Reduced expression of chlorophyllide a oxygenase (CAO) decreases the metabolic flux for chlorophyll synthesis and downregulates photosynthesis in tobacco plants

Abstract

Chlorophyll b is synthesized from chlorophyllide a, catalyzed by chlorophyllide a oxygenase (CAO). To examine whether reduced chlorophyll b content regulates chlorophyll (Chl) synthesis and photosynthesis, we raised CAO transgenic tobacco plants with antisense CAO expression, which had lower chlorophyll b content and, thus, higher Chl a/b ratio. Further, these plants had (i) lower chlorophyll b and total Chl content, whether they were grown under low or high light; (ii) decreased steady-state levels of chlorophyll biosynthetic intermediates, due, perhaps, to a feedback-controlled reduction in enzyme expressions/activities; (iii) reduced electron transport rates in their intact leaves, and reduced Photosystem (PS) I, PS II and whole chain electron transport activities in their isolated thylakoids; (iv) decreased carbon assimilation in plants grown under low or high light. We suggest that reduced synthesis of chlorophyll b by antisense expression of CAO, acting at the end of Chl biosynthesis pathway, downregulates the chlorophyll b biosynthesis, resulting in decreased Chl b, total chlorophylls and increased Chl a/b. We have previously shown that the controlled up-regulation of chlorophyll b biosynthesis and decreased Chl a/b ratio by over expression of CAO enhance the rates of electron transport and CO₂ assimilation in tobacco. Conversely, our data, presented here, demonstrate that-antisense expression of CAO in tobacco, which decreases Chl b biosynthesis and increases Chl a/b ratio, leads to reduced photosynthetic electron transport and carbon assimilation rates, both under low and high light. We conclude that Chl b modulates photosynthesis; its controlled down regulation/ up regulation decreases/ increases light-harvesting, rates of electron transport, and carbon assimilation.

Supplementary information: The online version contains supplementary material available at 10.1007/s12298-023-01395-5.

Keywords: Chlorophyll b; Chlorophyllide a oxygenase; Electron transport; Light intensity; Photosynthesis.

Fig. 1

Generation of CAO antisense (CAOas) plants in tobacco (Nicotiana tabacum). A Schematic representation of the construct used for the antisense expression of AtCAO in the tobacco plant. B PCR analysis revealed the integration of CaMV35S-AtCAO into tobacco plant’s genome. A likely, 1.7 kb DNA fragment, was observed in all the 5 transgenics used (listed above the data), when the PCR was done using 35S-promoter internal forward primer and AtCAO forward primer. Further, as expected, 1.7 kb band was absent in the wildtype (WT) genomic DNA. C Northern blot analysis of CAO of WT and CAOas tobacco plants (CAOas1, CAOas2, CAOas3) grown under 300 µmol photons m⁻² s⁻¹ light (natural sun light + metal halide lamps) for 30 days. D Phenotype of WT and CAOas plants grown for 30 days under 300 μmol photons m⁻² s⁻¹light

Fig. 2

Chlorophyll content of wild-type (WT) and CAO antisense (CAOas) tobacco plants grown under low-light (LL) and high-light (HL) in the greenhouse A chlorophyll b, B chlorophyll a/b ratio, C Total chlorophyll content, and, D Total chlorophyll content of the 2nd, 3rd and 4th leaf from the top, from WT and CAOas plants. Plants were grown for up to 30 days under light intensity of 300 μmol photons m⁻² s⁻¹; then, they were transferred to LL (70–80 μmol photons m⁻² s⁻¹) and HL (700–800 μmol photons m⁻² s⁻¹) for additional 4 weeks. Each data point is the average of seven replicates and error bars represent the ± SE; asterisks indicate significant differences determined by ANOVA-test along with Dunnett’s post hoc test compared to WT (*P < 0.05, **P < 0.01). FW: Fresh weight. Statistical tests were conducted between WT and mutant within the same treatment. Lines have been drawn between WT and transgenic lines to show statistical differences between them

Fig. 3

Metabolites of chlorophyll biosynthesis pathway of WT and CAOas plants grown under LL and HL. The content of tetrapyrrole intermediates, as measured from WT and CAOas plants grown under LL and HL, was measured as described under Materials and Methods; also see the legend of Fig. 2. A ALA, B Glutamate semialdehyde (GSA), C protoporphyrin IX (Proto IX), D Mg-protoporphyrin IX monoester {MP(E)}, and E protochlorophyllide (Pchlide). Each data point is the average of four replicates, error bars represent the mean ± SE; asterisks indicate significant differences determined by ANOVA-test along with Dunnett’s post hoc test compared to WT (*P < 0.05, **P < 0.01). Statistical tests were conducted between WT and mutant within the same treatment

Fig. 4

Enzyme activities of chlorophyll biosynthesis pathway of WT and CAOas plants grown under different light regimes. A ALA dehydratase, B PBG deaminase, C Protoporphyrinogen oxidase, D Mg-chelatase, E MP(E) cyclase, measured from both LL- and HL- grown WT and CAOas plants (see Material and Methods). Plastids from different plants were isolated, enzymatic assays were performed, and ProtoX, Mg-chelatase and MPE cyclase were expressed per mg protein per hour, F Protochlorophyllide oxidoreductase activity (POR) determined in dark. Both LL- and HL-grown WT and CAOas plants were incubated in dark for 8 h. Plants were exposed to light (100 μmol photons m⁻² s⁻¹) for 15 min after dark incubation, and their Pchlide concentration was measured, as well as phototransformation (%) of Pchlide to Chlide. Each data point is the average of four replicate, error bars represent the ± SE; asterisks indicate significant differences determined by ANOVA-test along with Dunnett’s post hoc test compared to WT (*P < 0.05, **P < 0.01). Statistical tests were conducted between WT and mutant within the same treatment

Fig. 5

Electron transport rate of WT and CAOas plants. LL- and HL-grown WT and CAOas plants were dark adapted for 20 min before measurements were made, using PAM 2100 fluorometer. ETR was estimated from data obtained by this fluorometer at different light intensities (up to 900 µmol photons m⁻² s⁻¹) as described in the methods. Each data point is the average of five replicates; error bars represent the ± SE; asterisks indicate significant differences determined by ANOVA-test along with Dunnett’s post hoc test compared to WT (*P < 0.05, **P < 0.01). Statistical tests were conducted between WT and mutant within the same treatment

Fig. 6

Electron transport reactions of thylakoid membranes isolated from wild-type and CAOas plants. A Electron transport through PSII (oxygen evolution; water to PD); B whole chain (water to MV; oxygen uptake); and C PSI (ascorbate to MV; oxygen uptake). The oxygen evolution/uptake was measured polarographically, using a Hansatech oxygen electrode. Each data point is the average of five replicates, error bars represent the mean ± SE; asterisks indicate significant differences determined by ANOVA-test along with Dunnett’s post hoc test compared to WT (*P < 0.05, **P < 0.01). Statistical tests were conducted between WT and mutant within the same treatment

Fig. 7

Net CO₂ assimilation rates of the attached leaves of WT and CAOas plants grown in LL and HL conditions. Net CO₂ assimilation rates were monitored with an infrared gas analyzer (Licor 6400-XT portable photosynthetic system) at ambient CO₂ at different light intensities (80, 400, 800 µmol photons m⁻² s⁻¹). These experiments were done three times, error bars represent the ± SE; asterisks indicate significant differences determined by ANOVA-test along with Dunnett’s post hoc test compared to WT (*P < 0.05, **P < 0.01). Statistical tests were conducted between WT and mutant within the same treatment.

Fig. 8

Schematic diagram demonstrating the central role of Chlorophyll b in the upregulation and the downregulation of photosynthesis and on the biomass of the plants