Tryptophan-independent auxin biosynthesis contributes to early embryogenesis in Arabidopsis

Abstract

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.

Keywords: Arabidopsis thaliana; IAA biosynthesis; embryogenesis; phytohormone; tryptophan.

Fig. 1.

INS and TSA display distinct subcellular localizations and different functions in tryptophan biosynthesis and IAA biosynthesis. (A) Subcellular localizations of INS-GFP, TSA-GFP, cTP-INS-GFP, and ∆cTP-TSA-GFP in protoplasts from stable transgenic plants. Ch, chlorophyll. (B) Phenotypes of 12-d-old wild-type (Col-0), ins-1, trp3-1, 35S:TSA/trp3-1, 35S:cTP-INS/trp3-1, and INS:INS/trp3-1 seedlings. (C) Tryptophan (Trp) contents in 7-d-old seedlings. gdw⁻¹, per gram dry weight. The Trp accumulation mutant trp5-1 was used as a control. Data are represented as mean ± SEM; **P < 0.01 by Student’s t test; n = 5. (D) Free IAA contents in 12-d-old seedlings (n = 5). gfw⁻¹, per gram fresh weight. Data are represented as mean ± SEM; *P < 0.05 and **P < 0.01 by Student’s t test; n = 5. (Scale bars, 10 μm in A and 0.5 cm in B.)

Fig. 2.

INS is a key component in the Trp-independent IAA biosynthetic pathway. (A) Schematic diagrams of ¹³C₆-anthranilic acid (¹³C₆-ANT) feeding experiment. Seedlings were transferred to liquid Murashige and Skoog (MS) media containing ¹³C₆-ANT and cultured for 24 h. In a parallel test, the MS media contained ¹³C₆-ANT and unlabeled Trp, which should increase the Trp pool size and decrease the biosynthesis of ¹³C₆-IAA only through the Trp-dependent pathway. (B) ¹³C₆-IAA contents in 12-d-old wild-type (Col-0), ins-1, and trp3-1 seedlings after ¹³C₆-ANT or ¹³C₆-ANT plus Trp treatment. gfw⁻¹, per gram fresh weight; ns, no significant difference. Data are represented as mean ± SEM; **P < 0.01 by Student’s t test; n = 5. (C) Phenotypes of 7-d-old wild-type (Col-0), ins-1, sur2, ins-1 sur2, trp3-1, and trp3-1 sur2 seedlings. (Scale bar, 0.5 cm.) (D) Hypocotyl length of 9-d-old wild-type (Col-0), ins-1, wei8-1, and ins-1 wei8-1 seedlings at 20 °C and 28 °C. Data are represented as mean ± SEM; different letters indicate significant difference by Tukey’s multiple comparison test (P < 0.05); n = 30.

Fig. 3.

Effects of INS and TSA on embryogenesis. Embryos of wild-type (Col-0) (A) and trp3-1 (B) plants at 1-, 4-, 8- or 16-cell globular (g), heart (h), torpedo (to), and cotyledon (c) stages. HY, hypophysis; LSC, lens-shaped cell; QC, quiescent center; and CSC, columella stem cells. (C–I) When normal sibling embryos reach the heart stage, defective embryos of ins-1 are arrested before the globular stage with normal body plans (C) or display abnormal cell division patterns (D–I). (J–P) When normal sibling embryos reach the heart stage, defective embryos from the ins-1 trp3-1^+/− siliques are arrested with normal body plans (J) or exhibit abnormal cell division patterns (K–P). Arrows indicate aberrant cell divisions in early embryogenesis, and brackets mark aberrant basal cell regions.

Fig. 4.

Trp-independent IAA biosynthesis contributes significantly to early embryogenesis. (A) Percentage of defective embryos in the wild type (Col-0) and mutants at the globular stage. Data are represented as mean ± SEM; **P < 0.01 by Student’s t test; n = 4, and each biological repeat contains about 250 embryos. (B) Free IAA contents in ovules from the wild-type (Col-0), ins-1, ins-1 trp3-1^+/−, and trp3-1 siliques. “ins-1 normal” refers to the ins-1 ovules with normal morphology, “ins-1 abnormal” refers to the ins-1 ovules containing defective embryos, and “ins-1 trp3-1” refers to the abnormal ovules from ins-1 trp3-1^+/− siliques that contain defective embryos of ins, ins trp3^+/−, and ins trp3-1^−/− together. Data are represented as mean ± SEM; *P < 0.05 and **P < 0.01 by Student’s t test; n = 5. (C) Expression patterns of INS and TSA proteins during embryogenesis. 1C, 2C, 4C, 8C, and 16C represent 1-, 2-, 4-, 8-, and 16-cell stages, respectively. (D–M) DR5 activities shown by GFP signals in wild-type embryos (D and E), defective ins-1 embryos (F and G), trp3-1 embryos (H and I), normal ins-1 embryos (J and K), and ins-1 trp3-1 embryos (L and M). (Scale bars, 15 μm in C–M.)

Fig. 5.

INS and YUCs act in parallel IAA biosynthetic pathways. (A–L) Morphologies of wild-type (Col-0) (A), ins-1 (B–F), yuc4 yuc6 (yuc4/6) (G), and ins-1 yuc4/6 (H–L) embryos at the globular stage. Arrows indicate aberrant cell divisions and brackets mark aberrant basal cell regions. (M) Percentage of defective embryos in Col-0, ins-1, yuc4/6, and ins-1 yuc4/6 at the globular stage. Data are represented as mean ± SEM; different letters indicate significant difference by Tukey’s multiple comparison test (P < 0.05); n = 4, and each biological repeat contains about 250 embryos. (N) Free IAA levels in 12-d-old Col-0, ins-1, yuc4/6, and ins-1 yuc4/6 seedlings. gfw⁻¹, per gram fresh weight. Data are represented as mean ± SEM; different letters indicate significant difference by Tukey’s multiple comparison test (P < 0.05); n = 5.

Fig. 6.

A model of the Trp-dependent and Trp-independent IAA biosynthetic pathways. Solid arrows refer to pathways with identified enzymes and dashed arrows to undefined ones. ANT, anthranilate; AS, anthranilate synthase; CHA, chorismic acid; IAAld, indole-3-acetaldehyde; IAM, indole-3-acetamide; IAN, indole-3-acetonitrile; IAOx, indole-3-acetaldoxime; IGP, indole-3-glycerol phosphate; IGs, indole glucosinolates; IGS, indole-3-glycerol phosphate synthase; IPyA, indole-3-pyruvic acid; PAT, phosphoribosylanthranilate transferase; TAA1, tryptophan aminotransferase of Arabidopsis1; TAM, tryptamine; TSA, tryptophan synthase α; TSB, tryptophan synthase β; YUC, YUCCA.