Effect of constitutive expression of bacterial phytoene desaturase CRTI on photosynthetic electron transport in Arabidopsis thaliana

Get Complete Project Material File(s) Now! »

Photoprotective mechanisms in higher plants

During the day photosynthetic organisms are exposed to different light intensities. The photosynthetic apparatus has evolved for both maximizing light capture in case of low light conditions and to guarantee protection during exposure to high light. In low light conditions the amount of light energy absorbed matches the amount utilized in photosynthesis. In high light conditions, the rate of incoming photons is higher than the rate of electron transfer through the photosynthetic apparatus, and the reaction centers become progressively saturated (closed) (figure 1.12; Ruban et al., 2012).

Photoprotective role of carotenoids

The communication between carotenoids and (Bacterio)chlorophyll molecules at level of the light harvesting complexes is bidirectional. Carotenoids not only are able to capture the light and transfer the excitation energy to chlorophylls, but they can also interfere with excited singlet and triplet states of chlorophyll to avoid photodamage. This action takes place in the nanosecond/microsecond timescale thus guaranteeing an extremely fast response. In high light conditions, the energy reaching the photoynthetic apparatus can exceed the normal level used for photoynthesis. The excess of energy can then provocate the persistence of excited states of chlorophylls. Triplet-excited chlorophylls can react with molecular oxygen to produce singlet O2, which is a powerful oxidizing agent and rapidly kills those cells exposed to it (Foote, 1976). Carotenoids are able to overcome this effect in one of two ways: 1) they can quench singlet oxygen directly through energy transfer or chemical reaction, or 2) they can quench the chlorophyll triplet itself via a rapid triplet-triplet energy transfer, preventing the production of singlet oxygen (Krinsky et al., 1971).
In vivo, the latter process is dominant. For this reaction to occur efficiently, the energy level of the carotenoid’s triplet state must be lower than that of chlorophyll and lower than that of singlet oxygen i.e. < 1eV (1274 nm-7849 cm-1- or 94 kj/mole; Foote et al., 1970) in order to prevent the triplet-excited carotenoid from reacting with molecular oxygen itself.
In practice, this means that only those carotenoids with 9 or more conjugated double bonds (n≥9) have the ability to photoprotect (Foote et al., 1970). This property could thus be one of the reasons why nature has selected only carotenoids of this length in the photosynthetic proteins. Moreover, because the carotenoid triplet state is lower in energy than singlet oxygen, it returns harmlessly to the ground state with the liberation of heat (Cogdell and Frank, 1987).
Carotenoids can also quenching the singlet excited state of chlorophyll, which could also represent a source of singlet oxygen, through a not yet clarified mechanism which plays an essential role in the non-photochemical quenching or NPQ (Truscott et al., 1973; Palozza and Krinsky, 1992; Niyogi et al., 1997; Pascal et al., 2005; Ruban et al., 2007; Britton et al., 2008). This function will be better described in paragraph 1.5.2.

Role of xanthophylls as qE quenchers

An important question to solve for qE researchers was: what is the physical cause of the reduction in fluorescence emission during the quenching phenomenon?.
During NPQ, chlorophyll excitation energy is mainly dissipated by internal conversion from to the ground state (loss as heat) or by energy transfer to another pigment. In LHC proteins chlorophylls are relatively close to each other and to the xanthophylls, thus allowing an efficient interaction and energy exchange potentially between all the pigments. The observation of the formation of singlet excited state (S1) of xanthophylls upon chlorophyll excitation in the NPQ state, support the idea that excitonic interactions between chlorophylls and xanthophylls are involved in quenching (Polivka et al., 2002, Ma et al., 2003; Ruban et al., 2007; Bode et al., 2009; Liao et al., 2010 a and b). Indeed the short lifetime and the close proximity of the S1 state energy to that of the lowest excited state of chlorophyll in the Qy band could account for this physical mechanism of quenching. The existence of energy transfer from a porphyrin ring to the S1 state of a carotenoid in an artificial conjugated dyad model system (see also chapter 4) further strengthens this hypothesis (Berera et al., 2006).

Possible quenching site(s) within LHCII

By observing the LHCII crystal structure (Liu et al., 2004; Standfuss et al., 2005), Pascal and coworkers (2005) proposed several possible quenching sites: terminal emitter domain consisting in chlorophylls Chl a611, Chl a612, Chl b608 and lutein 1; neoxanthin domain: neoxanthin, lutein 2, Chl b606, Chl b607; xanthophyll cycle binding domain: violaxanthin, Chl a611 and Chl a601. Time-resolved absorption spectroscopy applied to LHCII in different quenching states also showed the formation of a carotenoid excited state concomitant with the decay of the chlorophyll excited state and identified this carotenoid as lutein1 (Ruban et al., 2007). The authors proposed that the conformational change occurring in LHCII during NPQ gives rise to an increase in the rate of energy transfer to lutein 1 and, consequently, to energy dissipation (figure 1.14). Moreover, from a comparison of lutein 1 and lutein 2 in the crystal structure, it has been speculated how a change in the configuration of lutein 1 would bring it closer to chlorophyll a 612 (Yan et al., 2007), providing the key step in the switching on of quenching. This idea provides an explanation of the link between the observed changes in protein conformation and fluorescence quenching.

β-Carotene in PSII Reaction Centres

At low temperature, PSII-RCs exhibit two main peaks in the carotenoid absorption region, at 489 and 507 nm (figure 2.3). Linear dichroism experiments showed that these peaks correspond to distinct Car molecules, with different orientations relative to the membrane plane (27) and, considering their position, they must be attributed to the 0-0 sub-level of the absorption transition of each molecule. Although the resolution between these peaks becomes much lower at room temperature, the overall position of the carotenoid absorption transition appears not to shift by more than a few nm between low temperature and room temperature (see figure 2.3). This is not specific to PSII and was already observed in other photosynthetic complexes. For instance, in light-harvesting complexes from purple bacteria it was shown that, in general, the absorption transitions of the bound carotenoid molecules are very poorly dependent on temperature (43).

READ  Kinematics of Soft-continuum Manipulators using PH-Curves 

Mechanisms tuning carotenoid absorption in PSII-RC and LHCII

In both PSII-RC and LHCII, RR spectroscopy unambiguously shows that the position of the absorption transition of the blue-absorbing carotenoid molecule is mainly governed by the polarizability provided by the protein environment. Indeed, the position of this transition and the frequency of the ν1 mode of these molecules strictly obey the correlation obtained for both β-carotene and lutein according to solvent refractive index. The deduced average value of the environment polarizability of the blue-absorbing β-carotene in PSII-RC is lower than that of the blue absorbing lutein in LHCII. This is consistent with the environment deduced by analysis of X-ray crystallographic structures (30, 31, 37). In PSII-RCs, carotenoids are mainly surrounded by amino acids and are quite distant from other cofactors; they exhibit only low rates of energy transfer to the bound Chl molecules (27, 33, 34). On the other hand, the luteins in LHCII are in very close contact with the LHCII-bound Chl molecules, both at the levels of their end cycles and of the conjugated C=C chain (37). Some of these Chls, such as Chl a 603, are nearly in van der Waals contact with lut1 (closest distance 3.83 Å) and are likely to provide an environment of higher polarizability. By contrast, it is quite clear that the energy shifts between the blue- and the red-absorbing carotenoid molecules in both studied complexes are not induced by a variation in polarizability of their binding sites. If so, the position of the absorption transition of these carotenoids and their ν1 Raman frequency would obey a correlation similar to the blue/red lines in figure 2.2, whereas it is clear that they deviate from these lines (see figures 2.5 and 2.8). Again, this conclusion is consistent with the description of the environment of these molecules provided by the crystallographic structures of the two pigment-protein complexes: the blue and red luteins in LHCII, as well as the blue and red β- carotene molecules in PSII-RC, are embedded in quite similar protein environments, which are unlikely to display large changes in average polarizability (indeed in LHCII, the two binding pockets are related by the local 2-fold symmetry of the complex). Instead, the absorption transition of these molecules and the frequency of their ν1 Raman band behave as if the conjugated chain of the carotenoid molecules was increased by nearly one C=C double bond at constant polarizability (figures 2.5 and 2.8). Note that for both red-absorbing Cars, the main ν2 band is also seen to shift to lower frequency in parallel with the downshift in ν1 (figures 2.4 and 2.7); this is exactly as expected for an increase in conjugation length (see e.g. 47). Thus the apparent length of the conjugated chain of the red-absorbing lutein and β-carotene in LHCII and PSII-RC (at room temperature) become 10 and 10.2, respectively (figures 2.5 and 2.8). The external parameters susceptible to induce such changes are not documented in the literature and, again, there is no dramatic change in the environment of these pigments which could be at the origin of such a change. It was shown that the luteins of LHCII and the β-carotenes in PSII-RC experience different distortions at low temperature (3, 35) and we show in this work that these distortions also exist at room temperature. However, small distortions around C-C bonds are expected to have little influence on the structure of the C=C conjugated chain and, while in LHCII the red-absorbing lutein is distorted (48), in PSII it is the blue-absorbing carotenoid which exhibits the larger distortion (35).

Table of contents :

Title page
List of abbreviations
List of figures
List of tables
CHAPTER 1 Introduction
1.1 General introduction
1.2 Carotenoids
1.3 Chlorophylls
1.4 The proteins of the photosynthetic apparatus from higher plants
1.4.1 Peripheral Photosystem II Antenna Complexes
1.4.2 Photosystem II supercomplexes
1.4.3 Photosystem II
1.4.4 Cytochrome b6f
1.4.5 Photosystem I
1.4.6 ATP-synthase complex
1.4.7 Linear electron transport
1.4.8 Cyclic electron transport
1.5 Photoprotective mechanisms in higher plants
1.5.1 Photoprotective role of carotenoids
1.5.2 Non-photochemical quenching (NPQ)
1.6 Photoinhibition
1.7 Realizing artificial photosynthesis
1.8 Experimental approach
1.8.1 Resonance Raman spectroscopy
1.8.2 Carotenoid molecules
1.8.3 Chlorophylls and derivatives
1.8.4 Step-scan Fourier transform infrared spectroscopy
1.9 Project outline
CHAPTER 2 Mechanisms underlying carotenoid absorption in oxygenic photosynthetic proteins
2.1 Introduction
2.2 Experimental procedure
2.3 Results and discussion
2.3.1 Isolated β-Carotene and Lutein
2.3.2 β-Carotene in PSII Reaction Centres
2.3.3 Lutein Molecules in LHCII
2.3.4 Mechanisms tuning carotenoid absorption in PSII-RC and LHCII
CHAPTER 3 Effect of constitutive expression of bacterial phytoene desaturase CRTI on photosynthetic electron transport in Arabidopsis thaliana
3.1 Introduction
3.2 Materials and Methods
3.3 Results
3.3.1 Light sensitivity of CRTI expressing lines
3.3.2 Generation of H2O2-derived hydroxyl radicals in the CRTI-lines
3.3.3 Reduction state of the plastoquinone pool in CRTI-lines
3.3.4 Cyclic electron flow in the CRTI-lines
3.4 Discussion
CHAPTER 4 Carotenotetrapyrrole Dyads Mimic Photosynthetic Triplet-Triplet Energy Transfer
4.1 Introduction
4.2 Methods
4.3 Results
4.4 Discussion
CHAPTER 5Effect of the isomeric forms of dodecyl-maltoside detergent on LHCII spectroscopic properties
5.1 Introduction
5.2 Material and methods
5.3 Results
5.4 Discussion
CHAPTER 6 General discussion and future perspective
6.1 Introduction
6.2 Tuning of carotenoid absorption properties
6.3 Regulation of the photosynthetic electron flow in Arabidopsis thaliana
6.4 Reengineering photosynthesis: artificial antenna system
6.5 Dynamic and flexibility of LHCII
6.6 Conclusions


Related Posts