Genome-wide characterization and analysis of bHLH transcription factors related to anthocyanin biosynthesis in spine grapes (Vitis davidii)

As one of the largest transcription factor family, basic helix-loop-helix (bHLH) transcription factor family plays an important role in plant metabolism, physiology and growth. Berry color is one of the important factors that determine grape quality. However, the bHLH transcription factor family’s function in anthocyanin synthesis of grape berry has not been studied systematically. We identified 115 bHLH transcription factors in grape genome and phylogenetic analysis indicated that bHLH family could be classified into 25 subfamilies. First, we screened six candidate genes by bioinformatics analysis and expression analysis. We found one of the candidate genes VdbHLH037 belonged to III (f) subfamily and interacted with genes related to anthocyanin synthesis through phylogenetic analysis and interaction network prediction. Therefore, we speculated that VdbHLH037 participated in the anthocyanin synthesis process. To confirm this, we transiently expressed VdbHLH037 in grape and Arabidopsis transformation. Compared with the control, transgenic materials can accumulate more anthocyanins. These results provide a good base to study the function of the VdbHLH family in anthocyanin synthesis of grape berry.

protein 7 . Studies have shown that MYB is one of the most important transcription factors regulating anthocyanin biosynthesis 8 . The first MYB transcription factor in plants was found in maize, and was involved in the metabolic regulation of flavonoids 9 . MYB is the most widely distributed transcription factor family, with 138 members in grapes. It was found that MybA regulated anthocyanin synthesis through the expression of UFGT in Kyoho grape 10 . Kobayashi et al. found that white coloration is caused by retrotransposon insertion in MybA and that the same mutant allele has spread among most white grape cultivars in the world 11 . bHLH is also a major transcription factor regulating anthocyanin biosynthesis, which can directly activate DFR expression and can also promote anthocyanin accumulation by interacting with MYB. The WD40 transcription factor associated with coloration was also found in grapes. It was functionally verified and found to be expressed only on colored grapes 12 .
The bHLH domain consists of 50-60 amino acids that can be divided into basic region and HLH region 13 .The basic region at the N-terminus that contains approximately 15 amino acids is responsible for binding to DNA 14 . It has a highly conserved HER motif (His5-Glu9-Arg3) that recognizes a consensus hexanucleotide sequence E-box. Based on the different central bases, E-box is of different types, of which G-box is the most common type. Two amphipathic α-helices of the HLH region consisting of roughly 40 amino acids are separated by a loop of variable length 15 . It can promote the formation of homodimers or heterodimers through interacting with other bHLH proteins 16 .
The bHLH protein family is one of the largest families of TFs in plants; for example, there are 162 bHLH genes in Arabidopsis thaliana 17 , 192 in tobacco (Nicotiana tabacum) 18 , 159 in tomato (Solanum lycopersicum) 19 , 188 in apple (Malus × domestica) 20 , and 113 in strawberry (Fragaria × ananassa) 21 . It is usually divided into 15-26 subfamilies. For instance, 162 bHLH members of Arabidopsis are classified into 26 subfamilies. 230 bHLH proteins in Chinese cabbage (Brassica rapa ssp. pekinensis) are divided into 24 subfamilies 13 . Apple contains 18 subfamilies according to the phylogenetic tree analysis 20 . In plants, transcription factors belonging to the same subfamily have high similarity in structure, motif and protein function 22 . bHLH family are involved in many developmental and physiological processes in plants, such as anthocyanin biosynthesis 23,24 , growth and development 25,26 , and defense response to biotic and abiotic stress 27,28 . Different types of bHLH genes have different biological functions. It has been shown that bHLH III (d + e) subfamily members can enhance plant resistance and regulate anthocyanin synthesis through JA signaling pathway 29,30 . In addition, members of the III (f) subfamily have been proved to be involved in anthocyanin synthesis 31 .
The berry color of Chinese wild grapes is generally black, the spine grape has both black and white berry, which is of great significance for studying the coloring mechanism of grape berry. In this study, we analyzed 115 bHLH transcription factors comprehensively and systematically in spine grape. First, we mapped the bHLH transcription factors on to the 19 chromosomes of grape. Then we analyzed phylogenetic relationships, gene structural features, distribution of key amino acid residues in bHLH domain, conserved motifs and protein interaction network of bHLH transcription factors. Finally, six candidate genes were screened by combining with results of transcriptome sequencing and qRT-PCR validation. We verified VdbHLH037 of III (f) subfamily function by heterologous expression in Arabidopsis thaliana and transient expression in "Kyoho" grapes. The results show that VdbHLH037 plays a positive role in the regulation of anthocyanin synthesis.

Identification and annotation of bHLH transcription factors in grape.
A total of 137 bHLH transcription factors were obtained from EnsemblPlants database. Subsequently, we used the conserved domain to determine the existence of the conserved bHLH domain. Some members without the bHLH domain were removed. Then we analyzed the nucleotide and amino acid sequences through SMART and DNAStar. If the sequences were similar, only one member with the longest sequence was left. Finally, we screened 115 VdbHLH members for the further analysis according to the results of transcriptome sequencing 15 . We collated the information of 115 VdbHLH transcription factors from the Plant Transcription Factor Database (Supplementary  Table S1). We mapped the VdbHLH transcription factors on the 19 chromosomes according to their distributions on the chromosome (Fig. 1). Then we renamed them from VdbHLH001 to VdbHLH108 based on their location on the chromosome 1-19 from the Plant Transcription Factor Database. The remaining six unknown chromosomal loci were renamed from VdbHLH109 to VdbHLH115 in the order of position from the minimum to maximum. We will further study the biological functions of 115 VdbHLH genes, especially for anthocyanin synthesis.
Phylogenetic analysis of the bHLH transcription factor family in grape. In order to better understand the function of grape bHLH protein, we need to assess their evolutionary relationship. Based on 162 Atb-HLHs in Arabidopsis, we constructed an un-rooted phylogenetic tree with the nomenclature protocol according to the multiple sequence alignments of the conserved domains in grape and Arabidopsis 31 (Fig. 2). On the basis of Heim's method, we made appropriate adjustments. For example, I (a + b) subfamily was divided into I a, I b(1) and I b(2) subfamilies. III a subfamily and III c subfamily were merged into III (a + c) subfamily. Ultimately, the grape bHLH family was divided into 25 subfamilies. The members that were not assigned to the 25 subfamilies were classified as "orphans". The plant bHLH family consists of approximately 26 subfamilies 32,33 , whereas there is no bHLH member in the grape X IV subfamily, probably due to the long evolutionary process. We found that the number of members of different subfamilies varies greatly. For example, the largest subfamily X II contains 14 members, while the smallest subfamilies II, VIII a, VIII c(1) and X III contain only one member. The classification of the grape bHLH family provides evidence of their evolutionary relationship.
Gene structure and conserved motif analysis of VdbHLH genes. In order to obtain information about the VdbHLH family, we analyzed the structural characteristics of the VdbHLH family and labeled the  Fig. S1). The results of the structural analysis showed that the number of exons ranged from 1 to 11. Four members had no introns. The average number of exons in 25 subfamilies ranged from 1 (VIII b) to 9 (V a). All four members without introns belong to the (VIII. b) family. Introns can occur anywhere in the gene. If the intron is located between the third nucleotide of one codon and the first nucleotide of another codon, it is called phase 0 intron. Correspondingly, the introns between the first nucleotide and the second nucleotide of a codon are called the phase 1 introns. The introns between the second nucleotide and the third nucleotide of a codon are called the phase 2 introns. This is very important for the process of exon replication. Exons between two identical phases are called symmetric exons, whose nucleotide number is an integer multiple of three, which will not cause the reading frame to be shifted and can be copied. In contrast, asymmetric exons cannot be replicated. It is generally believed that symmetric exons and phase 0 introns can facilitate exon shuffling, recombinational fusion and protein domain exchange 34,35 . It is generally believed that motifs play an important role in the interaction of different modules in transcriptional complexes and transduction of signal 15,31 . In addition, the structure of a motif is closely related to gene classification. Therefore, we analyzed the 20 conservative motifs of the VdbHLH family and their distribution among different subfamilies using MEME ( Supplementary Fig. S2). Furthermore, we also counted the sequence, length and number of occurrences of motifs (Supplementary Table S2). The subfamily IV a has the most types of motifs (nine types) and subfamily X III has the least types of motif (one type). The average number of motifs The conserved amino acid residues in bHLH domain and their ability of DNA-binding. To further understand the function of the VdbHLH family, we performed multiple sequence alignment of 115 Vdb-HLH domains and calculated the percentage of conserved amino acids based on previous results (Fig. 3). The bHLH domain is made up of one basic region, two helix regions and one loop region. The results show that the conservation of basic region and helix region is higher than that of the loop region. The bHLH region of grape is composed of 65 amino acid residues, of which 21 conserved amino acid residues with a consensus rate of more than 50%. Among them, Arg-16, Leu-29 and Leu-65 are extremely conservative, with a consensus rate of more than 90% (Supplementary Table S3). Previous studies have shown that Glu-13, Arg-16 and Arg-17 in the basic region are critical for DNA binding, and Leu-29 and Leu-65 in the helix region are very important for dimerization activity 26 . By comparison, we found that seven amino acid residues (Ile-20, Leu-26, Gln-30, Met-54, Ile-59, Ile-62 and Leu-65) in plants are more conserved than those in animals, indicating that they play a very important role in plants.
It is generally believed that the basic region is critical for DNA binding and the bHLH family to achieve its biological function 36 . According to Atchley's research, the basic region of the bHLH protein has 13 amino acid residues, whereas the grape contains 17 amino acid residues, of which more than five are responsible for DNA binding 37 . The number of amino acid residues in the basic region of VdbHLH089 is less than six, which is considered incapable of DNA binding, so we designated it as a non-DNA binding protein 19 . As previously reported, we have divided the remaining 114 VdbHLH members into three categories: G-box (His/Lys-9, Glu-13 and  38,39 . Based on the conserved amino acid residues in the basic region, we predicted 60 for the G-box-binding proteins, 27 for the non-G-box-binding proteins and 27 for the non-E-box-binding proteins. In addition, one of them was classified as a non-DNA-binding protein (Supplementary Table S4).

The collinear correlations of bHLH genes in grape, Arabidopsis and tomato.
Comparative genomics is a powerful tool for quickly understanding, localizing and cloning unknown genes by comparing genes and genomic structures. The rationale is that there is interspecific and intraspecific synteny. The synteny refers to the similarity in location and sequence of genes on interspecific and intraspecific homologous chromosomes 40 . The synteny analysis of the genome shows a large number of collinear regions in the genome that can be used as direct evidence for the existence of whole-genome duplication (WGD) 41,42 . Ohno first proposed the hypothesis of polyploidization, that is, genome-wide replication, to explain the way in which diploid genomes evolve into tetraploids through WGD 43 . In 1997, Wolfe et al. confirmed the WGD hypothesis in Saccharomyces cerevisiae genome 44 . In 2000, the first plant genome-wide sketch was sequenced and analyzed. The evolutionary history of Arabidopsis genome suggests that it has undergone two whole-genome duplications (WGD: α and β) and one whole-genome triplication (WGT: γ) 45 . In 2007, the genome of grapes was sequenced. Studies have shown that the ancestral genome of grapes may be paleohexaploid 46 . The study of grape genome duplication events laid the theoretical foundation for hexaploid sharing of the ancestral genomes of dicotyledons and opened doors for the study of the evolutionary history of ancestral genomes in angiosperms. Subsequently, the whole-genome sequencing of 14 fruit trees species was completed. The whole-genome sequencing results of these fruit tree species have established a huge resource platform for fruit tree molecular biology research that not only helps in understanding their genome structure and functions but also has important guiding significance for exploring the origin and evolution of fruit trees, locating and cloning important functional genes and accelerating molecular breeding. Comparative genomics confirmed that the grapes did not undergo any additional genome-wide replication event, while the ancestors in Solanaceae experienced an independent WGT event after differentiation 47 . Therefore, we carried out a few synteny analyses of grape and tomato genomes and found that the number of tomato www.nature.com/scientificreports/ genes was less than three times that of grapes. These results indicate that a large number of gene losses occur in WGD of tomato. We use OrthoMcl to analyze the orthologous bHLH genes in grape, Arabidopsis and tomato genome. We found 138 bHLH orthologous gene pairs between grape and tomato, but only 69 bHLH orthologous gene pairs between grape and Arabidopsis (Supplementary Table S5). The result was consistent with the close relationship between grape and tomato. In the analysis of synteny, we found each grape bHLH gene has one to three orthologous tomato genes. This shows that with the triplication of genome, some bHLH genes replicate as well. Through collinear correlations of the bHLH gene, we found 54, 10 and 75 paralogous bHLH gene pairs in grape, Arabidopsis and tomato, respectively (Supplementary Table S6). We used software Circos to show the relationships of orthologous and paralogous bHLH genes among grape, Arabidopsis and tomato (Fig. 4).
The expression level of spine grape berry bHLH genes at different developmental stages. We sequenced and analyzed the berry skins 40 (before veraison), 80 (at veraison) and 120 (berry ripening period) days after flowering of black and white spine grape. Among the 115 members of the bHLH family, 95 bHLH genes distributed in 21 subfamilies are expressed at least one of the developmental stages of the spine grape berry (Fig. 5). In order to screen the bHLH family candidate genes associated with anthocyanin synthesis and accumulation in pericarp of spine grape, reads per kilobase per million (RPKM) values were used to estimate the expression levels of bHLH family members. We screened 6 bHLH candidate genes (VdbHLH003, VdbHLH004, VdbHLH033, VdbHLH037, VdbHLH062 and VdbHLH097) by differential gene expression pattern analysis. These genes were mainly expressed at 80 and 120 days after the flowering of the black spine grape, and the expression level was low or not expressed in the 40 days after the flowering of the black spine grape and in the white spine grape. We validated the candidate genes by qRT-PCR, which was consistent with the results of transcriptome sequencing ( Supplementary Fig. S3). Among them, the expression levels of VdbHLH033, VdbHLH037 and Vdb-HLH062 increased with the ripening of blackberries. According to the previous analysis, VdbHLH037 belongs to the subfamily III f. It is suggested that it may be related to anthocyanin synthesis in grapes.
Interaction network prediction and functional analysis of candidate gene. Interaction network analysis helps us understand gene function and efficiency 21 . We used STRING to predict the interaction network of candidate genes based on VdbHLH orthologs in Arabidopsis (Fig. 6). VdbHLH003 (AMS in Arabidopsis) plays a crucial role in tapetum development and is required for male fertility and pollen differentiation, especially during the post-meiotic transcriptional regulation of microspore development within the developing anther. The interaction between VdbHLH004 (BPEp in Arabidopsis) and ARF8 has been verified in Arabidopsis. Therefore, it is believed that BPEp influences cell size and ultimately controls petal size by regulating the expression of auxin response gene. VdbHLH033 (FRU in Arabidopsis) is related to iron absorption. VdbHLH037 (TT8 in Arabidopsis) synergizes with TT1, PAP1 and TTG1 to regulate the flavonoid pathway. In addition, it also affects the expression of DFR (dihydroflavonol 4-reductase). The complex of TT8, TT2 and TTG1 is crucial to BAN genes that regulate flavonoid synthesis in seed endothelium. The genes MYB113 and MYB114 are involved in regulating Overexpression of VdbHLH037 promoted anthocyanin accumulation of berry. Forty-five days after anthesis, green berry was used to inject Agrobacterium containing vector control (VC) and overexpression (OE) with a1-ml syringe. After eight days, the berries injected with OE showed faster color development than berries with VC (Fig. 7a). Compared with VC berries, OE berries had higher red color (a*), lower yellowness (b*) and similar lightness (L*) (Fig. 7c). The anthocyanin content of OE berries increased compared with VC www.nature.com/scientificreports/ berries (Fig. 7d). The number of red spots in OE berries was also more than that in VC berries after 8 days of infection (Supplementary Table S7). However, the number tended to be close after 16 days of injection (Supplementary Table S8). To further verify the results, RT-PCR was used to detect the infected berries. The results showed that the maker gene (hygromycin gene) could be detected 2, 4, 6, 8 days after infected by Agrobacterium containing OE and VC, but not by Agrobacterium alone (Fig. 7b). These results indicated that overexpression of VdbHLH037 could promote anthocyanin accumulation in berries.

Overexpression of VdbHLH037 can promote anthocyanin accumulation and upregulation of anthocyanin synthesis related genes in transgenic Arabidopsis. The T3-generation homozygous
transgenic Arabidopsis was used to further analyze the function of VdbHLH037 in anthocyanin synthesis. In four-week-old transgenic Arabidopsis, the leaves and petioles of the three OE lines (OE-2, OE-3 and OE-5) exhibited purple-black pigments when compared with VC lines (Fig. 8a). The anthocyanin content of three OE lines was also significantly higher than that of VC lines. There was no significant difference in rosette diameter and plant height between VC lines and OE lines (Fig. 8c). The transcription levels of 12 genes related to anthocyanin synthesis of VC and OE lines were analyzed. Compared with VC, the expression levels of AtCHI, AtCHS, AtF3H, AtDFR, AtLDOX (leucoanthocyanidin dioxygenase) and AtUGT78D2 in OE were significantly increased. The transcription level of OE-2 was the highest and that of OE-5 was the lowest. In VC, the expression level of these genes was low or barely detectable (Fig. 8d).

Discussion
As one of the most important horticultural crops in the world, grape can be used as table grape or for wine, drying, juicing and so on. Grape color is the key factor for the commerciality of berry and also a key selection index for grape breeding. The color of berry is determined by the composition and content of anthocyanin. Previous studies have shown that bHLH is involved in anthocyanin synthesis, but the function of bHLH in grape coloration is rarely reported 23,24 . Spine grape has strong adaptability and resistance and is the only Chinese wild grape with both black and white berry, which provides an excellent material for studying the coloring mechanism of grape. Based on the results of 'Pinot Noir' sequencing, We screened 137 bHLH genes, removed the pseudogenes through the analysis of the conserved domain and finally obtained 115 bHLH genes. We named them according to their positions on chromosomes (Fig. 1). Then we conducted a phylogenetic analysis of VdbHLH and divided it into 25 subfamilies, the results are similar to Arabidopsis and tomato 17,36 . According to the evolutionary relationship, VdbHLH037 belongs to III (f) subfamily (Fig. 2). Members of this subfamily have been found to be related to anthocyanin synthesis. In three strawberry varieties, seven candidate bHLH genes related to anthocyanin synthesis were screened by genome-wide analysis of the bHLH family. In addition, it is considered that the Fab-HLH29 gene of III (f) subfamily is related to anthocyanin synthesis through gene interaction network analysis and  48 . In chrysanthemums, CmbHLH2 of III (f) subfamily regulates anthocyanin content by binding to the promoter of CmDFR and interacting with CmMYB6 so that flowers appear red, pink and yellow 49 . In skins and seeds of grape, VvMYC1 regulates anthocyanin and proanthocyanidin synthesis through interacting with MYB5a, MYB5b, MYBA1/A2 and MYBPA1 to adjust genes of flavonoid pathway 50 . Through further analysis of gene structure and conserved motif, we added evidence supporting the phylogenetic relationship of the VdbHLH family (Supplementary Figs. S1 and S2). The basic region of the VdbHLH family determines the binding ability of DNA. The ratio of E-box-binding proteins in the VdbHLH family is lower than that in Arabidopsis and tomato, which indicates that binding motifs of the VdbHLH family are diverse (Fig. 3).
Through comparative genome analysis, we found that although some bHLH genes were lost during plant evolution and genome duplication events, the number of the bHLH gene was increasing (Fig. 4). In addition, it has been reported that there are more bHLH genes in higher plants than in lower plants 13 . These results indicated that bHLH genes play a key role in plant evolution. In order to further understand the function of bHLH genes in grape berry coloring, we analyzed the expression pattern of the bHLH family in the three periods of black and white spine grapes. The results showed that the expression level of VdbHLH003, VdbHLH004, VdbHLH033, VdbHLH037, VdbHLH062 and VdbHLH097 was significantly increased at the veraison stage of black spine grape, and was low or almost undetectable in the early development stage of black spine grape and the three development stages of white spine grape (Fig. 5). VdbHLH037 is the highest up-regulated gene among the six genes, which was subsequently confirmed by qRT-PCR (Supplementary Fig. S3). Previously it was found that bHLH proteins perform their function by forming homodimer with bHLH protein or heterodimer with non-bHLH protein 51 . Therefore, we used Arabidopsis orthologs to predict the regulatory network of these six genes. The predicted interaction genes F3H, DFR, LDOX and UF3GT of the VdbHLH037 gene were involved in the synthesis of anthocyanin. Besides, the interaction between MYB transcription factor and VdbHLH037 is predicted (Fig. 6). Previous studies have shown that bHLH regulates anthocyanin biosynthesis by forming MBW transcriptional complex with MYB and WD40 52 . In addition, the sequence of VdbHLH037 and AtTT8 is found to be highly similar. AtTT8 belongs to III (f) subfamily and regulates anthocyanin synthesis in Arabidopsis by forming MBW transcriptional complex with AtTT2 and AtTTG1 53 . The interaction network predicted by the remaining five genes had no direct relationship with the regulation process of anthocyanin synthesis. Accordingly, by the above results, it can be speculated that VdbHLH037 plays a positive role at the veraison period in berry. www.nature.com/scientificreports/ To understand the regulatory mechanism of VdbHLH037 in anthocyanin synthesis of grape berry, we used the transient expression technology in berry to study its function 54 . After infection, compared with the VC, the coloration of OE berry was improved due to higher a* value. In addition, OE berries could accumulate more anthocyanin than VC. However, as time went on, the difference gradually decreased, which may be due to transient expression. Meanwhile, we also used transgenic Arabidopsis to study the function of VdbHLH037 in the synthesis of anthocyanin. The results showed that OE-2, OE-3 and OE-5 could accumulate more anthocyanin than VC. The transcription level of AtCHI, AtCHS, AtF3H, AtDFR, AtLDOX and AtUGT78D2 in OE berries was significantly higher than that in VC berries, and the expression level of these genes was consistent with the phenotype of transgenic Arabidopsis. AtCHI and AtCHS participated in the synthesis of flavonoid. AtF3H could catalyze and modify flavonoid. The flavonoid synthesis pathway entered the anthocyanin synthesis pathway under the action of AtDFR and AtLDOX. The gene AtUGT78D2 was responsible for encoding the glycosyltransferase. These results suggested that VdbHLH037 could promote anthocyanin accumulation by regulating related genes in the anthocyanin synthesis pathway. This study provides a theoretical basis for further understanding the function of bHLH family members in the process of grape berry coloring.   Table S9). The recombinant plasmid and pCAMBIA3301 vector were transformed into Aabidopsis Columbia ecotype by the floral dip method 55 . The phosphinothricin resistance and qRT-PCR were used to screen seeds (Fig. 8B). Homozygous T3-generation plants were used for further analyses.

Methods
Color measurement. The anthocyanin content in grape skins was measured after inoculation for 8 days.
The leaf of four-week-old transgenic Arabidopsis with VC and OE were used to detect anthocyanin content. The samples (50 mg) was pulverized with liquid nitrogen and then each sample was homogenized in 1% (v/v) hydrochloric acid in methanol and shaken at 4 °C overnight. The anthocyanin content in grape andArabidopsis were determined by measuring the absorbance of the extract at 530 nm using an ultraviolet-visible (UV-Vis) spectrophotometer 56 . The color of grape berry skin was measured after inoculation for 8 days with a handheld colorimeter (CR-400, Konica Minolta, Tokyo, Japan). Color parameters were recorded as L * (lightness), a * (redness) and b * (yellowness).

Analysis of qRT-PCR and RT-PCR.
The 'Kyoho' skin of 2, 4, 6, 8 days after infected by Agrobacterium containing OE and VC were used to detect hygromycin gene. The berry skins at 40, 80 and 120 days after anthesis of black and white spine grape were used to identify candidate genes 57 . The RNA was extracted using TIANGEN RNAprep Pure Plant Kit (Tiangen Biotech). The four-week-old leaf of Arabidopsis was used to screen positive plants. The RNA was isolated from samples using TRIzol reagent (Invitrogen). Genomic DNA was removed by DNaseI (Thermo Scientific). The first-strand complementary DNA (cDNA) synthesis was performed with a RevertAid First-Strand cDNA Synthesis Kit (Thermo Scientific). qRT-PCR was performed in the presence of SYBR green qPCR Master Mix (Fermentas) and the amplification was performed in the Eco Real-Time PCR system (Illumina). All reactions were performed in triplicate 58 . The primers were designed using Oligo 7.0 and are listed in the Supplementary Table S9. The VlActin and VdActin were used as internal controls for grape. AtUBQ3 was used as internal controls for Arabidopsis.

Statistical analysis.
All experiments were replicated independently at least three times, and the data were shown as mean ± SD of three independent experiments. Data were subjected to a statistical analysis according to Student's t-test, and significant differences among the means were determined by the LSD (least significant difference), at*p < 0.05 and **p < 0.01.