Metabolomic, transcriptional, hormonal, and signaling cross-talk in superroot2

Abstract

Auxin homeostasis is pivotal for normal plant growth and development. The superroot2 (sur2) mutant was initially isolated in a forward genetic screen for auxin overproducers, and SUR2 was suggested to control auxin conjugation and thereby regulate auxin homeostasis. However, the phenotype was not uniform and could not be described as a pure high auxin phenotype, indicating that knockout of CYP83B1 has multiple effects. Subsequently, SUR2 was identified as CYP83B1, a cytochrome P450 positioned at the metabolic branch point between auxin and indole glucosinolate metabolism. To investigate concomitant global alterations triggered by knockout of CYP83B1 and the countermeasures chosen by the mutant to cope with hormonal and metabolic imbalances, 10-day-old mutant seedlings were characterized with respect to their transcriptome and metabolome profiles. Here, we report a global analysis of the sur2 mutant by the use of a combined transcriptomic and metabolomic approach revealing pronounced effects on several metabolic grids including the intersection between secondary metabolism, cell wall turnover, hormone metabolism, and stress responses. Metabolic and transcriptional cross-talks in sur2 were found to be regulated by complex interactions between both positively and negatively acting transcription factors. The complex phenotype of sur2 may thus not only be assigned to elevated levels of auxin, but also to ethylene and abscisic acid responses as well as drought responses in the absence of a water deficiency. The delicate balance between these signals explains why minute changes in growth conditions may result in the non-uniform phenotype. The large phenotypic variation observed between and within the different surveys may be reconciled by the complex and intricate hormonal balances in sur2 seedlings decoded in this study.

Figure 1.

Phenotype of 10-Day-Old Seedlings Grown on Vertical Agar Plates Showing Phenotypic Differences between sur2 Knockout Mutant and Wild-Type Seedlings. (A) The white arrow underlines the heterogeneity observed among the sur2 mutants. (B) Transverse section of plastic embedded hypocotyls of wild-type and sur2 showing radial expansion of cortical cells and close-up of adventitious root on the hypocotyls.

Figure 2.

Schematic Representation of Indole Glucosinolate Pathway Showing the Importance of Indole-3-Acetaldoxime as a Central Metabolite for Indole Secondary Metabolism in Arabidopsis thaliana. R can either be a glucose moiety or an amino acid. Known positive regulators of transcription involved in this grid are indicated in magenta.

Figure 3.

Biological Processes Affected in the sur2 Knockout Mutant Based on Expression Analysis using the Agilent 22K Array. The 301 genes most significantly affected in their expression were analyzed in BiNGO in order to identify the biological processes significantly affected. The figure is based on the data extracted from the BiNGO analysis (Supplemental Data 2). The representation includes sorting by p-value (color) and size of each group. When GO-groups only referred to a small number of genes, those genes were preferred.

Figure 4.

Metabolites Identified as Accumulating at Significantly Different Levels in the sur2 Mutant and Wild-Type Plants Based on Average Values. Columns represent the metabolites that were significantly detected as up- or down-regulated with a cut-off of two-fold and with a p-value of 0.0001 (see Supplemental File 2). Major metabolites subsequently identified are represented with a *. Sinapoyl glucose (M10) had less than a two-fold variation, but was kept because of its high intensity and because it is known to be co-regulated with sinapoyl malate (M3).

Figure 5.

Chemical Structures of the Metabolites Identified as Accumulating at Different Levels in the sur2 Mutant and Wild-Type Plants.

Figure 6.

Diagram Displaying the Major Intrinsic Metabolomic Network in sur2 Mutant.