Mutation of Arabidopsis Copper-Containing Amine Oxidase Gene AtCuAOδ Alters Polyamines, Reduces Gibberellin Content and Affects Development

Abstract

Polyamines (PAs) are essential metabolites in plants performing multiple functions during growth and development. Copper-containing amine oxidases (CuAOs) catalyse the catabolism of PAs and in Arabidopsis thaliana are encoded by a gene family. Two mutants of one gene family member, AtCuAOδ, showed delayed seed germination, leaf emergence, and flowering time. The height of the primary inflorescence shoot was reduced, and developmental leaf senescence was delayed. Siliques were significantly longer in mutant lines and contained more seeds. The phenotype of AtCuAOδ over-expressors was less affected. Before flowering, there was a significant increase in putrescine in AtCuAOδ mutant leaves compared to wild type (WT), while after flowering both spermidine and spermine concentrations were significantly higher than in WT leaves. The expression of GA (gibberellic acid) biosynthetic genes was repressed and the content of GA₁, GA₇, GA₈, GA₉, and GA₂₀ was reduced in the mutants. The inhibitor of copper-containing amine oxidases, aminoguanidine hydrochloride, mimicked the effect of AtCuAOδ mutation on WT seed germination. Delayed germination, reduced shoot height, and delayed flowering in the mutants were rescued by GA₃ treatment. These data strongly suggest AtCuAOδ is an important gene regulating PA homeostasis, and that a perturbation of PAs affects plant development through a reduction in GA biosynthesis.

Keywords: copper-containing amine oxidases; flowering; gibberellic acid; polyamines; putrescine; senescence.

Figure 1

The expression of AtCuAOδ and the effects of its perturbation on senescence: (a) gene expression in leaf 5 and 6 of rosettes during leaf development and senescence (n = 3, ± SE); (b) total number of leaves, number of yellow leaves, % of yellow leaves, and rosette fresh weight after 8 weeks growth of the two mutants C#4 and BIS#4 (n = 8, ± SE); (c) chlorophyll levels in leaves no. 5 and 6 of mutants C#4 and Bis#4 at three different stages (± SE, n = 6); (d) leaf yellowing of the nine oldest leaves (oldest to youngest, left to right) from 8-week-old rosettes (scale bar = 1 cm) of the two mutants C#4 and BIS#4; (e) over-expressor lines P9, P17, and P27; (f) yellowing as measured by changes in RGB (increased yellowing) during dark-induced senescence in C#4 and BIS#4; (g) over-expressor lines P17 and P27 (n = 9, ±SE). Significant differences in means are indicated by * at p ≤ 0.05 based on a T-test; different lettering is based on a one-way ANOVA followed by a Tukey’s test; DAS is days after sowing.

Figure 2

Perturbation of AtCuAOδ expression affects flowering time, stalk height, silique number, and their length: day of bolting and flowering and number of leaves at bolting in (a) mutants C#4 and BIS#4 (n = 8); (b) over-expressor lines P17 and P27; (c) line P9 (n = 8). Also shown are the primary stalk length, number of siliques, and rosette diameter after 8 weeks growth of (d) two mutants C#4 and BIS#4 (n = 8); (e) over-expressor lines P17 and P27; (f) line P9 (n = 8); (g) mean mature silique length (n = 100); (h) seed number per silique (n = 100 siliques); and (i) silique images (scale bar = 1cm) of the two mutants C#4 and BIS#4 siliques; means ± SE, significant differences are indicated by * at p ≤ 0.05 based on a Student t-test where data were normally distributed or Mann-Whitney test where data were not.

Figure 3

The effect of AtCuAOδ mutation on germination and leaf emergence mutant lines (C#4 and BIS#4) and over-expressor lines P17 and P27 compared to WT: (a) the germination rate of mutants C#4 and BIS#4 (n = 5; 30 seeds/replicate); (b) leaf emergence in mutant lines C#4 and BIS#4 (n = 8); (c) leaf emergence in over-expressor lines P17 and P27 (n = 8–16). Significant differences in means are indicated by * at p ≤ 0.05 based on a Student t-test. DAS is days after sowing, mean ± SE.

Figure 4

Free polyamine contents in leaves 5 and 6 of the two mutant lines (C#4 and BIS#4) and WT plants pre- and post-bolting and the effect of aminoguanidine hydrochloride on germination of WT. (a) The content of the di-amine Put and the tetra-amine Spm, (b) the content of the tri-amine Spd, (n = 6); (c) seed germination of WT on water agar medium containing 1 mM aminoguanidine hydrochloride, data from three biological replicates (~60 seeds/replicate); Asterisks indicate values significantly different from wild type (WT) or control at p ≤ 0.05 based on a Student t-test where data were normally distributed or Mann-Whitney test where data were not. Mean values ± SE.

Figure 5

The effect of exogenous application of GA₃ on AtCuAOδ mutant lines (C#4 and BIS#4) and wild type plants: (a) % germination on water agar medium containing 10μM GA₃; (b) day of bolting, number of leaves at bolting, and day of 1st flower opening with and without application of 50 μM GA3 (n = 12); (c,d) stalk length with and without application of 50 μM GA₃; scale bars = 5 cm. Error bars represent the standard error of the mean. Letters indicate a statistically significant difference between treatments within each character measured using one-way ANOVA (p ≤ 0.05). Asterisks indicate a significant difference from WT at p ≤ 0.05 based on a Student t-test. The experiment was repeated twice with similar results.

Figure 6

GA content and expression of GA biosynthetic genes in AtCuAOδ mutants compared to WT. (a) Quantitative analysis of endogenous gibberellins by GC-MS/MS extracted from 2-week-old seedlings. (b) Real-time PCR of GA biosynthetic genes, KS1, GA20ox-1, GA3ox-1, and GA2-ox-1. n = 3; means ± SE; asterisks indicate significant differences to WT (p ≤ 0.05), based on a Student t-test.

Figure 7

Diagram showing the effects of AtCuAOδ mutation on plant growth regulator balance and changes in development. The loss of AtCuAOδ function results in a premature increase in Put. This in turn down-regulates the expression of GA biosynthetic genes (specifically KS, GA20ox (less so), GA2ox and GA3ox) resulting in a reduction of the flow through the gibberellin pathway. This results in a delay in germination and bolting (black arrows). Delayed seed production results in delayed rosette senescence. Effects on silique length and number of seeds per silique may be directly mediated by the reduction in GAs or may occur via a different mechanism.