Expression of gibberellin 20-oxidase1 (AtGA20ox1) in Arabidopsis seedlings with altered auxin status is regulated at multiple levels

Abstract

Bioactive gibberellins (GAs) affect many biological processes including germination, stem growth, transition to flowering, and fruit development. The location, timing, and level of bioactive GA are finely tuned to ensure that optimal growth and development occur. The balance between GA biosynthesis and deactivation is controlled by external factors such as light and by internal factors that include auxin. The role of auxin transport inhibitors (ATIs) and auxins on GA homeostasis in intact light-grown Arabidopsis thaliana (L.) Heynh. seedlings was investigated. Two ATIs, 1-N-naphthylthalamic acid (NPA) and 1-naphthoxyacetic acid (NOA) caused elevated expression of the GA biosynthetic enzyme AtGA20-oxidase1 (AtGA20ox1) in shoot but not in root tissues, and only at certain developmental stages. It was investigated whether enhanced AtGA20ox1 gene expression was a consequence of altered flow through the GA biosynthetic pathway, or was due to impaired GA signalling that can lead to enhanced AtGA20ox1 expression and accumulation of a DELLA protein, Repressor of ga1-3 (RGA). Both ATIs promoted accumulation of GFP-fused RGA in shoots and roots, and this increase was counteracted by the application of GA(4). These results suggest that in ATI-treated seedlings the impediment to DELLA protein degradation may be a deficiency of bioactive GA at sites of GA response. It is proposed that the four different levels of AtGA20ox1 regulation observed here are imposed in a strict hierarchy: spatial (organ-, tissue-, cell-specific) > developmental > metabolic > auxin regulation. Thus results show that, in intact auxin- and auxin transport inhibitor-treated light-grown Arabidopsis seedlings, three other levels of regulation supersede the effects of auxin on AtGA20ox1.

Fig. 1.

Molecular analysis of the AtGA20ox1 mRNA expression by RT-PCR analysis (A) and AtGA20ox1::GUS reporter by fluorimetric GUS assay (B) in response to GA₄, PAC, ATIs or auxins treatment. Two-day-old Arabidopsis seedlings were transferred in liquid nutrient solution with or without GA₄, PAC, ATIs or auxins for 8 d. (C) Time-course of AtGA20ox1::GUS activity in 10-d-old Arabidopsis seedlings in response to treatment with PAC, NOA or NPA for 0.5 d (white bars), 1 d (grey bars), 2 d (dark grey bars), 4 d (light grey bars), or 6 d (black bars). Values shown are means ±SD (n=3 different experiments) compared to respective control normalized to 100%. Different letters on the bars represent means that are statistically different relative to control using the Student t test where (a) P ≤0.05 and (b) P ≤0.01.

Fig. 2.

Measurements of hypocotyl (A) and root (B) lengths of 10-d-old Arabidopsis seedlings grown on serial dilutions of nutrient as specified in the Materials and methods section. (C) Fluorimetric GUS assay of GA20ox1::GUS activity of 10-d-old Arabidopsis seedlings in reduced growth conditions and treated for 8 d with 0.1% ethanol (black bars), 5 μM PAC (grey bars) or 12.5 μM NPA (white bars). Values shown are means ±SD (n=3). Letters on the graph represent means that are statistically different relative to control using the Student t test where (a) P ≤0.05 and (b) P ≤0.01.

Fig. 3.

Fluorimetric GUS assay of DR5::GUS reporter construct of 10-d-old shoots (black bars) and roots (white bars) of Arabidopsis seedlings following 8 d treatment with GA₄, PAC, ATIs or auxins to whole seedlings. Values shown are means ±SD (n=3 different experiments) compared to their respective control (shoot or root) normalized to 100%. Different letters on the bars represent means that are statistically different relative to control using the Student t test where (a) P ≤0.05 and (b) P ≤0.01.

Fig. 4.

Light microscopy pictures of histochemical staining of the (A) DR5::GUS or (B) AtGA20ox1::GUS activity in 10-d-old Arabidopsis seedlings following treatment with ATIs or auxin.

Fig. 5.

Fluorimetric GUS assay of AtGA20ox1::GUS reporter construct following treatment with GA₄, PAC, ATIs or auxins in shoots (black bars) and root (white bars) of 10-d-old Arabidopsis seedlings as described in Fig. 3. Values shown are means ±SD (n=3 different experiments) compared to their respective control (shoot or root) normalized to 100%. Different letters on the bars represent means that are statistically different relative to control using the Student t test where (a) P ≤0.05 and (b) P ≤0.01.

Fig. 6.

(A) Confocal microscopy images of GFP-RGA reporter accumulation in response to treatment with or without PAC or ATIs in 5-d-old pRGA::GFP::RGA Arabidopsis seedlings. Three-day-old Arabidopsis seedlings were treated for 48 h with or without PAC or ATIs. GFP-RGA fluorescence was monitored in shoot tips, hypocotyls, primary roots, and primary root tips. For combination treatment, GA₄ was applied 4 h prior to acquiring the images. (B) Fluorimetric GFP assay of GFP-RGA reporter accumulation in 10-d-old pRGA::GFP::RGA Arabidopsis seedlings in response to 8 d treatment with PAC, ATIs or auxins alone (black bars) or with GA₄ (white bars). Values shown are means ±SD (n=3 different experiments) compared to control normalized to 100%. Letters on the bars represent means that are statistically different relative to control using the Student t test where (a) P ≤0.01.

Fig. 7.

Fluorimetric GUS assay of DR5::GUS (A) and AtGA20ox1::GUS (B) reporter activity in response to various treatments with ATIs or auxins alone (black bars) or in combination with GA₄ (grey bars) or PAC (white bars) in 10-d-old Arabidopsis seedlings. Values shown are means ±SD (n = at least 3 different experiments) compared to the 0.1% ethanol control normalized to 100%. Different letters on the bars represent means that are statistically different relative to control using the Student t test where (a) P ≤0.05 and (b) P ≤0.01.

Fig. 8.

Overview of AtGA20ox1 regulation in Arabidopsis seedlings. Organ-, tissue-, and cell-specific regulation supersedes developmental regulation of AtGA20ox, which encodes a multifunctional dioxygenase catalysing a rate-limiting step in the synthesis of bioactive GA₄. GA₄ can be deactivated by AtGA2ox or can bind to its receptor GID1a, b, c (low affinity) which triggers a conformational change to allow binding of DELLA proteins (RGA, GAI, RGLs). DELLA degradation via SCF^SLY1/GAR2, through the 26S proteosome, allows GA signalling and response, including metabolic regulation to down-regulate AtGA20ox expression. Lastly, auxin regulates GA pathways by affecting DELLA stability in roots and promoting GA 20-oxidation in shoots. Numbers in parentheses indicate the following references: (1) Phillips et al., 1995; (2) Xu et al., 1995; (3) Thomas et al., 1999; (4) Desgagné-Penix et al., 2005; (5) Griffiths et al., 2006; (6) Nakajima et al., 2006; (7) Willige et al., 2007; (8) Iuchi et al., 2007; (9) Fu and Harberd, 2003; (10) Frigerio et al., 2006; (11) Pufky et al., 2003; (12) Goda et al., 2004; (13) Rieu et al., 2008.