Cdna Libraries

Numerous enzyme sequences have been identified in Arabidopsis, but little headway has been made in matching specific enzymes with their function on herbicide substrates. Molecular information on enzymes and function identification towards herbicides has been studied based on cDNA libraries. cDNA libraries are prepared from the mRNA of interest that is synthesized to DNA (cDNA) strand, which contains a specific coding region of a genome that is inserted into vectors and cloned [30].

Studies on P450 metabolism of herbicides have reported the use of two yeast-expressed plant enzymes, CYP73A1 [31] and CYP81B1 [32], in which ring-methyl hydroxylation of chlortoluron was observed. In both cases, however, the reaction was extremely slow if compared to a previous investigation, where a very fast N-demethylation catalysis was observed with CYP76B1 [33]. It was found that CYP76B1 catalyzes the mono- and di-N-demethylation of both chlortoluron and isoproturon in higher plants, and plays a significant role in the detoxification of most phenylureas, including the methoxylated forms [33]. Other studies showed that 2,4-D treated S401 tobacco cells could metabolize chlorotoluron using four different P450 enzymes identified as CYP71A11, CYP81B2, CYP81C1 and CYP81C2 [34]. Because only CYP71A11 and CYP81B2 were highly induced, both cDNA clones were expressed in yeast S. cerevisiae, which exhibited enhanced chlorotoluron metabolism [34].

Recent studies have investigated specific GST isoform function in response to herbicides (2,4-dichlorophenoxyacetic acid and metolachlor) and other chemicals by overexpressing specific GST cDNAs from plants in E. coli [35]. Results from this research showed that three GST isoform genes (AtGSTF2, AtGSTF6 and AtGSTF8) are increased in the presence of metolachlor or 2,4-D [35]. Further literature studies with Arabidopsis plants exposed to chloroacetanilide herbicides have supported the information about AtGSTF2 induction not only for metolachlor but also for acetochlor [36]. However, it was also observed that both herbicides induced other GST isoform genes, such as AtGSTU24 and AtGSTUl [36]. The expression of Arabidopsis GSTs has also been studied in response to herbicide safeners - chemicals used to enhance herbicide tolerance of crop plants by a number of different mechanisms, specially by the induction of GST activity [37]. Studies from the proteomic analysis of Arabidopsis GSTs reported an effective strategy for examining the Arabidopsis GST family in response to the benoxacor safener, where it was observed a 6-fold induction of the gene AtGSTF9 [38].

Phase III transformation pathway in plants rely on the transport of xenobiotics to the vacuole and storage in this compartment. The transporters involved in this process are mainly the ATP-binding cassete (ABC), which contain a cytoplasmic domain (the ABC protein) that binds and hydrolyzes ATP to energize solute translocation accross the cytoplasmic membrane [39, 40]. Induction of transport activity in Arabidopsis treated with herbicides and herbicide safeners has been observed previously, indicating that several glutathione-conjugate transporters exist [40]. Recent studies have functionally analyzed a full length cDNA of Arabidopsis coding for a protein (AtMRP3) that exhibits high homologies to MPR1 and YCF1, two gluthathione-conjugate transporters of humans and yeast, respectively [40]. It was observed that AtMRP3 was not only a glutathione-conjugate but also a chlorophyll catabolite transporter [40]. This investigation confirmed the multifunctionality between ABC-transporters in plants and opens the need for further identification and functional analysis of other members of the MRP family. Additional studies have provided evidence that the oligopeptide transporter (OPT) family in Arabidopsis mediate the transport of glutathione derivatives and metal complexes, thus participation in stress resistance [41]. RNA blots on yeast cell suspensions and real-time reverse transcription-PCR on Arabidopsis plants indicated that AtOPT6 expression is strongly induced by the herbicide primisulfuron [41].

Body Detox Made Easy

Body Detox Made Easy

What exactly is a detox routine? Basically a detox routine is an all-natural method of cleansing yourbr body by giving it the time and conditions it needs to rebuild and heal from the damages of daily life and the foods you eat and other substances you intake. There are many different types of known detox routines.

Get My Free Ebook


Post a comment