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BZR1 Mediates Brassinosteroid-Induced Autophagy andNitrogen
Starvation in Tomato1
Yu Wang,a,b,2 Jia-Jian Cao,a,2 Kai-Xin Wang,a Xiao-Jian Xia,a
Kai Shi,a Yan-Hong Zhou,a Jing-Quan Yu,a,c
and Jie Zhoua,3,4
aDepartment of Horticulture/Zhejiang Provincial Key Laboratory
of Horticultural Plant Integrative Biology,Zhejiang University,
Hangzhou 310058, ChinabKey Laboratory of Southern Vegetable Crop
Genetic Improvement, Ministry of Agriculture, College
ofHorticulture, Nanjing Agricultural University, Nanjing 210095,
ChinacKey Laboratory of Horticultural Plants Growth, Development,
and Quality Improvement, AgriculturalMinistry of China, Hangzhou
310058, China
ORCID IDs: 0000-0001-5351-1910 (K.S.); 0000-0002-7860-8847
(Y.-H.Z.); 0000-0002-7626-1165 (J.-Q.Y.); 0000-0002-8797-7214
(J.Z.).
Autophagy, an innate cellular destructive mechanism, plays
crucial roles in plant development and responses to
stress.Autophagy is known to be stimulated or suppressed by
multiple molecular processes, but the role of phytohormone
signaling inautophagy is unclear. Here, we demonstrate that the
transcripts of autophagy-related genes (ATGs) and the formation
ofautophagosomes are triggered by enhanced levels of
brassinosteroid (BR). Furthermore, the BR-activated transcription
factorbrassinazole-resistant1 (BZR1), a positive regulator of the
BR signaling pathway, is involved in BR-induced autophagy.Treatment
with BR enhanced the formation of autophagosomes and the
transcripts of ATGs in BZR1-overexpressing plants,while the effects
of BR were compromised in BZR1-silenced plants. Yeast one-hybrid
analysis and chromatinimmunoprecipitation coupled with quantitative
polymerase chain reaction revealed that BZR1 bound to the promoters
ofATG2 and ATG6. The BR-induced formation of autophagosomes
decreased in ATG2- and ATG6-silenced plants. Moreover,exogenous
application of BR enhanced chlorophyll content and autophagosome
formation and decreased the accumulation ofubiquitinated proteins
under nitrogen starvation. Leaf chlorosis and chlorophyll
degradation were exacerbated in BZR1-silencedplants and the BR
biosynthetic mutant d^im but were alleviated in BZR1- and
BZR1-1D-overexpressing plants under nitrogenstarvation. Meanwhile,
nitrogen starvation-induced expression of ATGs and autophagosome
formation were compromised inboth BZR1-silenced and d^im plants but
were increased in BZR1- and BZR1-1D-overexpressing plants. Taken
together, ourresults suggest that BZR1-dependent BR signaling
up-regulates the expression of ATGs and autophagosome
formation,which plays a critical role in the plant response to
nitrogen starvation in tomato (Solanum lycopersicum).
Autophagy is an evolutionarily conserved andhighly regulated
self-degradation process that recyclescellular nutrients or breaks
down damaged compo-nents for the optimization of plant growth,
develop-ment, and stress responses (Qin et al., 2007; Liu
andBassham, 2012; Zhou et al., 2013). In plant cells,
autophagy is initiated by the formation of double-membrane
vesicles termed autophagosomes, whichengulf the intracellular
material and subsequently de-liver them to vacuoles for degradation
under stress,such as nutrient starvation (Bassham et al., 2006;
Hofiuset al., 2009; Araújo et al., 2011). The deficiency of
theautophagic genes is associated with susceptibility tonitrogen
(N) and carbon starvation and suppressedsenescence-induced
breakdown of mitochondria-residentproteins in plants (Zhou et al.,
2013; Li et al., 2014).Autophagy-deficient mutants had increased
the levelsof insoluble proteins that are highly ubiquitinatedunder
heat and oxidative stresses in Arabidopsis(Arabidopsis thaliana)
and tomato (Solanum lycopersicum;Zhou et al., 2014b). Furthermore,
autophagy has beenshown to interact with defense signaling pathways
andinduce plant resistance against pathogens (Liu et al.,2005; Lai
et al., 2011).In recent years, the identification of autophagy-
related genes (ATGs) has firmly established the occur-rence of
autophagosome formation. However, ourunderstanding of the
mechanisms and signaling cas-cades that regulate autophagy in
plants remains
1This work was supported by the National Key Research and
De-velopment Program of China (2018YFD1000800) and the
NationalNatural Science Foundation of China (31872089, 31430076,
and31801902).
2These authors contributed equally to the article.3Author for
contact: [email protected] author.The author responsible for
distribution of materials integral to the
findings presented in this article in accordance with the policy
de-scribed in the Instructions for Authors (www.plantphysiol.org)
is: JieZhou ([email protected]).
Y.W. and J.Z. planned and designed the research; Y.W. and
J.-J.C.performed experiments and analyzed data; K.-X.W., X.-J.X.,
K.S.,Y.-H.Z., and J.-Q.Y. performed molecular cloning and
analyzeddata; Y.W. and J.Z. wrote the article; all authors
reviewed, revised,and approved the article.
www.plantphysiol.org/cgi/doi/10.1104/pp.18.01028
Plant Physiology�, February 2019, Vol. 179, pp. 671–685,
www.plantphysiol.org � 2019 American Society of Plant Biologists.
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http://orcid.org/0000-0001-5351-1910http://orcid.org/0000-0001-5351-1910http://orcid.org/0000-0002-7860-8847http://orcid.org/0000-0002-7860-8847http://orcid.org/0000-0002-7626-1165http://orcid.org/0000-0002-7626-1165http://orcid.org/0000-0002-8797-7214http://orcid.org/0000-0002-8797-7214http://orcid.org/0000-0001-5351-1910http://orcid.org/0000-0002-7860-8847http://orcid.org/0000-0002-7626-1165http://orcid.org/0000-0002-8797-7214http://crossmark.crossref.org/dialog/?doi=10.1104/pp.18.01028&domain=pdf&date_stamp=2019-01-22mailto:[email protected]://www.plantphysiol.orgmailto:[email protected]://www.plantphysiol.org/cgi/doi/10.1104/pp.18.01028
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incomplete (Thompson and Vierstra, 2005; Michaeliet al., 2016).
Target of rapamycin (TOR), a PtdIns3K-related kinase that can
phosphorylate Atg13 and in-hibit the formation of the Atg1/13
complex, has beenidentified as a key regulator of autophagy in
plants (Liuand Bassham, 2010, 2012; Pérez-Pérez et al., 2010).
TORRNA interference Arabidopsis plants showed consti-tutive
activation of autophagy (Liu and Bassham, 2010).NBR1 (a neighbor of
the BRCA1 gene), the first identi-fied cargo receptor for selective
autophagy, interactswith both Atg8 and ubiquitin and mediates the
en-capsulation of ubiquitinated protein aggregates inautophagosomes
(Svenning et al., 2011; Zientara-Rytteret al., 2011; Zhou et al.,
2013). Arabidopsis nbr1mutantsare selectively hypersensitive to
specific abiotic stresses,including heat, oxidative stress, and
osmotic stress, butno difference is observed between nbr1 and
wild-typeplants in response to age- and darkness-induced
se-nescence or necrotrophic pathogens (Zhou et al., 2013).In
addition, heat shock transcription factor A1a wasreported to bind
to the promoters of ATGs and inducetheir expression, resulting in
autophagosome formationand, eventually, increased drought tolerance
in tomato(Wang et al., 2015). Notably, reactive oxygen speciesalso
are involved in the induction of autophagy inplants (Xiong et al.,
2007; Chen et al., 2015). Glyceral-dehyde-3-phosphate
dehydrogenase, an important en-zyme in the glycolytic pathway, is
thought to transducehydrogen peroxide signal and can
antagonisticallyregulate autophagy in plants (Guo et al., 2012;
Henryet al., 2015). The cytoplastic isoforms of
glyceraldehyde-3-phosphate dehydrogenase (GAPCs) interactwithAtg3to
inhibit its activity in Nicotiana benthamiana plants.Meanwhile,
reactive oxygen species weaken the interac-tion betweenGAPCs
andAtg3 but enhance theAtg3-Atg8interaction and autophagic
responses (Han et al., 2015).
In animal development and disease resistance, fineregulation of
autophagy relies on different hormonesignals (Sinha et al., 2012;
Tian et al., 2013; Chen et al.,2014). However, the regulation of
autophagy in plantsby phytohormones and the underlying
mechanismsare largely unknown. In Arabidopsis, drought toler-ance
is induced through ring finger E3 ligase-mediatedubiquitination
downstream of stress-responsive absci-sic acid signaling (Zhang et
al., 2007). Autophagosomescan engulf ubiquitinated proteins and
then transferthem to vacuoles for degradation by hydrolytic
en-zymes. Ethylene treatment increased the expression ofATG8
homologs in petals of petunia (Petunia hybrida),while pollination
induced the formation of autopha-gosomes accompanied by increasing
ethylene produc-tion (Shibuya et al., 2013). In addition, autophagy
wasinduced by an agonist of salicylic acid,
benzo-(1,2,3)-thiadiazole-7-carbothioic acid (Yoshimoto et al.,
2009).Taken together, these observations suggest that
phy-tohormones may be involved in the activation of au-tophagy.
However, the mechanism of autophagicinduction by phytohormone
signaling remains unclear.
Brassinosteroids (BRs) are phytohormones thatplay critical roles
in plant growth, development, and
responses to stress (Kim and Wang, 2010; Sun et al.,2010;
Albrecht et al., 2012). BRs are first perceived bythe receptor
brassinosteroid-insensitive1 (BRI1); this isfollowed by
autophosphorylation and activation ofthe BRI1 intracellular kinase
domain (Kinoshita et al.,2005; Wang et al., 2014). This activated
BRI1 triggersa downstream phosphorylation and dephosphoryla-tion
signal transduction cascade that results in thenuclear localization
of dephosphorylated brassinazole-resistant1 (BZR1) and
BRI1-EMS-suppressor1 tran-scription factors, which bind to the
E-boxes (CANNTG)and/or to the BR-response element (CGTGT/CG) ofthe
promoters of target genes (He et al., 2005; Kim andWang, 2010; Sun
et al., 2010; Jiang et al., 2015). A recentstudy demonstrated that
TOR signaling mediates au-tophagy to degrade BZR1, which is
involved in theregulation of the BR signaling pathway (Zhang et
al.,2016). Although BR is a type of multifunctional hor-mone, its
roles in regulating autophagic degradationare unclear. Genome-wide
microarray experiments in-dicated that the expression of
approximately 20% ofgenes is regulated by BR in Arabidopsis (Guo et
al.,2013). A number of studies also have demonstratedthat BRs
activate multiple signaling pathways to in-duce plant tolerance
against various environmentalstresses that have similar roles in
autophagy duringplant stress responses (Choudhary et al., 2012;
Lozano-Durán et al., 2013; Zhou et al., 2014a). However,
func-tional evidence regarding the involvement of BRsignaling in
autophagy pathways is absent.
Autophagy plays a vital role in N starvation inplants. The
atgmutants, such as atg4, atg5, and atg7, aremore sensitive to
nutrient-limited conditions than wild-type plants (Yoshimoto et
al., 2004; Phillips et al., 2008).atg10-1 plants show dysfunctional
accumulation ofautophagic bodies in vacuoles under
nutrient-deficientconditions and increased sensitivity to N
starvation,leading to elevated leaf senescence and programmedcell
death (Phillips et al., 2008). Furthermore, Atg11interacts with the
Atg1/13 protein kinase complex topromote the starvation-induced
phosphorylation ofAtg1 and the turnover of Atg1 and Atg13, which
pro-vides a dynamic mechanism that tightly connects au-tophagy to
the nutritional status of plants (Li et al.,2014). The atg12
mutants show altered autophagictransport and N remobilization,
leading to the inhibi-tion of seedling growth and plantmaturation,
increasedleaf senescence, and arrested ear development underN
starvation in maize (Zea mays; Li et al., 2015). In thisstudy, we
found that BR induced the BZR1-mediatedformation of autophagy in
tomato. Silencing of BZR1attenuated the transcript levels of ATGs
and the for-mation of autophagosomes, while these parameterswere
enhanced in BZR1-overexpressing plants after BRtreatment. Silencing
of ATG2 and ATG6 compromisedthe formation of BR-induced
autophagosomes. In ad-dition, silencing of BZR1 compromised the
resistance toN starvation, but this characteristic was enhanced
inBZR1-overexpressing and exogenous BR-treated plants.This report
demonstrates that the BR signaling pathway
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positively regulates autophagy in tomato plants throughBZR1
activation, while BR signal-induced autophagyplays a vital role in
response to N starvation.
RESULTS
BR Induces Autophagy and ATG Expression
To investigate whether BR can induce autophagy intomato plants,
we first analyzed the transcript levels of12 tomato ATGs after
treatment with exogenous bras-sinolide (BL; the most biologically
active member of theBR family). As shown in Figure 1A, the
transcript levelsof ATGs increased slightly as early as 3 h and
reachedthe highest levels at 12 h after BL treatment. However,the
expression levels of most of the ATGs decreasedto control levels
after BL application for 24 h (Fig. 1A).
To gain further insight into the role of BL in activat-ing
autophagy, we used the fluorescent dye mono-dansylcadaverine (MDC)
to detect autophagic activityin wild-type plants after BL
treatment. In the controlplants, only a few MDC-stained
autophagosomes wereobserved (Fig. 1, B and C). In contrast,
numerousMDC-stained autophagosomes were detected after BL
treat-ment (Fig. 1, B and C). Then, we used transmissionelectron
microscopy (TEM), which is a classic methodfor detecting autophagy
in most organisms, includ-ing plants, to confirm the MDC results.
Consistent withthe results of MDC staining, TEM showed only a
fewclassic autophagosomes with double membranes inthe cytoplasm and
single-membrane autophagic bod-ies in the vacuoles of the control
plants (Fig. 1, D andE). Nonetheless, the numbers of
autophagosomesand autophagic bodies increased by 9.3-fold at 12
hafter BL treatment (Fig. 1, D and E). Notably, during
Figure 1. Effects of BRs on the induction of autophagy in tomato
leaves. A, Heat map showing the expression profiles of ATGsafter BL
treatment at different time points. Six-week-old tomatowild-type cv
Condine Red plantswere treatedwith 500 nM BL, andtotal RNAwas
extracted from leaf samples at the indicated times. Transcript
levels were determined using real-time quantitativePCR (RT-qPCR),
and cluster analysis was performed usingMeV version 4.9. The color
bar at the top shows the levels of expression;3 h, 6 h, 12 h, and
24 h indicate the time course: 3, 6, 12, and 24 h, respectively,
after BL treatment. B, MDC-stained auto-phagosomes in the leaves of
wild-type plants. Six-week-old plants were treated with 500 nM BL,
and the leaves were stained withMDC and visualized at 12 h by
confocal microscopy. MDC-stained autophagosomes are in green. Bars
= 20 mm. C, Relativeautophagic activity normalized to the activity
of the wild-type control plants in B. The number of MDC-stained
autophagosomesper imagewas quantified to calculate the autophagic
activity relative towild-type control plants, whichwas set to
1.More than 20images for each treatment were used for the
quantification. D, Representative TEM images of autophagic
structures in the me-sophyll cells of wild-type plants.
Six-week-old plantswere treatedwith 500 nM BL, and themesophyll
cells were visualized at 12 hby TEM. Autophagic bodies are marked
by red arrows. S, Starch; V, vacuole. Bars = 1 mm. E, Relative
autophagic activity nor-malized to the activity of the wild-type
control plants in D. The number of autophagic bodies per image was
quantified to cal-culate the autophagic activity relative to
wild-type control plants, which was set to 1. More than 20 images
were used to quantifyautophagic structures. F, Atg8 protein levels
in the leaves of wild-type plants. The nonlipidated and lipidated
forms of Atg8 areindicated by Atg8 and Atg8-PE, respectively. Actin
was used as a loading control for the western-blot analysis. The
results in C andE represent means6 SE. Means with the same letter
did not differ significantly at P, 0.05 according to Duncan’s
multiple rangetest. Three independent experiments were performed
with similar results.
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autophagosome formation, the C terminus of Atg8 iscleaved by
Atg4 and conjugated to the membrane lipidphosphatidylethanolamine
(PE), which is monitored asa marker for autophagic activation
(Yoshimoto et al.,2004; Kwon et al., 2013). To verify our results,
we ana-lyzed the formation of Atg8-PE conjugates by
immu-noblotting. Significantly, the Atg8-PE bands weredetected in
the plants at 12 h after BL treatment, butthey were barely found in
the control plants (Fig. 1F).
To test the role of endogenous BR in the inductionof autophagy,
we compared the autophagic activity inthe wild type, d^im, a weak
allele mutant impaired inthe key BR biosynthetic gene DWARF (DWF),
andDWF-homozygous T2 progeny ofDWF-overexpressing(DWFOE) plants
from two independent lines (2# and3#) with accumulated high levels
of endogenous BL (Liet al., 2016). We detected low-punctate
fluorescent sig-nals in wild-type and d^im plants; however, the
numberof MDC-stained autophagosomes increased significantlyin
DWFOE-2# and DWFOE-3# plants (SupplementalFig. S1, A and B). The
Atg8-PE bands were weak inwild-type and d^im plants but were
prominent inDWFOE-2# and DWFOE-3# plants (Supplemental Fig.S1C).
Taken together, these results suggest that higherlevels of BR,
either through exogenous application orendogenous manipulation, can
induce the formation ofautophagosomes in tomato plants.
BZR1 Modulates BR-Induced Autophagy andATG Expression
The BZR1 transcription factor is a downstream com-ponent of the
BR signal transduction cascade, whichregulates thousands of nuclear
genes (Belkhadir andJaillais, 2015). To investigate the role of
BZR1 in the BR-induced formation of autophagosomes, we
comparedBZR1-silenced (TRV-BZR1) plants, which had approx-imately
20% of the BZR1 transcript level of the TRVcontrol plants
(Supplemental Fig. S2A),wild-type plants,and homozygous T2 progeny
of BZR1-overexpressing(BZR1OE) plants from two independent lines
(1# and2#). The expression levels of BZR1 in BZR1OE lines(1# and
2#) were 33.8 and 29.8 times higher than thosein wild-type plants,
respectively (Supplemental Fig.S2B). BZR1 protein was noticeably
phosphorylatedin BZR1OE plants without BL treatment, while
thedephosphorylated bands were increased after BLtreatment,
especially at 12 h (Supplemental Fig. S3A).Furthermore, the
expression levels of CPD and DWF,two key genes involved in BR
biosynthesis inBZR1OE plants, were 15.2% to 18.1% lower than
thosein wild-type plants in the absence of BL,
respectively(Supplemental Fig. S3, B and C). The expression
levelsofCPD andDWF inwild-type plants were decreased by48% and
53.6%, respectively, after BL treatment for 12h, but their
expression levels were much higher thanthose in the BZR1OE plants
(Supplemental Fig. S3,B and C). Strikingly, we observed few
MDC-stainedautophagosomes in plants grown in the absence of BL
(Fig. 2, A and B). However, silencing of BZR1 signifi-cantly
suppressed the formation of autophagosomes byBL treatment, as
evidenced by staining results at 12 h(Fig. 2, A and B). BZR1OE
plants had more MDC-stained autophagosomes than wild-type plants
afterBL treatment (Fig. 2, A and B). The TEM results wereconsistent
with the MDC staining results, as few auto-phagosomes and
autophagic bodies were observed inall the plants in the absence of
BL (Supplemental Fig. S4,A and B). However, the numbers of
autophagosomesand autophagic bodies increased from 9.5- to 10-fold
inTRV and wild-type plants after 12 h of BL
application(Supplemental Fig. S4, A and B). Meanwhile, increasesof
2.9- and 18-fold were observed in the TRV-BZR1plants and BZR1OE
plants, respectively (SupplementalFig. S4, A and B). To further
confirm our results, weused western blotting to detect the
abundance of Atg8-PE. In control plants, Atg8-PE bands were
barelydetected (Fig. 2, C and D), while Atg8-PE bands wereabundant
in BL-treated TRV and wild-type plants(Fig. 2, C and D).
Interestingly, the Atg8-PE band wasweak in TRV-BZR1 plants but was
more prominent inBZR1OE plants at 12 h after BL treatment (Fig. 2,
C andD). These results suggest the potential involvement ofBZR1 in
BR-induced autophagy.
To further investigate the role of BZR1 in BR-inducedautophagy,
we examined the transcript levels of sixATGs in TRV-BZR1 and BZR1OE
plants. In the absenceof BL, the expression levels of these ATGs in
TRV-BZR1or BZR1OE plants were not significantly different fromthose
in TRV or wild-type plants (Fig. 3). However, thetranscripts ofATG2
andATG6were induced by 1.5-foldin TRV plants after BL application
but decreased by28.1% and 26.2% in TRV-BZR1 plants compared withTRV
plants, respectively, after BL treatment (Fig. 3). Incomparison,
the expression levels of ATG5, ATG8h,ATG9, and ATG18f were not
different from those inTRV plants (Fig. 3A). Strikingly, BL
treatment resultedin a more significant increase in the expression
of ATGsin BZR1OE plants (Fig. 3B). Taken together, these re-sults
suggest that BR regulates autophagy by modu-lating BZR1-mediated BR
signaling and the expressionof ATGs.
To further validate the possible regulation of ATGsby BZR1, we
examined the promoters of ATG2 andATG6 and found that their
promoters contain E-boxes(CANNTG; Fig. 4A). We performed a yeast
one-hybridassay to determine whether BZR1 can bind directly tothe
ATG2 and ATG6 promoters in vitro. As shown inFigure 4B, yeast cells
containing only the bait vectorharboring ATG2 and ATG6 promoter
regions grew onselection medium when transformed with BZR1-AD,while
those transformed with empty pGADT7 vectordid not grow on the
selection medium. Meanwhile,yeast cells containing the bait vector
harboring mutatedATG2 and ATG6 promoter regions did not grow
onselection medium when transformed with BZR1-ADand pGADT7 vector
(Supplemental Fig. S5). These re-sults indicate that BZR1 binds
directly to the promotersof ATG2 and ATG6 in vitro. To determine
whether
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tomato BZR1 directly regulates the expression of ATG2and ATG6 in
vivo, we used chromatin immunoprecip-itation (ChIP) coupled with
qPCR assays to analyzeBZR1 protein binding to the promoters of both
geneswith or without BL treatment. Strikingly, only thepromoter
sequences of ATG2 and ATG6 were precipi-tated from the chromatin of
3-hemagglutinin (HA)-tagged BZR1OE plants with an anti-HA antibody
afterBL treatment, but this was not observed in BZR1OEplants
without BL treatment and wild-type plants(Fig. 4C). Furthermore,
IgG control antibody failed toprecipitate these gene promoter
sequences (Fig. 4C).Thus, BZR1 binds directly to the ATG2 and
ATG6promoters and may regulate their expression in re-sponse to BR
stimuli.
BR-Dependent ATGs Are Involved in the Formationof
Autophagosomes
To further investigate the role of the ATGs in BR-induced
autophagy, we silenced ATG2 and ATG6 inwild-type and BZR1OE plants,
respectively, and theexpression of these genes decreased by 60% to
85% ingene-silenced plants compared with the expressionlevels in
the TRV control plants (Supplemental Fig. S6).
BL treatment increased the number of MDC-stainedautophagosomes
by 8.2-fold in wild-type plants andby 15.8-fold in BZR1OE plants,
respectively (Fig. 5, Aand B). Furthermore, silencing of ATG2 and
ATG6compromised the BL-induced accumulation of MDC-stained
autophagosomes (Fig. 5, A and B). Moreover,BL treatment increased
the numbers of autophago-somes and autophagic bodies by 7.2- and
15.8-fold inwild-type and BZR1OE plants, respectively (Fig. 5, Cand
D). Importantly, BL failed to induce the formationof autophagosomes
and autophagic bodies in ATG2-and ATG6-silenced plants (Fig. 5, C
and D). Further-more, the abundance of Atg8-PE was not
significantlydifferent in any of the plants in the absence of
BL(Fig. 5E).While BL application increased the abundanceof Atg8-PE
in wild-type and BZR1OE plants, this in-crease was compromised in
both ATG2- and ATG6-silenced plants (Fig. 5E). These results
suggest thatBL-induced autophagosomes are largely dependent onATG2
and ATG6.
BR-Induced Autophagy Is Essential in N Starvation
Autophagy plays vital roles in nutrient recycling,which involves
the engulfment of damaged and
Figure 2. Accumulation of autophagosomes in BZR1-silenced and
BZR1OE plants after BL treatment. A, MDC-stained auto-phagosomes in
the leaves of TRV, TRV-BZR1, wild-type (WT), and BZR1OE plants.
Six-week-old plants were treated with 500 nMBL. After 12 h, the
leaves were stained with MDC and visualized by confocal microscopy.
MDC-stained autophagosomes areshown in green. Bars = 20 mm. B,
Relative autophagic activity normalized to the activity of the
TRVor wild-type control plants inA. The number of MDC-stained
autophagosomes per image was quantified to calculate the autophagic
activity relative to TRVorwild-type control plants, which was set
to 1. More than 20 images for each treatment were used for the
quantification. C and D,Atg8 protein levels in the leaves of TRV
and TRV-BZR1 or wild-type and BZR1OE plants. The nonlipidated and
lipidated forms ofAtg8 are indicated by Atg8 and Atg8-PE,
respectively. Actin was used as a loading control for the
western-blot analysis. The resultsin B representmeans6 SE.
Meanswith the same letter did not differ significantly at P, 0.05
according toDuncan’smultiple rangetest. Three independent
experiments were performed, with similar results. 1# and 2#
represent two lines of BZR1OE plants.
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unfolded proteins or cytoplasmic organelles and theirtransfer to
vacuoles for reuse (Liu and Bassham, 2012).To better understand the
role of BR-induced autoph-agy, we tested plant tolerance to N
starvation after BRtreatment. As shown in Figure 6A, no significant
dif-ference was observed under optimal growth condi-tions, while N
starvation dramatically attenuated plantgrowth, with leaves showing
chlorosis. The chlorophyllcontent in wild-type plants was decreased
by 52.2% atday 14 after N starvation but was increased after
foliarapplication of BL (Fig. 6B). As compromised formationof
autophagosomes promotes the accumulation ofubiquitinated protein
aggregates under abiotic stresses(Zhou et al., 2013; Wang et al.,
2015), we then deter-mined the changes in insoluble protein
content. WhileBL treatment did not affect the levels of insoluble
pro-tein aggregates under N-sufficient conditions (Fig. 6C),N
starvation increased the levels of insoluble protein by
103.2% in the absence of BL and by 57.5% in the pres-ence of BL
(Fig. 6C). To determine whether these in-soluble proteins were
ubiquitinated, we isolated total,soluble, and insoluble proteins
and separated them bySDS-PAGE to analyze their ubiquitination using
ananti-ubiquitin monoclonal antibody. No significantdifferences in
the total, soluble, and insoluble proteinlevels were observed in
any of the plants underN-sufficient conditions (Fig. 6D). N
starvation resultedin a reduced increase in the level of
ubiquitinated pro-teins in BL-treated plants (Fig. 6D). BL
increased thenumber of MDC-stained autophagosomes underN-starvation
conditions but not under N-sufficientconditions (Fig. 6, E and F).
In addition, there was an
Figure 3. Induction of ATGs by BL in BZR1-silenced and
BZR1OEplants. A, Expression of ATGs in TRV and TRV-BZR1 plants. B,
Ex-pression of ATGs in wild-type (WT) and BZR1OE plants.
Six-week-oldtomato plantswere treatedwith 500 nM BL, and total
RNAwas extractedfrom leaf samples harvested after 12 h. The
expression levels were de-termined using RT-qPCR. All data are
presented as means of five bio-logical replicates 6 SE. Means with
the same letter did not differsignificantly at P , 0.05 according
to Duncan’s multiple range test.Three independent experiments were
performed, with similar results.1# and 2# represent two lines of
BZR1OE plants.
Figure 4. BZR1 binds to the promoters of ATGs in vitro and in
vivo. A,E-boxes in the promoters of tomato ATG2 and ATG6. Numbering
isfrom predicted transcriptional start sites. B, Yeast one-hybrid
assayshowing the binding of BZR1-AD to ATG2 and ATG6 promoters.
Yeastcells with positiveDNA-protein interactionswere grown on Leu2
plateswith 100 ng mL21 aureobasidin A. C, Direct binding of BZR1 to
thepromoters of ATG2 and ATG6 was investigated using ChIP-qPCR
inBZR1OE plants. Six-week-old BZR1OE plants were treated with
wateror 500 nM BL, and input chromatin was isolated from leaf
samples at 12h. An anti-HA antibody was used to immunoprecipitate
the epitope-tagged BZR1-chromatin complex, while the control
reaction was per-formed in parallel with mouse IgG. Input and
ChIP-DNA samples wereanalyzed by qPCR using primers specific to the
promoters of the ATGs.The ChIP results are presented as percentages
of the input DNA. Meanswith the same letter did not differ
significantly at P, 0.05 according toDuncan’s multiple range test.
Three independent experiments wereperformed, with similar results.
WT, Wild type. 1# represents a line ofBZR1OE plant.
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increased accumulation of Atg8-PE in response to BLunder
N-starvation conditions (Fig. 6G).To examine whether BZR1-modulated
autophagy is
involved in nutrient remobilization, we used wild-typeplants,
BZR1OE plants, and BZR1-1D-overexpressing(BZR1-1DOE) plants, which
contain a BZR1 mutated inthe putative Pro-, Glu-, Ser-, and
Thr-rich domain (Tanget al., 2011), to promote the accumulation of
dephos-phorylated BZR1 (Supplemental Fig. S7) and the
BRbiosynthetic mutant d^im to detect their tolerance to N
starvation. As shown in Figure 7A, the dephosphory-lated band of
BZR1 was increased gradually in thefirst 5 d under N starvation but
began to decrease onday 7. Except for d^im plants, all other plants
exhibitedsimilar growth phenotypes under N-sufficient condi-tions
(Fig. 7B). In contrast, N starvation significantly inhibi-ted plant
growth (Fig. 7B). After 7 d of N starvation, thecotyledons of
BZR1-silenced plants began to lose theirgreen color, but TRV plants
remained green. After 14 d,the fourth fully expanded leaves of
TRV-BZR1 plants
Figure 5. Effects of BL on the autophagosome formation in ATG2-
and ATG6-silenced plants. A, MDC-stained autophagosomesin the
leaves of wild-type (WT) or BZR1OE TRV, TRV-ATG2, and TRV-ATG6
plants. Six-week-old plants were treatedwith 500 nMBL; after 12 h,
the leaves were stained with MDC and visualized by confocal
microscopy. MDC-stained autophagosomes areshown in green. Bars = 20
mm. B, Relative autophagic activity normalized to the activity of
the wild-type TRV control plants in A.The number of MDC-stained
autophagosomes per image was quantified to calculate the autophagic
activity relative to wild-typeTRV control plants, whichwas set to
1.More than 20 images for each treatmentwere used for the
quantification. C, TEM images ofautophagic structures in the
mesophyll cells of wild-type TRV, TRV-ATG2, and TRV-ATG6 or BZR1OE
TRV, TRV-ATG2, and TRV-ATG6 plants. Six-week-old plants were
treated with 500 nM BL, and the mesophyll cells were visualized
after 12 h by TEM.Autophagic bodies are indicated by red arrows.
Cp, Chloroplast; S, starch; V, vacuole. Bars = 1 mm. D, Relative
autophagicactivity normalized to the activity of the wild-type TRV
control plants in C. The number of autophagic bodies per image
wasquantified to calculate the autophagic activity relative to
wild-type TRV control plants, which was set to 1. More than 20
imageswere used to quantify autophagic structures. E, Atg8 protein
levels in the leaves of wild-type or BZR1OE TRV, TRV-ATG2,
andTRV-ATG6 plants. The nonlipidated and lipidated forms of Atg8
are indicated by Atg8 and Atg8-PE, respectively. Actin was usedas a
loading control for the western-blot analysis. The results in B and
D represent means6 SE. Means with the same letter did notdiffer
significantly at P, 0.05 according to Duncan’s multiple range test.
Three independent experiments were performed, withsimilar results.
1# represents a line of BZR1OE plant.
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showed chlorosis, but TRV plants showed less severesymptoms than
TRV-BZR1 plants (Fig. 7B). Similarly,the leaves of wild-type plants
were light green, whilethe leaves of BZR1OE and BZR1-1DOEplants
remainedgreen until day 14 after N starvation, but the leaves
ofd^im plants showed chlorosis (Fig. 7B). To confirm theobserved
phenotype, the chlorophyll contents in TRV,TRV-BZR1, wild-type,
BZR1OE, BZR1-1DOE, and d^implants were measured. The chlorophyll
content was notsignificantly different in these plants, except for
BZR1-1DOE plants, which showed higher chlorophyll
contents under N-sufficient conditions (Fig. 7C). After14 d of
N-starvation treatment, the chlorophyll contentin TRV plants
decreased by 61.6% compared with thatin TRV control plants, while
that in BZR1-silencedplants was 70.7% lower than that in TRV-BZR1
con-trol plants (Fig. 7C). Although N starvation decreasedthe
chlorophyll content in wild-type, BZR1OE, andBZR1-1DOE plants, its
content was far higher than thatin wild-type plants (Fig. 7C).
To further estimate the role of BZR1 in the formationof
autophagosomes under N starvation, we examined
Figure 6. Role of BRs in the response to N starvation in tomato
leaves. A, Exogenous BL increased tolerance to N starvation
intomato plants. Two-week-old plants were transferred to N-free
medium for 14 d. Bar = 10 cm. B, The chlorophyll content of
thefourth expanded leaveswas determined immediately on day 14 under
N starvation. FW, Fresh weight. C, Exogenous BL alleviatedthe
accumulation of insoluble proteins under N starvation. Leaf tissues
were collected on day 14 under N starvation for thepreparation of
total, soluble, and insoluble proteins as described in “Materials
and Methods.” Total proteins in the starting ho-mogenates and
insoluble proteins in the last pellets were determined to calculate
the percentages of insoluble proteins to totalproteins. D,
Exogenous BL inhibited the ubiquitination of insoluble protein
aggregates under N starvation. Proteins from thestarting
homogenates (T), first supernatant fractions (S), and last pellet
fractions (P) were subjected to SDS-PAGE and probed withan
anti-ubiquitin monoclonal antibody. E, MDC-stained autophagosomes
in BL-treated and control plants on day 7 under Nstarvation.
MDC-stained autophagosomes are shown in green. Bars = 20 mm. F,
Relative autophagic activity normalized to theactivity of the
wild-type (WT) control plants in E. The number of MDC-stained
autophagosomes per image was quantified tocalculate the autophagic
activity relative to wild-type control plants, which was set to 1.
More than 20 images for each treatmentwere used for the
quantification. G, Atg8 protein levels in the leaves of BL-treated
and control plants on day 7 under N starvation.The nonlipidated and
lipidated forms of Atg8 are indicated by Atg8 and Atg8-PE,
respectively. Actin was used as a loading controlfor the
western-blot analysis. The results in B, C, and F represent means6
SE. Meanswith the same letter did not differ significantlyat P ,
0.05 according to Duncan’s multiple range test. Three independent
experiments were performed, with similar results.
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the expression of BZR1-regulatedATG2 andATG6 genes.The
expression levels ofATG2 andATG6 in BZR1-1DOEplants were higher
than those in wild-type plants, whileboth gene expression levels in
the plants, except forBZR1-1DOE plants, did not differ from each
other underN-sufficient conditions (Supplemental Fig. S8). N
star-vation significantly increased the transcript levels ofATG2
and ATG6 in TRV and wild-type plants, but thiseffect was
compromised in BZR1-silenced and d^implants (Supplemental Fig. S8).
Importantly, the expres-sion levels of both genes in BZR1OE and
BZR1-1DOE
plants increased significantly compared with those inwild-type
plants at 7 d under N starvation (SupplementalFig. S8B).
Furthermore, we found that the number ofMDC-stained autophagosomes
in BZR1-1DOE plantswas higher than that in wild-type plants, while
no sig-nificant differencewas observed among TRV,
TRV-BZR1,wild-type, BZR1OE, and d^im plants under
N-sufficientconditions (Fig. 7, D and E). However, the numbers
ofMDC-stained autophagosomes increased by 21.3- to 24.5-fold in TRV
andwild-type plants on day 7 ofN starvation(Fig. 7, D and E). The
formation of autophagosomes in
Figure 7. Role of BZR1 in the response to N starvation in tomato
leaves. A, N starvation induced the dephosphorylation of BZR1.The
phosphorylated and dephosphorylated forms of BZR1 are indicated by
pBZR1 and dBZR1, respectively. Two-week-old plantswere transferred
to N-deficient nutrient solution to collect the leaf samples at the
indicated time points. Total proteins wereisolated, subjected to
12% SDS-PAGE, and probedwith an anti-HAmonoclonal antibody. Actin
was used as a loading control forthewestern-blot analysis. B,
Tolerance toN starvation in TRV, TRV-BZR1, wild type (WT), BZR1OE,
BZR1-1DOE, and d^im plants.The plantswere transferred
toN-freemedium for 14 d. Bars = 10 cm. C, The chlorophyll content
of the fourth expanded leaveswasdetermined immediately after 14 d
of control or N-deficient treatment in TRVand TRV-BZR1 plants or
wild-type, BZR1OE, BZR1-1DOE, and d^im plants. FW, Freshweight.
D,MDC-stained autophagosomes in the leaves of TRV, TRV-BZR1, wild
type,BZR1OE,BZR1-1DOE, and d^im plants. The plants were transferred
to N-free medium for 7 d, and the leaves were stained with MDC
andvisualized by confocal microscopy. MDC-stained autophagosomes
are shown in green. Bars = 20 mm. E, Relative autophagicactivity
normalized to the activity of the TRV or wild-type control plants
in D. The number of MDC-stained autophagosomes perimage was
quantified to calculate the autophagic activity relative to TRV or
wild-type control plants, which was set to 1. Morethan 20 images
for each treatment were used for the quantification. F and G, Atg8
protein levels in the leaves of TRV, TRV-BZR1,wild type, BZR1OE,
BZR1-1DOE, and d^im plants on day 7 under N starvation. The
nonlipidated and lipidated forms of Atg8 areindicated by Atg8 and
Atg8-PE, respectively. Actin was used as a loading control for the
western-blot analysis. The results in C andE represent means6 SE.
Means with the same letter did not differ significantly at P, 0.05
according to Duncan’s multiple rangetest. Three independent
experiments were performed, with similar results. 1# represents a
line of BZR1OE plants, and 6# rep-resents a line of BZR1-1DOE
plants.
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BZR1-silenced and d^im plants was suppressed signifi-cantly
after 7 d of N starvation (Fig. 7, D and E). Incontrast, the number
of MDC-stained autophagosomesincreased significantly in BZR1OE and
BZR1-1DOEplants compared with wild-type plants (Fig. 7, D andE).
While N starvation increased the level of Atg8-PE,BZR1 silencing
dramatically suppressed the accumu-lation of Atg8-PE compared with
that in TRV plants(Fig. 7, F and G). Importantly, BZR1OE and
BZR1-1DOE plants had higher abundance while d^im plantshad lower
abundance of Atg8-PE than wild-type plantsunder N-starvation
conditions (Fig. 7G). These resultsindicate that BR induces the
formation of autophago-somes, which then engulf the ubiquitinated
proteinaggregates for reuse, resulting in increased resistance toN
starvation.
DISCUSSION
Over the past two decades, studies on autophagy inplants have
established its crucial role in growth anddevelopment and the
response to abiotic and bioticstresses (Bassham et al., 2006).
However, our under-standing of the mechanism and regulation of
autoph-agy in plants remains obscure. In this study, wedemonstrated
that BR, a vital phytohormone associatedwith plant growth,
development, and stress responses,contributed to the formation of
autophagosomes intomato. BZR1, a downstream transcription factor
of
the BR signal transduction pathway, acted as an acti-vator of
autophagic genes to promote the formation ofautophagosomes, while
BR-induced autophagy wasinvolved in N remobilization. This study
provides ev-idence for the mechanisms of the BR-mediated
regula-tion of autophagy in plants.
BRs, a class of essential plant-specific steroidal
phy-tohormones, play important roles in plant growth, de-velopment,
and responses to various abiotic and bioticstresses (Yang et al.,
2011; Choudhary et al., 2012). BR-deficient and BR-insensitive
mutants usually exhibitsevere growth defects, including short
petioles andhypocotyls, delayed flowering and leaf senescence,
andreduced male fertility (Szekeres et al., 1996; Kim et al.,2005).
Treatment with BR enhances tolerance to pho-tooxidative and cold
stresses in cucumber (Cucumissativus; Xia et al., 2009). Similarly,
exogenous BR treat-ment also increases tolerance to oxidative and
heatstress in tomato, which is associated with the accumu-lation of
apoplastic hydrogen peroxide and the activa-tion of MPK1/2 (Zhou et
al., 2014a). Overexpressingthe key BR biosynthetic gene AtDWF4
increases toler-ance to cold, dehydration, and heat stresses (Divi
andKrishna, 2010; Sahni et al., 2016). Moreover, d^im plantshave
been found to have higher while DWFOE plantshave lower levels of
oxidized proteins and membranelipid peroxidation in response to
chilling stress (Xiaet al., 2018). Notably, abiotic stresses, such
as heat anddrought, result in severe damage to cellular
compo-nents, including protein denaturation and aggregation(Vinocur
and Altman, 2005), which are recognized byubiquitin and degraded
via autophagy for reuse (Zhouet al., 2013). In recent years,
autophagy has been dem-onstrated to be involved in the responses to
variousabiotic stresses (Bassham, 2007). Our previous studyshowed
that atg5 and atg7mutants aremore sensitive toheat, oxidative,
salt, and drought stresses in Arabi-dopsis than wild-type plants
(Zhou et al., 2013). Fur-thermore, silencing of ATG10 or ATG18f
compromisesthe tolerance to drought stress in tomato (Wang et
al.,2015). In this study, we found that enhanced levels ofBR,
either through exogenous application or endoge-nous manipulation,
induced the formation of auto-phagosomes in tomato leaves (Fig. 1;
Supplemental Fig.S1). Thus, the BR signaling pathway might induce
au-tophagy for the degradation of denatured and mis-folded proteins
to increase stress tolerance. Indeed,exogenous application of BL
increased the tolerance toN starvation along with increased
formation of auto-phagosomes and inhibited the accumulation of
insolubleprotein levels (Fig. 6). In insects, 20-hydroxyecdysone,
asteroid hormone, plays a critical role in activating au-tophagy
(Yin and Thummel, 2005; Ryoo and Baehrecke,2010). For example,
injection of 20-hydroxyecdysoneincreases the expression of ATGs and
inhibits the ac-tivity of TOR complex 1, leading to the induction
ofautophagy in the fat body of silkworm (Bombyx mori;Tian et al.,
2013). BR and 20-hydroxyecdysone are bothsteroidal hormones with
similar chemical structuresand induce autophagy. These results
imply that the
Figure 8. Proposed model for the induction of autophagy by BRs
intomato plants. Upon the perception of BR by BRI1, BR activates
thedownstream signal transduction cascades, leading to the
dephospho-rylation and nuclear localization of BZR1, which induces
the expres-sion of ATG2 and ATG6 to trigger the formation of
autophagosomes.Furthermore, BR-induced autophagy is involved in the
response to Nstarvation.
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up-regulation of ATGs and the induction of autophagyby steroid
hormones are conserved in both plants andanimals.BZR1, a
transcription factor downstream of the BR
signaling pathway, regulates the expression of numer-ous
BR-responsive genes (Sun et al., 2010). Recentstudies have
demonstrated that sugar activates the TORsignaling-dependent
autophagic pathway to regulatethe degradation of BZR1 to balance
growth and carbonavailability in Arabidopsis (Zhang et al., 2016).
In ad-dition, drought and carbon starvation induce
thebrassinosteroid insensitive2 phosphorylation of DSK2,a selective
autophagic receptor that promotes DSK2interaction with Atg8,
thereby targeting BRI1-EMS-suppressor1 for breakdown with the
attenuation ofplant growth (Nolan et al., 2017). These results
suggestthat autophagy is involved in the regulation of BRsignaling
through the degradation of BZR1 underdrought stress and carbon
starvation. However, tran-scriptome analysis with an Affymetrix
ATH1 arrayrevealed that numerous stress-related genes,
includingthose involved in proteinmetabolism
andmodification,defense responses, and calcium signaling, are BR
re-sponsive (Divi et al., 2016). Furthermore, BR
signaling-defective mutants were hypersensitive to salt stress(Cui
et al., 2012). Exogenous BR treatment not onlyenhanced plant
tolerance to drought and cold stressesbut also promoted seed
germination under salt stress(Kagale et al., 2007). In addition,
primary root growthwas inhibited dramatically and the accumulation
ofanthocyanin was increased significantly in wild-typeplants grown
on low-phosphate medium, but theroots of bzr1-D mutants grew well
and the leavesremained green (Singh et al., 2014), suggesting that
BRsignal participates in the response to low phosphateavailability.
These results indicate the multiple func-tions of BZR1 in response
to different stresses. In thisstudy, we found that the highest
level of BZR1 de-phosphorylation was observed after BL treatment
for12 h, and the expression of BR biosynthesis genes wasinhibited
(Supplemental Fig. S3), indicating that BLtreatment transiently
activated BR signaling. In addi-tion, silencing of the BZR1 gene
abolished the BL-induced formation of autophagosomes (Fig. 2, A
andC; Supplemental Fig. S4), while autophagic activitywas higher
after BL application in BZR1OE plants thanin wild-type plants (Fig.
2, A and D; SupplementalFig. S4). Yeast one-hybrid and ChIP-qPCR
assaysshowed that BZR1 bound directly to the promoters ofthe ATG2
and ATG6 genes (Fig. 4, B and C). Further-more, silencing of the
BZR1 gene compromised the in-duction of ATG2 and ATG6 genes by BL
treatment(Fig. 3A). These results indicate that the ATG2 andATG6
genes are target genes of the BZR1 transcriptionfactor and might
directly regulate the expression ofboth genes to induce
autophagy.Autophagy has been shown to play a critical role in
nutrient starvation. Arabidopsis atg mutants exhibitedpremature
senescence and were hypersensitive to Nand fixed carbon starvation
(Doelling et al., 2002;
Yoshimoto et al., 2004; Chung et al., 2010). The atg7 andatg9
plants showed accelerated senescencewhen grownon N-free medium,
characterized by the prematurechlorosis of the mature rosette
leaves (Doelling et al.,2002). Nutrient starvation induces the
formation ofautophagosomes to engulf the denatured proteins
andtransfer them to vacuoles for reuse, leading to
enhancedN-utilization efficiency. However,
autophagy-defectivemutants compromise the degradation of proteins
andthe generation of amino acids (Barros et al., 2017).Similarly,
BR treatment increased N uptake, which wasassociated with increased
activity of nitrate reductaseand increased levels of free amino
acids and solubleproteins (Dalio et al., 2013). Additionally, the
totalnodule number and the efficiency of N fixation werereduced in
BR-insensitive Medicago truncatula mutants(Cheng et al., 2017).
These results indicate that BR playsa critical role in N
utilization. Consistent with theseprevious studies, our study
showed that BL treatmentincreased the tolerance to N starvation,
which wasassociated with the increased formation of autophago-somes
to degrade the insoluble proteins (Fig. 6). Fur-thermore, BZR1
silencing increased sensitivity to Nstarvation and suppressed the
expression of ATGsand the formation of autophagosomes (Fig. 7, B,
D,and F). However, BZR1OE plants were more tolerantto N deficiency
than wild-type plants, which was as-sociated with the accumulation
of increased numbersof autophagosomes (Fig. 7, B, D, and G). We
also foundthat N starvation promoted the accumulation and
de-phosphorylation of BZR1 in tomato at the early stage ofN-free
treatment, while the abundance and dephos-phorylation of BZR1
gradually decreased after 7 d(Fig. 7A), which was consistent with
the results obtainedfor Arabidopsis under carbon starvation (Zhang
et al.,2016; Nolan et al., 2017). These results showed that, atthe
early stage of N starvation, the accumulation anddephosphorylation
of BZR1 was enhanced in plants topromote N recycling and increased
cellular energy byinducing the expression of ATG2 and ATG6 and
theformation of autophagosomes. As the stress of N star-vation
progresses, plant growth is inhibited and au-tophagy may break down
BZR1 to balance growth andthe stress response, leading to a
decrease in its accu-mulation, as observed in Arabidopsis in
response tocarbon starvation and drought (Zhang et al., 2016;Nolan
et al., 2017). These results suggest that autoph-agy and BZR1 can
regulate each other and that BZR1plays dual roles under starvation
stresses.In summary, in this study, we used comprehensive
genetic and molecular tools to provide insights into therole of
BR in the regulation of autophagy. Upon theperception of BR by
BRI1, BRs activate the downstreamsignal transduction cascades,
resulting in the dephos-phorylation and nuclear localization of
BZR1, whichcan induce the expression of ATGs to trigger
autopha-gosome formation. In addition, BR-induced autophagymediates
the response to N deficiency in tomato plants(Fig. 8). This report
systematically illustrates themechanism of autophagy induction by
BRs.
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MATERIALS AND METHODS
Plant Materials and Experimental Design
The tomato (Solanum lycopersicum) ‘Condine Red’ genotype was
used in allexperiments. Germinated seedswere grown in 250-cm3
plastic pots filled with amixture of peat and vermiculite (2:1,
v/v). The plants were watered daily withHoagland nutrition solution
in the chamber. The growth conditions weremaintained at 23°C/21°C
day/night temperatureswith a photoperiod of 14 h at600 mmol m22 s21
photosynthetic photon flux density.
For treatment with BL (Sigma-Aldrich B1439), 6-week-old plants
weresprayedwith 500 nM BL, and the control plants were
sprayedwithMilli-Qwatercontaining an equal amount of ethanol used
for the preparation of BL solution.Twelve hours after BL treatment,
the upper first fully expanded leaves wereexcised to detect
autophagic activity, or they were sampled and frozen quicklyin
liquid N and stored at280°C before using them for gene expression,
proteinanalysis, and biochemical analysis.
For N-starvation experiments, 2-week-old seedlings were grown in
N-freeliquid medium containing Murashige and Skoog micronutrient
salts (Sigma-Aldrich M0529), 3 mM CaCl2, 1.5 mM MgSO4, 1.25 mM
KH2PO4, 5 mM KCl, and2mMMES (pH 5.7). The plants were sprayedwith
500 nM BL or water every 2 d.The autophagic activity was monitored
on day 7, and the chlorophyll contentand protein levels were
measured on day 14 under N-deficient treatment.
Total RNA Isolation and Gene Expression Analysis
Total RNAwas extracted from tomato leaves by using the RNAsimple
TotalRNA Kit (Tiangen DP419) according to the manufacturer’s
instructions. Onemicrogram of total RNAwas used to reverse
transcribe to cDNA template usingthe ReverTra Ace qPCR RT Kit
(Toyobo FSQ-301).
TheRT-qPCRassayswere performed using the
SYBRGreenPCRMasterMix(Takara RR420A) in the LightCycler 480 II
Real-Time PCR detection system(Roche). The PCR conditions consisted
of denaturation at 95°C for 3 min fol-lowed by 40 cycles of
denaturation at 95°C for 15 s, annealing at 58°C for 15 s,and
extension at 72°C for 30 s. The Actin and Ubiquitin3 genes were
used asinternal controls. Gene-specific primers were designed based
on cDNA se-quences as described in Supplemental Table S1. Relative
gene expression wascalculated as described previously (Livak and
Schmittgen, 2001).
MDC Staining
Tomato leaves were stainedwithMDC as described previously (Wang
et al.,2015). Briefly, tomato leaves were excised and then
immediately vacuuminfiltrated with 100 mM MDC (Sigma-Aldrich 30432)
for 30 min, followed bytwo washes with phosphate-buffered saline
(PBS; Solarbio P1020). MDC-incorporated structures were excised by
a wavelength of 405 nm and detec-ted at 400 to 580 nm in the LSM
780 confocal microscope (Carl Zeiss).
TEM Analysis
To visualize the accumulation of autophagosomes by TEM, tomato
leaveswere cut into small pieces (;1 mm 3 4 mm) and fixed with 2.5%
(v/v) glu-taraldehyde in 0.1 M PBS buffer (pH 7) for 12 h in the
dark. Then, they werewashed with PBS buffer three times and again
fixed in 1% (v/v) osmium te-troxide at room temperature for 2 h;
the samples then were dehydrated in agraded ethanol series
(30%–100%, v/v) and embedded in Epon 812. Ultrathinsections (70 nm)
were prepared on an ultramicrotome (Leica EM UC7) with adiamond
knife and collected on Formvar-coated grids. The sections
weredetected using an H7650 transmission electron microscope
(Hitachi) at an ac-celerating voltage of 75 kV to observe
autophagosomes and autophagic bodies.
Protein Extraction and Western Blotting
For protein extraction, the harvested tomato leaf samples were
ground inliquidNandhomogenized in the extraction buffer (20mMHEPES,
pH7.5, 40mMKCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM
phenylmethylsulfonylfluoride, 5 mM DTT, and 25 mM sodium fluoride).
The soluble, insoluble, andubiquitinated proteins were detected as
described in our previous study(Zhou et al., 2013). The extracted
proteins were heated at 95°C for 15 min; thiswas followed by
separation using 10% SDS-PAGE. For Atg8 detection, thedenatured
proteins were separated on a 13.5% SDS-PAGE gel in the presence
of
6 M urea. For western blotting, the proteins on the SDS-PAGE gel
were trans-ferred to a nitrocellulose membrane. Then, the membrane
was blocked for 1 hin TBS buffer (20 mM Tris, pH 7.5, 150 mM NaCl,
and 0.1% Tween 20) with 5%skimmilk powder at room temperature and
then incubated for 1 h in TBS bufferwith 1% BSA (Amresco 0332)
containing a mouse anti-HA monoclonal anti-body (Pierce 26183),
mouse anti-ubiquitin monoclonal antibody (Sigma-Aldrich U0508),
rabbit anti-actin polyclonal antibody (Abcam ab197345), orrabbit
anti-Atg8 polyclonal antibody (Abcam ab77003 or Agrisera
AS142769).After incubation with a goat anti-mouse horseradish
peroxidase-linked anti-body (Millipore AP124P) or goat anti-rabbit
horseradish peroxidase-linkedantibody (Cell Signaling Technology
7074), the complexes on the blot werevisualized using the
SuperSignal West Pico Chemiluminescent Substrate(Thermo Fisher
Scientific 34080) by following the manufacturer’s instructions.
Vector Construction and Transformation
To obtain the tomato BZR1OE construct, the 981-bp full-length
coding DNAsequence (CDS) was amplified with specific primers
(Supplemental Table S2)using tomato cDNA as the template. The PCR
product was digested with AscIand KpnI and inserted behind the
Cauliflower mosaic virus 35S promoter in theplant transformation
vector pFGC1008-HA. To obtain the BZR1-1DOE con-struct, the CDS was
amplified using specific primers (Supplemental Table S2)and ligated
to pFGC1008-HA vector using the ClonExpress MultiS One StepCloning
Kit (Vazyme C113-01). The resulting plasmids were transformed
intoAgrobacterium tumefaciens strain EHA105 and transformed into
tomato seeds asdescribed previously (Fillatti et al., 1987).
Transgenic plants overexpressing theBZR1 and BZR1-1D transgene were
identified by RT-qPCR (Supplemental Fig.S2B). Two independent
homozygous lines of the T2 progeny were used inthe study.
Virus-Induced Gene Silencing Constructs and
A.tumefaciens-Mediated Virus Infection
The virus-induced gene silencing (VIGS) constructs for silencing
of theBZR1, ATG2, and ATG6 genes were generated by PCR
amplification usingspecific primers (Supplemental Table S3),
digested with SacI and XhoI, and li-gated into the same sites in
TRV2. The resulting plasmid was transformed intoA. tumefaciens
strain GV3101. A. tumefaciens-mediated virus infection was
per-formed as described previously (Ekengren et al., 2003). The
plants were kept at22°C and used for experiments after A.
tumefaciens infiltration for 3 weeks.Leaflets in the middle of the
fifth fully expanded leaves, which showed about20% to 40%
transcript levels of control plants, were used.
Yeast One-Hybrid Assay
The yeast one-hybrid experiment was performed as described
previously(Ravindran et al., 2017). The promoter sequences of ATGs
and the CDS of BZR1were amplified using specific primers
(Supplemental Table S4) and ligated intothe pAbAi and pGADT7
vectors, respectively. To generate the mutant of theE-boxes, the
sequence CANNTG was replaced by TCNNAA using the FastMultiSite
Mutagenesis System (TransGen FM201-01) according to the
manu-facturer’s instructions, and the primers used for plasmid
construction are listedin Supplemental Table S5. All constructs
were checked by DNA sequencing.The linearized constructs containing
ATG promoter fragments in pAbAi wereintegrated into the genome of
the Y1HGold yeast strain, and either BZR1-AD oran empty AD vector
was transformed into each. The transformed yeast cellswere selected
on Leu2 plates supplemented with 100 ng mL21 aureobasidin Ato
detect DNA-protein interactions.
ChIP
ChIP experiments were performed using the EpiQuik Plant ChIP
Kit(Epigentek P-2014) according to the manufacturer’s instructions.
Briefly, ap-proximately 1 g of leaf tissue was harvested from
BL-treated 35S-BZR1-HA andwild-type plants. Chromatin was
immunoprecipitated with an HA antibody(Pierce 26183), and goat
anti-mouse IgG (Millipore AP124P) was used as thenegative control.
ChIP-qPCR was performed with primers specific for theATG2, ATG5,
and ATG6 promoters (Supplemental Table S6).
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Chlorophyll Content
The chlorophyll in tomato leaves was extracted in 80% (v/v)
acetone, and itscontent was analyzed spectrophotometrically as
described previously (Chunget al., 2010).
Statistical Analysis
At least five independent replicates were used for each
determination. Sta-tistical analysis of the bioassays was performed
using the SPSS for Windowsversion 18.0 (CoHort Software)
statistical package. Experimental data wereanalyzed with Duncan’s
multiple range test at P , 0.05.
Accession Numbers
Sequence data from this article can be found in Solgenomics data
libraries(http://solgenomics.net/) according to the accession
numbers listed inSupplemental Tables S1 and S2.
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Relevance of endogenous BRs inducing
autoph-agy in tomato leaves.
Supplemental Figure S2. Relative mRNA abundance of BZR1 in
VIGS,BZR1OE, and BZR1-1DOE plants.
Supplemental Figure S3. BL induced the dephosphorylation of BZR1
andinhibited the expression of BR biosynthetic genes in BZR1OE
plants.
Supplemental Figure S4. Visualization of the accumulation of
autophago-somes in BZR1-silenced and BZR1OE plants with BL
treatment by TEM.
Supplemental Figure S5. BZR1 binds to the promoters of ATGs in
vitro.
Supplemental Figure S6. Relative mRNA abundance of ATG2 and
ATG6in ATG2- and ATG6-silenced wild-type or BZR1OE plants.
Supplemental Figure S7.. Induction of the dephosphorylation of
BZR1 byBL in BZR1OE and BZR1-1DOE plants.
Supplemental Figure S8. Expression of ATG2 and ATG6 in
BZR1-silenced,BZR1OE, BZR1-1DOE, and d^im plants.
Supplemental Table S1. Primers used for RT-qPCR assays.
Supplemental Table S2. Primers used for the construction of
BZR1OE andBZR1-1DOE vectors.
Supplemental Table S3. Primers used for VIGS vector
construction.
Supplemental Table S4. Primers used for yeast one-hybrid
assays.
Supplemental Table S5. Primers used for the construction of ATG2
andATG6 promoter mutant vectors.
Supplemental Table S6. Primers used for ChIP-qPCR assays.
Received August 23, 2018; accepted November 13, 2018; published
November27, 2018.
LITERATURE CITED
Albrecht C, Boutrot F, Segonzac C, Schwessinger B,
Gimenez-Ibanez S,Chinchilla D, Rathjen JP, de Vries SC, Zipfel C
(2012) Brassinosteroidsinhibit pathogen-associated molecular
pattern-triggered immune sig-naling independent of the receptor
kinase BAK1. Proc Natl Acad SciUSA 109: 303–308
Araújo WL, Tohge T, Ishizaki K, Leaver CJ, Fernie AR (2011)
Proteindegradation: An alternative respiratory substrate for
stressed plants.Trends Plant Sci 16: 489–498
Barros JAS, Cavalcanti JHF, Medeiros DB, Nunes-Nesi A,
Avin-Wittenberg T, Fernie AR, Araújo WL (2017) Autophagy
deficiency
compromises alternative pathways of respiration following
energydeprivation in Arabidopsis thaliana. Plant Physiol 175:
62–76
Bassham DC (2007) Plant autophagy: More than a starvation
response.Curr Opin Plant Biol 10: 587–593
Bassham DC, Laporte M, Marty F, Moriyasu Y, Ohsumi Y, Olsen
LJ,Yoshimoto K (2006) Autophagy in development and stress responses
ofplants. Autophagy 2: 2–11
Belkhadir Y, Jaillais Y (2015) The molecular circuitry of
brassinosteroidsignaling. New Phytol 206: 522–540
Chen J, Wang L, Wu C, Hu Q, Gu C, Yan F, Li J, Yan W, Chen G
(2014)Melatonin-enhanced autophagy protects against neural
apoptosis via amitochondrial pathway in early brain injury
following a subarachnoidhemorrhage. J Pineal Res 56: 12–19
Chen L, Liao B, Qi H, Xie LJ, Huang L, Tan WJ, Zhai N, Yuan LB,
Zhou Y,Yu LJ, et al (2015) Autophagy contributes to regulation of
the hypoxiaresponse during submergence in Arabidopsis thaliana.
Autophagy 11:2233–2246
Cheng X, Gou X, Yin H, Mysore KS, Li J, Wen J (2017) Functional
char-acterisation of brassinosteroid receptor MtBRI1 in Medicago
truncatula.Sci Rep 7: 9327
Choudhary SP, Yu JQ, Yamaguchi-Shinozaki K, Shinozaki K, Tran
LS(2012) Benefits of brassinosteroid crosstalk. Trends Plant Sci
17: 594–605
Chung T, Phillips AR, Vierstra RD (2010) ATG8 lipidation and
ATG8-mediated autophagy in Arabidopsis require ATG12 expressed
fromthe differentially controlled ATG12A and ATG12B loci. Plant J
62:483–493
Cui F, Liu L, Zhao Q, Zhang Z, Li Q, Lin B, Wu Y, Tang S, Xie Q
(2012)Arabidopsis ubiquitin conjugase UBC32 is an ERAD component
thatfunctions in brassinosteroid-mediated salt stress tolerance.
Plant Cell 24:233–244
Dalio RJD, Pinheiro HP, Sodek L, Haddad CRB (2013)
24-Epibrassinoliderestores nitrogen metabolism of pigeon pea under
saline stress. Bot Stud54: 9
Divi UK, Krishna P (2010) Overexpression of the brassinosteroid
biosyn-thetic gene AtDWF4 in Arabidopsis seeds overcomes abscisic
acid-induced inhibition of germination and increases cold tolerance
intransgenic seedlings. J Plant Growth Regul 29: 385–393
Divi UK, Rahman T, Krishna P (2016) Gene expression and
functionalanalyses in brassinosteroid-mediated stress tolerance.
Plant Biotechnol J14: 419–432
Doelling JH, Walker JM, Friedman EM, Thompson AR, Vierstra
RD(2002) The APG8/12-activating enzyme APG7 is required for
propernutrient recycling and senescence in Arabidopsis thaliana. J
Biol Chem277: 33105–33114
Ekengren SK, Liu Y, Schiff M, Dinesh-Kumar SP, Martin GB (2003)
TwoMAPK cascades, NPR1, and TGA transcription factors play a role
in Pto-mediated disease resistance in tomato. Plant J 36:
905–917
Fillatti JJ, Kiser J, Rose R, Comai L (1987) Efficient transfer
of a glyphosatetolerance gene into tomato using a binary
Agrobacterium tumefaciensvector. Biotechnology (N Y) 5: 726–730
Guo H, Li L, Aluru M, Aluru S, Yin Y (2013) Mechanisms and
networks forbrassinosteroid regulated gene expression. Curr Opin
Plant Biol 16:545–553
Guo L, Devaiah SP, Narasimhan R, Pan X, Zhang Y, Zhang W, Wang
X(2012) Cytosolic glyceraldehyde-3-phosphate dehydrogenases
interactwith phospholipase Dd to transduce hydrogen peroxide
signals in theArabidopsis response to stress. Plant Cell 24:
2200–2212
Han S, Wang Y, Zheng X, Jia Q, Zhao J, Bai F, Hong Y, Liu Y
(2015)Cytoplastic glyceraldehyde-3-phosphate dehydrogenases
interact withATG3 to negatively regulate autophagy and immunity in
Nicotianabenthamiana. Plant Cell 27: 1316–1331
He JX, Gendron JM, Sun Y, Gampala SSL, Gendron N, Sun CQ, WangZY
(2005) BZR1 is a transcriptional repressor with dual roles
inbrassinosteroid homeostasis and growth responses. Science
307:1634–1638
Henry E, Fung N, Liu J, Drakakaki G, Coaker G (2015) Beyond
glycolysis:GAPDHs are multi-functional enzymes involved in
regulation of ROS,autophagy, and plant immune responses. PLoS Genet
11: e1005199
Hofius D, Schultz-Larsen T, Joensen J, Tsitsigiannis DI,
Petersen NHT,Mattsson O, Jørgensen LB, Jones JDG, Mundy J, Petersen
M (2009)Autophagic components contribute to hypersensitive cell
death inArabidopsis. Cell 137: 773–783
Plant Physiol. Vol. 179, 2019 683
Brassinosteroid-Induced Autophagy in Tomato
Dow
nloaded from https://academ
ic.oup.com/plphys/article/179/2/671/6116478 by guest on 25 June
2021
http://solgenomics.net/http://www.plantphysiol.org/cgi/content/full/pp.18.01028/DC1http://www.plantphysiol.org/cgi/content/full/pp.18.01028/DC1http://www.plantphysiol.org/cgi/content/full/pp.18.01028/DC1http://www.plantphysiol.org/cgi/content/full/pp.18.01028/DC1http://www.plantphysiol.org/cgi/content/full/pp.18.01028/DC1http://www.plantphysiol.org/cgi/content/full/pp.18.01028/DC1http://www.plantphysiol.org/cgi/content/full/pp.18.01028/DC1http://www.plantphysiol.org/cgi/content/full/pp.18.01028/DC1http://www.plantphysiol.org/cgi/content/full/pp.18.01028/DC1http://www.plantphysiol.org/cgi/content/full/pp.18.01028/DC1http://www.plantphysiol.org/cgi/content/full/pp.18.01028/DC1http://www.plantphysiol.org/cgi/content/full/pp.18.01028/DC1http://www.plantphysiol.org/cgi/content/full/pp.18.01028/DC1http://www.plantphysiol.org/cgi/content/full/pp.18.01028/DC1http://www.plantphysiol.org/cgi/content/full/pp.18.01028/DC1
-
Jiang J, Zhang C, Wang X (2015) A recently evolved isoform of
the tran-scription factor BES1 promotes brassinosteroid signaling
and develop-ment in Arabidopsis thaliana. Plant Cell 27:
361–374
Kagale S, Divi UK, Krochko JE, Keller WA, Krishna P (2007)
Brassinos-teroid confers tolerance in Arabidopsis thaliana and
Brassica napus to arange of abiotic stresses. Planta 225:
353–364
Kim TW, Wang ZY (2010) Brassinosteroid signal transduction from
re-ceptor kinases to transcription factors. Annu Rev Plant Biol 61:
681–704
Kim TW, Hwang JY, Kim YS, Joo SH, Chang SC, Lee JS, Takatsuto S,
KimSK (2005) Arabidopsis CYP85A2, a cytochrome P450, mediates
theBaeyer-Villiger oxidation of castasterone to brassinolide in
brassinoste-roid biosynthesis. Plant Cell 17: 2397–2412
Kinoshita T, Caño-Delgado A, Seto H, Hiranuma S, Fujioka S,
Yoshida S,Chory J (2005) Binding of brassinosteroids to the
extracellular domain ofplant receptor kinase BRI1. Nature 433:
167–171
Kwon SI, Cho HJ, Kim SR, Park OK (2013) The Rab GTPase
RabG3bpositively regulates autophagy and immunity-associated
hypersensitivecell death in Arabidopsis. Plant Physiol 161:
1722–1736
Lai Z, Wang F, Zheng Z, Fan B, Chen Z (2011) A critical role of
au-tophagy in plant resistance to necrotrophic fungal pathogens.
Plant J66: 953–968
Li F, Chung T, Vierstra RD (2014) AUTOPHAGY-RELATED11 plays
acritical role in general autophagy- and senescence-induced
mitophagy inArabidopsis. Plant Cell 26: 788–807
Li F, Chung T, Pennington JG, Federico ML, Kaeppler HF, Kaeppler
SM,Otegui MS, Vierstra RD (2015) Autophagic recycling plays a
centralrole in maize nitrogen remobilization. Plant Cell 27:
1389–1408
Li XJ, Chen XJ, Guo X, Yin LL, Ahammed GJ, Xu CJ, Chen KS, Liu
CC, XiaXJ, Shi K, et al (2016) DWARF overexpression induces
alteration inphytohormone homeostasis, development, architecture
and carotenoidaccumulation in tomato. Plant Biotechnol J 14:
1021–1033
Liu Y, Bassham DC (2010) TOR is a negative regulator of
autophagy inArabidopsis thaliana. PLoS ONE 5: e11883
Liu Y, Bassham DC (2012) Autophagy: Pathways for self-eating in
plantcells. Annu Rev Plant Biol 63: 215–237
Liu Y, Schiff M, Czymmek K, Tallóczy Z, Levine B, Dinesh-Kumar
SP(2005) Autophagy regulates programmed cell death during the
plantinnate immune response. Cell 121: 567–577
Livak KJ, Schmittgen TD (2001) Analysis of relative gene
expression datausing real-time quantitative PCR and the 2(-D D
C(T)) method. Methods 25:402–408
Lozano-Durán R, Macho AP, Boutrot F, Segonzac C, Somssich IE,
ZipfelC (2013) The transcriptional regulator BZR1 mediates
trade-off betweenplant innate immunity and growth. eLife 2:
e00983
Michaeli S, Galili G, Genschik P, Fernie AR, Avin-Wittenberg T
(2016)Autophagy in plants: What’s new on the menu? Trends Plant Sci
21:134–144
Nolan TM, Brennan B, Yang M, Chen J, Zhang M, Li Z, Wang X,
BasshamDC, Walley J, Yin Y (2017) Selective autophagy of BES1
mediated byDSK2 balances plant growth and survival. Dev Cell 41:
33–46.e7
Pérez-Pérez ME, Florencio FJ, Crespo JL (2010) Inhibition of
target of ra-pamycin signaling and stress activate autophagy in
Chlamydomonasreinhardtii. Plant Physiol 152: 1874–1888
Phillips AR, Suttangkakul A, Vierstra RD (2008) The
ATG12-conjugatingenzyme ATG10 Is essential for autophagic vesicle
formation in Arabi-dopsis thaliana. Genetics 178: 1339–1353
Qin G, Ma Z, Zhang L, Xing S, Hou X, Deng J, Liu J, Chen Z, Qu
LJ, Gu H(2007) Arabidopsis AtBECLIN 1/AtAtg6/AtVps30 is essential
for pollengermination and plant development. Cell Res 17:
249–263
Ravindran P, Verma V, Stamm P, Kumar PP (2017) A novel
RGL2-DOF6complex contributes to primary seed dormancy in
Arabidopsis thaliana byregulating a GATA transcription factor. Mol
Plant 10: 1307–1320
Ryoo HD, Baehrecke EH (2010) Distinct death mechanisms in
Drosophiladevelopment. Curr Opin Cell Biol 22: 889–895
Sahni S, Prasad BD, Liu Q, Grbic V, Sharpe A, Singh SP, Krishna
P (2016)Overexpression of the brassinosteroid biosynthetic gene
DWF4 in Bras-sica napus simultaneously increases seed yield and
stress tolerance. SciRep 6: 28298
Shibuya K, Niki T, Ichimura K (2013) Pollination induces
autophagy inpetunia petals via ethylene. J Exp Bot 64:
1111–1120
Singh AP, Fridman Y, Friedlander-Shani L, Tarkowska D, Strnad
M,Savaldi-Goldstein S (2014) Activity of the brassinosteroid
transcriptionfactors BRASSINAZOLE RESISTANT1 and
BRASSINOSTEROID
INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1/BRASSINAZOLE
RESISTANT2 blocks developmental reprogramming inresponse to low
phosphate availability. Plant Physiol 166: 678–688
Sinha RA, You SH, Zhou J, Siddique MM, Bay BH, Zhu X, Privalsky
ML,Cheng SY, Stevens RD, Summers SA, et al (2012) Thyroid
hormonestimulates hepatic lipid catabolism via activation of
autophagy. J ClinInvest 122: 2428–2438
Sun Y, Fan XY, Cao DM, Tang W, He K, Zhu JY, He JX, Bai MY, Zhu
S, OhE, et al (2010) Integration of brassinosteroid signal
transduction with thetranscription network for plant growth
regulation in Arabidopsis. DevCell 19: 765–777
Svenning S, Lamark T, Krause K, Johansen T (2011) Plant NBR1 is
a se-lective autophagy substrate and a functional hybrid of the
mammalianautophagic adapters NBR1 and p62/SQSTM1. Autophagy 7:
993–1010
Szekeres M, Németh K, Koncz-Kálmán Z, Mathur J, Kauschmann
A,Altmann T, Rédei GP, Nagy F, Schell J, Koncz C (1996)
Brassinoste-roids rescue the deficiency of CYP90, a cytochrome
P450, controlling cellelongation and de-etiolation in Arabidopsis.
Cell 85: 171–182
Tang W, Yuan M, Wang R, Yang Y, Wang C, Oses-Prieto JA, Kim
TW,Zhou HW, Deng Z, Gampala SS, et al (2011) PP2A
activatesbrassinosteroid-responsive gene expression and plant
growth bydephosphorylating BZR1. Nat Cell Biol 13: 124–131
Thompson AR, Vierstra RD (2005) Autophagic recycling: Lessons
fromyeast help define the process in plants. Curr Opin Plant Biol
8: 165–173
Tian L, Ma L, Guo E, Deng X, Ma S, Xia Q, Cao Y, Li S (2013)
20-Hydroxyecdysone upregulates Atg genes to induce autophagy in
theBombyx fat body. Autophagy 9: 1172–1187
Vinocur B, Altman A (2005) Recent advances in engineering plant
toleranceto abiotic stress: Achievements and limitations. Curr Opin
Biotechnol 16:123–132
Wang J, Jiang J, Wang J, Chen L, Fan SL, Wu JW, Wang X, Wang ZX
(2014)Structural insights into the negative regulation of BRI1
signaling byBRI1-interacting protein BKI1. Cell Res 24:
1328–1341
Wang Y, Cai S, Yin L, Shi K, Xia X, Zhou Y, Yu J, Zhou J (2015)
TomatoHsfA1a plays a critical role in plant drought tolerance by
activating ATGgenes and inducing autophagy. Autophagy 11:
2033–2047
Xia XJ, Wang YJ, Zhou YH, Tao Y, Mao WH, Shi K, Asami T, Chen Z,
YuJQ (2009) Reactive oxygen species are involved in
brassinosteroid-induced stress tolerance in cucumber. Plant Physiol
150: 801–814
Xia XJ, Fang PP, Guo X, Qian XJ, Zhou J, Shi K, Zhou YH, Yu JQ
(2018)Brassinosteroid-mediated apoplastic H2O2-glutaredoxin 12/14
cascaderegulates antioxidant capacity in response to chilling in
tomato. PlantCell Environ 41: 1052–1064
Xiong Y, Contento AL, Nguyen PQ, Bassham DC (2007) Degradation
ofoxidized proteins by autophagy during oxidative stress in
Arabidopsis.Plant Physiol 143: 291–299
Yang CJ, Zhang C, Lu YN, Jin JQ, Wang XL (2011) The mechanisms
ofbrassinosteroids’ action: From signal transduction to plant
develop-ment. Mol Plant 4: 588–600
Yin VP, Thummel CS (2005) Mechanisms of steroid-triggered
programmedcell death in Drosophila. Semin Cell Dev Biol 16:
237–243
Yoshimoto K, Hanaoka H, Sato S, Kato T, Tabata S, Noda T, Ohsumi
Y(2004) Processing of ATG8s, ubiquitin-like proteins, and their
deconju-gation by ATG4s are essential for plant autophagy. Plant
Cell 16:2967–2983
Yoshimoto K, Jikumaru Y, Kamiya Y, Kusano M, Consonni C,
PanstrugaR, Ohsumi Y, Shirasu K (2009) Autophagy negatively
regulates celldeath by controlling NPR1-dependent salicylic acid
signaling duringsenescence and the innate immune response in
Arabidopsis. Plant Cell21: 2914–2927
Zhang Y, Yang C, Li Y, Zheng N, Chen H, Zhao Q, Gao T, Guo H,
Xie Q(2007) SDIR1 is a RING finger E3 ligase that positively
regulates stress-responsive abscisic acid signaling in Arabidopsis.
Plant Cell 19:1912–1929
Zhang Z, Zhu JY, Roh J, Marchive C, Kim SK, Meyer C, Sun Y, Wang
W,Wang ZY (2016) TOR signaling promotes accumulation of BZR1
tobalance growth with carbon availability in Arabidopsis. Curr Biol
26:1854–1860
Zhou J, Wang J, Cheng Y, Chi YJ, Fan B, Yu JQ, Chen Z (2013)
NBR1-mediated selective autophagy targets insoluble ubiquitinated
proteinaggregates in plant stress responses. PLoS Genet 9:
e1003196
684 Plant Physiol. Vol. 179, 2019
Wang et al.
Dow
nloaded from https://academ
ic.oup.com/plphys/article/179/2/671/6116478 by guest on 25 June
2021
-
Zhou J, Wang J, Li X, Xia XJ, Zhou YH, Shi K, Chen Z, Yu JQ
(2014a) H2O2mediates the crosstalk of brassinosteroid and abscisic
acid in tomatoresponses to heat and oxidative stresses. J Exp Bot
65: 4371–4383
Zhou J, Wang J, Yu JQ, Chen Z (2014b) Role and regulation of
autophagyin heat stress responses of tomato plants. Front Plant Sci
5: 174
Zientara-Rytter K, Lukomska J, Moniuszko G, Gwozdecki R,
Surowiecki P,Lewandowska M, Liszewska F, Wawrzy�nska A, Sirko A
(2011)Identification and functional analysis of Joka2, a tobacco
member ofthe family of selective autophagy cargo receptors.
Autophagy 7:1145–1158
Plant Physiol. Vol. 179, 2019 685
Brassinosteroid-Induced Autophagy in Tomato
Dow
nloaded from https://academ
ic.oup.com/plphys/article/179/2/671/6116478 by guest on 25 June
2021