CQAs content in sweet potato plant tissues
HPLC was performed to investigate the content of CQAs in the leafy sweet potato varieties EC16 and FS7-6. The analysis revealed the presence of six CQAs, including 3-CQA, 3,5-diCQA, 3,4-diCQA, 4,5-diCQA, 4-CQA and 5-CQA in different tissues of stem tips, stems, leaves and roots. These CQAs are highly accumulated in stems and leaves compared to stems tips and roots in both varieties (Fig. 1A). The 3,5-diCQA is the predominant acid in stems followed by 3-, 5- and 4-CQA. Meanwhile, in stem tips and leaves, 3,5-diCQA and 5-CQA are the main CQAs, whereas the roots accumulated more 4- and 5-CQA. Conversely, 3,4- and 4,5-diCQA are the lowest CQAs in all tissues of both varieties. Notably, 5-CQA is one of the major CQAs that is present in large quantity simultaneously in all tissues of EC16 and FS7-6. As shown in Fig. 1B, the total amount of CQAs is significantly higher in EC16 than in FS7-6. It could be speculated that the content of CQAs varied among cultivars and plant tissues.
Phylogenetic analysis of IbGST genes
For investigation of the evolutionary relationships, orthologs of IbGSTTCHQD and IbGSTT in other plant species were identified using NCBI BLASTP. Phylogenetic tree analysis indicated that IbGSTT protein is closely related to the GST from Nicotiana attenuata (XP_019264208.1:1-231), whereas IbGSTTCHQD protein is a homologous to the GSTprotein of Theobroma cacao (EOY15086.1:1-322) (Fig. 2). This result showed close homology and domain similarity between the IbGSTs and GST proteins from other plant species.
Analysis of IbGST promoter regions
To identify relevant factors that regulate the expression of the GST genes, the upstream 2 kb of transcription start site of IbGSTTCHQD and IbGST was analyzed using the PlantCARE online tool. Various cis-regulatory elements, including core promoter element of light-responsive elements (G-box), transcription start (TATA-box), abscisic acid responsive elements (ABRE) and other environmental stress responsive-elements such as DRE, MYB and MYC were identified in the promoters of the IbGST genes(Table 1). The ABRE and G-box elements were discovered in the promoter of IbGSTTCHQD gene, while the plant metabolism cis-acting regulatory element CAAT-box, TATA-box, and DRE and were found in the promoter of IbGSTT gene. Moreover, the transcription factors MYC and MYB, which play crucial roles in PPs metabolism, plant growth and defense mechanisms were found in both genes, indicating that the transcription of the IbGST genes might be regulated by diverse physiological and environmental factors, which are essential for the accumulation of CQAs in plant tissues.
Expression profiles of IbGST genes
Different transcripts of IbGST genes were found in stem tips, stems, leaves and roots of untransformed EC16 and FS7-6 plants. Compared to other tissues, leaves and stems accumulated more CQAs (Fig. 1B). The expression of IbGSTTCHQD washighly elevated in the leaves ofEC16 compared to the leaves of FS7-6 variety but was most concentrated in stem tips, stems and roots of FS7-6 (Fig. 3A). IbGSTT was highly expressed in leaves and roots of both varieties (Fig. 3B). The relative expression levels of these genes varied considerably among tissues and varieties. Our results suggest that the accumulation of CQAs in each of the leafy sweetpotato tissues might not be directly related to the expression pattern of the IbGST genes. However, we observed higher expressions of IbGSTTCHQD and IbGSTT in EC16 compared to FS7-6, which corresponded to the maximum concentration of CQAs in EC16 variety.
Comparison of IbGST gene sequences between EC16 and FS7-6
The comparison of protein sequences obtained after amplification of IbGST genes from EC16 and FS7-6, showed similarities of more than 95% between the two varieties for each gene. However, slight variations in amino acid sequences were also observed. For examples, the amino acids leucine (L) and tyrosine (Y) in IbGSTTCHQD synthesized from the EC16 variety were substituted with phenylalanine (F) and cysteine (C), respectively, in FS7-6 variety (Fig. 4A). Additionally, the arginine (R) and glycine (G) amino acids in IbGSTT gene from EC16 were replaced by histidine (H) and valine (V), respectively in FS7-6 (Fig. 4B). These molecular mutations could alter the functions of the target protein in both varieties, and probably the content of CQAs. Since the EC16 variety exhibited higher amount of CQAs, the amplified sequences from this variety were then used to determine whether the accumulation of CQAs would be correlated with the overexpression of IbGSTTCHQD and IbGSTT.
IbGSTTCHQD and IbGSTT enhance CQAs accumulation in Nicotiana transformed plants
Transient expression and genetic transformation of IbGSTTCHQD and IbGSTT genes in Nicotiana plants were employed to evaluate the implication of those two genes in the accumulation of CQAs. Suspensions of the Agrobacterium tumefaciens containing the constructed pCambia1300-IbGST and pCambia1300-IbGSTT overexpression vectors from the EC16 variety were transferred into the N. benthamiana leaves. Gene expression profiling revealed significant increases more than 4-fold in the expression levels of IbGSTTCHQD and IbGSTT genes in the transformed leaves compared to the untransformed leaves (Figs. 5A, B). Furthermore, the overexpression of IbGSTTCHQD and IbGSTT enhanced the levels of monoCQAs, while the diCQAs were less accumulated in comparison with the untransformed leaves (Figs. 5C, D). For genetic transformation, the coding sequences of IbGSTTCHQD and IbGSTT driven by the CaMV35S promoter were transformed into N. tabacum via Agrobacterium-mediated transformation system and kanamycin-resistant explants were generated within 3 months (Figs. 6A, B, C, D). Ten independent transgenic lines were randomly selected, and integration of the transgene was verified by PCR. All the ten selected lines produced the expected products of 981 bp and 723 bp for the integration of IbGSTTCHQD and IbGSTT, respectively (Figs. 6E, F), indicating that the IbGSTs were incorporated into the genome of N. tabacum. RT-qPCR analysis exhibited significant increases of IbGSTTCHQD and IbGSTT transcript levels (3-5-folds) in the transgenic lines compared to the wild type plants (Figs. 6G, H). The three lines that exhibited higher expression levels of each gene were further selected for the quantification of CQAs. Plants expressing the 35S::IbGSTTCHQD or 35S::IbGSTTtransgenes showed significant accumulation of monoCQAs compared to the wild types. Among those highly accumulated monoCQAs, 5-CQA was the highest (Figs. 6I, J). However, no significant increases were observed in the content of diCQAs except for lines 2 expressing the 35S::IbGSTTCHQD transgene and line 8 expressing the 35S::IbGSTT transgene. In general, the IbGST genes enhance the accumulation of monoCQAs up to 20% for IbGSTT transgene and 55% for IbGSTTCHQD transgene, but no association with the diCQAs accumulation was observed (Fig. 6K). These results indicated that the sweet potato GST gene expression profiles correlate with the accumulation of monoCQAs in transformed N. benthamiana leaves and transgenic N. tabacum plants overexpressing either IbGSTTCHQD or IbGSTT.
Expression profile of IbGST genes under abiotic stresses
Seedlings from the three tobacco transgenic lines selected previously based on the observed higher expression levels of IbGSTTCHQD and IbGSTT genes and wild types were subjected to salinity and oxidative stresses. Forty days after stress induction, total RNA was extracted from the leaves, cDNA was synthesized, and the gene expression profiles were analyzed. The IbGSTTCHQD wassignificantly upregulated in all lines except for line 4 under oxidative stress conditions (Figs. 7A, B). High expression levels of IbGSTT were detected in the transgenic lines 8 and 10 under both stress conditions, whereas no significant difference was detected in the transgenic line 2 (Figs. 7C, D). Our results showed that IbGSTTCHQD and IbGSTT responded positively to salinity and oxidative stress conditions.