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. 2024 Jan 17;25(2):1149.
doi: 10.3390/ijms25021149.

Disruption of the Arabidopsis Acyl-Activating Enzyme 3 Impairs Seed Coat Mucilage Accumulation and Seed Germination

Affiliations

Disruption of the Arabidopsis Acyl-Activating Enzyme 3 Impairs Seed Coat Mucilage Accumulation and Seed Germination

Ninghui Cheng et al. Int J Mol Sci. .

Abstract

The Acyl-activating enzyme (AAE) 3 gene encodes an oxalyl-CoA synthetase that catalyzes the conversion of oxalate to oxalyl-CoA as the first step in the CoA-dependent pathway of oxalate catabolism. Although the role of this enzyme in oxalate catabolism has been established, its biological roles in plant growth and development are less understood. As a step toward gaining a better understanding of these biological roles, we report here a characterization of the Arabidopsis thaliana aae3 (Ataae3) seed mucilage phenotype. Ruthidium red (RR) staining of Ataae3 and wild type (WT) seeds suggested that the observed reduction in Ataae3 germination may be attributable, at least in part, to a decrease in seed mucilage accumulation. Quantitative RT-PCR analysis revealed that the expression of selected mucilage regulatory transcription factors, as well as of biosynthetic and extrusion genes, was significantly down-regulated in the Ataae3 seeds. Mucilage accumulation in seeds from an engineered oxalate-accumulating Arabidopsis and Atoxc mutant, blocked in the second step of the CoA-dependent pathway of oxalate catabolism, were found to be similar to WT. These findings suggest that elevated tissue oxalate concentrations and loss of the oxalate catabolism pathway downstream of AAE3 were not responsible for the reduced Ataae3 seed germination and mucilage phenotypes. Overall, our findings unveil the presence of regulatory interplay between AAE3 and transcriptional control of mucilage gene expression.

Keywords: Arabidopsis; acyl-activating enzyme; oxalate; seed mucilage.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Deletion of AtAAE3 impairs seed germination, seed coat mucilage accumulation, and increases seed permeability. (A) Arabidopsis WT and Ataae3 seeds were germinated on 0.5× MS media with 0.5% sucrose for 4 days. (B) Arabidopsis WT and Ataae3 seeds were stained with ruthenium red solution to visualize mucilage accumulation on the seed coats. (C) Quantification of mucilage in Ataae3 and WT seeds. RR staining intensity was quantified using Image J (ij153-win-java8). Student’s t-test; n = 3; ** p < 0.01 indicates a statistically significant difference between mutant seeds and wild type controls. (D) Seeds were stained with 2,3,5-triphenyltetrazolium chloride to evaluate seed coat permeability. Scale bars = 500 µm.
Figure 2
Figure 2
Effect of AtAAE3 on mucilage-related gene expression. Disruption of AtAAE3 significantly suppresses seed coat mucilage gene expression, as revealed by q-RT-PCR. Student’s t-test; n = 3; *** p < 0.001 indicates a significant difference between mutant seeds and wild type controls.
Figure 3
Figure 3
Effect of AtAAE3 on mucilage-related transcription factor expression. Disruption of AtAAE3 significantly suppresses expression of regulatory transcription factor genes involved in seed mucilage formation and seed coat development. Student’s t-test; n = 3; * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate a significant difference between mutant seeds and wild type controls.
Figure 4
Figure 4
Effect of excess oxalate on seed coat mucilage and cell wall development. Overexpression of Obc1 in Arabidopsis increases calcium oxalate crystal accumulation in (A) leaf, and (B) seeds. Student’s t-test; n = 3; *** p < 0.001 indicates a significant difference between Obc1 seeds and wild type controls. (C) Arabidopsis WT and Obc1 seeds were stained with RR to visualize mucilage accumulation on the seed coats. (D) Seeds were stained with 2,3,5-triphenyltetrazolium chloride to evaluate the seed coat permeability. Scale bars = 500 µm. (E) Quantification of seed permeability (%). n > 100; a, b, and c indicate significant differences between wild type, Ataae3, and Obc1 seeds. Accumulation of oxalate content in Obc1 seeds does not change seed mucilage formation but significantly alters seed coat permeability. (F) Arabidopsis WT, Ataae3, and Obc1 seeds were germinated on a 0.5× MS medium, using 0.5% sucrose, for 4 days. Seed germination (%) was determined. n > 100; a and b indicate significant differences between WT, Ataae3, and Obc1 seeds. There was no difference in seed germination between WT and Obc1 seeds.
Figure 5
Figure 5
Effect of excess oxalate on mucilage gene expression. Q-RT-PCR analysis of RNA samples extracted from wild type and Obc1 siliques. The results indicate that accumulation of oxalate content does not change the expression of seed mucilage genes but does affect the expression of some regulatory genes involved in seed coat development. Student’s t-test; n = 3; ** p < 0.01 indicates a significant difference between wild type and Obc1 seeds.
Figure 6
Figure 6
AtAAE3 is critical for mucilage accumulation. (A) First two steps in the CoA-dependent pathway of oxalate catabolism. (B) Arabidopsis WT, Ataae3, and Atoxc seeds were stained with RR to visualize mucilage accumulation on the seed coats. The results show that the reduction in mucilage accumulation is specific to the Ataae3 and does not result from a lack of a particular downstream metabolic intermediate due to blockage of the CoA-dependent pathway of oxalate catabolism.
Figure 7
Figure 7
A working hypothesis for the role of AtAAE3 in seed mucilage formation and seed coat development. Shown in the left panel is the expression of the TF regulatory network and the genes involved in mucilage production and extrusion in WT mucilage secretory cells (MSCs). In the Ataae3 mucilage secretory cells (MSCs) (right panel), expression of the TFs and their target genes is significantly down-regulated, reducing mucilage production and mucilage extrusion.

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