Research Article: Systematic Isolation and Characterization of Cadmium Tolerant Genes in Tobacco: A cDNA Library Construction and Screening Approach

Date Published: August 31, 2016

Publisher: Public Library of Science

Author(s): Mei Zhang, Hui Mo, Wen Sun, Yan Guo, Jing Li, Jin-Song Zhang.


Heavy metal pollution is a major limiting factor that severely affects plant growth worldwide, and the accumulation of heavy metal in the plant may be hazardous to human health. To identify the processes involved in cadmium detoxification, we constructed a cDNA library of tobacco roots acclimated to cadmium (Cd) stress. According to the results of functional screening cDNA library with a yeast Cd-sensitive mutant, ycf1Δ, we obtained a series of candidate genes that were involved in Cd response. Sequence analysis and yeast functional complementation of 24 positive cDNA clones revealed that, in addition to antioxidant genes, genes implicated in abiotic and biotic stress defenses, cellular metabolism, and signal transduction showed Cd detoxification effects in yeast. The real time RT-PCR analyses revealed that some Cd tolerance/ detoxification genes may be able to anticipate in other stresses such as biotic defense and water balance in tobacco. Taken together, our data suggest that plants’ acclimation to Cd stress is a highly complex process associated with broad gene functions. Moreover, our results provide insights into the Cd detoxification mechanisms along with the antioxidant system, defense gene induction, and calcium signal pathway.

Partial Text

Plant growth requires different essential micronutrients, including heavy metals, such as Zn, Mn, Ni and Cu, for normal functioning of cellular metabolism. However, in nature, there are some heavy metal elements (such as Cd, Hg and Pb), which are non-essential for plant growth. Even at trace levels in environment, these non-essential metals may cause substantial damage to organisms. Cadmium, usually as a mimic of Zn [1], is a severe environmental pollutant with high toxicity to organisms [2]. With the development of industry and modern agriculture, the environmental pollution has become increasingly serious. The active free Cd ion is supplied to soil, air, and water mainly by electroplating, battery, alloy manufacturing, pigment, and mining processes, or by fertilization of land with phosphate or sewage sludge. Cadmium is easily taken up by plant roots and leaves, and then transferred from plants into the human body [3, 4]. In general, besides the main absorption by food chain from rice or vegetables, tobacco is another main source of Cd accumulation in human body, especially for smoking population [5].

Yeast is a single-celled organism and therefore is less complex to study for initial functional screening purposes. In the current research, given the Cd-accumulating characteristic of tobacco plants, we constructed a cDNA library with tobacco roots, and screened this library by functional complementary assay with the Cd-sensitive yeast mutant strain, ycf1Δ, for the purpose of cloning and isolating the genes related to Cd detoxification/tolerance in tobacco. The YCF1 gene was isolated according to its ability to confer Cd resistance from Saccharomyces cerevisiae, which shows extensive homology with the human multidrug resistance-associated protein (MRP1) gene [26]. The YCF1 gene encodes an ATP-binding cassette (ABC) transporter, which functions the Cd detoxification as transmembrane pump in yeast through glutathione S-conjugated metal ion combination and translocation [11]. The results of this study demonstrated that cDNA functional screening with a yeast deficient mutant strain was an efficient approach for isolating defective-element-related candidate functional genes. In our experiment, the screening criterion for the Cd-tolerant gene was to recover the Cd-sensitive phenotype of ycf1Δ, which involved searching multiple pathways or mechanism processes, and obtaining genes with different degrees of Cd tolerance according to recovery levels of the yeast clones. Our results clearly showed that a series of candidate genes related to Cd tolerance screened from the cDNA library of the tobacco roots were divided into different categories according to their predicted functions (Fig 2 and S3 Table). This is different from YCF1’s function category: encoding a transporter protein in yeast. This result also indicated that the Cd detoxification mechanisms in vivo might be involved in diverse metabolic pathways and signal transduction.

In summary, we have determined a series of candidate Cd tolerant/detoxification functional genes, and laid a good foundation for elucidating the molecular mechanisms of tobacco responding to Cd. Our study has provided the following new information: (1) the anti-oxidation system of tobacco plays an important role in Cd tolerant/detoxification; (2) plant response of Cd stresses signaling pathways are involved in cross-talks with other biological and abiotic stress signaling pathways, mainly through participation in oxidative stress responses. Further research will be performed to identify the biological functions of some candidate Cd-tolerant genes in plants based on transgenic techniques.