Shizue Matsubara

The lutein epoxide cycle in higher plants: its relationships to other xanthophyll cycles and possible functions

Several xanthophyll cycles have been described in photosynthetic organisms. Among them, only two are present in higher plants: the ubiquitous violaxanthin (V) cycle, and the taxonomically restricted lutein epoxide (Lx) cycle, whereas four cycles seem to occur in algae. Although V is synthesised through the β-branch of the carotenoid biosynthetic pathway and Lx is the product of the α-branch; both are co-located in the same sites of the photosynthetic pigment-protein complexes isolated from thylakoids. Both xanthophylls are also de-epoxidised upon light exposure by the same enzyme, violaxanthin de-epoxidase (VDE) leading to the formation of zeaxanthin (Z) and lutein (L) at comparable rates. In contrast with VDE, the reverse reaction presumably catalysed by zeaxanthin epoxidase (ZE), is much slower (or even inactive) with L than with antheraxanthin (A) or Z. Consequently many species lack Lx altogether, and although the presence of Lx shows an irregular taxonomical distribution in unrelated taxa, it has a high fidelity at family level. In those plants which accumulate Lx, variations in ZE activity in vivo mean that a complete Lx-cycle occurs in some (with Lx pools being restored overnight), whereas in others a truncated cycle is observed in which VDE converts Lx into L, but regeneration of Lx by ZE is extremely slow. Accumulation of Lx to high concentrations is found most commonly in old leaves in deeply shaded canopies, and the Lx cycle in these leaves is usually truncated. This seemingly anomalous situation presumably arises because ZE has a low but finite affinity for L, and because deeply shaded leaves are not often exposed to light intensities strong enough to activate VDE. Notably, both in vitro and in vivo studies have recently shown that accumulation of Lx can increase the light harvesting efficiency in the antennae of PSII. We propose a model for the truncated Lx cycle in strong light in which VDE converts Lx to L which then occupies sites L2 and V1 in the light-harvesting antenna complex of PSII (Lhcb), displacing V and Z. There is correlative evidence that this photoconverted L facilitates energy dissipation via non-photochemical quenching and thereby converts a highly efficient light harvesting system to an energy dissipating system with improved capacity to engage photoprotection. Operation of the α- and β-xanthophyll cycles with different L and Z epoxidation kinetics thus allows a combination of rapidly and slowly reversible modulation of light harvesting and photoprotection, with each cycle having distinct effects. Based on the patchy taxonomical distribution of Lx, we propose that the presence of Lx (and the Lx cycle) could be the result of a recurrent mutation in the epoxidase gene that increases its affinity for L, which is conserved whenever it confers an evolutionary advantage.

Populations of photoinactivated photosystem II reaction centers characterized by chlorophyll a fluorescence lifetime in vivo

Photosystem (PS) II centers, which split water into oxygen, protons, and electrons during photosynthesis, require light but are paradoxically inactivated by it. Prolonged light exposure concomitantly decreased both the functional fraction of PSII reaction centers and the integral PSII chlorophyll (Chl) a fluorescence lifetime in leaf segments of Capsicum annuum L. Acceleration of photoinactivation of PSII by a pretreatment with the inhibitors/uncoupler lincomycin, DTT, or nigericin further reduced PSII Chl a fluorescence lifetimes. A global analysis of fluorescence lifetime distributions revealed the presence of at least two distinct populations of photoinactivated PSII centers, one at 1.25 ns, and the other at 0.58 ns. Light treatment first increased the 1.25-ns component, a weak quencher, at the expense of a component at 2.22 ns corresponding to functional PSII centers. The 0.58-ns component, a strong quencher, emerged later than the 1.25-ns component. The strongly quenching PSII reaction centers could serve to avoid further damage to themselves and protect their functional neighbors by acting as strong energy sinks.

Non-invasive approaches for phenotyping of enhanced performance traits in bean

Plant phenotyping is an emerging discipline in plant biology. Quantitative measurements of functional and structural traits help to better understand gene-environment interactions and support breeding for improved resource use efficiency of important crops such as bean (Phaseolus vulgaris L.). Here we provide an overview of state-of-the-art phenotyping approaches addressing three aspects of resource use efficiency in plants: belowground roots, aboveground shoots and transport/allocation processes. We demonstrate the capacity of high-precision methods to measure plant function or structural traits non-invasively, stating examples wherever possible. Ideally, high-precision methods are complemented by fast and high-throughput technologies. High-throughput phenotyping can be applied in the laboratory using automated data acquisition, as well as in the field, where imaging spectroscopy opens a new path to understand plant function non-invasively. For example, we demonstrate how magnetic resonance imaging (MRI) can resolve root structure and separate root systems under resource competition, how automated fluorescence imaging (PAM fluorometry) in combination with automated shape detection allows for high-throughput screening of photosynthetic traits and how imaging spectrometers can be used to quantify pigment concentration, sun-induced fluorescence and potentially photosynthetic quantum yield. We propose that these phenotyping techniques, combined with mechanistic knowledge on plant structure-function relationships, will open new research directions in whole-plant ecophysiology and may assist breeding for varieties with enhanced resource use efficiency varieties.

Continuous Turnover of Carotenes and Chlorophyll a in Mature Leaves of Arabidopsis Revealed by 14CO2 Pulse-Chase Labeling

Carotenoid turnover was investigated in mature leaves of Arabidopsis (Arabidopsis thaliana) by 14CO2 pulse-chase labeling under control-light (CL; 130 μmol photons m−2 s−1) and high-light (HL; 1,000 μmol photons m−2 s−1) conditions. Following a 30-min 14CO2 administration, photosynthetically fixed 14C was quickly incorporated in β-carotene (β-C) and chlorophyll a (Chl a) in all samples during a chase of up to 10 h. In contrast, 14C was not detected in Chl b and xanthophylls, even when steady-state amounts of the xanthophyll-cycle pigments and lutein increased markedly, presumably by de novo synthesis, in CL-grown plants under HL. Different light conditions during the chase did not affect the 14C fractions incorporated in β-C and Chl a, whereas long-term HL acclimation significantly enhanced 14C labeling of Chl a but not β-C. Consequently, the maximal 14C signal ratio between β-C and Chl a was much lower in HL-grown plants (1:10) than in CL-grown plants (1:4). In lut5 mutants, containing α-carotene (α-C) together with reduced amounts of β-C, remarkably high 14C labeling was found for α-C while the labeling efficiency of Chl a was similar to that of wild-type plants. The maximum 14C ratios between carotenes and Chl a were 1:2 for α-C:Chl a and 1:5 for β-C:Chl a in CL-grown lut5 plants, suggesting high turnover of α-C. The data demonstrate continuous synthesis and degradation of carotenes and Chl a in photosynthesizing leaves and indicate distinct acclimatory responses of their turnover to changing irradiance. In addition, the results are discussed in the context of photosystem II repair cycle and D1 protein turnover.

Sun-shade patterns of leaf carotenoid composition in 86 species of neotropical forest plants

A survey of photosynthetic pigments, including 86 species from 64 families, was conducted for leaves of neotropical vascular plants to study sun-shade patterns in carotenoid biosynthesis and occurrence of α-carotene (α-Car) and lutein epoxide (Lx). Under low light, leaves invested less in structural components and more in light harvesting, as manifested by low leaf dry mass per area (LMA) and enhanced mass-based accumulation of chlorophyll (Chl) and carotenoids, especially lutein and neoxanthin. Under high irradiance, LMA was greater and β-carotene (β-Car) and violaxanthin-cycle pool increased on a leaf area or Chl basis. The majority of plants contained α-Car in leaves, but the α- to β-Car ratio was always low in the sun, suggesting preference for β-Car in strong light. Shade and sun leaves had similar β,ε-carotenoid contents per unit Chl, whereas sun leaves had more β,β-carotenoids than shade leaves. Accumulation of Lx in leaves was found to be widely distributed among taxa: >5 mmol mol Chl-1 in 20% of all species examined and >10 mmol mol Chl-1 in 10% of woody species. In Virola elongata (Benth.) Warb, having substantial Lx in both leaf types, the Lx cycle was operating on a daily basis although Lx restoration in the dark was delayed compared with violaxanthin restoration.

Diurnal and acclimatory responses of violaxanthin and lutein epoxide in the Australian mistletoe Amyema miquelii

This study investigated the chloroplast pigment content of the Australian mistletoe Amyema miquelii (Lehm. ex Miq.) Tiegh. over diurnal periods in sun- and shade-acclimated leaves. Amyema miquelii exhibited the typical higher plant complement of neoxanthin, the xanthophyll cycle pigments, lutein, chlorophylls a and b and β carotene. Substantial levels of lutein epoxide were also present. Interestingly, diurnal light exposure elicited a decrease in lutein epoxide that paralleled the decrease in violaxanthin. Compared with shade-acclimated leaves, sun leaves exhibited reduced lutein epoxide and violaxanthin levels and higher chlorophyll a/b ratios. It is clear that the pools of violaxanthin and lutein epoxide respond in parallel to both diurnal light changes and sun-shade acclimation, although there seemed to be some differences in the recovery characteristics. These results raise a question as to whether lutein and lutein epoxide cycling may provide an auxiliary means of energy dissipation for some species.

Short- and Long-Term Operation of the Lutein-Epoxide Cycle in Light-Harvesting Antenna Complexes

The lutein-5,6-epoxide (Lx) cycle operates in some plants between lutein (L) and its monoepoxide, Lx. Whereas recent studies have established the photoprotective roles of the analogous violaxanthin cycle, physiological functions of the Lx cycle are still unknown. In this article, we investigated the operation of the Lx cycle in light-harvesting antenna complexes (Lhcs) of Inga sapindoides Willd, a tropical tree legume accumulating substantial Lx in shade leaves, to identify the xanthophyll-binding sites involved in short- and long-term responses of the Lx cycle and to analyze the effects on light-harvesting efficiency. In shade leaves, Lx was converted into L upon light exposure, which then replaced Lx in the peripheral V1 site in trimeric Lhcs and the internal L2 site in both monomeric and trimeric Lhcs, leading to xanthophyll composition resembling sun-type Lhcs. Similar to the violaxanthin cycle, the Lx cycle was operating in both photosystems, yet the light-induced Lx → L conversion was not reversible overnight. Interestingly, the experiments using recombinant Lhcb5 reconstituted with different Lx and/or L levels showed that reconstitution with Lx results in a significantly higher fluorescence yield due to higher energy transfer efficiencies among chlorophyll (Chl) a molecules, as well as from xanthophylls to Chl a. Furthermore, the spectroscopic analyses of photosystem I-LHCI from I. sapindoides revealed prominent red-most Chl forms, having the lowest energy level thus far reported for higher plants, along with reduced energy transfer efficiency from antenna pigments to Chl a. These results are discussed in the context of photoacclimation and shade adaptation.

Photoinactivation of Photosystem II in leaves

Photoinactivation of Photosystem II (PS II), the light-induced loss of ability to evolve oxygen, inevitably occurs under any light environment in nature, counteracted by repair. Under certain conditions, the extent of photoinactivation of PS II depends on the photon exposure (light dosage, x), rather than the irradiance or duration of illumination per se, thus obeying the law of reciprocity of irradiance and duration of illumination, namely, that equal photon exposure produces an equal effect. If the probability of photoinactivation (p) of PS II is directly proportional to an increment in photon exposure (p = kΔx, where k is the probability per unit photon exposure), it can be deduced that the number of active PS II complexes decreases exponentially as a function of photon exposure: N = Noexp(−kx). Further, since a photon exposure is usually achieved by varying the illumination time (t) at constant irradiance (I), N = Noexp(−kI t), i.e., N decreases exponentially with time, with a rate coefficient of photoinactivation kI, where the product kI is obviously directly proportional to I. Given that N = Noexp(−kx), the quantum yield of photoinactivation of PS II can be defined as −dN/dx = kN, which varies with the number of active PS II complexes remaining. Typically, the quantum yield of photoinactivation of PS II is ca. 0.1μmol PS II per mol photons at low photon exposure when repair is inhibited. That is, when about 107 photons have been received by leaf tissue, one PS II complex is inactivated. Some species such as grapevine have a much lower quantum yield of photoinactivation of PS II, even at a chilling temperature. Examination of the longer-term time course of photoinactivation of PS II in capsicum leaves reveals that the decrease in N deviates from a single-exponential decay when the majority of the PS II complexes are inactivated in the absence of repair. This can be attributed to the formation of strong quenchers in severely-photoinactivated PS II complexes, able to dissipate excitation energy efficiently and to protect the remaining active neighbours against damage by light.

Sustained downregulation of photosystem II in mistletoes during winter depression of photosynthesis

Cold acclimation by sustained downregulation of PSII was studied in intact leaves of an Australian mistletoe Amyema miquelii (Lehm. ex Miq.) Tiegh. and its host Eucalyptus. The trends were followed from autumn to spring on leaves that had developed in summer and were exposed to different microclimates with progress of the seasons. In sun leaves of mistletoe, efficiency of excitation energy transfer from light-harvesting pigments to Chl a molecules in PSII core complexes was markedly reduced in winter. Concomitantly, a band in 77K fluorescence emission spectra emerged at 715 nm, in the same way as the cold-hard band found in overwintering snow gum seedlings (Gilmore and Ball 2000, Proceedings of the National Academy of Sciences USA 97, 11 098-11 101). Further, a distinct band, which presumably involves Chl-b-containing antennae complexes, appeared at 705 nm in -2°C fluorescence emission spectra with decreasing intensity of the PSII band. Much shorter PSII fluorescence lifetimes measured in sun leaves of mistletoe that were exhibiting sustained downregulation of PSII indicated enhanced thermal dissipation of excitation energy. Winter acclimation symptoms of the photosynthetic apparatus were more striking in mistletoe sun leaves compared with eucalypt sun leaves. Because the light and temperature environments of sun leaves are similar for the parasite and host, we primarily attribute the enhanced light-acclimation symptoms to the limited photosynthetic capacity of A. miquelii in winter.

Lutein epoxide cycle, light harvesting and photoprotection in species of the tropical tree genus Inga

Dynamics and possible function of the lutein epoxide (Lx) cycle, that is, the reversible conversion of Lx to lutein (L) in the light-harvesting antennae, were investigated in leaves of tropical tree species. Photosynthetic pigments were quantified in nine Inga species and species from three other genera. In Inga, Lx levels were high in shade leaves (mostly above 20 mmol mol(-1) chlorophyll) and low in sun leaves. In Virola surinamensis, both sun and shade leaves exhibited very high Lx contents (about 60 mmol mol(-1) chlorophyll). In Inga marginata grown under high irradiance, Lx slowly accumulated within several days upon transfer to deep shade. When shade leaves of I. marginata were briefly exposed to the sunlight, both violaxanthin and Lx were quickly de-epoxidized. Subsequently, overnight recovery occurred only for violaxanthin, not for Lx. In such leaves, containing reduced levels of Lx and increased levels of L, chlorophyll fluorescence induction showed significantly slower reduction of the photosystem II electron acceptor, Q(A), and faster formation as well as a higher level of non-photochemical quenching. The results indicate that slow Lx accumulation in Inga leaves may improve light harvesting under limiting light, while quick de-epoxidation of Lx to L in response to excess light may enhance photoprotection.