bzr1 mediates brassinosteroid-induced autophagy and … · bzr1 mediates brassinosteroid-induced...

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.

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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, dîm, 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 dîm 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 dîm 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 dîmplants (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 dîm 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 dîm 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 dîm plants. FW, Freshweight. D,MDC-stained autophagosomes in the leaves of TRV, TRV-BZR1, wild type,BZR1OE,BZR1-1DOE, and dîm 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 dîm 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 dîm 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 dîm 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, dîm 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.

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