An Oxalyl-CoA Synthetase Is Involved in Oxalate Degradation and Aluminum Tolerance

Abstract

Acyl Activating Enzyme3 (AAE3) was identified to be involved in the catabolism of oxalate, which is critical for seed development and defense against fungal pathogens. However, the role of AAE3 protein in abiotic stress responses is unknown. Here, we investigated the role of rice bean (Vigna umbellata) VuAAE3 in Al tolerance. Recombinant VuAAE3 protein has specific activity against oxalate, with K_m = 121 ± 8.2 µm and V_max of 7.7 ± 0.88 µmol min^-1 mg^-1 protein, indicating it functions as an oxalyl-CoA synthetase. VuAAE3-GFP localization suggested that this enzyme is a soluble protein with no specific subcellular localization. Quantitative reverse transcription-PCR and VuAAE3 promoter-GUS reporter analysis showed that the expression induction of VuAAE3 is mainly confined to rice bean root tips. Accumulation of oxalate was induced rapidly by Al stress in rice bean root tips, and exogenous application of oxalate resulted in the inhibition of root elongation and VuAAE3 expression induction, suggesting that oxalate accumulation is involved in Al-induced root growth inhibition. Furthermore, overexpression of VuAAE3 in tobacco (Nicotiana tabacum) resulted in the increase of Al tolerance, which was associated with the decrease of oxalate accumulation. In addition, NtMATE and NtALS3 expression showed no difference between transgenic lines and wild-type plants. Taken together, our results suggest that VuAAE3-dependent turnover of oxalate plays a critical role in Al tolerance mechanisms.

Figure 1.

Amino acid sequence alignment of AAE3 proteins from rice bean (VuAAE3; KX354978), Arabidopsis (AtAAE3; At3g48990), and M. truncatula (MtAAE3; XP_003599555.1). The conserved AMP binding domain and acetyl-CoA synthetase domain are indicated.

Figure 2.

Biochemical analysis of VuAAE3. A, SDS-PAGE gel of purified His-VuAAE3 protein (right) and molecular weight markers (left). B, Reaction rate against different organic anions. The reaction mixture contains 0.5μg/mL recombinant protein and 375 μm each of the tested substrates; the mixture was incubated for 1 h at room temperature. bars indicate ± se (n = 3). C, Visual inspection of NADH residue level indicated by nitroblue tetrazolium and l-Methoxy-5-methylphenazinium methosulfate. D, Kinetic analysis of VuAAE3 using a range of oxalate concentrations. K_m and V_max were determined from nonlinear regression to the Michaelis-Menten equation for concentrations up to 1500 µm oxalate.

Figure 3.

Subcelluar localization of VuAAE3 in transgenic plants. Both 35S::GFP and 35S::VuAAE3-GFP constructs were introduced individually into Arabidopsis. The homozygous seedlings of T4 generation were used to observe GFP fluorescence. Bar = 50 μm.

Figure 4.

Rice bean VuAAE3 expression analysis. A, Dose-dependent VuAAE3 expression in rice bean root tips (0–1 cm). The roots were exposed to various concentrations of Al for 8 h. B, Time-dependent VuAAE3 expression in rice bean root tips (0–1 cm). The roots were exposed to 25 µm Al for various times. C, Metal-dependent VuAAE3 expression in rice bean root tips (0–1 cm). The seedlings were subjected to nutrient solution as control (Ct.) or the same nutrient solution containing Al (25 µm), CdCl₂ (20 µm), LaCl₃ (10 µm), and CuCl₂ (0.5 µm) for 8 h. D, pH-dependent VuAAE3 expression in rice bean root tips (0–1 cm). Seedlings were grown in nutrient solution with different pH values for 8 h. All data were normalized relative to VuAAE3 expression in the absence of Al at pH 4.5. The expression was determined by real-time RT-PCR and 18S rRNA was used as an internal control. Values are expressed as means ± sd (n = 3). The asterisk indicates significant differences between treatment and control (pH 4.5 without Al stress).

Figure 5.

Tissue-specific expression of VuAAE3 in response to Al stress. A, Seedlings of rice bean (3 d old) were exposed to 0 or 25 µm Al for 8 h. Root tip (0–1 cm), basal root (1–2 cm), and leaves were sampled for RNA extraction. The expression was determined by real-time RT-PCR and 18S rRNA was used as an internal control. Values are expressed as means ± sd (n = 3). The asterisk indicates significant differences between treatments. B, Seedlings (7 d old) of transgenic Arabidopsis lines carrying P_VuAAE3::GUS construct were subject to 1:30 strength Hoagland nutrient solution at pH 5.0 with or without 4 µm Al for 6 h. Bar = 100 µm.

Figure 6.

The effect of Al stress on rice bean root tip oxalate content. Seedlings were exposed to nutrient solution containing 0 or 25 µm Al for different times. After treatment, the root tips were homogenized thoroughly in deionized water for oxalate analysis. Data are means ± sd (n = 3). Asterisk indicate significant difference between wild-type and transgenic lines within treatment at P < 0.05.

Figure 7.

The effect of exogenous oxalate on rice bean root growth and VuAAE3 expression. A, The effect of oxalate on rice bean root elongation. Seedlings were exposed to nutrient solution (pH 4.5) containing different concentrations of exogenous oxalate for 24 h. Root elongation was measured with a ruler before and after treatment (n = 12). Different letters indicate significant differences between treatments at P < 0.05. B, VuAAE3 expression in response to exogenous oxalate. After treatment, root tips (0–1 cm) were excised for RNA extraction and qRT-PCR analysis of VuAAE3 (n = 3). Different letters indicate significant differences between treatments at P < 0.05. C, VuAAE3 promoter activity assay of transgenic Arabidopsis lines carrying GUS reporter gene under the control of VuAAE3 promoter. Roots and leaves were excised from 3-week-old transgenic seedlings and subject to water or oxalate (1 mm) for 2 h. Bars represent 100 µm in roots and 1 mm in leaves.

Figure 8.

Overexpression of VuAAE3 enhances Al tolerance in tobacco. A, Detection of expression of VuAAE3 in the wild-type and VuAAE3 overexpression lines. RT-PCR analysis was performed to detect the mRNA expression of VuAAE3 (32 cycles) and the internal control NtACTIN (29 cycles). B, Root elongation of wild-type (WT) and the transgenic lines overexpressing VuAAE3 (OX-1 and OX-4). Data are expressed as means ± sd (n = 15). Asterisk indicates significant difference between wild-type and transgenic lines within treatment at P < 0.05. C, Representative seedlings of wild-type and overexpression lines grown in the 1:30 strength Hoagland nutrient solution at pH 5.0 for 6 d. D, Representative seedlings showing difference in Al sensitivity between wild-type and overexpression lines. Seedlings were grown in the 1:30 Hoagland nutrient solution containing 4 µm Al for 6 d.

Figure 9.

The effect of Al stress on oxalate content in wild-type and overexpression tobacco lines. The plants of wild-type and two independent transgenic lines were exposed to 1:30 strength Hoagland nutrient solution with 0 (−Al) or 4 µm Al (+Al) for 24 h. After treatment, root tips were homogenized thoroughly in deionized water for oxalate content analysis. Data are means ± sd (n = 3). Asterisk indicates significant differences between treatments at P < 0.05.