Docking analysis
To determine whether SiFPGS1, SiFPGS2 could bind THF as substrate, and SiFPGS3 could bind DHP as substrate, the molecular docking analysis was taken on. It was predicted that SiFPGS1 and SiFPGS2 had the lowest binding energy of -9.6 kcal·mol− 1 and − 9.2 kcal·mol− 1 with THF, respectively, SiFPGS3 had the lowest binding energy of -8.7 kcal·mol− 1 with DHP. THF had a strong hydrophobic interaction with Pro173, Thr154, Ser151, Thr171, Gly150, Lys147, Glu180, Ser172, His340, Ser422, Ala390, Cys385, Cys426, Asp415 and an electrostatic interaction with Cys385 of SiFPGS1. Three hydrogen bonds holding the SiFPGS1 to THF were formed at Arg183, Gln387, Arg389 with a 2.91 Å, 2.89 Å, 3.04 Å, respectively (Fig. S2a). Similarly, THF had a strong hydrophobic interaction with Ile417, Ala409, Ser414, His334, Gly144, Arg175, Pro167, Glu174, His168, Gln381, Ser166, Leu169, Leu380 and an electrostatic interaction with Asp407 of SiFPGS2. Five hydrogen bonds holding the SiFPGS2 to THF were formed at Lys141, Trp421, Arg383, Glu235, Thr165 with a 3.06 Å, 3.12 Å, 3.17 Å, 3.05 Å, 3.12 Å, respectively (Fig. 2). DHP had a strong hydrophobic interaction with Ser119, Gly387, Arg363, Ser393, Pro121, Lys97, His 324, Leu 123, Gly 98, Leu 360, Pro 361, Lys 95 of SiFPGS3 and an electrostatic interaction with Glu191, the hydrogen bond holding the SiFPGS3 to DHP was formed at Gly96 with a 2.96 Å (Fig. S2b). The results revealed that SiFPGS1, SiFPGS2 could dock with THF, SiFPGS3 could dock with DHP, which suggested that SiFPGS1, SiFPGS2 protein conformation were fit for binding THF as substrate, while SiFPGS3 protein conformation was fit for binding DHP as substrate.
Subcellular prediction analysis and promoter prediction analysis
Further, we predicted SiFPGSs protein subcellular localization by ProtComp software (http://linux1 softberry com), and the results showed that SiFPGS1 scored of 5.61, 4.25, 0.1, 0.04 in the chloroplast, cytoplasm, extracellular and cell membrane, respectively. Similarly, SiFPGS2 scored of 5.57, 4.24, 0.13 and 0.04 in the chloroplast, cytoplasm, extracellular and cell membrane, respectively. However SiFPGS3 made a score of 2.36, 2.14, 1.31, 1.29, 1.20, 1.09. 0.61 and 0.04 in extracellular, peroxisome, mitochondria, chloroplast, golgi apparatus, endoplasmic reticulum, cytoplasm and cell membrane, respectively (Table 1). The result revealed that SiFPGS1, SiFPGS2 made the highest score in the chloroplast, SiFPGS3 made the highest score extracellular, which suggested that maybe SiFPGS1, SiFPGS2 localize in the chloroplast, and SiFPGS3 localize extracellular.
Table 1
Subcellular localization analysis of SiFPGS proteins
Subcellular location | Integral |
SiFPGS1 | SiFPGS2 | SiFPGS3 |
Cytoplasm | 4.25 | 4.24 | 0.61 |
Chloroplast | 5.61 | 5.58 | 1.29 |
Extracellular | 0.10 | 0.13 | 2.36 |
Peroxisome | 0.00 | 0.00 | 2.14 |
Nucleus | 0.00 | 0.00 | 0.00 |
Mitochondria | 0.00 | 0.00 | 1.31 |
Endoplasmic reticulum | 0.00 | 0.00 | 1.09 |
Golgi apparatus | 0.00 | 0.00 | 1.20 |
Cell membrane | 0.04 | 0.04 | 0.04 |
Vacuole | 0.00 | 0.00 | 0.00 |
To further predict which signal pathway SiFPGSs may take part in, cis-acting element prediction analysis was carried on, and the result showed that the three genes shared the common necessary core elements, such as light response element (TCT-motif), MeJA response element (CGTCA-motif), anaerobic induction element (ARE motif) and abscisic acid response element (ABRE motif). SiFPGS1 specially possessed defense and stress response element (TC-rich repeats motif), seed-specific regulation element (RY-motif); SiFPGS2 specially possessed root specific element (motif I), meristem expression element (CAT-box motif); SiFPGS3 specially possessed low temperature responsive element (LTR motif) and anoxic specific inducibility element (GC-motif) (Fig. 3, Table 2).
Table 2
Cis-acting elements analysis of SiFPGS genes family of foxtail millet
cis-acting element | Position |
SiFPGS1 | SiFPGS2 | SiFPGS3 |
light response element | TCT-motif | 1060, 1079 | 55, 665, 885, 988, 1157, 1640 | 411, 623, 973, 1139, 1225, 1313, 1743, 1834 |
MeJA response element | CGTCA-motif | 1337, 1431 | 1223, 1676 | 45, 637, 1185, 1285 |
anaerobic induction element | ARE motif | 1301, 1977 | 444, 1025, 1945 | 1724 |
abscisic acid response element | ABRE motif | 769 | 1678 | 363, 1120, 1121, 1124, 1187 |
gibberellin response element | GARE motif | 1576 | 854, 1987 | - |
salicylic acid response element | TCA-motif | - | 1401,1420,1899, 1914 | 1711 |
zein metabolism regulation element | O2 motif | - | 1498 | 1180, 1188 |
defense and stress response element | TC-rich repeats motif | 1083 | - | - |
seed-specific regulation element | RY-motif | 867 | - | - |
meristem expression element | CAT-box motif | - | 1001, 1750 | - |
root specific element | motif I | - | 1174 | - |
anoxic specific inducibility element | GC-motif | - | - | 118 |
low temperature response element | LTR motif | - | - | 1020 |
Over-expression SiFPGS2 gene in Arabidopsis increased folates content
The phylogenetic tree analysis,the protein function analysis and docking analysis results suggested that SiFPGS1, SiFPGS2 homologue to each other and possessed the tetrahydrofolypolyglutamate synthase activity, belong to FPGS family; whereas,
SiFPGS3 was similar to DFA subfamily and possessed dihydrofolate synthase activity. Subcellular localization predicted that SiFPGS1, SiFPGS2 localized in the choloroplast, SiFPGS3 localized extracellular. Taken together, the bioinformation assays indicated that SiFPGS1 was homologue to SiFPGS2, while SiFPGS3 belong to DFA family, in the following research we choose SiFPGS2 as target gene to avoid gene redundancy. To further detect the gene function, SiFPGS2 were over-expressed in Arabidopsis, the transgenic lines were verified by genomic PCR (Fig. S3a), and the lines with highest expression level #3, was chosen out by qRT-PCR (Fig. S3b). The 5th -7th leaves one month old of wild type and over-expression lines were harvested and the folates content were measured, respectively, and the result showed that 5-CH3-THF, 5-CHO-THF, 5,10-CH = THF and THF content in wild type was 43.40, 6.06, 47.81, 8.04 µg·100g− 1, they were increased to 51.12, 6.24, 53.31, 9.23 µg·100g− 1 in SiFPGS2 over-expression line, respectively, which suggested that over-expression of SiFPGS2 could increase the THF and its derivatives content; whereas in SiFPGS2 over-expression line DHF was decreased to 0.75 µg·100g− 1 compared to 1.83 µg·100g− 1 in wild type (Fig. 4).
Over-expression SiFPGS2 in Arabidopsis promote root growth
It was reported that Atdfb/Atfpgs1 mutant show a short root phenotype (Srivastava et al. 2011b), above result showed that SiFPGS1, SiFPGS2 and AtFPGS1, AtFPGS2 possessed tetrahydrofolypolyglutamate synthase domain, it was possible that SiFPGS2 could function in root development, we tested the root length of SiFPGS2 over-expression lines. Firstly, fpgs1 (SALK_032554c), fpgs2 (SALK_008883c), fpgs3 (SALK_038762c) T-DNA insertion mutant was ordered from Arashare corporation (http://arashare.cn), and the homozygous lines were tested by genomic PCR (Fig. S4). As in Fig. S5, after grown in white light chamber for 7 days, fpgs2 mutant root length was shorter than Col-0, fpgs3 show a little shorter than Col-0, while there was no significant difference between fpgs1 and Col-0, so fpgs2 mutant was chosen as control. The root length in SiFPGS2 over-expressed lines reached 1.5 to 2 times compared to Col-0 (Fig. 5a, b), meanwhile the seedling weight in SiFPGS2 over-expressed lines #3 reached 1.6 times compared to Col-0, while in #1 transgenic lines there were no significant difference, the result above suggested that SiFPGS2 promote root development (Fig. 5c).
To further detect how SiFPGS2 function in root development, propidium iodide (PI) staining was performed according that PI, a red-fluorescent nuclear stain, could not cross intact plasma membrane but could penetrate damaged or dead cells, so it could accumulate at the viabile cells membrane. The major root of Col-0 and SiFPGS2 over-expressed lines grown in white light chamber for 4 days was cutted and stained with 180 µM PI, then the root architecture was detected by confocal, the root apical meristem zone length was measured and the division zone cell number was counted from the stem cell around the quiencent center to the cell that the cell length is the same as cell width in cortex (Fig. 6a), the result showed the root apical meristem zone length of Col-0, fpgs2, 35S::SiFPGS2/Col-0 was 0.3, 0.22, 0.31 mm, respectively (Fig. 6b), and there were 42, 32, 44 cells of root apical meristem zone in wild type, fpgs2 mutant and SiFPGS2 over-express lines, respectively (Fig. 6c), which suggested that SiFPGS2 accelerated the root apical meristem zone cell division and promoted the root growth.