Table 2 Labeling precursors used for different applications. Example labeling strategies employing different stable isotope-labeled precursors for studying IAA biosynthesis. These strategies can be used in combination with various inhibitors (Table 1) for targeted analysis of specific routes of IAA biosynthesis

3 mM [¹³C₃]Serine

[¹⁴ N] ATS

Traces synthesis of Trp and Trp-dependent pathway intermediates. [¹³C₃]Serine is condensed with indole to give [¹³C₃]Trp ([¹³C₃]-label is incorporated into Trp sidechain)

500 μM [¹³C₆]anthranilate and 500 μM [¹⁵N₁]indole

[¹⁴ N] ATS

Multiple auxin precursors upstream of Trp are applied to monitor label incorporation into various intermediates through multiple pathways

500 μM [¹³C₈¹⁵N₁]indole and 500 μM [¹⁵N₁]indole

[¹⁴ N] ATS

Multiple labeled forms of indole are applied to label indole-derived metabolites and potential IAA biosynthesis intermediates. LC–MS/MS analysis workflow for identifying candidate compounds is described in the Materials LC–MS analysis section

500 μM [¹³C₆]anthranilate and 500 μM [¹³C₈¹⁵N₁]indole

[¹⁵ N] ATS

Growing seedlings on [¹⁵ N] ATS media enables rapid [¹⁵ N]-labeling of newly synthesized IAA and biosynthesis intermediates during early seedling development (Fig. 3)

Unlabeled internal standards may be used for quantitation of IAA and biosynthetic intermediates in plant tissue grown on [¹⁵ N] ATS (reverse isotope dilution quantitation)