Exceptional transmission of plastids and mitochondria from the transplastomic pollen parent and its impact on transgene containment

Abstract

Plastids in Nicotiana tabacum are normally transmitted to the progeny by the maternal parent only. However, low-frequency paternal plastid transmission has been reported in crosses involving parents with an alien cytoplasm. Our objective was to determine whether paternal plastids are transmitted in crosses between parents with the normal cytoplasm. The transplastomic father lines carried a spectinomycin resistance (aadA) transgene incorporated in the plastid genome. The mother lines in the crosses were either (i) alloplasmic, with the Nicotiana undulata cytoplasm that confers cytoplasmic male sterility (CMS92) or (ii) normal, with the fertile N. tabacum cytoplasm. Here we report that plastids from the transplastomic father were transmitted in both cases at low (10 ؊4 -10 ؊5 ) frequencies; therefore, rare paternal pollen transmission is not simply due to breakdown of normal controls caused by the alien cytoplasm. Furthermore, we have found that the entire plastid genome was transmitted by pollen rather than small plastid genome (ptDNA) fragments. Interestingly, the plants, which inherited paternal plastids, also carried paternal mitochondrial DNA, indicating cotransmission of plastids and mitochondria in the same pollen. The detection of rare paternal plastid transmission described here was facilitated by direct selection for the transplastomic spectinomycin resistance marker in tissue culture; therefore, recovery of rare paternal plastids in the germline is less likely to occur under field conditions. Nicotiana tabacum ͉ organelle inheritance ͉ plastid transformation ͉ pollen transmission D NA in a plant cell is found in three cellular compartments: the nucleus, plastids, and mitochondria. Genes encoded in the nucleus are inherited biparentally, according to Mendel's rules. In contrast, plastids and mitochondria may be inherited maternally, paternally, or from both parents. In most crops, the maternal parent transmits plastids, because plastids are excluded from the sperm cell or, even if not excluded, are left behind in synergid cells during fertilization (1-3). In Chlamydomonas reinhardtii, a unicellular alga, in which maternal (mating-type ϩ ) and paternal (mating-type Ϫ ) chloroplasts fuse, only the maternal plastid genome (ptDNA) is inherited, because the maternal ptDNA is protected by methylation, whereas the nonmethylated paternal ptDNA is degraded (4). Once protocols for plastid transformation were developed (5, 6), localization of transgenes in the plastid genome was advocated as a means of transgene containment (7). Utility of plastid localization for containment became a hotly debated issue when the first herbicide-resistant transplastomic plants were obtained (8, 9). The reason for skepticism was the reported relatively high-frequency paternal ptDNA transmission in species in which plastids were assumed to be inherited strictly by the maternal parent. Paternal ptDNA transmission was detected in crosses by using streptomycin resistance (in 0.07-2.5%) (10, 11) and tentoxin resistance (0.5-2.5%) (12) in...

Inducible gene expression from the plastid genome by a synthetic riboswitch

“…Transgene expression from the plant's plastid genome has unique attractions to biotechnologists (28), including the plastids' potential to accumulate foreign proteins to extraordinarily high levels [to up to more than 70% of the plant's total soluble protein (29)] and the increased biosafety provided by the maternal mode of plastid inheritance, which greatly reduces unwanted transgene transmission via pollen (30,31). However, constitutive expression of pharmaceutical proteins or unique metabolic pathways from the plastid genome can result in mutant phenotypes and/or severe growth retardation of transplastomic plants due to metabolite toxicities, interference with photosynthesis or disturbance of the plastid endomembrane system (e.g., refs.…”

“…This is highly unfortunate, because tightly controllable expression systems represent versatile tools in both functional genomics and biotechnology. The possibility to express transgenes from the plastid genome has stirred considerable excitement among plant biotechnologists (23,27,28), mainly due to the attainable enormous expression levels (29) and the increased transgene containment provided by maternal inheritance of plastids in most crops (30,31). However, constitutive plastid transgene expression can cause severe mutant phenotypes (e.g., 32, 33) and, also for this reason, the development of tightly controllable systems for inducible gene expression is of utmost importance.…”

Mitochondrial mRNA Polymorphisms in Different Arabidopsis Accessions

“…In dicot plants, mtDNA is generally transmitted maternally, whereas the nuclear genome is inherited biparentally, with only rare exceptions from this rule having been reported recently for tobacco (Nicotiana tabacum; Svab and Maliga, 2007). Thus, the inheritance patterns of respective mRNAs in F 1 offspring obtained after reciprocal crosses of the analyzed accessions should allow discrimination, whether a polymorphism is due to differences in the mitochondrial or nuclear DNA.…”

High Levels of Bioplastic Are Produced in Fertile Transplastomic Tobacco Plants Engineered with a Synthetic Operon for the Production of Polyhydroxybutyrate

“…Plastid transformation systems allow the expression of multiple genes in polycistronic synthetic operons, and the maternal inheritance of plastid DNA in most plants provides a level of, although possibly not complete (Svab and Maliga, 2007;Sheppard et al, 2008), gene containment. Disadvantages of plastid transformation systems include the limited number of plants that can be routinely transformed and the extent of unwanted recombination that can occur if plastid transformation vectors contain sequences that are homologous to the host's plastome.…”