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1 Int. J. Mol. Sci. 2013, 14, ; doi: /ijms Article OPEN ACCESS Interntionl Journl of Moleculr Sciences ISSN Mitochondril Cytochrome c Oxidse nd F 1 F o -ATPse Dysfunction in Peppers (Cpsicum nnuum L.) with Cytoplsmic Mle Sterility nd Its Assocition with orf507 nd Ψtp6-2 Genes Jiojio Ji 1, Wei Hung 1,2, Chunchun Yin 1 nd Zhenhui Gong 1,2, * 1 2 College of Horticulture, Northwest A&F University, Yngling , Shnxi, Chin; E-Mils: jijio963@163.com (J.J.); xnknkn@163.com (W.H.); yinchunc35@163.com (C.Y.) Stte Key Lortory for Stress Biology of Arid Region Crop, Northwest A&F University, Yngling , Shnxi, Chin * Author to whom correspondence should e ddressed; E-Mil: gzhh168@yhoo.com.cn; Tel.: ; Fx: Received: 23 Novemer 2012; in revised form: 13 Decemer 2012 / Accepted: 28 Decemer 2012 / Pulished: 7 Jnury 2013 Astrct: Cytoplsmic mle sterility (CMS) in pepper (Cpsicum nnuum L.) hs een ssocited with novel genes in the mitochondri, such s orf507 nd Ψtp6-2. Plnt sterility hs een proved to result from the rerrngement of the mitochondril genome. Previous studies hve demonstrted tht orf507 is co-trnscried with the cox II gene, nd Ψtp6-2 is truncted t the 3' region of the tp6-2 tht is found in the mintiner line. Until this time, little hs een known out the reltionship etween the novel gene nd the function of its corresponding enzyme in mitochondri from the CMS pepper line. Moreover, the errnt function of the mitochondril enzymes is seldom reported in pepper. In this study, we oserved tht nther ortion occurred fter the tetrd stge in the CMS line (HW203A), which ws ccompnied y premture progrmmed cell deth (PCD) in the tpetum. The sptiotemporl expression ptterns of orf507 nd Ψtp6-2 were nlyzed together with the corresponding enzyme ctivities to investigte the interctions of the genes nd mitochondril enzymes. The two genes were oth highly expressed in the nther. The orf507 ws down-regulted in HW203A (CMS line), with nerly no expression in HW203B (the mintiner line). In contrst, the cytochrome c oxidse ctivity in HW203A showed the opposite trend, reching its highest pek t the tetrd stge when compred with HW203B t the sme stge. The Ψtp6-2 in the CMS line ws lso down-regulted, ut it ws up-regulted in the mintiner line. The corresponding F 1 F o -ATPse ctivity in

2 Int. J. Mol. Sci. 2013, the CMS line ws grdully decresed long with the development of the nther, which showed the sme trend for Ψtp6-2 gene expression. On the contrry, with up-regulted gene expression of tp6-2 in the mintiner line, the F 1 F o -ATPse ctivity shrply decresed fter the initil development stge, ut grdully incresed following the tetrd stge, which ws contrry to wht hppened in the CMS line. Tken together, ll these results my provide evidence for the involvement of errnt mitochondril cytochrome c oxidse nd F 1 F o -ATPse in CMS pepper nther ortion. Moreover, the novel orf507 nd Ψtp6-2 genes in the mitochondri my e involved in the dysfunction of the cytochrome c oxidse nd F 1 F o -ATPse, respectively, which re responsile for the ortion of nthers in the CMS line. Keywords: Cpsicum nnuum L.; cytoplsmic mle sterility; orf507; cytochrome c oxidse; Ψtp6-2; F 1 F o -ATPse 1. Introduction Cytoplsmic mle sterility (CMS) is n importnt crop trit tht is widely used in hyrid reeding to produce non-functionl pollen [1]. This trit hs proven to e relted to the norml function of the plnt s mitochondri [2]. In mny cses, this chrcteristic is consequence of intermoleculr rerrngements in the mitochondril DNA, which produce mny novel open reding frmes (ORFs) tht re co-trnscried with conventionl mitochondril genes [2]. Mny ORFs tht re ssocited with CMS hve een identified, such s orf239 in the common en [3], pcf in petuni [4], orf507 in pepper [5,6], orfb in Brssic junce [7], orfh522 in sunflower [8], orfh79 [9] nd orfz79 [10] in rice, to nme few. CMS phenotypes cn lso e induced y the differentil expression of mitochondril genes under the influence of vrious nucler ckgrounds [11] or due to the sence of RNA editing, s in tp9 of whet [12], nd sustoichiometric copy numer of chimeric gene. For exmple, the restortion of fertility in common en is relted to the suppression of the copy numer of sustoichiometric CMS-ssocited ORF y the ction of restorer gene [13]. In pepper, the sustoichiometric difference is lso present. Two mitochondril genes ssocited with CMS in pepper, nmely orf507 nd Ψtp6-2, hve een shown to exist even in the mintiner lines with low copy numer sustoichiometry, s is lso the cse with CMS-ssocited genes from rdish nd common en [5,13]. Norml nther development progress hs mny importnt steps. First, the microspore mother cell is differentited from the reproductive cells nd will then undergo meiosis to give rise to tetrd, t which time the tetrd cn relese the microspores. The mononucler microspore further undergoes two rounds of mitosis to form three-celled pollen grin consisting of lrger vegettive cell nd two sperm cells [14]. During this process, severl nther tissues undergo progrmmed cell deth (PCD) in precisely coordinted nd temporl progression [15,16]. Generlly, the tpetum undergoes cellulr degrdtion during the development of norml pollen grins. The tpetl cell cn provide nutrients for the development of the pollen cell nd menwhile relese lipid components for the formtion of the pollen exine [17]. In mny CMS plnts, the premture PCD of the tpetum cell is lso followed soon

3 Int. J. Mol. Sci. 2013, fter y the deth of the immture microspores. In some CMS plnts, the cllose envelopes persist until the microspore tetrd stge, so the single nucler microspore cnnot e relesed nd thus cnnot form pollen grin [18]. However, in other CMS plnts, individul microspore could e produced, wheres the development of single nucler microspore ws rrested ecuse of the sence of energy provided y the mitochondri nd nutrition from the tpetum cells [19]. Pollen formtion is highly energy-consuming process. Becuse pollen is non-photosynthetic tissue, the necessry energy is supplied y mitochondri, especilly in the cse of developing pollen grins [20]. The mitochondrion, which is the site of oth the tricroxylic cid cycle nd the oxidtive phosphoryltion pthwy, plys crucil role in energy nd cron metolism in eukryotic cells through its role in the oxidtive phosphoryltion complex (OPC) [21]. The OPC include five complexes tht re locted in the inner memrne of the mitochondri, which re complex I (NADH dehydrogense), complex II (succinte dehydrogense), complex III (cytochrome c1 oxidoreductse), complex IV (cytochrome c oxidse) nd complex V (F 1 F o -ATP synthse) [22]. Cytochrome c oxidse (EC ) plys crucil role in eroic life due to its specific cpility to ccept n electron from cytochrome c nd then trnsfer it to dioxygen to form wter while pumping protons cross the memrne. The electrochemicl potentil difference of protons is used to drive ATP synthesis [23]. The enzyme complex is composed of severl metl prosthetic sites nd 13 protein suunits in mmmls, of which three re synthesized in the mitochondri. COX II is firmly ound to suunit I nd is the only suunit to possess polr domin contining the inucler Cu A center. The Cu A inucler center is reduced y cytochrome c nd then psses n electron on to cytochrome, which in turn psses n electron on to the cytochrome 3 -Cu B inucler center nd, finlly, to the dioxygen [24]. In plnts, the mjority of ATP is synthesized y F 1 F o -ATP synthse [25]. Furthermore, in ddition to ATP synthesis, the mitochondril F 1 F o -ATP synthse lso engges in ATP hydrolysis, depending on the cell s physiologicl ph [26,27]. In yest, ATP hydrolysis ctivity is regulted y F 1 -ATPse inhiitor (INH1-IF) nd stilizer proteins [27 30]. These proteins cn inhiit the ATP hydrolysis of F 1 F o -ATP synthse when it is not needed to ensure sufficient supply of ATP for cell ctivities, nd when the impired memrne potentil is rectified [27 30]. CMS is ssocited with oxidtive phosphoryltion complex (OPC) dysfunction, resulting in insufficient ATP to meet energy needs for pollen development [31]. For instnce, the F A d suunit of mitochondril F 1 F o -ATP synthse in Aridopsis thlin, which is encoded y the MGP1 gene, is essentil for nther development [25]. In sunflower, the orf522, homology to tp8, encodes suunit of F 1 F o -ATP synthse nd induces mle sterility in the plnt [32]. Some novel ORFs re co-trnscried with the norml gene tht encodes the expected protein in mitochondri nd cn lso ffect the norml protein s function. For exmple, in rice Boro II cytoplsm, orf79 is co-trnscried with the B-tp6 gene nd encodes cytotoxic peptide, which leds to mle sterility [33]. A similr phenomenon hs lso een found in the homologous Honglin rice gene, which is clled orfh79. The protein tht is encoded y orfh79 in rice interferes with the construction of F 1 F o -ATPse, leding to decrese in ATPse ctivity nd the ortion of pollen grins [9]. In pepper, the CMS cytoplsm ws first isolted from n Indin C. nnuum ccession (PI164835) [34]. The cndidte genes orf456 or orf507 ndψtp6-2 hve een identified in CMS pepper [5,6,35]. The orf456 gene, which is co-trnscried with the coxii gene tht encodes suunit of cytochrome c

4 Int. J. Mol. Sci. 2013, oxidse in mitochondri, cn induce mle sterility in Aridopsis thlin, t lest when it is over-expressed [35]. Gulys [6] found tht ecuse of the deletion (cytosine, C), the originl stop codon of orf456 chnged to sprgine (N), so the trnslted frgment ws extended, ending with new termintion codon, giving totl length of 507 p, nmed orf507. The Ψtp6-2 is 3'-truncted form of wild-type tp6-2, which encodes suunit of F 1 F o -ATP synthse in norml cytoplsm [36]. Intct F 1 F o -ATP synthse contins 13 suunits, most of which re nucler-encoded, except for ATP6, ATP8, ATP9, ATP4 of F 0 nd the ATPA of F 1, which is encoded y mitochondril genes [37]. The two novel genes of interest hve een widely used s selection mrkers in pepper production, nd orf456 hs een shown to induce mle sterility in Aridopsis thlin, ut the definite function of the genes nd the reltionship of the novel gene nd its corresponding mitochondril enzyme ctivity re not cler. Menwhile, mitochondril enzyme dysfunction in pepper hs rely een explored to dte, though the CMS in pepper is elieved to e ssocited with this dysfunction [36]. In this study, we focused on investigting the reltionship etween novel mitochondril orf507, Ψtp6-2gene nd mitochondril cytochrome c oxidse nd F 1 F o -ATP synthse enzyme in the OPC system of CMS line, respectively. The gene expression ptterns nd the mitochondril enzyme ctivity were mesured, following the scertining of the nther ortion chrcteristics in pepper CMS line. 2. Results nd Discussion 2.1. Confirmtion of Pollen Phenotype in the Sterile nd Mintiner Lines The phenotypes of the flowers in oth the sterile nd mintiner lines were similr except for the numer of pollen grins. The nthers in the sterile line hrdly produced pollen grins, wheres the mintiner nthers were full of pollen grins (Figure 1). Figure 1. Morphologicl oservtion of flower in HW203A (CMS line) nd HW203B (mintiner line). () Flowers from the sterile nd fertile lines; () Anthers from the sterile nd fertile lines. Sterile Fertile

5 Int. J. Mol. Sci. 2013, Cytologicl Fetures of Anther Development in the Sterile nd Fertile Pepper Lines To study the reltionship of premture PCD on the ortion of pollen in HW203A (sterile line), we compred the chrcteristics of pollen development with those from the HW203B (mintiner line). So fr, studies of pepper nther development hve een performed in mny types of CMS system; moreover, the onset of norml development ws reported to e vried from one system to nother [18,38 40]. Shifriss nd Frnkel [38] nd Horner nd Rogers [18] oserved tht the sterile line could form n norml tetrd ut could not produce norml pollen for the development of tpetl cells. Kul [39] nd Bhrgw [40] oth thought tht pollen development ws norml t the eginning of meiosis. It is necessry to exmine the pollen-ortive stge of our CMS line to further nlyze gene expression nd enzyme ctivity. Most prior reports focused on the initil cytologicl normlities during nther development in the sterile line to understnd the potentil cuses of the CMS phenotype in pepper ecuse the tetrds cnnot then produce mononucler microspore. For our study, we first performed prffin section of the nther to oserve its development. All the flower uds were divided into eight grdes (Figure 2) ccording to the morphology of the uds [41], such s the length nd the width of the pepper florets. Figure 2. Grdes dividing of the flower uds in pepper. In histologiclly stined nther sections tht were oserved y using light microscopy, the norml development of CMS pepper ecme evident t the tetrd stge, when the tpetl cells strted to degenerte. At the tetrd stge (grde 4 nd 5), the microspores in the sterile line ppered norml reltive to those of the mintiner line, though the tpet egn to dispper from the sterile line t this stge, wheres they persisted in the mintiner line (Figure 3II). When the florets of the CMS lines hd reched length tht corresponded to grdes 6 nd 7, the tpetl cells thoroughly degenerted nd the centrl nucleus microspores were relesed from the tetrd. The microspores were compressed in the center of the locule y the enlrged middle lyer cells (Figure 3III(A3)). In the mintiner line, the tpet lso disppered, nd the microspores were uniformly distriuted in the locule (Figure 3III(B3)). At grde 8, the middle lyer cells further enlrged in the sterile line, nd the sterile microspores were degrded nd dhered to the residuum of the tpetl cells nd were crushed into lump of condensed mteril y the enlrged middle lyer (Figure 3IV(A4)). Mture pollen grins were produced in the mintiner line t the sme stge, nd the middle lyer cells were degrded (Figure 3IV(B4)).

6 Int. J. Mol. Sci. 2013, Figure 3. Anther development in florets of HW203A nd HW203B. Four stges of nther development in the fertile line nd the corresponding stges of development in the sterile line were compred using histologicl stining. The imges show cross-sections through single locules: I Microspore mother cell stge; II Tetrd stge; III Mononucler microspore stge; IV Mture pollen grin stge; A, Sterile line; B, Fertile line; T, tpetum; En, endothecium; ML, middle lyer; MMC, microspore mother cell; Tds, tetrds; MNM, mononucler microspore; D, degrded microspore; PG, pollen grin. Brs = 20 μm. To find if premture progrmmed cell deth (PCD) cused the precocious cllose degrdtion tht ws noted y Binh et l. [42], we stined the microspore with niline lue. As shown in Figure 4, the stined microspores of HW203A nd B hd prcticlly no differences etween them, which implied tht the premture PCD hd nothing to do with the cllose.

7 Int. J. Mol. Sci. 2013, Figure 4. Aniline lue stining of the pollen t tetrd nd mononucleus stges in HW203A () nd HW203B (). The white rrow indictes the cllose. Brs = 20 μm. Together, these results show tht the tetrd in HW203A could relese single nucleus microspores, wheres no norml pollen grins were produced in the end, just s descried y Wng et l. [43]. Although HW203A cn produce single nucleus microspores, the tpetl cells were degrded erly t the strt of meiosis nd hd nerly completed its process y the tetrd stge. During norml pollen development, the nther tpetum strts to degrde t the tetrd stge nd completely finishes when mture pollen forms [32]; the whole process is clled progrmmed cell deth (PCD). The progrmmed degrdtion of tpetl cells provides nutrients for pollen development such s proteins, lipids nd others. Finlly, the tpetl cell remnnts nd liposome re relesed to form cot round the pollen surfce. Therefore, we inferred tht the premture PCD in HW203A might led to lck of nutrients nd energy tht is needed for norml pollen development, especilly t the lter stge, s is lredy known in sunflower [8]. The nutrient nd energy deficiency of pollen might result in precocious degrdtion, which would led to the ortion of pollen t the mononucler microspore stge in HW203A. During the development of the microgmetophyte, the cllose plys n importnt role in protecting the microspore nd supporting the formtion of the pollen exine wll [44]. Similr roles for the cllose wll t the tetrd nd mononucler microspore stges in HW203A nd HW203B indicted tht the premture PCD did not ffect the cllose wll formtion, nd perhps the ortion of pollen in the sterile line hs wek reltionship to cllose Gene Expression Anlysis of Cox II-orf507 nd Ψtp6-2 Kim et l. [35] nd Gulys et l. [6] found tht orf507 nd the Ψtp6-2 [36] re ssocited with pollen formtion in the pepper CMS line. Within the CMS line, orf507, which is novel ORF t the 3'-end of the cox II gene, is co-trnscried with cox II, nd Ψtp6-2 is pseudogene tht is truncted t the C-terminl coding region when compred with the norml tp6-2 from the mintiner line. Kim [35] showed tht orf456 ws expressed in leves nd flowers, ut it is uncler if the two genes could not or could only e slightly expressed in some certin tissues of the flower, nd if they were temporlly expressed. In our experiment, we nlyzed the sptiotemporl expression ptterns of orf507 ndψtp6-2 in the sterile nd mintiner lines to find if the two genes were relted to the ortion of pollen grins y using reverse trnscription PCR (RT-PCR) nd rel time PCR (qrt-pcr).

8 Int. J. Mol. Sci. 2013, Gene expression nlysis ws then performed to investigte the differences in expression levels etween CMS nd the mintiner lines t different nther development stges, including the MMC (microspore mother cell), tetrd nd mononucler microspore stges. The cdnas were reverse-trnscried from the totl RNA of HW203A nd HW203B florets t the three different development stges listed ove. Pollen ortion strted t the tetrd stge. The results showed tht no or very fint expression products of orf507 were detected in CMS flowers t the mononucler microspore stge nd tht in the mintiner line, high-intensity expression ws found t the tetrd nd MMC stges (Figure 5). Gene expression levels in CMS line reched their highest peks t the tetrd stge, which were significntly higher thn t the mononucler microspore stge; while they were little higher thn t the MMC stge, ut with no significnt difference. The high intensity expression efore pollen ortion indicted tht orf507 ws trnscriptionlly down-regulted nd might e relted to the induction of ortion. Figure 5. RT-PCR () nd qrt-pcr () expression of orf507 t different development stges of the pollen cells in sterile nd fertile florets with coxii s the reference gene. Lnes F1~3 show florets t the microspore mother cell stge, tetrd stge nd mononucler microspore stge in the fertile line; S1~3 show florets t the corresponding stges in the sterile line. Dt represent the mens ± SD from three independent experiments. Sttisticlly significnt differences etween the mens were determined using Fisher s LSD test (p < 0.05). orf507 coxii 350 F1 F2 F3 S1 S2 S3 Reltive gene expression F1 F2 F3 S1 S2 S3 To scertin whether the gene ws expressed in specific plnt tissues, cdnas from the petls, nthers, stigms, plcents nd leves were used. The orf507 trnscripts were undetected or nerly so in the stigms from the CMS line (Figure 6). The expression of orf507 in the other four tissues hd no significnt difference, nd they were highest in the nthers nd plcents. This pttern indicted tht orf507 ws not constitutively expressed in the flower nd further demonstrted tht the expression of this novel gene might cuse injury to the nther or the microspore to some degree, with the highest expression in the nther.

9 Int. J. Mol. Sci. 2013, Figure 6. RT-PCR () nd qrt-pcr () expression of orf507 in different tissues from the pepper sterile line with cox II s the reference gene. Dt represent the mens ± SD from three independent experiments. Sttisticlly significnt differences etween the mens were determined using Fisher s LSD test (p < 0.05). orf507 coxii 50 Petl Anther Stigm Plcent Lef Reltive gene expression petl nther stigm plcent lef PCR primers were designed to mplify ψtp6-2 in CMS plnt. The primer set is expected to mplify tp6-2 in mintiner plnt. The expression of tp6-2 in the mintiner plnts ws highest t the mononucler microspore stge; however, it ws highest t the MMC stge in the CMS line, nd oth were significntly higher thn t other development stges. Menwhile, the chnging trend of gene expression in the CMS line ws more or less contrry to wht ws found in the mintiner line. In concert with pollen development, the gene expression level ws grdully decresed in the CMS, ut it ws drmticlly incresed in the fertile line fter the tetrd stge (Figure 7). The opposing trends indicted tht the Ψtp6-2 in CMS ws down-regulted, nd conversely, the tp6-2 ws up-regulted in the mintiner line. Furthermore, sptil expression nlysis showed tht the pseudogene trnscripts were not or were only wekly detected in the stigms, petls nd leves oth in the CMS nd mintiner lines. However, the expression of tp6-2 in nthers ws significntly higher thn in other tissues from the mintiner line. In contrst, the expression of Ψtp6-2 in nther ws reltively higher thn other tissues in the CMS line nd ws slightly lower thn in the plcent, s shown in Figure 8. We speculted tht the tp6-2 might hve something to do with reproductive development, such s pollen development, nd tht Ψtp6-2 might hve little opposite impct on pollen development when compred with tp6-2 in norml cytoplsm. In summtion, oth the CMS nd mintiner lines showed tht the two genes of interest were not, or were only wekly expressed in the stigm, which might imply tht they did not ffect the stigm during pollen development. Moreover, temporl expression study showed tht the two genes were down-regulted in the CMS line. In the mintiner line, orf507 ws not expressed, wheres the opposite expression trend occurred for the tp6-2 gene. Menwhile, orf507 nd Ψtp6-2 or tp6-2 in norml cytoplsm were ll highly expressed in the nther. Following these results, we speculted tht orf507 ndψtp6-2 might e minly expressed in the tpetl cell or microspore cell. Their exct tissue specificity remins to e seen nd could e further explored y RNA gel lot.

10 Int. J. Mol. Sci. 2013, Figure 7. RT-PCR () nd qrt-pcr () expression of the regulr, non-truncted gene (rtp6-2) t different developmentl stges of pollen cells in sterile nd fertile florets with cox II s the reference gene. Lnes F1~3 indicte florets t the microspore mother cell stge, tetrd stge nd mononucler microspore stge in the fertile line; S1~3 show florets t the corresponding stge in the sterile line. Dt represent the mens ± SD from three independent experiments. Sttisticlly significnt differences etween mens were determined using Fisher s LSD test (p < 0.05). r tp6-2 coxii Reltive gene expression F1 F2 F3 S1 S2 S3 F1 F2 F3 S1 S2 S3 Figure 8. RT-PCR () nd qrt-pcr () expression of rtp6-2 in different tissues of the sterile nd fertile pepper lines with cox II s the reference gene, wheres F indictes the mintiner line nd S is the CMS line. Dt represent the mens ± SD from three independent experiments. Sttisticlly significnt differences etween the mens were determined using Fisher s LSD test (p < 0.05). F-Petl F-Anther F-Stigm F-Plcent F-Lef S-Petl S-Anther S-Stigm S-Plcent S-Lef r tp6-2 coxii Reltive gene expression F-petl cd F-nther F-stigm cd F-plcent cd F-lef c cd d S-petl S-nther d S-stigm S-plcent cd S-lef 2.4. Evlution of Cytochrome c Oxidse nd ATPse Activity CMS is closely connected with mitochondril dysfunction in higher plnts [45], especilly with regrds to energy metolism. In rice, CMS-HL, orfh79 trnscripts hve een shown to reduce

11 Int. J. Mol. Sci. 2013, ATPse stility nd ctivity [9], ut in rice, the CMS-ZA nd orfz79 trnscripts re ssocited with deficient NADH dehydrogense ctivity ut norml ATPse ctivity [10]. In new orf220-type CMS line of Brssic junce with down-regulted ltered tpa gene, mitochondril ATP synthesis decresed more thn one-fold. [46]. However, few reports on mitochondril enzyme ctivity in the CMS line of pepper hve een pulished, lthough mny novel mitochondril genes tht re cused y the rerrngement of DNA hve een found nd identified in this line. Given our current level of knowledge, we did not know if the novel genes were ssocited with mitochondril function for the corresponding enzyme. Kim et l. [35] trnsformed orf456 into Aridopsis thlin y using tpetum-specific promoter TA29, nd they oserved resulting defect in pollen-like grins in the exine wll. They speculted tht the expression of this novel gene might cuse premture progrmmed cell deth in tpetl cells, which provided lipids nd protein components for the formtion of the pollen exine wll, especilly cllose. In ddition to high orf507 expression in uds efore the tpetum degrdtion, we supposed tht orf507 hd ering on premture PCD through n effect on the energy supply. To demonstrte the relevnce of this gene to its reltive enzyme ctivity, we nlyzed cytochrome c oxidse nd ATPse ctivity in the CMS nd mintiner lines t different pollen development stges. Zhng et l. [9] demonstrted tht orfh79 contriutes to reduction in ATPse ctivity when co-trnscried with tp6. In the pepper CMS line, orf507 is co-trnscried with cox II (which encodes suunit of cytochrome c oxidse clled COX II), just like the orfh79 in rice. As shown in Figure 9, the enzyme ctivity of cytochrome c oxidse in the mintiner line is miniml t the tetrd stge, which is significntly lower thn t the MMC nd mononucler microspore stges. An opposite trend ws found in the CMS line. When the tpetum strted to degenerte t the tetrd stge, the enzyme ctivity of cytochrome c oxidse reched the mtrix. All these findings implied tht the sterile line ws cytochrome c oxidse-deficient. By comprtive nlysis of orf507 gene expression level nd cytochrome c oxidse ctivity, we speculted tht the expression of orf507 my ffect the norml function of cytochrome c oxidse. Figure 9. Activity of mitochondril cytochrome c oxidse in pepper florets t different developmentl stges. Lnes F1~3 show florets t the microspore mother cell, tetrd nd mononucler microspore stges in the fertile line; S1~3 denote florets t the corresponding stges from the sterile line. Dt represent the mens ± SD from three independent experiments. Sttisticlly significnt differences etween mens were determined using Fisher s LSD test (p < 0.05). Activity mmol cyt c/min mg protein c F1 F2 F3 S1 S2 S3

12 Int. J. Mol. Sci. 2013, The function of COX II, with its Cu A inucler center, is to ccept n electron from reduced cyt c nd trnsfer it to cytochrome in the respirtory chin [24]. It ws hypothesized tht ORF507 might interfere with the electron trnsmission from reduced cyt c to cyt, or reduce the stility of the enzyme y ffecting the interction of COX II with other suunits of cytochrome c oxidse, such s the inding of COX II to COX I. Nevertheless, further evidence is still needed to define the reltionship etween this gene nd cytochrome c oxidse. In the future, we cn demonstrte the interctions etween the orf507 protein nd cytochrome c oxidse using yest two-hyrid ssy. In higher plnts, F 1 F o -ATP synthse produces the ulk of the ATP. The two F 1 nd F 0 prts re coupled in cells, nd the intct F 1 F o -ATP synthse functions s ATPse nd ATP synthse s regulted y the H + concentrtion [47]. ATP6 is suunit of F 1, wter-solule unit tht contins the ctlytic inding sites for ATP synthesis or hydrolysis [48,49]. This suunit plys n importnt role in the ssemly of F o with F 1 ecuse ATP6 hs five trnsmemrne domins with n N terminus on the periplsmic side of the plsm memrne nd C terminus in the mtrix comprtment [49]. It ws reported tht muttions of tp6, such s the tp6-orfh79 in Honglin rice nd single mino cid muttion in yest mitochondri, could induce some interction chnges etween ATP6 nd other suunits of F 1 F o -ATP synthse; moreover, these interctions re needed to stilize the ssemly of F o with F 1 [50]. There re two copies of tp6 in pepper, from which the sequence of tp6-1 in the CMS line is the sme s it is in the mintiner line. The other tp6-2 hs different sequence in the CMS line, with truncted 3' region (Ψtp6-2) nd n intct sequence in its counterprt. In previous study, we showed tht the trnscription of Ψtp6-2 ws down-regulted in different pollen development stges, wheres the tp6-2 ws up-regulted in the mintiner line. With respect to the ATPse ctivity t the initil microspore mother cell stge, the ATPse ctivity in the mintiner line ws the highest (F1) nd ws much higher thn in the sterile line (S1). The enzyme ctivities in oth the sterile nd mintiner lines were significntly decresed fter the MMC stge. After the tetrd stge, when the nther strted to ort in the CMS line, the enzyme ctivity in the CMS line still grdully decresed, ut it incresed in the mintiner line. To nlyze the trend s whole, the up-regulted pttern of tp6-2 nd the grdully decresed ATPse ctivity might indicte tht the norml gene might e involved in the inhiition of ATPse ctivity. The chnging ATPse ctivity tht ws down-regulted in the CMS line ws similr to the gene expression of Ψtp6-2, mening tht Ψtp6-2 might increse the ATPse ctivity nd promote ATP hydrolysis (Figure 10). These results showed tht tp6-2 nd Ψtp6-2 encoded different proteins with opposite effects on the function of ATPse. On the other hnd, the chnging ATPse ctivity hppened fter the eginning of the nther ortion of the CMS nd mintiner lines, which my demonstrte tht the mitochondril F 1 F o -ATPse ws errnt in the CMS line, compred with in the mintiner line, nd the dysfunction of the enzyme hd something to do with the expression of Ψtp6-2.

13 Int. J. Mol. Sci. 2013, Figure 10. Activity of mitochondril ATPse in pepper florets t different developmentl stges. Lnes F1~3 show florets t the microspore mother cell, tetrd nd mononucler microspore stges in the fertile line; S1~3 indicte florets t the corresponding stges in the sterile line. Dt represent the mens ± SD from three independent experiments. Sttisticlly significnt differences etween mens were determined using Fisher s LSD test (p < 0.05) A ctivity mmol P i/min mg protein c c F1 F2 F3 S1 S2 S3 The reverse ATP hydrolysis nd synthesis of F 1 F o -ATP synthse is regulted y memrne electrochemicl potentil [25]. In yest mitochondri F 1 F o -ATP synthse, the ATP6 of F o forms the proton-conducting interfce with the c9-15 ring. For functionl F 1 F o -ATP synthse, ATP6 nd suunits β nd α consist of sttic suunits nd the suunits γ, ε nd c consist of rotting suunits. The reversile rottion of γ ε c suunits reltive to the β nd α ctlytic suunits of F 1 tkes plce during ATP synthesis (clockwise) nd ATP hydrolysis (counterclockwise). The ATP6 nd suunit c work s sttor to nchor the ctlytic suunits of F 1 to the memrne [51]. In view of our results, we supposed tht ATP6-2 my e involved in switching ATP hydrolysis to ATP synthesis. The protein structure of F 1 F o -ATP synthse in the CMS line my hve een chnged y the expression of Ψtp6-2, nd the chnged structure my inhiit the coupled enzyme when switching to clockwise direction to perform ATP synthesis, ut still persists in hydrolyzing ATP. When compred with the incresed ATPse hydrolysis ctivity in the mintiner line, the decresed ATPse ctivity following the tetrd stge my led to insufficient ATP supplies for norml tetrd microspore or mononucler microspore development, which ultimtely results in the ortion of pollen in the CMS line. Nevertheless, more evidence is required to ddress this hypothesis. In view of the ide tht ATP hydrolysis is not simple reversile rection of ATP synthesis from F 1 F o -ATP synthse, further demonstrtions of ATP synthse ctivity etween the CMS nd mintiner lines will e performed in the future. 3. Experimentl Section 3.1. Plnt Mterils nd Growing Conditions Pepper CMS line HW203A nd its corresponding mintiner line HW203B, which re ner-isogenic lines with similr nucler genotypes nd different cytoplsms, were provided y the Horticulturl College of Northwest A&F University. Plnts were grown in n unheted greenhouse during the summer, nd the florets were hrvested fter flowering t different nther development stges.

14 Int. J. Mol. Sci. 2013, Anlysis of Pollen Development The florets of fertile line HW203B nd CMS line HW203A were divided into eight grdes ccording to the length nd width of the sepls nd petls. The different floret grdes were fixed, emedded, nd sectioned using procedure tht ws dpted from Peng et l. [41]. The prffin section smples were oserved under light microscope. After the tetrd stge the microspore ws stined with 0.02% niline lue in 0.1 M PBS to oserve the ccumultion of cllus round the microspore, which ws exmined with fluorescence microscope under UV light Extrction of Mitochondri The florets in the pollen mother cell, tetrd nd single nucler microspore stges were hrvested, ground with pestle in cold extrction uffer (0.4 M mnnitol, 1 mm EDTA, 25 mm MOPS-KOH (ph 7.8), 10 mm KH 2 PO 4, 0.1% BSA, 1% PVP 40 nd 0.2% β-mercptoethnol). The homogente ws filtered through 200 mesh nylon striner nd then sujected to grdient centrifugtion ccording to procedure y Millr et l. [52]. The mitochondri pellets were resuspended in 1 ml of medium (0.4 M mnnitol, 1 mm EDTA, 10 mm KH 2 PO 4, 0.1% BSA). Protein yield ws determined y UV-Vis spectroscopy Gene Expression Pttern Anlysis To nlyze gene-specific expression, totl RNA ws isolted from the florets in the pollen mother cell, tetrd nd single nucler microspore stges from different tissues including the petls, nthers, stigms, plcents nd leves using the Trizol method nd ws then purified with DNse I. The mrna from ech smple ws sujected to reverse trnscription using the PrimeScript RT-PCR Kit (Tkr Biotech., Shig, Jpn) y comining the use of rndom hexmers nd oligo dt primer. The primers tht were used for RT-PCR nd qrt-pcr were designed s indicted in Tle 1, wheres coxii ws used s the reference gene [6]. The primers tht were used for gene expression nlysis of Ψtp6-2 nd tp6-2, which is known here s rtp6-2, were designed ccording to the homologous sequences of the two genes. The qrt-pcr ws performed using SYBR Green Kit (Sigm, St. Louis, MO, USA). Reverse-trnscription PCR (RT-PCR) ws performed just like norml PCR. The PCR protocol ws s follows: n initil step of 94 C for 5 min, followed y 28 cycles of 94 C for 30 s, 55 C for 30 s, nd 72 C for 30 s, followed y finl elongtion step of 72 C for 10 min. The PCR products were detected y electrophoresis on 1.5% grose gel. Tle 1. Oligonucleotides used for RT-PCR nd qrt-pcr in this study. Oligonucleotide Sequence Reference orf507 (forwrd) 5'-ATGCCCAAAAGTCCCATGTA-3' [35] orf507 (reverse) 5'-AAAAGCGCTAAACAAATTGC-3' This study r tp6-2 (forwrd) 5'-GCTTCGCCTTCGTATAGTAGTTC-3' This study r tp6-2 (reverse) 5'-AGTCATAGTGCTCACCCTGTTTG-3' This study coxii (forwrd) 5'-ATGATTGTTCTAGAATGGCT-3' [6] coxii (reverse) 5'-TTATGGGATTAATTGATTGGATACCCG-3' [6]

15 Int. J. Mol. Sci. 2013, ATPse nd Cytochrome c Oxidse Activity The mitochondri pellets of florets t three different stges were lysed y freeze-thw cycles with the ddition of 0.05% Triton X-100 [32] nd the lyste ws used for the spectrophotometric nlyses of enzyme ctivities. ATPse ctivity ws estimted ccording to the method of Hns Bisswnger [53]. The ctivity of cytochrome c oxidse ws nlyzed s descried y Prsd et l. [54], wheres the rection mix contined 50 mm phosphte uffer, ph 7.0, 10 μm reduced cyt c (reduced with 10 mm sodium scorte) nd 10 μl of protein in finl volume of 1.5 ml. 4. Conclusions In conclusion, the ortion of the HW203A nther occurs fter the tetrd stge, wheres the nther of the CMS line cn produce mononucler microspore, ut cnnot form norml pollen grins. The expression of orf507 nd low enzyme ctivity in the CMS line hs shown tht orf507 my e involved in the errnce of the CMS cytochrome c oxidse, in ddition to the opposing enzyme ctivity trend tht ws oserved etween the CMS nd mintiner lines. Menwhile, the down-regulted expression of Ψtp6-2 nd ATPse ctivity in the CMS line might indicte tht the Ψtp6-2 pseudogene produces novel protein tht could prticipte in the promotion of ATPse nd cuse persistent hydrolyztion of ATP. In the up-regulted tp6-2 from the mintiner line, the ATPse ws generlly down-regulted, which suggests tht ATP6-2 plyed role in the inhiition of ATPse hydrolysis ctivity. The different chnging trends of ATPse ctivity fter nther ortion in oth lines demonstrted tht ATPse function ws deficient in the CMS line. Moreover, ATPse dysfunction my cuse the enzyme to hve difficulty in hydrolyzing ATP to supply sufficient energy for the development of pollen fter the tetrd stge. The dysfunction of the two mitochondril enzymes will consequently result in n nther mitochondril defect nd upset the energy lnce tht is necessry for proper nther development. The mitochondril enzyme dysfunction in the OPC system might cuse the nther ortion in the CMS line. Therefore, we deduced tht the novel orf507 nd Ψtp6-2gene might e ssocited with dysfunction of cytochrome c oxidse, F 1 F o -ATP synthse in this CMS type, respectively. By identifying the gene expression of orf507 nd Ψtp6-2 nd their corresponding enzyme functions, it is hoped tht this pper will help to revel the full mechnism of the CMS phenomenon in chili pepper. On the sis of these studies, we will perform Virus-Induced Gene Silencing (VIGS) nd genetic trnsformtion to further explore the reltionships of these two genes with mitochondril enzymes in oxidtive phosphoryltion systems nd CMS. Acknowledgments This work ws supported y the Ntionl Nturl Science Foundtion of Chin (No ), The Twelfth Five-Yer Pln of Ntionl Science nd Technology in Rurl Ares (No. 2011BAD12B03), the Shnxi Provincil Science nd Technology Development Project (No. 2011K02-09), Tng Zhongying Breeding Fund of Northwest A&F University, nd the Shnxi Provincil Science nd Technology Coordinting Innovtive Engineering Project (2012KTCL02-09). Lnguge help ws provided y Americn Journl Experts.

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