Functional Analysis of the Tandem-Duplicated P450 Genes SPS/BUS/CYP79F1 and CYP79F2 in Glucosinolate Biosynthesis and Plant Development by Ds Transposition-Generated Double Mutants

Abstract

A significant fraction (approximately 17%) of Arabidopsis genes are members of tandemly repeated families and pose a particular challenge for functional studies. We have used the Ac-Ds transposition system to generate single-and doubleknockout mutants of two tandemly duplicated cytochrome P450 genes, SPS/BUS/CYP79F1 and CYP79F2. We have previously described the Arabidopsis supershoot mutants in CYP79F1 that exhibit massive overproliferation of shoots. Here we use a cytokinin-responsive reporter ARR5::uidA and an auxin-responsive reporter DR5::uidA in the sps/cyp79F1 mutant to show that increased levels of cytokinin, but not auxin, correlate well with the expression pattern of the SPS/CYP79F1 gene, supporting the involvement of this gene in cytokinin homeostasis. Further, we isolated Ds gene trap insertions in the CYP79F2 gene, and find these mutants to be defective mainly in the root system, consistent with a root-specific expression pattern. Finally, we generated double mutants in CYP79F1 and CYP79F2 using secondary transpositions, and demonstrate that the phenotypes are additive. Previous biochemical studies have suggested partially redundant functions for SPS/CYP79F1 and CYP79F2 in aliphatic glucosinolate synthesis. Our analysis shows that aliphatic glucosinolate biosynthesis is completely abolished in the double-knockout plants, providing genetic proof for the proposed biochemical functions of these genes. This study also provides further demonstration of how gluconisolate biosynthesis, regarded as secondary metabolism, is intricately linked with hormone homeostatis and hence with plant growth and development.Plant growth and development requires coordination of networks of biological processes within the plant, as well as with responses to external environments. The control of shoot branching by auxin and cytokinin is a well-known example of hormone interactions in controlling plant development. Cytokinin plays a key role in promoting bud growth, whereas auxin has an inhibitory effect. Therefore, the outcome appears to depend on the ratio of the two hormones (for review, see Tamas, 1995;Li and Bangerth, 1992). The two plant hormones not only play opposite roles in controlling plant growth and development, but also influence each other hormone homeostasis (Binns et al., 1987;Palni et al., 1988;Bangerth, 1994;Zhang et al., 1995;Makarova et al., 1996). We and others have described Arabidopsis mutants called supershoot (sps) or bushy (bus), which are disrupted in the gene encoding the cytochrome P450 CYP79F1 (Reintanz et al., 2001;Tantikanjana et al., 2001). These mutants exhibit massive proliferation of shoots, together with other developmental defects. The quantification of hormone levels in sps/cyp79F1 mutant plants indicates that both auxin and cytokinins are higher in the mutants, but the phenotypes of the sps/cyp79F1 plants are consistent with higher levels of cytokinins rather than auxins as the key factor responsible for the change in branching pattern (Tantikanjana et al., 2001).Despite the fact that s...

Metabolic engineering of aliphatic glucosinolates in Chinese cabbage plants expressing Arabidopsis MAM1, CYP79F1, and CYP83A1

“…This result resembles the cytokinin overproduction phenotype, in that cytokinin is known to break apical dominance and increase the thickness of the stem (29,30). In Arabidopsis CYP79F1 knockout mutants, cytokinin accumulated to the highest level at the leaf axil, although the CYP79F1 overexpression lines did not show any phenotypic difference (15,25,26). Hence, it has been suggested that the CYP79F1 gene may modulate cytokinin level at the site of bud initiation and the changes in cytokinin levels at these sites are responsible for the altered shoot branching pattern (15).…”

“…For the process of core structure formation, the methionine-condensed products are first converted into aldoxime by CYP79F1 and CYP79F2, which metabolize the short and long-chain (Met1-6) and only the long-chain methionine (Met5, 6), respectively (14). Aliphatic glucosinolate biosynthesis is completely abolished in the double-knockout Arabidopsis mutant sps-7cyp79F2-1 (15). Aldoximes are generally unstable and toxic, and subsequently they do not accumulate within the cell.…”

Ac-Immobilized, a Stable Source of Activator Transposase That Mediates Sporophytic and Gametophytic Excision of Dissociation Elements in Maize

“…Ac/Ds tagging systems have been utilized to isolate a number of genes (Bancroft et al 1993;Jones et al 1994;James et al 1995;Bhatt et al 1996;Meissner et al 1999), to define promoter and enhancer elements (Sundaresan et al 1995;Chin et al 1999;Greco et al 2003;Wu et al 2003;Jin et al 2004), and to create genetic mosaics for clonal analysis (Peng and Harberd 1997;Jenik and Irish 2001). The tendency of Ds to transpose to linked sites has also been exploited to disrupt genes present in tandem arrays (Takken et al 1999;Tantikanjana et al 2004) and to perform localized saturation mutagenesis (Nishal et al 2005). Ac/Ds mutagenesis platforms in Arabidopsis (Sundaresan et al 1995;Ito et al 1999;Muskett et al 2003;Kuromori et al 2004), tomato (Healy et al 1993;Knapp et al 1994;Meissner et al 2000), tobacco (Scofield et al 1992), rice (Chin et al 1999;Zheng-Ge et al 2003;Kim et al 2004;Kolesnik et al 2004), and barley (Koprek et al 2000;Cooper et al 2004) have been developed.…”

The Plastidic Bile Acid Transporter 5 Is Required for the Biosynthesis of Methionine-Derived Glucosinolates inArabidopsis thaliana

“…The following cytosolic steps involve oxidation of the chainelongated amino acid to an aldoximine catalyzed by the cytochrome P450 monooxygenases CYP79F1 and CYP79F2 (Hansen et al, 2001;Reintanz et al, 2001;Chen et al, 2003;Tantikanjana et al, 2004). A second oxidation step leads to an aci-nitro compound, catalyzed by CYP83A1 (Hemm et al, 2003;Naur et al, 2003), which is then conjugated with a sulfur donor into S-alkyl thiohydroximate.…”