YUCCA auxin biosynthetic genes are required for Arabidopsis shade avoidance

Abstract

Plants respond to neighbor shade by increasing stem and petiole elongation. Shade, sensed by phytochrome photoreceptors, causes stabilization of PHYTOCHROME INTERACTING FACTOR proteins and subsequent induction of YUCCA auxin biosynthetic genes. To investigate the role of YUCCA genes in phytochrome-mediated elongation, we examined auxin signaling kinetics after an end-of-day far-red (EOD-FR) light treatment, and found that an auxin responsive reporter is rapidly induced within 2 hours of far-red exposure. YUCCA2, 5, 8, and 9 are all induced with similar kinetics suggesting that they could act redundantly to control shade-mediated elongation. To test this hypothesis we constructed a yucca2, 5, 8, 9 quadruple mutant and found that the hypocotyl and petiole EOD-FR and shade avoidance responses are completely disrupted. This work shows that YUCCA auxin biosynthetic genes are essential for detectable shade avoidance and that YUCCA genes are important for petiole shade avoidance.

Keywords: Auxin; Photomorphogenesis; Phytochrome; Shade avoidance.

Figure 1. EOD-FR induction of eDR5::LUC luminescence.

(A–C) Mean luminescence of 5-day-old seedlings (A), 3-week-old juveniles (B), or 3-week-old juveniles in the presence of NPA (C) moved to darkness (solid black line) or treated with a 30 minute EOD-FR pulse prior to transfer to darkness (dashed red line). Dotted lines indicate SEM. Time 0 indicates the beginning of the EOD-FR treatment. n = 4 − 11 plants for each treatment. Representative plots for one of three independent experiments are shown. (D–F) False-color images of eDR5::LUC plants. Representative DMSO treated plant 40 (D) or 240 (E) minutes after EOD-FR pulse showing increase in petiole luminescence after treatment. (F) NPA treated plants 240 minutes after EOD-FR do not have observable petiole luminescence but show increased luminescence in the leaves and apices. (G) Mean luminescence of 3-week-old juveniles treated with DMSO (compare with (C)). For growth conditions see “seedling stage EOD-FR analysis” (A) and “analysis of juvenile plants under EOD-FR” (B-G) in Materials and Methods.

Figure 2. Shade and EOD-FR induction of YUCCA genes.

(A) Expression levels of YUCCA genes from a published shade-induction microarray experiment (Sessa et al., 2005). (B) mRNA levels in EOD-FR treated wild-type plants. (C) mRNA levels in EOD-FR treated athb-2 mutant plants. For (B and C) plants were treated for five days with EOD-FR, and samples were taken 1 hour after the last EOD-FR treatment. mRNA levels shown are normalized to untreated plants. Results shown are averages of n = 5 − 6 ± SEM. Asterisks mark statistical significance of induction (* p-value ≤ 0.05, ** p-value ≤ 0.005) calculated by the REST-program (Pfaffl, Horgan & Dempfle, 2002).

Figure 3. Histochemical localization of GUS in transgenic Arabidopsis thaliana plants containing the YUCCA2::GUS,YUCCA5::GUS, YUCCA8::GUS or YUCCA9::GUS constructs.

(A–D) Whole plants. (E–H) Hypocotyls and shoot-apical meristems. (I–L) Leaves. (M–P) Roots.

Figure 4. YUCCA genes are required for shade avoidance.

(A–C) Hypocotyl (A, C) or petiole (B) measurements of short day grown plants with (dark red) or without (blue) EOD-FR pulses. Means of n = 17 − 137 plants ± SEM are shown. Representative data from one of three experiments is shown. (D) Petiole lengths of plants grown in long day high (red, simulated sun) or low (dark red, simulated shade) R:FR conditions. Means of n = 48 − 116 petioles ± SEM are shown. (E) Induction of eDR5::LUC2 expression in 15 day-old wild type and yucca2, 5, 8, 9 mutants moved from short day (8L:16D) conditions to darkness (blue line) or treated with a 30 minute EOD-FR pulse (dark red line). Shading indicates 95% confidence interval. Time 0 indicates the beginning of the EOD-FR treatment. Fourteen Col and 10 yucca2589 plants were measured. For growth conditions see “seedling stage WID-FR analysis” (A, C) and “analysis of juvenile plants under EOD-FR” (B, E), and “analysis of juvenile plants under high and low R:FR” (D) in Materials and Methods.

Figure 5. Hypocotyl length of additional lines in simulated sun and shade.

Four independent experiments were performed with a total of 35–150 plants per treatment/genotype combination. For growth conditions see “seedling stage high and low R:FR analysis” in Materials and Methods.

Figure 6. Adult wild-type and yucca mutant lines.

The mutant lines did not show severe morphological defects, although some showed reduced fertility. For growth conditions see “analysis of juvenile plants under high and low R:FR” in Materials and Methods. These plants were only grown in high R:FR.