Structural and functional analysis of GME1 homologous genes and ascorbate accumulation in cultivated and wild tomato species

Ascorbic acid (ascorbate, vitamin C) plays an important role in various metabolic processes both in plants and humans. Increasing the ascorbate content in plants using breeding approaches is important, both from the point of view of increasing the nutritional value of fruits, and from the point of view of plant resistance to stress. It is known that tomato has high potential as an ascorbate source in the human diet. Unfortunately, the ascorbate levels in ripe fruits of modern tomato ( Solanum lycopersicum ) cultivars and hybrids are low in comparison with relative wild tomato species. Use wild tomato accessions in breeding programs can significantly increase the ascorbate content of ripe fruits. However, for effective breeding for this trait, a more detailed study of the genetic determinants responsible for the ascorbate levels increase in in ripe fruits is necessary. In this study, we cloned and sequenced novel GME1 gene homologous, which plays a key role in ascorbate biosynthesis in cultivated tomato and 11 wild tomato species. Structural analysis showed a low GME1 variability level of in tomato species. In the GME1 coding sequences, 28 SNPs were identified, of which only two led to nonsynonymous aminoacid substitutions (G2E and E281D) in S. neorickii and S. peruvianum var. dentatum . Analysis of GME1 motifs and domains did not reveal any specific motifs either at the interspecific level or at more distant taxonomic levels. The high GME1 conservatism observed in quite evolutionarily distant tomato species is most likely due to the functional significance of this enzyme for the ascorbate synthesis and, indirectly, for protection from stress factors, primarily photostress. No correlation was found between amino acid or nucleotide substitutions and ascorbate levels in fruits. Expression analysis, including comparative interspecies organ-specific analysis and analysis of the dependence of the ascorbate content in mature fruits in tomato cultivars and wild species accessions and the GME1 expression level, also did not reveal a relationship between transcriptional levels and ascorbate concentration. It can also be assumed that the final ascorbate content in the ripe tomato fruit may be influenced not by the intensity of GME1 expression at the last stage of fruit ripening, but by how this gene was active at earlier stages of ripening.