Expression of Plasma Membrane Water Channel Genes under Water Stress in Nicotiana excelsior

Deduced amino acid sequences encoded by the cDNAs related tothe MIP gene family from Nicotiana excelsior were characterized.Phylogenetic characterization of the products of correspondinggenes named NeMip1, NeMip2, and NeMip3 strongly suggested thatthey are water channel proteins localized in the plasma membrane.Organ specificity of the gene expression was examined in leaves,roots, and reproductive organs. NeMip1 was expressed in rootsand reproductive organs; however, it was hardly detectable inleaves. Two other genes, NeMip2 and NeMip3, were expressed inall of organs examined. mRNA accumulation from the genes wasinvestigated in leaves under salt- and drought-stresses. Theresults demonstrated that mRNA accumulation from all three genesincreased under salt- and drought-stresses within one day. However,they showed different accumulation patterns. In addition totheir up-reg-ulation under salt- and drought-stresses, dailychanges in NeMip2 and NeMip3 mRNA accumulation was observedunder unstressed conditions in leaves. (Received May 2, 1997; Accepted September 3, 1997)     

The Effect of {alpha}-Naphthalene Acetic Acid (NAA) and Benzylaminopurine (BAP) on the Accumulation of Volatile Oil Components in Cell Cultures of Tarragon (Artemisia dracunculus)

Disorganized cells of A. dracunculus have been demonstratedto be competent to synthesize the four phenylpropene compoundscharacteristic of tarragon essential oil. Allylanisole and methyleugenol, the major components, are accumulated at particularlylow levels and are inhibited largely by the presence of exogenousNAA. Volatilization, active metabolism, and autotoxicity ofthese compounds are involved in limiting their accumulation.The data reported here suggest that both limited expressionof specific synthetic pathways, and the absence of suitable(inert) accumulation sites can restrict accumulation of phenylpropenesin disorganized cultures of A. dracunculus. Key words: Artemisia dracunculus, tarragon, tissue culture, essential oil, phenylpropenes, allylanisole, methyl eugenol     

Pigmented Plant Tissues in Culture II. Growth, Development and Decline of Chlorophyllous Tissues

During the phase of exponential growth in chlorophyllous calluscultures derived from Haplopappus gracilis, Hypochaeris radicata,and Acer pseudoplatamus, cells double their number on the average,and also their volume, in about 4.3, 6.6, and 9–2 daysrespectively. The two rates decline subsequently but cell expansioncontinues for a short time after division has ceased. With culturesof Oxalis dispar, however, which have an average cell generationtime of about 10 days, there is first a short exponential phasedominated by division, and this is followed by a series of phasesdominated alternately by either division or expansion. Chlorophyll accumulation does not occur in Haplopappus duringthe exponential phase (chlorophyll a decreases) but there isa slow accumulation of caro-tenoids. The bulk of the pigmentsaccumulate during the declining phase of growth mainly afterdivision has ceased. With Hypochaeris and Acer, on the otherhand, accumulation is most intense during the exponential phase,and few pigments are added later. With Oxalis, most of the accumulationoccurs after the exponential phase; carotenoids accumulate untilthe cessation of growth whereas chlorophylls start to declinebefore this. With all species, pigments decline after the cessationof growth. The loss is small in Haplopappus and the tissuesare still bright green when the medium dries out: Hypochaerisand Oxalis, in contrast, eventually become colourless. The data are discussed in relation to the changes in pigmentcontent that accompany the growth and development of a singlecell in each species.     

Accumulation of Betacyanin in Phytolacca americana Cells and of Anthocyanin in Vitis sp. Cells in Relation to Cell Division in Suspension Cultures

In suspension cultures of Vitis sp., maximal accumulation ofanthocyanin was observed during the stationary phase. Accumulationof anthocyanin occurred in parallel with the cessation of celldivision under conditions such as a reduction of the concentrationof phosphate in the medium, or the presence of aphidicolin,an inhibitor of DNA synthesis. By contrast, in suspension culturesof Phytolacca americana, aphidicolin inhibited the accumulationof betacyanin and cell division. When aphidicolin was removedfrom cells by washing, partially synchronized division of cellswas induced and the accumulation of betacyanin also occurred,in conjunction with cell division. In the absence of phosphatefrom the medium, cell division did not occur and accumulationof betacyanin also ceased. Readdition of phosphate to cellsstarved for phosphate induced both cell division and the accumulationof betacyanin. These results indicate a positive correlationbetween the accumulation of betacyanin and cell division inPhytolacca which contrasts with a negative correlation betweenthe accumulation of anthocyanin and cell division in Vitis. (Received April 17, 1989; Accepted December 23, 1989)     

Metallothioneins 2 and 3 contribute to the metal-adapted phenotype but are not directly linked to Zn accumulation in the metal hyperaccumulator, Thlaspi caerulescens

To study the role of metallothioneins (MTs) in Zn accumulation,the expression of TcMT2a, TcMT2b, and TcMT3 was analysed inthree accessions and 15 F₃ families of two inter-accession crossesof the Cd/Zn hyperaccumulator Thlaspi caerulescens, with differentdegrees of Zn accumulation. The highest expression levels werefound in the shoots of a superior metal-accumulating calamineaccession from St Laurent le Minier, with >10-fold TcMT3expression compared with another calamine accession and a non-metallicolousaccession. Moreover, F₃ sibling lines from the inter-accessioncrosses that harboured the MT2a or MT3 allele from St Laurentle Minier had higher expression levels. However, there was noco-segregation of TcMT2a or TcMT3 expression and Zn accumulation.To examine the functions of TcMTs in plants, TcMT2a and TcMT3were ectopically expressed in Arabidopsis. The transformantlines had reduced root length in control medium but not at highmetal concentrations, suggesting that the ectopically expressedproteins interfered with the physiological availability of essentialmetals under limited supply. The Arabidopsis transformant linesdid not show increased tolerance to Cd, Cu, or Zn, nor increasedCd or Zn accumulation. Immunohistochemical analysis indicatedthat in roots, MT2 protein is localized in the epidermis androot hairs of both T. caerulescens and Arabidopsis thaliana.The results suggest that TcMT2a, TcMT2b, and TcMT3 are not primarilyinvolved in Zn accumulation as such. However, the elevated expressionlevels in the metallicolous accessions suggests that they docontribute to the metal-adapted phenotype, possibly throughimproving Cu homeostasis at high Zn and Cd body burdens. Alternatively,they might function as hypostatic enhancers of Zn or Cd tolerance. Key words: Cd, crosses, metallothionein, protein, quantitative real-time PCR, Thlaspi caerulescens, Zn Received 14 August 2008; Revised 14 October 2008 Accepted 15 October 2008     

Time-course of Biosynthesis of Phenolics and Lignin in Roots of Wheat Genotypes Differing in Manganese Efficiency and Resistance to Take-all Fungus

Inhibition of lignin biosynthesis in Triticum aestivum L. rootsby Mn deficiency has been suggested as the mechanism of reducedresistance of Mn-deficient wheat roots to infection by the take-allfungus (Gaeumannomyces graminis var. tritici). This study evaluatedphenolics and lignin accumulation in roots of wheat genotypesdiffering in Mn efficiency (measured as growth and yield inMn-deficient soils) and take-all resistance. Seedlings of theMn-inefficient, take-all sensitive genotype Bayonet and theMn-efficient, more take-all resistant genotype C8MM were grownin nutrient solution without added Mn for 18 d and then transferredto a Mn-deficient sandy soil fertilized with Mn at 0 or 30 mgkg^-1. Both genotypes had Mn-deficient roots and shoots at thetime of transfer to the soil. Roots of both genotypes were inoculatedwith the take-all fungus 0, 1, 3 and 7 d after transfer. Twenty-fourhours after inoculation, take-all fungus penetrated the rootstele of take-all sensitive Bayonet but not of more resistantC8MM wheat. Rates of phenolics and lignin accumulation in rootsdeclined steadily during growth in soil for up to 8 d, werehigher in mature, fully differentiated parts of the root systemcompared to distal, younger root tissue, and were higher inBayonet than in C8MM. Manganese fertilization did not significantlyinfluence rates of phenolics and lignin accumulation but reduceddepth of radial penetration by hyphae in both genotypes. Therate of phenolics accumulation was positively (r = 0·91to 0·96) correlated with the rate of lignin accumulation.Mn-efficient C8MM had a higher rate of lignin accumulation perunit of phenolics than Mn-inefficient Bayonet over a wide rangeof phenolics synthesis rates. From this we suggest that C8MMhas a more efficient mechanism for conversion of phenolics tolignin, the trait which appears related to higher take-all resistanceof this genotype.Copyright 1994, 1999 Academic Press Gaeumannomyces graminis var. tritici, lignin, manganese, phenolics, resistance, roots, Triticum aestivum     

Accumulation of Pb and Zn in Gametophytes and Sporophytes of the Moss Funaria hygrometrica(Funariales)

Accumulation of lead and zinc was studied in the moss Funariahygrometrica Hedw. collected from mine tailings. Heavy metalaccumulation in gametophytes and sporophytes was quantifiedby graphite furnace atomic absorption spectrometry (GFAAS) andinductively coupled plasma-atomic emission spectrometry (ICP-AES).Pb and Zn accumulation in the placental zone was analysed byx-ray scanning electron microscopy (SEM) and transmission electronmicroscopy (TEM) microanalysis. Spectrometry showed that whilemoss gametophytes accumulated considerable concentrations ofheavy metals, sporophytes accumulated only small concentrationsof metals. X-ray SEM and TEM showed that the two metals accumulatedin placental transfer cells on both the gametophytic and sporophyticsides. To investigate the uptake pattern for both metals undercontrolled conditions, F. hygrometrica plants collected froma non-polluted site were treated in the laboratory with separatesolutions of Pb and Zn at two concentrations (10^-2and 10^-4 M)for 24 or 168 h. Metal accumulation was analysed separatelyin gametophytes and sporophytes using GFAAS and ICP–AES.Each generation had a different accumulation quotient for bothmetals, and gametophytes accumulated significantly more metalthan sporophytes. Concentrations of Zn in sporophytes were alwayshigher than concentrations of Pb. The findings are discussedin relation to the role performed by the gametophyte and theplacenta in the accumulation and sequestration of Pb and Zn.Copyright 2001 Annals of Botany Company Atomic spectroscopy, Funaria hygrometrica, gametophyte, Pb and Zn accumulation, sporophyte, x-ray TEM and SEM microanalysis     

The Ultrastructure and Analytical Microscopy of Silicon Deposits in the Roots of Saccharum officinarum (L.)

Silicon accumulation in the endodermis of the ‘set’and ‘shoot’ roots of Saccharum officinarum (L.)were investigated by scanning electron microscopy and electron-probemicroanalysis. Silicon microassay was also carried out by meansof the Corinth analytical microscope (CORA). Aggregates arelargely associated with the inner tangential wall (ITW) of theendodermis and their formation is basically similar to thoseseen in Sorghum bicolor (L.) Moench. In contrast to Sorghumthe earliest deposits in Saccharum appear in wall strata wellwithin the cell wall cytoplasm interface. An additional layerof silicon was also located along the endodermal pericycle boundaryextending some distance along the middle lamella of the radialwalls. The results are discussed in relation to those of previous studiesof silicon accumulation in endodermal cells and to possiblefactors affecting such accumulations.     

An Endogenous Suppressor of the Defense Response in Pisum sativum

Healthy pea plants contain a substance, tentatively called "endogenoussuppressor", which specifically suppresses the accumulationof pisatin in pea plants that is induced by treatment with CuCl₂or an elicitor from Mycosphaerella pinodes. This suppressorelicits the accumulation of phytoalexins in other legumes, suchas kidney bean, soybean and cowpea. The endogenous suppressorfunctions to delay the accumulation of pisatin, the activationof phenylalanine ammonialyase (PAL) and the accumulation ofmRNAs for PAL and chalcone synthase induced by the elicitorfrom M. pinodes. The substance specifically induces susceptibilityto nonpathogens, such as Mycosphaerella ligulicola and M. melonis,in pea out of four species of legume tested, but the effectis not cultivar-specific. Thus, the endogenous suppressor inhealthy pea plants suppresses a series of self-defense reactionsand induces susceptibility in pea plants in a species-specificmanner, being similar to the exogenous fungal suppressor fromthe pea pathogen, M.pinodes. (Received February 19, 1992; Accepted May 11, 1992)     

Simulation of water-bloom formation in the cyanobacterium Microcystis aeruginosa

A mechanism for buoyancy increases in the cyanobacterium Microcystisaeruginosa and the associated formation of surface water-bloomsis presented. The mechanism is based on considering a responsetime in the rate of carbohydrate accumulation. When irradianceincreases, the Microcystis cells may require time to increasetheir rate of carbohydrate accumulation. If irradiance decreasesbefore adjustment, the maximum rate of carbohydrate accumulationis not reached. Colony buoyancy increases during mixing whenthe time scales of the light fluctuations are shorter than theresponse time. To examine the mechanism, a model of Microcystisbuoyancy that incorporates the response time has been coupledwith a hydrodynamics model that simulates mixing. The modelwas applied to a shallow lake to show that a prolonged episodeof intense mixing caused the simulated Microcystis coloniesto become excessively buoyant. Once the mixing subsided, thecolonies accumulated at the surface. Decreases in carbohydratewere reduced in large colonies as their size afforded buoyancyforces that could readily overcome the entraining forces ofthe mixing.     

Effect of Light on Alkaloid Accumulation in Cell Cultures of Nicotiana Species

The effect of light on alkaloid accumulation in a range of cellcultures of tobacco was determined. Cell suspension culturesof Nicoriana rabacwn L. cv. Wisconsin-38 with differing degreesof photosynthetic activity, callus cultures of N. glauca Graham,root cultures of N. rustica L. and shoot cultures of N. tabacumwere used. The alkaloid content of green illuminated cultureswas greatly reduced compared with non-green cultures grown inthe dark, but decreased accumulation did not correlate withincreasing photosynthetic activity. The accumulation of allof the major alkaloids was affected, regardless of the speciesof tobacco used. Transfer of N. glauca callus from the darkinto the light caused a decrease in alkaloid accumulation, whilemoving cultures from the light into the dark resulted in anincrease in alkaloid content. In root cultures light causeda reduction in growth, which affected alkaloid synthesis. Inshoot cultures there were only traces of alkaloid detectable,regardless of whether or not cultures were illuminated. Lightappeared to cause a non-photosynthetic suppression of alkaloidaccumulation in visibly undifferentiated cultures, and thiseffect was modified in visibly differentiated cultures. Key words: Nicoriana spp, tobacco, alkaloid accumulation, cell culture     

Temporal and Spatial Heterogeneity in the Accumulation of Anthocyanins in Cell Cultures of Catharanthus roseus (L.) G.Don.

The accumulation of anthocyanins, a group of pigmented secondarymetabolites, in cell cultures of the Madagascar periwinkle Catharanthusroseus has been investigated. In these cultures it was foundthat anthocyanin accumulation was restricted to the post-divisionphase of the culture growth cycle, during which the culturesbecame deep purple in colour. As a result of anthocyanin visibilityit has been possible to ascertain that accumulation of thesemetabolites occurred in only a small proportion of the cellpopulation. Approximately 10% of cells regularly accumulateddetectable levels. Considerable variation within this ‘productive’population was observed and using a standard integrating microdensitometerit has been possible to quantify directly this heterogeneityand compare it with data obtained from whole plants. Analysishas revealed that the variation in both intracellular anthocyanincontent and concentration in cell cultures was much greaterthan that observed within tissues of mature plants. Significantdifferences in mean values were however found between the wholeplant tissues. The relevance of this temporal and spatial heterogeneityobserved in vitro to our understanding of the control of secondarymetabolite accumulation and to the potential use of tissue culturesystems as a means to produce these compounds is discussed. Key words: Heterogeneity, anthocyanins, cell culture     

Histochemical Localization of Citral Accumulation in Lemongrass Leaves (Cymbopogon citratus(DC.) Stapf., Poaceae)

Lemongrasses (Cymbopogonspp., Poaceae) are a group of commerciallyimportant C₄tropical grasses. Their leaves contain up to 1.5%(d.wt) essential oils with a typical lemon-like aroma, consistingmainly of citral (a mixture of the isomeric acyclic monoterpenealdehydes geranial and neral). To specifically locate the sitesof citral accumulation in lemongrass we employed Schiff's reagent,which reacts with aldehydes and gives a purple-red colorationwith citral. Using this technique, single oil-accumulating cellswere detected in the adaxial side of leaf mesophyll, commonlyadjacent to non-photosynthetic tissue, and between vascularbundles. Cell walls of these oil cells are lignified. Our resultssuggest that citral accumulation takes place in individual oilcells within the leaf tissues.Copyright 1998 Annals of BotanyCompany Lemongrass;Cymbopogon citratus; Poaceae; oil cells; histochemistry; citral; aldehydes; Schiff's-reagent.     

The Effect of Inhibitors of Photosynthetic and Respiratory Electron Transport on Nitrate Reduction and Nitrite Accumulation in Excised Z. mays L. Leaves

The assimilation of nitrate and nitrite under dark and lightconditions in Zea mays L. leaves was investigated. Nitrate wasassimilated under dark-aerobic conditions. Anaerobiosis stimulatednitrate reduction and nitrite accumulation under dark conditions.Vacuum infiltration of inhibitors of respiratory electron transport,antimycin A and rotenone, stimulated nitrate reduction and nitriteaccumulation under dark-aerobic conditions. Vacuum infiltrationof low concentrations of PCP, DNP and mCCCP depressed nitratereduction and nitrite accumulation under dark-aerobic conditions,whereas, infiltration of higher concentrations stimulated nitratereduction and nitrite accumulation. The greatest level of nitrateand nitrite reduction occurred under light conditions. The inhibitorof photosynthetic electron transport, DCMU, stimulated the accumulationof nitrite in the light, but decreased nitrate reduction. Whenthe inhibitors of respiratory electron transport antimycin Aand rotenone, were supplied together with DCMU in the light,nitrite accumulation was enhanced. Low concentrations of mCCCPdecreased both nitrate reduction and nitrite accumulation underlight conditions when supplied with DCMU. Key words: Nitrate reduction, Nitrite accumulation, Leaves     

The Effect of Low Temperature upon Starch, Sucrose and Fructan Synthesis in Leaves

Measurements on plants in a temperature gradient tunnel showthat diurnal accumulation of starch in illuminated leaves wasgreatly reduced at temperatures below 8 °C, whereas sucrosesynthesis was less affected under similar conditions. High chillingsensitivity for leaf starch accumulation was observed in a numberof chilling resistant temperate species. Enzymes of sucroseand fructan metabolism from mature leaves of Lolium temulentumwere less strongly inhibited at low temperatures than enzymesinvolved in starch synthesis. These results are discussed inrelation to carbon partitioning in species which grow and aremetabolically active at chilling temperatures. Lolium temulentum, starch, sucrose, fructan, temperature, enzyme activity, carbon partitioning     

Studies in the Physiological Action of 2, 2-Dichloropropionic Acid: I.: MECHANISMS CONTROLLING THE INHIBITION OF ROOT ELONGATION

The inter-relationships between time and concentration and thedegree of inhibition of root elongation have been examined forSorghum vulgare, Zea mays, Helianthus annuus, and Pisum sativum.For all species the inhibitory effect is cumulative but thereis a tenfold difference in the concentration required to halvethe elongation of the most sensitive (S. vulgare) and most resistantspecies (P. sativum). From a comparison of the growth of intactsroots and isolated segments, together with estimates of cellnumber, it has been established that the primary effect is tointerfere with meristematic activity in the root tip, wherethe mitotic cycle is arrested at prophase. Using 2, 2-dichloropropionic acid, containing chlorine-36, thecourses of uptake by both intact roots and isolated segmentshave been followes. In every instance uptake is cumulative withthe greatest accumulation in the root tip. There are again largespecific differences but of a reverse order; uptake is leastfor P. sativum and greatest for S. vulgare. For these two speciesand Z. mays, it is concluded that the magnitude of the equi-effectiveconcentration required to halve root elognation is dependenton the level of accumulation rather than on the reaction atcell level: the cells of H. annuus are more sensitive.     

Seed Water Relations and the Regulation of the Duration of Seed Growth in Soybean

Soybean [Glycine max (L.) Merrill] seeds and cotyledons weregrown in an in vitro culture system to investigate the relationshipsbetween cell expansion (net water uptake by the seed) and drymatter accumulation. Seeds or cotyledons grown in a completenutrient medium containing 200 mol m^–3 sucrose continueddry matter accumulation for up to 16 d after in planta seedsreached physiological maturity (maximum seed dry weight). Seedor cotyledon water content increased throughout the cultureperiod and the water concentration remained above 600 g kg^–1fresh weight. These data indicate that the cessation of seeddry matter accumulation is controlled by the physiological environmentof the seed and is not a pre-determined seed characteristic.Adding 600 mol m^–3 mannitol to the medium caused a decreasein seed water content and concentration. Seeds in this mediumstopped accumulating dry matter at a water concentration ofapproximately 550 g kg^–1. The data suggest that dry matteraccumulation by soybean seeds can continue only as long as thereis a net uptake of water to drive cell expansion. In the absenceof a net water uptake, continued dry matter accumulation causesdesiccation which triggers maturation. Key words: Glycine max (L.) Merrill, solution culture, duration of seed growth, water content, dry matter accumulation     

Regulation of Soybean Embryogenesis by Abscisic Acid

Abscisic Acid (ABA) stimulates growth and protein accumulationin soybean (Glycine max. L. Merr.) embryos during the earlyphases of embryogenesis. Growth of mid-stage embryos is suppressedby ABA, but protein accumulation is not impaired. Metabolitedistribution studies indicate that ABA alters partitioning ofsucrose in older embryos such that protein accumulation is sustainedat the expense of lipid accumulation. The responses of in vitrocultured embryos to ABA is consistent with the normal patternof ABA accumulation and disappearance that occurs during embryogenesisin situ. A close correlation exists between ABA levels and embryogrowth rates in situ in three cultivars of soybeans. Dependingon the age or stage of the developing embryo, ABA either servesto promote or inhibit embryo growth. Key words: Embryogenesis, ABA, Seeds, Soybean.     

Relationship between the Stability of Antenna Chl {alpha}-Binding Proteins CP43/47 and the Accumulation of the Extrinsic 30-kDa Protein in PS II in Euglena

The accumulation of the extrinsic 30-kDa protein of PS II inthylakoid membranes was studied in relation to the assemblyof PS II antenna Chl-proteins CP43/47 and light-harvesting Chia/b-protein (LHC) in greening cells of Euglena. We showed previouslythat the 30-kDa protein accumulates in the membrane later thanthe reaction center-binding Dl protein (Mizobuchi and Yamamoto1989). In the present study, the rate of accumulation of theapoproteins of LHC and CP43/47 was shown to be similar to thatof the Dl protein. By contrast, we found by non-denaturing lithiumdodecyl sulfate-polyacrylamide gel electrophoresis that, atthe early stages of greening, newly assembled antenna Chi a-bindingproteins CP43 and CP47 were more susceptible to detergent thanwere the other Chl-protein complexes. Prolonged illuminationwas required for the stabilization of CP43 and CP47, and thetime course of the light-dependent formation of stable CP43/47paralleled that of the accumulation of the extrinsic 30-kDaprotein. These results are discussed in terms of the relationshipbetween the stabilization of CP43/47 and the accumulation ofthe 30-kDa protein in the thylakoid membrane and the roles ofthese two processes in the assembly of the oxygen-evolving systemin Euglena are examined. (Received February 18, 1991; Accepted June 17, 1991)