Auxin and tryptophan homeostasis are facilitated by the ISS1/VAS1 aromatic aminotransferase in arabidopsis

Indole-3-acetic acid (IAA) plays a critical role in regulating numerous aspects of plant growth and development. While there is much genetic support for tryptophan-dependent (Trp-D) IAA synthesis pathways, there is little genetic evidence for tryptophan-independent (Trp-I) IAA synthesis pathways. Using Arabidopsis, we identified two mutant alleles of ISS1 ( I: ndole S: evere S: ensitive) that display indole-dependent IAA overproduction phenotypes including leaf epinasty and adventitious rooting. Stable isotope labeling showed that iss1, but not WT, uses primarily Trp-I IAA synthesis when grown on indole-supplemented medium. In contrast, both iss1 and WT use primarily Trp-D IAA synthesis when grown on unsupplemented medium. iss1 seedlings produce 8-fold higher levels of IAA when grown on indole and surprisingly have a 174-fold increase in Trp. These findings indicate that the iss1 mutant's increase in Trp-I IAA synthesis is due to a loss of Trp catabolism. ISS1 was identified as At1g80360, a predicted aromatic aminotransferase, and in vitro and in vivo analysis confirmed this activity. At1g80360 was previously shown to primarily carry out the conversion of indole-3-pyruvic acid to Trp as an IAA homeostatic mechanism in young seedlings. Our results suggest that in addition to this activity, in more mature plants ISS1 has a role in Trp catabolism and possibly in the metabolism of other aromatic amino acids. We postulate that this loss of Trp catabolism impacts the use of Trp-D and/or Trp-I IAA synthesis pathways.T32 AR059033 - NIAMS NIH HH

The Arabidopsis ATR1 Myb Transcription Factor Controls Indolic Glucosinolate Homeostasis

Plants derive a number of important secondary metabolites from the amino acid tryptophan (Trp), including the growth regulator indole-3-acetic acid (IAA) and defense compounds against pathogens and herbivores. In previous work, we found that a dominant overexpression allele of the Arabidopsis (Arabidopsis thaliana) Myb transcription factor ATR1, atr1D, activates expression of a Trp synthesis gene as well as the Trp-metabolizing genes CYP79B2, CYP79B3, and CYP83B1, which encode enzymes implicated in production of IAA and indolic glucosinolate (IG) antiherbivore compounds. Here, we show that ATR1 overexpression confers elevated levels of IAA and IGs. In addition, we show that an atr1 loss-of-function mutation impairs expression of IG synthesis genes and confers reduced IG levels. Furthermore, the atr1-defective mutation suppresses Trp gene dysregulation in a cyp83B1 mutant background. Together, this work implicates ATR1 as a key homeostatic regulator of Trp metabolism and suggests that ATR1 can be manipulated to coordinately control the suite of enzymes that synthesize IGs

Redirection of tryptophan metabolism in tobacco by ectopic expression of an Arabidopsis indolic glucosinolate biosynthetic gene

Indole-3-acetaldoxime (IAOx) is a branch point compound of tryptophan (Trp) metabolism in glucosinolate-producing species such as Arabidopsis, serving as a precursor to indole-glucosinolates (IGs), the defense compound camalexin, indole-3-acetonitrile (IAN) and indole-3-acetic acid (IAA). We synthesized [(2)H(5)] and [(13)C(10)(15)N(2)]IAOx and [(13)C(6)], [(2)H(5)] and [2\u27,2\u27-(2)H(2)]IAN in order to quantify endogenous IAOx and IAN in Arabidopsis and tobacco, a non-IG producing species. We found that side chain-labeled [2\u27,2\u27-(2)H(2)]IAN overestimated the amount of IAN by 2-fold compared to when [(2)H(5)]IAN was used as internal standard, presumably due to protium-deuterium exchange within the internal standard during extraction of plant tissue. We also determined that [(13)C(1)]IAN underestimated the amount of IAN when the ratio of [(13)C(1)]IAN standard to endogenous IAN was greater than five to one, whereas either [(2)H(5)]IAN or [(13)C(6)]IAN showed a linear relationship with endogenous IAN over a broader range of concentrations. Transgenic tobacco vector control lines did not have detectable levels of IAOx or IAN (limit of detection∼100 pg/gfr.wt), while lines expressing either the IAOx-producing CYP79B2 or CYP79B3 genes from Arabidopsis under CaMV 35S promoter control accumulated IAOx in the range of 1-9 μg/gfr.wt. IAN levels in these lines ranged from 0.6 to 6.7 μg/gfr.wt, and IAA levels were ∼9-14-fold above levels in control lines. An Arabidopsis line expressing the same CYP79B2 overexpression construct accumulated IAOx in two of three lines measured (∼200 and 400 ng/gfr.wt) and accumulated IAN in all three lines. IAN is proposed to be a metabolite of IAOx or an enzymatic breakdown product of IGs induced upon tissue damage. Since tobacco does not produce detectable IGs, the tobacco data are consistent with IAN being a metabolite of IAOx. IAOx and IAN were also examined in the Arabidopsis activation tagged yucca mutant, and no accumulation of IAOx was found above the limits of detection but accumulation of IAN (3-fold above wt) occurred. The latter was surprising in light of recent reports that rule out IAOx and IAN as intermediates in YUCCA-mediated IAA synthesis

Trp-dependent auxin biosynthesis in Arabidopsis: involvement of cytochrome P450s CYP79B2 and CYP79B3

The plant hormone auxin regulates many aspects of plant growth and development. Although several auxin biosynthetic pathways have been proposed, none of these pathways has been precisely defined at the molecular level. Here we provide in planta evidence that the two Arabidopsis cytochrome P450s, CYP79B2 and CYP79B3, which convert tryptophan (Trp) to indole-3-acetaldoxime (IAOx) in vitro, are critical enzymes in auxin biosynthesis in vivo. IAOx is thus implicated as an important intermediate in auxin biosynthesis. Plants overexpressing CYP79B2 contain elevated levels of free auxin and display auxin overproduction phenotypes. Conversely, cyp79B2 cyp79B3 double mutants have reduced levels of IAA and show growth defects consistent with partial auxin deficiency. Together with previous work on YUCCA, a flavin monooxygenase also implicated in IAOx production, and nitrilases that convert indole-3-acetonitrile to auxin, this work provides a framework for further dissecting auxin biosynthetic pathways and their regulation

Auxin and Tryptophan Homeostasis Are Facilitated by the ISS1/VAS1 Aromatic Aminotransferase in Arabidopsis

Indole-3-acetic acid (IAA) plays a critical role in regulating numerous aspects of plant growth and development. While there is much genetic support for tryptophan-dependent (Trp-D) IAA synthesis pathways, there is little genetic evidence for tryptophan-independent (Trp-I) IAA synthesis pathways. Using Arabidopsis, we identified two mutant alleles of ISS1 ( I: ndole S: evere S: ensitive) that display indole-dependent IAA overproduction phenotypes including leaf epinasty and adventitious rooting. Stable isotope labeling showed that iss1, but not WT, uses primarily Trp-I IAA synthesis when grown on indole-supplemented medium. In contrast, both iss1 and WT use primarily Trp-D IAA synthesis when grown on unsupplemented medium. iss1 seedlings produce 8-fold higher levels of IAA when grown on indole and surprisingly have a 174-fold increase in Trp. These findings indicate that the iss1 mutant's increase in Trp-I IAA synthesis is due to a loss of Trp catabolism. ISS1 was identified as At1g80360, a predicted aromatic aminotransferase, and in vitro and in vivo analysis confirmed this activity. At1g80360 was previously shown to primarily carry out the conversion of indole-3-pyruvic acid to Trp as an IAA homeostatic mechanism in young seedlings. Our results suggest that in addition to this activity, in more mature plants ISS1 has a role in Trp catabolism and possibly in the metabolism of other aromatic amino acids. We postulate that this loss of Trp catabolism impacts the use of Trp-D and/or Trp-I IAA synthesis pathways.T32 AR059033 - NIAMS NIH HH