Interplay between the NADP-linked thioredoxin and glutathione systems in Arabidopsis auxin signaling

Abstract

Intracellular redox status is a critical parameter determining plant development in response to biotic and abiotic stress. Thioredoxin (TRX) and glutathione are key regulators of redox homeostasis, and the TRX and glutathione pathways are essential for postembryonic meristematic activities. Here, we show by associating TRX reductases (ntra ntrb) and glutathione biosynthesis (cad2) mutations that these two thiol reduction pathways interfere with developmental processes through modulation of auxin signaling. The triple ntra ntrb cad2 mutant develops normally at the rosette stage, undergoes the floral transition, but produces almost naked stems, reminiscent of the phenotype of several mutants affected in auxin transport or biosynthesis. In addition, the ntra ntrb cad2 mutant shows a loss of apical dominance, vasculature defects, and reduced secondary root production, several phenotypes tightly regulated by auxin. We further show that auxin transport capacities and auxin levels are perturbed in the mutant, suggesting that the NTR-glutathione pathways alter both auxin transport and metabolism. Analysis of ntr and glutathione biosynthesis mutants suggests that glutathione homeostasis plays a major role in auxin transport as both NTR and glutathione pathways are involved in auxin homeostasis.

Figure 1.

Phenotype of the ntra ntrb cad2 Mutant at Different Developmental Stages. (A) Fifteen-day-old Col-0 (left) and ntra ntrb cad2 (right) grown in vitro. (B) Three-week-old Col-0 (left) and ntra ntrb cad2 (right) plants grown on soil. (C) and (D) Five-week-old Col-0 (C) and ntra ntrb cad2 (D) plants. Arrows indicate emergence of secondary rosette stems in ntra ntrb cad2, which were not observed in the wild type at this stage. (E) and (F) Seven-week-old Col-0 (left) and ntra ntrb cad2 (right) plants (E) and 10-week-old ntra ntrb cad2 plant (F). All plants were grown under a long-day regime (16 h light/8 h dark). At all developmental stages, cad2 and ntra ntrb plants (not represented) have a similar phenotype as Col-0 plants. (G) and (H) Five-week-old Col-0 (G) and ntra ntrb cad2 (H) rosette leaves. Collected leaves were cleared in Hoyer's solution and observed under a binocular microscope. Note the abnormal leaf shape and the less developed and irregular vasculature of ntra ntrb cad2 leaf. Bar = 1 mm.

Figure 2.

Structure of the Inflorescence Meristem of the ntra ntrb cad2 Mutant. (A) and (B) Stem apex of 5-week-old Col-0 (A) and ntra ntrb cad2 (B) plants. Bars = 1 mm. (C) and (D) Histological structure of the inflorescence meristem stained by toluidine blue. Col-0 inflorescence (C) and ntra ntrb cad2 meristem (D). Bars = 100 μ m. (E) and (F) Higher magnification of the inflorescence meristem of Col-0 (E) and ntra ntrb cad2 (F). Bars = 20 μ m.

Figure 3.

Root Growth and Emergence of Secondary Roots in the cad2, ntra ntrb, and ntra ntrb cad2 Mutants. (A) Daily root growth was measured on Col-0, cad2, ntra ntrb, and ntra ntrb cad2 plants grown on MS/2 vertical agar plates at 22°C, under a 16-h-light/8-h-dark regime. Primary root growth rate was calculated as the mean of daily root growth between the 8th and the 11th day after seeds were put under growth conditions. Data are means ± se; n ≥ 35. (B) The number of secondary root initials was measured after 10 d after germination. Col-0, cad2, ntra ntrb, and ntra ntrb cad2 plants were grown in the same conditions as in (A). Data are means ± se; n ≥ 35. (C) Phenotype of Col-0, cad2, ntra ntrb, and ntra ntrb cad2 plants cultivated as in (A) and observed 12 d after germination. Bars = 5 mm. (D) Phenotype of the primary root meristem of Col-0, cad2, ntra ntrb, and ntra ntrb cad2 plants cultivated as in (A) and observed 10 d after germination. The arrows show the approximate extent of the meristem based on elongation of the pericycle cell files. Bars = 40 μ m. [See online article for color version of this figure.]

Figure 4.

Auxin Transport Analysis in Mutants. Basipetal auxin transport was evaluated by measuring the radioactivity of stem segments of the plants after feeding of ³H-IAA. Each value represents the average of eight plants (± se, n = 8) of wild-type Col-0, homozygote mutants cad2, pad2, ntra ntrb, ntra ntrb cad2, pin1/pin1, and heterozygote PIN1/pin1. As a negative control, acropetal auxin transport was evaluated in stem segments of Col-0 plants.

Figure 5.

PIN Expression Is Influenced by Glutathione and NTR. (A) Five-day-old plantlets were transferred to a medium containing 2.5 mM BSO. Steady state levels of PIN1 (light green), PIN2 (dark green), PIN3 (light blue), PIN4 (dark blue), AUX1 (red), IAA1 (orange), and IBC7 (pink) mRNAs were analyzed by quantitative RT-PCR at different time points after treatment (0, 12, and 24 h). For each gene, the level of mRNA was normalized to that of EF1 α, which is constitutive under these conditions. Data are means of two technical replicates. The level of total glutathione was measured at the same time points and is reported in each panel (black lines). Data are means of six biological repetitions ± se, n = 6. (B) Five-day-old plantlets expressing PIN1-GFP, PIN2-GFP, and PIN3-GFP proteins under the control of their natural promoters were subjected to the same treatment as in (A). Plants either treated (BSO) or untreated (C) with 2.5 mM BSO for 24 h were analyzed by confocal microscopy. Compared with untreated plants, the abundance of PIN1-GFP, PIN2-GFP, or PIN3-GFP was reduced in plants treated by BSO. Higher magnification of the PIN1-GFP and PIN2-GFP signals (inset) suggests that the overall cellular localization of the fusion protein is not perturbed by the BSO treatment. A line expressing the plasma membrane–localized LTI6b (low temperature induced) protein fused to GFP (GFP-LTI6b) under the 35S promoter was used as a control (Cutler et al., 2000). The abundance of the GFP-LT16b protein was not affected by BSO treatment. Bar = 50 μ m. (C) and (D) PIN1 immunolocalization in Col-0 and ntra ntrb cad2. Abundance of PIN1 protein was analyzed in 5-d-old plantlets by immunolocalization and subsequently by confocal microscopy. Col-0 (C) and ntra ntrb cad2 (D). Arrowheads indicate some stellar cells in which a very low membrane signal persisted. Bar = 50 μ m.

Figure 6.

Auxin Levels Are Altered in the ntra ntrb cad2 Mutant. Localization of the Pro_DR5:GUS activity in wild-type Col-0 ([B] and [C]) and in ntra ntrb cad2 ([D] and [E]) shoots ([B] and [D]) and roots ([C] and [E]). (A) IAA levels were determined in wild-type Col-0 and homozygous ntra ntrb cad2 mutant seedlings 15 d after germination (left panel) grown as described in Methods and Figure 1A. Data are means of three biological repetitions ± se, n = 3. The IAA level is significantly lower in the ntra ntrb cad2 homozygotes versus wild-type seedlings. IAA levels were also measured in isolated wild-type Col-0 flower buds and in stem apex of adult ntra ntrb cad2 and pin1 homozygote plants grown in soil during 5 weeks as described in Methods and Figures 1C and 1D. Auxin content was also much lower in the ntra ntrb cad2 mutant but not in the pin1 apex. (B) to (E) Auxin reporter Pro_DR5:GUS gene expression in the ntra ntrb cad2 mutant.

Figure 7.

Rescue of ntra ntrb cad2 Floral Phenotype by Exogenous Glutathione. Induction of flower development in ntra ntrb cad2 by supplementation with exogenous glutathione. (A) After bolting, flowerless plants were regularly irrigated with water alone (−GSH) or with exogenous glutathione 0.4 mM (+GSH). (B) Higher magnification of ntra ntrb cad2 flowers. [See online article for color version of this figure.]

Figure 8.

The ntra ntrb cad2 Mutant Is Less Susceptible to the de Novo Shoot Induction System. (A) to (H) Shoot apical meristem ([A] and [E]), stem ([B] and [F]), leaf ([C] and [G]), and root explants ([D] and [H]) were harvested from 7-week-old wild-type Col-0 ([A] to [D]) or ntra ntrb cad2 ([E] to [H]) mutant plants and transferred to an auxin-rich callus-inducing medium, which induces cell proliferation and callus formation after 3 weeks. (I) to (K) In order to regenerate shoots, the calli induced from leaves for 3 weeks were transferred to a cytokinin-rich (10 μ M BAP) shoot-inducing medium containing increasing concentrations of auxin: 0 μ M IAA (I), 0.1 μ M IAA (J), and 1 μ M IAA (K). After 2 additional weeks, regenerated shoot meristems were observed. Note that shoot regeneration occurred in all media in Col-0 calli but only in media containing high concentrations of IAA in ntra ntrb cad2 calli. The black arrow indicated the regenerated shoots. (L) Counting of the number of regenerated shoot per callus in Col-0 and ntra ntrb cad2 (±se, n = 30). No IAA (white bars), 0.1 μ M IAA (yellow), 1 μ M IAA (orange), and 3 μ M IAA (red).

Figure 9.

ntra ntrb cad2 Root Development Phenotype Is Rescued by Exogenous Auxin. Col-0, cad2, pad2, ntra ntrb, and ntra ntrb cad2 plants grown on MS/2 vertical agar plates at 22°C, under a 16-h-light/8-h-dark regime. Five days after seeds were put under growth culture, plantlets were transferred on plates containing different concentrations of IAA. (A) Primary root growth rate was calculated as the mean of daily root growth measured between the 2nd and the 5th day after the transfer. Data are means ± se; n ≥ 30. (B) and (C) Number of secondary roots initials was measured 2 d (B) and 6 d (C) after the transfer. Data are means ± se; n ≥ 30.