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Phytohormones play key roles in many aspects of plant growth and development, as well as in resistance to abiotic stresses. The presence of heavy metal limits phytohormone endogenous level; however, the application of brassinosteroids (BRs) restores phytohormone homeostasis and reduces unfavorable consequences of heavy metal on plant growth. Therefore, the aim of this study was to (1) broaden previously published analyses/findings to study the effect of 24-epibrassinolide (EBL) and/or lead (Pb) on  Acutodesmus obliquus  phytohormone level after 5 days of cultivation using LC-QToF-MS quantification technique and (2) extend the total amount of quantified phytohormones in  A. obliquus . The study confirmed that exogenous EBL increased the contents of gibberellin A 3  (GA 3 ), auxins (AXs) (indole-3-acetic acid, phenylacetic acid), BRs brassinolide, 24-epibrassinolide (EBL), 28-homobrassinolide, castasterone, 24-epicastasterone, typhasterol, cathasterone, 6-deoxotyphasterol, as well as different types of cytokinins (CKs): free bases, ribosides, and conjugates ( N - and  O -glucosides). On the other hand, treatment with Pb had an opposite effect on BRs, GA 3 , AXs, and free bases, as well as ribosides of CKs. The abscisic acid (ABA) level decreased under EBL treatment but increased in response to Pb stress. The level of  N -glucosides, as well as  O -glucosides of CKs, also significantly increased in response to Pb alone. Interestingly, the co-application of EBL and Pb led to an increase in BRs, GA 3 , AXs, and CKs content in the algae. Principal component analysis revealed that based on treatments, increase of GA 3 , BRs, and AXs was found to be in the following order: 1 μM EBL &gt; 0.01 μM Pb + 1 μM EBL &gt; 500 μM Pb + 1 μM EBL &gt; control &gt; 0.01 μM Pb &gt; 500 μM Pb. Moreover, very strong significant linear relations between almost all studied AXs and BRs were noted. Summarizing, this research did not only allow to detect the occurrence of ABA, GA 3 , AXs, BRs, and CKs in  A. obliquus  for the first time but also to study the combined action of EBL and Pb, as well as compare it with treatment alone.

Hormonal response of Acutodesmus obliquus exposed to combined treatment with 24-epibrassinolide and lead