Mitochondrially-targeted expression of a cytoplasmic male sterility-associated orf220 gene causes male sterility in Brassica juncea
© Yang et al; licensee BioMed Central Ltd. 2010
Received: 11 May 2010
Accepted: 26 October 2010
Published: 26 October 2010
The novel chimeric open reading frame (orf) resulting from the rearrangement of a mitochondrial genome is generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). Both positive and negative correlations have been found between CMS-associated orfs and the occurrence of CMS when CMS-associated orfs were expressed and targeted at mitochondria. Some orfs cause male sterility or semi-sterility, while some do not. Little is currently known about how mitochondrial factor regulates the expression of the nuclear genes involved in male sterility. The purpose of this study was to investigate the biological function of a candidate CMS-associated orf220 gene, newly isolated from cytoplasmic male-sterile stem mustard, and show how mitochondrial retrograde regulated nuclear gene expression is related to male sterility.
It was shown that the ORF220 protein can be guided to the mitochondria using the mitochondrial-targeting sequence of the β subunit of F1-ATPase (atp2-1). Transgenic stem mustard plants expressed the chimeric gene containing the orf220 gene and a mitochondrial-targeting sequence of the β subunit of F1-ATPase (atp2-1). Transgenic plants were male-sterile, most being unable to produce pollen while some could only produce non-vigorous pollen. The transgenic stem mustard plants also showed aberrant floral development identical to that observed in the CMS stem mustard phenotype. Results obtained from oligooarray analysis showed that some genes related to mitochondrial energy metabolism were down-regulated, indicating a weakening of mitochondrial function in transgenic stem mustard. Some genes related to pollen development were shown to be down-regulated in transgenic stem mustard and the expression of some transcription factor genes was also altered.
The work presented furthers our understanding of how the mitochondrially-targeted expression of CMS-associated orf220 gene causes male sterility through retrograde regulation of nuclear gene expression in Brassica juncea.
Cytoplasmic male sterility (CMS), the maternally inherited trait of failure to produce functional pollen, exists in many plant species and has wide application for the production of hybrid crops. CMS can occur at different stages during reproductive development. It is generally believed that CMS is associated with the rearrangement of mitochondrial genomes, which, in many cases, is attributed to the generation of novel open reading frames (orfs) [1–5]. Some experimental evidence confirms the correlation between CMS-associated orfs and the occurrence of CMS. In some studies mitochondrially-targeted expression of novel orfs was shown to lead to male sterility or semi-sterility [6–9], while in others it did not [10–12]. A probable interaction between orfB and the ATP synthesis complex in CMS has been demonstrated in sunflower using 2-D electrophoresis and Western blot analysis . However, the specific role of mitochondrial novel orfs in causing male sterility is not yet clearly established and better evidence that mitochondrially-targeted expression of orfs causes male sterility is needed. In particular, how mitochondrial factor regulates the expression of the nuclear genes involved in male sterility is poorly understood. Is there any cross-talk between mitochondria and the nucleus that ultimately determines the abortion of pollen? If so, how do mitochondrial factors directly or indirectly halt the processes of pollen development, and through which pathway?
Recently, many studies have focused on mitochondrial regulation of nuclear gene expression in higher plants [14–18]. This communication pathway from mitochondria to the nucleus is defined as mitochondrial retrograde regulation (MRR), and has been documented mainly in yeast and animals [19, 20]. Some ABC model genes related to floral organ development, namely the nuclear MADS-box TF genes, have been shown to be targets for floral organ homeotic transformation regulated by MRR [21–26]. In addition, several other nuclear genes have recently been shown to be retrograde regulated by mitochondria in some CMS systems [27–29].
Previously, we isolated the CMS-associated orf220 gene from CMS stem mustard, Brassica juncea, . In the present study, we constructed transgenic stem mustard expressing the chimeric orf220 gene mediated by Agrobacterium tumefaciens. These transgenic stem mustard plants exhibited male sterility. Global changes in the expression of mitochondrial and nuclear genes in transgenic stem mustard were examined using oligoarray analysis.
Chimeric gene construction and transformation of stem mustard
The transformation system for stem mustard established in our laboratory is based on a regeneration system from cotyledons with proximal hypocotyls (Figure 1-B). We used this transformation system to produce transgenic stem mustard plants incorporating the alien chimeric orf220 gene. Candidate transgenic stem mustard plants were checked by PCR and RT-PCR (Figure 1-C) and 5 T0 plants were selected, all exhibiting male sterility. We selected one of these transgenic lines and obtained 28 T1 generation plants, which yielded 13 male-sterile and 15 male-fertile plants. We also used the orf220 gene as a marker to check that this gene was inherited and could be expressed in T1 transgenic stem mustard. The male-sterile phenotype was shown to be genetically transmitted to the T1 generation and associated with orf220 expression (Figure 1-A). All the 13 male-sterile T1 plants expressed the orf220 gene, and all the 15 male-fertile T1 plants lacked orf220 expression (data not shown).
We used the chimeric orf220 gene with GFP expression to check whether this chimeric gene was targeted to the mitochondria. It was shown that the orf220 gene with the mitochondrial-targeting sequence (atp2-1) was located in the mitochondria, however, the orf220 gene without the mitochondrial-targeting sequence (atp2-1) was only found in the nucleus (Figure 1-D).
Phenotypes of transgenic stem mustard
Activity of pollen produced from transgenic stem mustard
Global gene expression patterns in transgenic and WT stem mustard
Down-regulated expressed genes in transgenic stem mustard observed in this study
DNA-binding family protein
protodermal factor 1 (PDF1)
glycosyl hydrolase family 1 protein/anther-specific protein ATA27
cytochrome P450 family protein
cinnamoyl-CoA reductase family
protease inhibitor/seed storage/lipid transfer protein (LTP) family protein
calcium-binding EF hand family protein
ABC transporter family protein
glycosyl hydrolase family 17 protein
male sterility protein 2 (MS2)
major intrinsic family protein/MIP family protein
cytochrome P450 family protein
tRNA pseudouridine synthase family protein
sterol desaturase family protein
pectate lyase family protein/pectate lyase family protein
chalcone and stilbene synthase family protein
alcohol dehydrogenase (ATA1)
cytochrome c oxidase subunit 2
cytochrome c oxidase subunit 1
ATP synthase subunit 9
Up-regulated expressed genes in transgenic stem mustard observed in this study
glutathione S-transferase, putative/glutathione S-transferase, putative
diacylglycerol kinase, putative
sulfate transporter (Sultr1;2)
alcohol dehydrogenase, putative
pectate lyase family protein
calcineurin-like phosphoesterase family protein
beta-fructosidase (BFRUCT4)/beta-fructofuranosidase/invertase, vacuolar
RNA-binding protein, putative
RNA-binding protein, putative
superoxide dismutase (Fe), chloroplast (SODB)/iron superoxide dismutase (FSD1)
adenylylsulfate kinase 2 (AKN2)
homeobox-leucine zipper protein 12 (HB-12)/HD-ZIP transcription factor 12
protein kinase family protein
zinc finger (C3HC4-type RING finger) family protein
beta-galactosidase, putative/lactase, putative
pyruvate phosphate dikinase family protein
The precise mechanism by which mitochondria trigger male sterility is still unknown. It is well known that there is a relationship between novel orfs and the occurrence of CMS, in which orfs play an essential role in disrupting mitochondrial function [1–5]. Meanwhile, several recent studies using cDNA microarrays have identified some nuclear target genes downstream of the pathway of CMS occurrence [27, 28]. However, the exact mechanism underlying the occurrence of CMS, especially the downstream nuclear target genes and retrograde signaling pathway, remains to be exploited.
The evidence varies as to whether mitochondrially-targeted expression of novel orfs can induce male sterility or not. In some cases this leads to male sterility and in others to semi-sterility [6–9]. However, some mitochondrially-targeted expressions of such novel orfs fail to induce either male sterility or semi-sterility [10–12]. The failure of mitochondria-target expression of novel orfs to induce male sterility is probably due to problems of sub-mitochondrial location , the expression period  or the expression amount of ORF protein  in transgenic plants. In the present study, the reproductive phenotype of transgenic stem mustard was extremely similar to those observed in CMS stem mustard ( and supplementary data, Figure 1). From these observations, we concluded that ectopic expression of the chimeric orf220 gene causes male sterility in transgenic stem mustard. And this could be attributed to direct mitochondrial localization of ORF220 protein guided by a mitochondrial-targeting peptide. In transgenic plants, we observed reduced expressions of several mitochondrial genes related to respiratory complex, as caused by ectopic expression of chimeric orf220 gene, which may affect mitochondrial function, although we don't know how this happens.
Functional genes specifically related to pollen development have been well documented till now, of which mutation of any of these genes causes failure of microsporogenesis or abortion of pollen [32–34]. In our study, we observed that many genes related to pollen development were down-regulated in transgenic plants. Furthermore, many same genes in relation to mitochondrial respiratory complex and pollen development were also observed to be down-regulated in transgenic plants and CMS line as well, although we couldn't conclude that these identical down-regulated genes were the causal factor of producing similar phenotype in transgenic plant and CMS line from this study. It is unlikely that mitochondrial proteins alone could directly result in male sterility without a signal transduction from the mitochondria to the nucleus. Such signaling from the mitochondria to the nucleus, termed mitochondrial retrograde regulation, has been well described in yeast and mammals [19, 20], although it is less frequently reported in higher plants. Among the clusters of expressed genes in transgenic and WT plants, we also observed several transcriptional factor (TF) genes were induced by ectopic expression of chimeric orf220 gene, such as, homeobox-leucine zipper family protein (HD-ZIP), DNA/RNA binding protein and zinc finger family protein etc. These TF genes were reported to be involved in the regulation of developmental processes, the response of plants to environmental and redox regulation [35–39]. However, whether these TF genes are associated with mitochondrial retrograde regulation of nuclear gene expression or not need to be further substantiated.
In conclusion, the mitochondrially-targeted expression of orf220 gene was capable of inducing male sterility in transgenic stem mustard. We proposed that the transformation of orf220 gene in stem mustard impairs mitochondrial function, and this response is signaled by the mitochondria to nucleus through a particular signal transduction pathway. How the orf220 gene functions precisely to induce male-sterility and its biochemical characterization remains to be discovered. In addition, further research is worthy of being investigate to explore the signal pathway of mitochondrial retrograde regulation and how nuclear target genes are responsive in order, expecially what is the primary receptor gene in the nucleus, if any.
Construction and transformation of the chimeric orf220
Primers used in chimeric expression vector construction
Primers Sequence (5'-3')
Chimeric gene orf220
Chimeric gene atp2-1
Proximal portions of hypocotyls from cotyledons obtained from 5-day-old aseptic seedlings were pre-cultured (MS + 3 mg/L 6-BA + 0.5 NAA + 3% sucrose + 0.8% agar) for 2 days, and subsequently co-cultured with A. tumefaciens containing the chimeric gene in the dark for 2 days on differentiation medium (MS + 3 mg/L 6-BA + 0.5 NAA + 3% sucrose + 0.8% agar). Shoots were subsequently regenerated for resistance screening from the proximal portions of the cotyledon hypocotyls on differentiation media supplemented with kanamycin (MS + 3 mg/L 6-BA + 0.5 NAA + 20 mg/L kanamycin + 3% sucrose + 0.8% agar). Regenerating shoots (5 cm in length) were cut from explants and rooted in the following medium: 1/2 MS + 0.1 NAA + 10 mg/L kanamycin + 3% sucrose + 0.8% agar. This regeneration system from stem mustard cotyledons was developed in our laboratory .
Construction of GFP fusion vectors and transient expression
The chimeric atp2-1/orf220 and orf220 coding sequences were amplified from the chimeric vector obtained above using standard protocols with the LA Taq PCR system (Takara, Japan), and using specific primers flanked by Gateway recombination cassettes (Invitrogen, California, USA). The primers used here are listed in Table 3. PCR products were cloned into pDONR221 according to the manufacturer's instructions. Cloning into the final GFP vectors (pK7FWG2) was by LR reaction (Invitrogen, California, USA). The mt-RFP plasmid containing the pre-sequence of Arabidopsis thaliana ATPase delta-prime subunit and DsRed2 was provided by Dr. S. Arimura and Prof N. Tsutsumi (Laboratory of Plant Molecular Genetics, The University of Tokyo) .
Biolistic co-transformation of the GFP and RFP fusion vectors was performed on Arabidopsis leaves. In brief, GFP and RFP plasmids (5 μg each) were co-precipitated onto gold particles and transformed using a PDS-100/He biolistic transformation system (Bio-Rad, http://www.bio-rad.com). Healthy Arabidopsis leaves were placed on MS medium and bombarded. Leaves were then incubated for 48 hrs at 22°C before microscopy using a Nikon fluorescence microscope system.
Phenotypic evaluation of transgenic stem mustard
Regenerated maintainer lines of stem mustard plants from medium containing kanamycin (50 mg/L) were treated as putative candidates and were further screened by rooting them on medium containing kanamycin (25 mg/L). Putative transgenic stem mustard, designated as T0, with normal roots was identified using the orf220 gene in PCR and RT-PCR analysis. At flowering, they were pollinated with normal pollen from WT plants. Seeds from the T0 generation were grown as T1 generation and were further identified using the orf220 gene in PCR and RT-PCR analysis. The primers used in these analyses are listed in Table 3. Transgenic stem mustard plants were observed at flowering time. Seeds were set to study male fertility by self-pollination using a bag covering the flower. Female fertility of transgenic plants was confirmed by pollination with WT pollen.
Pollen morphology and activity evaluation
The morphology of pollen grains of transgenic and WT stem mustard was examined using a scanning electron microscope (KYKY-1000B). Pollen activity was evaluated using 2,3,5-triphenyl-2h-tetrazolium chloride (TTC) staining, in vitro germination and in-situ germination. In TTC staining, pollen grains were soaked in 0.1% TTC solution. Active pollen stains red because the NADH/NADPH produced deoxidizes TTC to TTF (which is red). Pollen in vitro germination was performed at 28°C and 100% relative humidity, during which pollen grains were cultured on a liquid medium consisting of boric acid (250 mg/L) and sucrose (10%). Pollen germination success was calculated and photographed after 4 hrs using a microscope (LEICA). To assess in-situ germination, pollen from transgenic and WT plants were placed onto the surface of the stigma and 12 hrs after pollination the pistils were removed and fixed rapidly in FAA fixing solution (ethanol: acetic acid, 3:1) for 2 hrs. The fixed pistils were washed three times with sterile water and treated overnight in softening solution (8 mol/L NaOH). The pistils were then washed in distilled water and stained in 0.1% aniline blue for 3 hrs in the dark. The stained pistils were observed and photographed with a Leica DMRA2 fluorescent microscope.
Floral buds of one inflorescence from WT, transgenic stem mustard, and CMS stem mustard were collected to compare the expression of genes during floral development. Bud samples were ground in liquid nitrogen, and total RNAs were prepared using Trizol reagent according to the manufacturer's protocol (Invitrogen). The oligoarrays used in this study were derived from the Arabidopsis thaliana ATH1 chip. All the hybridization procedures and data analysis were performed by CapitalBio Corp. (Bejing, China). Arrays were scanned with a confocal laser scanner, LuxScan™ 10 K (CapitalBio Corp.), and the resulting images analyzed with SpotData Pro 2.0 software (CapitalBio Corp.). Three biological replicates were performed. Differently expressed genes were identified using the t-test and multiple test corrections were performed using the False Discovery Rate (FDR) . Genes with an FDR <0.01 and a fold change of double or more were considered to be different in gene expression.
For gene annotation, we used the updated TAIR (The Arabidopsis Information Resource) annotation for the Arabidopsis Genome Genechip array http://www.arabidopsis.org and the CapitalBio Corp MAS 2.0 system http://bioinfo.capitalbio.com/mas/. All data were submitted to the CapitalBio Corp MAS 2.0 system http://bioinfo.capitalbio.com/mas/. Genes were classified into functional categories using Gene Ontology information available from TAIR. The putative pathways were identified through the known pathways in the KEGG database provided by the CapitalBio Corp MAS 2.0 system http://bioinfo.capitalbio.com/mas/.
This work was supported by a grant from the National Natural Science Foundation of China (NSFC30800749) and a grant from ZJNSF (Y3080082). We thank Prof. Mikio Nakazono, Prof. Rosine. de Paepe and Prof. Sally A. Mackenzie for providing critical comments on the paper. We thank Dr. Shinyichi Arimura for offering mitochondrially-targeted RFP and assistance in using the Laser Scanning Confocal Microscope.
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