Diel leaf growth cycles in Clusia spp. are related to changes between C3 photosynthesis and crassulacean acid metabolism during development and during water stress

This study reports evidence that the timing of leaf growth responds to developmental and environmental constraints in Clusia spp. We monitored diel patterns of leaf growth in the facultative C₃-crassulacean acid metabolism (CAM) species Clusia minor and in the supposedly obligate CAM species Clusia alata using imaging methods and followed diel patterns of CO₂ exchange and acidification. Developing leaves of well-watered C. minor showed a C₃-like diel pattern of gas exchange and growth, with maximum relative growth rate (RGR) in the early night period. Growth slowed when water was withheld, accompanied by nocturnal CO₂ exchange and the diel acid change characteristic of CAM. Maximum leaf RGR shifted from early night to early in the day when water was withheld. In well-watered C. alata , similar changes in the diel pattern of leaf growth occurred with the development of CAM during leaf ontogeny. We hypothesize that the shift in leaf growth cycle that accompanies the switch from C₃ photosynthesis to CAM is mainly caused by the primary demand of CAM for substrates for nocturnal CO₂ fixation and acid synthesis, thus reducing the availability of carbohydrates for leaf growth at night. Although the shift to leaf growth early in the light is presumably associated with the availability of carbohydrates, source–sink relationships and sustained diurnal acid levels in young leaves of Clusia spp. need further evaluation in relation to growth processes.     

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.     

Protein-heparin interactions measured by BIAcore 2000 are affected by the method of heparin immobilization

Surface plasmon resonance (SPR) biosensors such as the BIAcore 2000 are a useful tool for the analysis of protein-heparin interactions. Generally, biotinylated heparin is captured on a streptavidin-coated surface to create heparinized surfaces for subsequent binding analyses. In this study we investigated three commonly used techniques for the biotinylation of heparin, namely coupling through either carboxylate groups or unsubstituted amines along the heparin chain, or through the reducing terminus of the heparin chain. Biotinylated heparin derivatives were immobilized on streptavidin sensor chips and several heparin-binding proteins were examined. Of the surfaces investigated, heparin attached through the reducing terminus had the highest binding capacity, and in some cases had a higher affinity for the proteins tested. Heparin immobilized via intrachain bare amines had intermediate binding capacity and affinity, and heparin immobilized through the carboxylate groups of uronic acids had the lowest capacity for the proteins tested. These results suggest that immobilizing heparin to a surface via intrachain modifications of the heparin molecule can affect the binding of particular heparin-binding proteins.     

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.     

Bovine mammary gland UDP-GalNAc:GlcNAcbeta-R beta1-->4-N- acetylgalactosaminyltransferase is glycoprotein hormone nonspecific and shows interaction with alpha-lactalbumin

We have identified a novel N -acetylgalactosaminyltransferase activity in lactating bovine mammary gland membranes. Acceptor specificity studies and analysis of products obtained in vitro by 400 MHz1H-NMR spectroscopy revealed that the enzyme catalyses the transfer of N - acetylgalactosamine (GalNAc) from UDP-GalNAc to acceptor substrates carrying a terminal, beta-linked N -acetylglucosamine (GlcNAc) residue and establishes a beta1-->4-linkage forming a GalNAcbeta1-->4GlcNAc ( N, N '-diacetyllactosediamine, lacdiNAc) unit. Therefore, the enzyme can be identified as a UDP-GalNAc:GlcNAcbeta-R beta1-->4-N- acetylgalactosaminyltransferase (beta4-GalNAcT). This enzyme resembles invertebrate beta4-GalNAcT as well as mammalian beta4- galactosyltransferase (beta4-GalT) in acceptor specificity. It can, however, be clearly distinguished from the pituitary hormone-specific beta4-GalNAcT by its incapability of acting with an elevated activity on a glycoprotein substrate carrying a hormone-specific peptide motif. Furthermore, the GalNAcT activity appeared not to be due to a promiscuous action of a beta4-GalT as could be demonstrated by comparing the beta4-GalNAcT and beta4-GalT activities of the mammary gland, bovine colostrum, and purified beta4-GalT, by competition studies with UDP-GalNAc and UDP-Gal, and by use of an anti-beta4-GalT polyclonal inhibiting antibody. Interestingly, under conditions where mammalian beta4-GalT forms with alpha-lactalbumin (alpha-LA) the lactose synthase complex, the mammary gland beta4-GalNAcT was similarly induced by alpha-LA to act on Glc with an increased efficiency yielding the lactose analog GalNAcbeta1-->4Glc. This enzyme thus forms the second example of a mammalian glycosyltransferase the specificity of which can be modified by this milk protein. It is proposed that the mammary gland beta4-GalNAcT functions in the synthesis of lacdiNAc- based, complex-type glycans frequently occurring on bovine milk glycoproteins. The action of this enzyme is to be considered when aiming at the production of properly glycosylated protein biopharmaceuticals in the milk of transgenic dairy animals.      

Regulation of B-lymphocyte production in the bone marrow: mediation of the effects of exogenous stimulants by adoptively transferred spleen cells

The cellular mechanism by which an injection of sheep red blood cells (SRBC) results in an increased production of B lymphocytes in mouse bone marrow has been studied by adoptive cell transfer and the use of two in vivo assays of bone marrow B-cell genesis. Proliferation of B progenitor cells was examined by immunofluorescent labeling combined with mitotic arrest, while small lymphocyte renewal was measured by [3H]thymidine labeling and radioautography. In C3H/HeJ mice the administration of SRBC resulted in increased proliferation among bone marrow pre-B cells which contained cytoplasmic mu heavy chains but lacked kappa light chains and surface mu chains. The turnover of small lymphocytes also increased. These stimulatory effects were transferred to naive recipient mice by organ fragments and by cell suspensions harvested from the spleens of donor mice injected with SRBC. In contrast, spleen cells and thymus cells from saline-injected donors and thymus cells from SRBC-injected donors had no such stimulatory effects. The results demonstrate that spleen cells mediate the stimulatory effect of SRBC on bone marrow B-lymphocyte production. Spleen cell transfer provides a system to study further the cells and factors involved in the regulation by external environmental agents of the rate of primary B-cell genesis in vivo.     

Environmental effects on photosynthesis, nitrogen-use efficiency, and metabolite pools in leaves of sun and shade plants

Effects of varying light intensity and nitrogen nutrition on photosynthetic physiology and biochemistry were examined in the sun plant Phaseolus vulgaris (common bean) and in the shade plant Alocasia macrorrhiza (Australian rainforest floor species). In both Phaseolus and Alocasia, the differing growth regimes produced large changes in photosynthetic capacity and composition of the photosynthetic apparatus. CO₂-saturated rates of photosynthesis were linearly related to leaf nitrogen (N) content in both species but photosynthesis per unit leaf N was markedly higher for Phaseolus than for Alocasia. Photosynthetic capacity was also higher in Phaseolus per unit ribulose 1,5-bisphosphate (RuBP) carboxylase (RuBPCase) protein. The leaf content of RuBPCase was linearly dependent on leaf N content in the two species. However, the proportion of leaf N which was RuBPCase was greater in Phaseolus than in Alocasia and was more sensitive to growth conditions, ranging from 6% of leaf N at low light to 20% at high light. In Alocasia, this range was much less, 6 to 11%. However, chlorophyll content was much more sensitive to light intensity in Alocasia. Thus, the RuBPCase/chlorophyll ratio was quite responsive to N availability and light intensity in both species (but for different reasons), ranging from 6 grams per gram for Phaseolus and 2 grams per gram for Alocasia at high leaf N and 1.5 gram per gram for Phaseolus and 0.5 gram per gram for Alocasia at low leaf N. These large changes in the proportions of components of the photosynthetic apparatus had marked effects on the sensitivity of these species to photoinhibition. These environmental effects also caused changes in the absolute levels of metabolites of the photosynthetic carbon reduction cycle. Concentrations of RuBP and P-glycerate were approximately 2-fold higher in high light-grown than low light-grown Phaseolus and Alocasia when expressed on a leaf area basis. However, if metabolite pool sizes are expressed on the basis of the RuBPCase catalytic site concentration, then they were little affected by the marked changes in leaf makeup. There appears to be fundamental differences between these species in the mechanism of sun-shade adaptation and N partitioning in the photosynthetic apparatus that result in significant differences in the N-use efficiency of photosynthesis between Phaseolus and Alocasia but similar RuBPCase:substrate:product ratios despite these differences.     

Responses of Two CAM Species to Different Irradiances during Growth and Susceptibility to Photoinhibition by High Light

Two CAM species, Kalanchoë daigremontiana Hamet et Perrier and Hoya carnosa (L.) R. Br., were grown under a range of five photon flux area densitites (PFD) and then characterized. Significant acclimation to shade was indicated by progressive decreases in leaf thickness, rates of respiratory O₂ uptake, light compensation point, maximum rates of photosynthetic O₂ evolution, nocturnal acid accumulation, and δ¹³C values, and increases in chlorophyll concentration and absolute levels of room temperature (25°C) and 77K fluorescence. Quantum yields (as measured by O₂ exchange) and the ratio of variable 77K fluorescence over the maximum yield (F_v/F_m) were relatively constant across the treatments. The only significant deviation from the above characteristics was in H. carnosa grown under full glasshouse PFD, where it apparently experienced photoinhibition. Following a photoinhibitory treatment, K. daigremontiana exhibited increases in the light compensation point and progressively greater reductions in the quantum yield, maximum photosynthetic rate, F_v/F_m, and the variable component of room temperature fluorescence with increasing shade during growth. Thus although Crassulacean acid metabolism plants can adjust to shaded conditions, they are susceptible to photoinhibition when exposed to higher PFD than that experienced during growth.