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.

严重的维生素 E 缺乏会导致共济失调、神经病变、贫血和其他健康状况，维生素 E 状态不足在健康人群中普遍存在（Malik 等 人，2021 年）。与此同时，全球粮食生产无法提供足够的维生素 E，导致营养缺口达到 31%（Smith 等 人，2021 年）。尽管在作物种子中发现了各种生育色胺醇亚型，但只有 α-生育酚表现出最高的生物活性和肝组织浓度（Traber，2024）。然而，作物往往会积累丰富的γ-生育酚，并且α-生育酚含量低于γ-生育酚（Mène-Saffrané 和 Pellaud，2017）。因此，探索提高主食作物α-生育酚含量和 α/γ-生育酚比值的新基因具有吸引力。

玉米 （Zea mays L.） 作为全球重要的主食作物，为增强人类健康提供了丰富的生育酚。玉米中受两种关键酶 ZmVTE1 和 ZmVTE4 调控的 α-生育酚的生物合成（Li et al.， 2012;Sattler et al.， 2003）。在我们之前的研究中，我们在 1 号染色体上的 umc1177-bnlg1429 区间内确定了一个数量性状位点 （QTL），该基因位点有助于甜玉米中最高的 α/γ-生育酚比率 （41.16%） （Feng et al.， 2013）。ZmSPS2 （Zm00001d027694，根据基因组注释“Solanesyl diphosphate synthase 2 chloroplastic”命名）位于该基因组区域（表 S1），与维生素 E 生物合成基因（ZmVTE1 和 ZmVTE4）共表达（表 S2、S3）。此外，ZmSPS2 的表达谱与内核发育过程中 α/γ-生育酚比率的变化一致（图 1a）。此外，在玉米中获得三个具有完全保守结构域的 ZmSPS2 同源物（图 S1，表 S4）。这些 SPS2 同源物的表达谱与内核发育过程中 α/γ-生育酚比率的变化无关（图 S2）。这些发现表明通过 ZmSPS2 调节 α/γ-生育酚比率的可能性。在本研究中，获得了玉米突变体和过表达系;随后，与野生型植物相比，探索了生育酚含量。

我们通过 MaizeGDB 获得了转座子插入突变体 （UFMu-13 105， UFMu-7763），称为 mu-1 和 mu-2。使用 RT-qPCR 评估突变体的表达 （图 S3）。采用液相色谱-质谱联用 （LC-MS/MS） 测定 α-生育酚和 γ-生育酚含量。与野生型 W22 相比，突变型籽粒中 γ-生育酚和总生育酚的含量显著增加，而α-生育酚含量在两个突变型品系中未发生变化（图 1b）。此外，突变籽粒中 α/γ-生育酚比率降低了 37-42%。这一发现表明，敲低 ZmSPS2 负向调节 α/γ-生育酚比率并促进 γ-生育酚积累。因此，我们在自交玉米品系 B104 的背景中生成了过表达系。并通过 RT-qPCR 验证两个转基因株系的表达水平 （图 S4）。LC-MS/MS 分析表明，与野生型相比，转基因籽粒中 α-生育酚的含量增加了 1.45-1.54 倍，而γ-生育酚含量降低到 63-78%（图 1c）。有趣的是，ZmSPS2 过表达和野生型植物之间的总生育酚没有显著差异。此外，发现转基因品系中的 α/γ-生育酚比率升高了 1.85 至 2.44 倍（图 1c）。

我们进一步研究了超过 295 个玉米自交系中 ZmSPS2 gDNA 序列的自然变异。在编码区确定了 ZmSPS2 的两种主要单倍型（图 1d）。为了进一步研究单倍型差异是否影响 α/γ-生育酚比率和 α-生育酚积累，我们检测了 5 个 Hap1 和 5 个 Hap2 系的生育酚含量。在 Hap2 系中检测到高 α/γ-生育酚比率 （图 1e）。α-生育酚的平均水平在 Hap2 系中较高，而 γ-生育酚在 Hap2 系中较低（图 1f）。特别是，在 Hap2 系中检测到高 α-生育酚百分比 （图 S5）。因此，Hap2 被确定为与高 α/γ-生育酚比率相关的精英单倍型，可作为潜在的靶等位基因，培育出 α-生育酚含量更高的品种，以改善玉米营养品质。

植酰二磷酸是生育酚生物合成的重要前体之一（图 S6），植酰二磷酸供应的操纵可以改变生育酚的积累。叶绿素分解为叶酰二磷酸供应提供游离植物醇（图 S6）。原叶绿素氧化还原酶 B （PROB） 催化叶绿素 a 进行叶绿素转换和分解。先前的研究表明，过表达 ZmPROB2 显示总生育酚含量适度增加（Zhan et al.， 2019），并且 Zmprob1 敲低使玉米粒中 γ-生育酚略有降低（Liu et al.， 2024）。这些结果表明，增强前体生物合成或阻断竞争性代谢分支可以增强γ-生育酚的积累，这可能是由于 γ-生育酚是玉米粒中最丰富的生育酚成分。此外，植酰二磷酸可通过香叶基香叶基二磷酸还原酶从香叶基香叶基二磷酸来源（图 S6）。在我们的结果中，ZmSPS2 具有完整的 PLN02857 （八肾酰二磷酸合酶） 保守结构域 （表 S4），它可能催化香叶基香叶基二磷酸形成茄兰酰二磷酸 （C45 侧链） 用于质体醌-9 （PQ9） 生物合成。尽管发生了潜在的底物竞争，但 ZmSPS2 过表达系中的α-生育酚含量增加（图 1c），这与先前通过操纵植酰二磷酸供应改变生育酚含量的研究不一致。

此外，PQ9 通路与生育酚生物合成平行，这两个通路共享 VTE3 和 VTE1（图 S6）。然而，生育酚含量在 Zmhst1 突变体的胚胎中仅适度死亡 （Hunter et al.， 2018），这是 PQ9 通路中的第一个定型基因。因此，阻断 PQ9 途径不足以增加生育酚积累，尤其是增加玉米α-生育酚积累和 α/γ-生育酚比率。我们发现，与它们的 WT 植物相比，ZmVTE4 的表达在突变体和转基因品系中都没有显着变化（图 S7）。因此，我们倾向于潜在的竞争性代谢通量可能对促进 ZmSPS2 转基因植物中的生育酚积累产生影响，但它不是主要的。我们进一步在体外通过纯化的 ZmVTE4 和额外的 ZmSPS2 蛋白测试了 γ-生育酚到 α-生育酚的甲基转移酶反应。结果表明，ZmSPS2 显着增加 ZmVTE4 的酶活性（图 S8）。ZmSPS2 增加玉米粒中 α-生育酚积累和 α/γ-生育酚比值的详细机制仍有待进一步阐明。

综上所述，我们证明 ZmSPS2 调节 α/γ-生育酚比值以提高玉米α-生育酚含量，过表达 ZmSPS2 导致 α-生育酚含量增加和 α/γ-生育酚比值升高。此外，我们的结果还为玉米营养品质育种提供了 ZmSPS2 的优良单倍体。