Exploring the Contribution of Autophagy to the Excess-Sucrose Response in Arabidopsis thaliana

Abstract

Autophagy is an essential intracellular eukaryotic recycling mechanism, functioning in, among others, carbon starvation. Surprisingly, although autophagy-deficient plants (atg mutants) are hypersensitive to carbon starvation, metabolic analysis revealed that they accumulate sugars under such conditions. In plants, sugars serve as both an energy source and as signaling molecules, affecting many developmental processes, including root and shoot formation. We thus set out to understand the interplay between autophagy and sucrose excess, comparing wild-type and atg mutant seedlings. The presented work showed that autophagy contributes to primary root elongation arrest under conditions of exogenous sucrose and glucose excess but not during fructose or mannitol treatment. Minor or no alterations in starch and primary metabolites were observed between atg mutants and wild-type plants, indicating that the sucrose response relates to its signaling and not its metabolic role. Extensive proteomic analysis of roots performed to further understand the mechanism found an accumulation of proteins essential for ROS reduction and auxin maintenance, which are necessary for root elongation, in atg plants under sucrose excess. The analysis also suggested mitochondrial and peroxisomal involvement in the autophagy-mediated sucrose response. This research increases our knowledge of the complex interplay between autophagy and sugar signaling in plants.

Keywords: Arabidopsis thaliana; autophagy; mitochondria; peroxisome; reactive oxygen species (ROS); sucrose.

Figure 2

Less root length inhibition in atg mutants under sucrose and glucose but not fructose. WT, atg5-1, and atg7-2 plants were sown on Nitsch plates containing increasing concentrations of sugars (molar equivalent to sucrose concentration). (A) Germination percentage (GP) of WT, atg5-1, and atg7-2 seeds under 0% and 4% sucrose. Data are presented as the average ± SE. An asterisk denotes a significant difference from WT under the same treatment conditions and at the same time point, as determined by Dunnett’s test (p < 0.05, n = 4–6); light grey—atg5-1, dark grey—atg7-2. (B) Quantification of primary root length under various glucose treatment conditions. An asterisk denotes a significant difference from WT under the same treatment by Dunnett’s test (p < 0.05, n = 8–24). x indicates sample mean, and circles denote outliers. (C) Quantification of primary root length under various fructose concentrations. No significant difference from WT under the same treatment condition was found by Dunnett’s test (p < 0.05, n = 9–17). x indicates sample mean, and circles denote outliers. (D) Quantification of primary root length under various mannitol treatments. No significant difference from WT under the same treatment condition was found by Dunnett’s test (p < 0.05, n = 16–22). x indicates sample mean, and circles denote outliers.

Figure 1

atg mutants display reduced sensitivity to sucrose excess. (A,B). WT, atg5-1, and atg7-2 plants were sown on Nitsch plates containing increasing concentrations of sucrose, and grown vertically for 14 days. (A) Representative image of the plants. (B) Quantification of primary root length under various sucrose treatments. Asterisk denotes a significant difference from WT under the same treatment conditions, as determined by Dunnett’s test (p < 0.05, n = 10–30), x indicates sample mean, and circles denote outliers. (C) WT, atg5-1, and atg7-2 plants were sown on Nitsch plates containing 0% or 4% sucrose and grown vertically. The primary root length was measured every two days for 10 days after imbibition. Data are presented as the average ± SE. An asterisk denotes a significant difference from WT under the same treatment conditions and at the same time point, as determined by Dunnett’s test (p < 0.05, n = 12–31). Dark grey—atg5-1, light grey—atg7-2.

Figure 3

Sucrose excess induces autophagy in the shoots but not in the roots, and the root phenotype is not NBR1-dependent. (A,B) GFP-ATG8f plants were sown on Nitsch plates containing increasing concentrations of sucrose, and grown for 10 days. (A) Roots and (B) shoots were collected separately, and proteins were extracted and analyzed by Western blot using an anti-GFP antibody. Equal protein loading was verified by Coomassie brilliant blue staining. (C) WT, atg5-1, nbr1, or (D) NBR-OX (35S::NBR1) plants were sown on Nitsch plates containing increasing concentrations of sucrose and grown vertically for 14 days, after which the primary root length was quantified (top panel). An asterisk denotes a significant difference from WT under the same treatment, as determined by Dunnett’s test (p < 0.05, n = 10–30), x indicates sample mean, and circles denote outliers. Bottom panel—a representative image of the plants.

Figure 4

No difference in starch accumulation between WT and atg mutants under sucrose excess. WT, atg5-1, and atg7-2 plants were sown on Nitsch plates containing increasing concentrations of sucrose and grown for 14 days. (A) Representative images of IKI staining of starch. (B) Quantification of starch. No significant difference from WT under the same treatment was found by Dunnett’s test (p < 0.05, n = 4), x indicates sample mean, and circles denote outliers.

Figure 5

Sucrose excess affects the root metabolome, but the influence of autophagy on the response is mild. Roots from WT, atg5-1, and atg7-2 seedlings grown for 10 days under various sucrose concentrations were collected, and polar metabolites were extracted and analyzed by gas-chromatography mass-spectrometry (GC-MS) (A). Principal component analysis (PCA) performed on scaled values of metabolites measured in roots grown under 3% sucrose (B). Heatmap displaying the relative amounts of root metabolites under various sucrose concentrations. Results are presented as the log2 ratio normalized to WT under 0% sucrose. An asterisk denotes a significant difference from WT under the same treatment regimen, as determined by Dunnett’s test (p < 0.05, n = 5–6).

Figure 6

Proteomic analysis of roots revealed differences in protein levels during sucrose excess and between WT and atg5-1 plants. WT and atg5-1 were sown on Nitsch plates containing 1% or 3% sucrose and grown vertically for 10 days. Roots were then collected and subjected to proteomic analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS). (A) PCA of the label-free quantification (LFQ) values of the entire proteome. (B) Volcano plot for 1% sucrose—p values and fold-change relative to atg5-1. (C) Volcano plot for 3% sucrose—p values and fold-change relative to atg5-1. (D) Venn diagram of differentially accumulated proteins: http://bioinformatics.psb.ugent.be/webtools/Venn/, accessed on 26 December 2021.

Figure 7

atg5-1 shows alterations in many biological processes under high sucrose levels. Enriched biological processes identified from proteomics analyses performed using David functional annotation tools in GOTERM_BP_DIRECT. (A) Biological processes involving protein accumulation in atg5-1 compared to WT under 3% sucrose. (B) Biological processes involving protein depletion in atg5-1 compared to WT under 3% sucrose. The red dotted line indicates the threshold for significant enrichment of the given proteins for the biological process (p < 0.05).

Figure 8

Root peroxidase activity is increased in atg mutant plants under sucrose excess (A). Heatmap describing the fold-change in peroxidase levels between atg5-1 and WT under 1% and 3% sucrose. Black boxes indicate peroxidases that were not detected. An asterisk denotes a significant change between WT and atg5-1, as determined by Student’s t-test. (B). WT, atg5-1, and atg7-2 plants were sown on Nitsch plates containing 0%, 1%, or 3% sucrose and grown vertically for 10 days. Roots were collected and the peroxidase activity was assessed. An asterisk denotes a significant difference from WT under the same treatment, as determined by Dunnett’s test (p < 0.05, n = 4), x indicates sample mean, and circles denote outliers.

Figure 9

Proposed mechanism of action for autophagy during sucrose excess. In WT plants under high sucrose/glucose levels, primary root shortening occurs via the accumulation of ROS and reduced levels of auxin. In contrast, under similar conditions, atg mutant plants present reduced ROS accumulation, which is mediated via peroxidases, PUMP5, TCA cycle enzymes, and peroxisomal activity. There is also less auxin reduction, which is mediated by peroxisomal proteins and ILL activity. These changes in protein and organellar activity lead to reduced primary root shortening. Red arrows indicate upregulation, blew arrows indicate downregulation.