ZMS5H, A NOVEL ENZYME INVOLVED IN SALICYLIC ACID HYDROXYLATION

RIGHINI, S; SERRA, PALOMA; FALCONE FERREYRA, MARÍA LORENA; CASATI, PAULA

Salta

Congreso; Joint LV Annual SAIB Meeting and XIV PABMB Congress; 2019

Salicylic acid (SA) has been described as an important signaling molecule in plants, regulating growth, development, senescence and responses to biotic and abiotic stresses. Levels of salicylic acid are regulated not only by activation of its biosynthetic pathway, but also through its modification by metabolic modifications, such as glycosylation, methylation, amino acid conjugation, and hydroxylation. Hydroxylated SA is the major degradation product of SA. Recently, the A. thaliana enzyme catalyzing SA to 2,3-dihydroxybenzoic acid (2,3-DHBA; AtS3H) has been identified, and SA was found to accumulate in s3h mutants. In this study, we report the discovery and functional characterization of a novel maize salicylic acid 5-hydroxylase (ZmS5H), a 2-oxoglutarate dependent dioxygenase that catalyzes the formation of 2,5-DHBA by hydroxylating SA at the C5 position of its phenyl ring. Once identified, we carried out in vitro activity assays in order to kinetically characterize this enzyme. His-tagged ZmS5H was heterologously expressed in Escherichia coli and then purified. The reaction product 2,5-DHBA was identified by HPLC by comparison with authentic standards. Interestingly, according to sequence similarity analysis, ZmS5H and AtS3H are closely evolutionarily related, though we could not identify 2,3-DHBA as a product of the studied reaction. Kinetic parameters of the recombinant ZmS5H were also determined by HPLC. In addition, its activity in planta was demonstrated, as transgenic Arabidopsis plants expressing ZmS5H were more susceptible to P. syringae pv tomato DC3000 pathogen infection than WT plants, suggesting that these plants would have decreased SA levels due to higher hydroxylation of the hormone. In order to further confirm this, we investigated the expression level of three different genes modulated by SA in s3h, wild-type and transgenic Arabidopsis plants expressing ZmS5H that were treated with this hormone compared to plants treated with mock solution. PR1, EDS-1 and SAG13 showed a decreased expression level in plants overexpressing ZmS5H compared to s3h mutants and wild-type plants, suggesting that in fact ZmS5H hydroxylates SA in planta. We are now analyzing a possible crosstalk between SA hydroxylation and flavonoids synthesis, a model proposed in our lab based in previous results. So far, transgenic plants expressing two different maize flavone synthases recently characterized, (ZmFNSI and ZmFNSII), which accumulate flavones, in a mutant s3h background, show increased susceptibility towards infection with P. syringae compared to wild-type and mutant plants, suggesting that flavones regulate SA levels in vivo.