Effect of Overexpression of γ-Tocopherol Methyltransferase on α-Tocopherol and Fatty Acid Accumulation and Tolerance to Salt Stress during Seed Germination in Brassica napus L.

Rapeseed (Brassica napus L.) is an important oil crop and a major source of tocopherols, also known as vitamin E, in human nutrition. Enhancing the quality and composition of fatty acids (FAs) and tocopherols in seeds has long been a target for rapeseed breeding. The gene γ-Tocopherol methyltransferase (γ-TMT) encodes an enzyme catalysing the conversion of γ-tocopherol to α-tocopherol, which has the highest biological activity. However, the genetic basis of γ-TMT in B. napus seeds remains unclear. In the present study, BnaC02.TMT.a, one paralogue of Brassica napus γ-TMT, was isolated from the B. napus cultivar “Zhongshuang11” by nested PCR, and two homozygous transgenic overexpression lines were further characterised. Our results demonstrated that the overexpression of BnaC02.TMT.a mediated an increase in the α- and total tocopherol content in transgenic B. napus seeds. Interestingly, the FA composition was also altered in the transgenic plants; a reduction in the levels of oleic acid and an increase in the levels of linoleic acid and linolenic acid were observed. Consistently, BnaC02.TMT.a promoted the expression of BnFAD2 and BnFAD3, which are involved in the biosynthesis of polyunsaturated fatty acids during seed development. In addition, BnaC02.TMT.a enhanced the tolerance to salt stress by scavenging reactive oxygen species (ROS) during seed germination in B. napus. Our results suggest that BnaC02.TMT.a could affect the tocopherol content and FA composition and play a positive role in regulating the rapeseed response to salt stress by modulating the ROS scavenging system. This study broadens our understanding of the function of the Bnγ-TMT gene and provides a novel strategy for genetic engineering in rapeseed breeding.


Introduction
Rapeseed (Brassica napus L., AACC, 2n = 38) is one of the most important sources of edible oils worldwide. It is an allopolyploid species originating from the natural hybridisation between two ancestorial species, namely, B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18) [1]. The nutritional characteristics of rapeseed oil are mainly determined by its fatty acid (FA) composition and vitamin E content. The major FAs present in B. napus seeds (canola type) are oleic acid (C18:1), linoleic acid (C18:2), and linolenic acid (C18:3), with concentrations of 61%, 21%, and 11%, respectively [2]. Among these, C18:2 and C18:3 are polyunsaturated FAs (PUFAs) that cannot be biosynthesised in the human body. These PUFAs play an important role in the protection of eyesight and the prevention of various diseases, such as cancer and cardiovascular and inflammatory diseases [3][4][5]. Vitamin E, which is present in cold-pressed oil, is an essential dietary nutritional element for all mammals, and its deficiency leads to neurological disorders, ataxia, and even death [6,7]. Increasing the quantities of FAs and vitamin E has long garnered interest in rapeseed breeding [8]. Therefore, the characterisation of key genes regulating the biosynthesis of FAs and/or vitamin E in B. napus will be helpful in accurately designing new germplasms or varieties of crops with high PUFA and vitamin E contents using molecular biology techniques, thereby improving human health.
Vitamin E is an essential lipid-soluble antioxidant comprising tocopherols and tocotrienols. Tocopherols are mostly present in the seeds of common oil crops and the green-coloured components of higher-order plants, whereas tocotrienols are mainly found in cereal kernels and certain tropical fruits [9]. According to the number and position of the methyl substituents on the aromatic ring, tocopherols are classified into four forms: α, β, γ, and δ [10]. Among them, α-tocopherol is beneficial to human health, as it has the highest efficacy [11].
The major tocopherol functions are related to antioxidant properties because of their ability to interact with polyunsaturated acyl groups and protect membrane lipids (especially PUFAs) from oxidative damage by scavenging lipid peroxy radicals and quenching reactive oxygen species (ROS) in photosystem II. Furthermore, they also prevent damage due to the fact of oxidation during membrane lipid peroxidation [10,30]. Tocopherols donate a hydrogen atom to lipid-free radicals, thereby neutralising the radical and terminating the autocatalytic chain reaction of lipid peroxidation and protecting cell membranes [31,32]. In plants, both tocopherol and FA biosynthesis occur in the plastid, and the tocopherol functions as an antioxidant for stabilising PUFAs against lipid oxidation in vegetable oils [32]. The balance between vitamin E and PUFA content mainly determines the susceptibility to lipid peroxidation and storage stability of rapeseed oil. However, there is a paucity of studies examining the correlation between tocopherol and FA components in rapeseed oils. A positive correlation has been demonstrated between linoleic acid (18:2) and αtocopherol, linolenic acid (18:3) and γ-tocopherol from the vegetable oils of 14 different plant species [33]. In addition, upon studying a collection of maize hybrids, a relationship between FAs, particularly PUFAs, and tocochromanol content was observed [34]. Furthermore, the content of tocopherol and FA components in seeds from three Brassica oil crop species was analysed, and a significantly positive correlation was observed between αtocopherol and the sum of C18:1 and C18:2, whereas no association was observed between the γ-tocopherol and FA components [35]. a paucity of studies examining the correlation between tocopherol and FA components in rapeseed oils. A positive correlation has been demonstrated between linoleic acid (18:2) and α-tocopherol, linolenic acid (18:3) and γ-tocopherol from the vegetable oils of 14 different plant species [33]. In addition, upon studying a collection of maize hybrids, a relationship between FAs, particularly PUFAs, and tocochromanol content was observed [34]. Furthermore, the content of tocopherol and FA components in seeds from three Brassica oil crop species was analysed, and a significantly positive correlation was observed between α-tocopherol and the sum of C18:1 and C18:2, whereas no association was observed between the γ-tocopherol and FA components [35]. In addition to their antioxidant function, recent studies have suggested additional roles for tocopherols in diverse biological processes, including germination [36], cell signalling [37], and biotic [38] and abiotic stresses, such as those related to salt [39][40][41][42][43][44], low temperature [45,46], drought [41,47], and heavy metals [40,43,48,49]. The RNAi-mediated silencing of γ-TMT leads to a deficiency in α-tocopherol and elevated susceptibility to salt stress, whereas the overexpression of γ-TMT increases the salt and heavy metal tolerance in transgenic tobacco [39,43]. In addition, the increased sensitivity to salt stress in vitamin E-deficient mutants of A. thaliana has been observed. Tocopherols play a key role in salt In addition to their antioxidant function, recent studies have suggested additional roles for tocopherols in diverse biological processes, including germination [36], cell signalling [37], and biotic [38] and abiotic stresses, such as those related to salt [39][40][41][42][43][44], low temperature [45,46], drought [41,47], and heavy metals [40,43,48,49]. The RNAi-mediated silencing of γ-TMT leads to a deficiency in α-tocopherol and elevated susceptibility to salt stress, whereas the overexpression of γ-TMT increases the salt and heavy metal tolerance in transgenic tobacco [39,43]. In addition, the increased sensitivity to salt stress in vitamin E-deficient mutants of A. thaliana has been observed. Tocopherols play a key role in salt stress tolerance not only by reducing the extent of oxidative stress but also by improving ion homeostasis and the hormonal balance of leaves [42]. Furthermore, the overexpression of the γ-TMT gene in transgenic B. juncea plants enhances the tolerance to abiotic stresses, such as salt, heavy metals, and osmoticums [40].
In the present study, BnaC02.TMT.a, an Atγ-TMT orthologue in B. napus, was isolated using nested PCR and functionally characterised. To explore the biological functions of BnaC02.TMT.a in rapeseed, we measured the contents of tocopherols and FAs and conducted a germination assay on sodium chloride (NaCl) stress medium in transgenic overexpression plants. We showed that the overexpression of BnaC02.TMT.a increased the αand total tocopherol content, altered the proportion of FA composition, and promoted the germination rate after NaCl treatment. Under conditions involving salt-related stress, the accumulation of ROS is an important factor affecting the inhibition of seed germination, and hydrogen peroxide (H 2 O 2 ) and superoxide anion (O 2 -) are the main components of ROS [50,51]. To obtain a deeper insight into the mechanism underlying increased salt tolerance in transgenic plants, we analysed ROS accumulation, which was estimated using diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining and measured the H 2 O 2 and O 2 content. Our results suggest that BnaC02.TMT.a prevents oxidative damage by ROS scavenging during germination in B. napus seeds under conditions involving salt stress. This study revealed that the overexpression of BnaC02.TMT.a could affect tocopherols, FAs, and the response to salt stress, which will be useful for engineering rapeseed with improved vitamin E content, FA composition, and salt tolerance.

Sequence Analysis of Bnγ-TMT Paralogs
Upon performing the protein BLAST using A. thaliana Atγ-TMT in Brassica napus pangenome information resource (BnPIR), four Bnγ-TMT paralogs were predicted in the genome of the B. napus cultivar "Zhongshuang11 (ZS11)": BnaC02T0331100ZS, BnaC02T0197500ZS, BnaA02T0247300ZS, and BnaA02T0154300ZS, which were located on the chromosomes C02 and A02 and designated BnaC02.TMT.a, BnaC02.TMT.b, BnaA02.TMT.a, and BnaA02.TMT.b, respectively. The alignment of the amino acid sequences of the four Bnγ-TMT paralogs revealed high identities ranging from 96.5% to 98.8% ( Figure S1). The identity of the protein sequence between the four Bnγ-TMT proteins and Atγ-TMT was at least 88.4%, whereas BnaC02.TMT.a showed a higher identity with Atγ-TMT, reaching 89.3% ( Figure S1). Moreover, all Bnγ-TMT paralogs were predicted to possess four highly conserved S-adenosylmethionine (SAM)-binding domains, similar to Atγ-TMT ( Figure 2). This implies that all four paralogs of Bnγ-TMT may be functionally conservative. Phylogenetic analysis was performed to investigate the evolutionary relationships between the Bnγ-TMT and 11 γ-TMT proteins in A. thaliana and six other oil crops. As shown in Figure 3, BnaC02.TMT.b was most closely related to Atγ-TMT, whereas BnaC02.TMT.a was closely related to BnaA02.TMT.a. Compared with γ-TMT in other species, all four paralogs were closely related to Atγ-TMT.

Analysis of the Expression Pattern of Bnγ-TMT
To verify the function of Bnγ-TMT, quantitative reverse transcription PCR (RT-qPCR) was used to explore the spatiotemporal expression pattern of all Bnγ-TMT paralogs in various tissues of the B. napus cultivar "ZS11". As illustrated in Figure 4, Bnγ-TMT was widely expressed in various tissues, with higher expression levels in the flowers, leaves, and developing seeds (35 days after pollination (DAP)) than in the roots and stems. Notably, during seed development, the Bnγ-TMT expression remained relatively low at the embryogenesis stage from 10 to 15 DAP, decreased slightly from 20 to 26 DAP, and progressively increased at 29 DAP and 32 DAP, reaching a maximum at 35 DAP ( Figure 4).

BnaC02.TMT.a Increased α-Tocopherol Content in B. napus Seeds
To elucidate the role of Bnγ-TMT, we introduced the overexpression construct 35S:Bna C02.TMT.a-6 haemagglutinin (6HA) into the B. napus cultivar "ZS11" plants ( Figure 5A). Two representative independent T 2 homozygous lines (OE#3 and OE#5) were confirmed by the PCR amplification of BnaC02.TMT.a using the specific primers 35S_P/BnTMT_R ( Figure 5B). Furthermore, the expression level of the Bnγ-TMT gene was confirmed in these transgenic plants by RT-qPCR. The expression of Bnγ-TMT was significantly higher in these transgenic plants than in wild-type ZS11 ( Figure 5C).     To verify the function of Bnγ-TMT, quantitative reverse transcription PCR (RT-qPCR) was used to explore the spatiotemporal expression pattern of all Bnγ-TMT paralogs in various tissues of the B. napus cultivar "ZS11". As illustrated in Figure 4, Bnγ-TMT was widely expressed in various tissues, with higher expression levels in the flowers, leaves, and developing seeds (35 days after pollination (DAP)) than in the roots and stems. Notably, during seed development, the Bnγ-TMT expression remained relatively low at the embryogenesis stage from 10 to 15 DAP, decreased slightly from 20 to 26 DAP, and progressively increased at 29 DAP and 32 DAP, reaching a maximum at 35 DAP ( Figure 4).

BnaC02.TMT.a Increased α-Tocopherol Content in B. napus Seeds
To elucidate the role of Bnγ-TMT, we introduced the overexpression construct 35S:BnaC02.TMT.a-6 haemagglutinin (6HA) into the B. napus cultivar "ZS11" plants ( Figure  5A). Two representative independent T2 homozygous lines (OE#3 and OE#5) were confirmed by the PCR amplification of BnaC02.TMT.a using the specific primers 35S_P/BnTMT_R ( Figure 5B). Furthermore, the expression level of the Bnγ-TMT gene was confirmed in these transgenic plants by RT-qPCR. The expression of Bnγ-TMT was significantly higher in these transgenic plants than in wild-type ZS11 ( Figure 5C).
To investigate the function of BnaC02.TMT.a in mediating tocopherol accumulation, we measured the levels of total tocopherol and α-, β-, γ-, and δ-tocopherol in the mature seeds of wild-type ZS11 and the transgenic lines OE#3 and OE#5. The levels of α-tocopherol and total tocopherol were much higher in both of the transgenic lines than those in the wild-type seeds ( Figure 5D,E), whereas no apparent differences were observed in the levels of γ-and δ-tocopherol between the wild-type and transgenic plants ( Figure S2). In addition, no β-tocopherol was detected in either the control or transgenic plants.

BnaC02.TMT.a Promoted PUFA Biosynthesis in B. napus Seeds
To elucidate the biological functions of BnaC02.TMT.a in seed FAs, we measured the quantities of the major FA compositions and total FAs of mature seeds between the wildtype ZS11 plants and the BnaC02.TMT.a homozygous transgenic lines OE#3 and OE#5. In the mature seeds of the transgenic lines, a significant decrease was observed in the C18:1 (oleic acid) content along with a significant increase in the amount of C18:2 (linoleic acid) and C18:3 (linolenic acid) ( Figure 6A), suggesting that α-tocopherol might affect the conversion of oleic acid to linoleic acid and linoleic acid to linolenic acid. However, no significant differences were observed with respect to the total FA content ( Figure S3D) and several seed morphological traits, such as the seed coat colour ( Figure S3A), seed size ( Figure  S3B), and dry weight ( Figure S3C), between the mature seeds of the wild-type and trans- To investigate the function of BnaC02.TMT.a in mediating tocopherol accumulation, we measured the levels of total tocopherol and α-, β-, γ-, and δ-tocopherol in the mature seeds of wild-type ZS11 and the transgenic lines OE#3 and OE#5. The levels of α-tocopherol and total tocopherol were much higher in both of the transgenic lines than those in the wild-type seeds ( Figure 5D,E), whereas no apparent differences were observed in the levels of γand δ-tocopherol between the wild-type and transgenic plants ( Figure S2). In addition, no β-tocopherol was detected in either the control or transgenic plants.

BnaC02.TMT.a Promoted PUFA Biosynthesis in B. napus Seeds
To elucidate the biological functions of BnaC02.TMT.a in seed FAs, we measured the quantities of the major FA compositions and total FAs of mature seeds between the wildtype ZS11 plants and the BnaC02.TMT.a homozygous transgenic lines OE#3 and OE#5.
In the mature seeds of the transgenic lines, a significant decrease was observed in the C18:1 (oleic acid) content along with a significant increase in the amount of C18:2 (linoleic acid) and C18:3 (linolenic acid) ( Figure 6A), suggesting that α-tocopherol might affect the conversion of oleic acid to linoleic acid and linoleic acid to linolenic acid. However, no significant differences were observed with respect to the total FA content ( Figure S3D) and several seed morphological traits, such as the seed coat colour ( Figure S3A), seed size ( Figure S3B), and dry weight ( Figure S3C), between the mature seeds of the wild-type and transgenic plants. Three critical stages of seed oil accumulation (24, 28, and 32 DAP) were carefully selected to compare the expression profiles of BnFAD2 and BnFAD3 between the transgenic line OE#3 and the wild-type control. As expected, the expression of BnFAD2 was significantly higher at the seed maturation stage from 28 and 32 DAP in the transgenic seeds than in the wild-type seeds ( Figure 6B), and the expression of BnFAD3 was consistently upregulated in the developing seeds of the OE#3 transgenic plants at 24 and 32 DAP ( Figure 6B).  Three critical stages of seed oil accumulation (24, 28, and 32 DAP) were carefully selected to compare the expression profiles of BnFAD2 and BnFAD3 between the transgenic line OE#3 and the wild-type control. As expected, the expression of BnFAD2 was significantly higher at the seed maturation stage from 28 and 32 DAP in the transgenic seeds than in the wild-type seeds ( Figure 6B), and the expression of BnFAD3 was consistently upregulated in the developing seeds of the OE#3 transgenic plants at 24 and 32 DAP ( Figure 6B).

Functional Characterisation of BnaC02.TMT.a in the Seed Germination under Conditions Involving Salt Stress in B. napus
The effects of BnaC02.TMT.a on seed germination under conditions involving salt stress in the wild-type and transgenic lines OE#3 and OE#5 were examined in sterile redistilled water with or without 180 mM NaCl. As illustrated in Figures 7 and 8, OE#3, OE#5, and the control ZS11 showed similar seed germination rates in the medium without NaCl treatment.
Notably, under stress conditions involving 180 mM NaCl, the differences in germination between the wild-type and transgenic seeds were apparent (Figures 7 and 8). The seed germination was completely inhibited during the first 40 h after sowing (HAS) and from 40 to 192 HAS, which was significantly higher in the transgenic line than in the controls ( Figure 8). redistilled water with or without 180 mM NaCl. As illustrated in Figures 7 and 8, OE#3, OE#5, and the control ZS11 showed similar seed germination rates in the medium without NaCl treatment. Notably, under stress conditions involving 180 mM NaCl, the differences in germination between the wild-type and transgenic seeds were apparent (Figures 7 and  8). The seed germination was completely inhibited during the first 40 h after sowing (HAS) and from 40 to 192 HAS, which was significantly higher in the transgenic line than in the controls ( Figure 8).  Under control conditions, the ROS levels were similar between the wild-type and transgenic lines OE#3 and OE#5. In contrast, upon treatment with 180 mM NaCl, a lower DAB and NBT staining intensity, especially in cotyledons, was observed with the transgenic plants, suggesting that the ROS levels were significantly suppressed in the transgenic lines OE#3 and OE#5 compared to those in the wild-type ZS11 ( Figure 9A). Paralleling the observations described in Figure 9A, under control conditions, no significant differences were observed in the H 2 O 2 and O 2 − contents between the wild-type and transgenic plants. Compared to the controls, the H 2 O 2 and O 2 − contents were substantially decreased in both of the transgenic plants exposed to 180 mM NaCl ( Figure 9B). Therefore, our results revealed that the overexpression of BnaC02.TMT.a alleviated the oxidative damage to the seeds under conditions involving salt stress by ROS scavenging, thereby promoting seed germination under such conditions.   Under control conditions, the ROS levels were similar between the wild-type and transgenic lines OE#3 and OE#5. In contrast, upon treatment with 180 mM NaCl, a lower DAB and NBT staining intensity, especially in cotyledons, was observed with the transgenic plants, suggesting that the ROS levels were significantly suppressed in the transgenic lines OE#3 and OE#5 compared to those in the wild-type ZS11 ( Figure 9A). Paralleling the observations described in Figure 9A, under control conditions, no significant differences were observed in the H2O2 and O2 − contents between the wild-type and transgenic plants. Compared to the controls, the H2O2 and O2 − contents were substantially decreased in both of the transgenic plants exposed to 180 mM NaCl ( Figure 9B). Therefore, our results revealed that the overexpression of BnaC02.TMT.a alleviated the oxidative damage to the seeds under conditions involving salt stress by ROS scavenging, thereby promoting seed germination under such conditions.
Our results suggest that Bnγ-TMT plays a positive role in regulating the response of B. napus to salt stress, which might involve complex mechanisms, such as the ROS scavenging system.  Our results suggest that Bnγ-TMT plays a positive role in regulating the response of B. napus to salt stress, which might involve complex mechanisms, such as the ROS scavenging system.

Discussion
Rapeseed is one of the most important oil supply crops and contains a significant amount of tocopherols, which are beneficial for human health. Enhancing the quality and composition of FAs and tocopherols in seeds has long been a target for rapeseed breeding. In this study, we showed that BnaC02.TMT.a promoted the accumulation of αand total tocopherol, altered the FA composition in seeds, and enhanced the tolerance to salt stress during seed germination in B. napus.
B. napus is an allotetraploid species formed by the natural hybridisation between B. rapa and B. oleracea [1]. A large number of chromosome duplications, rearrangements, and deletions occurred during the evolutionary processes, resulting in an average of 2-8 paralogs of each A. thaliana locus in the B. napus genome [52]. Consistently, four paralogs to Atγ-TMT were found in the B. napus cultivar "ZS11" genome ( Figure 2). It was shown the overexpression of Atγ-TMT increased the α-tocopherol concentration and vitamin E activity with unaltered total tocopherol quantity in seeds, and the Atγ-TMT mutation exhibited an absent α-tocopherol but high levels of γ-tocopherol accumulation in the leaves of A. thaliana [20,21]. We found that the overexpression of BnaC02.TMT.a promoted α-tocopherol accumulation in B. napus seeds ( Figure 5D). BnaC02.TMT.a shared a high amino acid identity and a close evolutionary relationship with Atγ-TMT (Figures 2 and 3). In addition, four SAM binding domains involved in methyl transfer reactions [53] were highly conserved between BnaC02.TMT.a and Atγ-TMT (Figure 2). These results suggest that BnaC02.TMT.a may exhibit a conserved role in regulating the accumulation of α-tocopherol in A. thaliana. In rapeseed oil, the major tocopherol form is γ-tocopherol (65%), followed by α-tocopherol (35%). Furthermore, a small proportion of δ-tocopherol (<1%), and β-tocopherol is absent [54]. BnaA.VTE4.a1 (one γ-TMT paralogue from B. napus variety "Express") is able to increase the α-tocopherol content because of the shift from γto α-tocopherol, and the total tocopherol level is not altered in transgenic A. thaliana seeds [55]. Interestingly, we found that the overexpression of BnaC02.TMT.a resulted in higher levels of α-tocopherol ( Figure 5D); however, no obvious changes were observed in γ-tocopherol content in B. napus seeds ( Figure S2A), which may explain the higher levels of total tocopherol ( Figure 5E). The distinct roles of BnaA.VTE4.a1 and BnaC02.TMT.a in γand total tocopherol production might be attributed to their amino acid differences and functional divergence ( Figure S4). Notably, the functional characterisation of BnaA.VTE4.a1 was performed in a heterologous transgenic system, which may not truly reflect its function in B. napus. A possible reason for the unaltered γ-tocopherol content is that γ-tocopherol could be replenished by other mechanisms in the B. napus transgenic lines.
The levels of α-tocopherol are positively correlated with linoleic acid and PUFAs in vegetable oils [33,34]. Upon examining a collection of B. napus lines with low erucic acid, a significant positive correlation was found between the α-/γ-tocopherol ratio and C18:2 and PUFAs [35]. Accompanied by an increased α-tocopherol level, the FA composition was altered; that is, decreased levels of oleic acid (C18:1) and increased levels of linoleic acid (C18:2) and linolenic acid (C18:3) were observed in 35S:BnaC02.TMT.a-6HA plants ( Figure 6A). FAD2 [56,57] and FAD3 [58,59] are essential for the formation of C18:2 and C18:3, respectively, and higher transcript levels of BnFAD2 and BnFAD3 ( Figure 6B) may be helpful for the accumulation of C18:2 and C18:3 in 35S:BnaC02.TMT.a-6HA seeds. As previously reported, the association between tocopherol and FAs in oils is mostly attributed to the biochemical function of tocopherol in protecting against lipid oxidation, rendering it crucial for PUFA stability [33,34]. Furthermore, biochemical and genetic evidence indicates that tocopherols have a more limited role in photoprotection than previously assumed [45,46]. In the absence of tocopherols, the conversion of 18:2 to 18:3 in endoplasmic reticulum (ER) lipids is reduced under low temperatures, suggesting some plausible scenarios for the direct or indirect interaction of tocopherols during ER PUFA metabolism. FAD2 and FAD3 are ER-localised FA desaturases. Together with the findings indicating that BnaC02.TMT.a enhanced the expression of FAD2 and FAD3 in 35S:BnaC02.TMT.a-6HA developing seeds ( Figure 6B), we hypothesised that in addition to their antioxidant activity, the function of α-tocopherol in regulating FA formation may be attributed to the interaction between α-tocopherol and ER FA metabolism.
The role of tocopherols in abiotic stresses, such as salt [39][40][41][42][43][44], low temperature [45,46], drought [41,47], and heavy metals [40,43,48,49], has been observed. These results, together with our observations of 35S:BnaC02.TMT.a-6HA plants under conditions involving salt stress (Figures 7-9), suggest that tocopherols play a crucial role in the alleviation of salt stress. Stresses lead to excessive ROS levels causing oxidative damage [60,61]. H 2 O 2 and O 2 are the most common forms of ROS generated during photosynthesis [50]. In this study, the overexpression of BnaC02.TMT.a promoted seed germination (Figures 7 and 8) and reduced the production of H 2 O 2 and O 2 in B. napus under conditions involving salt stress (Figure 9), indicating that BnaC02.TMT.a alleviated the negative effects of NaCl toxicity by scavenging ROS. This was reminiscent of the expression of γ-TMT in chloroplasts, accelerating the conversion of γ-tocopherol to α-tocopherol, which contributes to a decrease in susceptibility to salt [43]. Tocopherol-deficient Arabidopsis mutants were particularly sensitive to salt stress, highlighting the role of α-tocopherol in maintaining cellular Na + /K + homeostasis and hormonal balance [42,47]. The overexpression of Msγ-TMT in alfalfa (Medicago sativa L.) can directly or indirectly affect the biosynthesis of multiple phytohormones that may play a comprehensive role in drought stress tolerance [47]. FAs or FA derivatives function as signalling molecules or hormones, carbon and energy storage materials, and surface layers that protect plants from environmental stresses [62][63][64]. In addition, C18:3 serves as a precursor for the biosynthesis of jasmonic acid, which is involved in the response to various stresses [65,66]. Overall, considering the changes in FA composition, especially with an increase in the proportion of C18:3 ( Figure 6A), other possible reasons for the better seed germination performance of the 35S:BnaC02.TMT.a-6HA plants in the present study might, in part, be attributed to signalling cascades connecting tocopherol levels with FAs, hormonal responses, ion homeostasis, and all aspects that warrant further investigation.
Plant growth and crop production are adversely affected by environmental stresses in nature. By uncovering the genes that underlie the tolerance adaptive trait, natural variation has the potential to be introgressed into elite cultivars [67]. Modern analytical approaches, such as genomic prediction, machine learning, and multi-trait gene editing, as well as genome-environment associations, were applied to speed up the identification and deployment of genotypic sources for climate change adaptation [67,68]. Indeed, genotypes from advanced interspecific congruity backcross exhibit promising responses to extreme conditions (i.e., heat and drought), which offers novel perspectives to breed traits of interest in the face of multiple climate changes [69]. Our study revealed that BnaC02.TMT.a acts as a positive regulator of tocopherol biosynthesis and salt stress response, providing a beneficial candidate for improving tocopherol activity and salt stress resistance in rapeseed breeding. Tocopherol-associated traits could be employed in traditional breeding for rapeseed improvement. Furthermore, advanced breeding strategies established in recent years have provided new avenues for harnessing tocopherols biosynthesis to improve the adaptation to stressful environments.

Plant Material and Growth Conditions
The B. napus cultivar "Zhongshuang11 (ZS11)" was used in this study. It was obtained through a material transfer agreement from the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan, China, and selfed for at least 10 generations. The rapeseed seeds were placed in a glass dish with three layers of filter paper soaked with sterile redistilled water and imbibed at 4 • C for one day in the dark. After imbibition, the seeds were transferred to 11 × 11 cm pots and grown in a greenhouse at 22 • C with a long day duration (LD, 16 h light/8 h dark) for six weeks. Subsequently, the plants were vernalised for four weeks at 4 • C under LD conditions in a cold chamber. After vernalisation, the plants were returned to the initial greenhouse conditions for ten weeks until harvest.

Protein Sequence and Phylogenetic Analysis
The protein sequences of γ-TMT were obtained from the National Center for Biotechnology Information (NCBI) database and B. napus pan-genome information resource (BnPIR) database (http://cbi.hzau.edu.cn/bnapus/index.php, accessed on 6 September 2022). The protein sequence alignment was performed using MUSCLE (http:// www.ebi.ac.uk/Tools/msa/muscle/, accessed on 8 September 2022). The S-adenosyl methionine-binding domain was predicted using the Conserved Domain Search program (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 8 September 2022). The phylogenetic analysis was performed using MEGA7 to confirm the evolutionary relationships between the γ-TMT protein sequences. A bootstrap analysis with 1000 replicates was performed to assess the statistical reliability of the tree topology.

Gene Cloning and Plasmid Construction
Owing to a high sequence similarity among the four paralogs of Bnγ-TMT, nested PCR was used for cloning BnaC02.TMT.a. Two pairs of PCR primers (specific and nested primers) were designed. The specific primers were designed based on the alignment of four Bnγ-TMT paralogs, and the nested primers were designed based on the full-length coding sequence (CDS) of BnaC02.TMT.a (BnaC02T0331100ZS) without a stop codon derived from the BnPIR database. To obtain full-length BnaC02.TMT.a cDNA, the total RNA was extracted from the developing seeds of the B. napus cultivar "ZS11" using the SteadyPure Plant RNA Extraction Kit (Accurate Biology, Changsha, China) and was subjected to reverse transcription to obtain first-strand cDNA according to the manufacturer's instructions (TransGen, Beijing, China).
The full-length BnaC02.TMT.a CDS was amplified by nested PCR using specific and nested primers. The first pair of the PCR primers amplified a specific fragment using the primers BnaC02.TMT.a_Fc and BnaC02.TMT.a_Rc. The second pair of primers, called nested primers, was used to clone the full-length CDS of BnaC02.TMT.a. The sequence was ligated into the binary vector pGreen-35S-6HA under the control of the CaMV35S (35S) promoter, which had been digested by Xho1 and Cla1 [70]. The primers used in this study are listed in Supplementary Table S1.

Generation of B. napus Transgenic Plants
To obtain the transgenic B. napus plants, the hypocotyl segments of the B. napus cultivar "ZS11" were infected with the plasmid 35S:BnaC02.TMT.a-6HA by Agrobacterium tumefaciens strain GV3101 [71]. Transgenic T 0 plants were selected on MS agar medium containing 10 µg/mL phosphinothricin, and a successful transformation was confirmed by DNA genotyping according to the PCR amplification performed using gene-specific primers. The transformants were selected by genotyping according to PCR amplification, and homozygous seeds from the T 2 generation were used for subsequent experiments after cultivation under the same aforementioned conditions.

Morphological Observation of Mature Seeds
The T 3 plants of the BnaC02.TMT.a homozygous transgenic lines (OE#3 and OE#5) and wild-type ZS11 controls were grown in a greenhouse under the aforementioned conditions. Siliques were harvested from the middle and basal parts of the main inflorescence. Subsequently, mature seeds were randomly selected from the different lines and observed using a stereomicroscope (Olympus SZ 61, Tokyo, Japan).

Measurement of the FA Content
The T 3 plants of the BnaC02.TMT.a homozygous transgenic lines (OE#3 and OE#5) and wild-type ZS11 controls were grown in a greenhouse under the aforementioned conditions. For the FA measurement, seeds were harvested from the middle and basal parts of the main stem of eight individual plants from each line. The FAs were extracted and analysed as previously described [72,73], with minor modifications. First, the seeds were ground in a mortar, and approximately 8 mg of seed powder was heated at 80 • C in a methanol solution containing 2.5% (v/v) H 2 SO 4 for 2 h, after which the total FAs were converted to FA methyl-esters. After cooling to room temperature, the FA methyl-esters were extracted with 1 mL hexane and 2 mL 0.9% (w/v) NaCl, and the organic phase was transferred to autoinjector vials. The gas chromatography (GC-2010 Plus, Shimadzu, Japan) analysis was performed using methyl heptadecanoate as an internal standard. The concentration of each FA species was normalised to that of the internal control.

HPLC Analysis of the Tocopherol Content
The tocopherol analysis was performed by Suzhou Michy Biology (http://www. michybio.com, accessed on 8 July 2021). The dried seeds from eight individual plants (transgenic lines and wild-type ZS11) were sent to the company. Briefly, 30-80 mg of mature seeds were ground, and the powder was mixed with 1 mL of ethyl alcohol extraction buffer containing 1% butylated hydroxytoluene (BHT) and 1 mL of 100 g/L potassium hydroxide solution. After incubation for 40 min (85 • C) and cooling to room temperature, 2 mL of petroleum ether was added. After mixing and centrifugation, the lower organic layer was recovered, dried under nitrogen, and resuspended in 0.4 mL methanol for the HPLC analysis.
The tocopherol content of the organic extract was determined using an Agilent 1100 HPLC system. A total of 10 µL of the sample was chromatographically analysed using a Compass C18 column (250 × 4.6 mm length, 5 µm particle size) and a solvent system consisting of methyl alcohol as the mobile phase with a flow rate of 1 mL/min. The column temperature was 20 • C, and the sample was scanned at 294 nm. The identification and quantification of tocopherols were performed by comparing the retention time and peak area to tocopherol standards (Supplementary Table S2).

ROS Quantification
The H 2 O 2 content was measured using the titanium-peroxide complex method, as described previously, with minor modifications [74][75][76]. A total of 0.1 g (fresh weight) of the seeds collected at 96 HAS were ground in liquid nitrogen and extracted with 1 mL of cooled acetone. After the vortex mixing, the samples were centrifuged for 10 min at 8000× g at 4 • C. The supernatant was then added to a solution comprising 0.1 mL of 5% (w/v) titanium sulphate and 0.2 mL of ammonia. The mixture was centrifuged for 10 min at 8000× g. After removing the supernatant, the precipitate was dissolved in 1 mL of 2 M H 2 SO 4 . The absorbance was immediately measured at 415 nm. The H 2 O 2 content was calculated using a standard curve generated using known concentrations of H 2 O 2 .
The O 2 − content was quantified as previously described, with minor modifications [74,76]. First, 0.1 g (fresh weight) of the seeds at 96 HAS was ground in liquid nitrogen and added to 1 mL of K-phosphate buffer (65 mM, pH 7.8). The homogenates were centrifuged at 12,000× g for 20 min (4 • C). A total of 1 mL 0.1 M hydroxylamine was added to the supernatant, and the mixture was incubated at 25 • C for 20 min. Then, 1 mL of 7 mM α-naphthalenamine and 1 mL of 58 mM 4-aminobenzenesulfonic acid were added, and the samples were centrifuged for 20 min at 12,000× g at 4 • C. Trichloromethane (1 mL) was added to eliminate the pigments, and the mixture was centrifuged at 10,000× g for 5 min. The absorbance of the supernatant was measured at 530 nm. The O 2 content was determined using a standard curve generated from known concentrations of NaNO 2 .

Germination Test
For the seed germination assay, 50 plump seeds from each treatment group were carefully picked and washed with sterile redistilled water thrice, evenly placed on a glass dish with three layers of filter paper soaked in the same volume of 180 mM NaCl solution or sterile redistilled water as the control, and imbibed at 4 • C for one day in the dark. After imbibition, the seeds were transferred to a growth chamber for germination. Every other day, the germinating seeds were transferred to a new glass dish with three layers of filter paper soaked in the same volume of 180 mM NaCl solution or sterile redistilled water until the seed germination experiment was completed. Three independent biological replicates were obtained for the control and salt treatments of the transgenic lines (OE#3 and OE#5) and ZS11. Seed germination was defined as the complete emergence of the radicle through the seed coat [77]. The number of germinated seeds was calculated every 8 h until the completion of the experiment.

Gene Expression Analysis
The roots, stems, flowers, and developing seeds for the spatial and temporal expression analysis were harvested from at least eight individual plants grown in different pots. The total RNA was isolated using the SteadyPure Plant RNA Extraction Kit (Accurate Biology, Changsha, China) and reverse-transcribed using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, Beijing, China) according to the manufacturer's instructions. Quantitative reverse transcription RT-PCR (RT-qPCR) was performed in 96well blocks using SYBR Green Master Mix (Cofitt, Hong Kong, China) with three biological replicates and three technical replicates. Considering the high sequence similarity among the paralogs of Bnγ-TMT, the primers were designed for the conserved regions within groups of paralogs to detect joint gene expression. The relative expression levels were calculated using a modified double delta method [78]. The housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase (BnGAPDH), a key enzyme in the process of glycolysis and gluconeogenesis, was considered as a reference, which is widely founds in various organisms and is regarded as an internal control [79]. The primers used for RT-qPCR are listed in Supplementary Table S1.

Statistical Analysis
A completely randomised design was used in this study. All data were classified using Win-Excel and reported as the mean ± standard deviation (SD). The data were analysed by one-way analysis of variance (ANOVA) using SPSS software (version 17.0, SPSS Inc., Chicago, IL, USA). Significant differences were determined by Student's t-test (p ≤ 0.05).

Conclusions
In summary, we discovered new functions for Bnγ-TMT in the regulation of tocopherol and FA biosynthesis and salt tolerance in rapeseed. Since it is an allopolyploid species, four paralogs of Bnγ-TMT were found in the B. napus genome. The protein sequence and phylogenetic analyses suggested that all four Bnγ-TMT paralogs might exhibit functions similar to those shown by Atγ-TMT for determining tocopherol accumulation. Together with the expression data, these results further suggest that Bnγ-TMT might regulate tocopherol accumulation mainly during the maturation stage in B. napus seeds. Overexpression of BnaC02.TMT.a promotes seed αand total tocopherol accumulation during seed maturation. Additionally, the fatty acid composition was altered. Our findings suggest that BnaC02.TMT.a promoted the conversion of oleic acid to linoleic acid and subsequently linoleic acid to linolenic acid by upregulating the expression of BnFAD2 and BnFAD3 in rapeseed. Furthermore, BnaC02.TMT.a plays a crucial role in mediating the response to salt stress during seed germination, which involves complex mechanisms, including ROS scavenging capability, signalling cascades connecting tocopherol with FAs, hormonal responses, and ion homeostasis. This study broadens our understanding of the function of the Bnγ-TMT gene and provides a novel strategy for genetic engineering to improve rapeseed breeding. MBPQ 2-methyl-6-phytyl-