Cloning And Functional Characterization Of Heavy Metal-responsive Genes From Garlic

Metallothioneins (MTs) and phytochelatins (PCs) are two best-characterized heavy metal-binding ligands in plant cells. MTs are products of mRNA translation, however, PCs are enzymatically synthesized from glutathione (GSH) by phytochelatin synthase (PC synthase). Now both MT and PC synthase genes were found in many plant species. It is valuable to investigate the expression pattern of these two genes in response to heavy metal stresses and characterize their functions by transferring them into plants. In this study, the cDNAs encoding a PC synthase (AsPCS1) and a type 2 MT (AsMT2a) were cloned from the seedlings of garlic. The transcript levels of AsPCS1 and AsMT2a, together with the contents of PCs, were determined in roots and leaves of garlic seedlings after exposure to Cd and other stresses. Furthermore, the heavy metal resistance of AsMT2a (AsPCS1) overexpressing Arabidopsis was evaluated. The results are as follows: 1. The full length of AsMT2a was 525 bp and contained 240 bp ORF encoding 79 amino acids with predicted molecular mass of 8020 Da. The deduced protein sequence indicated AsMT2a presented the characteristic structure of most plants MTs, i.e. two Cys-rich domains separated by a long, Cys-free spacer domain. The number and positions of the Cys residues in the N-and C-terminal domains were completely conserved with those in other plant type-2 MT proteins, suggesting the functional significance of these Cys residues. 2. The results of RT-PCR showed that a maximum increase of AsPCS1 expression levels in roots was observed after 1hr exposure to Cd, subsequently its expression levels dropped but were still much higher than that of untreated roots and maintained at a steady-state level. Upon exposure to Cd, PCs contents were increased with the increasing of incubation time in roots, while the strongest increase was detected within 1 hr and the maximal yield of PCs was observed at 24 hr. In response to Cd, AsMT2a exhibited an expression pattern distinct from AsPCS1. Its transcript levels in roots were increased very slightly within 10 hr of Cd treatment and evident increase occurred after that time. These results indicated that AsPCS1 might play a pivotal role in immediate detoxification of Cd (Fig. 4). Approximately 2-fold induction of PCs contents in Cd-stressed roots within 1 hr is consistent with its role in the instant effects in Cd detoxification. These results also suggested that AsMT2a and AsPCS1 could coordinately function in different phases of Cd treatment. AsMT2a might confer the long and persistent Cd tolerance on garlic seedlings, i.e. once an excess of Cd accumulation in roots over the chelating capacity of PCs happens, MTs synthesis is largely increased. 3. When AsPCS1 and AsMT2a were transformed into As and Cd sensitive yeast mutant FD236-6A, both genes could expressed stably by RT-PCR analysis and Cd/As tolerance of yeast could be restored by the expression of either AsMT2a or AsPCS1 genes. 4. AsMT2a and AsPCS1 were transformed into Arabidopsis mediated by Agrobacterium tumefaciens and these two targeting genes could be expressed in transgenic plants. It was interesting that two transcripts of AsPCS1 in AsPCS1or AsPCS1+AsMT2a overexpressing Arabidopsis seedlings were found. Both transcripts contained its own ORF and 38 amino acids discrepancy between them occurred according to the deduced protein sequences. These results indicated that partial AsPCS1 mRNA underwent accurate cutting and splicing, however the true mechanism of cutting and splicing remained unlear. 5. AsPCS1 overexpressing Arabidopsis showed stronger tolerance to Cd comparedwith wild type. More and longer roots of transgenic plants were found under Cd stresses. Whereas no obvious increase of As resistance of AsPCS1 transgenic plants was observed. In contrast to AsPCS1, As tolerance of AsMT2a overexpressing Arabidopsis was greatly improved and transgenic plants also possessed more and longer roots. The transgenic plants containing these two genes also showed enhanced resistance to Cd stress. 6. PCs and heavy metal content of AsPCS1 overexpressing Arabidopsis increased in response to Cd and As stress. Exposure to Cd resulted in 4-fold increase of cadmium content in transgenic plants compared with wild type, wherase only 1.2-fold increase of As content was found when AsPCS1 transgenic plantswere exposed to arsenate stress. Cd (or As) in AsMT2a transgenic Arabidopsis accumulated 1.4 (or 0.8) times over wild type at 500 μM CdCl2 (or 500 μM Na3AsO4). Cd content increased 2.4 fold over the seedlings overexpressing AsMT2a when the transgenic plants containing both AsPCS1 and AsMT2a genes were treated with 500 μM CdCl2, wherase, it was just slightly higher than that in the plants overexpressing AsPCS1. Moreover, we cloned a new member of metallothionein gene family from garlic seedlings and named AsMT2b. In this study, we observed the function of AsMT2b in garlic seedlings and transgenic Arabidopsis in response to heavy metals. 1. The full length of AsMT2b was 520 bp and contained 243 bp ORF encoding 80 amino acids (including 15 Cys residues). The alignment of the deduced AsMT2b amino acids with other plant type 2 MT protein sequences showed that the position and arrangement of Cys residues in AsMT2b were different from that of other type 2 MT protein. The structure of N terminal domain was CXXC——CXC——CXC——CXCC and the structure of C terminal domain was CXXC——CXC——CXC. It is possible that AsMT2b plays specific biological roles in garlic tolerance to heavy metal stresses. 2. In response to 200 μM Cd, the transcripts of AsMT2b decreased within shorterphases (24 hr), but its expression levels increased with the extending time (48 hr) of Cd stress or the increasing of Cd concentration (e.g. 500 μM), suggesting that AsMT2b may function until garlic seedlings suffered much stronger stress. 3. When AsMT2b was transformed into As/Cd sensitive yeast mutant FD236-6A, it was found that transformants showed no increase of As tolerance but appeared great increase of Cd tolerance. 4. AsMT2b overexpressing Arabidopsis challenged by CdCl2 showed stronger Cd resistance with longer roots and higher germination rates compared with wild type plants. However, there were no significant difference to growth between the transgenic plants and WT. Furthermore, the resistance of AsMT2b transgenic plants to heavy metals was obviously different from AsMT2a transgenic plants i.e. the former showed more stronger Cd tolerance but more weaker As tolerance than the latter. 5. Cadmium contents of AsMT2a overexpressing seedlings rose significantly under Cd exposure and averagely increased 70% over WT. However, variations of Cd content between different transgenic lines were found. In addition, we studied the oxidative stress and antioxidative ability of CdCl2-stressed garlic seedlings. The results showed that most Cd accumulated in roots, but some was also translocated and accumulated in leaves at longer exposure time (after 12 h) and higher concentrations (5 mM, 10 mM) of CdCl2. Cd (5 mM and 10 mM) initially inhibited the activities of SOD and CAT but thereafter recovered or even increased compared with control plants. POD activities at 5 mM and 10 mM of Cd increased more than 3-4 times over control plants within 12 h and then dropped but were still higher than controls at the end of experiment. These results suggested that in leaves of garlic seedlings challenged by CdCl2 at higher concentrations induction of these various enzymes is part of a general defense strategy to cope with overproduction of reactive oxygen. In conclusion, coordinated responses in heavy metal-stressed garlic seedlingsoccurred between AsPCS1 and AsMT2a,and between AsMT2 and AsMT2b. These responses may be one of the components of celluar homeostasis mechanism. Moreover, the enhanced metal-resistance and metal-accumulation in these transgenic Arabidopsis suggested that these genes were in expectation of phytoremediation for soil contaminated by heavy metals.