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 α- and β-carotene pools on the first day, but during long-term acclimation α-carotene pools changed noticeably. Nonetheless, the total change in α- and β-branch carotenoid pools was equal. We discuss the implications for regulation of metabolic flux through the α- and β-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.It has long been recognized that photosynthesis in plants must resolve two conflicting requirements, the need to ramp up maximum light-harvesting efficiency in dim light and to wind back to lower efficiency when light is in excess, in order to maintain high rates of growth and productivity in varying light environments (Björkman, 1981; Pearcy, 1990). A wealth of research has established that plants adjust through an array of morphological and molecular events that confer photoprotection, mitigate and repair photoinactivation of PSII, and facilitate acclimation of the photosynthetic apparatus over different time scales in response to variable light regimes in wild plants, crops, and algae (Osmond et al., 1999; Demmig-Adams et al., 2006). In the context of the light reactions, low light acclimation optimizes light harvesting and energy transfer to the photosystems, particularly PSII, via enlarged functional antennae, accumulation of accessory light-harvesting pigments, and down-regulation of unnecessary competing photoprotective processes. High light acclimation involves increased photoprotection and photorepair, downsized antennae, fewer photosystems, and sometimes changes in the PSI to PSII stoichiometry (Osmond et al., 1999; Förster et al., 2005). Along with their function in energy transfer to the photosynthetic reaction centers as accessory pigments to chlorophylls, the xanthophyll pigments violaxanthin (V), antheraxanthin (A), and zeaxanthin (Z) play a central role in these transformations of the photosynthetic apparatus, especially in thermal energy dissipation and detoxification of reactive oxygen species.Two xanthophyll cycles are now known in terrestrial plants, the lutein epoxide cycle (Lx cycle) based on interconversions of lutein-5,6-epoxide (Lx) and lutein (L) synthesized from α-carotene (α-C), and the violaxanthin cycle (V cycle) based on the interconversions of V and A+Z synthesized from β-carotene (β-C; García-Plazaola et al., 2007). Presumably, both cycles are catalyzed by the same enzymes, violaxanthin epoxidase (VDE) for deepoxidation in high light and zeaxanthin epoxidase (ZE) for the reverse reactions in low light or darkness (Latowski et al., 2004). Rediscovery of the Lx cycle in the parasitic angiosperm Cuscuta reflexa (Bungard et al., 1999) has led to growing interest in differing manifestations of this cycle in terrestrial plants and its relationships to the apparently universal V cycle (Demmig-Adams, 1998). A complete Lx cycle seems to function on a daily basis in both C. reflexa and the mistletoe Amyema miquelii (Matsubara et al., 2001), even though Lx conversion to L is sometimes slower than V to A+Z and dark recovery of Lx is usually slower than that of V. Intriguingly, in shade leaves of Inga sapindoides, high concentrations of Lx were seemingly irreversibly converted to L on exposure to strong light, in marked contrast to the co-occurring, fully reversible V cycle (Matsubara et al., 2005). Similar responses have been found in other woody plants with long-lived leaves in deeply shaded canopies, including Mediterranean oaks (Quercus spp.; García-Plazaola et al., 2003), sweet bay laurel (Laurus nobilis), and avocado (Persea americana; Esteban et al., 2007, 2008). This response type is known as a truncated Lx cycle (García-Plazaola et al., 2007).The functions attributed to the Lx cycle were initially based on structural analogies between Lx and A and between L and Z (Bungard et al., 1999; Pogson and Rissler, 2000; Matsubara et al., 2001). With increased evidence for the possible role of L in photoprotection (Pogson et al., 1996, 1998; Lokstein et al., 2002; Dall''Osto et al., 2006), additional functional analogies emerged. Furthermore, recent in vitro reconstitution studies with light-harvesting complex proteins and purified pigments also support a spatial overlap of the cycles, as some pigment-binding sites can be occupied by either α- or β-xanthophylls (Matsubara et al., 2007). An attractive hypothesis is that photoconversion of Lx to L might sustain or enhance photoprotection associated with the V cycle (Demmig-Adams and Adams, 1992; Niyogi, 2000). In support of this view, it has been demonstrated in leaves of Quercus rubra and in leaflets of Inga marginata that increasing amounts of photoconverted L, which persist even when A and Z are epoxidized to V, were associated with faster engagement and higher levels of nonphotochemical quenching (NPQ) of chlorophyll fluorescence (García-Plazaola et al., 2003; Matsubara et al., 2008). Furthermore, evidence from mammalian eye research as well as from plants suggests that L also acts as a highly efficient reactive oxygen species scavenger (Kim et al., 2006; Johnson et al., 2007).Broader issues, such as the roles of short-term dynamics of the two cycles in relation to long-term processes of shade and sun acclimation and in relation to leaf development and age, are poorly understood. Nonfruiting shoots of avocado trees constitute a very suitable model system in which to address these issues. Long-lived leaves of shade-grown avocado contain some of the highest levels of Lx thus far recorded (Esteban et al., 2007; García-Plazaola et al., 2007) and have two to four flushes of leaf initiation per year that exhibit a form of delayed greening in which leaf expansion precedes increases in stomatal conductance, chlorophyll content, and CO₂ assimilation. Expanding leaves remain sinks for up to 1 month until they reach about 70% to 80% of full expansion (Schaffer et al., 1991), and stomata do not become fully functional until leaves attain 90% of full expansion (Scholefield and Kriedemann, 1979). However, shoots also retain old leaves through several flushes, and leaves from the previous season contribute significantly to total plant carbon gain (Liu et al., 2002), with photosynthesis rates up to 50% of those in new, fully expanded leaves (Heath et al., 2005). These properties offer an array of opportunities for new research into the concurrent operation of the two xanthophyll cycles.Since there have been very few studies of these complex responses, we carried out a series of short- and long-term light treatments that are likely to reflect what leaves may experience in natural environments, with the aim to gain further insight into the physiological relevance of the Lx and V cycles under those circumstances. Four types of acclimation experiments were undertaken in this study. First, short-term acclimation from shade to sun addressed fast responses to a drastic increase in the light environment, simulating a prolonged sun fleck in shaded mature leaves or exposure to a bright sunny day in young leaves that had emerged during a prolonged overcast (shaded) growth period. These experiments revealed an unexpected loss of L prior to deepoxidation of Lx and V and a reverse Lx cycle in young leaves. Second, long-term acclimation of sun leaves to prolonged shade simulated normal processes of shading by further growth of outer canopy leaves. These treatments established the very slow accumulation of Lx in avocado leaves. Third, sequential sun exposures of mature leaves over several days, followed by continuous shade, were applied to simulate successive prolonged sun flecks, mimicking stochastic canopy disturbance during severe weather events, which confirmed many responses in the above experiments, particularly the very slow epoxidation of L to Lx in prolonged shade. Fourth, long-term acclimation of young and mature leaves to sun was examined. These experiments simulated sudden changes to canopy architecture as experienced during pruning and extended our understanding of the comparative rates and magnitude of Lx and V cycle engagement. We discuss the short-and long-term kinetics of both cycles in avocado leaves of different ages during acclimation, with particular attention to the stoichiometric relationships between xanthophyll and carotenoid pools and changing PSII efficiency.     

Influence of long-term stability conditions on microbicidal nucleoside prodrug (WHI-07)-loaded gel-microemulsion

The objective of this study was to evaluate the long-term stability of the antiretroviral spermicide WHI-07 (5-bromo-6-methoxy-5,6-dihydro-3′-azidothymidine-5′-(p-bromophenyl)-methoxyalaninyl phosphate) in a polymer-based microemulsion. The recovery and stability of WHI-07 in gelmicroemulsion was examined by a validated high-performance liquid chromatography (HPLC) method. The stability was examined over a period of 24 weeks at 3 controlled temperatures (4°C, 25°C, and 40°C). The recovery of the prodrug from 0.5% to 2.0% WHI-07-loaded gel-microemulsion was 99.8%. HPLC analysis revealed that a 2% WHI-07-loaded gel-microemulsion stored at room temperature and cold temperatures for 24 weeks retained >90% of the prodrug, whereas those stored at 40°C maintained 90% of initial WHI-07 for at least 10 weeks. The observed stability of WHI-07 in gel-microemulsion is of great importance for its widespread utility in various climatological conditions. Published: September 1, 2006     

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 -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.Abbreviations A antheraxanthin - - and -Car - and -carotene - Chl chlorophyll - -DM dodecyl--d-maltoside - DPS de-epoxidation state of the violaxanthin cycle (= [A+Z]/[V+A+Z]) - Lut lutein - Lx lutein epoxide - Caro total carotenoid concentration - V violaxanthin - VAZ pool size of the violaxanthin cycle (= V+A+Z) - VDE violaxanthin de-epoxidase - Z zeaxanthin - ZE zeaxanthin epoxidase     

A low-density single nucleotide polymorphism panel for brown trout (Salmo trutta L.) suitable for exploring genetic diversity at a range of spatial scales

The rivers of southern England and northern France which drain into the English Channel contain several genetically unique groups of trout (Salmo trutta L.) that have suffered dramatic declines in numbers over the past 40 years. Knowledge of levels and patterns of genetic diversity is essential for effective management of these vulnerable populations. Using restriction site-associated DNA sequencing (RADseq) data, we describe the development and characterisation of a panel of 95 single nucleotide polymorphism (SNP) loci for trout from this region and investigate their applicability and variability in both target (i.e., southern English) and non-target trout populations from northern Britain and Ireland. In addition, we present three case studies which demonstrate the utility and resolution of these genetic markers at three levels of spatial separation:(a) between closely related populations in nearby rivers, (b) within a catchment and (c) when determining parentage and familial relationships between fish sampled from a single site, using both empirical and simulated data. The SNP loci will be useful for population genetic and assignment studies on brown trout within the UK and beyond.     

Chlorophyll fluorescence in the resurrection plant Selaginella lepidophylla (Hook. & Grev.) Spring during high-light and desiccation stress,and evidence for zeaxanthin-associated photoprotection

The function of photosystem (PS)II during desiccation and exposure to high photon flux density (PFD) was investigated via analysis of chlorophyll fluorescence in the desert resurrection plant Selaginella lepidophylla (Hook. and Grev.) Spring. Exposure of hydrated, physiologically competent stems to 2000 mol · m^–2 · s^–1 PFD caused significant reductions in both intrinsic fluorescence yield (F_O) and photochemical efficiency of PSII (F_V/F_M) but recovery to pre-exposure values was rapid under low PFD. Desiccation under low PFD also affected fluorescence characteristics. Both F_V/F_M and photochemical fluorescence quenching remained high until about 40% relative water content and both then decreased rapidly as plants approached 0% relative water content. In contrast, the maximum fluorescence yield (F_M) decreased and non-photochemical fluorescence quenching increased early during desiccation. In plants dried at high PFD, the decrease in F_V/F_M was accentuated and F_O was reduced, however, fluorescence characteristics returned to near pre-exposure values after 24-h of rehydration and recovery at low PFD. Pretreatment of stems with dithiothreitol, an inhibitor of zeaxanthin synthesis, accelerated the decline in F_V/F_M and significantly increased F_O relative to controls at 925 mol · m^–2 · s^–1 PFD, and the differences persisted over a 3-h low-PFD recovery period. Pretreatment with dithiothreitol also significantly decreased non-photochemical fluorescence quenching, increased the reduction state of Q_A, the primary electron acceptor of PSII, and prevented the synthesis of zeaxanthin relative to controls when stems were exposed to PFDs in excess of 250 mol · m^–2 · s^–1. These results indicate that a zeaxanthin-associated mechanism of photoprotection exists in this desert pteridophyte that may help to prevent photoinhibitory damage in the fully hydrated state and which may play an additional role in protecting PSII as thylakoid membranes undergo water loss.Abbreviations and Symbols DTT dithiothreitol - EPS epoxidation state - F_O yield of instantaneous fluorescence at open PSII centers - F_M maximum yield of fluorescence at closed PSII centers induced by saturating light - F_M F_M determined during actinic illumination - F_V yield of variable fluorescence (F_M-F_O) - F_V/F_M photochemical efficiency of PSII - q_P photochemical fluorescence quenching - q_NP non-photochemical fluorescence quenching of Schreiber et al. (1986) - NPQ non-photochemical fluorescence quenching from the Stern-Volmer equation - PFD photon flux density - RWC relative water content This paper is based on research done while W.G.E. was on leave of absence at Duke University during the fall of 1990. We would like to thank Dan Yakir, John Skillman, Steve Grace, and Suchandra Balachandran and many others at Duke University for their help and input with this research. Dr. Barbara Demmig-Adams provided zeaxanthin for standard-curve purposes.     

Carbon assimilation patterns and growth of the introduced CAM plant Opuntia inermis in Eastern Australia

Summary The daily course of CO₂ and H₂O exchange in cladodes of Opuntia inermis was studied at four sites in Eastern Australia. On most occasions cladode water contents were high and nocturnal stomatal opening resulted in substantial uptake of CO₂ and synthesis of about 130 equiv cm^-2 of malic acid during the night. Under water stress nocturnal stomatal opening was confined to the latter part of the night and acid synthesis was reduced to about 40 equiv cm^-2. Night temperature had little effect on acid synthesis, which responded primarily to rainfall and changed from the stressed condition within 2–3 days in irrigation experiments. On many occasions following summer rainfall stomata opened for 4 h in the late afternoon permitting net CO₂ fixation which may contribute about 25% of the total carbon assimilated. This CO₂ fixation was insufficient to have a marked impact on the ₁₃C value of the Opuntia cladodes. CO₂ fixation in the light occurred in conjunction with maximum dark CO₂ fixation under mesic conditions. Dark CO₂ fixation rates were 3 to 5 times greater than those recorded in desert cacti under favorable conditions. Relative growth rates calculated on the basic of CO₂ exchange correspond to measured relative growth rates of 0.05 g g^-1 dry wt day^-1 which prevailed for 60–90 days in summer. The capacity for very active CO₂ fixation in the dark and light following summer rainfall and the capacity to persist at low levels of metabolic activity through summer drought are discussed in relation to the success of this introduced species in this habitat.     

Stomatal responses to humidity in Opuntia inermis in relation to control of CO2 and H2O exchange patterns

Summary At constant cladode temperature the stomatal resistance of O. inermis increased when the cladode-air vapor pressure difference was increased and stomatal resistance decreased when the cladode-air vapor pressure difference was lowered. Net CO₂ fixation in the dark was very responsive to these humidity dependent changes in stomatal resistance. Net CO₂ fixation and stomatal resistance in the light did not respond to changes in cladode-air vapor pressure differences in the light under the conditions tested. When temperature response functions for dark CO₂ fixation were examined at constant ambient humidity, the reduction in dark CO₂ fixation at higher temperatures was largely due to stomatal closure in response to the increased vapor pressure difference. The water requirement for net CO₂ fixation in the dark at typical nocturnal vapor pressure differences was about 10 times lower than that of net CO₂ fixation in the light at vapor pressure differences typical of the late afternoon. The role of the stomatal responses to humidity in determining the patterns and rates of net CO₂ exchange in the light or dark, and its possible ecological significance is discussed.     

Effect of oxygen on photosynthesis and biosynthesis of glycolate in photoheterotrophically grown cells of Rhodospirillum rubrum

Photosynthetic CO₂ fixation was studied using cells of Rhodospirillumrubrum grown heterotrophically on malate or butyrate. Ratesof CO₂ fixation were higher in the malategrown cells than inthe butyrate-grown bacteria but ribulosebisphosphate (RUP₂)carboxylase/oxygenase activities were higher in the extractsprepared from the butyrate-grown bacteria. The photosyntheticCO₂ fixation in the butyrate-grown R. rubrum cells was inhibitedby KCN, and the inhibitory effect of O₂ on CO₂ fixation wasreversed when cells were returned to an N₂ atmosphere. In themalate-grown cells, photosynthetic CO₂ fixation was insensitiveto KCN and the inhibitory effect exerted by O₂ was practicallyirreversible. ¹⁴CO₂ was not incorporated into glycolate by either malate-or butyrate-grown cells in an N₂ atmosphere, but small amountsof labeled glycolate were found in malate- and butyrate-growncells in air or 100% O₂. Glycolate excreted by these cells in100% O₂ was measured colorimetrically and its identity establishedby mass spectrometry. When the O₂ atmosphere was labeled with¹⁸O₂, only one of the carboxyl oxygens of the excreted glycolatewas labeled, and the enrichment of ¹⁸O in this carboxyl oxygenrelative to the ¹⁸O₂ provided was greater than 80%. These studiesshow that significant glycolate production by R. rubrum onlyoccurs in the presence of O₂ and that in both malateand butyrate-growncells, the glycolate so formed is presumably produced via RuP₂oxygenase. ¹ Paper No. 46 in the series "Structure and Function of ChloroplastProteins", and research supported in part by research grantsfrom the Japanese Ministry of Education (No. 211113), the TorayScience Foundation (Tokyo), and the Nissan Science Foundation(Tokyo). (Received August 19, 1978; )     

Photoinhibition of intact attached leaves of c(3) plants illuminated in the absence of both carbon dioxide and of photorespiration

When leaflets of bean and leaves of other species of C₃ plants are illuminated in the absence of CO₂ and at low O₂ partial pressure, the capacity for CO₂ assimilation at saturating light and its efficiency at low light intensities are inhibited. This photoinhibition is dependent on leaflet age and period of illumination. In young leaflets and following short exposure to these photoinhibitory conditions, some recovery of CO₂ assimilation capacity is observed immediately after treatment. Following substantial (70 to 80%) photoinhibition of CO₂ assimilation, recovery in fully expanded leaflets is observed only after 48 hours in normal air. The photoinhibition is largely prevented by providing CO₂ at partial pressures equivalent to the CO₂ compensation point, or by >210 millibars O₂ which permits internal CO₂ production by photorespiration. If leaflets are illuminated in 60 microbars CO₂ and 210 millibars O₂ (the CO₂ compensation point in air), no photoinhibition is observed. Electron transport processes and fluorescence emission associated with photosystem II are inhibited in chloroplast thylakoids isolated from leaflets after illumination in zero CO₂ and 10 millibars O₂. These studies support the hypothesis that CO₂ recycling through photorespiration is one means of effectively dissipating excess photochemical energy when CO₂ supply to illuminated leaves is limited.