The largely unused uracil-excision molecular cloning technique has excellent features in most aspects compared to other modern cloning techniques. Its application has, however, been hampered by incompatibility with proof-reading DNA polymerases. We have advanced the technique by identifying PfuCx as a compatible proof-reading DNA polymerase and by developing an improved vector design strategy. The original features of the technique, namely simplicity, speed, high efficiency and low cost are thus combined with high fidelity as well as a transparent, simple and flexible vector design. A comprehensive set of vectors has been constructed covering a wide range of different applications and their functionality has been confirmed.

BackgroundGlucosinolates are natural metabolites in the order Brassicales that defend plants against both herbivores and pathogens and can attract specialized insects. Knowledge about the genes controlling glucosinolate regulation is limited. Here, we identify three R2R3 MYB transcription factors regulating aliphatic glucosinolate biosynthesis in Arabidopsis by combining several systems biology tools.Methodology/Principal Findings MYB28 was identified as a candidate regulator of aliphatic glucosinolates based on its co-localization within a genomic region controlling variation both in aliphatic glucosinolate content (metabolite QTL) and in transcript level for genes involved in the biosynthesis of aliphatic glucosinolates (expression QTL), as well as its co-expression with genes in aliphatic glucosinolate biosynthesis. A phylogenetic analysis with the R2R3 motif of MYB28 showed that it and two homologues, MYB29 and MYB76, were members of an Arabidopsis-specific clade that included three characterized regulators of indole glucosinolates. Over-expression of the individual MYB genes showed that they all had the capacity to increase the production of aliphatic glucosinolates in leaves and seeds and induce gene expression of aliphatic biosynthetic genes within leaves. Analysis of leaves and seeds of single knockout mutants showed that mutants of MYB29 and MYB76 have reductions in only short-chained aliphatic glucosinolates whereas a mutant in MYB28 has reductions in both short- and long-chained aliphatic glucosinolates. Furthermore, analysis of a double knockout in MYB28 and MYB29 identified an emergent property of the system since the absence of aliphatic glucosinolates in these plants could not be predicted by the chemotype of the single knockouts.Conclusions/SignificanceIt seems that these cruciferous-specific MYB regulatory genes have evolved both overlapping and specific regulatory capacities. This provides a unique system within which to study the evolution of MYB regulatory factors and their downstream targets.

Phenotypic variation between individuals of a species is often under quantitative genetic control. Genomic analysis of gene expression polymorphisms between individuals is rapidly gaining popularity as a way to query the underlying mechanistic causes of variation between individuals. However, there is little direct evidence of a linkage between global gene expression polymorphisms and phenotypic consequences. In this report, we have mapped quantitative trait loci (QTLs)–controlling glucosinolate content in a population of 403 Arabidopsis Bay × Sha recombinant inbred lines, 211 of which were previously used to identify expression QTLs controlling the transcript levels of biosynthetic genes. In a comparative study, we have directly tested two plant biosynthetic pathways for association between polymorphisms controlling biosynthetic gene transcripts and the resulting metabolites within the Arabidopsis Bay × Sha recombinant inbred line population. In this analysis, all loci controlling expression variation also affected the accumulation of the resulting metabolites. In addition, epistasis was detected more frequently for metabolic traits compared to transcript traits, even when both traits showed similar distributions. An analysis of candidate genes for QTL-controlling networks of transcripts and metabolites suggested that the controlling factors are a mix of enzymes and regulatory factors. This analysis showed that regulatory connections can feedback from metabolism to transcripts. Surprisingly, the most likely major regulator of both transcript level for nearly the entire pathway and aliphatic glucosinolate accumulation is variation in the last enzyme in the biosynthetic pathway, AOP2. This suggests that natural variation in transcripts may significantly impact phenotypic variation, but that natural variation in metabolites or their enzymatic loci can feed back to affect the transcripts.

Characteristic for cruciferous plants is their production of N-and S-containing indole phytoalexins with disease resistance and cancer-preventive properties, previously proposed to be synthesized from indole independently of tryptophan. We show that camalexin, the indole phytoalexin of Arabidopsis thaliana, is synthesized from tryptophan via indole-3-acetaldoxime (IAOx) in a reaction catalyzed by CYP79B2 and CYP79B3. Cyp79B2͞cyp79B3 double knockout mutant is devoid of camalexin, as it is also devoid of indole glucosinolates [Zhao, Y., Hull, A. K., Gupta, N. R., Goss, K. A., Alonso, J., Ecker, J. R., Normanly, J., Chory, J. & Celenza, J. L. (2002) Genes Dev. 16, 3100 -3112], and isotope-labeled IAOx is incorporated into camalexin. These results demonstrate that only CYP79B2 and CYP79B3 contribute significantly to the IAOx pool from which camalexin and indole glucosinolates are synthesized. Furthermore, production of camalexin in the sur1 mutant devoid of glucosinolates excludes the possibility that camalexin is derived from indole glucosinolates. CYP79B2 plays an important role in camalexin biosynthesis in that the transcript level of CYP79B2, but not CYP79B3, is increased upon induction of camalexin by silver nitrate as evidenced by microarray analysis and promoter-␤-glucuronidase data. The structural similarity between cruciferous indole phytoalexins suggests that these compounds are biogenetically related and synthesized from tryptophan via IAOx by CYP79B homologues. The data show that IAOx is a key branching point between several secondary metabolic pathways as well as primary metabolism, where IAOx has been shown to play a critical role in IAA homeostasis.C haracteristic for cruciferous plants is the synthesis of a wide range of species-specific phytoalexins that structurally are sulfur-containing indole alkaloids (1) and the synthesis of glucosinolates (reviewed in ref.2). Both groups of natural products are involved in plant defense and have cancer-preventive properties (3, 4). Very little is known about the biosynthetic pathway of the S-containing indole phytoalexins. Their similar structure with N-and S-containing side chains at C-3 of the indole ring suggests a biogenetic relationship (5). Camalexin (3-thiazol-2Ј-yl-indole) is produced in the model plant Arabidopsis thaliana (6). It is induced by a variety of microorganisms, e.g., Pseudomonas syringae (6) and Alternaria brassisicola (7), and by abiotic factors, such as AgNO 3 (8). These findings make camalexin a good model compound for studying biosynthesis and regulation of cruciferous indole phytoalexins. In vivo feeding experiments where radiolabeled anthranilate and tryptophan were applied on leaves treated with AgNO 3 led to the suggestion that tryptophan was not a precursor in camalexin biosynthesis because tryptophan was much less efficiently incorporated into camalexin compared with anthranilate (8, 9). The data were further supported by labeling studies performed in three tryptophan mutants (8), where reduced levels of camalexin accumulated in ...

Genomic approaches have accelerated the study of the quantitative genetics that underlie phenotypic variation. These approaches associate genome-scale analyses such as transcript profiling with targeted phenotypes such as measurements of specific metabolites. Additionally, these approaches can help identify uncharacterized networks or pathways. However, little is known about the genomic architecture underlying data sets such as metabolomics or the potential of such data sets to reveal networks. To describe the genetic regulation of variation in the Arabidopsis thaliana metabolome and test our ability to integrate unknown metabolites into biochemical networks, we conducted a replicated metabolomic analysis on 210 lines of an Arabidopsis population that was previously used for targeted metabolite quantitative trait locus (QTL) and global expression QTL analysis. Metabolic traits were less heritable than the average transcript trait, suggesting that there are differences in the power to detect QTLs between transcript and metabolite traits. We used statistical analysis to identify a large number of metabolite QTLs with moderate phenotypic effects and found frequent epistatic interactions controlling a majority of the variation. The distribution of metabolite QTLs across the genome included 11 QTL clusters; 8 of these clusters were associated in an epistatic network that regulated plant central metabolism. We also generated two de novo biochemical network models from the available data, one of unknown function and the other associated with central plant metabolism.