Local auxin metabolism regulates environment-induced hypocotyl elongation

Abstract

A hallmark of plants is their adaptability of size and form in response to widely fluctuating environments. The metabolism and redistribution of the phytohormone auxin play pivotal roles in establishing active auxin gradients and resulting cellular differentiation. In Arabidopsis thaliana, cotyledons and leaves synthesize indole-3-acetic acid (IAA) from tryptophan through indole-3-pyruvic acid (3-IPA) in response to vegetational shade. This newly synthesized auxin moves to the hypocotyl where it induces elongation of hypocotyl cells. Here we show that loss of function of VAS2 (IAA-amido synthetase Gretchen Hagen 3 (GH3).17) leads to increases in free IAA at the expense of IAA-Glu (IAA-glutamate) in the hypocotyl epidermis. This active IAA elicits shade- and high temperature-induced hypocotyl elongation largely independently of 3-IPA-mediated IAA biosynthesis in cotyledons. Our results reveal an unexpected capacity of local auxin metabolism to modulate the homeostasis and spatial distribution of free auxin in specialized organs such as hypocotyls in response to shade and high temperature.

Figure 1. Identification of the VAS2 gene

a, Representative plants of Col WT, sav3 mutants and vas2 mutants. Plants were grown on 1/2 MS plates and kept under Wc for 5 days, and then maintained in Wc for 4 days (plant on the left of each panel) or transferred to shade for 4 days (plant on the right of each panel). b, Diagram of the VAS2 genomic DNA sequence, with exons indicated by boxes. Mutation of vas2-1 and the SALK T-DNA line vas2-2 are shown. c, Quantification of hypocotyl length of plants grown under similar conditions as described above. d, vas2-2, but not gh3.9, can suppress the short hypocotyl of the sav3-3 mutant under shade. In c,d the results are shown as mean ± s.e.m. ***P < 0.001 (two-tailed Student’s t-test). The comparisons are between WT plants and mutant plants under the same growth conditions and the same treatment. e, Glu is the most efficient co-substrate of IAA-amido synthetase VAS2. VAS2 protein was expressed in E. coli, purified to homogeneity and assayed for conjugation activities using free IAA and the 20 natural amino acids, respectively. V, velocity.

Figure 2. vas2 mutant accumulates more free IAA and IAA biosynthetic precursors, but less of amino acid conjugate IAA-Glu

a, Major Trp-dependent IAA biosynthetic and catabolic/conjugating pathways in A. thaliana. ANT, anthranilate; Trp, tryptophan; 3-IPA, indole-3-pyruvic acid; IAA, indole-3-acetic acid; oxIAA, 2-oxoindole-3-acetic acid; IAA-Glu, IAA-glutamate; Other GH3, seven additional GH3 IAA-amido synthetases that can conjugate IAA to various amino acids including Asp in vitro. b, vas2 mutants accumulate larger quantities of ANT. c, vas2 mutants contain elevated amounts of Trp. d, vas2 mutants possess larger amounts of 3-IPA. e, vas2 mutants amass increased quantities of free IAA. f, vas2 mutants accumulate similar concentrations of oxIAA as WT plants. g, vas2 mutants possess smaller amounts of IAA-Glu. h, vas2 mutants possess larger amounts of IAA-Asp. All results are shown as mean ± s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001 (two-tailed Student’s t-test). Comparisons are established between WT plants and mutant plants cultivated under the identical growth conditions and same treatments.

Figure 3. Shade-induced hypocotyl elongation of vas2, but not vas1, is largely independent of auxin transport from cotyledons to hypocotyls

a, Representative plants of WT, sav3-1 mutant, vas2 sav3-1 double mutant and vas2-1 mutant are shown. Plants were grown on 1/2 MS (−NPA) or 1/2 MS containing 4 μM NPA (+NPA) for 5 days, then maintained in Wc for 4 days (plant on the left of each panel) or transferred to shade for 4 days (plant on the right of each panel). b, Quantification of hypocotyl length. c, Representative plants of WT, sav3 mutants, pin3-4 mutants, vas2 mutants and vas2 pin3-4 double mutants are shown. Plants were grown in similar conditions to those described above without NPA treatment. d, Quantification of hypocotyl length. e, Shade-induced hypocotyl elongation of vas1-2 sav3-1 double mutant is strongly inhibited by NPA. In b,d,e the results are shown as mean ± s.e.m. ***P < 0.001 (two-tailed Student’s t-test). Comparisons are made between WT plants and mutants prepared using the same growth conditions and the same treatments.

Figure 4. vas2 mutation has no effect on the responses of the cotyledon’s angle and size of the sav3 mutant on treatment with a combination of NPA and shade

a, Representative plants of Col WT, sav3-1 mutant, vas2-1 sav3-1 double mutant and vas2-1 mutant are shown. Plants were grown on 1/2 MS without NPA under Wc (Wc, −NPA) for 9 days or 1/2 MS containing 4 μM NPA for 5 days, then transferred to shade for 4 days (shade, +NPA). The cotyledon angle—the angle between the cotyledon and petiole—is denoted by red lines and further represented with black lines for clarification. b, Quantification of the cotyledon angle and the ratio of the cotyledon angle with shade and NPA treatment to that without shade treatment (shade, +NPA/Wc, −NPA). c, Representative cotyledons of Col WT, sav3-1 mutant, vas2 sav3-1 double mutant and vas2-1 mutant are shown. Plant growth conditions are the same as described in Fig. 3a. d, Quantification of cotyledon size and the ratio of the cotyledon size with shade and NPA treatment to that without shade treatment (shade, +NPA/Wc, −NPA). Values are means ± s.e.m.

Figure 5. DR5::GUS intensity and distribution indicate that vas2 mutant accumulates more free IAA, which is distributed to the epidermis of hypocotyl under shade regardless of NPA treatment

DR5::GUS was introduced into sav3-1 mutant, vas2-2 mutant and vas2-2 sav3-1 double mutants by genetic cross. a–h, vas2 mutants exhibit stronger DR5::GUS activities under both Wc and shade. Plants harbouring the DR5::GUS transgene were grown on 1/2 MS plates (−NPA) under Wc for 5 days, and then kept in Wc (a–d) or transferred to shade (e–h) for 4 days. i–l, NPA treatment inhibits DR5::GUS accumulation in the hypocotyl of WT plants, but not of vas2 mutants. Plants harbouring the DR5::GUS transgene were grown on 1/2 MS plates supplemented with 4 μM NPA (+NPA) under Wc for 5 days, and then transferred to shade (i–l) for 4 days.

Figure 6. VAS2 regulates high temperature-induced hypocotyl elongation

a, Representative plants of WT, sav3-1 mutant, vas2 sav3-1 double mutant and vas2-1 mutant are shown. Plants were grown on 1/2 MS (−NPA) or 1/2 MS containing 4 μM NPA (+NPA) for 5 days at 22 °C, then kept at 22 °C for 4 days (plants on the left of each panel) or transferred to 29 °C for 4 days (plants on the right of each panel). b, Quantification of hypocotyl length. The results are shown as mean ± s.e.m. ***P < 0.001 (two-tailed Student’s t-test). Comparisons are made between WT plants and mutants prepared using the same growth conditions and the same treatments. c–h, vas2 mutants exhibit stronger DR5::GUS expression under both 22 and 29 °C. Plants harbouring the DR5::GUS transgene were grown on 1/2 MS plates (−NPA) at 22 °C for 5 days, and then kept at 22 °C (c,d) or transferred to 29 °C (e–h) for 4 days. g–h, NPA treatment inhibits DR5::GUS accumulation in the hypocotyl of WT plants, but not of vas2 mutants. Plants harbouring the DR5::GUS transgene were grown on 1/2 MS plates supplemented with 4 μM NPA (+NPA) at 22 °C for 5 days and then transferred to 29 °C for 4 days.