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.