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.

Figure 1

Translational inactivation of the ndhB gene in the pSSH1 plastid transformation plasmid. The pSSH1 plasmid insert spans the first 732 bp of the ndhB gene. An additional C-G bp was introduced into codon 206 of ndhB by oligonucleotide-directed mutagenesis. The resulting plasmid was called pSSH1M. This additional nucleotide generated a diagnostic SmaI site and, in addition to a frame shift, all three stop codons (only two of which are shown). A 300-bp portion of the ndhB coding region (identical in tobacco and nightshade) adjacent to the HindIII cloning site is shown as it appears in IR_A (5′–3′ direction, strand A), from position 143,798 in the tobacco plastid genome. Selected restriction enzyme sites are also shown.

Figure 2

Distribution of co-integration and recombination frequencies in the targeted region following transformation of tobacco plastids with the pSSH1M plasmid. The 7.8-kb donor insert of the pSSH1M plasmid is shown at the top of the figure. Arrows mark the location of the specific resistance and RFLP sites scored (brackets indicate the absence of the wild-type restriction enzyme site). Asterisks mark intron-containing genes. At the lower part of the figure the line is sectioned to show the major intervals between the donor-type marker sites investigated in the plastid transformants. The frequency of co-integration (int.) of the individual non-selected markers with the selected spectinomycin resistance locus is shown above the line. A 100% value represents the total number of spectinomycin-resistant transformants possessing a donor marker. The observed recombination frequency (rec.) in the individual internal sections, calculated as a percentage of the transformants recombined in the particular interval, is shown below the line. A 100% value represents the total number of transformants possessing a recombination event.

Figure 3

Site-specific inactivation of the ndhB gene in the plastid transformants. Gel electrophoresis of SmaI-digested plastid DNA of wild-type tobacco (a) and several plastid transformants (b–d) distinguishes a noninactivated transformant (b) from those possessing the inactivated ndhB gene (c–d). The smaller of the new, inactivation-specific fragments (5.68 and 5.45 kb) comigrates with the unchanged fragment number 9. On the left a HindIII digest of λ DNA is also shown (fragment sizes: 23.13, 9.42, 6.56, 4.36, 2.32, and 2.03 kb). Southern hybridization with a plastid DNA probe spanning the region containing the diagnostic restriction site in the 11.13-kb SmaI fragment number 4 reveals both the site-specificity and homoplasmy of the introduced mutation.

Figure 4

Homoplasmy of the plastid DNA population in the ndhB-inactivated transformants. Gel electrophoresis of SmaI-digested PCR product of wild-type tobacco (a), two ndhB-inactivated transformants (b and c), and a noninactivated transformant (d). The priming sites were located to cover the 5′ end of ndhB and flank the diagnostic SmaI site. The primer located inside the ndhB gene is outside the targeted plastid DNA region. The primers amplify a product of 966 bp, which is cut into 814- and 152-bp fragments by SmaI if the mutation introduced into ndhB is present. On the left a 100-bp DNA ladder is also shown (fragment sizes: 1,500 and 1,000–100 bp). The complete and correct cleavage of the PCR product in the ndhB-inactivated transformants reveals both the homoplasmy and site-specificity of the introduced mutation.

Figure 5

Absence of the NDH-H subunit in the ndhB⁻ plastid transformants. Separation of protein fractions (20 mg of protein per lane) derived from purified chloroplasts by fully denaturating PAGE reveals no obvious difference after Coomassie Brilliant Blue staining between wild-type (WT) and ndhB⁻ (1.2) tobacco plants. Western hybridization of the separated protein fractions electrotransferred onto nitrocellulose membranes by an anti-NDH-H antibody detects the protein in total thylakoid membranes and the stroma lamellae of the wild type. The absence of detectable NDH-H protein in the ndhB⁻ mutant indicates that no functional NDH complex can be assembled. Molecular mass markers: 96, 66.2, 45, 31, 21.5, and 14.4 kD.

Figure 6

Enhanced growth delay of the ndhB⁻ plastid transformants under humidity stress. Well-watered wild-type (left) and ndhB⁻ (right) plants were grown in low air humidity (30% and 40% relative humidity during the day and the night, respectively), following a month of growth under normal conditions (60% relative humidity). A visible growth difference was developed in less than a week under humidity stress. The development of freshly expanding leaves was specifically hindered in ndhB⁻ plants.

Figure 7

Differential reduction in photosynthesis of wild-type and NDH-inactivated plants grown in low air humidity. Photosynthesis and evapotranspiration of 5-week-old wild-type and ndhB⁻ plants are shown in normal (60 relative %) and low (30 relative %) air humidity on the day preceding and following the humidity transition, respectively. The whole-plant photosynthesis and evapotranspiration values recorded in one experiment were the sum of four to eight plants of the same type grown in one computer-controlled growth chamber recording CO₂ consumption and water vapor condensation. On the left net photosynthesis calculated for one plant is displayed as the mean ± sd of four independent experiments. On the right evapotranspiration calculated for one plant is displayed as the mean ± sd of four independent experiments. Asterisk indicates significant differences from controls (P < 0.05). Low air humidity generated a difference in photosynthesis of ndhB⁻ and wild-type plants, demonstrating an enhanced sensitivity of the ndhB⁻ transformants to humidity stress. Evapotranspiration of ndhB⁻ and wild-type plants showed a similar response, indicating that their stomata responded non-differentially to low air humidity.

Figure 8

ABA treatment provokes the mutant-specific stress phenotype under normal growth conditions. Wild-type (left) and ndhB⁻ (right) plants grown for a month under normal phytotron conditions were subsequently sprayed with 10 μm ABA solution. The growth difference was developed during 2 weeks of spraying of the leaves every 2nd d. The development of freshly expanding leaves was specifically hindered in ndhB⁻ plants.

Figure 9

Elevated external CO₂ concentration complements the differential photosynthesis reduction generated by humidity stress. Five-week-old wild-type and ndhB⁻ plantlets were grown under normal conditions in computer-controlled growth chambers recording CO₂ exchange. The effect of humidity stress (caused by decreasing the relative air humidity from 60% to 30%) and the additional effect of elevated CO₂ concentration (from ambient to 0.5%) was tested. Whole-plant photosynthesis in one chamber during illumination was recorded as the CO₂ consumption of six plants of the same type. Relative photosynthesis values are displayed on the y axis. For ease of comparison, photosynthesis of wild-type plants was taken to be 100%. The mean wild-type absolute photosynthesis values (from left to right) were the following: 15.55, 18.06, 54.26, and 31.51 mL⁻¹ h⁻¹ plant⁻¹. The relative decrease in whole-plant photosynthesis in NDH-inactivated plants was fully compensated during the transitory elevation of the CO₂ level. This result pinpoints limitation of CO₂ availability as a direct cause of the differential reduction in photosynthesis in wild-type and ndhB⁻ transformant plants under humidity stress conditions.