Characterization of the GGP gene family in potato (Solanum tuberosum L.) and Pepper (Capsicum annuum L.) and its expression analysis under hormonal and abiotic stresses

GDP-l-galactose phosphorylase (GGP) is a key rate-limiting enzyme in plant ascorbic acid synthesis, which plays an important role in plant growth and development as well as stress response. However, the presence of GGP and its function in potato and pepper are not known. In this study, we first identified two GGP genes in each potato and pepper genomes using a genome-wide search approach. We then analyzed their physicochemical properties, conserved domains, protein structures and phylogenetic relationships. Phylogenetic tree analysis revealed that members of the potato and pepper GGP gene families are related to eggplant (Solanum melongena L.), Arabidopsis (Arabidopsis thaliana L.), tobacco (Nicotiana tabacum L.) and tomato (Solanum lycopersicum L.), with tomato being the most closely related. The promoter sequences mainly contain homeopathic elements such as light-responsive, hormone-responsive and stress-responsive, with light-responsive elements being the most abundant. By analyzing the structure of the genes, it was found that there is no transmembrane structure or signal peptide in the GGP gene family of potatoes and peppers, and that all of its members are hydrophilic proteins. The expression profiles of different tissues show that StGGP1 has the highest expression levels in leaves, StGGP2 has the highest expression levels in stamens, and CaGGPs have the highest expression levels in the early stages of fruit development (Dev1). It was found that StGGPs and CaGGPs genes showed different response to phytohormones and abiotic stresses. Abscisic acid (ABA) treatment induced the most significant change in the expression of StGGPs, while the expression of CaGGPs showed the most pronounced change under methyl jasmonate (MeJA) treatment. StGGPs responded mainly to dark treatment, whereas CaGGPs responded mainly to NaCl stress. These results provide an important basis for a detailed study about the functions of GGP homologous genes in potato and pepper in response to abiotic stresses.

www.nature.com/scientificreports/pepper, CaGGP1 and CaGGP2.The distribution of GGP genes on chromosomes was visualized using the genome annotation files of potato and pepper.The potato GGP genes are unevenly distributed across two chromosomes, and the pepper GGP genes are located on the same chromosome, as shown in Fig. 1.The number of genes on each chromosome is independent of the size of the chromosome.

Analysis of conserved structural domains and gene structure of GGP family members in potato and pepper
Figure 2 shows the conserved motifs of the GGP gene family and visualization of evolutionary relationships, conserved structures and gene structures of potato and pepper GGP genes.The conserved structural domains showed that both potato and pepper GGP families contained 10 conserved motifs, and all members contained identical conserved motifs (Motif), i.e., Motif 1-Motif 10, and the specific sequence information is shown in Table 2.The structural analysis of the genes showed that the GGP genes of the pepper all contain five exons, in addition to CaGGP1, which has four introns, and CaGGP2, which has five introns.However, the gene structures of the potato GGPs are very different.StGGP1 has seven exons and six introns, while StGGP2 has five exons, four introns and a longer intron.In addition, the values of the selective evolutionary pressure showed that both the GGP genes of the potato and the pepper were calculated to be less than 1 and that both had a tendency to be purified (Fig. 2C).

Multiple series comparison and phylogenetic analysis of the GGP family of potato and pepper
The protein sequences of the potato and pepper GGP families have been analyzed.The most conserved sequences are annotated in dark blue, followed by red and blue.As shown in Fig. 3A, the homology between members of the potato and pepper GGP families was as high as 79.62%, indicating that the family is evolutionarily conserved.
To study the evolutionary relationship of potato and pepper GGPs with GGPs of the template crop Arabidopsis thaliana and other species, a phylogenetic tree of GGP protein sequences in Arabidopsis, tomato, eggplant, tobacco, potato, and pepper was constructed.The results showed that the 10 GGP proteins from these plants were categorized into three subgroups (A, B, and C), with StGGP2 and SlGGP1 being the most closely related subgroup (four members).Class B includes two members, and both compose of Arabidopsis GGP proteins.In addition, tomato is the most closely related to potato among the three subgroups, suggesting an evolutionary link between potato and tomato.Meanwhile, the GGP family members of potato and pepper are closely related to those of eggplant, Arabidopsis, and tomato (Fig. 3B).The results of collinearity analysis showed that two pepper genes are collinear with two potato genes, there are two homologous pairs between pepper and potato (Fig. 3C).

Analysis of the role of cis-elements of potato and pepper GGP genes
In order to understand the expression regulation of potato and pepper GGP genes, 2000 bp upstream of the potato and pepper GGP genomes were extracted and submitted to PlantCARE for promoter analysis.The potato predictions revealed seven light-responsive elements (Box 4, Box II, GA motif, GATA motif, G-box, GT1 motif and TCT motif); three hormone-responsive elements, including abscisic acid (ABRE), salicylic acid (TCA element) and (MeJA); and one stress element (LTR).The light-responsive elements were unevenly distributed in Pepper predictions revealed the presence of three light responsive elements (Box 4, GT1-motif, TCT-motif), four hormone responsive elements containing methyl jasmonate (CGTCA-motif, TGACG-motif), gibberellin (P-box) and growth hormone (TGA-element) and three stress responsive elements (TC-rich repeats, GC-motif, ARE).The TCT motif, the MeJA, the P box and the TGA element were present in every GGP of pepper.The stress response element TC-rich repeats were distributed in CaGGP1 and CaGGP2 of pepper, and the anaerobically induced response element GC-motif, ARE, was distributed in CaGGP1 (Fig. 4C, D).

Structural analysis of proteins from the GGP gene family
The analysis of the physicochemical properties of the potato and pepper GGP family revealed that the length of the potato and pepper GGP proteins ranges from 371 to 438aa, and the theoretical isoelectric point pI, except CaGGP2 which is greater than seven, are distributed between 4.71 and 5.79, which belonged to the acidic proteins.The molecular mass of the GGP proteins from the potato and the pepper ranges from 41.5 to 49.7 kDa.In addition, neither the potato nor the pepper GGP family has a signal peptide or transmembrane structure, according to both signal peptide and transmembrane structure predictions.Meanwhile, it was predicted that both potato and pepper GGP proteins may be hydrophilic.They contain more hydrophilic peaks (Table 3).
In addition, the potato and pepper GGP genes were found to be distributed mainly in chloroplasts, cytoplasm, nucleus and mitochondria by subcellular localization.However, the distribution of each member was different in different cellular fractions (Table 4).
The secondary structure of the potato and pepper family GGPs revealed that each member of the potato and pepper family GGPs has the highest percentage of irregular curl (pink line) between 44.29 and 49.87%;The percentage of alpha helices (blue line) is between 22.64 and 33.33%, the percentage of elongations (red line) is between 16.67 and 22.91%, and the percentage of beta folds (green line) is the smallest, between 4.58 and 5.71%    5).Based on the tertiary structure results, the structures of potato and pepper GGP family proteins are consistent with the structural components of the secondary predictions, and the members are modeled very similarly (Fig. 5).

Expression pattern analysis of potato and pepper GGP gene family in different tissues
In order to confirm the tissue-specific expression patterns of StGGPs and CaGGPs, we analyzed the expression levels of the GGPs genes at different stages of growth and in different tissues in potato and pepper.The results showed that the expression of StGGP1 was highest in the leaves, whereas the expression of StGGP2 was highest in the stamens.The pepper GGP gene is expressed in roots, stems, leaves, flowers and fruits, with leaves showing the lowest expression.In addition, the expression of GGPs in pepper was highest at the beginning of fruit development (Dev1), followed by a decrease, and the lowest expression was found in mature green fruits.The expression of the pepper GGP gene family in different tissues and during fruit expansion was extracted from previously published data.These results indicate that the GGPs genes are mainly active in the leaves of the potato, whereas in the pepper, the GGPs are mainly active in the fruits, especially in the pre-ripening of fruits (Fig. 6).
The heatmap is shown in blue/yellow/orange colors indicating low/medium/high expression, respectively.In the figure, ZL1-F-Dev1-ZL1-F-Dev4 represents fruits with lengths of 0-1 cm, 1-3 cm, 3-4 cm and 4-5 cm.In order to understand the expression patterns of the StGGPs and CaGGPs genes under different hormonal treatments and abiotic stresses, we treated potato and pepper seedlings with two hormones (ABA and MeJA) as well as with four stress factors (dark, cold, NaCl and PEG).As shown in Fig. 7, the expression of StGGP1, StGGP2, CaGGP1 and CaGGP2 was differentially upregulated after 6 h stimulation with ABA and MeJA, and their expression levels all peaked at 24 h under ABA treatment.When treated with MeJA, StGGP1 and StGGP2 peaked at 12 h, whereas CaGGP1 and CaGGP2 peaked at 24 h, and the expression levels of the genes gradually declined after having peaked.

Expression profiling of the GGP gene in potato and pepper under darkness, low temperature NaCl, and PEG treatments
Under abiotic stress treatments, both StGGPs and CaGGPs genes were differentially up-regulated after a certain period of stress.Under the duress of darkness, the level of expression of StGGP1 reached a maximum at 6 h and increased by a factor of 14 compared to the level at 0 h.However, the expression levels of StGGP2, CaGG1 and CaGG2 were gradually upregulated after dark stress treatment, and all of them reached the maximum at 24 h.Overall, the relative expression levels of the StGGPs were higher than those of the CaGGPs under the dark stress    treatment (Fig. 8A).The expression levels of StGGPs and CaGGPs were gradually upregulated with time after cold stress treatment, with the difference that the expression levels of StGGPs reached the highest after 12 h, while those of CaGGPs reached the highest after 24 h (Fig. 8B).Under NaCl and PEG stress treatments, the expression levels of both StGGPs and CaGGPs were differentially up-regulated.Under both stress treatments, the expression levels of StGGPs peaked at 24 h, whereas CaGGPs peaked at 12 h, and the expression levels at 48 h were similar to those at 0 h (Fig. 8C.D).

Discussions
AsA is found in a wide range of plant tissues and is a multifunctional metabolite involved in many physiological processes such as photosynthesis, cell wall biosynthesis, seed germination, flowering, fruit softening and ripening, post-harvest storage, and plant resistance to adverse environments 21 .The GGP gene has been identified in several species 13 .In this study, a more comprehensive and systematic analysis of the potato and pepper GGP gene families has been undertaken with the aid of bioinformatic techniques.Four GGP genes (two in potato and two in pepper) have been identified.Meanwhile, the selective evolutionary pressure indicated that there is a trend towards the purification of GGP members in both potato and pepper, and the trend towards the purification of GGP in potato is higher than that in pepper.Using a multi-species phylogenetic tree, we found that the GGPs of potato and pepper are more conserved in evolution than those of Arabidopsis, eggplant and tomato.In addition, most plant GGP genes have similar gene structures and motif patterns, this suggests that plant GGP genes have conserved functions 22 .The analysis of the physicochemical properties of the potato and pepper GGP gene family members showed that the protein sequences of the potato and pepper GGP families differ very little, with amino acid lengths ranging from 371aa to 440aa.With the exception of CaGGP2, which has an isoelectric point greater than 7, the isoelectric point of the rest proteins is less than 7.These results suggest that GGP proteins have a higher content of acidic amino acids.In addition, the instability indices of the potato and pepper GGP families are greater than 40.This indicates that potato and pepper GGPs are unstable proteins.Meanwhile, transmembrane structure analysis and signal peptide prediction indicated that potato and pepper GGP families do not have transmembrane structures as well as signal peptides.These results show the similarity between the StGGPs and the CaGGPs in terms of their physico-chemical properties as well as their gene structure.Previous study www.nature.com/scientificreports/had also shown that most plant GGP genes had similar gene structures and motif patterns 23 .These similarities suggest that the GGP gene may play the same role in growing and developing potato and pepper.
GGPs have different patterns of expression in different plant tissues.Overexpression of kiwifruit GGP in tomato led to a six-fold increase in AsA content in red fruit 15 .In potato we found that the StGGP1 is highly expressed in leaves.In addition, the expression levels of StGGP1 are higher than those of StGGP2 in all tissues except stamens.This leads us to speculate that StGGP1 is the major gene for AsA synthesis in potato.However, in contrast to this, the GGP family of peppers has the lowest expression in the leaves and the highest expression in the fruits.Meanwhile, the expression of CaGGPs genes gradually decreases as the peppers ripened.In addition, the expression of tomato GGP was low in fruits, and the expression tended to decrease with fruit ripening 24 .Similarly, previous study showed that most of the 12 banana varieties contained more AsA in the unripe stage than in the ripe stage 16 .
Study has shown that AsA is involved in photosynthesis as an electron carrier or electron donor, and that the pathway for the biosynthesis of l-galactose is closely linked to photosynthesis 25 .AsA synthesis and GGP expression in Arabidopsis thaliana were induced by high light stress 26 .Similarly, analysis of the roles of the homeopathic components in our study showed that the potato GGP family respondes mainly to light-responsive components (seven) and hormone-responsive components (three), in addition to stress-responsive components.There are also three light-responsive elements in the GGP family of pepper.These results suggest that the GGP family plays a major role in light response.In subsequent experiments we found a significant up-regulation of both StGGPs as well as CaGGPs after dark treatment.This is further evidence that the GGP gene family plays an important role in the response to light.In addition, we found that StGGP1 contains response elements that regulate the circadian rhythm.Meanwhile, previous study has shown that AsA content in leaves is two-fold higher during the day than at night, which may be related to circadian-regulated response elements 27 .Therefore, the potato and pepper GGP families may have an effect on AsA levels through participation in photosynthesis.
Previous study has shown that the overexpression of tomato GGP gene improved fruit quality and resistance 15 .Previous study has shown that the expression of the GGP gene in kiwifruit was induced by light and abiotic stress, and was significantly correlated with the concentration of AsA 28 .MeJA induces chemical defense in plants by stimulating the expression of plant defense genes.MeJA treatment increased the activity of the promoter, induced the expression of GGP in kiwifruit, and enhanced the AsA level 28 .ABA-responsive elements (ABRE) are also important for the enhancement of GGP transcript levels and GUS activities of GGP promoters.In both potatoes and pepper, MeJA-responsive elements were found.However, ABA response elements were found only in potato.Interestingly, in our experiments, after treatment with ABA and MeJA, the expression levels of both StGGPs and CaGGPs were significantly increased.This may be due to the fact that the ABA treatment regulates the expression of other genes in the pepper and thus promotes the expression of the CaGGPs.GGP plays an important role in protecting plants against chilling stress by maintaining the pool and redox state of ascorbate 29 .In addition, our study showed that StGGPs and CaGGPs may also have different responses to cold stress, salt stress and drought stress.Previous studies in rice, leek and lettuce have also shown that the GGP gene plays an important role under high light and abiotic stress conditions [30][31][32] .These results suggest that although potato and pepper are very different in both morphological and environmental characteristics, their GGP genes may play the same role under abiotic stress.However, more studies are needed to understand the specific functions of the GGP gene family in plant growth and response to abiotic stress.

Conclusions
In conclusion, we identified four GGP genes in the whole genomes of potato and pepper, of which potato and pepper each contains two, and all of their members show a trend of purified selection.Meanwhile, there are strong homology and conservation of the GGP family in potato, pepper, tomato, Arabidopsis, tobacco and eggplant.The potato and pepper GGP genes have a genetic basis for responding to a wide range of hormones and stresses.StGGPs and CaGGPs were expressed at different levels in a variety of tissues, with StGGP1 having the highest expression level in leaves, StGGP2 the highest in stamens, and CaGGPs having the highest expression at Dev1.The expression patterns of GGP genes indicate tissue-specific roles across different plant species.In addition, StGGPs and CaGGPs may be involved in the mitigation of abiotic stress and hormonal responses.Meanwhile, StGGPs responded mainly to dark stress, whereas CaGGPs responded mainly to NaCl stress.Therefore, this study may provide further research on the involvement of the GGPs gene family in growth regulation and stress response in potato and pepper.It may also provide a theoretical basis for further exploration of the functions of plant GGP members.

Identification of members of the GGP gene family in potato and pepper
The whole genome data and annotation files for potato and pepper were downloaded from NCBI (https:// www.NCBI.nlm.nih.gov/).Membership information and protein sequences of the tomato GGP gene family were downloaded from Solanaceae Genomics Network, and the tomato database was used to search for identified members of the GGP gene family as template genes.The Blast Compare Two Seqs function of the TBtools software was used for a preliminary comparison to obtain the transcriptome IDs of the candidate members of the potato and pepper GGP gene families.The structural domains of the potato and pepper GGP nominees were viewed using the NCBI Web CD search tool, and the TBtools visualization of NCBI CDD patterns.Candidate proteins with GGP structural domains were members of the potato and chili pepper GGP gene families, and the genes corresponding to these proteins were named StGGP (potato) and CaGGP (pepper) genes.The chromosomal location distribution of the GGP gene was analyzed using the "gene location visualize from GTF/GFF" function of the TBtools software.The GGP genes for potatoes and pepper were renamed based on their location on the chromosome.

Conserved motif and protein conserved domain analysis
The MEME website was used to conduct an online analysis of the shared conserved structural domains of potato and pepper GGP gene families.The structure of the potato and pepper GGP gene families was visualized using the TBtools "gene structure view (advanced) function".

GGP gene family multiple sequence comparison and phylogenetic tree construction
Potato and pepper GGPs were subjected to multiple sequence comparison analysis using DNAMAN Software.
The GGP protein sequences of Arabidopsis, cabbage (Brassica campestris), apple (Malus domestica cv.Gala), and tomato were downloaded from the Arabidopsis, Brassica, NCBI, and Solanaceae databases, respectively.The evolutionary tree was constructed using the MEGA11 software, using the neighbor joining method, with the number of replicates set to 1000 and the rest of the options set to default values.The evolutionary tree was then further modified using an online website (https:// www.evolg enius.info// evolv iew/# mytre es/ clcle/ 123).

Prediction of the role of homeopathic elements of the GGP gene family
TBtools was used to extract data 2000 bp upstream of the potato and pepper GGP genes from the potato and pepper gene databases.The data was submitted to the PlantCARE database (http:// bioin forma tics.psb.ugent.be/ webto ols/ plant care/) for gene homeotic element (promoter) analysis and was visualized using TBtools.

Analyzing protein structure and expression patterns
The Expasy online website (https:// web.expasy.org/ compu te_ pi/) was used to analyze the molecular weights, instability coefficients, isoelectric points and hydrophilicity of individual members of the potato and pepper GGP genes.The WoLF PSORT online website (https:// wolfp sort.hgc.jp/) was used to analyze the subcellular localization of each member of the potato and pepper GGPs.The signal peptides of the GGP proteins of potatoes and peppers were predicted using SignalP (https:// servi ces.healt htech.dtu.dk/ servi ce.php?Signa lP-5.0).The NPS online website (https:// npsa-prabi.ibcp.fr/ cgi-bin/ npsa_ autom at.pl? page= npsa_ sopma.html) was used to predict the secondary structure.The TMHMM online website (https:// servi ces.healt htech.dtu.dk/ servi ce.php?TMHMM-2.0) was used for transmembrane structural analysis.The UniPort online website (https:// www.unipr ot.org/) was used to extract expression data of the potato GGP family in different tissues.The pepper GGP expression profile was analyzed using the transcriptome data set obtained from the sequencing of the pepper genome (https:// www.ncbi.nlm.nih.gov/ geo/ query/ acc.cgi? acc= GSE45 037) and visualized using TBtools 33 .
The pepper variety "Qiang feng 101", provided by the Vegetable Breeding Laboratory, College of Horticulture, Gansu Agricultural University, was used as the test material for this experiment.The pepper seeds were soaked and allowed to germinate, then sown in hollow trays.The seedlings were grown under day/night temperatures of 25 °C/20 °C, relative humidity of about 60%, 14 h of light and 10 h of darkness.When the pepper seedlings had six true leaves, they were treated in the same way as potatoes.
The collected samples were immediately frozen with liquid nitrogen and stored in a vertical ultra-low-temperature refrigerator at −80 ℃ (Qingdao Haier Special Electric Appliance Co., Ltd., Qingdao, China).Each treatment contained three biological replicates.

RNA extraction and qRT-PCR fluorescence quantification
Total RNA was extracted from the samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), taking advantage of the FastQuant First Strand cDNA Synthesis Kit (Tiainen, Beijing, China) to synthesize cDNA (The reaction system was 2 µL RNA, 2 µL 5 × Evo M-MLV Reagent Premix and 6 µL ddH2O).Dilute the cDNA concentration to 500 ng.µL −1 .These reactions were carried out under the following conditions: 37 °C for 15 min, 85 °C for 5 s, and finally ending at 4 °C.LightCycler 480 real-time PCR system (Roche Applied Science, Penzberg, Germany) and SYBR Green Premix Pro Taq HS Premix kit were used for qRT-PCR.The reaction system was 2 × SYBR Green Pro Taq HS Premix 10 µL, primer (The concentration was 10 μ mol.L −1 ) F 0.4 μL, primer

Figure 1 .
Figure 1.Chromosomal location of the GGP gene.(A) Chromosomal location of the potato GGP gene, (B) chromosomal location of the pepper GGP gene, chromosomal numbers shown at top of each bar.Gene names are shown in black.Scale bars are shown on the left.

Figure 2 .
Figure 2. Motif, gene structure and selective evolutionary pressures in the potato and pepper GGP gene families.(A) motif and gene structure of the potato GGP gene family; (B) motif and gene structure of the pepper GGP gene family; on the left is the motif, with different colors representing different motifs; on the right is the gene structure; (C) selective evolutionary pressures (Ka/Ks).

Figure 3 .
Figure 3. Multiple sequence comparisons, collinearity analysis, and phylogenetic trees of the potato, pepper, tomato, eggplant, Arabidopsis, and tobacco GGP gene families.(A) Multiple sequence comparison of potato and pepper GGPs.(B) Phylogenetic trees of the GGP families of potatoes, pepper, tomatoes, eggplants, Arabidopsis and tobacco.The six different species are represented by the six different colored shapes.The red pentagram represents tobacco.The blue pentagram represents Arabidopsis thaliana.The green pentagram represent eggplant.The black squares represent potatoes.The red squares represent tomatoes.The white circles represent pepper.(C) The collinearity analysis of the GGP gene family was conducted in pepper and potato.Syntenic GGP gene pairs are represented by the marked red lines.

Figure 4 .
Figure 4. Analysis of cis-acting elements in potato and pepper.(A) Potato homeopathic component visualization.(B) Potato homeopathic component type statistics.(C) Pepper homeopathic component visualization.(D) Pepper homeopathic component type statistics.Different colored wedges represent different cis elements.The length and position of each GGP gene were mapped to scale.The scale bar represents the length of the DNA sequence.The length and position of each GGP gene were mapped to scale.The scale bar represents the length of the DNA sequence.

Figure 5 .
Figure 5. Three-stage structure model of potato and pepper GGP.Tertiary structural modeling of the potato GGP gene family.(C,D) Tertiary structural modeling of the GGP gene family in pepper.

Figure 6 .
Figure 6.Potato data from UniPort online site (https:// www.unipr ot.org/), pepper data from pepper genome sequencing (https:// www.ncbi.nlm.nih.gov/ geo/ query/ acc.cgi? acc= GSE45 037).Expression analysis of different tissues of potato and pepper GGPs gene families.(A) Expression pattern of potato GGP in different tissues.(B) Expression pattern of pepper GGP in different tissues.(C) GGP expression patterns at different times during the ripening of the fruit of the pepper.Color scale represents fold change normalized by log 2 -transformed data.

Figure 7 .
Figure 7. Relative expression levels of StGGPs and CaGGPs under two hormone treatments.(A) ABA treatment.(B) MeJA treatment.Expression values were normalized to the control (0 h) for each gene.An asterisk indicates a significant difference between the stress-treated and control groups.(p ≤ 0.05, **p ≤ 0.01, oneway ANOV A, Tukey's test).

Figure 8 .
Figure 8. Relative expression levels of StGGPs and CaGGPs under different hormone treatments.(A) Dark stress.(B) Cold stress.(C) NaCl stress.(D) PEG stress.Expression values were normalized to the control (0 h) for each gene.An asterisk indicates a significant difference between the stress-treated and control groups.(p ≤ 0.05, **p ≤ 0.01, one-way ANOV A, Tukey's test).

Table 1 .
Identification of potatoes and pepper GGP gene family.

Table 2 .
Potato and pepper GGP motif information.

Table 3 .
Analysis of physicochemical properties of potato and pepper GGP.

Table 4 .
Subcellular localization of GGP in potato and pepper.

Table 5 .
Distribution of secondary structural elements in potato and pepper GGP.