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Transcriptional and protein structural characterization of homogentisate phytyltransferase genes in barley, wheat, and oat

Abstract

Background

Homogentisate phytyltransferase (HPT) is the critical enzyme for the biosynthesis of tocopherols (vitamin E), which are the major lipid-soluble antioxidants and help plants adapt to various stress conditions. HPT is generally strictly conserved in various plant genomes; however, a divergent lineage HPT2 was identified recently in some Triticeae species. The molecular function and transcriptional profiles of HPT2 remain to be characterized.

Results

In this study, we performed comprehensive transcriptome data mining of HPT1 and HPT2 in different tissues and stages of barley (Hordeum vulgare), wheat (Triticum aestivum), and oat (Avena sativa), followed by qRT-PCR experiments on HPT1 and HPT2 in different tissues of barley and wheat. We found that the common HPT1 genes (HvHPT1, TaHPT1s, and AsHPT1s) displayed a conserved transcriptional pattern in the three target species and were universally transcribed in various tissues, with a notable preference in leaf. In contrast, HPT2 genes (HvHPT2, TaHPT2, and AsHPT2) were specifically transcribed in spike (developmentally up-regulated) and shoot apex tissues, displaying a divergent tissue-specific pattern. Cis-regulatory elements prediction in the promoter region identified common factors related to light-, plant hormone-, low temperature-, drought- and defense- responses in both HPT1s and HPT2s. We observed the transcriptional up-regulation of HvHPT1 and HvHPT2 under various stress conditions, supporting their conserved function in environmental adaption. We detected a clear, relaxed selection pressure in the HPT2 lineage, consistent with the predicted evolution pattern following gene duplication. Protein structural modelling and substrate docking analyses identified putative catalytic amino acid residues for HvHPT1 and HvHPT2, which are strictly conserved and consistent with their function in vitamin E biosynthesis.

Conclusions

We confirmed the presence of two lineages of HPT in Triticeae and Aveninae, including hexaploid oat, and characterized their transcriptional profiles based on transcriptome and qRT-PCR data. HPT1s were ubiquitously transcribed in various tissues, whilst HPT2s were highly expressed in specific stages and tissue. The active transcription of HPT2s, together with its conserved cis-elements and protein structural features, support HPT2s’ role in tocopherol production in Triticeae. This study is the first protein structural analysis on the membrane-bound plant HPTs and provides valuable insights into its catalytic mechanism.

Peer Review reports

Background

Tocopherols, together with tocotrienols, are the major forms of vitamin E, which are produced exclusively by photosynthetic organisms, such as plants, cyanobacteria, and algae [1]. Vitamin E is the major lipid-soluble antioxidant synthesized in plants and essential human diet nutrients [2, 3]. Despite its crucial nutritional value, studies show that the recommended dietary amount of vitamin E (15–30 mg/day) was often not met [4]. There are four isomers of tocopherols (α, β, γ, and δ), which differ only in the number and positions of methyl substituents on the chromanol ring [5]. Of these, α-tocopherol has the highest vitamin E activity, protecting polyunsaturated fatty acids from lipid peroxidation by quenching free radicals in cell membranes and other lipophilic environment, thus enhancing plant tolerance to biotic and abiotic stresses, such as cold, drought, salinity, heavy metal, high light, and bacterial infection [5,6,7,8,9,10,11,12,13].

The biosynthesis of tocopherols starts with the formation of homogentisate (HGA) via the shikimate pathway. Homogentisate phytyltransferase (HPT), together with homogentisate geranylgeranyl transferase (HGGT) are the key enzymes catalyzing the condensation of HGA and the saturated C20 isoprenoid phytyl diphosphate (PDP) and geranylgeranyl disphosphate to form 2-methyl-6-phytylbenzoquinone [14, 15]. Next, different isomers tocopherols were synthesized via methylations and cyclization of the aromatic ring, which are sequentially catalyzed by 2-methyl-6-phytylbenzoquinone methyltransferase, tocopherol cyclase, and γ-tocopherol methyltransferase [16,17,18]. Among the candidate genes in the tocopherol biosynthesis pathway, the biological function of HPT has been well elucidated in plants. In Arabidopsis, HPT is highly expressed in almost all of tissues and organs, including the root, leaf, flower and the embryo of caryopsis [18]. Overexpression of Arabidopsis HPT (VTE2_1) resulted in up to a 4.4-fold increase in total tocopherol levels in leaves and up to a 2-fold increase in seeds [19, 20]. On the other hand, the targeted mutation of VTE2_1 completely eliminated tocopherol production, indicating this gene is the gene limiting tocopherol biosynthesis in Arabidopsis [21]. Tocopherol deficiency in vte2 mutant resulted in severe defects in seed longevity, germination and early seedling growth [21] and increased sensitivity to low temperatures [12, 22]. In addition to Arabidopsis, the biological function of HPT genes in tocopherol production has also been confirmed in major crops such as barley [23], rice [24], oat [25], apple [26], and oil palm [27].

Despite the universal conservation of HPT in plants, the number of paralogous HPT genes can vary greatly across different species. In most cases, this is mainly caused by variations in their ploidy levels. For example, a single copy of HPT has been found in Arabidopsis [21], rice [28], tomato [29], oil palm [30], whilst 3 HPTs were reported in hexaploidy oat [25], 3 HPTs in hexaploid wheat [31], and 2 HPTs in rapeseed [32]. These observations indicate that the copy number of HPT tends to be highly conserved. However, our recent studies [23, 31] showed that a divergent lineage of HPT2 is present in Triticeae plants such as barley and wheat, representing an interesting observation for this gene family. Particularly, the retention of the divergent HPT2 in barley and wheat were suggested to be related to environmental adaption, such as cold tolerance [31]. In previous studies, the transcription pattern of HPT has been directly associated tocopherol accumulation in various crops [23, 26, 30, 33], which may play a critical role in plant growth under various stress conditions. Indeed, up-regulation of genes related to vitamin E biosynthesis and vitamin E accumulation has been reported in several species under low temperature [22, 34,35,36,37], light intensity [28, 38], drought [39], and bacterial infection [10]. Therefore, it would be interesting to investigate the transcriptional and molecular profiles of these divergent HPTs in Triticeae, which may have important implications for understanding their biological function and potential use in crop breeding for improved vitamin E content and stress tolerance.

The present study aims to characterize the transcriptional and protein structural profile of HPT in barley, bread wheat, and oat, and explore their impact on vitamin E synthesis. We searched HPT genes in 7 cereal crops and confirmed the presence of the divergence HPT2 lineage in Trticeae and Aveninae. Transcriptional analyses based on qRT-PCR and public RNAseq data were performed for HPT1 and HPT2 in barley, bread wheat, and oat. Natural selection analyses identified potential amino acid sites under positive selection. Comprehensive protein structural modelling and substrate-docking analyses were performed to investigate the active sites responsible for tocopherol production. The potential application of this divergent HPT2 lineage in crop breeding for enriched vitamin E content was discussed.

Results

The presence of a divergent lineage of homogentisate phytyltransferase (HPT2) in Triticeae and Avenidae

Using HvHPT1 as the query sequence, we performed genome-wide screening of HPT homologues in 4 Triticeae (H. vulgare, T. aestivum, T. turgidum subsp. dicoccoides, T. intermedium), 2 Avenidae (A. eriantha, A. sativa), plus a reference species B. distachayon. The total gene number ranged from 2 in B. distachayon, 3 in H. vulgare and A. eriantha, 6 in T. intermedium, 7 in T. aestivum, to 9 in A. sativa. A maximum likelihood phylogeny was developed to examine their evolutionary pattern. As shown in Fig. 1, the target genes were divided into two major clusters, representing the HPT and HGGT lineages, respectively. In the HGGT lineage, the numbers of HGGT genes in the target species were generally consistent with their ploidy levels with the exception of A. sativa, which displayed a species-expansion with 5 HGGT genes, whilst T. turgidum has one only. In the HPT lineage, the single HPT gene in B. distachayon diverged first in accordance with the species phylogeny. Then, the HPT genes in Triticeae and Avenidae species diverged into 2 lineages, corresponding to a duplication event in their common ancestor as indicated in our previous study [31]. The first lineage HPT1 (highlighted in blue) displayed a relatively shorter phylogeny branch and corresponds to the ancestral HPT gene in these species. The second lineage HPT2 (highlighted in red) had a relatively longer phylogeny branch, indicating a more divergent sequence profile. In term of gene copy number, HPT2 is preserved as a single copy in the 6 target species, while the number of HPT1 is generally in agreement with species ploidy levels, with the exception of T. intermedium (tetraploid), which has 3 HPT1 (Fig. 1). Noteworthy, Triticeae and Aveninae HPTs separated into 2 distinct clusters in the HPT1 lineage, whilst barley HvHPT2 (HORVU.MOREX.r3.2HG0208140.1) and T. intermedium TiHPT2 (Thint.V1245000.1) seem to have a relatively closer relationship (Fig. 1; branch support 0.84) with A. eriantha AeHPT2 (AE040009.mRNA1) and A. sativa AsHPT2 (AVESA.00010b.r2.2CG0326330.1), implying an unusual amino acid sequence profile.

Fig. 1
figure 1

Displays the phylogeny of HPT and HGGT homologues in Triticeae and Avenidae. The phylogenetic tree was developed using ML method based on the amino acid sequence alignment. Branch supports based on 1000x bootstrapping were indicated above each branch. HPT1 and HPT2 lineages in Triticeae and Aveninae were highlighted in blue and red, respectively. Sequence alignment and phylogenetic tree are available at Supplementary Data S1. Natural selection pressure (dN/dS) for different lineages were indicated accordingly

Transcriptome analyses of HPT2 and HPT1 in barley, wheat, and oat

To explore the transcriptional profiles of HPT1 and HPT2, public transcriptome databases in barley (https://ics.hutton.ac.uk/eorna/index.html), wheat (http://www.wheat-expression.com/), and oat (https://wheat.pw.usda.gov/GG3) covering various tissues and developmental stages were consulted. In barley, HvHPT1 (6 alternative transcripts in Fig. 2A) was universally transcribed in all tissues, including root, shoot, leaf, spike, and grain. The highest expression of HvHPT1 was observed in the leaf, followed by root and shoot, with relatively lower expression in grain. In contrast, HvHPT2 (Fig. 2B) displayed a clear tissue-specific pattern and was only actively transcribed in the shoot apex and spike, and relatively weaker in the leaf. Furthermore, HvHPT2 has a relatively weaker expression than HvHPT1. In bread wheat reference genome (Chinese Spring, EnsemblPlants release 56, https://plants.ensembl.org/Triticum_aestivum/Info/Index), where only HPT1s (TaHPT1_7A, TaHPT1_7B, TaHPT1_7D; Fig. 2C) were present, all three HPT1s were widely expressed in various tissues: the highest in leaf, followed by shoot, root, and spike, and the lowest in grain, which is similar to that observed for HvHPT1. Particularly, TaHPT1_7D seemed to be transcribed relatively higher than TaHPT1_7A and TaHPT1_7B, implying a potential sub-genome bias in gene transcription. In hexaploid oat, the three HPT1 genes (AsHPT1_1, AsHPT1_2, AsHPT1_3; Fig. 2D) were also found widely transcribed in various tissues: exceptionally high in leaf, followed by root and spike, and the lowest in grain. In contrast, their HPT2 homologue (AsHPT2; Fig. 2E) was specifically transcribed in the spike (glume) and weakly in the leaf. In summary, we observed a conserved transcription pattern for HPT1 and HPT2 across barley, wheat, and oat. Particularly, we found the divergent lineage HPT2 was specifically transcribed in the spike (potentially in the husk or glume part), whilst HPT1 was universally expressed in various tissues with leaf as the highest.

Fig. 2
figure 2

Transcriptional atlas of HPT1 and HPT2 in barley, wheat, and oat. A The transcriptional profile of HvHPT1 (6 alternative transcripts). B The transcriptional profile of HvHPT2. C The transcriptional profile of HPT1s in bread wheat. D The transcriptional profile of HPT1s in oat. E The transcription profile of AsHPT2 in oat. Transcriptome data for barley, wheat, and oat were obtained from EORNA [40], Wheat Expression Browser [41], and GrainGenes [42] databases, respectively. Corresponding transcriptional data was available in Supplementary Data S2

qRT-PCR verification of the transcription of HPT1 and HPT2 in barley and wheat

Due to tocopherols’ proven function under various stress conditions, the specific transcription of HPT2 in spike tissue above is particularly interesting. To gain more insights and also verify the transcriptional profiles of HPT1 and HPT2, qRT-PCR experiments were performed in barley and bread wheat lines containing the HPT2 gene. In barley, three tissues (FL: flag leaf; ST: stem; SP: spike/inflorescence) at two stages (S1: 3 cm inflorescence; S2: spike heading) for three cultivars (Hindmarsh, Latrobe, RGT_planet) were analysed. Results (Fig. 3A) showed that HvHPT1 was upregulated from stage S1 to S2 and was expressed the highest in FL, followed by ST, and least in SP. Among the three cultivars, Hindmarsh displayed consistently higher HvHPT1 transcription, implying transcriptional variation across cultivars. In contrast to HvHPT1, HvHPT2 (Fig. 3B) was preferentially transcribed in the spike tissue at both stages in all cultivars, consistent with public transcriptome data. Overall, HvHPT2 seemed to display a relatively weaker transcription level than that of HvHPT1, consistent with the transcriptome data, although the qRT-PCR primer amplification efficiency needs to be calibrated to directly compare the expression levels of these two genes.

In wheat cv. Lancer, which contains the HPT2 gene, inflorescence at five different stages (1 cm, 2 cm, 4 cm, 6 cm, and 8 cm) and flag leaf, stem, and husk (at the anthesis stage) was were studied. Overall, wheat HPT1 genes (TaHPT1_7a, 7b, 7d; Fig. 3C and E) displayed conserved expression profiles across tissues and were all actively transcribed at comparable levels, the highest being in flag leaf, relatively lower in husk and stem. The preferential expression of wheat HPT1 genes was similar to barley HvHTP1 and was also consistent with the above transcriptome data. Compared to HPT1s, TaHPT2 (Fig. 3F) also displayed relatively weaker transcription: the highest expression in flag leaf, followed by husk, but barely expressed in stem and inflorescences. The active transcription of TaHPT2 in wheat husk and leaf is similar to that observed for HvHPT2 and public transcriptome data.

Fig. 3
figure 3

qRT-PCR analyses of HPT1 and HPT2 in barley and bread wheat. A HvHPT1 and B HvHPT2 in 3 tissues (FL: flag leaf; ST: stem; SP: spike) at 2 stages (S1: 3 cm inflorescence; S2: spike heading) in 3 cultivars. C TaHPT1_7a, D TaHPT1_7b, E TaHPT1_7d, and F TaHPT2 in wheat cultivar Lancer (inf: indicate inflorescence; DPA: days post anthesis). Primer sequences are available at Supplementary Data S3

Cis-acting element, gene structure, and stress response analyses of HPT1 and HPT2

The transcriptional divergence between HPT1 and HPT2 indicated potential genetic variation in their regulatory regions. Therefore, in this study, we extracted the putative promoter regions of the HPT1 and HPT2 genes across the four Triticeae and two Aveninae species in this study and predicted the cis-acting elements. Results (Fig. 4A) showed that the promoter regions of both HPT1 and HPT2 were enriched with various cis-elements related environment adaption, particularly high in light-responsive and anaerobic induction, followed by low-temperature responsive and drought-inducibility, which is consistent with vitamin E’s well-known function in various stress tolerance. In addition, various cis-elements related to plant hormones, including abscisic acid, gibberellin acid, jasmonic acid, auxin, and salicylic acid were also identified. Noteworthy, for both HPT1 and HPT2 lineages, we noticed that abscisic acid elements were more prevalent in Aveninae than in Triticeae. Within the HPT2 lineage, low-temperature element was only detected in HvHPT2 and TaHPT2, whilst it is more enriched in most HPT1 genes, indicating varied regulatory binding sites for these two lineages. Overall, we found generally conserved cis-elements in the putative promoter region of HPT2s, such as light responsiveness, anaerobic induction, drought-inducibility, and jasmonic acid responsiveness, supporting their potential function in stress adaption. Regarding gene structure, results (Fig. 4B) showed that HPT1 and HPT2 contain 12 exons with conserved exon length, supporting their close relationship as gene duplicates. The exceptions are TdHPT1a and TdHPT1b from T. turgidum subsp. Dicoccoides, which contained partial gene fragments compared to other HPTs, may be caused by gene annotation error.

To verify the transcriptional responses of HPT1 and HPT2 under stress conditions, the public barley transcriptome data with various stress treatments was consulted. Results (Supplementary Data S2, HvHPT_stress) showed that HvHPT1 was ubiquitously transcribed in most barley tissues and were generally up-regulated under various stresses, such as disease infection (spot blotch), cold stress, waterlogging, drought, salinity, heat, and continuous light regimen. Consistent with our above transcriptional analyses, significant expression of HvHPT2 was only observed in inflorescence and spike tissues of those varieties where it is present, such as Bowman (Supplementary Data S2). Notably, the transcription of HvHPT2 could reach a comparable level with that of HvHPT1 at specific tissues and stages, such as the shoot apex, awn primordium (5 mm), white anther (10 mm), apical, and inflorescence tissues (Project ID: PRJEB39672, PRJEB34648, and PRJEB8748; Supplementary Data S5), further supporting its critical role in inflorescence development. Interestingly, we also observed clear up-regulation of HvHPT2 in the spike and inflorescence tissues under stress conditions, including long day (PRJEB8748), and drought (PRJEB12540), which are consistent with the cis-element prediction and support HvHPT2’s positive role in stress adaption.

Fig. 4
figure 4

Cis-acting elements and gene structures of HPTs in 6 Triticeae and Aveninae species. The predicted cis-acting elements (A) and gene structure (B) were displayed using HPT phylogeny as the order. CDS: code region sequence; UTR: untranslated region. Corresponding promoter region sequences and cis-acting elementary prediction results are available in Supplementary Data S4

Natural selection analyses

To assess the evolution rates of HPT1 and HPT2 in Triticeae and Aveninae, we calculated the ratio (ω) of non-synonymous (dN) to synonymous (dS) substitutions using the branch-specific models based on the developed ML phylogeny (Fig. 1) in this study. Three lineages were specified for ω calculations: HPT1, HPT2, and background HPTs, corresponding to ωHPT1, ωHPT2, and ωBG, respectively. Under the three-ratio model (ωHPT2 ≠ ωHPT1 ≠ ωBG), ωHPT1, ωHPT2, and ωBG were estimated at 0.20821, 0.60389, and 0.39162, respectively (Fig. 1; Supplementary Data S5), which showed that the selection pressure on HPT2 is significantly higher than that from HPT1, indicating clearly relaxed selection pressure, whilst the latter is under relatively higher purifying selection.

Protein structural modelling

To investigate if there were any potential functional divergence for HPT2 at the protein structural level, we performed amino acid sequence alignment for HPT1 and HPT2 in Triticeae and Aveninae (Fig. 5). Overall, HPT2s have 73.7% aa identity with HPT1s, whilst HPT2s and HPT1s share 80.7% and 90.7% identity with themselves, respectively, implying HPT2s are relatively more relaxed than HPT1s. The strong similarity between HPT2 and HPT1 implied that they may still share a potentially conserved function. Consistently, a highly conserved UbiA prenyltransferase family domain (aa125-aa386; PF01040) could be identified, which included two conserved aspartic-acid-enriched motifs DX[D/E]XD and DXXDXXXD (indicated by black arrows in Fig. 5), which are widely conserved in the UbiA superfamily [43]. Protein domain analyses indicated that both HvHPT1 and HvHPT2 are membrane-bound proteins, with an N-terminal cytoplasmic domain (aa 1-113 using HvHPT2 as reference), belonging to the HOMOGENTISATE SOLANESYLTRANSFERASE, CHLOROPLASTIC (PTHR43009) family. Among the 15 amino acid sites previously identified to be under positive selection in HPT2, most of them displayed clear amino acid substitutions in our sequence alignment (underscored by solid star symbols in Fig. 5).

Fig. 5
figure 5

Amino acid sequence alignment of HPT homologues in Triticeae and Avenidae. Sequence alignment was trimmed and numbered using HvHPT2 as a reference. The red highlight indicates the conservation level at each site. HPT1s and HPT2s were separated by a black line. Amino acid sites under positive selection were underscored by a solid star symbol. Two conserved D-enriched motifs were indicated by downward arrows. Major helices were indicated by blue dashed lines above

To assess the potential effect of these amino acid substitutions on enzyme activity, 3-dimensional structural models for HvHPT1 and HvHPT2 were created without the N-terminal cytoplasmic regions, which lack structural template. Substrate molecules homogentisate (HGA) and phytyl diphosphate (PDP) for HPT were also docked to the protein models to determine the substrate binding site. The overall folds of HvHPT1 and HvHPT2 are highly conserved, consisting of 10 main transmembrane helices (light orange in Fig. 6A-left; blue dash lines in Fig. 5) and connecting loops, similar to the archaeal UbiA protein (Fig. 6A-right; PDB: 4OD5) for which the protein structure has been experimentally determined [43, 44]. Based on structural superimposition with 4OD5, the substrate binding sites were determined to be located at the central cavity between the helix bundles. Notably, three short helices (Fig. 6A; highlighted in light pink) on top of the substrate binding pocket which cap the active sites after substrate binding [44] seem to be well conserved in our models. The two highly conserved aspartate-rich motifs (Fig. 6B; highlighted in pink) were located in these helix cap, coordinating the Mg2+ ion, playing an essential role in HPT function. The spatial location of the 15 selected amino acid sites were displayed (red) in Fig. 6B. Most of these residues are located on the transmembrane helices exposed to the exterior surface, except for 164S and 167D, which are positioned next to the conserved DXXXD motif (Figs. 5 and 6B). The electrostatic and hydrophobicity profile of HPT models were displayed in Fig. 6C and D, which showed that HvHPT1 and HvHPT2 have a conserved positively charged (thus hydrophilic) substrate binding pocket, which become more hydrophobic as it extends deeper into the transmembrane helices (Fig. 6C and D). Substrate docking in HvHPT1 and HvHPT2 indicated that the phytyl donor PDP may first bind deep into the active site, followed by HGA binding at the entrance.

Detailed examination of the interacting amino acid residues with the docked substrates revealed putative amino acid sites responsible for substrate binding in barley HPT. As shown in Fig. 6E (HvHPT1) and F (HvHPT2), 20 amino acid residues (109S, 110R, 112H, 154N, 161N, 165D, 166I, 167D, 169D, 173K, 225Y, 228D, 233R, 235K, 236R, 249R, 297P, 298D, 305F, 380Y) within 3 Å of the docked substrates were identified with putative substrate binding interactions, all of which were found be conserved between HvHPT1 and HvHPT2. The hydrophobicity profiles of these putative substrate binding residues are shown in Fig. 6G, where two modelled Mg2+ ions were coordinated by 165D and 169D, while 110R, 161N, 173K, 225Y, and 235K may be critical for the positioning of the di-phosphate groups in PDP, facilitating the cleavage and transfer of the phytyl group to HGA. The highly conserved substrate binding site for HvHPT1 and HvHPT2 supports the conserved function of HvHPT2 in tocopherol biosynthesis. In addition to those highly-conserved substrate-binding residues, amino acid substitutions between HvHPT1 and HvHPT2 were generally observed away from the substrate-binding sites, which may have little effect on substrate-binding. Indeed, under the optimal binding formations in our substrate docking analyses, the binding affinities for HGA and PDP to HvHPT1 and HvHPT2 were estimated at -10.72 and − 10.71 kcal/mol, respectively, similar to each other.

Fig. 6
figure 6

Protein structural modelling of HvHPT1 and HvHPT2. A Displays the overall structures of HvHPT1 and HvHPT2 (left) and 4OD5. B Displays the spatial positions of docked substrates Mg, HGA, and PDP, the interacting aspartic acid residues (pink), and positively selected amino acid sites (red) in HvHPT1 (left) and HvHPT2 (right). C Displays the electrostatic profiles of HvHPT1 and HvHPT2. D Displays the hydrophobicity profiles of HvHPT1 and HvHPT2. E Displays the substrate-interacting amino acids of HvHPT1 (numbering in HvHPT1). F Displays the substrate-interacting amino acids of HvHPT2 (numbering in HvHPT2). G Displays the hydrophobicity profile of the conserved substrate binding sites in HvHPT1 and HvHPT2 (numbering in HvHPT2). All amino acid site numberings were according to HvHPT2 unless otherwise specified. The generated protein models with docked substrate molecules are available in Supplementary Data S6

Discussion

HPT is the crucial enzyme responsible for tocopherols biosynthesis in plants. Previous studies have shown that this gene is strictly conserved in various plant genomes [21, 28,29,30]. Despite varied copy numbers of HPT have been reported in some species, most of which are caused by different ploidy levels [25, 31], whilst species-specific duplication and divergent evolution of HPT have rarely been observed. However, in our recent studies [23, 31], we noticed a divergent lineage of HPT2 present in Triticeae and Avenieadeae, which resulted from a dispersed gene duplication in their common ancestor. Most importantly, HPT2 is only partially retained in some pangenome lines, which seems to be closely related to environmental adaption [31], implying an active role for HPT2 in these plants. In this study, we confirmed the presence of HPT2 lineage and found that HPT2 is also present in hexaploid A. sativa cv. Sang, which has not been found before. The failure to identify HPT2 in hexaploidy A. sativa was due to the use of A. sativa cv. OT3098 (V2), in which HPT2 was not annotated. This further highlighted the limitation of using a single reference genome in genomic research.

The presence of a divergent lineage of HPT2 in Triticeae and Aveninae motivated us to investigate its biological function. At the transcriptional level, HvHPT2 was previously shown to be transcribed specifically in the husk tissue of barley grain [31]. In this study, we extracted public transcriptome data covering various tissues and stages and found that HvHPT2 is not only transcribed in spike but also in shoot apex, suggesting additional biological functions for this gene. In addition, TaHPT2 and AsHPT2 were also found to be specifically expressed in the spike/glume, displaying a conserved expression pattern with HvHPT2. Considering that the major function of vitamin E in low temperature and other stress responses [45], the specific transcription of HPT2s in spike in this study is particularly interesting, indicating that HPT2 may play a particular role in protecting these vulnerable tissues under stress conditions. Indeed, we found various plant hormones (auxin, GA, JA) responsive cis-regulatory elements in the promoter regions of HPT2s. Low-temperature responsive cis-regulatory elements were also detected for both HvHPT2 and TaHPT2, albeit not for the other HPT2s. In barley, we confirmed the preferential transcription of HvHPT2 in the young developing spike tissues using qRT-PCR in three Australian barley cultivars, which also showed clear transcriptional variations of HvHPT2 across different cultivars. Future studies may be needed to identify high-expression alleles for HvHPT2, which may be exploited for breeding and genetic engineering purpose. Particular attention should be given to those wild barley lines collected from Tibet, in which HvHPT2 was found to be generally conserved [31]. The absence of HPT2 in the bread wheat reference genome Chinese Spring limited our ability to uncover its complete expression profile in different tissues. However, qRT-PCR in one wheat cultivar, Lancer containing TaHPT2, showed that TaHPT2 was preferentially transcribed in the husk and flag leaf tissues, implying both shared and divergent expression profiles with HvHPT2. Further study may investigate its transcription in other wheat lines containing HPT2. In addition to HPT2s, we also examined the transcriptional profiles of HPT1s in barley and wheat, which displayed a conserved and wide-spread expression pattern, consistent with those reported in other species and studies [18, 25, 27, 28, 46].

Due to HPTs’ functioning as membrane-bound proteins, the experimentally determined structure of proteins in the UbiA superfamily is very limited, which hindered our understanding of the enzyme function of HPT at the protein structural level. To date, only two membrane-bound prenyltransferases from archaeal organisms have been structurally characterized [43, 44]. As far as the authors are concerned, our study is the first to create 3D models for plant HPTs and identified their putative substrate binding sites by substrate docking, revealing valuable structural insights into plant HPTs in tocopherol biosynthesis. Identifiying substrate-interacting amino acid residues may allow future engineering highly specific and efficient HPT mutants, which can be used for improved vitamin E production in crop breeding and genetic engineering. Indeed, transgenic expression of both HPT and HGGT has been widely performed to improve vitamin E content in many important crops such as cotton [47], barley [23], soybean [48, 49], tomato [26, 50], and lettuce [51]. The catalytic efficiency of HPTs from different species is clearly different. Our structural models confirmed the critical role of the conserved DXXDXXXD and DX[D/E]XD motifs in substrate binding in the UbiA superfamily. We found these two motifs form short helices on top of the catalytic cavity and are responsible for Mg2+ coordination, similar as that observed for their archaeal counterparts [43, 44]. Despite of only 73.7% amino acid identity between HPT1 and HPT2, we found that all of the putative substrate binding sites were strictly conserved between these two lineages of proteins, consistent with our previous transgenic expression analyses of HvHPT2, which was shown to be functional in tocopherol biosynthesis [31]. Moreover, our substrate docking analyses indicated that HvHPT1 and HvHPT2 may have similar substrate binding affinity, providing further support that HPT2 is a fully functional enzyme in tocopherol production.

Amino acid substitutions and transcriptional divergence are common observations during gene functional divergence following gene duplication [52]. Particularly, amino acid substitutions at the protein active sites could lead to immediate changes in either substrate binding specificity and/or binding affinity and play an important role in plant phenotypic diversification [53]. Well-known examples include the divergence between F3’H and F3’,5’H responsible for the red and blue anthocyanin productions, respectively [54], and the divergence between sorbitol dehydrogenase and L-idonic acid dehydrogenase for the sorbitol and tartaric acid productions, respectively [55]. Notably, within the vitamin E biosynthesis pathway, HPT and HGGT displayed varied substrate preferences [18], responsible for the production of tocopherols and tocotrienols, are also believed to be caused by amino acid substitutions in the active binding sites. Despite that we found no obvious substrate specificity changes between HPT1 and HPT2, the structural models created in this study may lay the foundation for future studies to uncover the molecular basis for the varied enzyme activities between HPT and HGGT. Many protein divergences after gene duplication lead to the accumulation of novel metabolites and confer environmental advantages are driven by natural selection [53, 55, 56]. We previously detected 15 amino acid sites in HPT2 under positive selection compared to HPT1 [31], which suggested that the emergence and retention of HPT2 in Triticeae and Aveninae may be associated with plants’ environmental adaption. In this study, we examined the spatial location of these amino acid sites and found that these amino acid changes may not directly affect substrate binding, but instead are likely related to membrane interaction due to their generally presence in the exterior surface of the protein structure. The biological significance of these selected amino acid changes in HPT2, together with its potential use in crop breeding and genetic engineering need to be examined with further in-depth functional analyses.

Conclusions

We confirmed the presence of two lineages of HPT in Triticeae and Aveninae, including hexaploid oat and characterized their transcriptional profiles based on transcriptome and qRT-PCR data. We found that HPT1s were ubiquitously transcribed in various tissues and species, whilst HPT2s were only highly expressed in specific stages and parts of the spike tissue. We revealed the transcriptional up-regulation of HvHPT1 and HvHPT2 under various stress conditions in barley. The active transcription of HPT2, together with its conserved cis-elements and protein structural features, support HPT2s’ active role in tocopherol production in Triticeae. will facilitate future breeding for cereal crops with improved tocopherol content and nutritional values. This study is the first protein structural analysis on the membrane-bound plant HPTs and provides valuable insights into its catalytic mechanism.

Methods

Identification of HPT homologues and phylogeny development

Genomic datasets for barley (H. vulgare cv. Morex V3), wild emmer (Triticum turgidum subsp. dicoccoides Zavitan V2), bread wheat (T. aestivum cv. Lancer V2.1), Thinopyrum intermedium (V3.1), Avena eriantha (id53381), A. sativa cv. Sang (V1.1), and Brachypodium distachyon (V3.1) were downloaded from Phytozome 13 (https://phytozome-next.jgi.doe.gov/), GrainGenes (https://breadwheat.pw.usda.gov/GG3/), EnsemblPlants (release 56, http://plants.ensembl.org/info/index.html), or the species-specific website and were used for gene identification. Genuine HPT homologues were identified using the method as described in our previous study. Amino acid sequence alignment was performed using MUSCLE program (8 iterations) [57]. Phylogeny was developed using IQ-TREE program [58] with JTT + G4 substitution model. Branching support was calculated based on bootstrapping for 1000 times. Final tree annotation was performed in Figtree (v1.4.3, http://tree.bio.ed.ac.uk/software/figtree).

RNA extraction and qRT-PCR analyses

Barley and wheat plants were grown in pots under a natural light glasshouse. Plant tissues were frozen in liquid nitrogen immediately after sampling and were ground into a fine powder using a pre-cooled pestle and a mortar. Total RNA was extracted using 100 mg of grounded powder using Trisure® (Bioline, Australia). SensiFAST™ cDNA Synthesis Kit (Bioline, Australia) was used for cDNA library construction following product instructions.

SensiFAST™ SYBR No-ROX Kit (Bioline, Australia) was used for RT-qPCR experiments with a reaction volume of 10 µl, containing 5 SensiFAST mix, 4.2 µl cDNA sample, 0.8 µl primers (500 nM). For each sample, three biological replicates with 2 technical replications were included. ViiA7 Real-Time PCR System (Thermo Fisher Scientific, United States) was used for the RT-qPCR reaction. RT-qPCR primers specificity was validated by melting curve analyses. Reference genes HvActin in barley and TaACT-1 in wheat [59] were used. Gene expression values were calculated using the comparative Ct method (2–ΔCt).

Transcriptome data mining

The transcriptional data for HPT-encoding genes were extracted from EORNA RNAseq database (https://ics.hutton.ac.uk/eorna/index.html) and BarleyExpDB (http://barleyexp.com/index.html) for barley [40, 60], Wheat Expression Browser (http://www.wheat-expression.com/) for bread wheat [41], and GrainGenes (https://wheat.pw.usda.gov/GG3/). Raw transcriptional value in transcripts per million read was plotted using Microsoft Excel software.

Promoter binding motif and gene structure analyses

Cis-regulatory elements were predicted using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) website. The 2000 bp sequences upstream of the translation start site for target HPT genes were used as input for PlantCARE prediction. Gene model information was extracted from each species’ corresponding genome annotation files. The identified cis-elements and gene structures were visualized using TBtools program [61] together with the developed phylogeny as a guide.

Natural selection analyses

Natural selection pressure was assessed by measuring the ratio of non-synonymous to synonymous substitutions (ω = dN/dS). Codon-based maximum-likelihood estimates of ω was performed using codeml in PAML4.7 [62]. Codon-based alignment of conserved domain sequences was carried out using MUSCLE. Alignment was trimmed manually using HvHPT2 as the reference. The sub-tree covering HPT only was used as input for codeml. Branch pattern specification was implemented using Treeview1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html).

Protein structural modelling and substrate docking

Protein structural modelling was performed using Google AlphaFold tool [63] using an online interface at https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb. The amino acid sequences of HvHPT1 and HvHPT2 were used as input. For each protein, five models were generated and examined. Only the top-ranking model was selected for downstream analysis. Autodock Vina tool [64] was employed to dock small molecules to the selected model with docking parameters energy_range = 4 kcal/mol, and exhaustiveness = 8. MGLTools (https://ccsb.scripps.edu/mgltools/ ) was used for docking file preparation. For each receptor and ligand combination, the top 9 ranking conformations were evaluated. PyMol (Schrödinger, LLC. Version 2.4.0, http://www.pymol.org/pymol) was employed for model visualizations in this study.

Availability of data and materials

Supplementary files deposited at figshare (DOI: https://doi.org/10.6084/m9.figshare.22983752).

Abbreviations

HPT:

Homogentisate phytyltransferase

HGGT:

Homogentisate geranylgeranyl transferase

PDP:

Phytyl diphosphate

FL:

Flag leaf

ST:

Stem

SP:

Spike/inflorescence

d N :

Non-synonymous substitutions

d S :

Synonymous substitutions

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Acknowledgements

The authors acknowledge the relevant plant community for making the genomic data available to the public.

Funding

This work was supported by the Natural Science Foundation of Zhejiang Province (LQ23C130003), National Science Foundation of China (32301872 and 31971932), China Agriculture Research System of MOF and MARA (CARS-05-05A), Key Research Foundation of Science and Technology Department of Zhejiang Province of China (2021C02064-3).

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CL, HB, NH and TX supervised the study and were involved in data analysis. ZZ, YJ, XH and ZC performed the analyses of gene expression, phylogenetic, natural selection, and protein structure. ZZ and YJ wrote the manuscript.

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Correspondence to Chengdao Li.

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Supplementary Information

Additional file 1: Supplementary Data S1.

Sequence alignment and phylogenetic tree files. Supplementary Data S2. Retrieved transcriptome data for the target HPT genes. Supplementary Data S3. Primer sequences used in qRT-PCR. Supplementary Data S4. Putative promoter region sequences and cis-acting element prediction results. Supplementary Data S5. Natural selection output files. Supplementary Data S6. Generated protein models of HvHPT1 and HvHPT2 with docked substrates HGA and PDP in the optional conformation.

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Zeng, Z., Jia, Y., Huang, X. et al. Transcriptional and protein structural characterization of homogentisate phytyltransferase genes in barley, wheat, and oat. BMC Plant Biol 23, 528 (2023). https://doi.org/10.1186/s12870-023-04535-x

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  • Published:

  • DOI: https://doi.org/10.1186/s12870-023-04535-x

Keywords