Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants

Abstract

Plants grown at high densities perceive a decrease in the red to far-red (R:FR) ratio of incoming light, resulting from absorption of red light by canopy leaves and reflection of far-red light from neighboring plants. These changes in light quality trigger a series of responses known collectively as the shade avoidance syndrome. During shade avoidance, stems elongate at the expense of leaf and storage organ expansion, branching is inhibited, and flowering is accelerated. We identified several loci in Arabidopsis, mutations in which lead to plants defective in multiple shade avoidance responses. Here we describe TAA1, an aminotransferase, and show that TAA1 catalyzes the formation of indole-3-pyruvic acid (IPA) from L-tryptophan (L-Trp), the first step in a previously proposed, but uncharacterized, auxin biosynthetic pathway. This pathway is rapidly deployed to synthesize auxin at the high levels required to initiate the multiple changes in body plan associated with shade avoidance.

Figure 1. Phenotypic Characterization of sav3 Mutants

(A) Hypocotyl phenotype of sav3 mutants. 5-day old seedlings were treated with Wc or simulated shade for 3 days. Representative seedlings of shade-treated WT Col-0 and sav3-2 are shown on the left; hypocotyl length is quantified on the right. phyB9 is a null allele of PHYB. (B) Leaf area and petiole length phenotypes of sav3-2. 7-day-old seedlings were treated with Wc or simulated shade for two weeks. Petiole length and leaf area of the first set of true leaves were measured. (C) Leaf hyponasty phenotype of sav3-1. Four-week-old plants were cultivated in a greenhouse and then exposed for 10 days to supplemental FR irradiation (see supplement for details).Error bars represent standard error of the mean (SEM).

Figure 2. SAV3 Encodes an Enzyme with an Alliinase C-Terminal/Aminotransferase Domain

(A) Protein alignment of Arabidopsis SAV3 family. The alliinase C-terminal domain is marked by a blue line. K217 is the PLP binding site. (B) Cytoplasmic localization of SAV3-YFP in root meristem cells. SAV3-YFP was expressed stably in sav3-1 under the control of the CaMV 35S promoter. The YFP fusion protein was visualized using a Leica confocal microscope. (C) Complementation test using SAV3 genomic DNA fragment (SAV3) or mutant forms of SAV3 (K217G, K217R or an N-terminal truncation containing the first 130 a.a.). At least 3 independent transgenic lines were used for characterization of SAV3 localization and phenotypes. Mean values of more than 12 seedlings are shown; error bars represent SEM.

Figure 3. Sav3 Mutants are Defective in Auxin Biosynthesis

(A) Responses of sav3 to the auxin analog, picloram. Seedlings were grown in Wc on plates with picloram for 3 days and then moved to Wc or simulated shade for 3 days. Error bars represent SEM. (B) sav3 seedlings have reduced IAA levels in Wc and shade light. WT and sav3 were grown in Wc and moved to Wc or shade for 1 hour. (C) sav3 has lower rates of IAA biosynthesis in the shade. Seedlings were grown for 5 days in Wc and then incubated in ½ MS with 30% ²H₂O and treated with Wc or shade for 2 hours. Samples with the same letter are not significantly different based on one-way ANOVA followed by a two-sided t-test at P<0.01.

Figure 4. Global Expression Analysis of sav3 Implicates a Role for SAV3 in Auxin Response

(A) Expression pattern of shade-induced genes. (B) Co-response analysis of SAV3-dependent, shade up-regulated genes. Expression data of each gene was normalized and medium centered using Cluster, and visualized by Treeview (http://rana.lbl.gov/EisenSoftware.htm). Green and red represent lower and higher expression level as compared to the median value, respectively. (C) Quantification of IAA19 and IAA29 expression using quantitative RT-PCR. Relative expression level as compared to a reference gene (At2G39960) is shown. Data are presented as mean values from 3 replicates +/− SEM.

Figure 5. SAV3 Expression is Dynamic

(A) SAV3 is expressed predominantly in the leaf margins. Expression patterns of P_SAV3n::SAV3-GUS and DR5::GUS are shown. 5-day-old seedlings were treated with Wc or shade for 8 hours. (B) and (C) in situ hybridization results show SAV3 is expressed during the heart (B) and torpedo (C) stages of embryogenesis. D) Shade-induced increase in DR5-GUS expression is dependent on SAV3 expression in leaves and functional auxin transport. 5-day old seedlings were pre-treated with 5 µM of NPA by submerging roots in NPA solution for 30 min and then subjected to Wc or shade for 4 hrs. Relative GUS activity was calculated by normalizing to the chlorophyll content. Mean values from 3 replicates are shown; error bars represent SEM. T-test assuming equal variance was carried out for Wc and shade-treated sample pairs. Comparing Wc and shade treated samples, only NPA treated hypocotyls shows no significant difference (using P<0.05 as cut-off).

Figure 6. SAV3 is a Trp Aminotransferase Involved in Auxin Biosynthesis

(A) Schematic diagram of the proposed IAA biosynthetic pathways. (B) Identification by LC/MS of indole pyruvic acid (IPA) as the product of SAV3 when L-Trp is used as substrate. Shown are the UV-chromatogram profiles of IPA control (1), reaction mixture (2) and reaction mixture without SAV3 (3). The number shown is the calculated mass of IPA.

Figure 7. Enzymatic Characterization of SAV3

(A) Determination of Km and Vmax of SAV3 to L-Trp. (B) sav3-2 is hypersensitive to 5-MT. Seedlings were grown on ½ MS medium supplemented with 20 µM 5-MT for 9 days in Wc. (C) Superimposed structure of SAV3 and alliinase active sites. SAV3 monomers are represented as green and cyan ribbons. Alliinase monomers are represented in grey and wheat ribbons (PDB code: 2hox). Labels are those of SAV3 residues. Pyridoxamine phosphate (PMP) as observed in SAV3 structure is represented by yellow sticks. aminoacrylate-PLP as observed in alliinase structure is represented by orange sticks. Trp-PLP from an in silico docking experiment is represented as magenta sticks.