Plant Carotenoids: functional genomicof xanthophylls biosynthesis and rolein Arabidopsis thaliana

The term “Xanthophylls” defines a group of molecules present in all oxygenic autotrophes, ranging from diatoms to the higher plants. Xanthophylls are also present in cyanobacteria. In higher plants, a peculiar characteristic of this class of compounds emerges: the high level of conservation of these carotenoids among several plant taxa, both in term of molecular species synthesized and of their relative amounts. In a biologist’s view a so high degree of evolutionary conservation strongly suggests a specific and peculiar function for each of the molecular species described in higher plants. Thus, in this phD work, I decided to undertake the (ambitious) task to attribute a function for each xanthophyll species in higher plants, in order to improve existing knowledge in this research field. Here below the major results obtained are summarized. Section A. Xanthophyll cycle and photooxidative stress: insights on the molecular basis of zeaxanthin-dependent photoprotection. In this section, zeaxanthin is analysed in terms of its capacity of binding to Lhc proteins and of modulating their spectroscopic properties. The effects of zeaxanthin in protecting thylakoid membranes from photooxidative stress is also analysed. In the first section (A.1), the first article summarizes the knowledge of the dynamics of zeaxanthin at the beginning of my phD period. We present an overview on the mechanistic aspects in the framework of the highly conserved xanthophylls composition in higher plants, and propose a model for Lhcs-xanthophyll cycle interaction. Experimental results evidence that xanthophylls undergo dynamic changes both in composition and distribution among Lhcs. It is shown that structural determinants control xanthophyll exchange in Lhc proteins: violaxanthin vs. zeaxanthin exchange rate is determined by the structure of individual antenna subunits; the exchange efficiency is dependent on lumenal pH, while it does not require the activated state of violaxanthin de-epoxidase enzyme (VDE). Xanthophyll exchange affects light-harvesting efficiency of PSII and increases protection from lipid peroxidation: zeaxanthin accumulation, thus acts as a signal transduction system, for A) regulation and B) protection of photosynthetic machinery. In the following parts, the above-mentioned points are examined in detail. In section A.2, the characterization of npq2lut2 mutant of A. thaliana, allowed evaluation of the in vivo effect of zeaxanthin as the only xanthophyll. Mutant plants show a strong modifications in the supramolecular organization of PSII: LHCII can not form trimers, as well as PSII supercomplexes are not detectable; abundance of LHCII and CP26 subunits in thylakoid membranes is strongly reduced. Zeaxanthin accumulation causes severe changes in the function of photosynthetic apparatus: plants undergo a suppression of state I-state II transitions, a reduction of photochemical efficiency in low light, moreover, they reach the photosynthetic saturation point at higher light intensity than control plants. On the whole, mutant show a photosynthetic phenotype approaching that of high-light acclimated plants. When exposed to strong photooxidative conditions, npq2lut2 plants show a lower degree of photoinhibition and lipid peroxidation, thus confirming the antioxidant role of zeaxanthin in vivo. In section A.3, the effect of zeaxanthin binding to Lhc subunits is analysed. Mutant npq2 of A. thaliana has a constitutive accumulation of zeaxanthin; main consequence on Lhc subunits is a lower fluorescence yield of chlorophyll in vivo than WT, that comes from a change in pigment-protein interactions. The CP26 subunit (minor antenna of PSII) has a peculiar behaviour upon binding zeaxanthin: CP26, isolated both from npq2 and from WT plants upon a treatment leading to zeaxanthin accumulation, undergoes a change in its pI. This is the first biochemical evidence for the existence of multiple conformations into Lhc proteins; switch from one conformation to the other is regulated by zeaxanthin binding. The rate of carotenoid to chlorophyll energy transfer in isolated LHCII has been measured by femtosecond fluorescence upconversion; results obtained are examined in section A.4. This new technical approach allows to estimate fraction of energy transferred from Cars to Chls either via carotenoid S1 or S2 state. LHCII isolated from npq2 mutant, binding zeaxanthin, has a lower amount of excitation energy transferred to Chls through the carotenoid S2 state; instead, the energy transferred through S1 state is less affected by xanthophyll composition. Section B: Xanthophylls in photosynthesis: biochemical and physiological analysis towards understanding the significance of their phylogenetic conservation. In vascular plants, carotenoid composition of thylakoid membranes represents a conserved feature and suggests a specific role for each molecular species. Isopentenyl diphosphate (IPP) is the starting point for all reactions leading to the variety of xanthophylls that we find in higher plants: several condensation and reduction steps generate lycopene; it represents a bifurcation point in the pathway: one β- and one ε-cyclization of lycopene ends leads to biosynthesis of lutein, while two β-cyclization originate the branch of β-carotene: zeaxanthin, violaxanthin and neoxanthin. In this work, I have used a functional genomic approach on A. thaliana in order to specifically block accumulation of one or more xanthophylls,and characterize the phenotypes in vivo. In section B.1, mutant lut2, specifically depleted of lutein, is analyzed. Lack of lutein has several effect on the organization and function of the photosynthetic machinery: violaxanthin level strongly increases to replace the missing lutein pool; LHCII trimerization is prevented; functional antenna size is decreased; the ability to perform state transition is reduced, as well as regulatory mechanism such as NPQ. The higher sensitivity of the lut2 mutant to photooxidative stress was evaluated through the level of photodamage when plants grow in stressing conditions (i.e. high light and low temperature). Spectroscopic analysis shows that this effect, can be ascribed to the lower efficiency of violaxanthin as chlorophyll triplet quencher: this is confirmed by results of photobleaching in vitro and time-resolved spectroscopy of carotenoid triplet formation, both measured on Lhcs isolated from lut2. The strongly increased photosensitivity of the npq1lut2 double mutant, lacking both zeaxanthin and lutein, highlights the overlapping function of these xanthophylls in photoprotection of thylakoid membranes. The role of neoxanthin in photosynthesis was analysed in section B.2. Aba4 mutant of A. thaliana accumulates less than 20% of WT levels of neoxanthin, retaining all other xanthophyll species. Neoxanthin deficiency does not affect PSII performance in light nor the activation of the thermal energy dissipation mechanisms. In isolated Lhcb proteins, the compensatory substitution of neoxanthin with violaxanthin within antenna proteins yields a lower photoprotection ability than WT lightharvesting proteins. Photooxidative stress in vivo, as well as thylakoid treatments with photosensitizers agents producing specific ROS molecules, shows an (unexpected) antioxidant role of neoxanthin and suggests a fundamental role of this xanthophyll in preserving photosynthetic machinery by reactive oxygen species produced from structures outside PSII. Section B.3 describes the complete elucidation of β-carotene hydroxylation pathway in A. thaliana. In addition to the previously described β-hydroxylases of the biosynthetic pathway of carotenoids, named Chy1 and Chy2, a third β-hydroxylase is described: Cyp97a3, a close paralog of Lut1, and has a function in β-ring hydroxylation. Indeed, triple mutant chy1chy2cyp97a3 completely lacks β-β- xanthophylls (violaxanthin, zeaxanthin and neoxanthin), while it retains lutein as the only xanthophyll. Comparison of this triple mutant with npq1lut2, a mutant lacking zeaxanthin and lutein, shows that both genotypes are completely depleted of qE. Nevertheless, photooxidative treatment induces a higher extent of both lipid peroxidation and photoinhibition in chy1chy2cyp97a3 with respect to npq1lut2. These evidences suggest a specific role of neoxanthin and/or violaxanthin in preserving thylakoid system from ROS. Section C: Thermal energy dissipation in photosynthesis: investigation on activation mechanisms. Non-photochemical quenching (NPQ), also termed ΔpH-dependent quenching (qE), is a rapidly inducible mechanism for the harmless thermal dissipation of excess absorbed photons in PSII. It works as a feed-back regulation mechanism for light harvesting: in fact, it is directly controlled by thylakoid ΔpH, that is formed by photosynthetic electron transport. In this section, results obtained studying the induction of NPQ both in higher plants (H. vulgare) and cyanobacteria (C. reinhardtii) are presented. In part C.1, a characterization of a novel kind of zeaxanthin-independent, non-photochemical quenching is described. In higher plants, a transiently induced non-photochemical quenching measured during transition from dark to low light, is well known and previously described. This mechanism is further analysed, showing that the transient chlorophyll quenching is not associated with zeaxanthin synthesis, while it proceeds through inactivation of a fraction of PSII reaction centers. The phenomenon measured is compatible with the origin of a quenched state inside the PSII, that relaxes upon activation of the Calvin cycle. The above-mentioned PSII reaction centers quenching is also detected at saturating light intensities, and maintains its reversibility character. Following relaxation of this RC associated quenching, the ΔpH-dependent, antenna-associated quenching fully replaces reaction center contribution and leads to full amplitude of energy dissipation. In section C.2, capacity of C. reinhardtii for triggering thermal energy dissipation (NPQ) is analyzed. The moderate amplitude of non-photochemical quenching measured in this alga comes mostly from state transitions (qT). A significant increase in ΔpH-dependent quenching (qE) is observed by: a) lowering temperature of the medium during fluorescence measurements, or b) blocking energy utilization by mutating genes encoding Rubisco. Thus, qE of C. reinhardtii is observed in conditions that strongly slow down electron transport. In optimal growth conditions, this alga seems to produce a trans-membrane ΔpH not sufficient for the triggering of qE. Thereforethe ΔpH-dependent quenching response is somehow less efficient than higher plants. Section D: Characterization of ELIPs physiological function. ELIPs are proteins belonging to Lhc-superfamily, accumulated into thylakoids at the early stage of greening after etiolated-to-light transition. ELIP transcripts and proteins appear during de-etiolation, before starting Lhc gene expression, and disappear before chloroplast development is completed. The physiological role of ELIPs is not well understood. The early expression upon illumination of etiolated seedlings suggests that ELIPs have a role in development of chloroplast. Moreover, since ELIPs are accumulated in conditions of excess light, they could also have a role in the acclimation to photoxidative stress, possibly through pigments interaction: because of their structural similarity with the LHCs, ELIPs are assumed to bind pigments, especially chlorophylls and xanthophylls. In section D.1, an investigation on the physiological role of ELIPs is presented. Production of transgenic lines of A. thaliana overexpressing ELIP2 gene resulted in a pale-green phenotype caused by a marked reduction of the pigment content of the chloroplasts. Chloroplast ultrastructure and density, as well as photosystems composition and functioning, were not affected by constitutive ELIP2 accumulation; reduction of pigment content comes from a decreased number of photosystems assembled in thylkoids. Further analysis showed that chlorophyll precursors were strongly reduced in ELIP2 transgenic plants, while levels of chlorophyll degradation products were not affected. These evidences suggests a role of ELIPs for regulation of chlorophyll accumulation in the thylakoids.