Selenium Biofortification in Maize Kernels by Knockout of a Nuclear Localised Serine Hydroxymethyltransferase

Selenium (Se) is an essential micronutrient for humans. Se deficiency often occurs in human diets and affects approximately one billion people worldwide (Tan et al. 2002), elevating risks of impaired antioxidant function, disrupted thyroid hormone metabolism, compromised immune responses, and heightened cancer susceptibility (Harthill 2011). The risk of Se deficiency is particularly high in sub-Saharan Africa, where diets rely heavily on maize—a staple crop providing over 30% of daily calories—yet maize kernels generally contain low Se levels (Gashu et al. 2021). Although conventional strategies such as nutritional supplementation, dietary diversification and commercial food fortification have been employed to improve micronutrient intake, such implementation in developing countries has faced persistent limitations because of economic constraints and low public acceptance. In contrast, genetic biofortification of crops to enrich mineral nutrients in edible tissues, such as maize kernels, offers a more sustainable and cost-effective strategy. However, previous efforts to increase Se accumulation in maize kernels through biofortification have achieved limited success. Selenate is the primary inorganic form of Se absorbed by plants, sharing uptake, assimilation and metabolic pathways with sulfate (Sors et al. 2005). We previously showed that knockout of nuclear localised serine hydroxymethyltransferases MSA1/AtSHMT7 in Arabidopsis thaliana or OsCADT1/OsSHMT4 in rice activates sulfur (S) deficiency response and enhances S and Se accumulation (Chen et al. 2020; Huang et al. 2016). To evaluate the potential for Se biofortification in maize, we characterised the orthologous enzyme serine hydroxymethyltransferase 5 (ZmSHMT5). Phylogenetic analysis revealed ZmSHMT5 shares 82.09% amino acid identity with OsCADT1 and 65.29% with MSA1 (Figure 1a; Table S1). ZmSHMT5 was closely related to ZmSHMT3 and shared 95.55% amino acid identity and 96.41% similarity (Figure S1a). Transient expression of ZmSHMT5–eYFP in Nicotiana benthamiana leaves or in maize leaf protoplasts confirmed its nuclear localization (Figure 1b; Figure S2; Appendix S1). Complementation of the Arabidopsis msa1-1 mutant with ZmSHMT5 driven by the native MSA1 promoter fully restored shoot S and Se levels to wild-type (WT, Col-0) levels (Figure 1c,d), suggesting the conserved function of ZmSHMT5 with MSA1. We obtained two independent zmshmt5 mutants in the inbred line B37 background (Liang et al. 2019), harbouring maize Mutator insertions in the 5′-UTR and first exon of ZmSHMT5, respectively (Figure 1e). Quantitative RT-PCR confirmed the significant reduction of ZmSHMT5 expression in both roots and shoots of zmshmt5 mutants, but knockout of ZmSHMT5 had no effect on ZmSHMT3 expression in either roots or shoots (Figure 1f; Figure S1b; Table S2). At the seedling stage, zmshmt5 mutants exhibited a growth phenotype comparable to WT (Figure 1g; Figure S3a–c). Ionomic profiling analysis revealed that total S concentrations in the shoots of zmshmt5 were increased to 117%–127% compared to the WT (Figure 1h). Similarly, zmshmt5 accumulated 160%–162% higher total Se in shoots than the WT (Figure 1i). The root S/Se concentrations remained unaffected in zmshmt5 (Figure 1h,i), indicating that enhanced accumulation of S/Se only occurs in aboveground tissues. There is no significant change for other elements in zmshmt5 except for the reduction of molybdenum in roots (Figure S4). Sulfur-containing metabolite analysis revealed significantly elevated concentrations of sulfate, cysteine and glutathione in mutant roots compared to WT, with respective increases of approximately 66%, 43% and 140% (Figure 1j–l). The concentration of sulfate and glutathione in the shoots of zmshmt5-1 and zmshmt5-2 was also significantly increased (to 1.4-fold and 1.5-fold, respectively), whereas shoot cysteine concentration was unchanged (Figure 1j–l). Se speciation analysis demonstrated significant increases in selenate (123%–150%), selenocystine (SeCys2; 179%–190%) and methyl-selenocysteine (MeSeCys; 146%–192%) in zmshmt5 shoots, with no changes in root Se metabolites or shoot selenomethionine (SeMet) (Figure 1m–p). Gene expression analysis indicated that several genes involved in S/Se uptake and assimilation were upregulated in zmshmt5. The gene expressions of sulfate transporter genes ZmSULTR1;1 and ZmSULTR3;4 were increased to 2 to 4-fold in the roots of zmshmt5 and the expression of ZmSULTR1;2 was increased to 13-fold in the shoots of zmshmt5-1 and 3-fold in zmshmt5-2 (Figure 1q; Figure S5). Expressions of adenosine 5′-phosphosulfate reductase (APR), which represents the rate-limiting step in the S/Se assimilation pathway, were also increased in zmshmt5, including the 4 – 7-fold upregulation of the APR2 gene in the roots (Figure 1q). These results suggested that the mutation of ZmSHMT5 enhances S/Se accumulation through transcriptional activation of S/Se uptake and assimilation. Field trials were conducted to assess the impact of ZmSHMT5 mutation on Se accumulation in maize kernels. Field-grown zmshmt5 plants exhibited no morphological differences from WT (Figure 1r) but showed a modest reduction in ear weight and 100-kernel weight compared to WT plants (Figure S6a,b). Ionomic profiling analysis demonstrated that kernel Se content increased to 2.8-fold in zmshmt5-1 and to 1.2-fold in zmshmt5-2 compared to WT (Figure 1s). The total S concentration was also significantly higher in kernels of zmshmt5-1 but not in zmshmt5-2 (Figure 1t), which may be due to distinct Mutator transposon insertion sites that cause differential residual expression and transcript structures (Ellison et al. 2023). Concentrations of other elements were not consistently changed in the kernels of zmshmt5 (Figure S7). No significant difference in total Se and S concentrations was observed in the kernels of WT sibling control plants without the Mutator transposon insertion (Figure S8a,b). Previous studies have reported the disruption of OsSHMT4/OsCADT1 resulting in alteration of storage protein biosynthesis and amino acid composition in rice (Chen et al. 2020; Matsusaka et al. 2021; Yan et al. 2022). Amino acid profile analysis suggested that concentrations of multiple amino acids were significantly increased in the kernels of zmshmt5 compared to the WT, including aspartic acid, threonine, serine, glutamic acid, alanine, valine, isoleucine, leucine, tyrosine and phenylalanine (Figure 1u). The protein content in kernels of two zmshmt5 mutants was also significantly higher than that of the WT (Figure S6c). In conclusion, our study demonstrated that knockout of the nuclear localised ZmSHMT5 enhances S and Se accumulation in shoots and kernels, concurrently improving kernel amino acid composition. These results establish ZmSHMT5 as a novel genetic target for genetic engineering and molecular breeding towards Se biofortification in maize, offering a dual benefit of Se enrichment and nutritional quality improvement. Notably, this work provides unique insights into maize-specific metabolic regulation of S and Se homeostasis, emphasising the translational potential of manipulating ZmSHMT5 to biofortify a globally important staple crop with critical micronutrients. X.-Y.H., F.-J.Z. and J.C. conceived and designed the project; J.C. and T.-Y.Y. performed the experiments; J.C. and X.-Y.H. prepared the manuscript with contribution from F.-J.Z. This work was supported by the Natural Science Foundation of China (32202592), the Natural Science Foundation of Jiangsu Province (BK20210389), the Hainan Provincial Natural Science Foundation of China (325CXTD613) and the China Postdoctoral Science Foundation (2021M691612). The data that supports the findings of this study are available in the Supporting Information of this article. Appendix S1–S2. Figures S1–S8. Tables S1–S2. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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