Abstract Salicylic acid (SA) is a central signaling molecule in development and defense, therefore its levels are tightly controlled. One control mechanism is conjugation with sugar moieties by UDP glucosyltransferases (UGTs). In Arabidopsis, UGT76B1, UGT74F1, and UGT74F2 are known to glucosylate SA. We show that these are the main SA UGTs in leaves, since only marginal levels of SA glucosides were found in a triple loss-of-function mutant. Analyzing transcriptomes, metabolite levels, and phenotypes of a full combinatorial set of loss-of-function mutants, we resolved the mutual relationships and the individual roles of these enzymes in SA homeostasis. The strongest gene expression changes were observed for the ugt76b1 ugt74f1 double mutant, which downregulated developmental genes and most pronouncedly upregulated cell death-related genes. Among the single mutants, ugt76b1 specifically exhibited increased production of reactive oxygen species, increased resistance to infection, and early senescence. Likewise, higher-order mutations confirmed the dominant role of UGT76B1 in controlling SA levels and thereby the expression of biotic stress response genes. Both UGT74F1 and UGT74F2 affected UGT76B1 expression. However, while UGT76B1 and UGT74F1 produced SA-2- O -β-glucoside, UGT74F2 did not contribute there substantially. Instead, UGT74F2 acted independently of UGT74F1, decreasing steady-state SA levels by producing salicyloyl glucose ester. Remarkably, this did not restrict defense responses. In contrast, UGT74F1 interacted with UGT76B1 in suppressing defense responses. Nevertheless, a benzothiadiazole-triggered defense scenario induced only UGT76B1, whereas UGT74F1 was linked to controlling abiotic stress responses. All three enzymes form a network that, in concert with other UGTs, regulates expression of developmental and stress-related genes. One sentence summary The salicylic acid glucosylating enzymes of Arabidopsis leaves are crucial for salicylic acid homeostasis and combinatorially impact defense responses and developmental processes.

A fine-tuned interplay of three A. thaliana UDP glucosyltransferases orchestrates salicylic acid homeostasis