Tryptophan-independent auxin biosynthesis contributes to early embryogenesis in Arabidopsis

The phytohormone auxin regulates nearly all aspects of plant growth and development. Tremendous achievements have been made in elucidating the tryptophan (Trp)-dependent auxin biosynthetic pathway; however, the genetic evidence, key components, and functions of the Trp-independent pathway remain elusive. Here we report that the Arabidopsis indole synthase mutant is defective in the long-anticipated Trp-independent auxin biosynthetic pathway and that auxin synthesized through this spatially and temporally regulated pathway contributes significantly to the establishment of the apical–basal axis, which profoundly affects the early embryogenesis in Arabidopsis. These discoveries pave an avenue for elucidating the Trp-independent auxin biosynthetic pathway and its functions in regulating plant growth and development.The phytohormone auxin plays critical roles in almost every aspect of plant development including embryogenesis, architecture formation, and tropic growth. One of the most fascinating topics in plant biology is how auxin can have so many diverse and context-specific functions (1). Dynamic auxin gradients, which are regulated mainly by local auxin synthesis, catabolism, conjugation, and polar auxin transport, are essential for integration of various environmental and endogenous signals (2, 3). Auxin perception and signaling systems are responsible for a read-out of the auxin gradients (1).Local auxin biosynthesis is important for leaf development, shade avoidance, root-specific ethylene sensitivity, vascular patterning, flower patterning, and embryogenesis (4). Indole-3-acetic acid (IAA), the naturally occurring principal auxin in plants, is biosynthesized from tryptophan (Trp) through four proposed routes according to their key intermediates, namely indole-3-acetaldoxime (IAOx), indole-3-pyruvic acid (IPyA), indole-3-acetamide (IAM), and tryptamine (TAM) (5). The TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA)/YUCCA (YUC) linear pathway has been considered as a predominant Trp-dependent auxin biosynthetic pathway (4, 6–8). However, labeling studies and analyses of Trp auxotrophic mutants have long predicted the existence of a Trp-independent IAA biosynthetic pathway (9–13). When the Arabidopsis and maize seedlings were fed with isotope-labeled precursor, IAA was more enriched than Trp, and the incorporation of label into IAA from Trp is low, suggesting that IAA can be produced de novo without Trp as an intermediate (11, 12). The Trp biosynthetic mutant trp1, defective in phosphoribosylanthranilate transferase (PAT), and indole-3-glycerol phosphate synthase (IGS) antisense plants are deficient in steps earlier than indole-3-glycerol phosphate (IGP) formation and display decreased levels of both IAA and Trp (10, 14). However, the trp3 and trp2 mutants, defective in tryptophan synthase α (TSA) and tryptophan synthase β (TSB) subunits, respectively, accumulate higher levels of IAA than the wild type despite containing lower Trp levels (10, 11, 15, 16). These data strongly suggested the existence of the Trp-independent IAA biosynthetic pathway that might branch from IGP and/or indole (10). Further studies suggested that a cytosol-localized indole synthase (INS) may be involved in the Trp-independent biosynthesis of indole-containing metabolite (17). However, the molecular basis, biological functions, and spatiotemporal regulation of the Trp-independent IAA biosynthetic pathway have remained a mystery.In higher eukaryotes, embryogenesis initiates the generation of the species-specific body plan. During embryogenesis, a single-celled zygote develops into a functional multicellular organism with cells adopting specific fates according to their relative positions. In higher plants, essential architecture features, such as body axes and major tissue layers, are established in embryogenesis, and auxin plays a vital role in apical-basal patterning and embryo axis formation (18, 19). Local auxin biosynthesis, polar auxin transport facilitated by PIN-FORMED1/3/4/7 (PIN1/3/4/7), and auxin response coordinately regulate apical–basal pattern formation during embryogenesis (7, 20–22). The TAA and YUC families in Trp-dependent IAA biosynthesis predominantly regulate embryogenesis at or after the globular stage (7, 22). However, the auxin source for embryogenesis before the globular stage remains elusive. In this study, we provide, to our knowledge, the first genetic and biochemical evidence that the cytosol-localized INS is a key component in the long predicted Trp-independent auxin biosynthetic pathway and is critical for apical–basal pattern formation during early embryogenesis in Arabidopsis.