Isolation, Properties and a Possible Function of a Water-Soluble Chlorophyll a/b-Protein from Brussels Sprouts

A water-soluble Chl a/b-protein (CP673) was isolated and purifiedfrom Brussels sprouts (Brassica oleracea L. var. gemmifera DC).The protein had a molecular mass of 78 kDa and an isoelectricpoint of 4.7, consisted of three or four subunits of 22 kDaand was extremely heat-stable. Although CP673 contained aboutone Chl a per protein, the blue and red absorption bands ofChl a that consisted of three or four Chl a forms with differentabsorption maxima suggested that there are several differentmodes or sites of binding for Chl a. Chl a/b ratio of largerthan 10 also indicated that Chl b is present only in a smallfraction of CP673. The heterogeneity of CP673 in terms of compositionand binding of Chl suggests that Chl is not an intrinsic componentof the Chl-protein. Homology search showed that the N-terminalamino acid sequence of CP673 is highly homologous with thatof a 22 kDa protein that accumulates in water-stressed leavesof two Brassicaceae plants, rapeseed and radish, but not withthose of the light-harvesting Chl a/b-proteins of photosynthesis.A possible function of the water-soluble Chl-protein was discussed. (Received September 17, 1996; Accepted November 18, 1996)     

Enzymatic Conversion of Pheophorbide a to the Precursor of Pyropheophorbide a in Leaves of Chenopodium album

Soluble proteins extracted from leaves of Chenopodium albumcatalyzed the conversion of pheophorbide a to a precursor ofpyropheophorbide a, putatively identified as C-13²-carboxyl-pyropheophorbidea. The precursor was then decarboxylated non-enzymatically toyield pyropheophorbide a. Soluble proteins and pheophorbidea, as the substrate, were required for the formation of theprecursor, and boiled proteins were enzymatically inactive.The maximum rate of conversion of pheophorbide a to the precursoroccurred at pH 7.5. The K_m for pheophorbide a was 12.5 µMat pH 7.0. Both pheophorbide b and bacteriopheophorbide a couldserve as substrates, but protopheophorbide a could not. Formationof methanol was detected during the enzymatic reaction, an indicationthat the enzyme is an esterase. Among seven alcohol analogstested, only methanol inhibited the enzymatic activity uncompetitively,with a K₁ of 71.6 mM. Mass-spectrometric (MS) analysis of theprecursor yield a peak at m/z 579 that indicated the releaseof a methyl group from pheophorbide a. It appears thereforethat the enzyme catalyzes the demethylation of the carbomethoxygroup at C-13² of pheophorbide a by hydrolysis to yield methanoland the precursor, C-13²-carboxyl-pyropheophorbide a, whichis converted to pyropheophorbide a by spontaneous decarboxylation.We have tentatively designated the enzyme "pheophorbidase".The presence of the enzyme was dependent on plant species andit was expressed constitutively. ¹Present address: Faculty of Science, Shizuoka University, Ohya,Shizuoka, 422 Japan     

Changes in the structure of a zooplankton community during a Ceratium (dinoflagellate) bloom in a eutrophic fishless pond

The community structure of zooplankton was studied in a eutrophic,fishless Japanese pond. The ecosystem was dominated by a dinoflagellate,Ceratium hirundinella, two filter-feeding clado-cerans, Daphniarosea and Ceriodaphnia reticulata, and an invertebrate predator,the dipteran Chaoborus flavicans. The midsummer zooplanktoncommunity showed a large change in species composition (theDaphnia population crashed) when a heavy Ceratium bloom occurred.It is shown that (i) the rapid density decline of D.rosea inmid-May was mainly caused by a shortage of edible phytoplankton,which was facilitated by the rapid increase in Chirundinellaabundance; (ii) the low density of D.rosea in June-July wasconsidered to be mainly caused by the blooming of Ceratium hirundinella(which may inhibit the feeding process of D.rosea), while predationby Cflavicans larvae, the changing temperature, the interspecificcompetition and the scarcity of edible algae were not judgedto be important; (iii) the high summer biomass of the planktonicCflavicans larvae was maintained by the bloom of C.hirundinella,because >90% of the crop contents of C.flavicans larvae wereC.hirundinella during this period. The present study indicatesthat the large-sized cells or colonies of phytoplankton arenot only inedible by most cladocerans, but the selective effectof the blooming of these algae can also influence the compositionand dominance of the zooplankton community, especially for thefilter-feeding Cladocera, in a similar way as the selectivepredation by planktivorous fish. The large-sized phytoplanktoncan also be an important alternative food for ominivorous invertebratepredators such as Chaoborus larvae, and thus may affect theinteractions between these predators and their zooplanktonicprey. In this way, such phytoplankton may play a very importantrole in regulating the dynamics of the aquatic food web, andbecome a driving force in shaping the community structure ofzooplankton.     

Transpiration Stream of Desert Species: Resistances and Capacitances for a C3, a C4, and a CAM Plant

Transpiration rates and water potentials of three sympatricdesert perennials, a C3 subshrub (Encelia farinosa), a C₄ bunchgrass(Hilaria rigida), and a CAM succulent (Agave deserti), wereanalysed using an electrical circuit analogue that includedresistances and capacitances for the leaves, stems, and roots.The water storage capability of the organs differed considerably,capacitance ranging over 1000-fold from the thin leaves of H,rigida to the massive leaves of A. deserti, although the capacitanceper unit volume varied only 1.9-fold. The diurnal changes inwater storage could support maximum transpiration rates of H.rigida for 4 min, E. farinosa for 7 min, and A. deserti for16 h. The time constant for equilibration of water from storageto the xylem ranged from 29 s for roots of H. rigida to 52 minfor leaves of A. deserti. Resistances for such movement wererelatively low for the succulent leaves of A. deserti and wereup to about 50-fold higher for the three organs of E. farinosa.Xylem resistances calculated using the Hagen-Poiseuille lawand measured xylem dimensions were 2.1- to 2.1-fold lower thanresistances estimated from observed water potential drops, adiscrepancy which is in agreement with other published data.Contrary to data on other plants, the xylem resistances in theroots and leaves of E. farinosa and H. rigida averaged only15% of the stem xylem resistance. Key words: Capacitance, Xylem resistance, Transpiration stream, Desert     

Energy-dependent redox state of heme a+a3 and copper of cytochrome oxidase in perfused rat brain in situ

Using theblood-free perfused rat brain, we examined the redox behavior ofcytochrome oxidase of two chromophores, hemea + a₃ and copper.When perfusate inflow was stopped to induce global ischemia,the reduction of heme a + a₃ was triphasic,with a rapid phase, a slow phase, and a second rapid phase. Incontrast, the reduction of copper was monophasic after the rapid phaseof heme a + a₃. The triphasicreduction of heme a + a₃ was diminished by energy-depleting treatments, such as addition of an uncoupler. Thetime course of the reduction of copper was not affected by the energydepletion. During global ischemia the decrease in creatine phosphate nearly paralleled the reduction of hemea + a₃, whereas ATPremained at the control level until ~60% of hemea + a₃ was reduced inthe rapid phase. In the slow phase, ATP started to decrease with thereduction of copper. The redox behavior of copper was similar to theslow phase of the reduction of heme a + a₃ because ofthe higher oxygen affinity of copper than of hemea + a₃. Therefore,the rapid phase of the reduction of hemea + a₃ can be used asan alarm before a decrease in ATP, whereas the reduction of copperindicates a decrease in ATP under severe hypoxia. Thus the coppersignal in noninvasive near-infrared spectroscopy is a useful parameterfor the clinical setting.

     

Isolation of a Tobacco cDNA Encoding Sar1 GTPase and Analysis of Its Dominant Mutations in Vesicular Traffic Using a Yeast Complementation System

The cDNA clone of NtSARl, a gene encoding the small GTPase Sar1pwhich is essential for vesicle formation from the endoplasmicreticulum (ER) membrane in yeast, has been isolated from Nicotianatabacum BY-2 cells. NtSAR1 as well as AtSAR1 cDNA isolated fromArabidopsis thaliana [d'Enfert et al. (1992) EMBO J. 11: 4205]could complement the lethality of the disruption of SARI inyeast cells in a temperature-sensitive fashion. They also suppressedyeast sec12 and sec16 temperature-sensitive mutations as yeastSARI does. Using this complementation system, we analyzed thephenotypes of several mutations in plant SAR1 cDNAs in yeastcells. The expression of NtSAR1 H74L and AtSAR1 N129I showeddominant negative effect in growth over the wild-type SARI,which was accompanied by the arrest of ER-to-Golgi transport.Such dominant mutations will be useful to analyze the role ofmembrane trafficking in plant cells, if their expression canbe regulated conditionally. (Received October 29, 1997; Accepted March 17, 1998)     

Photosynthesis and Formation of Fats in a Diatom

A study has been made, using C¹⁴ as a tracer, of the variationswhich occur according to the physiological condition of thecells in the distribution of carbon fixed, both in the lightand in the dark, by the diatom Navicula pelliculosa (Bréb.)Hilse during periods of 30 to 300 seconds. Fixation into thefollowing cell fractions was determined: (A) material solublein 80 per cent. ethanol but insoluble in benzene, (B) materialsoluble both in 80 per cent. ethanol and in benzene, and (C)material insoluble in 80 per cent. ethanol. Carbon fixed inphotosynthesis was incorporated, rapidly and in amounts representingup to 70 per cent. of the total fixation, into fractions B andC, as well as into fraction A. Considerable variation was foundin the proportions of carbon entering the three fractions inthe light; in actively growing cells the proportion enteringfraction C preponderated over that in B, corresponding to thesynthesis of protein, whereas in nitrogen-deficient cells fixationin B was the greater, corresponding to the synthesis of fat.These patterns changed only slowly, over periods of days, followingthe transfer of cells to altered conditions of nitrate supply.However, when ammonium nitrogen was supplied to nitrogen-deficientcells a marked change in distribution of carbon fixed occurredwithin 5 minutes, fixation in fraction B falling to a low valueand that in A rising correspondingly. In cells subjected toprolonged nitrogen-deficiency, fixation in fraction B fell toa relatively low value but the proportion which this fractionformed of the total dry matter in the cells rose as a resultof an increased rate of loss from the cells of constituentsother than lipides. The distribution of carbon fixed was alsodependent on light intensity. Fixation in fractions B and Crose relatively to that in A as light intensity was increasedup to 100 foot-candles but fell again at the highest intensityused, 2,000 foot-candles. These results are discussed with particularreference to the relationship between fat accumulation and photosynthesisin algae.     

Midsummer succession of rotifer plankton in a shallow eutrophic pond

Temporal changes in the density of rotifer plankton were examinedin a shallow eutrophic pond during July to September 1990, whenabiotic environmental variables were relatively stable. In earlyJuly when Daphnia similis was abundant, rotifer populationsremained at a low density. This is probably due to interferenceby the large cladoceran, because possible food was abundantand the rotifers showed high egg ratio. After late July, whenthe large cladoceran disappeared, a striking succession wasfound in the rotifer plankton. In late July, when Filinia longisetaand Conochilus dossuarius dominated, and in mid- to late Septemberwhen F.opoliensis and Brachionus falcatus dominated, there wasa causal relationship between the density and egg ratio, suggestingthat temporal changes in rotifer density were regulated mainlyby food abundance. However, in August, changes in the densityof most rotifers were not necessarily related with those inthe egg ratio. Brachionus angularis, B.forficula and Keratellaspp. decreased or remained at a low density regardless of theegg ratio, when the population of Asplanchna brightwelli wasdeveloped. However, Polyarthra vulgaris, B.calyciflorus andH.intermedia increased or maintained a high density at thistime. Stomach contents of A.brightwelli revealed that B.angularis,B.forficula and Keratella spp. were the preferred prey, whereasP.vulgaris, B.calyciflorus and H.intermedia were not. Theseresults provide strong evidence that the structure of rotiferplankton can change strikingly within a season due to species-specificdifferences not only in diet, but also in the ability to escapepredation, even if abiotic environmental variables are stable.     

Reproductive Isolation in a Native Population of Petunia sensu Jussieu (Solanaceae)

Reproductive isolation was studied in four syntopic speciesof Petunia sensu Jussieu (Solanaceae) at a site in Rio Grandedo Sul State, Brazil. Reciprocal artificial crossing experimentsconfirmed that a genetic barrier exists between Petunia(P. axillarisand P. integrifolia) andCalibrachoa (C. parviflora and C. heterophylla),and also between C. parviflora andC. heterophylla . Petuniaaxillaris(white, nocturnally scented flower) is geneticallycompatible with the syntopic and allotopic P. integrifolia(coloured,unscented flower). Reproductive isolation appears to be maintainedby the two species having different pollinators: nocturnallyactive hawkmoths (Manduca contracta andM. diffissa subsp. petuniae)pollinate P. axillaris while a diurnally active bee (Hexanthedasp.) pollinates P. integrifolia. Flowers of P. integrifoliaexhibit diurnal opening and closing movements synchronous withthe activity period of the bee. Other than a probable nectarrobber (a carpenter bee, Xylocopa augusti), no insect visitedflowers of P. axillaris in the day. Amounts of floral nectarin P. axillaris and P. integrifolia were within the range ofhawkmoth- and bee-pollinated flowers, respectively. Reproductiveisolating mechanisms in the genus Petunia sensu Jussieu arediscussed. Copyright 2001 Annals of Botany Company Calibrachoa, hawkmoth, Petunia, Petunia axillaris, Petunia integrifolia, pollinator, reproductive isolation, Solanaceae     

Identification of a Novel Triterpenoid Saponin from Pisum sativum as a Specific Inhibitor of the Diguanylate Cyclase of Acetobacter xylinum

A specific and highly potent inhibitor of diguanylate cyclase,the key regulatory enzyme of the cellulose synthesizing apparatusin the bacterium Acetobacter xylinum, was isolated from extractsof etiolated pea shoots (Pisum sativum). The inhibitor has beenpurified by a multistep procedure, and sufficient amounts ofhighly purified compound (3-8 mg) for spectral analysis wereobtained. The structure of this compound was established as3-O-a-L-rhamnopyranosyl-(l     

Touch-Inducible Genes for Calmodulin and a Calmodulin-Related Protein Are Located in Tandem on a Chromosome of Arabidopsis thaliana

Genes for calmodulin and calmodulin-related proteins in Arabidopsisare up-regulated by a variety of physical stimuli, which includerain, wind and touch [Braam and Davis (1990) Cell 60: 357].We have isolated five genes for calmodulin (AtCALl, 2, 3, 5,6) and one gene for a calmodulin-related protein (AtCAL4) froman Arabidopsis genomic library. Touch stimulus of Arabidopsisplants induces the accumulation of mRNA transcribed from AtCAL4andAtCAL5, but not from the other isolated genes. The two touch-induciblegenes are arrayed in tandem with a short intergenic region of700 bp but they show different organ-specific patterns of expression. (Received April 27, 1995; Accepted July 20, 1995)     

Comparison of Plant Telomere Locations using a PCR-generated Synthetic Probe

We have generated a telomere-specific probe by the polymerasechain reaction and used it to localize chromosome telomeresof ten unrelated angiosperm species in in situ. Concatenationof the simple monomers, 5'-(TTTAGGG)-3', derived from the sequenceof Arabidopsis thaliana telomeres, yielded a stable, versatileand reliable probe that gave a signal of high intensity followingfluorescence in situ hybridization. Most species, includingthose with known karyotype rearrangements, showed telomere labelonly at chromosome termini. These findings are discussed inthe context of the chromosomal events responsible for generatingand stabilizing karyotype change in plants.Copyright 1993, 1999Academic Press PCR, telomere repeat sequence (TRS), in situ hybridization, comparative physical mapping, karyotype evolution, genome organization, Lathyrus sativus L., Vicia sativa L. subsp. nigra (L.) Ehrh., Vigna radiata (L.) R. Wilczek cv. Berken, Nicotiana sylvestris Speg et Comes, Haplopappus gracilis (Nutt.) A. Gray, Gibasis pulchella (Kunth) Rafin., Tradescantia commelinoides Schultes. fil., Hordeum vulgare L. cv. Sultan., Hordeum vulgare L. cv. Tuleen 346, Milium vernale Bieb., Paphiopedilum insigne (Wall.) Pfitz     

Efficiency as a foraging currency in animals attaining a gain below the energetic ceiling

Previous research has found that efficiency, or, more precisely,the foraging gain ratio (FGR), is a valid currency in foragingtheory when (1) there is a limit to the energy that can beassimilated by the forager and (2) a forager is trying to meetan energy requirement. The FGR is b/ (c — c_r), whereb is the rate of metabolizable energy intake, and c and c_rare the rates of energy expenditure while foraging and resting,respectively. Here I show that, when energy expenditure hasa cost besides energy, animals should also choose the optionwith the highest FGR when they are aiming at a given positivedaily gain. The next question is which gain they should aimfor? Researchers have shown that observed intake levels ofgrowing ruminants are close to the levels predicted by maximizationof the efficiency of oxygen utilization. This currency can be approximated by (B — C + C_r) / C, where B is the daily metabolizable energy intake, and C and C_r are the total andbasal daily energy expenditures, respectively. By simulatinggrowth at different intake levels, I found that mass-specificoxygen consumption rate is indeed minimal at the observed intakelevels. This is the first study in which these efficiency measures(FGR and the efficiency of oxygen utilization) are combined.     

Conditional use of honest signaling by a Batesian mimic

Jumping spiders (Salticidae) usually avoid ants, but some specieswithin this family single out ants as preferred prey, whileothers (especially the species in the genus Myrmarachne) areBatesian mimics of ants. Field records show that ant-eatingsalticids sometimes prey on Myrmarachne, suggesting that theunwanted attention of predators that specialize on the modelmay be an important, but poorly understood, cost of Batesianmimicry. By staging encounters in the laboratory between livingant-eating salticids and Myrmarachne, we determined that ant-eatingsalticids attack Myrmarachne. However, when Myrmarachne detectsa stalking ant-eating salticid early enough, it adopts a distinctivedisplay posture (legs almost fully extended, elevated 45°,and held out to the side 45°), and this usually deters thepredator. When Myrmarachne detects an ant-eating salticid beforestalking begins, Myrmarachne makes preemptive displays thatappear to inhibit the initiation of stalking. Using immobilelures made from dead Myrmarachne that were either in a displayposture or a nondisplay posture, we ascertained that specificallythe display posture of Myrmarachne deters the initiation ofstalking (ant-eating salticids stalked nondisplaying more oftenthan displaying lures). In another experiment, we ascertainedthat it is specifically the interjection of display posturethat deters stalking. When ant-eating salticids that had alreadybegun stalking experienced lures that switched from a nondisplayto a display posture, they stopped stalking. Although the unwantedattentions of its models' predators may be, for Myrmarachne,a hidden cost of Batesian mimicry, Myrmarachne appears to havean effective defense against these predators.     

Simulations of the Ontogeny of Spongiophyton, a Devonian Plant

Mathematical representations of the cellular organization anddimensions of Spongiophyton Krusel, a Middle Devonian thallophytewere projected by means of a computer to simulate patterns ofdevelopment and organization. Extrapolation of the cellularpatterns observed on the surface of the fossil may be comparedwith those derived from living plants of which the ontogenycan be directly observed. Spongiophyton is compared in thisrespect with the growth of Protosalvinia (an Upper Devonianplant of enigmatic affinity), Pellia (a thallose liverwort)and Cutleria (a brown alga). The growth pattern of Spongiophytondeveloped by computer shows a closer similarity to that of thepseudoparenchymatous alga Cutleria than to the truly parenchymatousPellia or the fossil Protosalvinia. Computer simulations ofthe growth process throw light on the affinity of Spongiophytonwhich cannot be derived from direct observation of the fossil.Broader applications of computer simulations of tissue organizationand gross morphology are suggested with regard to the studyof living and fossil plants.     

Origin and evolution of a myxozoan worm

Buddenbrockia plumatellae is an active, muscular, worm-shapedparasite of freshwater bryozoans. This rare and enigmatic animalhas been assigned to the Myxozoa on the basis of 18S ribosomalDNA sequences and the presence of malacosporean spores. Herewe report cloning of four homologous protein-coding genes fromBuddenbrockia worms, the putatively conspecific sac-shaped parasiteoriginally described as Tetracapsula bryozoides and the relatedsac-shaped parasite Tetracapsuloides bryosalmonae, the causativeagent of proliferative kidney disease in salmonid fish. Analysesare consistent with the hypothesis that Buddenbrockia is indeeda malacosporean myxozoan, but do not provide support for conspecificitywith either T. bryozoides or T. bryosalmonae. Implications forthe evolution of worm-like body plans in the Myxozoa are discussed.     

Impacts of a native parasitic plant on an introduced and a native host species: implications for the control of an invasive weed

Background and Aims: While invasive species may escape from natural enemies in thenew range, the establishment of novel biotic interactions withspecies native to the invaded range can determine their success.Biological control of plant populations can be achieved by manipulationof a species' enemies in the invaded range. Interactions weretherefore investigated between a native parasitic plant andan invasive legume in Mediterranean-type woodlands of SouthAustralia. Methods: The effects of the native stem parasite, Cassytha pubescens,on the introduced host, Cytisus scoparius, and a co-occurringnative host, Leptospermum myrsinoides, were compared. The hypothesisthat the parasitic plant would have a greater impact on theintroduced host than the native host was tested. In a fieldstudy, photosynthesis, growth and survival of hosts and parasitewere examined. Key Results: As predicted, Cassytha had greater impacts on the introducedhost than the native host. Dead Cytisus were associated withdense Cassytha infections but mortality of Leptospermum wasnot correlated with parasite infection. Cassytha infection reducedthe photosynthetic rates of both hosts. Infected Cytisus showedslower recovery of photosystem II efficiency, lower transpirationrates and reduced photosynthetic biomass in comparison withuninfected plants. Parasite photosynthetic rates and growthrates were higher when growing on the introduced host Cytisus,than on Leptospermum. Conclusions: Infection by a native parasitic plant had strong negative effectson the physiology and above-ground biomass allocation of anintroduced species and was correlated with increased plant mortality.The greater impact of the parasite on the introduced host maybe due to either the greater resources that this host providesor increased resistance to infection by the native host. Thisdisparity of effects between introduced host and native hostindicates the potential for Cassytha to be exploited as a controltool.     

Structure and expression of a cDNA encoding a putative neoxanthin cleavage enzyme (NCE), isolated from a wilt-related tomato (Lycopersicon esculentum Mill.) library

Oxidative cleavage of a 9-cis xanthophyll probably representsthe key regulatory step in abscisic acid (ABA) biosynthesis.A transposon tagged maize (Zea mays) mutant vp14, provided theoriginal DNA sequence data needed to design a VP14 fusion proteincapable of catalysing this reaction in vitro. A cDNA encodinga similar protein has now been isolated from a wilt-relatedtomato (Lycopersicon esculentum Mill.) library. The tomato cDNAand derived amino acid sequence have been compared to thoseof maize and of other enzymes catalysing broadly similar oxidativecleavage reactions. The results of Northern analysis in tomatoindicated that mRNA levels of this vp14 homologue increaseddramatically in response to water stress. Key words: ABA biosynthesis, oxidative cleavage step, tomato     

Characterization of waldmeister, a novel developmental mutant in Arabidopsis thaliana

A novel Arabidopsis thaliana (L.) Heynh. developmental mutant,waldmeister (wam), is described. This mutant was found in theprogeny arising from an Ac-Ds tagging experiment, but does notappear to be tagged by an introduced transposon. This recessivenuclear mutation maps between GAPB and ap1 on chromosome 1 andshows extreme morphological and physiological changes in bothfloral and vegetative tissues. Changes to the vegetative phenotypeinclude altered leaf morphology, multiple rosettes, stem fasciation,retarded senescence and disturbed geotropic growth. Changesto the floral phenotype include delayed flowering, increasednumber of inflorescences, determinate inflorescences, alterednumber and morphology of floral organs, chimeric floral organs,and ectopic ovules . wam was crossed to a number of previouslydescribed floral mutants: apetela 2, apetela 3, pistillata,agamous, and leafy. The phenotype of the double mutant was ineach case additive. In the case of agamous, however, the indeterminaterepetitive floral structure of agamous was lacking, emphasizingthe determinate inflorescence growth of wam. The extreme phenotypeof the wam mutant is suggestive of a disturbance to a gene ofglobal importance in the regulation of plant growth and development. Key words: Arabidopsis thaliana, waldmeister, developmental mutant, flower mutant