Effects of StAAP1 and StAAP8 in Potato ATFs Transcription Factor Family on Plant Development CURRENT STATUS: UNDER REVIEW

Background: Amino acid transporters mediated the transport of various amino acids in plants, which were essential for plant growth and development. ATFs (the amino acid transporter family) was one of amino acid transporters in plants and AAPs (amino acid permease) subfamily belongs to the ATFs family. Results: In this research, eight AAP genes were identified in the potato and divided into two subgroups. The first subgroup is the StAAP gene containing six microporous transport channels (Pore-lining), and the second subgroup are the StAAP gene containing seven microporous transport channels. The subcellular location and overexpression vectors were built by StAAP1 and StAAP8 . The results indicated these two genes were localized on the cell membrane. Observing the phenotype of the transgenic plants, StAAP1 and StAAP8 promoted the growth of leaves, StAAP1 changed the shape of tubers and enhanced their weight and StAAP8 was no significant influence in tubers but decreased the weight a little. Conclusions: In this study, the expression pattern showed StAAP8 might regulate the transport of amino acids in potato roots and affect the osmotic potential of potato root cells. Thereby, the tolerance of plants to abiotic stress and StAAP1 and StAAP8 promote the amino acid transport into leaves and StAAP1 had effected on tuber development and StAAP8 might resist the weight of tubers.

The AAPs family had been participated a variety of activity pathways, such as absorbing amino acids, loading phloem amino acids, transferring amino acids, storing seeds and other aspects [17]. AtAAP1 was expressed in root tip, root epidermis, and root hair cells and it had important for uptake of acidic and neutral amino acids during the soil solution concentration exceeds 50 mM [18]. On the contrary, AtAAP5 played an important role in the intake of basic amino acids during the low soil solution concentration [19]. OsAAP3 and OsAAP6 also could promote the absorption of amino acids in the roots [20,21].
Tan showed the loading of phloem amino acids was very important for the entire physiological process of plants [22]. Although the amino acid transporters involved in this process had not been identified, but related research suggests that it might be AAPs [23]. The tracer experiments indicated that AtAAP2 was key for xylem to phloem transfer and sink N and C supply which according to expression studies of genes of N/C transport and metabolism in source and sink, and by phenotypic and metabolite analysis of AtAAP2 mutant [24]. And the decreased amino acid levels in phloem in AtAAP6 mutant also explained the important role of AAP genes in the transfer of amino acids from xylem to phloem.
Moreover, the growth and development of AtAAP8 mutant was affected due to reduced loading of amino acids in the phloem and content in the pool. And Hammers showed embryos of AtAAP1 mutant seeds had low levels of amino acids and protein levels in seeds after ingesting amino acids, and AtCAT6 remedied the lost function of AtAAP1 to avoid seed development [25]. Overexpression of VfAAP1 could be enhancing the protein levels and increasing the weight of seeds in cotyledons of broad beans and wild peas [26]. Overexpression OsAAP3 could increase some amino acid concentration but the total amino acids has not changed. And knock out OsAAP3 could enhance grain number per spike, yield per plant and protein content in rice seeds [20].
In recent years, some researchers indicated the expression of AAP was affected by plant growth and environmental factors [27][28][29]. The expression of AtAAP4 and AtAAP6 were decreased during the salt stress [30], and the expression of AAP genes could be affected when the plant attacked by pathogens.
Similarly, the expression levels of some genes were significantly up-regulated or down-regulated during some abiotic stresses in rice [16].
As an important crop, there were few related researches on potato amino acid absorption and transport. The growth of potato needed a plenty of nitrogen and these were influenced the tubers formation and yields. Zahir indicated that tryptophan could significantly promote the absorption of nitrogen by potatoes, and could also increase the concentration of potassium, nitrogen and phosphorus in tubers [31]. To further clarify the AAP genes, we provided support to the function of StAAP1 and StAAP8 in potato development.

Identification of StAAPs and analysis of their physical and chemical parameters
The result of blast showed 8 genes were belonged to AAPs, and these proteins had uniquely transcript and gene sequences. In addition, each annotation report for each protein were amino acid transporter on the PGSC website. these 8 AAP transcript factors were StAAP1 (PGSC0003DMP400008841), StAAP2 (PGSC0003DMP400010690) and StAAP8 (PGSC0003DMP400020470).
The basic physical and chemical parameters of StAAPs could be acquired from ProtParam website and these information which included Number of amino acids, theoretical pI, aliphatic index, instability index, grand average of hydropathicity (GRAVY) were showed in Table 1. The amino acid composition and some other information were not shown in this table. But we could clearly observe that 3 kinds of amino acid, alanine, isoleucine and valine, had higher frequency in StAAPs. The theoretical pI were greater than 8 which meant they were all basic proteins. Otherwise, the aliphatic index more than 95 and GRAVY more than 0.40 indicated they were hydrophobin proteins and instability index showed they were stable proteins. Plant-mPloc website could predict the proteins function sites and almost every StAAPs were located on cell membrane except StAAP5, it was consistent with the characteristics of AAP proteins were a hydrophobic protein. Only StAAP5 might be located in Golgi apparatus to play biological functions.

Analysis of StAAPs transmembrane structures
The results from NCBI-CDD showed that StAAPs all belonged to SdaC (cl26160) superfamily which was amino acid permease and the function of amino acid transport and metabolism. Through PSIPRED Workbench website to predict the transmembrane structures in each proteins and 8 StAAPs all had 9 to 10 transmembrane domains (Fig 1). Analysis of gene structure showed that the N-terminus of 3 genes, StAAP3, StAAP5 and StAAP6, was extracellular and C-terminus of all AAP genes was intracellular. According to the transmembrane domain prediction results, we could divide StAAPs into two categories. one was StAAPs with 6 micro Pore-linings, including StAAP1, StAAP2, StAAP3, StAAP4, StAAP7 and StAAP8. And the other was 7 micro Pore-linings, including StAAP5 and StAAP6. The prediction of membrane protein topology and signal peptides showed that no signal peptide sequence existed in the polypeptide chain of 8 StAAPs. These results further explanation that StAAP proteins were related to transport and amino acid channel proteins which located on the cell membrane.

Analysis of StAAP Protein Structure
The predication result displayed that 8 StAAPs had similar secondary structures (Fig 2A). we analyzed the protein secondary structures showed a-helix was the highest proportion in all structures in each protein. Among them, the polypeptide chain with the highest α-helix was StAAP1, which was ratio reached 46.99%. And the lowest was StAAP6,which was also reached 43.01%. The number of involved in the a-helix composition was greater than 200 amino acids in 8 polypeptide chains. on the contrary, b-turn was lowest proportion in secondary structures, and it was existed less than 4% in each protein.
The ratio of other secondary structures were random coil and extended strand from high to low.
We also predicted tertiary structure of StAAPs by Phyre2.0 website (Fig 2B). The result showed that accurately predicted ratio of amino acid residues greater than 90%, which meant the result of Phyre 2.0 website were credible and the proportion of accurately predicted amino acid sites is greater than 95% in 8 StAAPs. So that, the prediction of protein tertiary structure was high reliable. We analysis of protein secondary structure and tertiary structure indicated, a-helix played an important role in the spatial structure of AAP and it might be a core element of the transmembrane domain, which formed the amino acid transport channel on cell membrane. The predictions and simulations illustrated that 8 StAAPs were amino acid permease enzymes which located on the cell membrane, and they were mainly responsible for absorption and efflux of amino acids between cells.

Collinear analysis and Phylogenetic tree construction about StAAPs
The collinear analysis showed that StAAP1, StAAP2, StAAP3 and StAAP8 had colinearity genes and some genes were not belonged to StAAPs (Fig 3). Besides, StAAP1/StAAP8 and StAAP2/StAAP4 were colinearity genes and these 2 pair genes were also segmental duplication gene pair, respectively. This phenomenal might indicate these genes were relatively conservation and had important functions to maintain plant adaptation in hole evolution. Interestedly, StAAP2 had 6 colinearity genes in hole potato genome, and these genes were annotated Proline transporter, Isoform 2 of Lysine histidine transporter, Amino acid transporter and some other annotation. That could be identified StAAP2 was a transporter from the other way and its family members also were transporter.
We used 5 plant species' AAP genes to construct phylogenetic tree, which including S. tuberosum, N. tabacum, S. lycopersicum, Z. mays and model plant A. thaliana (Fig 3). According to the results of phylogenetic tree, the AAP genes could be divided into two subgroups, namely Subgroup I and Subgroup II. For the StAAP gene family, subgroup II contained only StAAP5 and StAAP6, which was consistent with the predicted results of protein transmembrane structure. At the same time, the AAP family genes in 5 plants were distributed in both subgroups, which further indicates that the AAP genes can be divided into two types according to the structural and functional characteristics of the genes that guided the production of proteins. These two types of AAP genes might perform different functions in plants.

The expression analysis of StAAPs
The potato RNA-seq expression database was acquired from Massa [32] which contained expression changes not only between different plant tissues but also under biotics, abiotics, and hormone treatments. We selected StAAPs to analyzed their expression patterns in various tissues and treatments and processed the RNA-seq database and generated a heatmap (Fig 4). Twelve different tissues, leaves, tubers, sepals, stamens, stolons, flowers, petioles, petals, roots, shoots, callus and carpels were used to analyze the expression sites in different genes (Fig 4). the results showed that This indicated that StAAP8 gene, as an amino acid transport channel protein, was also involved in the response of potato to abiotic stress. Combining with potato tissue-specific gene expression data, it was known that StAAP8 gene might affect the osmotic potential of potato root cells by regulating amino acid transport in potato roots, thereby increasing the plant's tolerance to abiotic stress such as drought. Based on the above analysis results, it could be seen that StAAP8 was more important in the other StAAP genes, and because the StAAP1 gene was a colinear relationship with the StAAP8 gene.
Thus, we chosen StAAP1 and StAAP8 for gene function research.

Subcellular location of StAAP1 and StAAP8 proteins
In order to determine the subcellular localization of StAAP1 and StAAP8 genes, pEGFP-StAAP1 and pEGFP-StAAP8 subcellular localization expression vectors were constructed, and agrobacterium injection was used to infect tobacco leaves after 36 hours to observe the StAAP1 and StAAP8 genes expression localization with pCEGFP empty vector as control (Fig 5). The distribution of GFP signals in the cells of the control group was not specific, while StAAP1 and StAAP8 were localized on the cell membrane.
Subcellular localization analysis found that StAAP1 and StAAP8 genes were localized on the cell membrane, indicating that these two genes mainly played a role in the cell membrane, and it was speculated that they might participate in the transmembrane transport of amino acids. For the followup studied of the biological functions of StAAP1 and StAAP8 provided the foundation.

Overexpression of StAAP1 and StAAP8
We selected pCambia1300 vector which contained CAMV35S promoter to rebuild the overexpression vectors. And using these vectors could study the function of StAAP1 and StAAP8 genes through agrobacterium-mediated potato genetic transformation method. Figure 6A showed the callus formation, differentiation and rooting, and this method had high conversion efficiency. We selected some plants which had normally rooting and good growth potential for identification from a molecular way.
In order to obtain the real transgenic plants, the PCR tested the DNA could distinguish the positive plants, primarily (Fig 6B). Due to pCambia1300 vector contained hygromycin resistance gene

The expression of transgenic plants detection
In order to determine the expression changing of transgenic plants, we used qRT-PCR to identify the expression of 12 transgenic plants and analyzed their change to ensure the higher one was selected to observe their phenotypes (Fig 7). StAAP1 and StAAP8 transgenic plants had up-regulated expression compared to wild Desiree. Figure 7A indicated the highest enhance was OXStAAP1-5 which was increased about 22 times. Similarly, OXStAAP8-10 was increased about 28 times from figure 7B.
Thus, we could confirm these transgenic plants were positive and some plants have good expression enhancement.

Analysis of the phenotypic changes in transgenic plants
We respectively selected three StAAP1 and StAAP8 transgenic plants planted into soil in the greenhouse. According to our observation showed no significant difference in plant height, but the leaves of two kinds of genes' transgenic plants were obviously bigger than wild plant. The numbers of leaves were existed some difference, the overexpression StAAP8 transgenic plants were less than wild and the StAAP1 plants were not different ( Fig 8A).
Moreover, we acquired the tubers and collected them to analyze the size and shape (Fig 8B). The results showed the number of wild and transgenic potato tubers were 10 to 12, which were not obviously difference. But StAAP1 tubers were bigger than wild tubers and StAAP1 tubers were longer than wild, but the total weight was no significant change. Unlike StAAP1 tubers, StAAP8 tubers were smaller and lighter than wild and the shape of tubers were not changed. Their homologous gene, AtAAP6, had opposite phenotype. The AtAAP6 mutant had larger rosette width than the wild type and seeds from the AAP6 mutant were also significantly larger than those from the wild-type plants [33].
Eventhough they had different results but they all influenced the leaves and StAAP1 and StAAP8 were related to potato tubers formation.

Bioinformatics analysis of StAAP
In this study, the StAAP gene family transmembrane domain analysis, protein spatial structure prediction, and phylogenetic tree analysis were used to preliminary classify the StAAP gene family members into two groups. This classification was consistent across tobacco, tomato, Arabidopsis and corn.
Combining the location of StAAP genes and their colinearity genes on chromosomes showed that the members of the StAAP genes and their corresponding collinear genes were not evenly distributed on the chromosomes. this phenomenon might be because they were some segmental duplications and the functional of each gene duplication pairs might existed some difference. Among the members of the StAAP gene family, there was a collinear relationship between the two pairs of genes StAAP1 / StAAP8 and StAAP2 / StAAP4, which indicated that these genes might have gene duplication in the genome during the evolution process and have important physiological functions in the life process of potatoes.

Expression pattern analysis of StAAP
Expression pattern analysis showed that the expression of StAAP8 gene was significantly increased under ABA treatment, salt stress and mannitol treatment which indicated that StAAP8 not only as an amino acid transport channel protein but also involved in the response of potatoes to abiotic stress.
On the other hand, the tissue-specific gene expression showed that StAAP8 gene might affect the osmotic potential of potato root cells by regulating the transfer of amino acids in potato roots, thereby the tolerance of plants to osmotic stress-related abiotic stresses were increased.

Identification of StAAPs and analysis of their physicochemical properties
Download potato proteins sequences to blast the StAAP proteins which using AtAAPs as a query and review the descriptions to each protein in order to preliminarily predict the transcription factor family members in potato 1 . some other information were predicted from ProtParam 2 and Plant-mPloc 3 .

Predictive analysis of transmembrane and protein structures
We can use the MEMSAT-SVM model in PSIPRED Workbench website 4 to predicted the protein transmembrane domain. TOPCPNS website 5 was predicted membrane protein topology and signal peptides. SOPMA website 6 and Phyre 2.0 website 7 are using to predict protein secondary structure and tertiary structure of protein.

Collinear analysis and Phylogenetic tree construction about StAAPs
Eight StAAP proteins are searching for their collinearity in hole potato protein sequences by MCScanX.
Simultaneously, 27 AAPs, from S. lycopersicum, N. tabacum, Z. may, S. tuberosum and A.thaliana, are used to construct the phylogenetic tree by MEGA 7 software and these protein sequences are downloaded from Sol Genomics Network 8 and Phytozome 9 . Finally, these results are combined and showed through circos-0.69 10 .

The expression analysis of StAAPs based on RNA-seq data
We select 12 tissues, 4 phytohormone treatments, 4 biotic treatments and 3 abiotic treatments from transcriptome sequencing results. And reference the data treatment from Zhang to visualize the expression [34].

Plant material growth conditions
The potato material (Desiree, which is self-preserved by the laboratory's resource library) was grown with 20 g/L sugar on solid MS medium with vitamins (MS, USA) as culture medium for four weeks in a plant incubator at 25±1°C under 10 000 Lx in light for 16 h and 20±1°C under 0 Lx for 8h.
The seeds of N. tabacum, which is self-preserved by the laboratory's resource library, was planted in the greenhouse for 4-6 weeks at 23°C under 10 000 Lx in light for 16 h and 20°C under 0 Lx for 8h.

Subcellular location of StAAP1 and StAAP8
Designing the primer consisted of some vector sequences which were next to the restriction enzyme cutting site, SpeI, and some purpose gene sequences which include initiation codon but no

Overexpression of StAAP1 and StAAP8
Using same method to construct the recombine vectors and the primers were (StAAP1-OE-

Ethics approval and consent to participate
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Consent for publication
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Availability of data and materials
All data generated or analyzed during this study are included in this published article.   Figure 1 Prediction of transmembrane domain of potato AAP gene family members. The gray rectangle indicated cytomembrane, upside was extracellular and downside was cytoplasm.
The yellow rectangles were transmembrane regions and the lines means pore-lining regions.
StAAP3, StAAP5 and StAAP6 had 10 transmembranes and others had 9 transmembranes.       weeks. One month cultivated from greenhouse to observe the growth situation. B: Phenotypes of the transgenic potato. Collected potato tubers after planting in the greenhouse for three months to observe their potato type, size and numbers.

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