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Peroxisomal Acyl-CoA oxidase 4 activity differs between Arabidopsis accessions

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Abstract

In plants, peroxisomes are the primary site of fatty acid β-oxidation. Following substrate activation, fatty acids are oxidized by Acyl-CoA Oxidase (ACX) enzymes. Arabidopsis has six ACX genes, although ACX6 is not expressed. Biochemical characterization has revealed that each ACX enzyme acts on specific chain-length targets, but in a partially overlapping manner, indicating a degree of functional redundancy. Genetic analysis of acx single and double mutants in the Columbia (Col-0) accession revealed only minor phenotypes, but an acx3acx4 double mutant from Wassileskija (Ws) is embryo lethal. In this study, we show that acx3acx4 Col and acx1acx3acx4 Col mutants are viable and that enzyme activity in these mutants is significantly reduced on a range of substrates compared to wild type. However, the triple mutant displays only minor defects in seed-storage mobilization, seedling development, and adult growth. Although the triple mutant is defective in the three most active and highly-expressed ACX proteins, increases in ACX2 expression may support partial β-oxidation activity. Comparison of acx mutant alleles in the Col-0 and Ws accessions reveals independent phenotypes; the Ws acx4 mutant uniquely shows increased sensitivity to propionate, whereas the Col-0 acx4 allele has sucrose-dependent growth in the light. To dissect the issues between Col-0 and Ws, we generated mixed background mutants. Although alleles with the Col-0 acx4 mutant were viable, we were unable to isolate an acx3acx4 line using the Ws acx4 allele. Reducing ACX4 expression in several Arabidopsis backgrounds showed a split response, suggesting that the ACX4 gene and/or protein functions differently in Arabidopsis accessions.

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Notes

  1. Because mutants from different backgrounds will be discussed in this work, the subscript Col or Ws will be used to distinguish the alleles. For examples that are true in multiple backgrounds, no subscript will be used.

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Acknowledgments

This research was supported by the National Science Foundation (IOS-0845507), a University of Missouri-St. Louis Research Award, and University of Missouri-St. Louis start-up funds. We are grateful to Ian Graham for supplying acx mutants in the Ws background for comparison and the Arabidopsis Biological Resource Center for the ACX4 clone. We gratefully acknowledge the assistance of Amanda Tawfall and Xuemin Wang in the performance of the GC analysis and Anthony Fischer, Melanie Miller, Wendy Olivas, and Joe Russo for assistance with northern blots. We also thank Robert Barlow for assistance in optimizing the ACX enzyme assays, Anupama Vijayaraghavan, Amanda Tulin, and Tad Wood for assistance in genotyping the acx double and triple mutants, and Steven Jarvis for help isolating RNA interference lines. We are grateful to Robert Barlow, Shelly Boyer, Ying Li, Lisa Schechter, Gretchen Spiess, Amanda Tulin, and Anupama Vijayaraghavan for critical comments on the manuscript.

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Authors and Affiliations

  1. Department of Biology, University of Missouri, St. Louis, R223 Research Building, One University Boulevard, St. Louis, MO, 63121, USA

    Bibi Rafeiza Khan & Bethany K. Zolman

  2. Department of Biochemistry and Cell Biology, Rice University, MS-140, 6100 Main Street, Houston, TX, 77005, USA

    A. Raquel Adham

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  1. Bibi Rafeiza Khan
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Correspondence to Bethany K. Zolman.

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Supplementary material 1 (DOCX 31 kb)

11103_2011_9843_MOESM2_ESM.eps

Supplemental Fig. 1 ACX gene expression during plant growth and development. a Levels of ACX1-ACX5 mRNA expression in plant organs. b Levels of ACX1-ACX5 mRNA expression in flowers tissues. All flower values were from stage 15 tissue. Graphs were constructed using publically-available microarray data from the Arabidopsis eFP Browser data at http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi (Winter et al. 2007). Data was retrieved on January 22, 2010. Supplementary material 2 (EPS 901 kb)

11103_2011_9843_MOESM3_ESM.eps

Supplemental Fig. 2 ACX gene expression levels in acx mutants. RNA was isolated from 3 d old Col-0 (C), acx3acx4 Col (DM), and acx1acx3acx4 Col (TM) seedlings. cDNAs were generated and amplified with ACX1, ACX3, or ACX4 specific primers spanning the T-DNA insertions. Col-0 genomic DNA (G) was included as a control to confirm the purity of the cDNA synthesis and all samples were amplified with gene-specific primers upstream of the ACX4 insertion as a control to show successful amplification. Although the acx1acx4 double mutant is not shown, the line was used to generate the acx1acx3acx4 Col triple mutant. Supplementary material 3 (EPS 884 kb)

11103_2011_9843_MOESM4_ESM.eps

Supplemental Fig. 3 Acyl-CoA levels in acx mutant seedlings. Long-chain acyl-CoAs (vs. total fatty acids) retained in 6 d old light-grown seedlings. Data were collected with C20:1 levels shown in Figure 2C. Error bars represent the standard error of the means of three biological replicates. Supplementary material 4 (EPS 634 kb)

11103_2011_9843_MOESM5_ESM.eps

Supplemental Fig. 4 acx Col mutants are resistant to IBA and 2,4-DB. a Root lengths of 8 d old Col-0 (Wt) and acx Col single, double and triple mutant seedlings grown under continuous yellow light on medium supplemented with the indicated concentration of IBA. The graph here repeats Fig. 8a, adding the single mutant phenotypes for comparison. Error bars represent the standard error of the means (n ≥ 12). b-d Root lengths of seedlings grown on medium with (b) 2,4-DB, (c) IAA, or (d) 2,4-D. aux1-7 is an auxin resistant mutant (Pickett et al. 1990) shown as a control. Error bars represent the standard error of the means (n ≥ 12). Supplementary material 5 (EPS 1787 kb)

11103_2011_9843_MOESM6_ESM.eps

Supplemental Fig. 5 Acyl-CoA oxidase activity in Col-0 and Ws wild-type samples. Extracts from embryos (a, b), 46 hr (c, d, e) and 60 hr old (f, g, h) light grown Col-0 (black bars) and Ws (gray bars) seedlings were tested for ACX activity with (a, c, f) n-hexanoyl-CoA (C6:0), (b, d, g) lauroyl-CoA (C12:0), and (e, h) oleoyl-CoA (C18:1) substrates. Error bars represent the standard error of rates from three independent experiments. Supplementary material 6 (EPS 912 kb)

11103_2011_9843_MOESM7_ESM.eps

Supplemental Fig. 6 The acx Col triple mutant has normal growth and development. Col-0 (Wt) and acx Col mutant plants were grown under continuous white light at 22°C with normal watering two times per week. Plants were measured for (a) rosette diameter and (b) plant height at 38 d. Error bars represent the standard error of the means (n ≥ 18). Values of statistical significance relative to wild-type controls are indicated: * P < 0.05, ** P < 0.01, *** P < 0.001. Supplementary material 7 (EPS 767 kb)

11103_2011_9843_MOESM8_ESM.eps

Supplemental Fig. 7 Alignment of ACX1 from Col-0 and Ws. ACX1 (At4g16760) sequences were aligned with the MegAlign program (DNAStar; Madison, WI, USA) using the ClustalW method. Amino acids that differ are shaded in black boxes. Supplementary material 8 (EPS 1627 kb)

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Khan, B.R., Adham, A.R. & Zolman, B.K. Peroxisomal Acyl-CoA oxidase 4 activity differs between Arabidopsis accessions. Plant Mol Biol 78, 45–58 (2012). https://doi.org/10.1007/s11103-011-9843-4

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