Isolation and nucleotide sequence of MT-I and MT-II cDNA clones from sweet potato storage roots
MT-I and MT-II cDNA clones from sweet potato storage roots were isolated. We have completed the sequencing of the clones, which were named MT-I and MT-II (MT-1, GenBank Accession Number AF116845 and MT-II, GenBank Accession Number FJ418632). The open reading frames in these two cDNAs encode pro-proteins of 66 and 81 amino acids, respectively, with a predicted molecular mass of 6,614 Da (pI 4.64) and 8,068 Da (pI 4.81). A comparison of the deduced amino acid sequence of MT-I and MT-II indicates 25% identity.
In plants, the members of MT family have been divided into four types according to the location and distribution of Cys residues. MT types 1–3 contain two Cys-rich clusters respectively at their N- and C-terminal regions, separated by a central Cys-free spacer of 30–40 residues. The type 4 which is known as Ec-type, has three Cys-rich clusters each separated by 10–15 residues (Nezhad, et al., [2013]). In this manuscript, amino acid sequences of MT-I and MT-II were compared at their N-terminal (domain 1) and C-terminal (domain 2) regions. The result showed that the deduced amino acid sequence of MT-I have a high degree of similarity with type 1 MT-like proteins from other plants, including a central hydrophobic domain flanked by conserved cysteine-rich motifs (conserved domain 1 region: CxCxxxCxCxxCxC and conserved domain 2 region: CxCxxxCxCxxCxC). In addition, deduced amino acid sequence of MT-II also exhibits a high degree of similarity with type 2 plant MT-like sequences, with the typical cysteine-rich domains at the N-terminal (CCxxxCxCxxxxCxCxxxCxxC) and C-terminal region (CxCxxxCxCxxCxC), respectively (Branislav, et al., [2013]). The data of gene structure analysis also agreed with the data from the comparison of amino acid sequences (Figure 1).
Copy numbers of MT-I and MT-II sequences in sweet potato
We performed Southern blot hybridization with Eco RI (E), Bam HI (B) and Hind III (H) digests of sweet potato Tainong 57 DNA, using probe derived from 3′-noncoding sequence of the cDNAs to estimate the copy number of the gene. Tainong 57, an elite sweet potato cultivar derived from a cross between Tainong 27 and Nancy Hall, has a hexaploid number of chromosome (2n = 6x = 90). The results suggest that MT-I and MT-II belong to a small multigene family in sweet potato (Figure 2A).
MT-I and MT-II mRNA levels were developmentally regulated
The presence and amounts of different sweet potato MT-I and MT-II mRNAs were examined in various organs and tissues by northern blot analysis. MT-I and MT-II were obtained from sweet potato storage roots. Figure 2B shows that MT-I and MT-II probe hybridized to mRNA species of approximately 1.0 kb. MT-I mRNA levels were the highest in the storage roots, followed by that in sprouted roots, fully expanded green leaves and vein; while it was the lowest in sprout. MT-II mRNA levels were the highest in the storage roots, followed by that in fully expanded green leaves; while it was the lowest in sprouted roots and vein.
Expression of MT-I and MT-II in E. coli
SDS-PAGE analysis of MT-I and MT-II crude extracts from the transformed E. coli (M15) showed high amounts of a polypeptide with the expected molecular mass (ca. 6.5 and 8 kDa) (Figure 3A and 3B). Each polypeptide was found as a soluble protein in the supernatant (Figure 3A and 3B, lane 2), and was absent in protein extracts obtained from E. coli transformed with pQE-31 vector (Figure 3A and 3B, lane 1). The expressed protein was highly purified from crude extracts as His-tagged pQE-MT-1 and pQE-MT-II (Figure 3A and 3B, lane 3), respectively. The polypeptides of MT-I and MT-II were analyzed by western blot assay. As shown in Figure 3C and 3D, MT-I and MT-II proteins expressed in the transformed E. coli (M15).
Effect of pH (6.0 and 7.0) on dehydroascorbate reductase activity of MT-I and MT-II proteins
The purified MT-I and MT-II were used to examine DHA reductase activity. Figure 4 shows AsA regeneration (ΔΑ 265 nm) from DHA at both pH 6.0 and 7.0 with (A) or without (B) GSH. Figure 4A shows that MT-I and MT-II exhibited DHA reductase activity and could reduce DHA back to AsA. The specific activities of DHA reductase for MT-I and MT-II in the presence of GSH were 3.45 and 5.52 nM AsA produced/min/mg protein at pH 7.0, respectively. However, in the absence of GSH, very low DHA reductase activities of MT-I and MT-II were found (Figure 4B): only 0.01 and 0.02 nM AsA produced/min/mg protein at pH 7.0, respectively. In addition, the specific activities of DHA reductase for MT-I and MT-II in the presence of GSH were 1.86 and 1.28 nM AsA produced/min/mg protein at pH 6.0, respectively. However, in the absence of GSH, very low DHA reductase activities of MT-I and MT-II were found (Figure 4B): only 0.006 and 0.018 nM AsA produced/min/mg protein at pH 6.0, respectively. MT-I and MT-II act as a GSH-dependent DHA reductase (Figure 5), and the rate of reduction was closely proportional to the concentration of GSH.
DHA is generated from the disproportionation of the MDA radical produced following the oxidation of ASA. DHA reductase catalyses the reduction of DHA to ASA using GSH as the reductant (Wu, et al., [2009]). If DHA is not recycled to ASA, it undergoes irreversible hydrolysis to 2, 3-diketogulonic acid. Expression of DHA reductase in plant, responsible for regenerating AsA from an oxidized state, regulates the cellular AsA redox state, which in turn affects cell responsiveness and tolerance to environmental reactive oxygen species (ROS). Because of its role in AsA recycling, we examined whether DHA reductase is important for plant growth (Wang, et al., [2010]). In its reaction with ROS, ASA is oxidized to the short-lived radical, MDA, which can rapidly disproportionate non-enzymatically to produce DHA and ASA. Alternatively, MDA can reduce DHA to ASA using NADPH as the reductant. Therefore, plants have evolved several mechanisms by which the oxidized forms of ASA can be recycled (Kerchev, et al., [2012]).
The most critical advance in MTs research is the demonstration of the redox regulation of Zn-S interaction and the coupling of zinc and redox metabolism (Oteiza, [2012]). The cluster structure of Zn-MT provides a chemical basis by which the cysteine ligand can induce oxidoreductive properties. The hypothesis that MT functions as an antioxidant against ROS and reactive nitrogen species has received extensive experimental support from many of the in vitro studies. Studies using a cell-free system have demonstrated the ability of MT as a free radical scavenger. MT has been shown to scavenge hydroxyl radical in vitro, because of its cysteinyl thiolate groups (Miura, et al., [1997]). In ad dition, there are possible reasons to explain the apparent low DHAR activity of MT-I and MT-II. Zinc (II) is an important regulator of GSH synthesis. The importance of zinc in the metabolism of GSH underscores the finding that, as zinc deficiency is accompanied by oxidant increase, many studies reveal a deficiency of GSH under such conditions (Hernandez, et al., [2012]). Therefore, MT-I and MT-II may be less reduced by GSH resulting in low DHAR activity comparing to other DHAR.
Effect of pH (6.0 and 7.0) on monodehydroascorbate reductase activity of MT-I and MT-II proteins
MDA was reduced to AsA in coupling with NADH oxidation (Δ A340 nm) at pH 6.0, and 7.0 when MT-I and MT-II proteins was used as MDA reductase. The MT-I and MT-II proteins exhibited MDA reductase activity at both pH 6.0 and 7.0 (Figure 6), with higher activity at pH 6.0 than pH 7.0. Therefore, the specific MDAR activity of MT-I and MT-II proteins was 0.18 and 0.17 unit/mg protein in pH 6.0, respectively.
Protein and diaphorase activity stainings in 15% SDS–PAGE gels for detection of monodehydroascorbate reductase activity of MT-I and MT-II proteins
MDA reductase activity staining of MT-I and MT-II was done for diaphorase activity (Kaplan and Beutler, [1967]) on SDS-PAGE gels (Figure 7). Comparing Figure 7 (A, lane 1 and B, lane 1) (protein staining) with Figure 7(A, lane 2 and B, lane 2) of MT-I and MT-II one can see that the diaphorase activity staining for MDA reductase activity came from 6 or 8 kD MT-I or MT-II. MDA reductase and DHA reductase were shown to contain free thiol groups in their catalytic sites (Trümper, et al., [1994]). When AsA is the sole hydrogen donor, the AsA peroxidase, guaiacol peroxidase, and AsA oxidase can produce MDA (Hou, et al., [1999]). Nonenzymatic oxidations of AsA also produce MDA when cells were under oxidative stress (Hossain, et al., [1984]). DHA reductase that catalyses the reduction of DHA by GSH have been purified from rice, spinach, and potato (Dipierro and Borranccino, [1991]). Several other proteins such as glutaredoxins (thiol transferases), protein disulphide isomerases, defensin, thioredoxin, and even a Kunitz-type trypsin inhibitor have been shown to have DHAR activity (Huang et al., [2008]; Huang et al., [2008]b). Plant Kunitz-type trypsin inhibitor has slight DHAR activity in its reduced form (Trümper et al. [1994]). Thioltransferase (glutaredoxin) and protein disulfide isomerase from animal cells also have DHAR activity (Wells et al. [1990]). Nevertheless, the amino acid sequence of the MT is quite distinct from these other DHAR enzymes.