Abstract
The low level of methionine, an essential sulfur-containing amino acid, limits the nutritional quality of seeds. Two main factors can control the level of protein-bound methionine: the level of free methionine that limits protein accumulation and the methionine residues inside the storage proteins. To reveal the main limiting factor, we generated transgenic Arabidopsis thaliana seed-specific plants expressing the methionine-rich sunflower seed storage (SSA) protein (A1/A2). The contents of protein-bound methionine in the water-soluble protein fraction that includes the SSA in A1/A2 were 5.3- and 10.5-fold, respectively, compared to control, an empty vector (EV). This suggests that free methionine can support this accumulation. To elucidate if the level of free methionine could be increased further in the protein-bound methionine, these lines were crossed with previously characterized plants having higher levels of free methionine in seeds (called SSE). The progenies of the crosses (A1S, A2S) exhibited the highest level of protein-bound methionine, but this level did not differ significantly from A2, suggesting that all the methionine residues of A2 were filled with methionine. It also suggests that the content of methionine residues in the storage proteins is the main limiting factor. The results also proposed that the storage proteins can change their content in response to high levels of free methionine or SSA. This was assumed since the water-soluble protein fraction was highest in A1S/A2S as well as in SSE compared to EV and A1/A2. By using these seeds, we also aimed at gaining more knowledge about the link between high free methionine and the levels of metabolites that usually accumulate during abiotic stresses. This putative connection was derived from a previous analysis of SSE. The results of metabolic profiling showed that the levels of 29 and 20 out of the 56 metabolites were significantly higher in SSE and A1, respectively, that had higher level of free methionine, compared A1S/A2S, which had lower free methionine levels. This suggests a strong link between high free methionine and the accumulation of stress-associated metabolites.
Highlights
The low levels of methionine, an essential sulfur-containing amino acid, limit the nutritional quality of a plant-based diet (Hesse and Hoefgen, 2003; Amir et al, 2012)
Quantitative real-time PCR analyses showed that A2 has 2.1-fold higher expression levels of the transgene seed storage (SSA) compared to A1 (Figure 2B), which is in accordance with the results obtained from the immunoblot analysis (Figure 2A)
The results showed that free methionine levels in Arabidopsis seeds can increase the protein-bound methionine in seeds as shown in the seeds of SSE
Summary
The low levels of methionine, an essential sulfur-containing amino acid, limit the nutritional quality of a plant-based diet (Hesse and Hoefgen, 2003; Amir et al, 2012). We tested a combination of the first two approaches in order to determine the rate-limiting factor of protein-bound methionine in Arabidopsis thaliana seeds that were used as a model plant. In order to achieve a high expression in seeds during the production of proteins, the heterologous genes were fused to the promoters of seed-storage proteins that express the genes in the last stage of seed development when most of the amino acids are formed (e.g., Karchi et al, 1993; Kim et al, 2012; Nguyen et al, 2012; Matityahu et al, 2013; Amir et al, 2018). The highest level of free methionine (sixfold) was detected in the seeds of A. thaliana [called SSE plants (Cohen et al, 2014)], expressing the feedbackinsensitive form of AtCGS, AtD-CGS (Hacham et al, 2006)
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