Postembryonic Seedling Lethality in the Sterol …Postembryonic Seedling Lethality in the Sterol-Deficient Arabidopsis cyp51A2 Mutant Is Partially Mediated by the Composite Action
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Postembryonic Seedling Lethality in the Sterol-DeficientArabidopsis cyp51A2 Mutant Is Partially Mediated bythe Composite Action of Ethylene and ReactiveOxygen Species1[W][OA]
Ho Bang Kim2*, Hyoungseok Lee2, Chang Jae Oh, Hae-Youn Lee, Hyang Lan Eum, Hyung-Sae Kim,Yoon-Pyo Hong, Yi Lee, Sunghwa Choe, Chung Sun An, and Sang-Bong Choi
Natural Science Research Institute (H.B.K.) and Department of Biological Sciences (H.-S.K., S.-B.C.), MyongjiUniversity, Yongin 449–728, Korea; Polar BioCenter, Korea Polar Research Institute, Incheon 406–840, Korea(H.L.); School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151–747,Korea (C.J.O., H.-Y.L., S.C., C.S.A.); Fruit Research Division, National Institute of Horticultural and HerbalScience, Rural Development Administration, Suwon 440–706, Korea (H.L.E., Y.-P.H.); and Department ofIndustrial Plant Science and Technology, Chungbuk National University, Cheongju 361–763, Korea (Y.L.)
Seedling-lethal phenotypes of Arabidopsis (Arabidopsis thaliana) mutants that are defective in early steps in the sterolbiosynthetic pathway are not rescued by the exogenous application of brassinosteroids. The detailed molecular andphysiological mechanisms of seedling lethality have yet to be understood. Thus, to elucidate the underlying mechanism oflethality, we analyzed transcriptome and proteome profiles of the cyp51A2mutant that is defective in sterol 14a-demethylation.Results revealed that the expression levels of genes involved in ethylene biosynthesis/signaling and detoxification of reactiveoxygen species (ROS) increased in the mutant compared with the wild type and, thereby, that the endogenous ethylene levelalso increased in the mutant. Consistently, the seedling-lethal phenotype of the cyp51A2 mutant was partly attenuated by theinhibition of ethylene biosynthesis or signaling. However, photosynthesis-related genes including Rubisco large subunit,chlorophyll a/b-binding protein, and components of photosystems were transcriptionally and/or translationally down-regulated in the mutant, accompanied by the transformation of chloroplasts into gerontoplasts and a reduction in bothchlorophyll contents and photosynthetic activity. These characteristics observed in the cyp51A2 mutant resemble those of leafsenescence. Nitroblue tetrazolium staining data revealed that the mutant was under oxidative stress due to the accumulation ofROS, a key factor controlling both programmed cell death and ethylene production. Our results suggest that changes inmembrane sterol contents and composition in the cyp51A2 mutant trigger the generation of ROS and ethylene and eventuallyinduce premature seedling senescence.
Sterols are isoprenoid-derived molecules that playessential roles in all eukaryotes. The squalene produc-tion pathway is well conserved in all organisms syn-thesizing sterols de novo; however, postsqualenepathways differ among biological kingdoms and lead
to the production of different end products. Ergosteroland cholesterol are major sterol forms in fungi andmammals, respectively. Higher plants synthesize acomplex mixture of sterols in which sitosterol, cam-pesterol, and stigmasterol are predominant forms(Benveniste, 2002, 2004). As integral components ofthe membrane lipid bilayer, sterols not only play afunctional role in regulating membrane fluidity andpermeability but also modulate the activity and dis-tribution of membrane-bound proteins such as recep-tors, enzymes, and components of the signalingpathway (Hartmann, 1998). Sterols are also precursorsfor the synthesis of diverse bioactive compoundsinvolved in important developmental processes, suchas steroid hormones (in animals), antheridiol (infungi), and ecdyson (in insects). Especially in higherplants, campesterol is a direct precursor for synthesisof the plant hormone brassinosteroids (BRs; Hartmann,1998), which function in postembryonic growth anddevelopment (Clouse and Sasse, 1998). Previous re-sults also demonstrated that sterols play a crucial role
1 This work was supported by a Korean Research Foundationgrant funded by the Korean Government (Basic Research PromotionFund; grant no. KRF–2006–312–C00402 to H.B.K.) and by the Bio-Green21 Program, Rural Development Administration, Republic ofKorea (grant nos. 20070301034028 to H.B.K. and 20080401034045 toS.-B.C.).
2 These two authors contributed equally to the article.* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Ho Bang Kim ([email protected]).
[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-
192 Plant Physiology�, January 2010, Vol. 152, pp. 192–205, www.plantphysiol.org � 2009 American Society of Plant Biologists www.plantphysiol.orgon August 12, 2020 - Published by Downloaded from
in cellulose biosynthesis during cell wall formation(Peng et al., 2002; Schrick et al., 2004).In addition to their structural function as membrane
components and their role as biosynthetic precursors,sterols have been known to play various regulatoryfunctions in biological systems. In animal systems,sterols have been implicated in transcriptional andposttranscriptional regulation, control of cholesterolbiosynthesis, meiosis, developmental patterning, andprotein cleavage and degradation (Edwards andEricsson, 1999). For instance, SCAP bearing sterol-sensing domain is an escort protein for endoplasmicreticulum-bound transcription factors, the sterol reg-ulatory element-binding proteins (SREBPs) that acti-vate genes for cholesterol synthesis and uptake aftertheir proteolytic maturation in the Golgi apparatus.Cholesterol has been known to play a key role inregulating the trafficking of SREBPs between the en-doplasmic reticulum and the Golgi (Goldstein et al.,2006). Cholesterol also plays important roles in thematuration of HEDGEHOG proteins, which transducesignals to adjacent cells and regulate many develop-mental processes, including neuronal specification andembryo development (Edwards and Ericsson, 1999).In plant systems, phytosterols are also believed to
act as signaling molecules to regulate diverse plantdevelopmental processes (Clouse, 2000, 2002). Thesterol/lipid-binding (Steroidogenic Acute RegulatoryTransfer [StART]) domain has been found in a numberof signaling proteins, including homeodomain pro-teins (Kallen et al., 1998; Ponting and Aravind, 1999).For example, the StART domain was found in thehomeodomain HD-ZIP family of putative transcrip-tion factors, including PHABULOSA (ATHB14) andGLABRA2, which are involved in leaf morphogenesisand trichome and root hair development, respectively(Ponting and Aravind, 1999; Schrick et al., 2004). Heet al. (2003) showed that sterols affect the expression ofgenes involved in cell expansion and cell division.Similarly, the transcription of a number of genes hasbeen shown to be activated in response to exogenouscholesterol treatments in animal systems (Edwardsand Ericsson, 1999), indicating that sterols could per-form common regulatory functions in both animal andplant development.The identification and characterization of Arabidop-
sis (Arabidopsis thaliana) mutant lines for the genesencoding structural enzymes of the postsqualenesterol biosynthetic pathway have revealed that thepathway can be divided into two domains, in termsof mutant phenotypes and BR responsiveness: BR-independent (sterol-specific) and BR-dependent path-ways (Clouse, 2002; Lindsey et al., 2003; Schaller, 2004).Mutants defective in the BR-dependent pathway, from24-methylene lophenol to the pathway end products(i.e., sitosterol, campesterol, and stigmasterol), typicallydisplay a BR-deficient dwarf phenotype that can berescued by an exogenous BR application. However,detailed analyses of Arabidopsis mutants defective inthe sterol-specific pathway, from mevalonate-derived
squalene to 24-methylene lophenol, have revealed thatsterols play essential roles in embryogenesis, hormonesignaling, pattern formation, organized cell divisionand expansion, chloroplast biogenesis and plant viabil-ity, and the correct localization of membrane-boundproteins (Lindsey et al., 2003; Willemsen et al., 2003;Schaller, 2004; Kim et al., 2005; Babiychuk et al., 2008;Pose et al., 2009). The functional roles of the afore-mentioned sterols have not been assigned to those ofBRs, indicating the BR-independent function of plantsterols.
Arabidopsismutants defective in the early sterol bio-synthetic pathway have displayed an early seedling-lethal phenotype as well as altered developmentalpatterns (Clouse, 2002; Lindsey et al., 2003; Schaller,2004; Kim et al., 2005). Studies on underlying mo-lecular mechanism of the seedling lethality still havebeen limited. Recently identified Arabidopsis sterol-biosynthetic mutant lines might give us plausible cluesto the fundamental mechanism of seedling lethality.The Arabidopsis cycloartenol synthase1 (cas1) mutantdefective in CAS1 showed an albino or cell-death phe-notype with severe defects in chloroplast biogenesis.Chloroplasts of the cas1 mutant displayed a disinte-gration of internal thylakoid membrane structures,with an accumulation of electron-dense lipid dropletscalled plastoglobuli. Chlorophyll and carotenoid pig-ment levels were also reduced in the cas1 mutant(Babiychuk et al., 2008). Changes in sterol content andcomposition due to a mutation on the ArabidopsisSQUALENE EPOXIDASE1 (SQE1) gene resulted in themislocalization of membrane-targeted RDH2/AtrbohNADPH oxidase, which is responsible for the gener-ation of apoplastic space-derived reactive oxygen spe-cies (ROS); these changes thereby induced an ectopicaccumulation of ROS in root hair cells (Pose et al.,2009). ROS is one of the key regulators controlling theoxidative stress-induced programmed cell death(PCD) pathway (Overmyer et al., 2003).
Previously, we isolated T-DNA insertion mutantsfor the Arabidopsis CYP51A2 gene that mediates the14a-demethylation step in the de novo sterol biosyn-thetic pathway. The loss-of-function mutant displayeda postembryonic seedling-lethal phenotype (Kim et al.,2005). Although multiple developmental defects, in-cluding reduced cell elongation and abnormal skoto-morphogenesis, were also observed in the mutants,these phenotypes could be largely attributed to BRdeficiency stemming from the depletion of its biosyn-thetic precursor campesterol (Kim et al., 2005); thesedefects were also observed in BR biosynthetic/signal-ing mutants (Choe, 2004). To gain insight into theunderlying molecular mechanism of seedling lethalityin the Arabidopsis cyp51A2 mutant, we performedfurther detailed molecular, biochemical, and physio-logical analyses on the basis of comparative transcrip-tome and proteome profilings between the wild typeand the cyp51A2 mutant. According to the results,changes in sterol content and composition in themutant likely activated the production of two key
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players involved in senescence-associated PCD (i.e.,ethylene and ROS). These changes in the sterol-deficientmutant thereby triggered a genetically encoded se-nescence program and eventually induced prema-ture PCD.
RESULTS
Global Gene Expression in the cyp51A2 Mutant
According to our previous results (Kim et al., 2005),the Arabidopsis cyp51A2 mutant that was defective inthe 14a-demethylation step of the sterol biosyntheticpathway displayed a seedling-lethal phenotype withreduced cell elongation. To gain insight into thesephenotypic characteristics, we performed three inde-pendent whole-genome microarray experiments usingtotal RNA from 9-d-old light-grown seedlings.
Campesterol, one of the major plant sterols, is adirect precursor for the production of BRs. The ex-pression of BR biosynthetic genes, including DWF4
and CPD, was up-regulated in the biosynthetic/sig-naling mutants and down-regulated by an exogenousBR application (Choe, 2004, 2007; Goda et al., 2004).Our microarray data revealed that the expression ofsterols and BR biosynthetic genes was increased in thecyp51A2 mutant (Table I; i.e., a homolog of sterol-C5-desaturase [HDF7], C-22 sterol desaturase [CYP710A4],steroid 22a-hydroxylase [DWF4], and BR-6 oxidase[CYP85A1 and CYP85A2]). Up-regulation of DWF4and CPD genes in the cyp51A2-3 mutant was furtherconfirmed byGUS histochemical analyses for promoter-GUS lines crossed to the mutant (Supplemental Fig.S1). This gene expression pattern also further supportsthe previous data indicating that the negative feedbackregulation system normally operates in the mutant,even under conditions of defective membrane integ-rity caused by sterol deficiency (Kim et al., 2005).However, the expression level of the CYP51A2 gene(At1g11680) encoding obtusifoliol 14a-demethylasewas greatly decreased in the mutant background(Supplemental Table S1), demonstrating the fidelityof the microarray experiment.
Table I. Up- or down-regulation of selected genes in the cyp51A2-3 mutant
Genes down-regulated in the mutant are listed in Functional Category photosynthesis related. AGI,Arabidopsis Genome Initiative.
Auxin-responsive genes, including AUX/IAA andSmall Auxin Up RNAs (SAUR), are known to be spe-cifically or commonly regulated by indole-3-acetic acid(IAA) and BR (Goda et al., 2004). Those genes weregenerally down-regulated in the cyp51A2 mutant(Supplemental Table S1); the down-regulation ofauxin-responsive genes such as AUX/IAA and SAURcould be attributed to a reduction in BR level or adefect in the auxin signaling pathway in the mutant.The expression of anthranilate synthase and anthrani-late phosphoribosyltransferase involved in the biosyn-thesis of Trp, an auxin precursor (Normanly et al.,2004), was up-regulated in the cyp51A2 mutant (Sup-plemental Table S1).One of the gene groups up-regulated in the cyp51A2
mutant is ethylene biosynthesis/signaling-related genes.These include 1-aminocyclopropane-1-carboxylic acid(ACC) synthase (ACS11 and ACS7), ACC oxidase(ACO4 and ACO2), two ethylene receptors (ETR2and ERS1), and a signaling component (CTR1; TableI). The transcription levels of ethylene-inducibledefense-related genes such as chitinase and defensinwere also increased in the mutant compared with thewild type (Supplemental Table S1), indicating thatthe endogenous ethylene level could be increased inthe mutant seedlings.The transcription levels of genes involved in ROS
detoxification generally increased in the mutant com-pared with the wild type (Table I), although severalgenes were down-regulated. The up-regulated genesincluded catalase, Fe-superoxide dismutase (Fe-SOD),glutathione S-transferase (GST), thioredoxin, and per-oxiredoxin (Mittler et al., 2004; Ahmad et al., 2008).The down-regulation of photosynthesis-related
genes was a prominent molecular feature culledfrom the global gene expression profiling betweenthe wild type and the cyp51A2mutant. These includeda number of CAB genes encoding chlorophyll a/b-binding proteins (Table I; Supplemental Table S1),genes encoding components of the PSI and PSII reac-tion centers (Table I; Supplemental Table S1), genesencoding proteins comprising an O2-evolving com-plex, a gene encoding Rubisco activase, and a geneencoding a thylakoid lumenal protein (Table I).
Proteome Analysis of the cyp51A2 Mutant
To better understand the molecular features of steroldeficiency with regard to plant growth and develop-ment, we compared the protein profiles of wild-typeand cyp51A2 mutant seedlings using two-dimensional(2-D) gel electrophoresis and then identified the pro-tein spots up- and down-regulated in the mutant com-pared with the wild type using matrix-assisted laserdesorption/ionization-time of flight (MALDI-TOF).Microarray data showed that ethylene biosynthetic
genes, including two genes encoding ACC oxidase,were up-regulated in the cyp51A2 mutant comparedwith the wild type. One of them, the protein level ofACC oxidase (ACO4), also increased in the mutant
(Table II). Another protein group that increased in themutant included ROS-scavenging and -detoxifyingenzymes such as GST, ascorbate peroxidase, Fe-SOD,ferritin, and thioredoxin (Table II; Mittler et al., 2004;Ahmad et al., 2008). Some of the genes encoding GSTs,Fe-SOD, and thioredoxin were also transcriptionallyactivated in the mutant (Table I; Supplemental TableS1). Salvage pathway-related proteins were also abun-dant in the cyp51A2 mutant compared with the wildtype. These include proteasome subunits and enzymesinvolved in the mobilization of lipids (pyruvate, or-thophosphate dikinase), proteins (Gln synthetase, as-partate aminotransferase [AAT], etc.), and nucleicacids (adenine phosphoribosyltransferase). Amongthem, mRNA levels of pyruvate, orthophosphate di-kinase, and AAT were increased in the mutant (Sup-plemental Table S1).
A comparison of the 2-D gel electrophoresis patternsof the wild type and cyp51A2 mutant showed thatthe protein level of Rubisco large subunit (RbcL;AtCG00490) was greatly reduced in the mutant com-pared with the wild type (Supplemental Fig. S2). Inaddition to RbcL, several proteins involved in photo-synthesis were also decreased in the mutant comparedwith the wild type (Table II), such as PSI reactioncenter subunit V (a chloroplast precursor) and photo-synthesis pathway enzymes such as glyceraldehyde-3-phosphate dehydrogenase, Fru-bisphosphate aldolase,and carbonic anhydrase. In the case of carbonic anhy-drase and Fru-bisphosphate aldolase (At4g26530), themRNA levels were reduced in the mutant (Supple-mental Table S1).
Ethylene Biosynthesis and Signaling in thecyp51A2 Mutant
Microarray and proteome data revealed that ethyl-ene biosynthesis and signaling could be enhanced inthe cyp51A2 mutant compared with the wild type. Tofurther confirm these data, we performed semiquanti-tive and/or quantitative reverse transcription (qRT)-PCR analysis for the selected genes involved in ethylenebiosynthesis and signaling, such as ETR1, ETR2, andERS1 (for ethylene signaling) and ACS7, ACS11, andACO4 (for ethylene biosynthesis). We also verified theexpression level of BASIC CHITINASE as an ethylene-responsive marker gene. The expression levels of allthe genes investigated in the experiment increased inthe mutant compared with the wild type (Fig. 1).
These results strongly suggest that both ethylenesignaling and biosynthesis are activated in the sterol-deficient cyp51A2 mutant. In this context, we hypoth-esized that inhibition of either ethylene biosynthesis orsignaling may partially alleviate seedling lethality ofthe mutant. To test this hypothesis, we used twoapproaches: treatment with chemical inhibitors thatblock ethylene biosynthesis or signaling, and geneticblocking of the ethylene signaling pathway. Treatmentwith a biosynthesis inhibitor (AVG) or a signaling
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inhibitor (silver nitrate [AgNO3]) on the cyp51A2 mu-tant not only delayed early seedling senescence butalso resulted in increases in fresh and dry weight andchlorophyll content, both in a dose-dependent man-ner, compared with the mock treatment control (Fig.2). These results further support the assertion thatsterol deficiency induces the activation of ethylene
biosynthesis/signaling. To investigate the effect ofgenetic blocking of the ethylene signaling pathway inthe cyp51A2 mutant, we genetically crossed cyp51A2-3heterozygote plants to an ethylene signaling mutant,etr1-1. Seedlings of the double homozygous mutantcyp51A2-32/2/etr1-1were dark green in color, whereasthe cyp51A2-3 mutant seedlings showed pale green
Table II. Up- or down-regulated proteins in the cyp51A2-3 mutant
Arabidopsis Genome Initiative (AGI) code numbers with asterisks indicate that the transcription level was also changed in the cyp51A2-3 mutantcompared with the wild type. Proteins down-regulated in the mutant are listed in Functional Categories photosynthesis related and ATPDX1.1 undernitrogen metabolism.
Functional Category Identified Protein AGI CodeFold Change
6 SE
ROS related ATGSTF2, GST (belonging to the w class) At4g02520* 1.83 6 0.69ATGSTF6, GST At1g02930 4.82 6 1.43ATGSTF7, GST (belonging to the w class) At1g02920 3.58 6 1.66ATGSTU20, GST (belonging to the t class) At1g78370* 2.26 6 0.6FSD1, Fe-superoxide dismutase At4g25100* 2.19 6 0.67APX1, L-ascorbate peroxidase At1g07890 1.59 6 0.89ATFER1, ferritin (chloroplast-localized) At5g01600 2.13 6 0.08ATTRX3, thioredoxin At5g42980 2.75 6 4.31
color (Fig. 3). These genetic crossing data are consis-tent with the results of the signaling inhibitor treat-ment, further supporting the assertion that theethylene signaling pathway is activated in the sterol-deficient cyp51A2 mutant.RT-PCR data for ethylene biosynthetic genes (ACS
and ACO) and an ethylene-responsive gene (BASIC
CHITINASE) indicated that ethylene content might beincreased in the mutants compared with the wild type(Fig. 1). This notion was further supported by the datathat treatment with ethylene biosynthesis inhibitorpartly delayed the early seedling senescence of themutant compared with the nontreatment control (Fig.2). In this context, we measured the ethylene content of
Figure 1. Expression levels of ethylene signaling/biosynthesis-related genes in the cyp51A2-3 mutant.A, RT-PCR analysis of ethylene signaling/biosynthesis-related genes in cyp51A2-3 mutants. Total RNA wasisolated from 9-d-old light-grown seedlings and usedfor RT-PCR. Actin-2 transcripts were amplified as apositive control for PCR. B, qRT-PCR analysis ofselected ethylene signaling/biosynthesis-related genesin cyp51A2-3 mutants. Ws-2, Wassilewskija-2.
Figure 2. Effect of ethylene biosynthesis or signaling inhibition on a seedling-lethal phenotype of the cyp51A2-3 mutant. A,Morphological change of the cyp51A2-3 mutant treated with AVG or AgNO3. Ten-d-old seedlings were transferred to eachtreatment condition and further cultured for 15 d under a dark/light cycle. Bar = 0.5 cm. B, Changes in fresh weight (left), dryweight (middle), and chlorophyll (Chl.) content (right) in cyp51A2-3 mutants treated with AVG or AgNO3.
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the wild type, the weak mutant allele cyp51A2-2, andthe strong mutant allele cyp51A2-3. The strong mutantallele produced approximately 4-fold more ethylenethan the wild type (Table III), whereas the weakmutant allele produced only slightly more than thewild type.
Accumulation of ROS in the cyp51A2 Mutant
Microarray and proteomic data showed that ROS-scavenging and -detoxifying enzymes were tran-scriptionally and/or translationally activated in thecyp51A2mutant compared with the wild type (Tables Iand II). Based on these results, we hypothesized thatROS could accumulate to higher levels in the mutantthan in the wild type. To test this hypothesis, weperformed nitroblue tetrazolium chloride (NBT) his-tochemical staining to detect superoxide radicals inlight-grown seedlings. A dark blue insoluble formazancompound was hardly detected in the wild type,whereas the mutants were densely stained with darkblue at the whole-plant level (Fig. 4). The staining levelwas denser in the strong mutant allele cyp51A2-3 thanin the weak mutant allele cyp51A2-2. This resultstrongly suggests that ROS highly accumulate in thesterol-deficient cyp51A2 mutant.
Photosynthetic Activity in the cyp51A2 Mutant
The sterol-deficient cyp51A2 mutant displayed aseedling-lethal phenotype (Kim et al., 2005). Accord-
ing to our microarray and proteomic data (Tables I andII), the seedling-lethal phenotype could be partiallyattributed to the greatly reduced photosynthetic activ-ity in the mutant compared with the wild type. Toverify the results, we performed several experimentalapproaches: qRT-PCR analysis for photosynthesis-related genes, western analysis for the proteins in-volved in photosynthesis, electron microscopy analysisfor chloroplast structure, and the measurement of di-rect photosynthetic activities.
qRT-PCR analyses were performed for two genesencoding either the PSI reaction center subunit VI(At1g52230) or the chlorophyll a/b-binding protein(At3g54890). The expression levels of both genes wereremarkably reduced in the mutant alleles comparedwith the wild type; the reduction was much morepronounced in the strong mutant allele than in theweak allele (Fig. 5, A and B).
Western analysis using antibodies against RbcL,components of the light-harvesting complex, and pho-tosystem components clearly showed that the levels ofthese photosynthesis core proteins were greatly down-regulated in the sterol-deficient cyp51A2 mutant com-pared with the wild type (Fig. 5C). The levels of theseproteins were much lower in the strong mutant allelecyp51A2-3 than in the weak mutant allele cyp51A2-2(Fig. 5C).
Electron microscopy analysis revealed that thecyp51A2 mutant displayed an aberrant plastid devel-opmental pattern compared with the wild type. Thechloroplasts of the wild type showed a highly orderedarrangement of thylakoid membrane structures andhad smaller electron-dense lipid droplets, plastoglo-buli (Fig. 6A), whereas the number of thylakoid mem-brane structures was reduced in the weak allelecyp51A2-2 mutant, which had an arrangement similarto that of the wild type (Fig. 6B). However, the chlo-roplasts of the strong mutant allele cyp51A2-3 lackedintact thylakoid membrane structures. Plastoglobuli inthe strong mutant allele were larger than those in boththe wild type and the weak mutant allele (Fig. 6C).
The expression levels of photosynthesis-related geneswere transcriptionally and/or translationally reducedin the cyp51A2 mutant compared with the wild type(Tables I and II; Fig. 5). Microscopy analysis alsorevealed that chloroplast development was severelyaffected by sterol deficiency (Fig. 6). In this context, we
Figure 3. Morphological phenotype of the cyp51A2-3/etr1-1 doublemutant. Heterozygous cyp51A2-3 plants were genetically crossed toan ethylene signaling mutant, etr1-1. The cyp51A2-3 mutant andcyp51A2-3/etr1-1 double mutant were cultured under light conditionsfor 2 weeks to compare seedling growth. Bar = 1 mm.
Table III. Ethylene contents of the wild type and cyp51A2-2 andcyp51A2-3 mutants
compared photosynthetic activity between the wild-type and mutant plants. First, the wild-type andmutant seedlings were measured for their chlorophyllcontent. Total chlorophyll content was slightly de-creased in the weak mutant allele compared with thewild type but noticeably reduced in the strong mutantallele (Fig. 7A). We also verified photosynthetic oxy-gen evolution as a measurable photosynthetic activity.In the weak mutant, the photosynthetic oxygen evo-lution value was comparable to that of the wild type,whereas the oxygen evolution rate of the strong mu-tant was less than 50% of that of the wild type (Fig. 7B).
DISCUSSION
Arabidopsis mutants defective in the early sterolbiosynthetic pathway that produces sterol interme-diates between squalene and 24-methylene lophenoldisplayed a seedling-lethal phenotype as well as analtered developmental pattern (Lindsey et al., 2003;Schaller, 2004; Kim et al., 2005). These mutants includefackel/hydra2, hydra1, and cyp51A2. Recently, the Arab-
idopsis cas1mutant defective in cycloartenol synthase,which mediates the first committed step of the post-squalene pathway, displayed albino or cell-death phe-notypes, depending on mutation. The cas1mutant alsoexhibited severe defects in chloroplast development(Babiychuk et al., 2008). However, the underlyingmolecular mechanisms of the lethality observed insterol-deficient mutants have not yet been studied. Inthis paper, to address the mechanism of seedlinglethality in sterol biosynthetic mutants, we performedmolecular, biochemical, and physiological analysesusing the Arabidopsis cyp51A2 mutant, which is de-fective in the 14a-demethylation step of the early sterolpathway (Kim et al., 2005). In those analyses, we foundthat the production of ethylene and ROS, key factorsof senescence-associated PCD, was activated in thecyp51A2 mutant and that the ethylene signaling path-way might also be influenced in the mutant. Based onthese findings, we conclude that the production ofethylene and ROSwas perturbed in the sterol-deficientcyp51A2mutant and that changes in these endogenousfactors partially caused premature seedling senes-cence and ultimately led to cell death.
Figure 4. Accumulation of ROS in cyp51A2mutants.In situ histochemical detection of ROS production inthe wild type (left), a weak allele cyp51A2-2 mutant(middle), and a strong allele cyp51A2-3 mutant(right). Nine-d-old seedlings were used for stainingwith NBT, which reacts with superoxide to produce adark blue insoluble formazan compound. Bars = 2mm (left and middle) and 1 mm (right). Ws-2,Wassilewskija-2.
Figure 5. Expression levels of photosynthesis-relatedgenes in cyp51A2 mutants. A, RT-PCR analysis ofphotosynthesis-related genes in cyp51A2mutants. B,qRT-PCR analysis of selected photosynthesis-relatedgenes in the cyp51A2-3 mutant. C, Western analysisof photosynthesis-related proteins in cyp51A2 mu-tants. Ws-2, Wassilewskija-2.
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Transcriptome and Proteome Analyses Reveal Activationof Ethylene Biosynthesis/Signaling in the
cyp51A2 Mutant
A comparison of transcriptomes of the wild typeand the cyp51A2 mutant revealed that the genes in-volved in ethylene biosynthesis and perception/sig-naling were generally up-regulated in the mutant(Table I; Fig. 1). The protein level of ACO4, one ofthe ethylene-forming enzymes, was also increased inthe mutant compared with the wild type (Table II).These molecular data were further confirmed by theinhibition of either ethylene biosynthesis or signaling(Figs. 2 and 3) and more directly by the measurementof endogenous ethylene levels (Table III). The acti-vation of ethylene biosynthesis and its signalingpresumably resulted in the up-regulation of defense-related genes such as defensin and chitinase (Fig. 1A;Supplemental Table S1). Previous microarray analysesshowed that the expression levels of ethylene recep-tor/signaling genes increased in wild-type plants thathad been exogenously treated with ethylene and/orthe constitutive ethylene-responsive mutant ctr1-1(Schenk et al., 2000; Alonso et al., 2003; Zhong andBurns, 2003; De Paepe et al., 2004), indicating thatethylene regulates its own signaling. Hua et al. (1998)also previously showed that expression of the ethylenereceptor genes ERS1, ERS2, and ETR2 was induced byethylene.
Ethylene biosynthesis can be altered by develop-mental or environmental factors (Lelievre et al., 1997;Wang et al., 2002; Argueso et al., 2007). The control ofethylene production can be achieved via a transcrip-tional or posttranscriptional regulation mechanism forbiosynthetic genes, such as ACS or ACO (Wang et al.,
2002; Argueso et al., 2007; McClellan and Chang,2008). ROS triggered by environmental stresses suchas ozone, wounding, and UV-B light induce ethyleneproduction by regulating the transcription of ethylenebiosynthetic genes (Wang et al., 2002; Argueso et al.,2007). NBT staining data led us to conclude that thecyp51A2 mutant is under oxidative stress due to animbalance between ROS generation and scavenging(Fig. 4). Therefore, ROS accumulation in the cyp51A2mutant is likely responsible for the up-regulation ofethylene biosynthetic genes, which in turn increasesendogenous ethylene level.
Ethylene Signaling in the cyp51A2 Mutant
Sterols have been shown to modulate the activitiesof membrane-bound proteins (Hartmann, 1998). Sev-eral pieces of direct and indirect evidence have dem-onstrated that changes in plant sterol content andcomposition affect the activities of H+-ATPase,NADPH oxidase, hormone signaling, and the polardistribution of auxin efflux carriers (Grandmougin-Ferjani et al., 1997; Souter et al., 2002; Willemsen et al.,2003; Men et al., 2008; Pose et al., 2009). Arabidopsishydra1 and fackel/hydra2 mutants displayed pheno-types reminiscent of abnormal ethylene signaling(i.e., an abnormal root hair formation pattern andreduced root growth). Inhibition of ethylene signalingthrough the application of silver ions, or the geneticcrossing to the ethylene-resistant mutant etr1-1, re-sulted in the rescue of root hair formation and rootgrowth. Based on these findings, the authors proposedthat the ethylene receptor ETR1 is altered constitu-tively in activity to promote the ethylene signaling
Figure 6. Plastid morphology in cyp51A2 mutants.A, Thewild type. B, cyp51A2-2mutant. C, cyp51A2-3mutant. Cotyledons from 9-d-old seedlings were usedfor transmission electronmicroscopy. Bars = 1 mm (A)and 0.5 mm (B and C).
Figure 7. Comparison of photosynthetic activity be-tween the wild type and cyp51A2 mutants. A, Chlo-rophyll (Chl.) contents. B, Photosynthetic oxygenevolution (Pmax value). Ws-2, Wassilewskija-2.
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pathway as a consequence of changes to the mem-brane environment, which is caused by reduction insterol content and incorrect sterol composition (Souteret al., 2002). In the cyp51A2 mutant described here, theinhibition of ethylene signaling by chemical treatmentor genetic crossing to etr1-1 partially suppressed theearly seedling-lethal phenotype (Figs. 2 and 3). There-fore, our data also serve as indirect evidence thatcorrect sterol composition is essential for regulatedethylene signaling, as demonstrated previously inhydra mutants (Souter et al., 2002). The effects ofaltered sterol content and composition on the activityof ethylene receptors need to be directly demonstrated.
Accumulation of ROS in the cyp51A2 Mutant
ROS are continuously generated as toxic by-productsof many essential metabolic reactions, including pho-tosynthesis. Because they are highly reactive, ROScause irreversible damage to major cellular compo-nents such as lipids, proteins, and nucleic acids (Apeland Hirt, 2004; Ahmad et al., 2008). A reduction inRbcL protein level was observed in the cyp51A2 mu-tant (Fig. 5C; Table II; Supplemental Fig. S2) and couldbe partially attributed to the breakdown of the abun-dant protein by highly reactive ROS. Evidence isaccumulating that ROS also play important roles assignaling molecules in controlling various biologicalprocesses, including PCD, stomatal closure, and de-fense responses to environmental stresses (Apel andHirt, 2004; Mittler et al., 2004). Chloroplasts have beenshown to be one of the major sites of ROS production.Recent studies revealed that nucleus-encoded plastidproteins EXECUTER1 and EXECUTER2 mediate theretrograde transfer of the ROS signal from the plastidto the nucleus and thereby modulate ROS-dependentnuclear gene expression (Lee et al., 2007; Kim et al.,2008).To avoid oxidative damage to cellular components,
the cellular ROS level should be stringently main-tained at nontoxic levels by enzymatic and nonen-zymatic antioxidant systems in several subcellularcompartments (Apel and Hirt, 2004; Ahmad et al.,2008). However, the cellular ROS level can be acutelyor chronically changed by environmental stimuli orduring the execution of normal developmental pro-cesses. The imbalance between ROS production andscavenging results in the triggering of oxidative stress,thereby perturbing production at the cellular level ofplant hormones such as ethylene and ultimately pro-moting cell death. NBT staining results showed accu-mulation of ROS such as superoxide radical in themutant comparedwith low levels in the wild type (Fig.4). This result indicates that the cyp51A2 mutant iscertainly under oxidative stress due to an imbalancebetween ROS production and its removal. As a com-pensatory mechanism against challenged oxidativestress, the enzymes or genes involved in ROS detox-ification were up-regulated in the cyp51A2 mutant(Tables I and II; Supplemental Table S1), as has been
observed in oxidatively stressed plants (Desikan et al.,2001; Vranova et al., 2002; Apel and Hirt, 2004).
Among the diverse sites of ROS production, plasmamembrane-localized NADPH oxidases are responsiblefor ROS generation in the apoplastic space. Severalreports have demonstrated that sterols modulate ac-tivity or the asymmetric distribution of membrane-bound proteins such as receptors, carriers, and enzymes(Grandmougin-Ferjani et al., 1997; Souter et al., 2002;Willemsen et al., 2003; Men et al., 2008). In theArabidopsis dry2/sqe1-5mutant defective in SQE1, outof six putative SQE genes (Benveniste, 2002), themislocalization of RHD2/AtrbohC NADPH oxidaseresulted in an ectopic ROS accumulation in the roothairs of the mutant. Based on this result, Pose et al.(2009) proposed that sterols play an essential role inthe localization of NADPH oxidases in root hair cells.Changes in sterol composition and content in thecyp51A2 mutant could modify the membrane environ-ment and thereby alter the stability or activity ofNADPH oxidases localized in the plasma membrane,by which stringent maintenance of the cellular ROSlevel was broken and ROS accumulated in the mutant(Fig. 4).
Premature Seedling Senescence of the cyp51A2 MutantResembles Leaf Senescence
Arabidopsis mutants defective in the early sterolbiosynthetic pathway displayed premature seedlingsenescence or a cell-death phenotype (Lindsey et al.,2003; Schaller, 2004; Kim et al., 2005; Babiychuk et al.,2008). Plant senescence is a highly regulated develop-mental process and a type of PCD, rather than a simpleand passive type of degeneration. PCD can occur at thecell, tissue, organ, or organism level and can be initi-ated by environmental, developmental, or hormonalfactors (Dangl et al., 2000). Leaf senescence is a well-characterized PCD that is accompanied by changes incell structure, metabolism, and gene expression. Thesechanges include the redifferentiation of chloroplastsinto gerontoplasts, a reduction of chlorophylls andcarotenoids, the activation of salvage pathways, andthe down-regulation of photosynthesis-related genes(Buchanan-Wollaston, 1997; Biswal et al., 2003; Limand Nam, 2007; Lim et al., 2007). The molecular,cellular, and physiological changes observed in thecyp51A2 mutant are quite similar to those of thesenescing leaf; for example, a number of genes in-volved in the structure of the photosynthetic appara-tus and photosynthetic pathway were down-regulatedin the mutant (Tables I and II; Supplemental Table S1).During senescence, chloroplasts redifferentiate intopremortal plastid-type gerontoplasts, which havelost thylakoid membrane structures and accumulatedelectron-dense lipid droplets known as plastoglobuli(Biswal et al., 2003). These gerontoplasts were ob-served in the cyp51A2 mutant, whereas wild-typeplants have a normal chloroplast structure containingintact grana (Fig. 6). This change in chloroplast structure
Seedling Lethality by Ethylene and ROS in the cyp51A2 Mutant
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was also observed in a sterol-specific cas1 mutant(Babiychuk et al., 2008). However, the Arabidopsiscas1 mutant is not seedling lethal, probably due to thepresence of a homologous gene (At3g45130) sharing62% identity to the cas1 gene (Benveniste, 2002). Con-sidering that the BR biosynthetic dwf4 mutant hadnormal plastid structure (Azpiroz et al., 1998), we canassume that sterols might play specific roles in plastiddevelopment. The changes in chloroplast structure inthe cyp51A2 mutant were also accompanied by a lossof chloroplast proteins, including RbcL, CAB, photo-system components, and photosynthetic pathway en-zymes (Table II; Fig. 5C); a reduction in chlorophylllevel (Fig. 7A); and ultimately a severe reduction inphotosynthetic oxygen evolution (Fig. 7B). Thesechanges indicate that premature senescence occurredin the sterol-deficient mutant.
During the degenerative senescence process, thesalvage pathway is highly geared toward the remobi-lization of hydrolyzed macromolecules such as lipids,proteins, and nucleic acids (Buchanan-Wollaston,1997; Dangl et al., 2000; Lim and Nam, 2007). Gluco-neogenesis leading from lipids to sugars is known tobe activated during the senescence of photosynthetictissues. mRNA or protein levels for malate synthase,phosphoenolpyruvate carboxy kinase, and pyruvate,orthophosphate dikinase were activated in thecyp51A2 mutant (Table II; Supplemental Table S1).Enzymes for the biosynthesis of amide forms of aminoacids were also up-regulated in the cyp51A2 mutant.These include Glu dehydrogenase, AAT, and Glnsynthetase (Table II; Supplemental Table S1). Theinduction of these enzymes is related to the remobili-zation of amino acids from the breakdown of proteinsduring leaf senescence. The breakdown of nucleicacids, in particular rRNA, occurs in senescing cells.An RNase gene (RNS1) responsible for the breakdownof nucleic acids during senescence was induced in thecyp51A2 mutant (Supplemental Table S1).
Among the plant hormones, ethylene is a strongpromoter of leaf senescence, although it is not anessential factor for progression of the highly organizeddevelopmental process (Lim and Nam, 2007; Limet al., 2007). Treatment with exogenous ethylene in-duces senescence, whereas inhibition of ethylene bio-synthesis, perception, or signal transduction increasesleaf longevity, primarily due to the delayed onset ofthe senescence program. As a promoter of senescence,ethylene inhibits photosynthesis, which is achieved bydecreasing the expression of photosynthesis-relatedgenes and increasing the expression of senescence-associated genes (Dangl et al., 2000; Gan, 2004). Severalmicroarray approaches have revealed that ethylenedown-regulates genes involved in the structure of thephotosynthetic apparatus and the photosyntheticpathway (Schenk et al., 2000; Alonso et al., 2003;Zhong and Burns, 2003; De Paepe et al., 2004). Thismolecular feature was also observed in the microarrayand proteomic data for the cyp51A2 mutant (Tables Iand II; Supplemental Table S1). Therefore, chronic
overproduction of ethylene in the sterol-deficientcyp51A2 mutant led to the early activation of thesenescence pathway and resulted in a reduction inphotosynthetic activity due to the down-regulation ofphotosynthesis-related genes. Ultimately, this led topremature seedling senescence in the mutant.
Ethylene and ROS Mediate Premature SeedlingSenescence in the cyp51A2 Mutant
ROS closely interacts with plant hormones, includ-ing ethylene, to control the PCD pathway. In theoxidative cell-death pathway, ROS not only activatesits own generation but also increases ethylene synthe-sis by inducing ethylene biosynthetic genes such asACS and ACO; an increased ethylene level servesas a positive feedback signal for ROS production(Overmyer et al., 2003). According to previous results,environmental factors that destroy the cellular redoxstate induce ROS production and consequently acti-vate ethylene production (Wang et al., 2002). Based onprevious reports, oxidative stress induced by an im-balance between ROS accumulation and scavengingcould be one of the causal agents that promote in-creased ethylene production in the cyp51A2mutant, byup-regulating the ACS and ACO genes (Tables I–III).Oxidative stress causes a rapid inhibition of photo-synthetic activity, which is reflected by changes in theactivity or synthesis of chloroplast proteins. A reduc-tion in chloroplast proteins was observed in thecyp51A2 mutant (Table II; Fig. 5C). Recent microarraydata further support the assertion that oxidative stresscan suppress the transcription level of photosynthesis-related genes such as CAB and photosystem compo-nents (Desikan et al., 2001; Vranova et al., 2002;Mahalingam et al., 2006), as reported in wild-typeArabidopsis that had been exogenously treated withethylene or the ethylene-responsive mutant ctr1-1(Schenk et al., 2000; Alonso et al., 2003; Zhong andBurns, 2003; De Paepe et al., 2004). Therefore, down-regulation in photosynthesis-related genes could becaused by oxidative stress-mediated ethylene produc-tion. Microarray and proteome data for the cyp51A2mutant also showed that a number of genes involvedin the photosynthetic apparatus, as well as the result-ing biochemical reactions, were down-regulated in themutant (Tables I and II; Supplemental Table S1). Incomparison with previous reports, these changes ingene expression might result from the accumulation ofROS in the mutant, which causes an increase in eth-ylene biosynthesis that is, in turn, followed by areduction in the expression of photosynthesis-relatedgenes and, eventually, by seedling lethality.
MATERIALS AND METHODS
Plant Materials and Growth Conditions
The Arabidopsis (Arabidopsis thaliana) ecotype Wassilewskija-2 was used
as the wild type. T-DNA-tagging mutants for CYP51A2, cyp51A2-2, and
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