) accumulation under low-temperature stress. These results align with transcriptomic data showing the upregulation of genes involved in pathogen response, chloroplast function, and metabolic pathways (such as like lipid, and amino acid metabolism) related to membrane maintenance.

At the molecular level, our multi-omics analyses revealed that the riboswitch edits triggered extensive transcriptional and metabolic reprogramming. RNA-seq profiling revealed the upregulation of genes associated with vitamin metabolism, photosynthesis, amino acid and lipid biosynthesis, and stress responses, collectively supporting the observed phenotypic advantages. Importantly, enzymatic assays confirmed the increased activities of TPP-dependent enzymes such as pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (2-OGDH), corroborating the role of TPP as a central metabolic coordinator. The resulting shifts in metabolite pools, including elevated levels of pyruvate, succinate, chorismate, essential amino acids, and the second messenger cAMP—further illustrate the profound metabolic rewiring induced by increased TPP availability.

Our mutational analysis revealed a clear structure-function relationship in the TPP riboswitch regulatory mechanism of the TPP riboswitch. The large fragment complete deletion of the riboswitch sequence in trs8 and the specific mutation in trs3 resulted in the strongest constitutive expression, indicating near-complete loss of TPP responsiveness. The other mutant lines exhibited graded responses corresponding to their specific alterations: trs7 (6 bp deletion), trs5 (2 bp deletion), trs4 (4 bp deletion), trs2 (3 bp deletion), trs6 (241 bp insertion), and trs1 (1 bp deletion) showed progressively weaker effects on luciferase activity. This stepwise pattern of reporter activity directly correlates with the extent of structural perturbation to the conserved aptamer domain. The consistent structure-activity relationship across the mutant series provides strong evidence that the phenotypic differences arise from specific, targeted alterations to the riboswitch rather than nonspecific genetic changes, demonstrating that even single-nucleotide alterations can significantly impair TPP binding and regulatory function.

Notably, we also validated the cross-species applicability of this approach by editing the homologous riboswitch in tomato. The resulting mutants exhibited similar pleiotropic benefits, including elevated nutrient levels, improved photosynthetic performance, and enhanced resistance to Botrytis cinerea. This consistency across distantly related species underscores the evolutionary conservation of TPP riboswitch function and suggests broad utility in dicot and monocot crops.

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 vigour.

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 evaluations of long-term agronomic performance, environmental impact, and socioeconomic feasibility.

In conclusion, our work establishes riboswitch-mediated metabolic rewiring as a powerful strategy for breaking the yield–quality–resilience trade-off in crops. By elevating cellular TPP levels through a single genetic intervention, we achieved coordinated improvements in nutrition, productivity, and stress adaptation—a triple benefit that has proven elusive in conventional breeding and transgenic approaches. We propose that this strategy could be extended to other vitamins and metabolic pathways, offering a general paradigm for the next generation of biofortified, climate-resilient crops.

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 (SCAA-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 analysis^52,53.

Metabolites quantification

For metabolites content analysis, samples were analysed using a high-performance liquid chromatography (HPLC) staple targeted method^54,55. 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:

$${{\rm{Fv}}}/{{\rm{Fm}}}=({{\rm{Fm}}}-{{\rm{Fo}}})/{{\rm{Fm}}}$$

(1)

$${{\rm{Y}}}({{\rm{II}}})=({{\rm{Fm}}}{{\mbox{'}}}-{{\rm{Fs}}})/{{\rm{Fm}}}{{\mbox{'}}}$$

(2)

$${{\rm{Y}}}({{\rm{NO}}})={{\rm{Fs}}}/{{\rm{Fm}}}$$

(3)

$${{\rm{NPQ}}}=({{\rm{Fm}}}-{{\rm{Fm}}}{{\mbox{'}}})/{{\rm{Fm}}}{{\mbox{'}}}$$

(4)

$${{\rm{qP}}}=({{\rm{Fm}}}{{\mbox{'}}}-{{\rm{Fs}}})/({{\rm{Fm}}}{{\mbox{'}}}-{{\rm{Fo}}}{{\mbox{'}}})$$

(5)

$${{\rm{ETR}}}={{\rm{Y}}}({{\rm{II}}})\times {{\rm{PAR}}}\times 0.84\times 0.5$$

(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:

$${{\rm{Electrolyte\; leakage}}}=({{\rm{R}}}1)/({{\rm{R}}}1+{{\rm{R}}}2)$$

(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 technology⁵⁹. 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.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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 and OMIX009554, respectively. Source data are provided with this paper.

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Acknowledgements

This work was supported by the key project of regional joint fund of the National Natural Science Foundation of China (U22A20476), Basic Research Project in 2023-2310LJ01 of Yazhouwan National Laboratory, the National Natural Science Foundation of China (32401887, 32560151, 32300452, 32500233, 32501942), Science and Technology Innovation 2030 Major Project (2024ZD0408004), the Project of National Key Laboratory for Tropical Crop Breeding (NO. PT2400008492), “111” Project (No. D20024). We thank Guotian Li from Huazhong Agricultural University and Gan Sha from South China Agricultural University for offering the M. oryzae strains ES67, LN3 and LN13. We also extend our sincere thanks to Chuanying Fang from Hainan University for his valuable guidance on the research concept.

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These authors contributed equally: Yufei Li, Kang Li, Jiazhi Lu, Mustafa Bulut.

Authors and Affiliations

Yazhouwan National Laboratory, Sanya, China
Yufei Li, Jiazhi Lu, Huanteng Hou, Qamar U. Zaman, Ran Zhang, Chenkun Yang, Chuansong Zhan, Guan Wang, Tong Chen, Shuangqian Shen & Jie Luo
School of Breeding and Multiplication (Sanya Institute of Breeding and Multiplication), Hainan University, Sanya, China
Yufei Li, Kang Li, Huanteng Hou, Ran Zhang, Zhuang Yang, Tong Chen, Xianqing Liu & Jie Luo
School of Tropical Agriculture and Forestry, Hainan University, Danzhou, China
Kang Li
Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany
Mustafa Bulut & Alisdair R. Fernie
Program Center MetaCom, Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany
Mustafa Bulut
Shenzhen Key Laboratory of Synthetic Genomics, Guangdong Provincial Key Laboratory of Synthetic Genomics, Key Laboratory of Quantitative Synthetic Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China
Qiao Zhao

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Contributions

J.L. and AR.F. conceived the project and supervised this study. Y.L., K.L., J.Z.L., M.B. and S.S. performed the experiments; H.H., QU.Z., R.Z., C.Y., C.Z., G.W., T.C., and X.L. prepared the materials; Y.L., K.L., J.Z.L, S.S., Z.Y., and J.L. analysed the data; Y.L., K.L., J.Z.L, S.S., AR.F., Q.Z., and J.L. wrote and revised the manuscript. All authors discussed the results and commented on the manuscript. All authors read and approved of its content.

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Correspondence to Shuangqian Shen, Alisdair R. Fernie or Jie Luo.

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Li, Y., Li, K., Lu, J. et al. Modulating TPP riboswitch activity simultaneously enhances crop yield, nutritional quality and stress tolerance. Nat Commun 17, 3328 (2026). https://doi.org/10.1038/s41467-026-69730-4

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Received: 17 March 2025
Accepted: 09 February 2026
Published: 28 February 2026
Version of record: 09 April 2026
DOI: https://doi.org/10.1038/s41467-026-69730-4