3 Discussion
Global temperatures are increasing due to global warming, which greatly increases the probability of extreme high temperatures happening, these changes substantially disturb normal crop growth and yield around the world, which seriously affects human food security (Hasanuzzaman, Nahar, Alam, Roychowdhury, & Fujita, 2013). Facing the changeable and repetitive stress environment, plants have evolved ‘thermopriming’ mechanisms which enable them to respond more effectively to a second stress exposure. The molecular ‘memory’ can last for several days and this stress-free period is so-called the thermomemory phase. HS memory involves the regulation of transcription factors, epigenetic modification of chromatin, post-transcriptional modifications of proteins, metabolic control and coordinated regulation at multiple organ level (Balazadeh, 2021). Priming of stress‐induced gene expression is critical for stress priming (Brzezinka, Altmann, & Baurle, 2019). In this study, we reported that BES1 is crucial for long-term maintenance of thermal memory genes (Fig. 5). MiRNA156 sustains expression of thermomemory genes such as HSA32 and APX2 in a post-transcriptional regulation manner (Stief et al., 2014b). In addition, BRUSHY1 (BRU1)/TONSOKU/MGOUN3 maintains the sustained induction of memory genes through mediating the epigenetic inheritance of chromatin states (Brzezinka et al., 2019). Our work provides evidence for BRs signal enhanced control module for these memory genes continuous induction, thus uncover a new sight of phytohormone molecule-affected transcription cascade control of thermomemory.
Although the importance of Brassinosteroids in elevating thermotolerance in Arabidopsis and crops has already been reported, the details of the molecular mechanisms BRs signal plays in thermomemory remain largely unexplored. We still do not fully understand how BRs control a large number of heat response genes, when this regulation occurs, which master regulator orderly controls the responses to environmental heat cues (Nawaz et al., 2017). A recent study indicates that a defect in BES1 showed serious sensitive characteristics to heat stress compared to wild type. Heat shock rapidly activates BES1 and ABA promotes the dephosphorylation of BES1 through repressing the activity of PP2C-type phosphatases in HS process (Albertos et al., 2022). HSP90 forms a complex with BES1 and assists the compartmentalized cycle between active and inactive BES1 (Samakovli et al., 2020; Shigeta et al., 2015). In this study, we further discovered that BES1 accumulation in the nucleus in response to heat priming and sustained for another 2-3 days into the memory phase (Fig. 2). The molecular mechanism was further supported by genetic evidence of the heat-resistant phenotype of bes1-D and heat-intolerant BES1-RNAi mutant. Thus, we additionally found the new performance of elaborate regulator BES1.
We observed that BES1 was still degraded slowly under the combined treatment of CHX and MG132, especially later in the memory phase when the memory response is gradually weakening (Fig. 2F), indicating other protein degradation pathways also target BES1 for degradation, such as the lysosome and autophagy pathway. Since autophagy plays a crucial role in nutrient cycling and tolerance to various biotic and abiotic stress (Nishad & Nandi, 2021), it is interesting to explore the interaction between autophagy and BRs signals in response to heat stress, especially in controlling the balance between BES1 degradation and stability. The phosphorylated and dephosphorylated BES1 were degraded mutually in the majority of cases. Thus an increased de novo protein synthesis is required for BES1 accumulation in the nucleus during heat stress. BZR1 accumulates in the nucleus at high temperature, inducing the expression of growth-promoting genes such as PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) thus facilitating plant growth under high temperature (Ibanez et al., 2018). Similarly, our research found that heat priming induces the transcription of BES1 and finally contributes to BES1 accumulation in the nucleus during thermomemory phase (Fig. 2B, C). Most research focuses on the phosphorylation and degradation of BES1, given the importance of BES1 transcript for protein accumulation, it is crucial to elucidate the upstream signal that establishment and controls the transcriptional activation of BES1 in heat stress and thermomemory response.
BRs act to mitigate heat stress via compensation and priming of gene expression. BR treatment enhances reactive oxygen species scavenging to alleviate the impacts of high temperatures by inducing production of SOD and POD (Yin et al., 2018), but specific molecular mechanisms that activate the expression of these enzymes are largely unknown. A research performed exogenous EBR treatment with Arabidopsis BRs mutants and found loss-of-function in either BRs biosynthesis or signaling exhibit increased ROS accumulation (Setsungnern et al., 2020). Nevertheless, the concrete molecular mechanism by which BRs signal transduction activates ROS scavenging remains to be revealed. According to the ChIP–chip results, Ascorbate Peroxidase 1(APX1) and APX3 are two putative targets of BES1 (Yu. et al., 2011). In this study, we identified APX2 directly regulated by BES1 during thermomemory phase, providing a possible molecular mechanism for BR-regulated ROS scavenging when plants suffer from heat attack.
Studies in Arabidopsis and tomato demonstrate that downstream of BRs signaling, but not BRs level, make sense in heat stress response (Mazorra et al., 2011; Setsungnern et al., 2020). Similarly, a latest study reveals that BES1 is rapidly dephosphorylated and activated by heat stress in a manner independent of BRs signaling (Albertos et al., 2022). It seems like a paradox that BRs activate BES1/BZR1 but endogenous BRs levels are indispensable for thermal tolerance. Interestingly, cytosolic BES1/BZR1 can be recruited to the nucleus following activation of BRs, whereas environmental factors affect the protein abundance of BES1/BZR1, thus BRs-BES1 regulatory module is critical for plants to integrate environmental factors and endogenous signals to maximize survival (R. Wang et al., 2021; Yang et al., 2017). BRs treatment significantly improved heat tolerance in tomato (Nie et al., 2013), maize (Yadava, Kaushal, Gautam, Parmar, & Singh, 2016) and barley (Janeczko et al., 2011). Our study provides a BR independent but enhanced thermomemory response. The increasing frequencies of heatwaves poses a great threat to crop yields and food supplies, fortunately, exogenous supply of stress-signaling phytohormones like BRs benefit to mitigate the negative effects of high temperature in plants. Thus, the knowledge BRs function in thermomemory is helpful for breeding or editing genomes of agricultural crops.
In summary, our results provide insight into BR-enhanced thermomemory in plants. Although endogenous BRs level is indispensable for thermotolerance, exogenous application of BRs can certainly enhance basal thermotolerance and thermomemory, in a BES1 dependent manner. BES1 responds to heat priming and accumulates in the nucleus to activate the expression of HS-associated memory genes. This finding is beneficial for manipulating the BR pathway in crops and other plants so that plants can accommodate the heat stress created by frequent harmful high temperature fluctuations in these environmental parameters.