ABSTRACT
During leaf senescence, autophagy is essential for nutrient recycling and remobilization, and for plant productivity. Metabolome and transcriptome studies performed on autophagy mutants revealed major disorders in nitrogen, carbon, and redox metabolisms. Analysis showed that autophagy mutants are depleted of antioxidant anthocyanin molecules. Transcriptome analysis revealed that the depletion of anthocyanin is due to the downregulation of the master genes encoding the enzymes and regulatory proteins involved in the flavonoid pathway. The hyperaccumulation of salicylic acid and the depletion of anthocyanin in autophagy mutants might result from the rerouting of carbon resources in the phenylpropanoid pathway and amplify oxidative stress in autophagy mutants.
Keywords: Anthocyanin, metabolism, metabolome, plant senescence, transcriptome
In plants, autophagy is induced in leaves during senescence. Leaf senescence is a key developmental step dedicated to nutrient remobilization from senescing tissues to younger sink tissues and to seeds. As plants cannot move and are often nutrient limited, leaf senescence is essential to recycle nutrients and ensure individual survival as well as progeny production. During this process, a high quantity of nitrogen-containing compounds such as proteins and nucleic acids are disassembled and transformed into amino acids. In that context the role of autophagy is dual. On the one hand, autophagic activity facilitates the removal of oxidized proteins and maintains cell longevity, and on the other hand autophagy contributes to the degradation of cell components and to the recycling of nutrients that will be mobilized to sink organs needing them. Plants certainly cope with the cell degradation/survival dilemma of senescence by fine-tuning autophagy activity in order to maintain cells alive long enough to facilitate nutrient recycling, mobilization, and reallocation in other organs.
Using 15N isotope and tracing experiments, we showed that autophagy is essential for nitrogen remobilization from senescing leaves to seeds and contributes significantly to the plant nitrogen use efficiency and productivity. Using Arabidopsis atg mutants we showed that autophagy is especially required during leaf senescence and when plants are grown under low nitrate availability. Autophagy contributes to maintain of cell homeostasis, C:N balance, and redox metabolism.
Compared to Arabidopsis wild type, atg mutants accumulate all sorts of nitrogen compounds in their leaves. They accumulate soluble proteins, amino acids, ammonium, and nitrate. They are by contrast carbon depleted and contain low concentrations of glucose, fructose, sucrose, and starch. Carbon depletion also consists in lower contents of flavonoids like anthocyanin. These metabolic defects are likely to be due to the deregulation of the whole plant metabolism that seems to concentrate activity around the management of oxidative stress. Indeed we observed in atg mutants large amounts of glutathione and related amino acids as glutamate, cysteine, and methionine. The atg mutants overaccumulate also other molecules related to oxidative stress signaling like raffinose and salicylic acid (SA), which is a plant hormone involved in the cell death program. The analysis of the transcriptome of atg mutants confirmed the presence of oxidative stress hallmarks and the overexpression of genes that are also overexpressed in catalase mutants and in SA hyper accumulators.
The most surprising finding was that while oxidative stress responses are overstimulated in the atg mutants, anthocyanin compounds are significantly and dramatically lower in atg mutants compared to wild type. Anthocyanins are usually considered in plants as important antioxidant molecules involved in plant resistance to stress. Anthocyanins overaccumulate in plants in response to stresses like nitrogen depletion or light excess. They may act as sunscreens in plants protecting them from photo-oxidation. Depletion of anthocyanin in Arabidopsis atg mutants had already been reported, and previous reports proposed that autophagy would play a role in the specific trafficking of anthocyanin precursors and molecules from the endoplasmic reticulum or cytosol where they are synthesized to the vacuole, where they are stored. We quantified anthocyanin molecules and their precursors using LC-MS in the leaves of atg mutants and wild type and monitored the expression of the genes involved in their biosynthesis and regulatory pathways. We found that genes involved in the anthocyanin pathway are actually downregulated in atg mutants. In order to determine whether autophagy mutants are defective in anthocyanin trafficking or not and can store anthocyanin in their central vacuole, we overexpressed the ATMYB75/PAP1-positive regulator of the anthocyanin pathway in autophagy mutants, and observed then the recovery of anthocyanin production and accumulation inside the vacuole.
The reason why autophagy mutants downregulate anthocyanin production remains, however, to be determined. As anthocyanins are carbon storage molecules, the unbalance of C:N resources in atg mutants might be one of the reasons. SA and anthocyanin biosynthetic pathways are connected and we know that due to oxidative stress, atg mutants overaccumulate SA. The possibility that in response to oxidative stress, atg mutants reroute their carbon resources from anthocyanin to salicylic acid is another hypothesis (Fig. 1). This model proposes that carbon skeletons, originating from phenylalanine and used for both anthocyanin and SA production, would be preferentially directed to SA biosynthesis rather than to anthocyanin production. The depletion of anthocyanin antioxidants would then amplify oxidative stress and maintain SA production until cells die. The carbon shift between SA and anthocyanin remains to be explored. Tracing 13C phenylalanine molecules to analyze carbon fluxes in autophagy mutants would be helpful.
Figure 1.
Modification of carbon, nitrogen, and redox homeostasis in atg mutants affects anthocyanin and salicylic acid contents, influencing plant response to stress.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.