An Oxalyl-CoA Dependent Pathway of Oxalate Catabolism Plays a Role in Regulating Calcium Oxalate Crystal Accumulation and Defending against Oxalate-Secreting Phytopathogens in Medicago truncatula

Abstract

Considering the widespread occurrence of oxalate in nature and its broad impact on a host of organisms, it is surprising that so little is known about the turnover of this important acid. In plants, oxalate oxidase is the most well studied enzyme capable of degrading oxalate, but not all plants possess this activity. Recently, an Acyl Activating Enzyme 3 (AAE3), encoding an oxalyl-CoA synthetase, was identified in Arabidopsis. AAE3 has been proposed to catalyze the first step in an alternative pathway of oxalate degradation. Whether this enzyme and proposed pathway is important to other plants is unknown. Here, we identify the Medicago truncatula AAE3 (MtAAE3) and show that it encodes an oxalyl-CoA synthetase activity exhibiting high activity against oxalate with a Km = 81 ± 9 μM and Vmax = 19 ± 0.9 μmoles min-1mg protein-1. GFP-MtAAE3 localization suggested that this enzyme functions within the cytosol of the cell. Mtaae3 knock-down line showed a reduction in its ability to degrade oxalate into CO2. This reduction in the capacity to degrade oxalate resulted in the accumulation of druse crystals of calcium oxalate in the Mtaae3 knock-down line and an increased susceptibility to oxalate-secreting phytopathogens such as Sclerotinia sclerotiorum. Taken together, these results suggest that AAE3 dependent turnover of oxalate is important to different plants and functions in the regulation of tissue calcium oxalate crystal accumulation and in defense against oxalate-secreting phytopathogens.

Fig 1. Biochemical Analysis of MtAAE3.

(A) SDS-PAGE gel of nickel-affinity purified His-MtAAE3 protein (right) and molecular weight markers (left). (B) Optimum pH for MtAAE3 was performed with 300 μM oxalate using two buffer systems; potassium phosphate pH 6–8 and Tris-HCl pH 7.5–10. (C) Kinetic analysis of MtAAE3 using a range of oxalate concentrations. K_m and V_max were determined from non-linear regression to the Michaelis-Menten equation for concentrations up to 400 μM oxalate.

Fig 2. Measurement of oxalate degradation to CO₂.

(A) Schematic of the proposed pathway of oxalate catabolism in M. truncatula. Asterisks denote enzymatic steps remaining to be validated. (B) Radiolabeled CO₂ measurements. WT and Mtaae3 knock-down leaf pieces were fed with 2.5 μCi of [¹⁴C]-oxalate along with 300 μM non-labeled oxalate. The ¹⁴CO₂ evolved was captured using 1M KOH and the relative radioactivity was measured. (C) Relative MtAAE3 transcript levels in leaves of Mtaae3 knock-down lines compared to WT as measured by qRT-PCR. Ubiquitin was used as the reference gene.

Fig 3. Subcellular localization of MtAAE3.

(A) Transient expression of free GFP (top row) and GFP-MtAAE3 (bottom row) in mesophyll leaf cells of N. benthamiana. GFP fluorescence images (left), chloroplast autofluorescence (middle), and merge of GFP and autofluorescence (right). (B) Transient expression of free GFP (top) and GFP-MtAAE3 (bottom) in N. benthamiana epidermal leaf cells. Bar = 20 μm

Fig 4. Complementation of Ataae3 with MtAAE3 under the control of the AtAAE3 promoter.

(A) Calcium oxalate crystal leaf phenotype of WT, Ataae3, and Ataae3/pAtAAE3::GFP-MtAAE3 Arabidopsis. Crystals are bright spots designated by arrows. (B) Oxalate concentrations in leaves from WT, Ataae3, and Ataae3/pAtAAE3::GFP-MtAAE3 Arabidopsis. (C) Complementation of Ataae3 calcium oxalate crystal seed phenotype through expression of MtAAE3. Crystals are bright spots designated by arrows. (D) Germination of WT, Ataae3, and Ataae3/pAtAAE3::GFP-MtAAE3 seeds. Bar = 50 μm.

Fig 5. Assessment of MtAAE3 in protection against oxalate-secreting phytopathogens.

(A) Measurements of fungal spread in leaves of WT and Mtaae3-1 knock-down line inoculated with S. sclerotiorum. Lesion areas were measured 48 h after inoculation. Data are mean ± SE. (B) Quantitative RT-PCR of MtAAE3 expression in roots from control and oxalate-treated plants over time. (C) Quantitative RT-PCR of MtAAE3 expression in shoots from control and oxalate-treated plants over time. The cycle threshold values were normalized using the ubiquitin as a reference gene.

Fig 6. Assessment of MtAAE3 in the regulation of calcium oxalate accumulation.

(A) Comparison of the crystal phenotypes in leaf and root from WT and Mtaae3 knock-down plants. Crystals are bright spots denoted by arrows. (B) Total oxalate in leaf and root from WT and Mtaae3 RNAi knock-down plants. Bar = 50 μm.