Variation in Storage α-Polyglucans of Red Algae: Amylose and Semi-Amylopectin Types in <Emphasis Type="Italic">Porphyridium</Emphasis> and Glycogen Type in <Emphasis Type="Italic">Cyanidium</Emphasis>

Red algae are widely known to produce floridean starch but it remains unclear whether the molecular structure of this algal polyglucan is distinct from that of the starch synthesized by vascular plants and green algae. The present study shows that the unicellular species Porphyridium purpureum R-1 (order Porphyridiales, class Bangiophyceae) produces both amylopectin-type and amylose-type alpha-polyglucans. In contrast, Cyanidium caldarium (order Porphyridiales, class Bangiophyceae) synthesizes glycogen-type polyglucan, but not amylose. Detailed analysis of alpha-1,4-chain length distribution of P. purpureum polyglucan suggests that the branched polyglucan has a less ordered structure, referred to as semi-amylopectin, as compared with amylopectin of rice endosperm having a tandem-cluster structure. The P. purpureum linear amylose-type polyglucan, which has a lambda(max) of 630 nm typical of amylose-iodine complex and is resistant to Pseudomonas isoamylase digestion, accounts for less than 10% of the total polyglucans. We produced and isolated a cDNA encoding a granule-bound starch synthase (GBSS)-type protein of P. purpureum, which is probably the approximately 60-kDa protein bound tightly to the starch granules, resembling the amylose-synthesizing GBSS protein of green plants. The present investigation indicates that the class Bangiophyceae includes species producing both semi-amylopectin and amylose, and species producing glycogen alone.     

Identification of chlorophyllide a oxygenase in the Prochlorococcus genome by a comparative genomic approach

Chl b is a major photosynthetic pigment of peripheral antenna complexes in chlorophytes and prochlorophytes. Chl b is synthesized by chlorophyllide a oxygenase (CAO), an enzyme that has been identified from higher plants, moss, green algae and two groups of prochlorophytes, Prochlorothrix and Prochloron. Based on these results, we previously proposed the hypothesis that all of the Chl b synthesis genes have a common origin. However, the CAO gene is not found in whole genome sequences of Prochlorococcus although a gene which is distantly related to CAO was reported. If Prochlorococcus employs a different enzyme, a Chl synthesis gene should have evolved several times on the different phylogenetic lineages of Prochlorococcus and other Chl b-containing organisms. To examine these hypotheses, we identified a Prochlorococcus Chl b synthesis gene by using a combination of bioinformatics and molecular genetics techniques. We first identified Prochlorococcus-specific genes by comparing the whole genome sequences of Prochlorococcus marinus MED4, MIT9313 and SS120 with Synechococcus sp. WH8102. Synechococcus is closely related to Prochlorococcus phylogenetically, but it does not contain a Chl b synthesis gene. By examining the sequences of Prochlorococcus-specific genes, we found a candidate for the Chl b synthesis gene and introduced it into Synechocystis sp. PCC6803. The transformant cells accumulated Chl b, indicating that the gene product catalyzes Chl b synthesis. In this study, we discuss the evolution of CAO based upon the molecular phylogenetic studies we performed.     

Intra-vacuolar reserves of membranes during stomatal closure: the possible role of guard cell vacuoles estimated by 3-D reconstruction

Stomatal apertures are regulated by morphological changes in guard cells which have been associated with guard cell vacuolar structures. To investigate the contribution of guard cell vacuoles to stomatal movement, we examined the dynamics of vacuolar membrane structures in guard cells and evaluated the changes in vacuolar volumes and surface areas during stomatal movement. Using a transgenic Arabidopsis line expressing green fluorescent protein (GFP)-AtVAM3, we have found that the guard cell vacuolar structures became complicated during stomatal closure with the appearance of numerous intra-vacuolar membrane structures. A three-dimensional (3-D) reconstruction using our originally developed software, REANT (reconstructor and analyzer of 3-D structure), and photobleaching analysis revealed the continuity of the vacuolar structures, even when they appeared to be compartmented in confocal images of closed stomata. Furthermore, calculations of the surface area by REANT revealed an increase in vacuolar surface area during stomatal closure but a decrease in the surface area of the guard cells. Movement of a vital staining dye, FM4-64, to the vacuolar membrane was accelerated during ABA-induced stomatal closure in Vicia faba. These results suggest that the guard cell vacuoles store some portion of the excess membrane materials produced during stomatal closure as intra-vacuolar structures.     

Elicitor-induced cytoskeletal rearrangement relates to vacuolar dynamics and execution of cell death: in vivo imaging of hypersensitive cell death in tobacco BY-2 cells

Disintegration of the vacuolar membrane (VM) has been proposed to be a crucial event in various types of programmed cell death (PCD) in plants. However, its regulatory mechanisms are mostly unknown. To obtain new insights on the regulation of VM disintegration during hypersensitive cell death, we investigated the structural dynamics and permeability of the VM, as well as cytoskeletal reorganization during PCD in tobacco BY-2 cells induced by a proteinaceous elicitor, cryptogein. From sequential observations, we have identified the following remarkable events during PCD. Stage 1: bulb-like VM structures appear within the vacuolar lumen and the cortical microtubules are disrupted, while the cortical actin microfilaments are bundled. Simultaneously, transvacuolar strands including endoplasmic microtubules and actin microfilaments are gradually disrupted and the nucleus moves from the center to the periphery of the cell. Stage 2: cortical actin microfilament bundles and complex bulb-like VM structures disappear. The structure of the large central vacuole becomes simpler, and small spherical vacuoles appear. Stage 3: the VM is disintegrated and a fluorescent dye, BCECF, leaks out of the vacuoles just prior to PCD. Application of an actin polymerization inhibitor facilitates both the disappearance of bulb-like vacuolar membrane structures and induction of cell death. These results suggest that the elicitor-induced reorganization of actin microfilaments is involved in the regulation of hypersensitive cell death via modification of the vacuolar structure to induce VM disintegration.     

Comparative biochemistry of alpha-glucan-utilization in Pseudomonas amyloderamosa and Pseudomonas saccharophila: physiological significance of variations in the pathway.

Growth patterns on and utilization of various alpha-glucans were investigated in Pseudomonas amyloderamosa and P. saccharophila. Maltose, maltodextrins (average chain length 7 glycosyl units) and glycogen supported excellent growth of both organisms and were extensively metabolized, although glycogen utilization in P. saccharophila was preceded by a prolonged lag phase. P. amyloderamosa produced limited growth on amylopectin and the carbohydrate was only partly degraded. It seemed likely that many of the unit chains liberated from amylopectin had a length exceeding the substrate range accepted by the maltodextrin permease (transport) system. A correlation was established between the pH of the medium and the utilization of glycogen and amylopectin for growth in P. amyloderamosa. The carbohydrates were at least partly utilizable at pH 6.0, whereas they could not support any growth at pH 6.5. Most likely, the lack of growth at the higher pH reflected the low activity of isolamylase at this pH. The enzyme patterns of maltodextrin catabolism in the two bacteria were established. Intracellularly, maltodextrin phosphorylase and 4-alpha-glucanotransferase occurred in both. Degradation of extracellular alpha-glucans was mediated by a mainly intracellular isoamylase in P. amyloderamosa, whereas P. saccharophila possessed an extracellular alpha-amylase and a firmly cell-bound pullulanase.     

Absence of the PsbZ subunit prevents association of PsbK and Ycf12 with the PSII complex in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

PsbZ (Ycf9) is a membrane protein of PSII complexes and is highly conserved from cyanobacteria to plants. We deleted the psbZ gene in the thermophilic cyanobacterium, Thermosynechococcus elongatus. The mutant cells showed photoautotrophic growth indistinguishable from that of the wild type under low and standard light conditions, while they showed even better growth than the wild type under high light. The mutant accumulated less carotenoids and more phycobiliproteins than the wild type under high light, suggestive of tolerance to photoinhibition. The mutant cells evolved oxygen at a rate comparable with the wild type, while the PSII complex isolated from the mutant retained much lower activity than the wild type. N-terminal sequencing revealed that Ycf12 and PsbK proteins were almost lost in the PSII complex. These results indicate that PsbZ is involved in functional integrity of the PSII complex by stabilizing PsbK and Ycf12. We suggest that Ycf12 is an unidentified membrane-spanning polypeptide that is placed near PsbZ and PsbK in the crystal structure of PSII.     

Potential role of annexin AnnAt1 from Arabidopsis thaliana in pH-mediated cellular response to environmental stimuli

Plant annexins, Ca(2+)- and membrane-binding proteins, are probably implicated in the cellular response to stress resulting from acidification of cytosol. To understand how annexins can contribute to cellular ion homeostasis, we investigated the pH-induced changes in the structure and function of recombinant annexin AnnAt1 from Arabidopsis thaliana. The decrease of pH from 7.0 to 5.8 reduced the time of the formation of ion channels by AnnAt1 in artificial lipid membranes from 3.5 h to 15-20 min and increased their unitary conductance from 32 to 63 pS. These changes were accompanied by an increase in AnnAt1 hydrophobicity as revealed by hydrophobicity predictions, by an increase in fluorescence of 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS) bound to AnnAt1 and fluorescence resonance energy transfer from AnnAt1 tryptophan residues to TNS. Concomitant lipid partition of AnnAt1 at acidic pH resulted in its partial protection from proteolytic digestion. Secondary structures of AnnAt1 determined by circular dichroism and infrared spectroscopy were also affected by lowering the pH from 7.2 to 5.2. These changes were characterized by an increase in beta-sheet content at the expense of alpha-helical structures, and were accompanied by reversible formation of AnnAt1 oligomers as probed by ultracentrifugation in a sucrose gradient. A further decrease of pH from 5.2 to 4.5 or lower led to the formation of irreversible aggregates and loss of AnnAt1 ionic conductance. Our findings suggest that AnnAt1 can sense changes of the pH milieu over the pH range from 7 to 5 and respond by changes in ion channel conductance, hydrophobicity, secondary structure of the protein and formation of oligomers. Further acidification irreversibly inactivated AnnAt1. We suggest that the pH-sensitive ion channel activity of AnnAt1 may play a role in intracellular ion homeostasis.     

Revisiting the structure of the anti-neoplastic glucans of Mycobacterium bovis Bacille Calmette-Guerin. Structural analysis of the extracellular and boiling water extract-derived glucans of the vaccine substrains

The attenuated strain of Mycobacterium bovis Bacille Calmette-Guérin (BCG), used worldwide to prevent tuberculosis and leprosy, is also clinically used as an immunotherapeutic agent against superficial bladder cancer. An anti-tumor polysaccharide has been isolated from the boiling water extract of the Tice substrain of BCG and tentatively characterized as consisting primarily of repeating units of 6-linked-glucosyl residues. Mycobacterium tuberculosis and other mycobacterial species produce a glycogen-like alpha-glucan composed of repeating units of 4-linked glucosyl residues substituted at some 6 positions by short oligoglucosyl units that also exhibits an anti-tumor activity. Therefore, the impression prevails that mycobacteria synthesize different types of anti-neoplastic glucans or, alternatively, the BCG substrains are singular in producing a unique type of glucan that may confer to them their immunotherapeutic property. The present study addresses this question through the comparative analysis of alpha-glucans purified from the extracellular materials and boiling water extracts of three vaccine substrains. The polysaccharides were purified, and their structural features were established by mono- and two-dimensional NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the enzymatic and chemical degradation products of the purified compounds. The glucans isolated by the two methods from the three substrains of BCG were shown to exhibit identical structural features shared with the glycogen-like alpha-glucan of M. tuberculosis and other mycobacteria. Incidentally, we observed an occasional release of dextrans from Sephadex columns that may explain the reported occurrence of 6-substituted alpha-glucans in mycobacteria.     

EARLY STARVATION1 specifically affects the phosphorylation action of starch‐related dikinases

Starch phosphorylation by starch‐related dikinases glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) is a key step in starch degradation. Little information is known about the precise structure of the glucan substrate utilized by the dikinases and about the mechanisms by which these structures may be influenced. A 50‐kDa starch‐binding protein named EARLY STARVATION1 (ESV1) was analyzed regarding its impact on starch phosphorylation. In various in vitro assays, the influences of the recombinant protein ESV1 on the actions of GWD and PWD on the surfaces of native starch granules were analyzed. In addition, we included starches from various sources as well as truncated forms of GWD. ESV1 preferentially binds to highly ordered, α‐glucans, such as starch and crystalline maltodextrins. Furthermore, ESV1 specifically influences the action of GWD and PWD at the starch granule surface. Starch phosphorylation by GWD is decreased in the presence of ESV1, whereas the action of PWD increases in the presence of ESV1. The unique alterations observed in starch phosphorylation by the two dikinases are discussed in regard to altered glucan structures at the starch granule surface.     

Responses to desiccation stress in bryophytes and an important role of dithiothreitol-insensitive non-photochemical quenching against photoinhibition in dehydrated states

The effects of air drying and hypertonic treatments in the dark on seven bryophytes, which had grown under different water environments, were studied. All the desiccation-tolerant species tested lost most of their PSII photochemical activity when photosynthetic electron transport was inhibited by air drying, while, in all the sensitive species, the PSII photochemical activity remained at a high level even when photosynthesis was totally inhibited. The PSI reaction center remained active under drying conditions in both sensitive and tolerant species, but the activity became non-detectable in the light only in tolerant species due to deactivation of the cyclic electron flow around PSI and of the back reaction in PSI. Light-induced non-photochemical quenching (NPQ) was found to be induced not only by the xanthophyll cycle but also by a DeltapH-induced, dithiothreitol-insensitive mechanism in both the desiccation-tolerant and -intolerant bryophytes. Both mechanisms are thought to have an important role in protecting desiccation-tolerant species from photoinhibition under drying conditions. Fluorescence emission spectra at 77K showed that dehydration-induced quenching of PSII fluorescence was observed only in tolerant species and was due to neither state 1-state 2 transition nor detachment of light-harvesting chlorophyll protein complexes from PSII core complexes.The presence of dehydration-induced quenching of PSI fluorescence was also suggested.     

Inhibitory effects of microalgae on the activation of hyaluronidase

The inhibitory effects of seven microalgae, Nostoc flagelliforme, Spirulina platensis, Porphyridium purpureum, Rhodosorus marinus,Chlorella pyrenoidosa, Dunaliella salina and Pleurochrysiscarterae on the activation of hyaluronidase were evaluated. Theinhibitory effect of the ethanol-insoluble fraction of each water extract frommicroalgae was stronger than that of the ethanol-soluble fraction. The50% inhibitory concentration (IC₅₀) of the ethanol-insolublefraction of S. platensis, P. purpureum, R. marinus, C.pyrenoidosa, D. salina and P. carterae was 0.15, 0.18, 0.26,0.94, 0.15 and 0.41 mg mL^-1, respectively. The IC₅₀ ofN .flagelliforme was not calculated, because there was no detectableinhibitory effect of this alga. The IC₅₀ of disodium cromoglycate(DSCG) used as the anti-allergic medicine was 0.14 mg mL^-1. The IC₅₀ of S. platensis, P. purpureum and D. salinawere almost the same as that of DSCG. This suggests that theethanol-insoluble fraction of S. platensis, P. purpureum and D. salina might be an anti-allergic substance. The ethanol-insoluble fractionof S. platensis and D. salina was ultrafiltered through a membranehaving a molecular exclusion limit of 20 kDa. The IC₅₀ of theresidue was stronger than that of the filtrate. These results suggest that theanti-allergic substance(s) of these microalgae may be polysaccharides.     

Bi-parental cytoplasmic DNA inheritance in Wisteria (Fabaceae): evidence from a natural experiment

Cytoplasmic inheritance was investigated in interspecific hybrids of Wisteria sinensis and W. floribunda. Species-specific nuclear, mitochondrial and plastid DNA markers were identified from wild-collected plants of each species in its native range. These markers provide evidence for the bi-parental transmission of plastids in hybrid swarms of these two species in the southeastern USA. These population level molecular data corroborate previous cytological evidence of this phenomenon in Wisteria.     

General function and endocrine control of the posterior lymph hearts in Bufo marinus and Rana catesbeiana

The effects of hypervolemia and graded increases in arginine vasotocin (AVT), angiotensin II (ANGII), and atrial natriuretic peptide (ANP) on lymph heart pressure (P(lh)) and rate (f(lh)) were examined in Bufo marinus and Rana catesbeiana. The P(lh) and f(lh) for normally hydrated B. marinus at rest were 1.45+/-0.01 kPa and 52.8+/-0.38 beats min(-1). The P(lh) and f(lh) were significantly lower in R. catesbeiana, 1.05+/-0.01 kPa and 48.4+/-0.35 beats min(-1). Hypervolemia, induced by intravenous infusion of isotonic saline, stopped the lymph hearts at volumes of 0.48%+/-0.06% and 0.32%+/-0.04% body mass in B. marinus and R. catesbeiana, respectively, equivalent to an 8% increase of their respective plasma volumes. ANP had no effect on P(lh) or f(lh) at any of the dosages tested. ANGII decreased f(lh) in both species, approximating the physiological range of concentrations. AVT, at physiological concentrations, increased P(lh) 48% in B. marinus and 38% in R. catesbeiana without changing f(lh) in either species. At higher than physiological dosages, P(lh) and f(lh) in both species declined. The results suggest that AVT, normally released during hemorrhage and dehydration, would increase lymph heart output and help compensate for the hypovolemia. This is a contrary result to previous work using supraphysiologic doses of AVT.     

The Clara cell: a comparative ultrastructural study in mammals.

Clara cells in the terminal bronchoiles of mouse, rat, rabbit, calf and human were compared by light, transmission and scanning microscopy, and species-differences were clearly present. Mouse Clara cells were most numerous and mouse and rabbit Clara cells had large dense mitochondria. Rabbit and calf had glycogen in Clara cells and rat Clara cells had the most variability in secretory granules, some of which had a crystalline structure. Calf Clara cells had deeply indented nuclei. Human Clara cells had the most prominent nucleoli and lacked smooth endoplasmic reticulum, which was a prominent feature of most other species. No evidence of apical extrusion or apocrine secretion of Clara cell secretory granules was observed.     

How did glycogen structure evolve to satisfy the requirement for rapid mobilization of glucose? A problem of physical constraints in structure building

Optimization of molecular design in cellular metabolism is a necessary condition for guaranteeing a good structure–function relationship. We have studied this feature in the design of glycogen by means of the mathematical model previously presented that describes glycogen structure and its optimization function [Meléndez-Hevia et al. (1993), Biochem J 295: 477–483]. Our results demonstrate that the structure of cellular glycogen is in good agreement with these principles. Because the stored glucose in glycogen must be ready to be used at any phase of its synthesis or degradation, the full optimization of glycogen structure must also imply the optimization of every intermediate stage in its formation. This case can be viewed as a molecular instance of the eye problem, a classical paradigm of natural selection which states that every step in the evolutionary formation of a functional structure must be functional. The glycogen molecule has a highly optimized structure for its metabolic function, but the optimization of the full molecule has meaning and can be understood only by taking into account the optimization of each intermediate stage in its formation. Received: 23 October 1996 / Accepted: 21 April 1997     

Multiple intracellular locations of Lon protease in Arabidopsis: evidence for the localization of AtLon4 to chloroplasts

Arabidopsis contains four Lon protease-like proteins (AtLon1-AtLon4), predicted to be localized in different cellular organelles, including mitochondria, peroxisomes and plastids. A notable question is whether Lon is present in chloroplasts, since it is absent from cyanobacteria and thus appears to have been lost during the evolution of photosynthetic organisms. Based on in vivo transient assays, we found that AtLon4 is dually targeted to both mitochondria and chloroplasts. Furthermore, immunoblot analysis localized AtLon4 to the thylakoids. Thus, in spite of its absence from basal photosynthetic organisms, our results suggest the presence of Lon in plant plastids.     

Spectroscopic and biochemical analysis of regions of the cell wall of the unicellular 'mannan weed', Acetabularia acetabulum

Although the Dasycladalean alga Acetabularia acetabulum has long been known to contain mannan-rich walls, it is not known to what extent wall composition varies as a function of the elaborate cellular differentiation of this cell, nor has it been determined what other polysaccharides accompany the mannans. Cell walls were prepared from rhizoids, stalks, hairs, hair scars, apical septa, gametophores and gametangia, subjected to nuclear magnetic resonance and Fourier transform infrared spectroscopy, and analyzed for monosaccharide composition and linkage, although material limitations prevented some cell regions from being analyzed by some of the methods. In diplophase, walls contain a para-crystalline mannan, with other polysaccharides accounting for 10-20% of the wall mass; in haplophase, gametangia have a cellulosic wall, with mannans and other polymers representing about a quarter of the mass. In the walls of the diplophase, the mannan appears less crystalline than typical of cellulose. The walls of both diploid and haploid phases contain little if any xyloglucan or pectic polysaccharides, but appear to contain small amounts of a homorhamnan, galactomannans and glucogalactomannans, and branched xylans. These ancillary polysaccharides are approximately as abundant in the cellulose-rich gametangia as in the mannan-rich diplophase. In the diplophase, different regions of the cell differ modestly but reproducibly in the composition of the cell wall. These results suggest unique cell wall architecture for the mannan-rich cell walls of the Dasycladales.