Targeted Inactivation of the Plastid ndhB Gene in Tobacco Results in an Enhanced Sensitivity of Photosynthesis to Moderate Stomatal Closure

Abstract

The ndh genes encoding for the subunits of NAD(P)H dehydrogenase complex represent the largest family of plastid genes without a clearly defined function. Tobacco (Nicotiana tabacum) plastid transformants were produced in which the ndhB gene was inactivated by replacing it with a mutant version possessing translational stops in the coding region. Western-blot analysis indicated that no functional NAD(P)H dehydrogenase complex can be assembled in the plastid transformants. Chlorophyll fluorescence measurements showed that dark reduction of the plastoquinone pool by stromal reductants was impaired in ndhB-inactivated plants. Both the phenotype and photosynthetic performance of the plastid transformants was completely normal under favorable conditions. However, an enhanced growth retardation of ndhB-inactivated plants was revealed under humidity stress conditions causing a moderate decline in photosynthesis via stomatal closure. This distinctive phenotype was mimicked under normal humidity by spraying plants with abscisic acid. Measurements of CO 2 fixation demonstrated an enhanced decline in photosynthesis in the mutant plants under humidity stress, which could be restored to wild-type levels by elevating the external CO 2 concentration. These results suggest that the plastid NAD(P)H:plastoquinone oxidoreductase in tobacco performs a significant physiological role by facilitating photosynthesis at moderate CO 2 limitation.

High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5′ and 3′ regulatory sequences

“…In contrast, no apparent reductions in transformation frequencies were observed in tobacco and tomato using homologous or homeologous N. tabacum and S. nigrum ptDNA sequences in transformation vectors (Kavanagh et al 1999;Horváth et al 2000;Nugent et al 2005;Nugent et al 2006), or in transformations of N. benthamiana plastids with tobacco-derived vectors (Davarpanah et al 2009), indicating that species-specific vectors are not always necessary. In the tobacco and tomato studies, it was demonstrated that integration of the cloned homeologous ptDNA sequences in the host plastome occurred by multiple recombination events in a similar region of the inverted repeats (IRs), that in comparison with ''Single Copy'' regions of the plastid genome, generally show a higher gene order conservation and a lower sequence divergence (Maier et al 1995).…”

Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance

“…The frequent homologous recombinations during plastid DNA transformation (Kavanagh et al 1999) may also decrease the co-integration frequency of the resistance mutation and the transgene. Nevertheless, it has been used in several instances with different Nicotiana genotypes (Golds et al 1993;O'Neill et al 1993;Kavanagh et al 1999;Horváth et al 2000;Rumeau et al 2004;Buhot et al 2006) and also with tomato (Nugent et al 2005), exploiting either homologous or homeologous recombination for transgene integration.…”

Plastid transformants of tomato selected using mutations affecting ribosome structure

“…However, the point mutation conferring antibiotic insensitivity cannot be subsequently removed, as it has become part of an essential plastid gene associated with the production of functional ribosomes. The versatility of derivatives of pSSH1 has also been demonstrated in targeted inactivation of the plastid ndhB gene (Horvath et al 2000) and integration and expression of genes for β-glucuronidase (GUS) and the bar gene for bialophos resistance in tobacco (McGrath-Curran et al 2003). Transgenes carried on one vector can also be targeted to another region of the plastid genome with a cointegration strategy with a vector similar to pSSH1 targeted to the 16S rRNA region (Rumeau et al 2004).…”