A Cytoplasmic Male Sterility-Associated Mitochondrial Protein Causes Pollen Disruption in Transgenic Tobacco (Callose Deposition/Common Bean) SHICHUAN HE*T, ANDRE R
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Proc. Natl. Acad. Sci. USA Vol. 93, pp. 11763-11768, October 1996 Genetics A cytoplasmic male sterility-associated mitochondrial protein causes pollen disruption in transgenic tobacco (callose deposition/common bean) SHICHUAN HE*t, ANDRE R. ABAD*, STANTON B. GELVINt, AND SALLY A. MACKENZIE*§ Departments of *Agronomy and tBiological Sciences, Purdue University, West Lafayette, IN 47907 Communicated by Charles C. Levings, III, North Carolina State University, Raleigh, NC, June 24, 1996 (received for review January 25, 1996) ABSTRACT In higher plants, dominant mitochondrial product of orf239, 27 kDa in size, is present only in the mutations are associated with pollen sterility. This phenom- reproductive tissues (12), implying that it is the gene respon- enon is known as cytoplasmic male sterility (CMS). It is sible for the pollen disruption event. Immunogold labeling thought that the disruption in pollen development is a con- studies using polyclonal antibodies against the ORF239 pro- sequence of mitochondrial dysfunction. To provide definitive tein have shown that the protein localizes to the callose layer evidence that expression of an abnormal mitochondrial gene and primary cell wall of developing pollen (12). Because pollen can interrupt pollen development, a CMS-associated mito- disruption in CMS common bean can be accompanied by the chondrial DNA sequence from common bean, orf239, was failure of normal callose deposition, it is possible that the introduced into the tobacco nuclear genome. Several trans- specific localization of the ORF239 peptide at the periphery of formants containing the orf239 gene constructs, with or the cell may interfere with normal cell wall deposition, leading without a mitochondrial targeting sequence, exhibited a semi- to incomplete cytokinesis, whereas the mitochondria serve sterile or male-sterile phenotype. Expression of the gene only as the sites of ORF239 protein synthesis. To provide fusions in transformed anthers was confirmed using RNA gel decisive evidence of ORF239 as the causative agent in the blotting, ELISA, and light and electron microscopic immu- disruption of pollen development and to investigate the bio- nocytochemistry. Immunocytological analysis showed that the logical implications of the observed extracellular localization 0RF239 protein could associate with the cell wall of aberrant of ORF239 to the cell wall, we have conducted transformation developing microspores. This pattern of extracellular local- experiments in Nicotiana tabacum, an unrelated plant species. ization was earlier observed in the CMS common bean line containing orf239 in the mitochondrial genome. Results pre- MATERIALS AND METHODS sented here demonstrate that 0RF239 causes pollen disrup- Transgene Constructions. For the development of trans- tion in transgenic tobacco plants and may do so without gene constructions, we used two different plant gene promot- targeting of the protein to the mitochondrion. ers: a pollen-specific promoter, PLat52 (13, 14), and a strong constitutive promoter, Psp (15). The mitochondrial transit The mitochondrion, as a cellular center for energy metabolism, sequence used was derived from the Nicotiana plumbaginifolia serves essential functions in the development of eukaryotic X3 subunit of mitochondrial F1-ATPase (atp2-1; ref. 16). The organisms. In plants, one of the developmental transitions that pollen-specific promoter PLatL2, the mitochondrial transit se- appears to be particularly influenced by mitochondrial func- quence, and the orf239 sequences used in the constructions tion is male reproductive (pollen) development. Mutations in were amplified by PCR to facilitate cloning. Promoter Psp is the mitochondrial genome most commonly result in the in- composed of three tandem copies of theAgrobacterium tume- ability of the plant to shed viable pollen. This phenomenon, faciens octopine synthase activator, one copy of the mannopine known as cytoplasmic male sterility (CMS) and observed in synthase 2' activator, and the mannopine synthase 2' promoter more than 150 plant species, has presented the opportunity to (15). It was cloned into a modified pBI101 transferred DNA investigate the special functions provided by mitochondria at binary vector, with XbaI-NotI-SacI cloning sites at its 3' end, this crucial developmental stage. followed by a poly(A) tail signal. This vector is designated The association of CMS with abnormal mitochondrial gene pATC940. PLat52, composed of 666 bp immediately upstream expression has been established in many plant species includ- to the ATG codon, was PCR-amplified from tomato total ing maize (1, 2), petunia (3, 4), sunflower (5-8), and common genomic DNA using two primers that were designed to intro- bean (9, 10). In all cases, pollen disruption in CMS lines is duce a HindIII site at the 5' end and anXbaI site at the 3' end. accompanied by the expression of novel mitochondrial DNA The primer sequences are 5'-CGCAAGCTTGGATAAGGG sequences that contain protein-coding open reading frames. TAGCTCTATCTA-3', and 5'-CCTCTAGATTGGAATT To date, there are two important issues regarding CMS yet to TTTTTTTTTGGTGT-3', respectively. The mitochondrial be resolved. (i) Definitive evidence that these aberrant mito- transit sequence was the N-terminal 60-aa sequence of atp2-1. chondrial genes actually cause pollen disruption is not yet This peptide targets foreign proteins to the mitochondria and available in any plant system, and (ii) the molecular mecha- is cleaved between the 54th and 55th amino acids after nisms effecting the disruption of pollen development are still translocation in transgenic tobacco (16). The DNA sequence not understood. was amplified from N. plumbaginifolia total genomic DNA In CMS common bean, male-sterile plants contain a novel using two primers introducing an XbaI site at the 5' end and mitochondrial DNA sequence (designated pvs) of unknown a KpnI site at the 3' end. The primer sequences are 5'- origin that is associated with the pollen sterility phenotype by CCTCTAGACCATGGCTTCTCGGAGGCTTCT-3' and 5'- various genetic criteria (10, 11). The 3.7-kb pvs sequence CCGGTACCGCTGCGGAGGTAGCGTACTG-3', respec- contains at least two open reading frames, ocf239 and orf98. tively. The mitochondrial pvs-orf239 DNA sequence was am- Immunohistochemical studies have shown that the protein Abbreviations: CMS, cytoplasmic male sterility; W38, Wisconsin 38. The publication costs of this article were defrayed in part by page charge tPresent address: Section of Genetics and Development, Cornell payment. This article must therefore be hereby marked "advertisement" inI University, Ithaca, NY 14853. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 11763 Downloaded by guest on September 28, 2021 11764 Genetics: He et al. Proc. Natl. Acad. Sci. USA 93 (1996) plified from a CMS bean mitochondrial cDNA clone Wisconsin 38 (W38)] were transformed by a leaf disk trans- containing the pvs sequence. With use of specially designed formation method (19). Shoots were rooted and propagated as primers, convenient cloning sites were introduced to the described by Ni et al. (15). orf239 fragment. The 5' primer 5'-CCGGTACCCATGTTC Transgenic Phenotype Evaluation. Putative transformants CTCCCATTCAACCCA-3' contains a KpnI site that, upon were characterized using several criteria. Regenerated seed- ligation, forms an in-frame chimeric gene with the 183-bp ling leaf tissues were processed for PCR, DNA gel blot, and transit sequence of atp2-1 (Fig. 1). The 61st amino acid of RNA gel blot analyses to test for presence of the transgene and ATP2-1 is changed from Ala to Val followed immediately by its transcripts. Total genomic DNA was prepared following the the first AUG condon of ORF239. protocol described by Vallejos et al. (20), and the RNA The procedures for cloning into manipulating vectors are as preparation procedure was taken from Powlowski et al. (21). follows. A PCR-amplified orf239 fragment was digested with DNA and RNA gel blot analyses were according to standard KpnI and Sacl, and then ligated to the KpnI-SacI site of the procedures (22). Phagemid vector pBS(+) (Stratagene), resulting in the con- Regenerated tobacco plants were grown to flowering. At struction conl. The PCR-amplified atp2-1 fragment was di- flowering, sterile plants were classified based on plant mor- gested with XbaI and KpnI, and ligated to XbaI- and KpnI- phology, flower morphology, and seed set. Male-sterile plants digested conl. The resulting construction was designated con2. were classified as those that demonstrated a compacted floral To clone the LatS2 promoter, the PCR-amplified fragment was head (also evident in shorter plant stature); collapsed, gray digested with HindlIl and XbaI and ligated to HindIII-XbaI- anthers recessed below the level ofthe stigma (Fig. 2); and little digested pBS(+), forming the construction con5. The HindIll- or no seed set within capsules. Semisterile plants were those XbaI LatS2 fragment from con5 was then ligated to the that demonstrated recessed anthers but intermediate pheno- HindlIl- and XbaI-digested conl and con2, respectively, re- types for all other characters. Pollinations using wild-type sulting in constructions con6 and con7. The DNA sequences of pollen (W38) allowed us to confirm female fertility. these constructions were then confirmed. To place the chi- ELISA Assays. We experienced certain technical difficulties in meric genes under the control of Psp in pATC940, the insert extracting enough ORF239