Characterization of the role of BRL3 in arabidopsis seed germination

Hormonal signaling is a crucial regulatory mechanism required for proper seed germination. Gibberellic acid (GA) plays a central role in promoting dormancy release, as evidenced by its enhancement of specific enzymatic activities during the early stages of germination. In contrast, abscisic acid (ABA) is essential for maintaining dormancy, actively suppressing metabolic pathways necessary for germination. Brassinosteroids (BRs) are steroidal plant hormones that have been found to influence germination. There are three functional brassinosteroid (BR) receptors in Arabidopsis thaliana: BRASSINOSTEROID INSENSITIVE 1 (BRI1), BRI1-LIKE 1 (BRL1), and BRI1-LIKE 3 (BRL3). When BR is present, it latches on to one of the three known BR receptors, alongside the co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1). What results in is the ubiquitination of BRASSINOSTEROID-INSENSITIVE 2 (BIN2), which causes subsequent degradation of BIN2 via the 26S proteasome pathway. BIN2 is a repressor of BR signaling, but it promotes ABA responses by physically interacting with ABSCISIC ACID-INSENSITIVE 5 (ABI5). ABI5 directly represses EXPANSIN family genes and likely contributes to the repression of GA-responsive genes, including certain GA-stimulated Arabidopsis (GASA) family proteins, to prevent seed germination. The characterization of the role of the BRL3 receptor in seed germination has not been described fully yet. To examine how BRL3 affects this process, germination phenotype of the BRL3 mutants brl3-2 and brl3-5 and relative expression of BR receptors and select downstream genes were analyzed. The baseline germination results revealed distinct germination phenotypes across the wild type Col-0, the BRI1 mutant bri1-6, and the BRL3 mutants. High germination frequencies of the BRL3 mutants stood out, while low germination frequency of the BRI1 mutant coincided with the characteristic response expected of this mutant. qPCR analysis revealed that compared to bri1-6, both brl3-2 and brl3-5 upregulate BRI1 and BRL1 expression at 20 hours after imbibition (HAI). This suggests that BRI1 and BRL1 overexpression in the brl3 mutants may compensate for the lack of BRL3 in germination, whereas the germination phenotype of bri1-6 is not compensated by BRL1 and BRL3 overexpression. BRL3 mutants were insensitive to exogenous ABA and 24-epibrassinolide (EBR) applications, but GA did augment germination in both mutants alongside the WT. GA application leads to the destruction of RGLA2, which allows for GASA6 to be expressed. Both BRL3 mutants had GASA6 upregulated at 20 HAI when compared to bri1-6. In contrast, GASA6 was downregulated in the bri1-6 mutant dry seeds compared to that of WT. CPD and DWF4 were also upregulated in brl3-2 and brl3-5 when compared to bri1-6 at 20 HAI. Higher expression of BRI1 and BRL1 likely resulted in higher GASA6 expression in brl3-2 and brl3-5 mutants. Taken together, these findings suggest that BRL3 controls a negative feedback loop for BR receptors and BR biosynthesis.