Decreased photochemical efficiency of photosystem II following sunlight exposure of shade-grown leaves of avocado: because of, or in spite of, two kinetically distinct xanthophyll cycles?

This study resolved correlations between changes in xanthophyll pigments and photosynthetic properties in attached and detached shade-grown avocado (Persea americana) leaves upon sun exposure. Lutein epoxide (Lx) was deepoxidized to lutein (L), increasing the total pool by ΔL over 5 h, whereas violaxanthin (V) conversion to antheraxanthin (A) and zeaxanthin (Z) ceased after 1 h. During subsequent dark or shade recovery, de novo synthesis of L and Z continued, followed by epoxidation of A and Z but not of L. Light-saturated nonphotochemical quenching (NPQ) was strongly and linearly correlated with decreasing [Lx] and increasing [L] but showed a biphasic correlation with declining [V] and increasing [A+Z] separated when V deepoxidation ceased. When considering [ΔL+Z], the monophasic linear correlation was restored. Photochemical efficiency of photosystem II (PSII) and photosystem (PSI; deduced from the delivery of electrons to PSI in saturating single-turnover flashes) showed a strong correlation in their continuous decline in sunlight and an increase in NPQ capacity. This decrease was also reflected in the initial reduction of the slope of photosynthetic electron transport versus photon flux density. Generally longer, stronger sun exposures enhanced declines in both slope and maximum photosynthetic electron transport rates as well as photochemical efficiency of PSII and PSII/PSI more severely and prevented full recovery. Interestingly, increased NPQ capacity was accompanied by slower relaxation. This was more prominent in detached leaves with closed stomata, indicating that photorespiratory recycling of CO(2) provided little photoprotection to avocado shade leaves. Sun exposure of these shade leaves initiates a continuum of photoprotection, beyond full engagement of the Lx and V cycle in the antenna, but ultimately photoinactivated PSII reaction centers.

Remote chlorophyll fluorescence measurements with the laser-induced fluorescence transient approach.

The interaction of plants with their environment is very dynamic. Studying the underlying processes is important for understanding and modeling plant response to changing environmental conditions. Photosynthesis varies largely between different plants and at different locations within a canopy of a single plant. Thus, continuous and spatially distributed monitoring is necessary to assess the dynamic response of photosynthesis to the environment. Limited scale of observation with portable instrumentation makes it difficult to examine large numbers of plants under different environmental conditions. We report here on the application of a recently developed technique, laser-induced fluorescence transient (LIFT), for continuous remote measurement of photosynthetic efficiency of selected leaves at a distance of up to 50 m. The ability to make continuous, automatic, and remote measurements of photosynthetic efficiency of leaves with the LIFT provides a new approach for studying the interaction of plants with the environment and may become an important tool in phenotyping photosynthetic properties in field applications.

De novo synthesis and degradation of Lx and V cycle pigments during shade and sun acclimation in avocado leaves.

The photoprotective role of the universal violaxanthin cycle that interconverts violaxanthin (V), antheraxanthin (A), and zeaxanthin (Z) is well established, but functions of the analogous conversions of lutein-5,6-epoxide (Lx) and lutein (L) in the selectively occurring Lx cycle are still unclear. We investigated carotenoid pools in Lx-rich leaves of avocado (Persea americana) during sun or shade acclimation at different developmental stages. During sun exposure of mature shade leaves, an unusual decrease in L preceded the deepoxidation of Lx to L and of V to A+Z. In addition to deepoxidation, de novo synthesis increased the L and A+Z pools. Epoxidation of L was exceptionally slow, requiring about 40 d in the shade to restore the Lx pool, and residual A+Z usually persisted overnight. In young shade leaves, the Lx cycle was reversed initially, with Lx accumulating in the sun and declining in the shade. De novo synthesis of xanthophylls did not affect alpha- and beta-carotene pools on the first day, but during long-term acclimation alpha-carotene pools changed noticeably. Nonetheless, the total change in alpha- and beta-branch carotenoid pools was equal. We discuss the implications for regulation of metabolic flux through the alpha- and beta-branches of carotenoid biosynthesis and potential roles for L in photoprotection and Lx in energy transfer to photosystem II and explore physiological roles of both xanthophyll cycles as determinants of photosystem II efficiency.

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.

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 ß-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 ß-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.

The effect of elevated CO2, soil and atmospheric water deficit and seasonal phenology on leaf and ecosystem isoprene emission.

Two cottonwood plantations were grown at different CO2 concentrations at the Biosphere 2 Laboratory in Arizona to investigate the response of isoprene emission to elevated [CO2] and its interaction with water deficits. We focused on responses due to seasonal variation and variation in the mean climate from one year to the next. In fall and in spring, isoprene emission rate showed a similar inhibition by elevated [CO2], despite an 8-10°C seasonal difference in mean air temperature. The overall response of isoprene emission to drought was also similar for observations conducted during the spring or fall, and during the fall of two different years with an approximate 5°C difference in mean air temperature. In general, leaf-level isoprene emission rates, measured at constant temperature and photon-flux density, decreased slightly, or remained constant during drought, whereas ecosystem-level isoprene emission rates increased. The uncoupling of ecosystem- and leaf-scale responses is not due to differential dependence on leaf area index (LAI) as LAI increased only slightly, or decreased, during the drought treatments at the same time that ecosystem isoprene emission rate increased greatly. Nor does the difference in isoprene emission rate between leaves and ecosystems appear to be due solely to increases in canopy surface temperature during the drought, though some increase in temperature was observed. It is possible that still further factors, such as increased penetration of PPFD into the canopy as a result of changes in leaf angle, reduced sink strength of the soil for atmospheric isoprene, and decreases in the mean Ci of leaves, combined with the small increases in canopy surface temperature, increased the ecosystem isoprene emission rate. Whatever the causes of the differences in the leaf and ecosystem responses, we conclude that the overall shape of the leaf and ecosystem responses to drought was constant irrespective of season or year.

Improved survival of very high light and oxidative stress is conferred by spontaneous gain-of-function mutations in Chlamydomonas.

Investigations into high light and oxidative stress in photosynthetic organisms have focussed primarily on genetic impairment of different photoprotective functions. There are few reports of "gain-of-function" mutations that provide enhanced resistance to high light and/or oxidative stress without reduced productivity. We have isolated at least four such very high light resistant (VHL(R)) mutations in the green alga, Chlamydomonas reinhardtii, that permit near maximal growth rates at light intensities lethal to wild type. This resistance is not due to an alteration in electron transport rate or quantity and functionality of the two photosystems that could have enhanced photochemical quenching. Nor is it due to reduced excitation pressure by downregulation of the light harvesting antennae or increased nonphotochemical quenching. In fact, photosynthetic activity is unaffected in more than 30 VHL(R) isolates. Instead, the basis of the VHL(R) phenotype is a combination of traits, which appears to be dominated by enhanced capacity to tolerate reactive oxygen species generated by excess light, methylviologen, rose bengal or hydrogen peroxide. This is further evidenced in lower levels of ROS after exposure to very high light in the VHL(R)-S9 mutant. Additionally, the VHL(R) phenotype is associated with increased zeaxanthin accumulation, maintenance of fast synthesis and degradation rates of the D1 protein, and sustained balanced electron flow into and out of PSI under very high light. We conclude that the VHL(R) mutations arose from a selection pressure that favors changes to the regulatory system(s) that coordinates several photoprotective processes amongst which repair of PSII and enhanced detoxification of reactive oxygen species play seminal roles.

Distinctive diel growth cycles in leaves and cladodes of CAM plants: differences from C3 plants and putative interactions with substrate availability, turgor and cytoplasmic pH.

Distinct diel rhythms of leaf and cladode expansion growth were obtained in crassulacean acid metabolism (CAM) plants under water-limited conditions, with maxima at mid-day during phase III of CO2 assimilation. This pattern coincided with the availability of CO2 for photosynthesis and growth during the decarboxylation of malic acid, with maximum cell turgor due to the nocturnally accumulated malic acid, and with the period of low cytoplasmic pH associated with malic acid movement from vacuole to cytosol. Maximum growth rates were generally only 20% of those in C3 plants and were reached at a different time of the day compared with C3 plants. The results suggest that malic acid, as a source of carbohydrates, and a determinant of turgor and cytoplasmic pH, plays a major role in the control of diel growth dynamics in CAM plants under desert conditions. The observed plasticity in phasing of growth rhythms under situations of differing water availability suggests that a complex network of factors controls the diel growth patterns in CAM plants and needs to be investigated further.

Occurrence of the lutein-epoxide cycle in mistletoes of the Loranthaceae and Viscaceae.

The lutein-epoxide cycle (Lx cycle) is an auxiliary xanthophyll cycle known to operate only in some higher-plant species. It occurs in parallel with the common violaxanthin cycle (V cycle) and involves the same epoxidation and de-epoxidation reactions as in the V cycle. In this study, the occurrence of the Lx cycle was investigated in the two major families of mistletoe, the Loranthaceae and the Viscaceae. In an attempt to find the limiting factor(s) for the occurrence of the Lx cycle, pigment profiles of mistletoes with and without the Lx cycle were compared. The availability of lutein as a substrate for the zeaxanthin epoxidase appeared not to be critical. This was supported by the absence of the Lx cycle in the transgenic Arabidopsis plant lutOE, in which synthesis of lutein was increased at the expense of V by overexpression of epsilon-cyclase, a key enzyme for lutein synthesis. Furthermore, analysis of pigment distribution within the mistletoe thylakoids excluded the possibility of different localizations for the Lx- and V-cycle pigments. From these findings, together with previous reports on the substrate specificity of the two enzymes in the V cycle, we propose that mutation to zeaxanthin epoxidase could have resulted in altered regulation and/or substrate specificity of the enzyme that gave rise to the parallel operation of two xanthophyll cycles in some plants. The distribution pattern of Lx in the mistletoe phylogeny inferred from 18S rRNA gene sequences also suggested that the occurrence of the Lx cycle is determined genetically. Possible molecular evolutionary processes that may have led to the operation of the Lx cycle in some mistletoes are discussed.

Crassulacean acid metabolism photosynthesis: ;working the night shift'.

Crassulacean acid metabolism (CAM) can be traced from Roman times through persons who noted a morning acid taste of some common house plants. From India in 1815, Benjamin-Heyne described a 'daily acid taste cycle' with some succulent garden plants. Recent work has shown that the nocturnally formed acid is decarboxylated during the day to become the CO(2) for photosynthesis. Thus, CAM photosynthesis extends over a 24-hour day using several daily interlocking cycles. To understand CAM photosynthesis, several landmark discoveries were made at the following times: daily reciprocal acid and carbohydrate cycles were found during 1870 to 1887; their precise identification, as malic acid and starch, and accurate quantification occurred from 1940 to 1954; diffusive gas resistance methods were introduced in the early 1960s that led to understanding the powerful stomatal control of daily gas exchanges; C(4) photosynthesis in two different types of cells was discovered from 1965 to approximately 1974 and the resultant information was used to elucidate the day and night portions of CAM photosynthesis in one cell; and exceptionally high internal green tissue CO(2) levels, 0.2 to 2.5%, upon the daytime decarboxylation of malic acid, were discovered in 1979. These discoveries then were combined with related information from C(3) and C(4) photosynthesis, carbon biochemistry, cellular anatomy, and ecological physiology. Therefore by approximately 1980, CAM photosynthesis finally was rigorously outlined. In a nutshell, 24-hour CAM occurs by phosphoenol pyruvate (PEP) carboxylase fixing CO(2)(HCO(3) (-)) over the night to form malic acid that is stored in plant cell vacuoles. While stomata are tightly closed the following day, malic acid is decarboxylated releasing CO(2) for C(3) photosynthesis via ribulose bisphosphate carboxylase oxygenase (Rubisco). The CO(2) acceptor, PEP, is formed via glycolysis at night from starch or other stored carbohydrates and after decarboxylation the three carbons are restored each day. In mid to late afternoon the stomata can open and mostly C(3) photosynthesis occurs until darkness. CAM photo-synthesis can be both inducible and constitutive and is known in 33 families with an estimated 15 to 20 000 species. CAM plants express the most plastic and tenacious photosynthesis known in that they can switch photosynthesis pathways and they can live and conduct photosynthesis for years even in the virtual absence of external H(2)O and CO(2), i.e., CAM tenaciously protects its photosynthesis from both H(2)O and CO(2) stresses.

Assessment of photoprotection mechanisms of grapevines at low temperature.

The photosynthetic response of grapevine leaves (Vitis vinifera L. cv. Riesling) to low temperature was studied in the field and laboratory. Light-saturated rates of photosynthetic electron transport were lower and non-photochemical energy dissipation was higher when leaves were subject to low morning temperatures than to high afternoon temperatures under field conditions. These responses to low temperatures occurred without sustained reduction of quantum efficiency of PSII as measured by the variable to maximum chlorophyll fluorescence yield ratio, Fv/Fm, after dark adaptation. The temperature dependence of light-saturated apparent electron transport rate, gas exchange and non-photochemical quenching (NPQ) was also examined in laboratory experiments with glasshouse-grown material. NPQ reached saturation at lower light intensity with decreasing temperature. The relationship between the quantum efficiency of PSII and CO2 fixation at 25°C (2-21% O2) and 10°C (2-21% O2) indicated a decreased dependence of electron transport on both photorespiration and the Mehler reaction at the lower temperature. The calculated percentage of electron flow to the Mehler reaction declined faster than photorespiration at low temperature. Warm- and cold-treated leaf discs under saturating light showed very little photoinhibition as measured by sustained reduction in Fv/Fm, which was linearly related to the percentage of functional PSII reaction centres. However, the addition of dithiothreitol greatly enhanced the rate of photoinhibition, indicating a potentially strong dependence on xanthophyll de-epoxidation for photoprotection at low temperature.

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.

Contrasting patterns of photosynthetic acclimation and photoinhibition in two evergreen herbs from a winter deciduous forest.

The relationship between the microclimate within an Oak-Hickory forest and photosynthetic characters of two resident evergreen herbs with contrasting leaf phenologies was investigated on a monthly basis for 1 full year. Heuchera americana has leaf flushes in the spring and fall, with average leaf life spans of 6-7 months. Hexastylis arifolia produces a single cohort of leaves each spring with a leaf life span of 12-13 months. We predicted that among evergreen plants inhabiting a seasonal habitat, a species for which the frequency of leaf turnover is greater than the frequency of seasonal extremes would have a greater annual range in photosynthetic capacity than a species that only produced a single flush of leaves during the year. Photosynthetic parameters, including apparent quantum yield, maximum photosynthetic capacity (Pmax), temperature of maximum photosynthesis, photochemical efficiency of PSII and leaf nitrogen (N) and chlorophyll concentrations, were periodically measured under laboratory conditions in leaves sampled from natural populations of both species. Mature leaves of both species acclimated to changing understory conditions with the mean seasonal differences being significantly greater for Heuchera than for Hexastylis. Area based maximum photosynthetic rates at 25°C were approximately 250% and 100% greater in winter leaves than summer leaves for Heuchera and Hexastylis respectively. Nitrogen concentrations were highest in winter leaves. Chlorophyll concentrations were highest in summer leaves. Low Pmax/N values for these species suggest preferential allocation of leaf nitrogen into non-photosynthetic pools and/or light-harvesting function at the expense of photosynthetic enzymes and electron transport components. Despite the increase in photosynthetic capacity, there was evidence of chronic winter photoinhibition in Hexastylis, but not in Heuchera. Among these ecologically similar species, there appears to be a trade-off between the frequency of leaf production and the balance of photosynthetic acclimation and photoinhibition.

Changes of fluorescence and xanthophyll pigments during dehydration in the resurrection plantSelaginella lepidophylla in low and medium light intensities.

The changes in photosynthetic efficiency and photosynthetic pigments during dehydration of the resurrection plantSelaginella lepidophylla (from the Chiuhahuan desert, S.W. Texas, USA) were examined under different light conditions. Changes in the photosynthetic efficiency were deduced from chlorophyll a fluorescence measurements (Fo, Fm, and Fv) and pigment changes were measured by HPLC analysis. A small decrease in Fv/Fm was seen in hydrated stems in high light (650 µmol photons·m-2·s-1) but not in low light (50 µmol photons·m-2·s-1). However, a pronounced decline in Fv/Fm was observed during dehydration in both light treatments, after one to two hours of dehydration. A rise in Fo was observed only after six to ten hours of dehydration. Concomitant with the decrease in photosynthetic efficiency during dehydration a rise in the xanthophyll zeaxanthin was observed, even in low-light treatments. The increase in zeaxanthin can be related to previously observed photoprotective non-photochemical quenching of fluorescence in dehydrating stems ofS. lepidophylla. We hypothesize that under dehydrating conditions even low light levels become excessive and zeaxanthin-related photoprotection is engaged. We speculate that these processes, as well as stem curling and self shading (Eickmeier et al. 1992), serve to minimize photoinhibitory damage toS. lepidophylla during the process of dehydration.