P-HYDROXYPHENYLPYRUVATE DIOXYGENASE from Medicago sativa is involved in vitamin E biosynthesis and abscisic acid-mediated seed germination

P-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) is the first committed enzyme involved in the biosynthesis of vitamin E, and is characterized by catalyzing the conversion of p-hydroxyphenyl pyruvate (HPP) to homogentisic acid (HGA). Here, an HPPD gene was cloned from Medicago sativa L. and designated MsHPPD, which was expressed at high levels in alfalfa leaves. PEG 6000 (polyethylene glycol), NaCl, abscisic acid and salicylic acid were shown to significantly induce MsHPPD expression, especially in the cotyledons and root tissues. Overexpression of MsHPPD was found to significantly increase the level of β-tocotrienol and the total vitamin E content in Arabidopsis seeds. Furthermore, these transgenic Arabidopsis seeds exhibited an accelerated germination time, compared with wild-type seeds under normal conditions, as well as under NaCl and ABA treatments. Meanwhile, the expression level of several genes associated with ABA biosynthesis (NCED3, NCED5 and NCED9) and the ABA signaling pathway (RAB18, ABI3 and ABI5) were significantly down-regulated in MsHPPD-overexpressing transgenic lines, as well as the total free ABA content. Taken together, these results demonstrate that MsHPPD functions not only in the vitamin E biosynthetic pathway, but also plays a critical role in seed germination via affecting ABA biosynthesis and signaling.

MsHPPD expression in different tissues. Quantitative reverse transcription-PCR (qRT-PCR) was performed to determine the expression pattern of MsHPPD in different organs of M. sativa. The results showed MsHPPD transcripts were detectable in all tested organs, with the highest expression in rosette leaves and the lowest expression in early flowers ( Fig. 2A). This implies that MsHPPD may play a more active role in leaves.
To further confirm the transcriptional expression pattern of MsHPPD, a pHPPD::GUS construct was transformed into Arabidopsis. GUS expression was detected in cotyledons, primary roots, sepals, petals, stigma, filament tubes, pollens, and the ends of seeds in transgenic lines. However, no GUS activity was observed in the root tip or hypocotyls (Fig. 2B).
MsHPPD expression under various conditions. The promoter sequence of MsHPPD was analyzed using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to better investigate the expressional regulation of MsHPPD 26 . The results revealed cis-elements, which have been shown to respond to defense and stress signals including salicylic acid, gibberellin, and light signals are presented in the promoter region of MsHPPD (Fig. S1). To explore the possible roles of these cis-elements with regard to regulating the expression of MsHPPD, expressional analysis of MsHPPD was executed under various stress treatments. These results showed that MsHPPD transcripts were gradually up-regulated in seedlings treated with increasing treatment period when seedlings were exposed to NaCl, PEG, and ABA ( Fig. 3A-C). Most notably, the expression of MsHPPD increased 500-fold after a 24 h treatment with NaCl, relative to the levels seen in the mock treatment. Likewise, treatments utilizing extended periods of darkness revealed that the expression of MsHPPD rose gradually as the darkness treatment time increased; however, upon exposure to light, the expression levels of MsHPPD dropped sharply, and this decaying trend continued with prolonged exposure to light (Fig. 3D). The expression levels of MsHPPD increased moderately, albeit significantly, subsequent to salicylic acid treatment, until the 8 h time point, at which point MsHPPD expression returned to the control level (Fig. 3E). No significant differences were observed after methyl jasmonic acid treatment (Fig. 3F).
pHPPD::GUS was used to approximate the expression levels of MsHPPD in response to NaCl, PEG, and ABA treatments. Seedlings treated with NaCl, PEG, or ABA showed increase GUS activity across the plants, especially in the roots and cotyledons. This observation is consistent with the qRT-PCR results (Fig. 3G). Expression of MsHPPD and endogenous vitamin E pathway genes in transgenic Arabidopsis.
qRT-PCR was used to examine the expression levels of MsHPPD in MsHPPD-overexpressing lines and wild-type Arabidopsis. This data showed that MsHPPD was more-highly expressed in transgenic Arabidopsis lines, to varying degrees, as compared to wild-type expression levels (Fig. 4A). Likewise, all of the pHPPD::GUS transgenic Arabidopsis lines exhibited higher levels of GUS activity compared with the corresponding wild-type plants (Fig. 4B), which was consistent with the transcriptional levels of MsHPPD. To test whether the overexpression of exogenous MsHPPD alters the endogenous vitamin E biosynthetic pathway, the expression levels  MsHPPD expression in three MsHPPD-overexpressing transgenic lines and wild-type, as measured by qRT-PCR. Total RNA was extracted from two-week-old MsHPPD-overexpressing transgenic Arabidopsis (OE2, OE6, and OE7) and control seedlings, before being subjected to qRT-PCR analysis. The expression levels of MsHPPD were normalized to At4g26410. Data are presented as mean ± SD, each with three biological replicates and three technical replicates. Statistical analyses were carried out via a two-tailed Student's t-test with a significance of P < 0.05. (B) Histochemical GUS staining of MsHPPD-overexpressing lines (OE2, OE6, and OE7) and wild-type seedlings with pBI121 empty vector in the Col-0 background. Twoweek-old transgenic and control Arabidopsis were used for GUS staining. Bar: 1 cm. (C) Expression analysis of endogenous genes involved in vitamin E biosynthesis in MsHPPD-overexpressing Arabidopsis (OE2, OE6, and OE7) and wild-type control. Two-week-old Arabidopsis seedlings were used for qRT-PCR. Data are shown as the mean ± SD with three biological replicates and three technical replicates. The expression levels of the genes detected in the experiment were normalized to At4g26410. Statistical analyses were carried out via a two-tailed Student's t tests with a significance of P < 0.05.
Scientific RepoRts | 7:40625 | DOI: 10.1038/srep40625 of critical endogenous vitamin E pathway genes were quantified. There are several genes that have been shown to regulate vitamin E biosynthesis. HPPD and HOMOGENTISATE PHYTYLRANSFERASE (HPT) have been found to play roles pertaining to determining the total vitamin E content, while 2-METHYL-6-PHYTYLBENZ OQUINONEMETHYLTRANSFERASE (MPBQMT) and γ-TOCOPHEROL METHYL TRANSFERASE (γ-TMT) play roles in determining the vitamin E composition 18 . A significant decrease of AtHPT, which plays a role in determining vitamin E content and is also the first gene downstream of HPPD in vitamin E biosynthesis, was observed in the generated MsHPPD-overexpressing lines, but the expression levels of AtMPBQMT, AtTMT, AtTC, AtHPPD and AtVTE5 were not in the wild-type. Since HPPD is also involved in plastoquinone-9 and plastochromanol-8 biosynthesis, the expression level of AtHST, which is the first gene downstream of HPPD in plastoquinone-9 and plastochromanol-8 biosynthesis, was examined, and no significant difference was observed. This suggests that the plastoquinone-9 and plastochromanol-8 biosynthetic pathway is not altered (Fig. 4C).

Analysis of vitamin E levels in transgenic Arabidopsis seeds and leaves. To test whether MsHPPD
is a functional gene involved in the production of tocotrienols and tocopherols, all vitamin E forms were quantified in the seeds and leaves of MsHPPD-overexpressing lines and wild-type Arabidopsis. β -tocotrienol and δ -tocopherol content were found to have increased by 12-36% and 10-57%, respectively, in transgenic Arabidopsis seeds compared to wild-type seeds (Fig. 5). However, δ -tocotrienol was undetectable in all lines and the content of the other forms of vitamin E was not altered (Fig. S2A). Total vitamin E content was significantly higher in the seeds of MsHPPD-overexpressing lines OE2 and OE7 than the corresponding content in the seeds of the control line (Fig. 5). This corresponds to the previously obtained MsHPPD expression levels, in which lines OE2 and OE7 had the most significantly enhanced MsHPPD expression, compared with OE6 and wild-type Seeds from MsHPPDoverexpressing lines (OE2, OE6 and OE7) and wild-type Arabidopsis were collected at the same time, air dried for two weeks and used for this measurement. Data are presented as mean ± SD with four biological replicates. Statistical analyses were carried out via a two-tailed Student's t-test with a significance of P < 0.05.

Effect of MsHPPD on seed germination in Arabidopsis.
To explore the potential role of the increased β -tocotrienol in transgenic Arabidopsis seeds, seed germination rate was examined under standard conditions, as well as under various stress conditions. Under normal conditions, the seed germination rate of MsHPPD-overexpressing transgenic lines was significantly higher than that of wild-type at 24 h. However, as the time after imbibition increased, the difference between the germination rates of transgenic and wild-type lines diminished, and so the difference in germination rate was less pronounced after 36 h, and by 48 h, both wild-type Seeds of transgenic and control Arabidopsis were harvested at the same time, dried at room temperature for two weeks, and germinated on half-strength MS medium, either with or without the applicable treatments. Seed germination rate was calculated based on four biological replicates (~80 seeds per replicate). Data are presented as mean ± SD. Statistical analyses were carried out via a two-tailed Student's t-test with a significance of P < 0.05. and transgenic lines reached equivalent germination rates under standard growth conditions (Fig. 6A). Under NaCl treatment, the seed germination rate of both transgenic and wild-type Arabidopsis lines was inhibited, however, the seed germination rate was significantly higher in the seeds of all transgenic lines compared to the corresponding rate observed in wild-type seeds, and this trend holds up at all tested time points. Ultimately, after 84 h, the germination rate of both the transgenic and control lines was determined to be 99% and 88%, respectively (Fig. 6B). Under 5 μ M ABA treatment, the seed germination rate of MsHPPD-overexpressing transgenic lines at 120 h was approximately 86%, and this was significantly higher than the germination rate of the control line, which was found to be 40% (Fig. 6C). Furthermore, when the ABA concentration was doubled to 10 μ M, the seed germination rate of the transgenic lines at 120 h decreased slightly to around 72%, but the germination rate of the wild-type lines decreased much more drastically to 18% (Fig. 6D).
Expression of genes involved in the ABA biosynthetic and signaling and total ABA content are significantly reduced in seeds of MsHPPD-overexpressing Arabidopsis. Since seed germination rate was significantly affected in the MsHPPD-overexpressing lines and ABA is an important phytohormone involved in seed dormancy and germination, we quantified the free ABA level in dry seeds from MsHPPD-overexpressing lines and wild-type. In addition, since a significant difference was observed in the seed germination rate between MsHPPD-overexpressing lines and wild-type imbibed seeds at 36 h, the ABA level in imbibed seeds were also measured. While no significant difference was observed in dry seeds, ABA content was imbibed MsHPPD-overexpressing Arabidopsis (OE2, OE6, and OE7) and wild-type seeds. Transgenic and control Arabidopsis seeds were harvested at the same time, dried at room temperature for two weeks, and used for ABA measurement in dry seeds (upper plot). Alternatively, they were submerged in water for 36 h and used for ABA measurement in imbibed seeds (lower plot). Data are presented as mean ± SD using three biological replicates. Statistical analyses were carried out via a two-tailed Student's t-test with a significance of P < 0.05. (B) Expression rate of MsHPPD in imbibed seeds relative to dry seeds of MsHPPD-overexpressing transgenic and control Arabidopsis. The samples used here were the same as those in A. Data are presented as mean ± SD using three biological replicates and three technical replicates. Statistical analyses were carried out via a two-tailed Student's t-test with a significance of P < 0.05.
Scientific RepoRts | 7:40625 | DOI: 10.1038/srep40625 significantly decreased in imbibed MsHPPD-overexpressing Arabidopsis seeds compared to imbibed wild-type seeds (Fig. 7A). Meanwhile, the expression levels of MsHPPD in MsHPPD-overexpressing lines were significantly higher in imbibed seeds than they were in dry seeds (Fig. 7B). Among the three MsHPPD-overexpressing lines, OE6 showed the dramatically highest MsHPPD expression level when imbibed with water and significantly reduced ABA content providing us with a suitable transgenic line to evaluate the relationship between MsHPPD expression level and ABA content. To determine whether the decrease of ABA levels in transgenic lines was due to changes in ABA biosynthesis or in ABA metabolism, qRT-PCR was performed to examine the expression levels of the ABA biosynthetic and signaling pathway genes in OE6. The results showed that the expression of the ABA biosynthetic genes ABA1, NCED3, NCED5 and NCED9 was not altered in dry seeds of the MsHPPD-overexpressing lines; however, in imbibed seeds, NCED3, NCED5 and NCED9 expression was significantly reduced, compared to the expression in wild-type seeds. Meanwhile, the expression levels of two other ABA biosynthetic genes, AAO3 and ABA3, were both significantly lower in the MsHPPD-overexpression lines than they were in wild-type seeds, regardless of whether the seeds were dried or imbibed. Likewise, the expression levels of the ABA signaling pathway genes RAB18, ABI3, and ABI5 were not changed in dry seeds, but were significantly down-regulated in imbibed seeds of the MsHPPD-overexpressing lines compared to wild-type seeds (Fig. 8).

Discussion
HPPD catalyzes the first committed step in vitamin E biosynthesis 2 . Although it has been studied in many organisms [27][28][29][30] ; the function of the HPPD gene in the economically important forage plant alfalfa remains unknown.  Fig. 7. Data are presented as mean ± SD using three biological replicates and three technical replicates. The expression levels of the genes detected in the experiment were normalized to At4g26410. Statistical analyses were carried out via a two-tailed Student's t-test with a significance of P < 0.05.
Scientific RepoRts | 7:40625 | DOI: 10.1038/srep40625 In this study, full-length HPPD cDNA from alfalfa was isolated, and subsequent amino acid sequence alignment showed that MsHPPD from alfalfa shared high similarity with LsHPPD from Lactuca sativa, MtHPPD from Medicago truncatula, and AtHPPD from Arabidopsis thaliana. Among these HPPDs, LsHPPD and AtHPPD have been previously implicated as being able to alter vitamin E content in transgenic plants 29,31 . Phylogenetic tree construction also revealed that MsHPPD was evolutionally closest to HPPD sequences from these dicotyledonous plants.
Expression pattern analyses showed that NaCl and PEG treatment rapidly and robustly induced MsHPPD expression, suggesting that MsHPPD is involved in multiple stress responses in alfalfa. Darkness was also found to induce the expression of MsHPPD, and this is in agreement with the results from Falk et al. 32 , who found that HPPD expression levels increased during dark-induced leaf senescence. This may be due to the breakdown of chlorophyll-released free phytol, a precursor for tocopherol synthesis, and thus increased the demand for HPPD 33,34 . When plants were transferred from darkness to light, MsHPPD expression dropped sharply and continued to decrease (Fig. 3D), which suggests that light acts as a negative regulator of MsHPPD expression.
ABA induced the expression of MsHPPD modestly, albeit significantly. MsHPPD expression levels were initially high in primary roots and cotyledons, and increased significantly after the ABA induction. It has been shown that salicylic acid (SA), methyl jasmonic acid (MeJA), and high light can induce the expression of both SmHPPD from Salvia miltiorrhiza and also AtHPPD 30,35 . MeJA treatment was not found to alter the expression levels of MsHPPD, which might be a manifestation of the lack of a MeJA-responsive element in the promoter region of MsHPPD (Fig. S1). However, in agreement with the previous study, SA has significantly induced the expression of MsHPPD. Taken together, our results suggest that HPPD could be induced by multiple conditions, and the flexible expression levels of MsHPPD in response to stresses implied that MsHPPD plays multiple important roles with regards to responding to stresses.
In the leaves of MsHPPD-overexpressing transgenic lines, no difference was recorded to either vitamin E composition or vitamin E content (Fig. S2). This was contradictory to the result obtained by Tsegaye et al. 31 , who reported that overexpressing AtHPPD increased tocopherol by around 37% in transgenic Arabidopsis leaves. However, overexpressing barley HPPD had no effect on the tocopherol content in transgenic tobacco leaves 11 . This discrepancy may be attributable to subtle differences between the different organisms used in these studies. To gain a deeper insight into the reason for this discrepancy, we tested the expression levels of endogenous vitamin E biosynthetic genes in Arabidopsis to examine whether MsHPPD overexpression affected the flux of the entire vitamin E biosynthetic pathway. Interestingly, we observed a significant decrease only in the expression level of endogenous AtHPT in transgenic lines, suggesting that the vitamin E biosynthesis pathway is tightly controlled. The increased expression levels of MsHPPD appear to have somehow inhibited the expression of AtHPT, which also plays a role in determining vitamin E accumulation along with HPPD, and the inhibition of AtHPT may counteract the effects caused by the enhancement of MsHPPD expression on vitamin E to an unknown degree. As such, perhaps this explains why no significant differences were observed regarding the vitamin E content of the leaves.
As was previously reported, the content and composition of vitamin E varies in different tissues and different developmental stages 8 . α -tocopherol is the dominant form found in plant leaves, while γ -tocopherol and tocotrienols mainly exist in seeds 36 . This phenomenon made us question the function of elevated β -tocotrienol in seeds; therefore, we tested the seed germination rate of seeds from MsHPPD-overexpressing Arabidopsis lines and also wild-type seeds, under both normal and stressful conditions. Under normal conditions, the transgenic lines germinated earlier than wild-type plants. Meanwhile, under NaCl treatment, the germination of both transgenic and wild type lines was inhibited compared to normal conditions; however, the germination rate of the seeds from transgenic lines was significantly higher than that of wild type seeds. These results suggest that MsHPPD plays a positive role in regulating seed germination.
Seed eFP browser 37 showed that HPPD belongs to the ABA group of genes and that it might be involved in ABA biosynthesis or the ABA signaling pathway. Here, we showed that transgenic Arabidopsis lines which overexpressing MsHPPD were less sensitive to ABA treatment, in terms of seed germination rate (Fig. 6C,D). Additionally, the ABA content was significantly lower in imbibed transgenic Arabidopsis seeds than it was in imbibed wild-type seeds. However, the expression level of MsHPPD was significantly higher in imbibed transgenic Arabidopsis seeds. Since vitamin E and ABA are both downstream of GGDP, overexpression of MsHPPD could increase in vitamin E content, thus decreasing the amount of GGDP substrate available for ABA biosynthesis. Expression of the ABA biosynthetic genes NCED3, NCED5 and NCED9 was found to be unaltered in dry seeds; but was significantly reduced in imbibed transgenic Arabidopsis seeds (Fig. 8), suggesting that the reduced GGDP pool size decreased the expression level of ABA biosynthetic genes. In addition, expression of the ABA signaling pathway genes ABI3 and ABI5, which are important for seed germination, decreased significantly in imbibed transgenic Arabidopsis seeds. Based on the above data, it is possible that vitamin E and ABA compete for GGDP, and, thus, elevated vitamin E necessitate reduced ABA levels, and indeed, this is what we observed (Fig. S3). In turn, the reduced ABA content led to a weakening of ABA signaling, ultimately resulting in either a faster-than-normal or higher-than-normal germination rate in the seeds of the transgenic MsHPPD-overexpressing lines, compared with the germination rate of the corresponding control, under various conditions. Moreover, vitamin E has been demonstrated to have additional biological activities besides its antioxidant functions in mammalian systems. Specific tocopherols have been found to modulate signal transduction pathways 38,39 . For instance, tocopherol-binding proteins can regulate gene transcriptional activities 40 . Although a regulatory activity for tocopherols has not yet been directly proven in land plants, the present data raises the possibility that vitamin E also modulates signal transduction pathways in plants. Specifically, tocopherol may modulate seed germination and other hormone responses by binding with other proteins to regulate the ABA signaling pathway. Additional work is necessary for these hypotheses to be investigated in future studies.
Scientific RepoRts | 7:40625 | DOI: 10.1038/srep40625 In conclusion, an HPPD gene that encodes a 434 amino acid protein, named MsHPPD, was isolated from alfalfa. MsHPPD was shown to be induced by multiple treatments, and overexpression of MHPPD in Arabidopsis increased the total vitamin E content. Seed germination of the transgenic lines was accelerated under normal conditions, and was less strongly inhibited under stress treatments. Overexpression of MsHPPD was also found to significantly reduce the free ABA content, as well as the expression of ABA biosynthetic genes and ABA signaling pathway genes. This represents the first evidence that vitamin E is involved in regulating germination rate via affecting the ABA biosynthesis and the subsequent ABA signaling pathway.

Methods
Plant materials and growth conditions. Arabidopsis (Columbia-0) was used for these present experiments. Seeds were surface sterilized with 70% ethanol for 2 min and 5% sodium hypochlorite for 5 min, and then washed three times with water. The sterilized seeds were planted on half-strength MS medium (2.2 g/L Murashige and Skoog salts, pH 5.7, and 8 g/L agar), and kept at 4 °C for 2 days to stratify the seeds and facilitate seed germination. The Arabidopsis plants were grown under long-day photoperiod conditions (16 h light/8 h dark at 25 °C). Seeds from Medicago sativa L. cv Zhongmu No. 1 were germinated on wet filter paper, and the seedlings were grown under long-day photoperiod conditions as above, after the appearance of the first true leaf.  Quantitative real-time PCR. Total RNA was isolated using TRIzol (Invitrogen, USA) and treated with DNase I to eliminate DNA contamination. One microgram of total RNA was then used for cDNA synthesis using the SuperScript III First-Strand Synthesis System (Invitrogen, USA). qRT-PCR was performed with an ABI 7500 Real-Time PCR System (Bio-Rad, USA) using the SYBR Green PCR Master Mix (Takara, Japan) and the appropriate primers ( Table 1). The relative gene expression levels were calculated by 2 −ΔΔCt 44 . At4g26410 45 and Actin 25 were used as reference genes to normalize the expression levels of the target genes in Arabidopsis and alfalfa, respectively. All experiments were performed using three biological replicates and three technical replicates.

Extraction and HPLC analyses of tocopherols and tocotrienols.
Extractions and analyses of tocopherols and tocotrienols were performed according to the method described before 25 .
Seed germination. MsHPPD-overexpressing and wild-type Arabidopsis seeds were surface-sterilized as described above and planted on half-strength MS medium with the presence or absence of 100 mM NaCl, 5 μ M ABA, or 10 μ M ABA. After being stratified at 4 °C for 3 days, the seeds were transferred to a growth chamber (16 h light/8 h dark at 25 °C) and germination rate was monitored every 12 hours.
ABA measurement in Arabidopsis seeds. For each replicate, approximately 20 mg (dry weight) of seeds were homogenized under liquid nitrogen, weighed, and extracted for 24 h in cold methanol and 2H6-ABA (internal standard, OlChemIm, Czech Republic). Endogenous ABA was purified and measured as previously described 47 with some modifications to the detection conditions. LC-MS/MS analysis was performed on a UPLC system (Waters) coupled to the 5500 QTRAP system (AB SCIEX, USA), and a BEH C18 column (1.7 mm, 2.1 × 150 mm; Waters) was used for LC separation with mobile phase 0.05% HAc (A) and 0.05% HAc in ACN (B). The gradient was set with initial 20% B and increased to 70% B within 6 min. ABA was detected in MRM mode with transition 263/153. Finally, quantitation was performed using the isotope dilution method.
Statistical analysis. Statistical analyses were performed by two-tailed Student's t-test. The difference was considered significant at P < 0.05. Data presented are mean ± SD.