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Three cDNAs encoding purple acid phosphatase (PAP) were cloned from potato (Solanum tuberosum L. cv. Desiree) and expression of the corresponding genes was characterised. StPAP1 encodes a low-molecular weight PAP clustering with mammalian, cyanobacterial, and other plant PAPs. It was highly expressed in stem and root and its expression did not change in response to phosphorus (P) deprivation. StIPAP2 and StPAP3 code for high-molecular weight PAPs typical for plants. Corresponding gene expression was shown to be responsive to the level of P supply, with transcripts of StPAP2 and StPAP3 being most abundant in P-deprived roots or both stem and roots, respectively. Root colonisation by arbuscular mycorrhizal fungi had no effect on the expression of any of the three PAP genes. StIPAP1 mRNA is easily detectable along the root axis, including root hairs, but is barely detectable in root tips. In contrast, both StPAP2 and StPAP3 transcripts are abundant along the root axis, but absent in root hairs, and are most abundant in the root tip. All three PAPs described contain a predicted N-terminal secretion signal and could play a role in extracellular P scavenging, P mobilisation from the rhizosphere, or cell wall regeneration
The presence of partially folded intermediates along the folding funnel of proteins has been suggested to be a signature of potentially aggregating systems. Many studies have concluded that metastable, highly flexible intermediates are the basic elements of the aggregation process. In a previous paper, we demonstrated how the choice between aggregation and folding behavior was influenced by hydrophobicity distribution patterning along the sequence, as quantified by recurrence quantification analysis (RQA) of the Myiazawa-Jernigan coded primary structures. In the present paper, we tried to unify the "partially folded intermediate" and "hydrophobicity/charge" models of protein aggregation verifying the ability of an empirical relation, developed for rationalizing the effect of different mutations on aggregation propensity of acyl-phosphatase and based on the combination of hydrophobicity RQA and charge descriptors, to discriminate in a statistically significant way two different protein populations: (a) proteins that fold by a process passing by partially folded intermediates and (b) proteins that do not present partially folded intermediates
There is concern about the lack of recruitment of Acacia trees in the Negev desert of Israel. We have developed three models to estimate the frequency of recruitment necessary for long-term population survival (i.e. positive average population growth for 1,000 years and <10% probability of extinction). Two models assume purely episodic recruitment based on the general notion that recruitment in and environments is highly episodic. They differ in that the deterministic model investigates average dynamics while the stochastic model does not. Studies indicating that recruitment episodes in and environments have been overemphasized motivated the development of the third model. This semi-stochastic model simulates a mixture of continuous and episodic recruitment. Model analysis was done analytically for the deterministic model and via running model simulations for the stochastic and semi-stochastic models. The deterministic and stochastic models predict that, on average, 2.2 and 3.7 recruitment events per century, respectively, are necessary to sustain the population. According to the semi-stochastic model, 1.6 large recruitment events per century and an annual probability of 50% that a small recruitment event occurs are needed. A consequence of purely episodic recruitment is that all recruitment episodes produce extremely large numbers of recruits (i.e. at odds with field observations), an evaluation that holds even when considering that rare events must be large. Thus, the semi- stochastic model appears to be the most realistic model. Comparing the prediction of the semi-stochastic model to field observations in the Negev desert shows that the absence of observations of extremely large recruitment events is no reason for concern. However, the almost complete absence of small recruitment events is a serious reason for concern. The lack of recruitment may be due to decreased densities of large mammalian herbivores and might be further exacerbated by possible changes in climate, both in terms of average precipitation and the temporal distribution of rain
Herbivore populations are commonly restricted by resource limitation, by predation or a combination of the two. Food supplement experiments are suitable for investigating the extent of food limitation at any given time. The main part of this study was performed in an extremely acidic lake (pH 2.7) where the food web consists of only a few components and potential food sources for herbivores are restricted to two flagellates. Life table experiments proved that Chlamydomonas was a suitable food source whereas Ochromonas was an unsuitable food source. The two flagellates and the two rotifers exhibit a pronounced vertical distribution pattern. In this study, a series of food supplement experiments were performed in order to: (1) quantify and compare potential resource limitation of two primary consumers (Cephalodella hoodi and Elosa worallii, Rotatoria) over time, (2) compare their response at different temperatures, (3) evaluate the effect of having an unsuitable food source alongside a valuable one, (4) estimate the effect of predation on rotifers by Heliozoa, and (5) compare the results with those from other acidic lakes. Additionally, the spatio- temporal population dynamics of both species were observed. The field data confirmed a vertical separation of the two species with E. worallii dominating in the upper water layers, and C. hoodi in the deeper, cooler water layers. The results from the food supplement experiments in which Chlamydomonas served as the supplemented suitable food source showed that the two rotifers were food limited in the epilimnion throughout the season to different extents, with Cephalodella being more severely food limited than Elosa. The experiments at different temperatures provided evidence that Elosa had a higher optimum temperature for growth than Cephalodella. When the unsuitable food algae Ochromonas was added alongside the suitable food source Chlamydomonas, C. hoodi was unaffected but E. worallii was negatively affected. Predation of Heliozoa on rotifers was observed but the total effect on the rotifer dynamics is probably low. The comparison with other lakes showed that resource limitation also occurred in one other lake, although to a lesser extent. Overall, the vertical separation of the two rotifers could be explained by both their differential extent of resource limitation and differential response to temperature.
When we think of extremophiles, organisms adapted to extreme environments, prokaryotes come to mind first. However, the unicellular red micro-alga Galdieria sulphuraria (Cyanidiales) is a eukaryote that can represent up to 90% of the biomass in extreme habitats such as hot sulfur springs with pH values of 0-4 and temperatures of up to 56 degreesC. This red alga thrives autotrophically as well as heterotrophically on more than 50 different carbon sources, including a number of rare sugars and sugar alcohols. This biochemical versatility suggests a large repertoire of metabolic enzymes, rivaled by few organisms and a potentially rich source of thermo-stable enzymes for biotechnology. The temperatures under which this organism carries out photosynthesis are at the high end of the range for this process, making G. sulphuraria a valuable model for physical studies on the photosynthetic apparatus. In addition, the gene sequences of this living fossil reveal much about the evolution of modern eukaryotes. Finally, the alga tolerates high concentrations of toxic metal ions such as cadmium, mercury, aluminum, and nickel, suggesting potential application in bioremediation. To begin to explore the unique biology of G. sulphuraria, 5270 expressed sequence tags from two different cDNA libraries have been sequenced and annotated. Particular emphasis has been placed on the reconstruction of metabolic pathways present in this organism. For example, we provide evidence for (i) a complete pathway for lipid A biosynthesis; (ii) export of triose-phosphates from rhodoplasts; (iii) and absence of eukaryotic hexokinases. Sequence data and additional information are available at http://genomics.msu.edu/galdieria
The aim of this study was to determine the impact of lentiviral transduction on primary murine B cells. Studying B cell activities in vivo or using them for tolerance induction requires that the cells remain unaltered in their biological behavior except for expression of the transgene. As we show here, murine B cells can efficiently be transduced by lentiviral, VSV-G-pseudotyped vectors without the necessity of prior activation. Culture with LPS gave enhanced transduction efficiencies but led to the upregulation of CD86 and proliferation of the cells. Transduction of naive B cells by lentiviral vectors was dependent on multiplicity of infection and did not lead to a concomitant activation. Furthermore, the transduced cells could be used for studies in the NOD mouse system without altering the onset of diabetes. We conclude that lentiviral gene transfer into naive B cells is a powerful tool for manipulation of B cells for therapeutic applications.
Offenhaltung durch Feuer
(2004)
Food quality controls egg quality of the zebra mussel Dreissena polymorpha : The role of fatty acids
(2004)
We investigated the investment of adult Dreissena polymorpha to the eggs by sampling mussels monthly from 4- and 15-m water depth. The fatty acid composition of eggs differed significantly between depths and over time. To assess whether temperature and food conditions led to the differences observed for mussels sampled from the two depths, mussels were reared in the laboratory under two different 3-month temperature regimes, simulating the temperature of the lake at 4- and 15-m depth. Possible effects of food quality were tested in each simulation using four diets differing in fatty acid composition: Cryptomonas erosa, Nannochloropsis limnetica [rich in polyunsaturated fatty acids (PUFAs) and long- chained PUFAs (.C18)], Scenedesmus obliquus, and the cyanobacterium Aphanothece sp. (deficient in long-chained PUFAs). In newly released eggs, specific (n-3) and (n-6) long-chained PUFAs increased when these fatty acids were available in the natural seston or in the laboratory diets. Mussels fed organisms deficient in long-chained PUFAs were still able to allocate arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid into eggs, which suggests that minimum levels of particular fatty acids were maintained in eggs by transfer from internal reserves of the female mussels to oocytes. In contrast to the diet, there were no effects of the temperature on the fatty acid composition of eggs.
Animals depend on a large set of essential compounds in their food. However, not all units of food are equal from a nutritional point of view. A reduced supply of protein and calcium might be critical for the growth, reproduction and survival of herbivorous gastropods. We experimentally examined the effects of different protein and calcium supplies on the growth and survival of the land snail Arianta arbustorum. Groups of snails were reared on agar-based diets with each of three levels of protein and calcium (nine treatments). Snails fed a high-protein diet grew faster and reached adulthood earlier and at a larger adult size than snails fed intermediate and low-protein diets. Surprisingly, the calcium concentration did not affect shell growth and adult size. Snails reared on intermediate- and low-calcium diets increased their consumption rates, but, despite this compensatory feeding, these snails were unable to take up the amount of calcium required for metabolism and shell growth and had a higher mortality. The calcium deficiency could partly be mitigated by the snails' internal calcium storage and reallocation capacity. A depletion of internal calcium reserves adversely affects the entire nutrient metabolism and was the probable cause for the high mortality rates associated with the intermediate- and low calcium diets observed in the present study
Predicting evolution of floral traits associated with mating system in a natural plant population
(2004)
Evolution of floral traits requires that they are heritable, that they affect fitness, and that they are not constrained by genetic correlations. These prerequisites have only rarely been examined in natural populations. For Mimulus guttatus, we found by using the Riska-method that corolla width, anther length, ovary length and number of red dots on the corolla were heritable in a natural population. Seed production (maternal fitness) was directly positively affected by corolla width and anther size, and indirectly so by ovary length and number of red dots on the corolla. The siring success (paternal fitness), as estimated from allozyme data, was directly negatively affected by anther-stigma separation, and indirectly so by the corolla length-width ratio. Genetic correlations, estimated with the Lynch-method, were positive between floral size measures. We predict that larger flowers with larger reproductive organs, which generally favour outcrossing, will evolve in this natural population of M. guttatus
Local adaption of the clonal plant Ranunculus reptans to flooding along a small-scale gradient.
(2004)
Catalysis by ChiB, a family 18 chitinase from Serratia marcescens, involves a conformational change of Asp142 which is part of a characteristic D140XD142XE144 sequence motif In the free enzyme Asp142 points towards Asp140, whereas it rotates towards the catalytic acid, Glu144, upon ligand binding. Mutation of Asp142 to Asn reduced k(cat) and affinity for allosamidin, a competitive inhibitor. The X-ray structure of the D142N mutant showed that Asn142 points towards Glu144 in the absence of a ligand. The active site also showed other structural adjustments (Tyr10, Ser93) that had previously been observed in the wild-type enzyme upon substrate binding. The X-ray structure of a complex of D142N with allosamidin, a pseudotrisaccharide competitive inhibitor, was essentially identical to that of the wild-type enzyme in complex with the same compound. Thus, the reduced allosamidin affinity in the mutant is not caused by structural changes but solely by the loss of electrostatic interactions with Asp142. The importance of electrostatics was further confirmed by the pH dependence of catalysis and allosamidin inhibition. The pH-dependent apparent affinities for allosamidin were not correlated with k(cat), indicating that it is probably better to view the inhibitor as a mimic of the oxazolinium ion reaction intermediate than as a transition state analogue. (C) 2003 Elsevier B.V. All rights reserved
Pectins are major components of primary plant cell walls and the seed mucilage of Arabidopsis. Despite progress in the structural elucidation of pectins, only very few enzymes participating in or regulating their synthesis have been identified. A first candidate gene involved-in the synthesis of pectinaceous rhamnogalacturonan I is RHM2, a putative plant ortholog to NDP-rhamnose biosynthetic enzymes in bacteria. Expression studies with a promoter beta-glucuronidase construct and reverse transcription PCR data show that RHM2 is expressed ubiquitously. Rhm2 T-DNA insertion mutant lines were identified using a reverse genetics approach. Analysis of the rhm2 seeds by various staining methods and chemical analysis of the mucilage revealed a strong reduction of rhamnogalacturonan I in the mucilage and a decrease of its molecular weight. In addition, scanning electron microscopy of the seed surface indicated a distorted testa morphology, illustrating not only a structural but also a developmental role for RGI or rhamnose metabolism in proper testa formation
Beweidung mit Wildtieren
(2004)
1. Lakes formed in mining pits often contain high concentrations of dissolved ferric iron and sulphate (e.g. 2 and 16 mmol L)1, respectively) and the pH is buffered between 2.5 and 3.5. Efforts to neutralise their water are based on the stimulation of lake internal, bacterial iron- and sulphate reduction. Electron donors may be supplied by organic carbon compounds or indirectly by enhancement of primary production. Here, we investigated the function of mixotrophic algae, which can potentially supplement or deplete the organic carbon pool, in the carbon metabolism and alkalinity budget of an acidic mining lake. 2. Two weeks after organic substrates had been added in a large in situ mesocosm of 30 m diameter, a bloom of Chlamydomonas occurred, reaching a biovolume of 80 mm3 L)1. Growth experiments using filtered lake water showed that the alga reduced the overall dissolved organic carbon (DOC) concentration despite significant photosynthetic activity. However, when Chlamydomonas were grown together with natural bacterioplankton, net DOC consumption did not increase. 3. Uptake experiments using [14C]-glucose indicated that bacteria dominated glucose uptake and remineralisation. Therefore, the DOC leached in the water column was processed mainly by planktonic bacteria. Leached DOC must be regarded as loss, not transferred by larger organisms to the sediment, where reduction processes take place. 4. From phytoplankton biomass and production 2 years after fertilisation we estimated that pelagic photosynthesis does not supply an electron donor capacity capable of reducing more than 2% of actual stock of acidity per year. We estimated that only the benthic primary production was in a range to compensate for ongoing inputs of iron and sulphate.
To unravel the postglacial colonization history and the current intercolony dispersal in the common eider, Somateria mollissima, we analysed genetic variation at a part of the mitochondrial control region and five unlinked autosomal microsatellite loci in 175 eiders from 11 breeding colonies, covering the entire European distribution range of this species. As a result of extreme female philopatry, mitochondrial DNA differentiation is substantial both among local colonies and among distant geographical regions. Our study further corroborates the previous hypothesis of a single Pleistocene refugium for European eiders. A nested clade analysis on mitochondrial haplotypes suggests that (i) the Baltic Sea eider population is genetically closest to a presumably ancestral population and that (ii) the postglacial recolonization progressed in a stepwise fashion via the North Sea region and the Faroe Islands to Iceland. Current long-distance dispersal is limited. Differentiation among colonies is much less pronounced at microsatellite loci. The geographical pattern of this nuclear genetic variation is to a large extent explained by isolation by distance. As female dispersal is very limited, the geographical pattern of nuclear variation is probably explained by male-mediated gene flow among breeding colonies. Our study provides genetic evidence for the assumed prominent postglacial colonization route shaping the present terrestrial fauna of the North Atlantic islands Iceland and the Faroes. It suggests that this colonization had been a stepwise process originating in continental Europe. It is the first molecular study on eider duck populations covering their entire European distribution range
Structural and animal species diversity in arid and semi-arid savannas of the southern Kalahari
(2004)
Gauntlets : Garbon and nutrient signalling : test systems, and metabolite and transcript profiles
(2004)
Negative effects of P-buffering and pH on photosynthetic activity of planktonic desmid species
(2004)
The photosynthetic activities of three planktonic desmid species (Staurastrum brachiatum, Staurodesmus cuspidatus var. curvatus, and Staurastrum chaetoceras) were compared after adaptation to medium enriched with either a 20 mM Na+- phosphate (P) or HEPES buffer. Incubations up to 2 d were carried out at pH 6 or 8 under normal air or air enriched with 5 % CO2. Gross maximum photosynthetic rate (Pmax) and growth rate were decreased in both S. brachiatum and Std. cuspidatus at higher pH when using the HEPES buffer and this effect was independent of CO2 concentration, indicating that pH had an inhibitory effect on photosynthesis and growth in these species. The P-buffer at pH 8 caused a large decrease in Pmax and quantum yield for charge separation in photosystem 2 (PS2), compared to HEPES-buffered algae. This effect was very large in both S. brachiatum and Std. cuspidatus, two species characteristic of soft water lakes, but also significant in S. chaetoceras, a species dominant in eutrophic, hard water lakes. The decreased Pmax in P- buffer could not be related to a significant increase in cellular P content known to be responsible for inhibition in isolated chloroplasts. Experiments at pH 6 and 8 showed that two conditions, high pH and high Na+ concentration, both contributed to the decreased Pmax and quantum yield in the desmids. Effects of a P-buffer were less pronounced by using K+-P buffer. The use of P-buffer at pH 8 possibly resulted in high irradiance stress in all species, indicated by damage in the PS2 core complex. In the soft water species pH 8 resulted in increased non-photochemical quenching together with a high de-epoxidation state of the xanthophyll cycle pigments.
A method for construction of biosensors with membranous cytochrome P450 isoenzymes was developed based on clay/ detergent/protein mixed films. Thin films of sodium montmorillonite colloid with incorporated cytochrome P450 2134 (CYP2B4) with nonionic detergent were prepared on glassy carbon electrodes. The modified electrodes were electrochemically characterized, and bio-electrocatalytic reactions were followed. CYP2B4 can be reduced fast on clay- modified glassy carbon electrodes in the presence of the nonionic detergent Tween 80. In anaerobic solutions, reversible oxidation and reduction is obtained with a formal potential between -0.292 and - 0.305 V vs Ag/AgCl 1 M KCl depending on the preparation of the biosensor. In air-saturated solution, bio-electrocatalytic reduction currents can be obtained with the CYP2B4-modified electrode on addition of typical substrates such as aminopyrine and benzphetamine. This reaction was suppressed when methyrapone, an inhibitor of P450 reactions, was present. Measurement of product formation also indicates the bioelectrocatialysis by CYP2B4
Environmental heterogeneity is a major determinant of plant population dynamics. In semi-arid Kalahari savannas, heterogeneity is created by savanna structure, i.e. by the spatial arrangement and temporal dynamics of woody plant and open grassland microsites. We formulate a conceptual model describing the effects of savanna dynamics on the population dynamics of the animal-dispersed shrub Grewia flava. From empirical results we derive model rules describing effects of savanna structure on several processes in Grewia's life cycle. By formulating the model, we summarise existing information on Grewia demography and identify gaps in this knowledge. Despite a number of such gaps, the model can be used to make certain quantitative predictions. As an example, we apply the model to investigate the role of seed dispersal in Grewia encroachment on rangelands. Model results show that cattle promote encroachment by depositing substantial numbers of seeds in open areas, where Grewia is otherwise dispersal-limited. Finally, we draw some general conclusions about Grewia's life history and population dynamics. Under natural conditions, concentrated seed deposition under woody plants appears to be a key process causing the observed association between Grewia and other woody plants. Furthermore, low rates of recruitment and high adult survival result in slow-motion dynamics of Grewia populations. As a consequence, Grewia populations interact with savanna dynamics on long temporal and short to intermediate spatial scales.
Ecologists increasingly use spatial statistics to study vegetation patterns. Mostly, however, these techniques are applied in a purely descriptive fashion without a priori statements on the pattern characteristics expected. We formulated such a priori predictions in a study of spatial pattern in a semi-arid Karoo shrubland, South Africa. Both seed dispersal and root competition have been discussed as processes shaping the spatial structure of this community. If either of the two processes dominates pattern formation, patterns within and between shrub functional groups are expected to show distinct deviations from null models. We predicted the type and scale of these deviations and compared predicted to observed pattern characteristics. As predicted by the seed dispersal hypothesis, small-scale co-occurrence within and between groups of colonisers and successors was increased as compared to complete spatially random arrangement of shrubs. The root competition predictions, however, were not met as shrubs of similar rooting depth co- occurred more frequently than expected under random shrub arrangement. Since the distribution of rooting groups to the given shrub locations also failed to match the root competition predictions, there was little evidence for dominance of root competition in pattern formation. Although other processes may contribute to small-scale plant co-occurrence, the sufficient and most parsimonious explanation for the observed pattern is that its formation was dominated by seed dispersal. To characterise point patterns we applied both cumulative (uni- and bivariate K-function) and local (pair- and mark-correlation function) techniques. Based on our results we recommend that future studies of vegetation patterns include local characteristics as they independently describe a pattern at different scales and can be easily related to processes changing with interplant distance in a predictable fashion.
Serotonin and histamine produce different spatiotemporal Ca2+ signals in blowfly salivary glands
(2004)
The influence of interleukin 4 (IL-4) on antibody titer in serum and spleen culture supernatant in mice immunized with spinach (Spinacia oleracea L.) Rubisco was investigated. Therefore, we boosted one mouse additionally to the antigen with recombinant mouse IL-4. We found that the Rubisco-specific antibody titer in serum as well as in spleen cell culture supernatant was significantly enhanced in the IL-4 mouse. Most of the antibodies were of the IgG1 subclass. After hybridoma generation, Rubisco-specific antibodies were found in more than 95% of the wells tested compared to about 12% of the control mouse.
Biosensorik / Bioanalytik
(2004)
From gene to life
(2004)
The development of the cardiovascular system in embryoid bodies deriverd from embryonic stem cells
(2004)
Transcription factors, growth factors and signal cascades capable of priming morphogenesis of heart
(2004)
Carbohydrate-binding modules (CBMs) are the most common non-catalytic modules associated with enzymes active in plant cell-wall hydrolysis. Despite the large number of putative CBMs being identified by amino acid sequence alignments, only few representatives have been experimentally shown to have a carbohydrate-binding function. Caldicellulosiruptor strain Rt8B.4 Man26 is a thermostable modular glycoside hydrolase beta-mannanase which contains two non-catalytic modules in tandem at its N terminus. These modules were recently shown to function primarily as beta- mannan-binding modules and have accordingly been classified as members of a novel family of CBMs, family 27. The N- terminal CBM27 (CsCBM27-1) of Man26 from Caldicellulosiruptor Rt8B.4 displays high-binding affinity towards mannohexaose with a K-a of 1 x 10(7) M-1. Accordingly, the high-resolution crystal structures of CsCBM27-1 native and its mannohexaose complex were solved at 1.55 Angstrom and 1.06 Angstrom resolution, respectively. In the crystal, CsCBM27-1 shows the typical beta-sandwich jellyroll fold observed in other CBMs with a single metal ion bound, which was identified as calcium. The crystal structures reveal that the overall fold of CsCBM27-1 remains virtually unchanged upon sugar binding and that binding is mediated by three solvent-exposed tryptophan residues and few direct hydrogen bonds. Based on binding affinity and thermal unfolding experiments this structural calcium is shown to play a role in the thermal stability of CsCBM27-1 at high temperatures. The higher binding affinity of CsCBM27-1 to mannooligosaccharides when compared to other members of CBM family 27 might be explained by the different orientation of the residues forming the "aromatic platform" and by differences in the length of loops. Finally, evidence is presented, on the basis of fold similarities and the retention of the position of conserved motifs and a calcium ion, for the consolidation of related CBM families into a superfamily of CBMs. (C) 2004 Elsevier Ltd. All rights reserved
The starch excess phenotype of Arabidopsis mutants defective in the starch phosphorylating enzyme glucan, water dikinase (EC 2.7.9.4) indicates that phosphorylation of starch is required for its degradation. However, the underlying mechanism has not yet been elucidated. In this study, two in vivo systems have been established that allow the analysis of phosphorylation of transitory starch during both biosynthesis in the light and degradation in darkness. First, a photoautotrophic culture of the unicellular green alga Chlamydomonas reinhardtii was used to monitor the incorporation of exogenously supplied P-32 orthophosphate into starch. Illuminated cells incorporated P-32 into starch with a constant rate during 2 h. By contrast, starch phosphorylation in darkened cells exceeded that in illuminated cells within the first 30 min, but subsequently phosphate incorporation declined. Pulse-chase experiments performed with P-32/P-31 orthophosphate revealed a high turnover of the starch-bound phosphate esters in darkened cells but no detectable turnover in illuminated cells. Secondly, leaf starch granules were isolated from potato (Solanum tuberosum) plants grown under controlled conditions and glucan chains from the outer granule layer were released by isoamylase. Phosphorylated chains were purified and analyzed using high performance anion-exchange chromatography and matrix-assisted laser desorption/ionization mass spectrometry. Glucans released from the surface of starch granules that had been isolated from darkened leaves possessed a considerably higher degree of phosphorylation than those prepared from leaves harvested during the light period. Thus, in the unicellular alga as well as in potato leaves, net starch degradation is accompanied with an increased phosphorylation of starch