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Chlorosis of Ogura-CMS Brassica rapa is due to down-regulation of genes for chloroplast proteins
J Plant Biotechnol 2017;44:115-124
Published online June 30, 2017
© 2017 The Korean Society for Plant Biotechnology.

Seok-Won Jeong, Hankuil Yi, Hayoung Song, Soo-Seong Lee, Youn-Il Park, and Yoonkang Hur*

BioBreeding Institute, Business Incubation, Chung-Ang University, Ansung, Gyonggi Province 17546, Korea,
Department of Biological Science, College of Biological Science and Biotechnology, Chungnam National University, Daejeon 34134, Korea
Correspondence to: e-mail:
Received June 13, 2017; Revised June 19, 2017; Accepted June 20, 2017.
cc This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Cytoplasmic male sterility (CMS) is a maternally inherited trait leading to loss of the ability to produce fertile pollen and is extensively used in hybrid crop breeding. Ogura- CMS was originally generated by insertion of orf138 upstream of atp8 in the radish mitochondrial genome and transferred to Brassica crops for hybrid breeding. Gene expression changes by dysfunctional mitochondria in Ogura-CMS result in pollen developmental defects, but little is known about gene expression patterns in vegetative tissue. To examine the interaction between nuclear and organellar regulation of gene expression, microarray and subsequent gene expression experiments were conducted with leaves of F1 hybrid Chinese cabbage derived from self- incompatible (SI) or Ogura-CMS parents (Brassica rapa ssp. pekinensis). Out of 24,000 genes deposited on a KBGP24K microarray, 66 genes were up-regulated and 26 genes were down-regulated by over 2.5 fold in the CMS leaves. Up-regulated genes included stress-response genes and mitochondrial protein genes, while genes for ascorbic acid biosynthesis and thylakoid proteins were down-regulated. Most of the major component genes for light reactions of photosynthesis were highly expressed in leaves of both SI and CMS plants, but most of the corresponding proteins were found to be greatly reduced in leaves of CMS plants, indicating posttranscriptional regulation. Reduction in thylakoid proteins and chlorophylls led to reduction in photosynthetic efficiency and chlorosis of Ogura- CMS at low temperatures. This research provides a foundation for studying chloroplast function regulated by mitochondrial signal and for using organelle genome introgression in molecular breeding.

Keywords : Ogura-CMS, microarray, chlorosis, Chinese cabbage, photosynthesis

Cytoplasmic male sterility (CMS) is a maternally-inherited trait that produces either aborted or infertile pollen grains. CMS is a consequence of miscoordination between nuclear and cytoplasmic gene products from different origins (Aviv and Galun 1980). These changes are usually caused by mutations, rearrangements, and/or recombinations in the mitochondrial genome, but not by nuclear gene mutations (Carlsson and Glimelius 2011). At least 14 mitochondrial genes that induce CMS have been characterized in plants (Chase 2007; Kojima et al. 2010). CMS is a valuable tool for commercial production of hybrid seeds in crops (Pelletier and Budar 2007), and is an excellent subject for the study of anterograde and retrograde signaling (Fujii and Toriyama 2008).

Ogura-CMS, originally identified in wild radish (Raphanus sativus) (Ogura, 1968), is controlled by a mitochondrial orf138 locus that consists of two co-transcribed open reading frames: orf138 and orfB (also called atp8, encoding ATP synthase subunit 8) (Bonhomme et al. 1991; Bonhomme et al. 1992; Krishnasamy and Makaroff 1993; Grelon et al. 1994). Brassica napus that contains Ogura-type CMS was originally produced by protoplast fusion (Pelletier et al. 1983) and transferred to Chinese cabbage in the 1980s (Yamagishi and Bhat 2014). Its first F1 hybrid seeds were produced from the CMS lines (Ke et al. 1992); however, these seeds have not been widely used because F1 plants showed a negative effect, chlorosis at low temperature (LT), instead of heterosis. To eliminate these undesirable effects, B. rapa breeders produced new hybrids by protoplast fusion and repeated backcrossing successful in B. napus and B. juncea (Yamagishi and Bhat 2014).

To understand mechanisms of Ogura-CMS in B. rapa, omics approaches have been recently conducted. Using B. rapa 300K microarray, Dong et al. (2013) analyzed genes specific for pollen development stage and concluded that the retrograde signal from Ogura-CMS mitochondria delays expression of large number of nuclear genes involved in pollen development. Wei et al. (2015) identified important miRNAs and their target genes in Ogura-CMS Chinese cabbage using several omics data. However, these two researches have focused on pollen development in floral buds and no omics approaches have been applied to dissect gene expression profiles in vegetative tissues, such as leaf of Ogura-CMS Chinese cabbage.

Mitochondrial influence on the nuclear gene expression is referred to as mitochondrial retrograde regulation (MRR) and it occurs in CMS lines via CMS-inducing genes (Carlsson and Glimelius 2011), making CMS a useful system to study MRR (Chase 2007). Chloroplastic retrograde signaling changes both nuclear (Fernández and Strand 2008; Liao et al. 2016; Woodson 2016) and mitochondrial gene expression (Liao et al. 2016). However, little is known about the regulation of chloroplast genes and nuclear genes for chloroplast proteins by mitochondrial retrograde signaling. Especially, the role of retrograde pathway specific for CMS has never been described for plant vegetative tissues.

Chinese cabbage (B. rapa ssp. pekinensis) is one of the most important leafy vegetables in Asia and exhibits strong heterosis. Application of Ogura-CMS to produce F1 seeds in Chinese cabbage has a high economic potential in seed industry, once accompanying problems like chlorosis have been resolved. To understand chlorosis development in Ogura-CMS Chinese cabbage under LT, we have examined gene expression profiles using KBGP24K microarray and compared chloroplast gene expression and photosynthetic activity. We concluded that chloroplast function was greatly inhibited in Ogura-CMS leaves due to the reduction of chloroplast gene expression by dysfunctional mitochondria.

Materials and Methods

Plant materials

Chinese cabbages (B. rapa ssp. pekinensis) were F1 hybrids obtained using either SI or Ogura-CMS (CMS) in BioBreeding Institute, Korea. Seeds were sown in pots on Aug. 10 and 3 week seedlings were transplanted to bigger pots and field. At 10-leaf stage (Fig. 1A) before the exposure to low temperature (LT) (the mid-September), 7th to 9th leaves were sampled from three individual plants and frozen in liquid nitrogen until use. Leaves from same developmental and environmental conditions were used for measurement of photosynthesis and western blot analysis.

Fig. 1.

Morphology of Chinese cabbage F1 hybrids derived from SI or Ogura-CMS. A, Plants before low-temperature (LT) exposure (mid-September); CMS shows light chlorosis on 7th to 9th leaf, which are indicated by red arrows. B, Plants after LT exposure (mid-October); CMS shows severe chlorosis in young developing leaves

KBGP-24K microarray (Version 1)

Using approximately 24,000 unigenes derived from EST analysis, oligomeric microarray was designed with 12 probes (six sense and six antisense) per gene (Lee et al. 2008). A set of 180,156 probes were designed, and duplicated in two separated block on a single chip. The 60-nucleotide probes with Tm values of 75 to 85°C were synthesized on the slide using NimbleGen System ( Random GC probes to monitor the hybridization efficiency and four corners to overlay the grid on the image were included.

Two biological replicates of total RNA were prepared from each plant sample and 10 μg of total RNA were used for cDNA synthesis with Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen, USA). Subsequent procedures for chip assay were followed as described (Lee et al. 2008). After normalization of probe intensity (Cy3 intensity), perfect match (PM) values of the six probes were used for selection of responsive genes. After removing genes with less than 1,000 PM value at all time points, genes specifically expressed or up-regulated in either tissue were selected and analyzed.

Determination of chlorophyll fluorescence parameters

Changes in in vivo chlorophyll fluorescence were monitored through Xe-pulse amplitude modulated fluorometry (Walz, Germany) using cabbage leaf disc that were dark-adapted for 20 min before measurement. The Fv/Fm value, which is an indicator for maximum PS II efficiency, was calculated as (Fm-F0)/Fm, where Fv is the dark-adapted variable fluorescence, Fm is the maximum fluorescence and F0 is the dark-adapted fluorescence. The actual quantum yield of PSII photochemistry in light-adapted cabbage leaf was calculated as 1–F/Fm′, where F is steady-state fluorescence and Fm′ is maximal fluorescence under illumination. Fluorescence quenching parameters were determined by qP, the coefficient of photochemical quenching, as defined by Schreiber et al. (1994), and NPQ (non-photochemical quenching: (Fm/Fm’ – 1) during illumination at 800 μmol photons m-2s-1).

Determination of photosynthetic O2 evolution

Light-response curves of photosynthetic O2 evolution during illumination were determined with a leaf-disc O2 electrode (Oxygraph system, Hansatech, UK) in air with 5% CO2 at 25°C. Various irradiances (50 and 800 μmol photons m-2s-1) were provided using neutral density filters. The temperature was kept constant at 25°C. The Chlorophyll content in leaf segments was determined from aqueous buffered 80% acetone extracts (25 mM Hepes, pH 7.5), as in Porra et al. (1989).

Analysis of proteins related to photosynthesis

Thylakoid protein components were measured immunochemically after isolation of the thylakoid membranes. Intact chloroplasts were isolated from leaves by homogenization (Robinson and Barnett 1988). Thylakoid membranes were resuspended in 10 mM Tricine-NaOH (pH 7.0), 300 mM sucrose, and 5 mM MgCl2. For protein gel blots, the membrane proteins were solubilized in 60 mM Tris-HCl (pH 7.8), 12% (w/v) sucrose, 2% (w/v) SDS, 1 mM EDTA, and 58 mM DTT. Protein gel electrophoresis was performed according to Laemmli (1970). The separated proteins were electrophoretically transferred to Immobilon-P membrane (Millipore). Chemiluminescence detection using antibodies was performed according to the manufacturer’s instructions (Amersham Pharmacia Biotech.: ECL + Plus). Polyclonal antibodies raised against specific photosynthetic components were purchased from Agrisera Co (Vännäs, Sweden) (Table 1). The soluble protein contents were measured using BioRad protein assay reagents according to the manufacturer’s instructions.

List of polyclonal antibodies used in this study. All antibodies were purchased from Agrisera Co. Ltd (Vännäs, Sweden)

Gene locationAntibody nameProtein nameClassification
PlastidPsbAPhotosystem II protein D1PSII
CytfCytochrome f protein (PetA) of thylakoid Cytb6/f-complexElectron transport
PsaAPhotosystem I P700 chlorophyll a apoprotein A1PSI
PsaCPhotosystem I iron-sulfur centerPSI

NucleusLhcB1LHCII chlorophyll a/b binding protein 1-(1-5)LHCII
LhcB2LHCII type II chlorophyll a/b-binding proteinLHCII
LhcA1PSI type I chlorophyll a/b-binding proteinLHCI
LhcA2PSI type II chlorophyll a/b-binding proteinLHCI


Morphology of Chinese cabbage F1 hybrids derived from SI or Ogura-CMS

Since cultivated Chinese cabbage varieties are F1 hybrids, we have focused on leaves of F1 hybrids derived from SI and Ogura-CMS. As shown in Figure 1, CMS Chinese cabbage exhibited slight pale green before the exposure to LT for long period of time (the mid-September in Daejeon, Korea), but severe chlorosis in young developing leaves after the exposure to LT (the mid-October). These phenomena appear to be similar to that of previous work (Pelletier et al. 1983) and imply defective in photosynthetic efficiency or assembly of photosynthetic electron transport. All experiments were performed with slight pale green leaves (minor chlorosis).

Analysis of differentially expressed genes (DEGs)

To identify DEGs in leaves of Ogura-CMS Chinese cabbage, transcriptomics experiment was carried out with KBGP24 oligomeric chips (Supplementary Table 1). Out of 24,000 genes, 66 genes and 26 genes were up-regulated and down- regulated over 2.5 fold in the CMS, respectively (Table 2 and 3). Many up-regulated genes, such as HSP70s and HSP90s, are stress-related genes. Interestingly, genes encoding mitochondrial components were also up-regulated in CMS: mitochondrial respiratory chain complex I (BRAS0001S00 003192), mitochondrial ATPase subunit 8 (BRAS0001S00 010278) (Table 2). The highest up-regulated gene was BRAS 0001S00026533, which is related to a cis-cinnamic acid responsive gene (AT2G01520) in Arabidopsis thaliana. AT2G01520 is a member of the major latex protein-like gene family, and plays a role in promoting vegetative growth or delaying flowering. On the other hand, down-regulated genes in CMS included a cytoplasmic phosphomannomutase–like gene (BRAS0001S00017904) and putative components for photosynthesis light reaction, such as YCF4 (BRAS000 1S00017830) and LHC2 (BRAS0001S00000039) (Table 3). These results suggest that protection for photosystems and light reaction efficiency could be greatly reduced in CMS Chinese cabbage. One more interesting finding was EPITHIOSPECIFIER MODIFIER1 (ESM10) genes (BRAS0001 S00010846 and BRAS0001S00013286) were highly down- regulated in CMS, altering glucosinolate hydrolysis and increasing insect feeding (Zhang et al. 2006).

Genes up-regulated in CMS by over 2.5-fold

Br_SEQ_IDAt_Locus    Gene DescriptionFold Change (CMS/SI)
BRAS0001S00026533AT2G01520MLP328 (MLP-like protein 328)21.04
BRAS0001S00022245No_HitA09 sequence (3’UTR)13.67
BRAS0001S00003192AT3G08610Unknown (mitochondrial respiratory chain complex I)13.56
BRAS0001S00000806AT4G24420RNA-binding (RRM/RBD/RNP motifs) family protein9.38
BRAS0001S00010278AT2G07707Plant mitochondrial ATPase, F0 complex, subunit 8 protein7.36
BRAS0001S00022734No_HitBra002978: Brassicarapa putativebeta-glucosidase41(LOC103844910)7.09
BRAS0001S00024268AT5G56010Bra035593: HSP90.3 (heat shock protein 81-3)6.44
BRAS0001S00015630AT4G24450GWD2; PWD (phosphoglucan, water dikinase) (involved in phosphorylation)6.29
BRAS0001S00017181AT3G48000Aldehyde dehydrogenase 25.69
BRAS0001S00017773AT2G07708Unknown protein (mitochondrion)5.40
BRAS0001S00004814AT1G70850MLP-LIKE PROTEIN 34 (MLP34)5.02
BRAS0001S00002171AT2G29460GSTU4 (Glutathione S-transferase 22)4.90
BRAS0001S00016599AT2G25140CLPB-M (Casein lytic proteinase B4)/HSP98.74.90
BRAS0001S00017434AT1G66130NAD(P)-binding Rossamann-fold superfamily protein4.64
BRAS0001S00018422AT3G49620DIN11 (Dark inducible 11)4.54
BRAS0001S00022560No_HitBra030240 (no_hit_found)4.51
BRAS0001S00023080No_HitBra015764 (Brassicarapanucleolin2-like:LOC103832086)4.41
BRAS0001S00006395AT2G40280S-adenosyl-L-methionine-dependent methyltransferases superfamily protein4.04
BRAS0001S00006011AT3G09350FES1A (Encodes one of the Arabidopsis orthologs of HspBP-1 and yeast Fes1p:Fes1A)4.01
BRAS0001S00003420AT3G56060Glucose-methanol-choline (GMC) oxidoreductase family protein3.66
BRAS0001S00019692AT2G18860Bra038827; Syntaxin/t-SNARE family protein3.64
BRAS0001S00000758AT1G18540Ribosomal protein L6 family protein3.60
BRAS0001S00019993AT4G19840PP2-A1 (PHLOEM PROTEIN 2-A1)3.52
BRAS0001S00018407AT4G19645TRAM, LAG1 and CLN8 (TLC) lipid-sensing domain containing protein3.52
BRAS0001S00017767AT5G65070Agamous-like 69 (AGL69, FCL4, MAF4)3.48
BRAS0001S00013824AT5G56010Hsp90.3 (Hsp81.3)3.46
BRAS0001S00029134AT5G60200Dof-type transcription factor (DOF5.3)3.40
BRAS0001S00017206AT3G06880Transducin/WD40 repeat-like superfamily protein3.27
BRAS0001S00006213AT5G40240Nodulin MtN21-like transporter family protein3.17
BRAS0001S00029341AT4G17910Acyl transferase3.06
BRAS0001S00018291AT2G43650SAS10/C1D family protein (Embryodfective 2777)3.04
BRAS0001S00016302AT1G02820Late embryogenesis abundant 3 (LEA3)3.02
BRAS0001S00028858AT5G64040Encodes the only subunit of photosystem I located entirely in the thylakoid lumen2.99
BRAS0001S00023256AT3G22380Time for Coffee2.98
BRAS0001S00025328AT5G36230ARM repeat superfamily protein2.92
BRAS0001S00001224AT1G23260MMZ1/UEV1A (DNA damage response)2.88
BRAS0001S00005028AT1G49600RNA-binding protein 47A (RBP47A)2.82
BRAS0001S00010541AT1G70830MLP-like protein 28 (MLP28)2.79
BRAS0001S00001764AT2G28000Chaperonin-60 alpha2.77
BRAS0001S00013300AT1G07790Histone 2B (HTB21)2.75
BRAS0001S00015262AT5G59480Haloacid dehalogenase-like hydrolase (HAD) superfamily protein2.72
BRAS0001S00010375AT5G09590Heat shock protein 70 (Hsc70-5)2.71
BRAS0001S00014652AT1G76860Small nuclear ribonucleoprotein family protein (LSM3B)2.66
BRAS0001S00011885AT3G61620Exonuclease RRP412.65
BRAS0001S00011316AT5G40160Ankryin repeat protein EMB5062.63
BRAS0001S00008586AT1G06720P-loop containing nucleoside triphosphate hydrolases superfamily protein2.63
BRAS0001S00004433AT3G19170Preseqeunce protease 12.57
BRAS0001S00002521AT2G37990Ribosome biogenesis regulatory protein (RRS1) family protein2.56
BRAS0001S00009108AT3G29200Chorismate mutase 1, chloroplast (CM1)2.56
BRAS0001S00017531AT4G31210DNA topoisomerase family protein2.56
BRAS0001S00011599AT4G23760COX19-like CHCG family protein2.51
BRAS0001S00017175AT1G19730Thioredoxin-type r (TRX4)2.50
BRAS0001S00003312AT3G48000Aldehyde dehydrogenase 22.50

Genes down-regulated in CMS over 2.5 fold

Br_SEQ_IDAt_LocusGene DescriptionFold Change (CMS/SI)
BRAS0001S00017904AT2G45790Cytoplasmic phosphomannomutase (ascorbate biosynthesis)-4.97
BRAS0001S00017830ATCG00520YCF4 (Encodes a protein required for photosystem I assembly and stability)-4.53
BRAS0001S00013286AT3G14210ESM1 (epithiospecifier modifier 1)-4.06
BRAS0001S00000044AT5G48850SDI1 (sulphur deficincy-induced 1)-3.30
BRAS0001S00009785AT1G52190NPF1.1 (nitrate transporter 1.11)-3.29
BRAS0001S00022937AT1G74670GASA6 (GA-stimulated arabidopsis 6)-2.94
BRAS0001S00018403AT1G25440BBX15 (B-box type zinc finger protein with CCT damain)-2.89
BRAS0001S00006406AT2G44080ARL (ARGOS-LIKE)-2.80
BRAS0001S00003430AT1G65310XTH17 (Xyloglucan-endotransglucosylase/hydrolase 17)-2.77
BRAS0001S00006587AT5G37300WSD1 (wax ester synthase(WS) and diacylglycerol acyltransferase (DGAT)-2.72
BRAS0001S00017873AT3G20370TRAF-like protein-2.56
BRAS0001S00019530AT2G34620Mitochondrial tranascription termination factor family protein-2.55
BRAS0001S00000993AT5G14030Transiocon-associated protein beta (TRAPB) family protein-2.54
BRAS0001S00010320AT4G03560CCH1 (Calcium channel 1)-2.53
BRAS0001S00008089AT5G19530ACL5 (Acaulis 5)-2.53
BRAS0001S00004281AT5G57800CER3 (Eceriferum 3)(similar to sterol desaturase family)-2.51
BRAS0001S00019903AT1G28290AGP31 (Arabinogalactan protein 31)(vascular tissue function)-2.50

Expression of photosynthesis-related genes

Expression of photosynthesis-related genes (encoding proteins for photosystem, electron transport and CO2 fixation) was strongly expressed in general, but there was no significant difference detected between SI and CMS Chinese cabbage at the transcript level (Table 4). Among LIGHT HARVESTING COMPLEX B (LHCB) 2.4 (= CYCLIN-DEPENDENT KINASE E1; CDKE1) paralogs, only BRAS0001S00000039 was highly up-regulated in SI compared to that of CMS.

Expression of photosynthesis-related genes from SI and CMS F1Brassica rapa

ClassificationAt_LocusGene AnnotationBrSEQ_IDProbe IntensityFold Change

AT3G22370LHCb6 proteinBRAS0001S0001378337311289831.30.8
AT5G63610LHCa2 protein 2BRAS0001S0000042333626326221.01.0
AT1G60950Putative LHCa2 proteinBRAS0001S00010799160214521.10.9
AT4G30650Chlorophyll a/b-binding protein CP26 in PS IIBRAS0001S0001156455770535361.01.0
AT1G29910Chlorophyll a/b binding proteinBRAS0001S0002122955707554731.01.0
AT5G38420Putative chlorophyll a/b-binding proteinBRAS0001S0000005655233528431.01.0
ATCG00740Chlorophyll a/b binding proteinBRAS0001S0000003154957559971.01.0
AT3G63160Photosystem II chlorophyll-binding protein PsbSBRAS0001S0001341954853483281.10.9
AT1G29930Chlorophyll a/b binding proteinBRAS0001S0001351850797538930.91.1
AT2G39730Chlorophyll a/b-binding protein CP26 in PS IIBRAS0001S0001365449470436641.10.9
AT1G44575PSI type III chlorophyll a/b-binding proteinBRAS0001S0000042149460412221.20.8
ATCG00530Chlorophyll a/b binding proteinBRAS0001S0000021745484495440.91.1
AT3G22120Chlorophyll a/b-binding protein-likeBRAS0001S0000038543766330511.30.8
ATCG01100Chlorophyll A-B binding protein / LHCI type I (CAB)BRAS0001S0001137541492312801.30.8
AT1G29910Putative chlorophyll a/b-binding proteinBRAS0001S0000068641411266741.60.6
AT2G07727LHCb3 chlorophyll a/b binding proteinBRAS0001S0001346941059327601.30.8
AT4G29350Chlorophyll a/b binding proteinBRAS0001S0001349439981400651.01.0
AT5G54770Chlorophyll a/b-binding protein-likeBRAS0001S0002613538668414530.91.1
AT2G06520Photosystem II chlorophyll-binding protein PsbSBRAS0001S0000018637576471640.81.3
AT3G04120Chlorophyll a/b binding proteinBRAS0001S0000006433412327561.01.0
AT2G15970Chlorophyll a/b-binding protein CP29BRAS0001S0000068530857286071.10.9
AT1G54780PSI type III chlorophyll a/b-binding proteinBRAS0001S0000042225078261561.01.0
AT1G08380Putative chlorophyll a/b binding proteinBRAS0001S0001419119691224350.91.1
AT3G62030Chlorophyll A-B binding family proteinBRAS0001S00015167152327270.61.8
AT1G55670Chlorophyll a/b-binding proteinBRAS0001S000239797947231.10.9
AT5G54160Putative chlorophyll a/b binding proteinBRAS0001S000103843766120.61.6
AT4G10340Chlorophyll a/b-binding protein (cab-12)BRAS0001S000001803363131.10.9
AT1G61520PSI type III chlorophyll a/b-binding proteinBRAS0001S000136792892981.01.0

Electron TransportAT3G16670Cytochrome b561BRAS0001S0000177827306282351.01.0
AT5G38420Cytochrome b559BRAS0001S0000731211509155510.71.4
AT5G17870Cytochrome b5BRAS0001S0001336610695108761.01.0
ATCG00480Cytochrome b5BRAS0001S00000053574858531.01.0
AT2G43560Cytochrome b6BRAS0001S00008540435658010.81.3
ATCG00630Putative cytochrome b5BRAS0001S00000068416857720.71.4
AT5G02380Cytochrome b5BRAS0001S00018351410160010.71.5
ATCG00140Cytochrome b5BRAS0001S00018217380347620.81.3
AT5G38410Cytochrome b-561DBRAS0001S00009511263319041.40.7
AT2G02100Cytochrome b-561DBRAS0001S00017536208517181.20.8
AT1G12090Cytochrome b5 domain-containing proteinBRAS0001S00010516125215140.81.2
AT1G08380Putative cytochrome b5BRAS0001S000055588446651.30.8
AT4G10340Putative cytochrome b561BRAS0001S000053357256311.10.9
AT1G29910Cytochrome b apoenzymeBRAS0001S000223683683611.01.0
AT3G56940Putative cytochrome b5BRAS0001S000119292372680.91.1
AT5G18070Plastocyanin-like domain-containing proteinBRAS0001S00019135384233331.20.9

CO2 FixationAT1G20620Ribulose bisphosphate carboxylase /oxygenase small subunitBRAS0001S0001958164284631881.01.0
AT5G38420Ribulose bisphosphate carboxylase /oxygenase small subunitBRAS0001S0001335859841607231.01.0
AT4G29350Ribulose bisphosphate carboxylase /oxygenase small subunitBRAS0001S0001353559037619721.01.0
AT1G08380Ribulose bisphosphate carboxylaseBRAS0001S0000018857299605600.91.1
AT1G07920Ribulose bisphosphate carboxylase /oxygenase small subunitBRAS0001S0000003249140467261.11.0
AT4G37210Ribulose bisphosphate carboxylaseBRAS0001S00013364925495981.01.0

To answer whether mitochondrial signal in Ogura-CMS affects expression of chloroplast and nucleus-encoded thylakoid proteins, expression levels of 8 proteins listed in Table 1 were examined by western blot analysis (Fig. 2). Except PsaA and PsaC, expressions of all other proteins showed a great reduction in CMS, suggesting that expression of these genes are regulated at the post-transcriptional levels. This result also revealed that mitochondria in Ogura-CMS affect plastid gene expression, along with nuclear gene expression.

Fig. 2.

Western blot analysis of thylakoid membrane components involved in the light reactions of photosynthesis

Photosynthesis efficiency and chlorophyll content

Since protein levels associated with light reaction of photosynthesis were greatly reduced in CMS leaves (Fig. 2), we suspected that the pigment contents for photosynthetic reaction center would also be low in CMS-leaves. As shown in Figure 3, both chlorophyll a and b levels were low in CMS- leaves, possibly related to the observation that Ogura-CMS Chinese cabbage develops chlorosis in LT (Fig. 1). Reduction in thylakoid proteins and chlorophyll a/b has caused a lower photosynthetic efficiency in CMS-leaves (Fig. 4). It was found that both chlorophyll fluorescence parameter and O2 evolution were low in CMS-leaves. Yield expressed as electron flux through PSII was also low in CMS-leaves (Fig. 4A) and these results were consistent with the rate of oxygen evolution under the high light (Fig. 4B). The higher excitation pressure combined with lower non-photochemical quenching detected in CMS-leaves may be responsible for less resistance to high light in certain stress conditions, such as LT.

Fig. 3.

Chlorophyll content of leaves from SI- and CMS-derived F1 hybrids of Chinese cabbage

Fig. 4.

Photosynthetic efficiencies of SI- and CMS-derived F1 hybrids: Chlorophyll fluorescence parameters (A) and O2 evolution (B). A, Electron flux through PSII (Yield), excitation pressure (1- qP), and non-photochemical quenching parameter (NPQ) in Chinese cabbage leaves under irradiance of 800 μmol photons m-2s-1. B, Light-response curves of photosynthetic O2 evolution in Chinese cabbage leaves.a


CMS is important for hybrid breeding of crop plants and Ogura-CMS from wild radish can be an option for Chinese cabbage, which is an important leafy vegetable in Korea. However, F1 hybrids B. rapa derived from Ogura-CMS could not be widely used because F1 plants did not show heterosis but instead developed severe chlorosis under low temperature (Ke et al. 1992). This undesirable effect is due to the incompatibility between chloroplast and nucleus, and the problem could be overcome by chloroplast substitution, which involved somatic hybridization and repeated backcrossing to cabbage (Dey et al. 2013). With similar approaches being tried, more detailed understanding of the mechanism by which leaf chlorosis is induced can accelerate breeding efforts for Chinese cabbage.

From transcriptome analysis, it was found that DEGs in Ogura-CMS leaves (Table 2 and 3) are less obvious in gene numbers and fold changes in expression compared to those observed with male gametophyte (Dong et al. 2013; Wei et al. 2015). At the transcript level, most genes involved in photosynthesis were highly expressed in leaves of both SI- and CMS-derived F1 hybrid (Table 4 and Supplementary Table 1). However, accumulation of the proteins showed clear difference between two genotypes (Fig. 2), implying that expression of these genes are regulated at the post- transcriptional level. With reduced levels of thylakoid components, the amount of photosynthetic pigments and photosynthesis efficiency were also decreased (Fig. 3 and 4). In addition, heat stress-related protein genes were highly up-regulated in CMS-leaves (Table 2), suggesting the CMS mimics the effects of oxidative stress conditions. Particularly, down-regulation of phosphomannomutase gene in Ogura-CMS leaves implied that the protective capability of photosystem under oxidative stress is decreased. This gene is involved in ascorbate biosynthesis, which is related to high temperature tolerance (Hoeberichts et al. 2008). Ogura-CMS chloroplasts appear to be impaired in removal of excess energy absorbed by photosystems under high light (Fig. 4A), leading to the loss of chlorophyll pigments (Fig. 4B).

Both mitochondria and chloroplasts are important to maintain metabolic and energy homeostasis in the plant cell. Therefore, extensive researches on the interaction between these organelles have been carried out with respect to photosynthesis and respiration at physiological levels. But, only one paper (Liao et al. 2016) has mentioned that dysfunctional chloroplast is related to the up-regulation of mitochondrial gene expression in Arabidopsis. Our results is the first report for the chloroplast gene expression change by dysfunctional mitochondria, showing accumulation of chloroplast proteins can be regulated by mitochondrial signal.

Plant mutations responsible for mitochondrial dysfunction result in change of nuclear gene expression (Newton et al. 2004; Dong et al. 2013), and several candidate signals have suggested: redox sensors and signals, kinases/phosphatases, hormones, and other sensors (Rhoads 2011). Recently, key genes for retrograde signaling for chloroplast and mitochondria have been identified (Giraud et al. 2009; Blanco et al. 2014; Saha et al. 2016). The expression of these four key genes in our experiments using Chinese cabbage was not similar to Arabidopsis data (Table 5). Only one paralog (BRAS000 1S00000039) corresponding to Arabidopsis AT3G27690 (LHCb) was differentially expressed between SI- and CMS leaves. These results may suggest that retrograde signaling or organelle interaction is regulated at the protein level or different signaling components unique to species are used. Considering that finding of the best combination between nucleus and organelles is prerequisite for CMS-based breeding, molecular mechanisms associated with CMS need to be further elucidated.

Expression of key retrograde signaling genes for chloroplast and mitochondria

At_LocusGene AnnotationBrSEQ_IDPI (Probe intensity)

 AT3G27690 LIGHT HARVESTING COMPLEX B (LHCB) 2.4 BRAS0001S00000036 57,49848,062

AT3G22370ALTERNATIVE OXIDASE1aBRAS0001S000000621,3781,372

AT5G63610CYCLIN-DEPENDENT KINASE E1 (CDKE1)BRAS0001S000273743,7883,714



This work was supported by Research Fund of Chungnam National University (CNU), Daejeon, Korea, to Yoonkang Hur (2016).

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