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\begin{document}

\title{A Brassinosteroids-BES1 Regulatory Module Manipulate Thermomemory in
Arabidopsis thaliana}



\author[1]{xiuhong yao}%
\author[1]{Juan Chen}%
\author[1]{Wen Yu}%
\author[1]{Zuxu Zhou}%
\author[1]{Fabin Yang}%
\author[1]{Yanling Li}%
\author[1]{Dawei Zhang}%
\author[1]{HONG-HUI LIN}%
\affil[1]{Sichuan University State Key Laboratory of Hydraulics and Mountain River Engineering}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Heat stress (HS) caused by ambient high temperature pose a threat to
plants. In the natural and agricultural environment, plants often
encounter repeated and changeable HS. Moderate HS primes plants to
establishment of a molecular `thermomemory' that enables plants to
withstand a later-and possibly more extreme-HS attack. Recent years,
brassinosteroids (BRs) have been implicated in HS response whereas
little is known about whether BRs signal transduction modulates
thermomemory. Here, we uncover the positive role of BRs signaling in
thermomemory of \emph{Arabidopsis thaliana}. Heat priming induces de
novo synthesis and nuclear accumulation of BRI1-EMS-SUPPRESSOR (BES1),
the key regulator of BRs signaling. BRs promote the accumulation of
dephosphorylated BES1 during memory phase, blocking BRs synthesis
impairs dephosphorylation. During HS memory, BES1 is required to
maintain sustained induction of HS memory genes and directly targets
\emph{APX2} and \emph{HSFA3} for activation. In summary, our results
reveal a BES1-required, BRs-enhanced transcriptional control module of
thermomemory in \emph{Arabidopsis thaliana}.%
\end{abstract}%



\sloppy


\textbf{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 (H\textsubscript{2}O\textsubscript{2})
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 \emph{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 \emph{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 (\emph{Hordeum vulgare L} .), improved
functioning of PSII was observed under HS upon 24-epibrassinolide (EBR)
application (Janeczko, Oklekov\selectlanguage{ngerman}á, Pociecha, Kocielniak, \& Mirek, 2011).
In rice (\emph{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\selectlanguage{english}-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.

\textbf{2 RESULTS}

\textbf{2.1 BRs play a positive role in thermomemory}

To elucidate the role of BRs in thermomemory in \emph{Arabidopsis} , we
performed a thermotolerance assay under primed and unprimed conditions
towards wild-type Col-0. Heat priming and triggering protocol was
outlined in Fig. 1A. Seedlings grown on standard half-strength
Murashige-Skoog (1/2MS) medium with or without 10 nM BL (brassinolide, a
kind of BRs with high physiological activity) were vibrant before HS.
Upon HS triggering and recovery, seedlings grown on the medium
containing BL exhibited significantly enhanced thermotolerance,
accompanied with comparably elevated survival rate and decreased
electrolyte leakage. Thermomemory induced by heat priming effectively
improved the thermotolerance of seedlings and the application of BL
further enhanced the vitality of thermomemory (Fig. 1). These
observations suggest that BRs positively modulate thermomemory process.

\textbf{2.2 BES1 is specifically required for thermomemory}

Given that BRs act positively in thermomemory, we then investigated
whether the defect in BRs synthesis weakened memory response. Previous
studies reported that BR-deficient and -overproduction seedlings
represented similar thermal tolerance (Albertos et al., 2022; Mazorra et
al., 2011), to further investigate whether endogenous BRs levels affect
thermomemory, we subject 5-day-old \emph{det2} and
\emph{DET2-OX}seedlings to our priming and triggering stimulus protocol.
Surprisingly, both \emph{det2} and \emph{DET2-OX} represented enhanced
thermal tolerance (Supplementary Fig. 1), indicating that thermomemory
is also independent of BR homeostasis, this promotes us to explore
downstream of BRs signaling. We next access the role BES1 acts in
thermomemery since the effect of BRs is largely mediated by the
BES1/BZR1 subfamily transcription factors. Considering the functional
redundancy between BES1 and BZR1, we chose \emph{BES1-RNAi} (a mutant
BES1/BZR1 mRNA almost undetectable caused by RNA interference) to carry
out thermomemory assay (Yin et al., 2005). As shown in Fig. 1, BES1
deficiency caused seedlings hypersensitive to heat, as comparably
declined survival rate and increased electrolyte leakage was detected in
\emph{BES1-RNAi} . In contrast to the elevated thermotolerance by
priming stimulation in Col-0, primed \emph{BES1-RNAi} seedlings
represent similar thermotolerance to the unprimed, indicating that
BES1/BZR1 is required for thermomemory. Application of 10 nM BL
increased the survival rate of Col-0 seedlings, however, it had no
obvious effect on \emph{BES1-RNAi}under both primed and unprimed
conditions. These results suggest that BES1 is indispensable for
BRs-enhanced thermomemory.

\textbf{2.3 BES1 accumulates during the memory phase}

To explore the expression pattern of \emph{BES1} in the memory phase, we
carried out a time course towards seedling collected at different time
points indicated in Fig. 2A. The transcriptional level of
\emph{BES1}remained high until 48 h into the thermomemory phase, and
still more than 6 fold compared with the control in 48 h (Fig. 2B).
Consistent with this, GUS analysis also demonstrates that heat priming
inducible expression of BES1 (Fig. 2C). Next, we assessed whether the
increased mRNA level in the memory phase leads to the accumulation of
BES1. We investigated the abundance of BES1 during thermomemory assay
and observed more than 2-fold accumulation in the memory phase compared
with control (prior priming), and BES1 protein remains abundant until 48
h into the thermomemory phase (Fig. 2D). To test whether the sustained
high level of BES1 protein at later memory time points are due to the
rise of transcriptional level and requires de novo protein synthesis, we
applied cycloheximide (CHX) to inhibit protein translation. Col-0
seedlings were treated with CHX after priming and BES1 protein level was
determined by immune blotting at 12 h, 24 h and 48 h into the memory
phase. Compared with mock treatment (DMSO), BES1 protein level declined
in the memory phase (Fig. 2E), supporting our suspect that increases in
BES1 mRNA contribute to the accumulation of BES1 protein during the
memory phase. In addition, we also concerned about the effect caused by
protein degradation, since the increased BES1 may also come from the
contribution of weakening degradation. Compared with mock treatment,
treatment with MG132 resulted in an increase of BES1 within the memory
phase (Fig. 2F), indicating that the degradation of BES1 weakened during
that time. Furthermore, we carried out a combined treatment with Col-0
seedling using both CHX and MG132. Not surprisingly, BES1 abundance in
these seedlings was slightly higher than that of CHX treatment but
obvious lower than MG132 treatment (Fig. 2G). These results demonstrate
that de novo synthesis and weakened degradation mutually contribute to
the accumulation of BES1 during the memory phase.

\textbf{2.4 Heat priming and BRs promote the accumulation of
dephosphorylated BES1 during memory phase}

HS activates BES1 via dephosphorylation (Albertos et al., 2022), so we
are curious about whether the state can be sustained in the memory
phase. We executed a transient expression experiment on \emph{N.
benthamiana} leaves to explore the subcellular distribution of BES1
after heat priming. As shown in Fig. 3A, BES1 was diffused in the
nucleus and cytoplasm prior priming, after exposure to 44\selectlanguage{english} for 90 min,
more BES1-GFP was shuttled to the nucleus, and the accumulation of BES1
in the nucleus could be maintained for another 2-3 d. BRs signal
transduction activates BES1/BZR1 through releasing the inhibition of
BIN2 thus promotes dephosphorylation of BES1, so we suppose BRs mediate
thermomemory through promoting the accumulation of activated BES1 during
the memory phase. We detected the abundance of de-phosphorylated BES1 in
seedlings grown on solid plate medium with or without BL application
during the memory phase. As shown in Fig. 3B, BL application strengthens
the accumulation of dephosphorylated BES1, whereas BRZ (brassinazole, a
specific inhibitor of BR synthesis) treatment impairs dephosphorylation.
These results indicate that heat priming promotes the accumulation of
dephosphorylated BES1 in the nucleus, BRs signal transduction further
enhances this process.

\textbf{2.5 The abundance of dephosphorylated BES1 is responsible for
BR-enhanced thermomemory}

To elucidate the critical function of BES1 accumulated during thermal
memory phase for BR signal, we detected BES1 protein by immunoblot
analysis in \emph{det2} and \emph{BES1-RNAi} seedlings at 48 h of memory
phase. As shown in Supplementary Fig. 2, the abundance of
dephosphorylated BES1 was higher in \emph{det2} , whereas it was almost
undetectable in \emph{BES1-RNAi} . This finding provides further
evidence for BES1 involved in the regulation of thermomemory, increased
BES1 level in \emph{det2} could explain the enhanced thermotolerance
in\emph{det2} mutant (Supplementary Fig. 1) and a lack of BES1 could be
responsible for the thermomemory function-loss in \emph{BES1-RNAi} (Fig.
2).

We next subjected 5-day-old \emph{bes1-D} mutant and Col-0 seedlings to
our priming and triggering stimulus protocol, since the amino acid
mutation in \emph{bes1-D} results in an increase in the total amount of
BES1 protein. Not surprisingly, dephosphorylated BES1 in \emph{bes1-D}is
comparably more abundant than that of Col-0 during the whole memory
phase. Consistent with this observation, \emph{bes1-D} seedlings
revealed better thermotolerance than Col-0 in both unprimed and primed
conditions, as the survival rate was comparably higher than Col-0 (Fig.
4C), accompanied with lower electrolyte leakage in \emph{bes1-D} (Fig.
4D), underscoring the contribution of bio activated BES1 to elevating
the thermolerance and enhance thermomemory.

\textbf{2.6 BES1 is required for sustained activation of HS-associated
memory genes}

To analyze whether BES1 mediates thermomemery through affecting the
expression of HS-associated memory genes, we examined the transcript
level of memory genes in thermomemory assay at the indicating time
point. The HS memory\selectlanguage{english}-associated genes \emph{APX2} (Stief et al.,
2014a),\emph{HSFA3} (Friedrich et al., 2021), \emph{HSA32} (Wu et al.,
2013),\emph{HSP21} (Sedaghatmehr, Mueller-Roeber, \& Balazadeh, 2016)
were analyzed (include both type I and type \selectlanguage{english}II HS-associated memory gene)
and a non\selectlanguage{english}-memory but HS\selectlanguage{english}-inducible gene \emph{HSP101} (McLoughlin et al.,
2019) was set as contrast. These genes did not show dramatic differences
among Col-0, \emph{bes1-D} and \emph{BES1-RNAi} prior priming (Fig. 5A),
indicating that BES1 has a weak regulatory effect on these genes under
normal growth conditions. The expression of \emph{APX2} ,
\emph{HSFA3}and \emph{HSP21} was induced several hundred-fold (Fig. 5B,
D and E) and\emph{HSA32} for several dozen fold (Fig. 5C) early in the
memory phase and sustained relative high transcript level 3 d after
priming in wild-type and \emph{bes1-D} . These genes were less induced
in\emph{BES1-RNAi} mutant early in the memory phase and later decreased
faster than Col-0 and \emph{bes1-D} . With the deepening of memory
phase, the expression of these memory genes started to decrease between
12 and 24 h and declined dramatically 48 h after priming in
\emph{BES1-RNAi}when it was still relatively high in Col-0 and
\emph{bes1-D} . For the non\selectlanguage{english}-memory gene \emph{HSP101} , no significant
difference was found among Col-0, \emph{bes1-D} and \emph{BES1-RNAi}
during the memory phase (Fig. 5F). These results indicate that BES1 is
required to a great extent for sustained activation of HS
memory\selectlanguage{english}-associated genes.

\textbf{2.7 BES1 directly targets \emph{APX2} and \emph{HSFA3} in the
memory phase}

Since BES1 is critical for the sustained activation of HS-associated
memory genes, we further examine whether BES1 directly target these
genes for activation. Yeast one-hybrid (Y1H) assays indicate that BES1
directly activates the expression of \emph{APX2} and \emph{HSFA3} (Fig.
6A)\emph{.} We next conducted an electrophoretic mobility shift assay
(EMSA) towards the fragment of \emph{APX2} and \emph{HSFA3} promoter
containing an E-box, the potential BES1 protein binding site. As
expected, the EMSA assay showed that MBP-BES1 proteins could bind to
APX2 and HSFA3 probes, but MBP did not (Fig. 6B, C). We further
confirmed that BES1 directly binds to the promoter of \emph{APX2}
and\emph{HSFA3} in vivo using a chromatin immunoprecipitation
(ChIP)-qPCR assay. Obvious enrichment was found in the promoter of
\emph{APX2} and\emph{HSFA3} (Fig. 6D). These results suggest that
\emph{APX2} and\emph{HSFA3} are transcriptional targets of BES1 proteins
during the thermomemory process.

\textbf{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\selectlanguage{english}-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 \emph{HSA32} and \emph{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 \emph{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 \emph{bes1-D} and
heat-intolerant \emph{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 \emph{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 \emph{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.

\textbf{4 MATERIALS AND METHODS}

\textbf{4.1 Plant materials}

\emph{DET2-OX} , \emph{bes1-D} and \emph{det2} used in this study were
previously reported (Zhang et al., 2021) and \emph{BES1-RNAi} (Li et
al., 2020). \emph{35S::GFP, 35S::BES1-GFP} and \emph{proBES1::GUS} were
introduced into Col-0 by \emph{Agrobacterium tumefaciens} -mediated
floral transformation. All the plants were in the Col-0 ecotype.

\textbf{4.2 Thermotolerance assay}

Seeds were sown on 1/2 MS plates with 0.70\% agar and stratified at 4\selectlanguage{english}
for 3 d. Then the plates were moved to the long day growth chamber (16-h
light and 8-h dark, 120
\selectlanguage{greek}μ\selectlanguage{english}mol.m\textsuperscript{--2}\selectlanguage{ngerman}·s\textsuperscript{--1}, LED fluorescent
tube) at 22\selectlanguage{english}. Priming treatment was performed as described previously
with subtle modifications(Brzezinka et al., 2019). Briefly, Seedlings
grown in a chamber for 5 d were subjected to priming protocol: 37degC for
60 min, followed by 120 min at 22degC, then 45 min at 44degC followed 2 d at
22\selectlanguage{english}, then seedlings were subjected to 44degC for 90 min in a
constant-temperature incubator for HS triggering. For the unprimed
group, triggering was applied to seedlings grown at 22degC for 7 d. After
triggering, seedlings were transferred to normal growth conditions until
analysis.

\textbf{4.3 Electrolyte leakage assay}

To measure electrolyte leakage caused by high temperature, seedlings
were harvested before or after heat triggering and rinsed twice with
deionized water, placed into 10-mL tubes containing 8 mL of deionized
water. Samples were placed at room temperature for 24 h and measured the
conductivity using an Electro-conductivity meter (DDSJ-308A, Lei-ci,
Shanghai, China), recorded as R1. Samples were then boiled at 100degC for
15 min and shaken at room temperature for another 1 h before the
electrical conductivity was measured again, recorded as R2. The
electrolyte leakage was calculated as: R1/R2. Three biological
replicates were performed for per simple.

\textbf{4.4 RNA isolation and RT-qPCR analysis}

Total RNA was isolated from seedlings using an RNeasy Mini Kit (Qiagen)
according to the manufacturer's instructions, and the extracted RNA was
treated with DNase to remove potential genomic DNA contamination.
First-strand cDNA was synthesized using M-MLV Reverse Transcriptase
(Invitrogen) with approximately 2 \selectlanguage{greek}μ\selectlanguage{english}g RNA. RT-qPCR reactions were
conducted on a 20 \selectlanguage{greek}μ\selectlanguage{english}l total reaction volume containing 10 \selectlanguage{greek}μ\selectlanguage{english}l of SYBR
Green Real-Time PCR Master Mix (Transgene, Beijing, China) and 2 \selectlanguage{greek}μ\selectlanguage{english}l
cDNA. The CFX96 Real-Time PCR Detection system (Bio-Rad, Hercules, CA,
USA) was used for qRT-PCR analysis. The relative expression levels of
genes were normalized to the reference gene \emph{ACTIN2} (AT3G18780).
For each sample, three biological replicates were performed and each
biological replicate contained three technical replicates.

\textbf{4.5 GUS staining}

The promoter of bes1 was cloned into PBI121 vector and introduced into
Col-0 by Agrobacterium tumefaciens-mediated floral transformation to
generate \emph{proBES1::GUS} transgenic plant.
Five-day-old\emph{proBES1::GUS} was grown in long day growth chamber
(16-h light and 8-h dark, 120
\selectlanguage{greek}μ\selectlanguage{english}mol.m\textsuperscript{--2}\selectlanguage{ngerman}·s\textsuperscript{--1}, LED fluorescent
tube) at 22\selectlanguage{english}, then seedlings were subjected to priming assays and
sampled at different time points. Seedlings were stained at 37\selectlanguage{english} in the
dark for 2.5 h using GUS Staining Solution (Sangon Biotech, Cat no.
A610085-0025, Shanghai). After staining, the seedlings were washed with
75\% ethanol and the image was captured using a fluorescence microscope
(Leica, Wetzlar, Germany).

\textbf{4.6 Protein extraction and western blot analysis}

For protein extraction, about 50 mg of tissue samples were placed in a 2
ml plastic centrifugal pipe and grinded into fine powder in liquid
nitrogen, then added 150 \selectlanguage{greek}μ\selectlanguage{english}L protein loading buffer (5\% sodium dodecyl
sulphate, 0.25\% bromophenol blue, 2.5\% \selectlanguage{greek}β-\selectlanguage{english}mercaptoethanol, 25\%
glycerol) to the powder and boiled at 100\selectlanguage{ngerman}°C for 10 min followed by
centrifugation at 13000 g for 10 min at 4°C. Clear supernatants was used
as total protein extract. The extracted protein solution was
electrophoresed on 10\% sodium dodecyl sulphate (SDS)-gel and
transferred to the nitrocellulose membrane to incubate with antibody.
For BES1 protein accumulation analysis, Proteins from different samples
were detected by anti-BES1 (Yin's lab) at a dilution of 1:5000.

\textbf{4.7 Subcellular localization}

The whole-genome sequence of BES1 was amplified and cloned into the
pCAMBIA1302-GFP vector to generate proBES1::BES1-GFP. The vector was
transformed into \emph{Agrobacterium tumefaciens} strain (GV3101) and
transient transformation into \emph{N. benthamiana} leaves. After 48 h,
the leaves were subjected to heat priming treatment, and the GFP
fluorescence was detected at different time points using a fluorescence
microscope (Leica, Wetzlar, Germany).

\textbf{4.8 Electrophoretic mobility shifts assay (EMSA)}

EMSA were performed as previously described (Lei et al., 2020).
Constructs for MBP-BES1 (Li et al., 2020) were transformed
into\emph{Escherichia coli} strain BL21 (DE3) and recombinant proteins
were induced by IPTG. MBP-BES1 was purified using Amylose resin (Cat no.
E8021; New England Biolabs, Ipswich, MA, USA). Purified MBP-BES1 was
incubated with the probe listed in supplemental table, the mixture was
reaction in binding buffer (25 mM HEPES-KOH, pH 8.0, 50 mM KCl, 1 mM
dithiothreitol and 10\% glycerol) for 30 min and shift in polyacrylamide
gel. The shifted probe and protein were tested with EMSA Kit (Thermo
Fisher).

\textbf{4.9 Yeast one-hybrid (Y1H) assays}

The full-length CDS of \emph{BES1} was cloned into the PGAD-T7 vector
and the promoter of \emph{APX2, HSFA3, HSA32, HSP21} were cloned into
pHIS2 vector. The constructs and empty vector controls were transformed
into yeast strain Y187 and grown on SD/-Leu-Trp-His dropout plates for
interaction analysis.

\textbf{4.10 Chromatin immunoprecipitation (ChIP)-qPCR}

ChIP was performed as previously described (Zhang et al., 2021). In
brief, \emph{Arabidopsis} seedlings harvest at 12 h after priming were
collected and crosslinked with formaldehyde and used for chromatin
isolation. After ultrasonic crushing, the complex of chromatins and
GFP-BES1 or GFP were immunoprecipitated by anti-GFP antibody and
harvested by protein A beads. Then the purified DNA was quantified by
RT-qPCR using specific primer listed in supplemental table.

\textbf{ACCESSION NUMBER}

\emph{BES1} (AT1G193500) \emph{DET2} (AT2G38050) \emph{HSA32}
(AT4G21320)\emph{HSP21} (AT4G27670) \emph{HSP22} (AT4G10250)
\emph{HSP101} (AT1G74310)\emph{APX2} (AT3G09640) \emph{HSFA3}
(AT5G03720)

\emph{ACTIN2} (AT3G18780)

\textbf{ACKNOWLEDGMENTS}

We thank Yanhai Yin (Iowa State University) for providing anti-BES1.
This work was supported by grants from the National Natural Science
Foundation of China (grant no. 31970263 to H.H.L; 32070213 to D.W.Z);
the Institutional Research Fund of Sichuan University (2020SCUNL212);
and the Fundamental Research Funds for the Central Universities
(SCU2020D003).

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\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1. BES1 is required for BR-enhanced thermotolerance and
memory response} .

(A). Phenotype of Col-0 and \emph{BES1-RNAi} under unprimed and primed
conditions with or without BL application. Control: seedlings prior
triggering; unprimed: seedlings were subjected to heat stress triggering
and extended recovery; primed: seedlings were subjected to heat priming
and stress triggering then extended recovery. The heat stress priming
and triggering protocol was represented as schematic on the 554 right.
Scale bar, 15 mm.

(B). Percentage of seedlings grown on 1/2 MS medium with or without 10
nM BL in different phenotype classes indicated in A. Phenotype classes
were counted reference to(Sedaghatmehr et al., 2016), in short, `green'
represents the entire plant was almost green, `weak' represents plants
that were largely pale. Data shown are averages of three independent
biological replicates each containing at least 100 individuals per
experience group.

(C). Electrolyte leakage assay of Col-0 and \emph{BES1-RNAi} grown on
1/2 MS with or without 10 nM BL in thermotolerance assay indicated in A.
Values are mean \selectlanguage{ngerman}± SEM of three biological replicates. Different letters
indicate statistically significant differences (P\textless{} 0.05)
compared with the control group (unprimed) exposed to the same treatment
time as determined by two-way ANOVA followed by Fisher's LSD test.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2. De novo synthesis and weakened degradation contribute
to the accumulation of BES1 during the memory phase.}

(A). Schematic representation of the thermomemory experimental set-up
in\emph{Arabidopsis} .

(B). Quantitative reverse transcription--PCR revealed enhanced
transcript of BES1 in the memory phase, transcripts of BES1 were
normalized to \emph{ACTIN2} . Values are mean \selectlanguage{ngerman}± SEM of three biological
replicates. Different letters indicate statistically significant
differences (P\textless{} 0.05) compared with the control group
(unprimed) exposed to the same treatment time as determined by two-way
ANOVA followed by Fisher's LSD test.

(C). BES1 promoter-GUS expression pattern in
transgenic\emph{Arabidopsis} plants during thermomemory assays.

(D-G). Immunoblot analysis of BES1 protein in Col-0 seedlings during the
thermomemory experiment. Seedlings were treated with DMSO (C), 100 \selectlanguage{greek}μ\selectlanguage{english}M
CHX (D), 50 \selectlanguage{greek}μ\selectlanguage{english}M MG132 (E), 100 \selectlanguage{greek}μ\selectlanguage{english}M CHX+50 \selectlanguage{greek}μ\selectlanguage{english}M MG132 (F) after priming,
samples were collected at the indicated time points. Quantified relative
band intensity of BES1 protein was listed below using Image J. \selectlanguage{greek}β-\selectlanguage{english}ACTIN
in total protein extracts was used as a loading control.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3. Heat priming and BRs promote the accumulation of
dephosphorylate BES1 during thermomemory phase.}

(A). BES1-GFP is accumulated to the nucleus during memory phase in leaf
epidermal cells of \emph{N. benthamiana} transformed with
proBES1::BES1-GFP vector.

(B). Immuno-blot analysis of dephosphorylated BES1 abundance using
\selectlanguage{greek}α-\selectlanguage{english}BES1 during thermomemory experiment in Col-0 plants grown on different
medium. Quantified relative band intensity of dephosphorylated BES1
protein was listed below using Image J. \selectlanguage{greek}β-\selectlanguage{english}ACTIN in total protein
extracts was used as a loading control.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4. Increased BES1 accumulation in \emph{bes1-D} enhances
thermal memory.}

(A). Immuno-blot analysis of BES1 abundance using \selectlanguage{greek}α-\selectlanguage{english}BES1 in col-0
and\emph{bes1-D} during memory phase. Quantified relative band intensity
of dephosphorylated BES1 protein was listed below using Image J. \selectlanguage{greek}β-\selectlanguage{english}ACTIN
in total protein extracts was used as a loading control.

(B). Phenotype of Col-0 and \emph{bes1-D} under unprimed and primed
conditions after recovery. Control: seedlings prior triggering;
unprimed: seedlings were subjected to heat stress triggering and
extended recovery; primed: seedlings were subjected to heat priming and
stress triggering then extended recovery. Scale bar, 15 mm.

(C). Percentage of seedlings in different phenotype classes indicated in
A. Phenotype classes were counted reference to (Sedaghatmehr et al.,
2016), in short, `green' represents the entire plant was almost green,
`weak' represents plants that were largely pale. Data shown are averages
of three independent biological replicates each containing at least 100
individuals per experience group.

(D). Electrolyte leakage assay of seedlings indicated in A. Values are
mean \selectlanguage{ngerman}± SEM of three biological replicates. Different letters indicate
statistically significant differences (P\textless{} 0.05) compared with
the control group (unprimed) exposed to the same treatment time as
determined by two-way ANOVA followed by Fisher's LSD test.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5. Sustained induction of several memory genes after
priming is impaired in \emph{BES1-RNAi} but enhanced in \emph{bes1-D}.}

(A). Transcript levels of memory-associated genes (\emph{APX2, HSA32,
HSP21, HSFA3} ) and non-memory gene \emph{HSP101} prior priming in
Col-0,\emph{bes1-D} and \emph{BES1-RNAi} . Values are mean \selectlanguage{ngerman}± SEM of
three biological replicates. Asterisks indicate statistically
significant differences (*P\textless{} 0.05, **P\textless{}0.01, ***P
\textless{}0.001) compared with the wild-type determined student's t
test, ns indicates no significance.

(B-F). Transcript levels of HS memory\selectlanguage{english}-associated genes (\emph{APX2,
HSA32, HSP21, HSFA3} ) and non\selectlanguage{english}-memory gene \emph{HSP101} after priming
in Col-0, \emph{bes1-D} and \emph{BES1-RNAi} . Transcript levels
determined by qRT\selectlanguage{english}-PCR were normalized to the reference gene
\emph{ACTIN2} and the respective non-HS control was harvested at the
same time point. Values are mean +- SEM of three biological replicates.
Asterisks indicate statistically significant differences (*P\textless{}
0.05, **P\textless{}0.01, ***P \textless{}0.001) compared with the
wild-type when seedlings exposed to the same treatment time as
determined by student's t test, ns indicates no significance.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6\emph{. APX2} and \emph{HSFA3} are two direct binding
targets of BES1.}

(A). Activation of BES1 against promoters of HS-associated memory genes
was tested in Y1H experiment. Growth of yeast transformants carrying the
HIS3 reporter gene under the control of each promoter region was
examined in the medium lacking leucine (L), Tryptophane (W) and
histidine (H) with or without 3-AT.

(B). Electrophoretic mobility shift assay. MBP-BES1 protein binds
specifically to the BES1 binding site within the APX2 promoter. 2+
represents twice the amount of protein, m represents the probe mutated
BES1 binding site.

(C). Electrophoretic mobility shift assay. MBP-BES1 protein binds
specifically to the BES1 binding site within the HSFA3 promoter. 2+
represents twice the amount of protein, m represents the probe mutated
BES1 binding site.

(D). ChIP-qPCR assays indicate enrichment of \emph{APX2} and\emph{HSFA3}
promoter in the immunoprecipitation products of BES1 protein. The ChIP
assays were performed with chromatin prepared from\emph{35S::GFP} and
\emph{35S::BES1-GFP} plants harvest at 12 h after heat priming, using an
anti-GFP antibody. The precipitated DNA was analyzed by qPCR using the
primer pairs of HS-associated memory genes. P-ACTIN2 was used as the
negative control. Values are means \selectlanguage{ngerman}± SD of three independent biological
replicates.

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