1 INTRODUCTION
Due to climate change, the global temperature will continue rising in the future, which will cause great challenges to crop production (Lesk, Rowhani, & Ramankutty, 2016). Ambient temperature of plants increases rapidly by 10℃ will pose a threat to plants and cause heat stress (HS). In order to survive, plants form a complex and efficient regulatory network to resist and adapt to HS, understanding these protective responses has important agronomic value for maximizing agricultural production and maintaining yield (Huang, Zhao, Burger, Wang, & Chory, 2021). Under HS, heat shock transcription factors (HSFs) are induced rapidly to active the expression of heat shock proteins (HSPs). HSPs mainly function as molecular chaperones to protect against thermal denaturation of substrates and facilitate refolding of denatured substrates, by regulating heat shock proteins, plants can tolerate certain heat levels (McLoughlin, Kim, Marshall, Vierstra, & Vierling, 2019).
In the natural environment, plants often suffer from repeated and changeable heat shock, these sub-lethal HS induce plants to produce thermal memory (Balmer, Pastor, Gamir, Flors, & Mauch-Mani, 2015). HS priming endows plants with a stronger thermal response to a second stress exposure. Hs memory involves an initial priming phase and a memory period that lasts for several days (Ohama, Sato, Shinozaki, & Yamaguchi-Shinozaki, 2017). The initial phase of the HS memory involves interacting transcriptional regulatory networks, HSFs play fundamental role among these transcription factors. In addition, specific modifications in the chromatin enable these memory genes to be induced rapidly to cope with next stress in the memory phase (Lamke, Brzezinka, Altmann, & Baurle, 2015). During the HS memory state, several HSPs with chaperone activity maintain high protein levels for several days after priming (Wu et al., 2013).
As efficient and accurate signal molecules, phytohormones actively participate in HS response. Exogenous application of abscisic acid (ABA) increases hydrogen peroxide (H2O2) accumulation by inducing the expression of respiratory burst oxidase Homologs (RBOHs), and thus enhances antioxidant capacity (Suzuki et al., 2011). In addition, ABA also induces the expression levels of HSPs to maintain protein in functional conformations (X. Wang, Zhuang, Shi, & Huang, 2017). In flower tissue of Arabidopsis , cytokinin treatment increases resistance in response to harmful high temperature, protecting developing flowers exposed to HS (K. Wang, Zhang, & Ervin, 2012). Salicylic acid (SA) reduces heat stress-induced membrane damage, enhancing the activities of antioxidant enzymes (Shah Jahan et al., 2019). In addition, SA treatment maintains the level of HSP21 protein in chloroplast, facilitates the recovery of photosynthesis after stress (L. J. Wang et al., 2010). High temperatures lead to accumulation of jasmonate (JA), loss of function in JA signaling or biosynthesis impairs the thermotolerance of Arabidopsis seedlings, indicating that JA is required for effective heat tolerance (Clarke et al., 2009). JA treatment did not induce the expression of HSPs, and a defect in JA signaling has no impact on HSPs transcription level (Manvi & Ashverya, 2016). In contrast to JA, ethylene signal transduction pathway promotes the expression of HSFs and HSPs via ETHYLENE RESPONSE FACTOR 95/97 (ERF95/97), the direct target of ETHYLENE INSENSITIVE 3 (EIN3) (Huang et al., 2021). Despite overwhelming evidences supporting the vital role phytohormones act in HS response, limited knowledge of the molecular mechanism restricts the application in crops, it is urgent to identify pivotal factors in response to heat shock in phytohormones signaling.
Brassinosteroids (BRs) are a class of plant steroid hormones that play a central role in the control of plant growth and development (Nolan, Nemanja, Liu, Eugenia, & Yin, 2019). BRs are perceived by the transmembrane receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1), and the binding of BRs activates BRI1, thus triggering a phosphorylation cascade and finally inactivating BR-INSENSITIVE 2 (BIN2), a GSK3-like kinase that negatively modulates BRs signal by phosphorylating BRI1-EMS-SUPPRESSOR 1 (BES1) and BRASSINAZOLE RESISTANT 1 (BZR1), the key regulators of BR-regulated gene expression (Guo, Li, Aluru, Aluru, & Yin, 2013). Transcriptome analysis showed that BES1/BZR1 up- or down-regulate thousands of BR-responsive genes (Sun et al., 2010; Yu. et al., 2011). In the spring barley (Hordeum vulgare L .), improved functioning of PSII was observed under HS upon 24-epibrassinolide (EBR) application (Janeczko, Okleková, Pociecha, Kocielniak, & Mirek, 2011). In rice (Oryza sativa ), EBR or 7,8-dihydro-8-20-hydroxyecdysone (DHECD) application improves photosynthetic efficiency and raises the rice yield under HS (Diaz et al., 2021). In tomato, epi-brassinolide (EBL) induces thermotolerance and peroxidase (POD) activity (Mazorra, Holton, Bishop, & Nunez, 2011). HS promotes the dephosphorylation of BES1 through repressing the activity of type 2C phosphatases (PP2Cs), activated BES1 directly binds to heat shock elements (HSEs) thus contribute to HS signaling (Albertos et al., 2022). These studies demonstrate that BRs signal play a positive role in HS response. Nevertheless, little is known about whether BRs signal transduction modulates thermal memory process, so far there are no reports involved in BR-mediated thermomemory.
In this study, we report that BRs act positively in HS memory and that BES1 is required for BRs-enhanced thermomemory. Heat priming induces the nucleus accumulation of BES1 and BES1 protein sustains for high levels several days after priming. BRs treatment further activates BES1 during the memory phase. The ability to maintain high levels of BES1 protein in the nucleus after priming is required for sustained activation of HS memory‐associated genes and BES1 activates memory genes in a direct or indirect manner. Our results thus demonstrate that BES1 lays the foundation of thermomemory, while BRs further pull up the upper limit of it.