Overexpression of ZmSPS2 increases α/γ‐tocopherol ratio to improve maize nutritional quality

Severe vitamin E deficiency causes ataxia, neuropathy, anaemia and other health conditions, and inadequate vitamin E status is prevalent in healthy population (Malik et al., 2021). Meanwhile, global food production falls short in delivering sufficient vitamin E, resulting in a nutrient gap of 31% (Smith et al., 2021). Although various tocochromanol isoforms are found in crop seeds, only α-tocopherol exhibits the highest biological activity and liver tissue concentration (Traber, 2024). However, crop tend to accumulate abundant γ-tocopherol and α-tocopherol content is lower than that of γ-tocopherol (Mène-Saffrané and Pellaud, 2017). Therefore, exploring new genes to enhance α-tocopherol content and α/γ-tocopherol ratio in staple crop is attractive. As a globally significant staple crop, maize (Zea mays L.) provides abundant tocopherols for enhancing human health. The biosynthesis of α-tocopherol regulated by two key enzymes ZmVTE1 and ZmVTE4 in maize (Li et al., 2012; Sattler et al., 2003). In our previous study, we identified a quantitative trait locus (QTL) within the umc1177–bnlg1429 interval on chromosome 1 that contributes to the highest α/γ-tocopherol ratio (41.16%) in sweet corn (Feng et al., 2013). ZmSPS2 (Zm00001d027694, named according to the genome annotation 'Solanesyl diphosphate synthase 2 chloroplastic'), located in this genomic region (Table S1), is co-expressed with vitamin E biosynthesis genes (ZmVTE1 and ZmVTE4) (Tables S2, S3). Furthermore, the expression profile of ZmSPS2 is consistent with changes in α/γ-tocopherol ratio during the kernel development (Figure 1a). In addition, three ZmSPS2 homologues with complete conserved domain were obtained in maize (Figure S1, Table S4). And the expression profile of these SPS2 homologues is not correlated with changes in α/γ-tocopherol ratio during the kernel development (Figure S2). These findings suggest the possibility of modulating α/γ-tocopherol ratio through ZmSPS2. In the present study, both maize mutants and overexpression lines were obtained; subsequently, the tocopherol contents compared to the wild-type plants were explored. We obtained the transposon insertion mutants (UFMu-13 105, UFMu-7763) via MaizeGDB, referred as mu-1 and mu-2. The expression of mutants was assessed using RT-qPCR (Figure S3). The α-tocopherol and γ-tocopherol contents were determined by liquid chromatography coupled with mass spectrometry (LC-MS/MS). Compared to the wild-type W22, the contents of γ-tocopherol and total tocopherols increased significantly in mutant kernels, while α-tocopherol contents are not changed in the two mutant lines (Figure 1b). Moreover, α/γ-tocopherol ratio decreased by 37–42% in mutant kernels. This finding indicated that knockdown ZmSPS2 negatively regulates α/γ-tocopherol ratio and boosts γ-tocopherol accumulation. Therefore, we generated the overexpression lines in the background of inbred maize line B104. And the expression levels of two transgenic lines were validated by RT-qPCR (Figure S4). LC-MS/MS analysis showed that content of α-tocopherol increased 1.45–1.54-fold in the transgenic kernels compared to the wild type, while γ-tocopherol content was decreased to 63–78% (Figure 1c). Interestingly, there was no significant difference in total tocopherols between ZmSPS2 overexpression and wild-type plants. Additionally, the α/γ-tocopherol ratio was found to be elevated 1.85- to 2.44-fold in the transgenic lines (Figure 1c). We further investigated the natural variation in ZmSPS2 gDNA sequence across over 295 maize inbred lines. Two major haplotypes of ZmSPS2 in the coding region were identified (Figure 1d). To further investigate whether the haplotype differences affect the α/γ-tocopherol ratio and α-tocopherol accumulation, we examined the tocopherol contents among five Hap1 and five Hap2 lines. High α/γ-tocopherol ratio was detected in Hap2 lines (Figure 1e). The average level of α-tocopherol was high in Hap2 lines, and γ-tocopherol was low in Hap2 lines (Figure 1f). Especially, high α-tocopherol percentage was detected in Hap2 lines (Figure S5). Thus, Hap2 was identified as the elite haplotype that associated with the high α/γ-tocopherol ratio, which could serve as a potential target allele to breed varieties with enhanced α-tocopherol content for improving maize nutritional quality. Phytyl diphosphate is one of the important precursors for tocopherol biosynthesis (Figure S6), and the manipulation of phytyl diphosphate supply can change tocopherol accumulation. Chlorophyll breakdown provides free phytol for phytyl diphosphate supply (Figure S6). Protochlorophyllide oxidoreductase B (PROB) catalyses chlorophyllide a for chlorophyll turnover and breakdown. Previous studies showed that overexpression ZmPROB2 shows a moderate increase of total tocopherol contents (Zhan et al., 2019), and Zmprob1 knockdown decreases γ-tocopherol slightly in the maize kernels (Liu et al., 2024). These results imply that enhancing the precursor biosynthesis or blocking the competing metabolic branches can enhance γ-tocopherol accumulation, which might be due that γ-tocopherol is the most abundant tocopherol component in the maize kernels. In addition, phytyl diphosphate is alternatively origin from geranylgeranyl-diphosphate by geranylgeranyl diphosphate reductase (Figure S6). In our results, ZmSPS2 has the complete PLN02857 (octaprenyl-diphosphate synthase) conserved domain (Table S4), which might catalyse geranylgeranyl-diphosphate to form solanesyl diphosphate (a C45 side chain) for the plastoquinone-9 (PQ9) biosynthesis. Although the potential substrate competition occurs, α-tocopherol content increased in the ZmSPS2 overexpression lines (Figure 1c), which is inconsistent with previous studies that altering tocopherol content through manipulation of phytyl diphosphate supply. Furthermore, the PQ9 pathway is parallel with tocopherol biosynthesis, and these two pathways share VTE3 and VTE1 (Figure S6). However, tocopherol content is just modestly deceased in the embryo of Zmhst1 mutant (Hunter et al., 2018), which is the first and committed gene in the PQ9 pathway. Thus, blocking PQ9 pathway is not sufficient to increase tocopherol accumulation, especially to increase α-tocopherol accumulation and α/γ-tocopherol ratio in maize kernels. We found that the expression of ZmVTE4 is not significantly changed in both the mutant and transgenic lines compared with their WT plants (Figure S7). Therefore, we favour that potential competing metabolic flux might have an impact in boosting tocopherol accumulation in ZmSPS2 transgenic plants, but it is not the dominant one. We further tested the methyltransferase reaction from γ-tocopherol to α-tocopherol by the purified ZmVTE4 and the additional ZmSPS2 protein in vitro. Results showed that ZmSPS2 significantly increases the enzyme activity of ZmVTE4 (Figure S8). The detailed mechanism of ZmSPS2 to increase the α-tocopherol accumulation and α/γ-tocopherol ratio in maize kernels remains to be further elucidated in the future. In summary, we demonstrated that ZmSPS2 regulated the α/γ-tocopherol ratio for enhancing α-tocopherol content in maize, and overexpression of ZmSPS2 resulted in an increase in α-tocopherol content and high α/γ-tocopherol ratio. Furthermore, our results also provide the elite haploid of ZmSPS2 for maize nutritional quality breeding. We are thankful to Prof. Wang Zhoufei for suggestions and Dr. Yang Chong for LC-MS/MS technique supports. This work was supported by the Natural Science Foundation of Guangdong Province (Grant Number: 2022A1515010707). No conflict of interests to declare. F.F. and B.W. contributed to the project design. Y.Y., Q.Y., D.L. and J.Y. performed the experiments and data analysis. K.L. provide the technique supports for haploid analysis. F.F. and B.W. wrote the manuscript and revised the article. The data that supports the findings of this study are available in the supplementary material of this article. Figure S1 Phylogeny tree of ZmSPS candidates using the amino acid sequences. Figure S2 Expression of other three ZmSPSs during the kernel development. Figure S3 Expression of ZmSPS2 in the mutant lines. Figure S4 Expression of ZmSPS2 in the overexpression maize lines. Figure S5 Percentage of α-tocopherol and γ-tocopherol in kernels of 10 maize inbred lines. Figure S6 Phytyl diphosphate supply pathway and the parallel pathways for plastoquinone-9 and tocopherol biosynthesis. Figure S7 Expression of ZmVTE4 in the mutant and overexpression maize lines. Figure S8 The effect of ZmSPS2 protein on the enzyme activity of ZmVTE4. Table S1 Candidate genes in the target QTL intervals on chromosome 1. Table S2 Top100 coexpressed genes with ZmVTE1 and ZmVTE4. Table S3 Top100 coexpressed genes with ZmVTE1. Table S4 Sequences analysis of ZmSPS2 and its homologues. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.