Role of the methionine cycle in the temperature-sensitive responses of potato plants to potato virus Y

Abstract

Plant-virus interactions are greatly influenced by environmental factors such as temperatures. In virus-infected plants, enhanced temperature is frequently associated with more severe symptoms and higher virus content. However, the mechanisms involved in such regulatory effects remain largely uncharacterized. To provide more insight into the mechanisms whereby temperature regulates plant-virus interactions, we analysed changes in the proteome of potato cv. Chicago plants infected with potato virus Y (PVY) at normal (22 °C) and elevated temperature (28 °C), which is known to significantly increase plant susceptibility to the virus. One of the most intriguing findings is that the main enzymes of the methionine cycle (MTC) were down-regulated at the higher but not at normal temperatures. With good agreement, we found that higher temperature conditions triggered consistent and concerted changes in the level of MTC metabolites, suggesting that the enhanced susceptibility of potato plants to PVY at 28 °C may at least be partially orchestrated by the down-regulation of MTC enzymes and concomitant cycle perturbation. In line with this, foliar treatment of these plants with methionine restored accumulation of MTC metabolites and subverted the susceptibility to PVY at elevated temperature. These data are discussed in the context of the major function of the MTC in transmethylation processes.

Keywords: isobaric tags for relative and absolute quantitation (iTRAQ); methionine cycle; potato virus Y; proteomic analysis; temperature-dependent antivirus defence; virus susceptibility.

FIGURE 1

Accumulation of PVY RNA (measured using quantitative reverse transcription PCR) in systemically infected leaves of potato plants over 3–21 days postinoculation (dpi) time periods at 22 or 28 °C as shown. Analysis of variance and Tukey's HSD post hoc tests were performed for quantitative reverse transcription PCR data. ***p < .001

FIGURE 2

(a) The strategy for comparative quantitative analysis of protein expression in potato leaves by 8‐plexisobaric tags for relative and absolute quantitation (iTRAQ). For detailed iTRAQ data see Tables S2 and S3. (b) Protein–protein interaction networks of differentially expressed proteins (DEPs). Proteins are indicated with nodes, and interactions between proteins are represented by edges. M1CQ02‐MS; M1BBU4‐MTHFR; M0ZZV7–SAHH; Q307Y9‐SAMS 1; M1BAA6‐SHM; M1AYK4‐S‐formylglutathione hydrolase. (c) GO enrichment analysis of the up‐regulated DEPs in infected potato plants at 14 days postinoculation (dpi) at 28 °C. (d) Heatmap showing the changes in abundance of key enzymes of the methionine cycle (MTC) and MTC‐related folate cycle. PVY22_8dpi, infected potato plants at 8 dpi at 22 °C; PVY28_8dpi, infected potato plants at 8 dpi at 28 °C; PVY22_14dpi, infected potato plants at 14 dpi at 22 °C; PVY28_8dpi, infected potato plants at 14 dpi at 28 °C

FIGURE 3

Expression level of methionine cycle (MTC) and MTC‐related protein genes: StMS (a) and StSAMS (b), StSAHH (c), StSHM (d) and StMTHFR (e) (measured using quantitative reverse transcription PCR [RT‐qPCR]) in systemically infected leaves of potato plants at 22 or 28 °C at 5, 7, and 8 days postinoculation (dpi) as shown. Analysis of variance and Tukey's HSD post hoc tests were performed for RT‐qPCR data. **p < .01, ***p < .001

FIGURE 4

Content of methionine cycle (MTC) metabolites in systemically infected leaves of potato plants at 22 or 28 °C at 8, 10, and 14 days postinoculation (dpi) as shown. (a) Content of S‐adenosylmethionine (SAM), S‐adenosylhomocysteine (SAH), and homocysteine (HCY) was measured by ELISA. Content of methionine (MET) was measured using methionine assay kit (fluorometric). (b) The ratios of SAM to SAH. Analysis of variance and Tukey's HSD post hoc tests were performed for data. ***p < .001

FIGURE 5

Effect of treatment with l‐methionine (MET) on accumulation of PVY RNA (measured using quantitative reverse transcription PCR [RT‐qPCR]) in systemically infected leaves of mock‐ and PVY‐inoculated potato plants at 22 or 28 °C at 5–21 days postinoculation (dpi) as shown. Analysis of variance and Tukey's HSD post hoc tests were performed for RT‐qPCR data. ***p < .001

FIGURE 6

Effect of treatment with l‐methionine (MET) on accumulation of S‐adenosylmethionine (SAM), S‐adenosylhomocysteine (SAH), and SAM to SAH ratios in systemically infected leaves of potato plants at 22 or 28 °C at 8, and 14 days postinoculation (dpi) (measured by ELISA) as shown. Analysis of variance and Tukey's HSD post hoc tests were performed for data. ***p < .001

FIGURE 7

A tentative model showing possible mechanisms underlying the temperature‐sensitive susceptibility of potato plants to PVY under elevated temperature. (a) Schematic representation of the methionine cycle (MTC) in healthy cells. SAMS converts methionine (MET) to S‐adenosylmethionine (SAM), which serves as a methyl donor for transmethylation reactions of various substances including proteins, DNAs, and small interfering (si) RNAs and is also a key component for the synthesis of ethylene. The methylation reaction by‐product S‐adenosylhomocysteine (SAH) is consequently hydrolysed by SAHH to adenosine and homocysteine (HCY). Then HCY is converted back to MET by MS. (b) PVY infection at higher temperature causes down‐regulation of key MTC (MS, SAMS, SAHH) and MTC‐related folate cycle (SHM, MTHFR) enzymes, leading to the decrease of SAM and increase of SAH. This would suppress SAM‐dependent methylation reactions, including HEN1‐directed methylation of siRNAs reducing stability of antiviral siRNAs. Inhibition of DNA and protein methyltransferases may also contribute to the susceptibility response to PVY. Finally, a decrease in ethylene production may be another factor conferring increased susceptibility to PVY at higher temperatures. Decreasing and increasing levels of MTC and MTC‐related enzymes and metabolites are indicated by arrows