Symposium 04 Light Harvesting Aerobic Systems 131 Modulation of Chlorophyll b Biosynthesis and Photosynthesis by Overexpression of Chlorophyllide a Oxygenase (CAO) in Tobacco1 Ajaya K Biswal2,a,, Gopal K Pattanayak2,a,, Sadhu Leelavathib, Vanga S Reddyb, Govindjeec , Baishnab C Tripathya* aSchool of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India; bInternational Center for Genetic Engineering and Biotechnology, New Delhi 110067, India; 2 AKB and GKP contributed equally to this article; c Department of Plant Biology, University of Illinois, Urbana, IL 61801, USA. *Corresponding author. E-mail: [email protected]. 1 A full length article will be published elsewhere Abstract: Chlorophyll (Chl) b is synthesized by oxidation of a methyl group on the B ring of the porphyrin molecule to a formyl group by chlorophyllide (Chlide) a oxygenase (CAO). The overexpression of Arabidopsis thaliana full length CAO (AtCAO) in tobacco (Nicotiana tabacum) resulted in an increased Chl synthesis and a decreased Chl a/b ratio in low-light-grown (LL) as well as in high-light-grown (HL) tobacco plants, where the effect was more pronounced. In HL-plants, increased [Chl b] resulted in efficient capture of solar energy and enhanced (40%–80%) electron transport rates at both limiting and saturating light intensities. Keywords: Chlophyll b biosynthesis; Chlorophyllide a Oxygenase; Nocotiana tabacum; Photosynthesis Introduction Chlide a to Chlide b (Oster et al., 2000). We have previously reported that overexpression of full length Chlorophyll (Chl) b is a closed Mg-tetrapyrrole AtCAO results in increased Chl b synthesis and found in plants, green algae and some decreased Chl a/b ratio in low-light, but more so in prochlorophytes. The main function of Chl b is to high-light-grown tobacco plants (Pattanayak et al., gather light energy and transfer it with 100% 2005). In the present study, we show that the efficiency to Chl a. Chl b is synthesized from Chl a by overexpression of full length AtCAO modulates the oxidation of methyl group on the B ring to a formyl flux of Chl biosynthesis pathway leading to increased group at that position. The genes encoding Chl b synthesis both in low-light- and high-light- chlorophyllide a oxygenase (CAO), responsible for grown transgenic tobacco plants. We further show Chl b synthesis, have been isolated from several that increased Chl b biosynthesis in full-length CAO- different species (Tanaka et al., 1998; Espineda et al., overexpressing (CAOx) plants results in efficient 1999; Nagata et al., 2004). The CAO enzyme is capture of solar energy and increased electron localized in chloroplast envelope and thylakoid transport mostly at limiting light intensities. membranes and contains domains for a [2Fe-2S] Rieske center and for a mononuclear nonheme iron- binding site (Eggink et al., 2004). The conserved Materials and Methods Rieske center and non-heme-iron binding motifs of CAO are likely to be involved in the electron Plant Materials and Growth Conditions transport from ferredoxin to moleclular oxygen. The Wild-type (WT) and CAO overexpressing recombinant CAO protein catalyzes the oxidation of (CAOX) tobacco (Nicotiana tabacum cv. Petit 132 Photosynthesis: Research for Food, Fuel and Future—15th International Conference on Photosynthesis Havana) plants (Pattanayak et al., 2005) were grown 4 Chl a + b in greenhouse in natural photoperiod for 25–30 days Chl b –2 –1 Chl a/b under light intensity of 200 mol photons m s at -1 3 25 ± 2 C. In our studies, these plants were transferred either to low light (LL) (70–80 mol photons m–2 s–1) 2 or to high light (HL) (700–800 mol photons m–2 s–1) for additional 18–20 days in a greenhouse. 1 Chlorophyll, mg (g FW) mg Chlorophyll, Chlorophyll a Fluorescence Measurements Chl a fluorescence was measured with a PAM- 0 2001 Chl fluorometer (Walz, Germany) at room WT-LL CAOx1-LL WT-HL CAOx1-HL temperature, from the front surface of leaves (see e.g., Fig. 1 Chlorophyll content and the leaf phenotype of wild-type Dutta et al., 2009). Before each measurement, the leaf (WT) and CAO overexpressing (CAOx1) tobacco plants grown was dark-adapted for 20 min. under low-light (LL) and high-light (HL). Total chlorophyll (Chl a+b) content, chlorophyll b (Chl b) and chlorophyll a/b (Chl a/b) ratio of WT and CAOx1 plants grown in LL (WT-LL, CAOx1-LL) and HL (WT-HL, CAOx1-HL). Plants grown for Results up to 20–25 days under light intensity of 200 mol photons m–2 s–1 –2 –1 were transferred to LL (70–80 mol photons m s ) and HL CAO Overexpressed (CAOx) Plants Grown in Low –2 –1 (700–800 mol photons m s ) for additional 18–20 days in Light or High Light Regimes had Altered Chlorophyll the greenhouse. The 2nd leaf was harvested from each plant a/b Ratio types for pigment analysis. Each data point is the average of The CAOx (CAOx1 and CAOx2) plants five replicates. accumulated higher amounts of Chl in low light (LL; 70–80 mol photons m–2 s–1) as well as in high light Photosynthetic Responses of CAOx Plants Grown in Low Light and High Light Regimes (HL; 700–800 mol photons m–2 s–1) as compared to To ascertain if increased Chl b content had the that in the wild-type (WT) plants grown under expected effect on photosynthetic apparatus, Chl a identical light regimes. As expected, WT-LL plants fluorescence of leaves of both WT and CAOx tobacco had higher Chl content and reduced Chl a/b ratio than plants grown in LL (70 µmol photons m–2 s–1) or HL the WT-HL plants (Fig. 1). The leaves of CAOx1- (700–800 µmol photons m–2 s–1) was measured. HL-plants were greener and accumulated 28% more Chl than WT-HL plants. The CAOx2-HL-plants 120 WT-LL accumulated 20% more Chl than the WT-HL plants CAOx-LL 100 WT-HL ) (data not shown). Due to increased (70%) synthesis of -1 CAOx-HL s -2 Chl b in the CAOx plants grown in HL (CAOx1-HL), 80 they showed an ~30% decline in Chl a/b ratio as compared to WT-HL plants. Similarly, CAOx2-HL- 60 plants showed ~50% increase in Chl b synthesis as mol electrons m 40 compared to WT-HL plants (data not shown). The ETR ( CAOx plants grown in LL (CAOx1-LL) were greener 20 than LL-grown WT plants (WT-LL) and had 15% 0 lower Chl a/b ratio (Fig. 1). The Chl a/b ratio and Chl 0 200 400 600 800 1000 -2 -1 content of WT and transgenic plants showed some PAR (moles photons m s ) minor variations in different growth seasons. Since Fig. 2 Electron transport rate (ETR) of WT and CAOx plants. LL and HL-grown WT and CAOx plants were dark adapted for CAOx1 plants had high amount of Chl b and reduced 20 min before readings were taken by PAM 2100 fluorometer. Chl a/b ratio as compared to that of CAOx2, we The ETR values were calculated from these fluorescence data characterized the CAOx1 plants in detail, henceforth (Table 1). The experiment was repeated thrice and each data referred simply as CAOx. point is the average of ten replicates. Photosynthesis: Research for Food, Fuel and Future—15th International Conference on Photosynthesis 133 Chl a fluorescence is used as a nondestructive and Discussion noninvasive signature of photosynthesis, particularly of Photosystem II (for reviews, see Papageorgiou and Studies from other research groups and that of Govindjee, 2005). The minimal fluorescence (F0), the ours have demonstrated that high light decreases total maximum fluorescence (Fm) and the maximum Chl and Chl b content. Overexpression of AtCAO in photochemical efficiency of PSII in dark-adapted tobacco or Arabidopsis results in increased total Chl leaves, measured as Fv/Fm (where Fv = Fm – Fo), and Chl b content, decreased Chl a/b ratio both in LL- were not substantially affected in LL- or HL- grown and HL-grown plants (Fig. 1) (Pattanayak et al., 2005; WT or CAOx plants (Table 1). The electron transport Tanaka and Tanaka, 2005). As expected from WT rate (ETR, µmol electrons m–2 s–1) was estimated from plants, the ETR values, calculated from yield fluorescence parameters. The light-response curves of ETR parameters of PAM fluorometry (Schreiber, 2004), suggest that in limiting (up to 70 µmol photons m–2 s–1) as were higher in HL-grown plants than that in LL- well as saturating light intensities, the ETR was grown plants (Fig. 2). As compared to that of HL- higher in CAOx plants than that in WT plants grown grown plants, the ETR saturated at a relatively lower light intensity (400 µmol photons m–2 s–1) in LL- in LL- or HL-regimes suggesting better light grown plants (Fig. 2). Due to increase in the antenna absorption and utilization, especially in Photosystem size, the ETR values at limiting light intensities of II, in the transgenic plants (Fig. 2). In WT, at limiting LL-grown WT-plants were higher than that of HL- light intensities, the rate of increase in ETR in LL- grown WT plants (Fig. 2). At higher light intensities, grown plants was essentially the same as that in HL- the HL-grown CAOx plants had elevated ETR than grown plants (Fig. 2) even though the latter have less that of HL-grown WT plants. Chl (Fig. 1). The ETR in LL-grown WT and CAOx Our studies reveal that regulated increase in Chl b plants saturated at lower light intensity, around biosynthesis by over-expression of full length CAO 400 µmol photons m–2 s–1 than HL-grown plants that –2 –1 increases the light-harvesting potential.