Influence of Complexation on the Uptake by Plants of Iron, Manganese, Copper and Zinc: I. EFFECT OF EDTA IN A MULTI-METAL AND COMPUTER SIMULATION STUDY

After growing barley (Hordeum vulgare L.) in nutrient solutionscontaining EDTA, uptake of the nutrient metals was determinedat three harvests and concentrations of the various chemicalspecies of each metal in the growth solutions was modelled bycomputer simulation. Complexation with EDTA had different effectson the uptake of the ions Fe³⁺, Mn²⁺, Cu²⁺, and Zn²⁺. At thehighest EDTA level (EDTA/Fe=2/l) the plants were chlorotic andgrowth was inhibited. This is attributed to a deficiency inZn rather than in Fe. The critical level of free Zn²⁺ requiredin nutrient solutions for healthy growth was found to be approximately10^–1010^–10 mol dm^–3, which is consistent withthat found by earlier workers for other plant species. Barleytolerated much lower levels of the free ions of copper and ironwithout exhibiting any obvious adverse effects. Key words: EDTA, micronutrients, trace metals, computer simulation, deficiencies, absorption, iron, manganese, copper, zinc     

Effects of Chloramphenicol on the Uptake and Incorporation of Amino-acids by Carrot Root Tissue

The uptake of uniformly labelled L-glutamic acid-¹⁴C, glycine-¹⁴C,and L-proline-¹⁴C by carrot root tissue is inhibited by chloramphenicolat a concentration of 2 g./l. L-glutamic acid absorption isaffected to a much greater extent than is the uptake of theother two amino-acids. It is shown that the ¹⁴C from the labelledamino-acids is incorporated into protein of the carrot slices,and that the amount of incorporation from glycine and L-prolineis about six times as large as that from L-glutamic acid undercomparable conditions. Almost all the ¹⁴C detected in the proteinafter feeding labelled L-proline or glycine remains in the amino-acidsupplied; in the case of L-glutamic acid some of the label istransferred to other amino-acid residues, but most of it remainsin glutamic acid. Chloramphenicol is found to inhibit ¹⁴C-incorporationfrom L-proline and glycine in short-term, but not in long-term,experiments whereas incorporation from L-glutamic acid is inhibitedin both long- and short-term experiments. Chloramphenicol alsoinhibits incorporation of ¹⁴C from L-glutamic acid into proteinsof preparations of isolated subcellular particles from carrotroot tissue. Under some conditions, ¹⁴C incorporation into proteinproceeds for some time after the slices have been removed fromthe radioactive solution. Such incorporation is not inhibitedsignificantly by chloramphenicol when ¹⁴C-labelled L-prolineis supplied, but it is reduced by about 50 per cent. when glycineand L-glutamic acid are used. It is shown that chloramphenicolinhibits net protein synthesis in carrot root slices suspendedin aerated solutions. It is concluded that amino-acid incorporationinto the protein of carrot slices proceeds by at least two mechanisms,one of which is related to protein synthesis and is sensitiveto chloramphenicol and the other is unrelated to protein synthesisand insensitive to the drug. Most of the L-proline and glycineincorporation into carrot root tissue protein seems to occurby the chloramphenicol-insensitive mechanism while much of theL-glutamic acid-¹⁴C incorporation seems to take place by thechloramphenicol-sensitive one.     

Long-term Partitioning, Storage and Re-mobilization of 14C Assimilated by Lolium perenne (cv. Melle)

The youngest fully expanded leaves of single tillers of vegetativeperennial ryegrass plants were exposed to ¹⁴CO₂. Thereafter,quantitative and fractional analysis of the partitioning, storageand re-mobilization after defoliation of the ¹⁴C-labelled assimilatewas sequentially conducted over a 22 d period. In undefoliated plants, most ¹⁴C reached its final destinationwithin 5–6 of feeding. Forty per cent of assimilated ¹⁴Cwas subsequently lost through respiration, while 13.5, 8.5 and34 per cent remained in roots, stem bases and tops respectively.At least some ¹⁴C was distributed to tillers throughout theplant, but secondary tillers subtended by the fed tiller madethe greatest demand on ¹⁴C translocated from the fed tiller. A small, but significant portion of ¹⁴C was invested into longterm storage in undefoliated plants, four per cent of the totalassimilated still being present in a labile chemical form inroots and stem bases 22 d after feeding. In plants that wereseverely defoliated 4 d after feeding, depletion of reserve¹⁴C was observed relative to undefoliated plants. The depletiontook place from stem bases, not roots, and both low and highmolecular weight storage compounds were involved. A portionof the depleted ¹⁴C was incorporated into new growth after defoliation. Lolium perenne, perennial ryegrass, assimilate partitioning, storage, re-mobilization, defoliation     

木荷属和柃木属植物的嘌呤代谢及嘌呤碱合成研究

以［8-¹⁴C］标记的腺嘌呤和黄嘌呤为底物,对两种可以合成少量咖啡碱和茶叶碱的木荷属和柃木属植物（Schima mertensiana,Eurya japonica）叶片的嘌呤代谢进行了检测研究。发现木荷属和柃木属植物中嘌呤代谢相似,¹⁴C标记的腺嘌呤可以整合到嘌呤核苷酸、RNA、酰脲（包括尿囊素和尿囊酸）、二氧化碳中。经过24 h培养,在叶片吸收的放射能中,仅有6%～7%用于甲基黄嘌呤类化合物的合成（3-甲基黄嘌呤、7-甲基黄嘌呤核苷、7-甲基黄嘌呤、茶叶碱）。和其他植物一样,绝大多数¹⁴C标记的黄嘌呤整合到嘌呤的分解代谢物中（二氧化碳和酰脲）,少量的放射能分布在3-甲基黄嘌呤及茶叶碱中。根据结果可以推断木荷属和柃木属植物具有N-甲基转移酶活性,可以用来合成咖啡碱和茶叶碱,相对于茶树而言,活性不高。综上,本文对木荷属和柃木属植物的嘌呤代谢以及嘌呤碱合成进行了研究。     

Absorption of Some Amino Acids by Sporophytes Isolated from Polytrichum formosum and Ultrastructural Characteristics of the Haustorium Transfer Cells

The absorption of ¹⁴C-labelled amino acids (glycine, threonineand -aminoisobutyric acid) by the isolated sporophyte of Polytrichumformosum takes place mainly in the haustorium. The isolationof the sporophyte does not alter the absorption capacity ofits haustorium nor its ultrastructure, in particular that ofits peripheral transfer cells. amino acids, transfer cells, sporophyte, Polytrichum formosum, haustorium     

The Strategy of Carbon Utilization in Uniculm Barley: II. THE EFFECT OF CONTINUOUS LIGHT AND CONTINUOUS DARK TREATMENTS

After a photoperiod of 8.25 h during which the youngest fullyexpanded leaf of uniculm barley plants was allowed to assimilate¹⁴CO₂ for 30 min, groups of plants were transfered either tocontinuous light or to continuous dark. Plants were harvestedover a 72 h period to examine the effect of the treatments (comparedwith control plants growing in normal light/dark cycles) onthe transport of ¹⁴C from the exposed leaf, the distributionof ¹⁴C assimilates to the rest of the plant, and the chemicalfate of assimilated ¹⁴C. In continuous light a substantial quantity (22% at 72 h) ofthe ¹⁴C assimilated by the leaf remained in that leaf in theform of starch and neutral sugars compared with only 4% in thecontrol fed leaf. Also the total amount of ¹⁴C respired fromplants maintained in continuous light was significantly less(c. 18% of the total originally fixed by 24 h) than that respiredfrom control plants (c. 36%). The result was that approximatelyequal amounts of ¹⁴C were accumulated in the growing leavesand roots of plants given continuous light or normal light/darkcycles. In continuous dark the fate of ¹⁴C was similar to that of controlplants. This is probably because the two treatments shared acommon light/dark environment for the first 22 h, during whichtime almost complete distribution and utilization of ¹⁴C occurred.     

Genetic evidence that induction of root Fe(III) chelate reductase activity is necessary for iron uptake under iron deficiency†

Reduction of Fe(III) to Fe(II) by Fe(III) chelate reductase is thought to be an obligatory step in iron uptake as well as the primary factor in making iron available for absorption by all plants except grasses. Fe(III) chelate reductase has also been suggested to play a more general role in the regulation of cation absorption. In order to experimentally address the importance of Fe(III) chelate reductase activity in the mineral nutrition of plants, three Arabidopsis thaliana mutants (frd1-1, frd1-2 and frd1-3), that do not show induction of Fe(III) chelate reductase activity under iron-deficient growth conditions, have been isolated and characterized. These mutants are still capable of acidifying the rhizosphere under iron-deficiency and accumulate more Zn and Mn in their shoots relative to wild-type plants regardless of iron status. frd1 mutants do not translocate radiolabeled iron to the shoots when roots are presented with a tightly chelated form of Fe(III). These results: (1) confirm that iron must be reduced before it can be transported, (2) show that Fe(III) reduction can be uncoupled from proton release, the other major iron-deficiency response, and (3) demonstrate that Fe(III) chelate reductase activity per se is not necessarily responsible for accumulation of cations previously observed in pea and tomato mutants with constitutively high levels of Fe(III) chelate reductase activity.     

The Effect of Direct Electric Current on Transport of Chloride Ions, Sucrose and Assimilates in Chenopodium rubrum

Treatment with DC (6 µA plant^-1) increased uptake andtranslocation of ³⁶C1^- ions, both to the leaves and to the stemsof 15-d-old Chenopodium rubrum plants. In leaves the distributionof these ions remained unchanged in comparison with untreatedcontrols, however, in stems a decreasing gradient from the shootapex to the base of both ions was formed as a consequence ofDC treatment. Neither uptake nor distribution of ^l4C-sucroseand ¹⁴C-labelled assimilates were affected by DC treatment. (Received April 10, 1995; Accepted September 18, 1995)     

The Distribution and Identity of Assimilates in Tomato with Special Reference to Stem Reserves

Much of the work on the distribution of ¹⁴C-labelled assimilatesin tomato has been done in winter under low light intensities,and consequently the reported distribution patterns of ¹⁴C maynot be representative of plants growing in high light. Further,there are several somewhat conflicting reports on patterns ofdistribution of ¹⁴C-assimilates in young tomato plants. We soughtto clarify the situation by studying the distribution of ¹⁴C-assimilatesin tomato plants of various ages grown in summer when the lightintensity was high. In addition, the role of the stem as a storageorgan for carbon was assessed by (a) identifying the chemicalfractions in the stem internode below a fed leaf and monitoring¹⁴ C activity in these fractions over a period of 49 d, and(b) measuring concentrations of unlabelled carbohydrates inthe stem over the life of the plant. The patterns of distribution of ¹⁴C-assimilates we found fortomato grown under high light intensity confirmed some of thosedescribed for plants grown under low light, but export of ¹⁴Cby fed leaves was generally higher than reported for much ofthe earlier work. Lower leaves of young plants exported over50% of the ¹⁴C they fixed, although export fell sharply as theplants aged. Initially, the roots and apical tuft were strongsinks for assimilates, but they had declined in importance bythe time plants reached the nine-leaf stage. On the other hand,the stem became progressively more important as a sink for ¹⁴C-assimilates.Older, lower leaves exported more of their ¹⁴C-assimilates tothe upper part of the plant than to the roots, whereas youngleaves near the top of the plant exported more of their assimilatesto the roots. The stem internode immediately below a fed leafhad about twice the ¹⁴C activity of the internode above theleaf. Mature leaves above and below a fed leaf rarely importedmuch ¹⁴C, even when in the correct phyllotactic relationshipto the fed leaf. In the first 3 d after feeding leaf 5 of nine-leaf plants, theorganic and amino acid pools and the neutral fraction of theinternode below the fed leaf had most of the ¹⁴C activity, butby 49 d after feeding, the ethanolic-insoluble, starch and lipidfractions had most of the ¹⁴C activity. Glucose, fructose andsucrose were the main sugars in the stem. Although concentrationsof these sugars and starch declined in the stem as the plantsmatured, there was little evidence to indicate their use infruit production. Stems of plants defoliated at the 44-leafstage had lower concentrations of sugars and starch at maturity,and produced less fruit than the controls. It was concludedthat tomato is sink rather than source limited with respectto carbon assimilates, and that the storage of carbon in thestem for a long period is possibly a residual perennial traitin tomato.Copyright 1994, 1999 Academic Press Lycopersicon esculentum, tomato, assimilate distribution, ¹⁴C, internode storage, sink-source relationships, starch, stem reserves, sugars     

Short-Term Fixation of 14Carbon by the Submerged Aquatic Angiosperm Potamogeton pectinatus

In ¹⁴C fixation experiments, 3-phosphoglyceric acid was thefirst product of carbon assimilation in the light in Potamogetonpectinatus. The pattern of early ¹⁴C-labelled compounds wasthe same over a range of pH values of the medium from 3.5 to8.1. Rates of ¹⁴C incorporation declined with increasing pHof the medium indicating that free CO₂ is the major exogenoussource of carbon for photosynthesis in Potamogeton pectinatus.     

EDTA-Facilitated Phytoremediation of Soil with Heavy Metals from Sewage Sludge

The objective of this research was to determine the effect of the chelate EDTA (ethylenediaminetetraacetic acid), which is used in phytoremediation, on plant availability of heavy metals in liquid sewage sludge applied to soil. Sunflower (Helianthus annuus L.) was grown under greenhouse conditions in a commercial potting soil; the tetrasodium salt of EDTA (EDTA Na₄) was added at a rate of 1 g kg^-1 to half the pots. Immediately after seeds were planted, half of the pots with each soil (with or without EDTA) were irrigated with 60 ml sludge, and half were irrigated with 60 ml tap water. For the subsequent five irrigations, plants in soil with EDTA received either sludge or tap water containing 0.5 g EDTA Na₄ per 1000 ml, and plants in soil without EDTA received sludge or tap water without EDTA. Of the four heavy metals whose extractable concentrations in the soil were measured (Cu, Fe, Mn, and Zn), only Zn had a higher concentration in sludge-treated soil with EDTA compared to sludge-treated soil without EDTA. The concentrations of Fe, Cu, and Mn were similar in sludge-treated soil with and without EDTA. Of the three heavy metals whose total concentrations in the soil were measured (Cd, Pb, Cr), Pb (<10 mg kg^-1) and Cd (< 1 mg kg^-1) were below detection limits, and Cr was unaffected by treatment. The concentration of all measured elements in plants (Cd, Cu, Fe, Zn, Pb) was higher than the concentrations measured in the soil. With no EDTA, sludge-treated plants had a higher concentration of the five heavy elements than plants grown without sludge. Cadmium was lower in sludge-treated plants with EDTA than plants with EDTA and no sludge. After treatment with EDTA, the concentrations of Cu, Fe, and Zn were similar in plants with and without sludge. Lead was higher in plants with EDTA than plants without EDTA, showing that EDTA can facilitate phytoremediation of soil with Pb from sewage sludge.     

Filter types, filtration and post-filtration treatment in phytoplankton production studies

Acid rinsing, used to decontaminate filters in ¹⁴C productionstudies, caused cell rupture and resulted in elevated ¹⁴C countsin the filtrates of six out of seven phytoplankton samples.Large volume (50 ml) rinses using only water caused some, butlesser, damage. Comparing the recovery of ¹⁴C-labelled cellsand chlorophyll a on glass-fibre, polycarbonate and celluloseester filters revealed unaccountable losses at times with allthree filter types. These losses could not be explained by cellrupture, attachment to the filter funnel wall, filter treatmentor self-absorption during scintillation counting. Compared tothe whole sample acid bubbling method, recovery on the glass-fibrefilters was highest. Results for the polycarbonate filters weremore variable, while, in all cases, recovery on cellulose esterfilters was much lower.     

Environmental Factors Affecting the Loss of Carbon from the Roots of Wheat and Barley Seedlings

The amounts of carbon released into soil from roots of wheatand barley seedlings grown under three environmental conditionsfor 3 weeks with shoots in constant specific activity ¹⁴CO₂are reported. This carbon loss was measured as respired ¹⁴CO₂from both the root and the accompanying microbial populationand as root derived ¹⁴C-labelled organic C compounds in thesoil. With a 16 h photoperiod, growth at 15 ?C constant or 18?C day/14 ?C night gave a loss of 33–40% of the totalnet fixed carbon (defined as ¹⁴C retained in the plant plus¹⁴C lost from the root). The proportion of ¹⁴C translocatedto the roots that was released into the soil did not changewith temperature, so carbon distribution within the plant musthave changed. With a 12 h photoperiod and a temperature regimeof 18 ?C/14 ?C carbon loss from the roots was decreased to 17–25%of the total fixed carbon. Key words: Cereals, Roots, Carbon loss     

Metabolism of Inorganic Carbon Taken Up by Roots in Salix Plants

The metabolic products of inorganic carbon taken up throughthe roots from nutrient solution were studied in willow plants.Willow cuttings (Salix cv. Aquatica gigantea) were suppliedwith unlabelled or ¹⁴C-labelled NaHC0₃ for 1, 5, 10, and 24h in light or in darkness. After feeding, the plants were dividedinto six samples (upper and lower leaves and corresponding stems,cuttings and roots), which were frozen in liquid N₂. Freeze-driedground samples were extracted into water-soluble, chloroform-solubleand insoluble fractions. The water-soluble fraction was furtherseparated into basic, acidic, and neutral fractions by ion-exchangechromatography. In the light experiment pronase treatment wasused to separate the insoluble fraction into proteins and insolublecarbohydrates. After I h feeding time, most of the ¹⁴C was fixed into organicacids and amino acids both in light and in darkness in all partsof the plants. In the roots a large part of the ^l4C-carbon wasincorporated into the protein and insoluble fractions alreadyduring short feeding times, and the amounts incorporated increasedwith time. In the leaves, after 1 and 5 h the main labelledcompounds were the organic acids and amino acids, but after10 h about half of the total ¹⁴C was in protein and in the insolublefraction. A further analysis of amino acids and organic acidswith HPLC showed that C-4 acids were labelled initially andthat over time the proportion of different acids changed. These results indicate that the metabolism of carbon in rootsmight take place via ß-carboxylation of PEP. Partof the fixed ¹⁴C is transported from the roots, probably asamino acids and organic acids, to the shoot. In roots the C-4acids are metabolized further into structural compounds (proteinsand insoluble carbohydrates). Key words: DIC, Salix, roots, metabolism, HPLC     

Factors related to iron absorption by enzymically isolated leaf cells

The rate of Fe absorption by cells enzymically isolated from tobacco leaves is correlated with the age of the leaves from which the cells are derived. The cells obtained from younger leaves absorb Fe more rapidly than those from older ones. Ca inhibits Fe and Mn absorption. Fe and Mn are mutually antagonistic in their absorption by leaf cells. Ca enhances the inhibition of Mn absorption by Fe, but reduces the inhibition of Fe absorption by Mn. The affinity constant for Fe absorption by leaf cells is low. The chelate EDDHA (ethylenediamine di(o-hydroxyphenylacetate) competitively inhibits Fe absorption.     

Metabolism of Caffeine and Related Purine Alkaloids in Leaves of Tea (Camellia sinensis L.)

Purine alkaloid catabolism pathways in young, mature and agedleaves of tea (Camellia sinensis L.) were investigated by incubatingleaf sections with ¹⁴C-labelled theobromine, caffeine, theophyllineand xanthine. Incorporation of label into CO₂ was determinedand methanol-soluble metabolites were analysed by high-performanceliquid chromatography-radiocounting and thin layer chro-matography.The data obtained demonstrate that theobromine is the immediateprecursor of caffeine, which accumulates in tea leaves becauseits conversion to theophylline is the rate limiting step inthe purine alkaloid catabolism pathway. The main fate of [8-¹⁴C]theophyllineincubated with mature and aged leaves, and to a lesser extentyoung leaves, is conversion to 3-methylxanthine and onto xanthinewhich is degraded to ¹⁴CO₂ via the purine catabolism pathway.However, with young leaves, sizable amounts of [8-¹⁴C]-theophyllinewere salvaged for the synthesis of caffeine via a 3-methylxanthine     

The Metabolism of Abscisic Acid

The light-catalysed isomerization of (+)-abscisic acid (ABA)to its trans isomer during isolation from leaves was monitoredby the addition of (±)-[2-¹⁴C]ABA to the extraction medium.(+)Trans-abscisic acid (t-ABA) was found to occur naturallyin rose (Rosa arvensis) leaves at 20µg/kg fresh weight;(+)-ABA was present at 594µg/kg. (±)-[2-¹⁴D]Trans-abscisicacid was not isomerized enzymically to ABA in tomato shoots. (±)-Abscisic acid was converted by tomato shoots to awater-soluble neutral product, ‘Metabolite B’, whichwas identified as abscisyl-ß-D-glucopyranoside. When(±)-[2-¹⁴C]trans-abscisic acid in an equimolar mixturewith (±)-[2-¹⁴C}ABA was fed to tomato shoots it was convertedto its glucose ester 10 times faster than was ABA. Trans-abscisyl-ß-D-glucopyrano8ide only was formedfrom (±)-[2-¹⁴C]t-ABA in experiments lasting up to 30h. Glucosyl abscisate was formed slowly from ABA and the freeacid fraction contained an excess of the unnatural (–).ABAas did the ABA released from abscisyl-ß-D-glucopyranosideby alkaline hydrolysis. The (+).ABA appeared to be the solesource of the acidic ‘Metabolite C" previously noted. The concentrations of endogenous (+)-, (+)-[2-¹⁴C]-, and (–)-[2-¹⁴C]ABAremaining as free acid, and also in the hydrolysate of abscisyl-ß-D-glucopyranoside,were measured by the ORD, UV absorption, and scintillation spectrometryof highly purified extracts of ABA from tomato shoots whichhad been supplied with racemic [2-^l4C]ABA.     

The synthesis of magnesium-protoporphyrin IX by etiochloroplast membrane preparations

Magnesium-protoporphyrin IX (or its monomethyl ester) is the first committed intermediate in the biosynthesis of chlorophyll in green plants. Membranes from lysed washed cucumber etiochloroplasts synthesized small amounts of ¹⁴C-labelled magnesium-protoporphyrin IX from [¹⁴C]protoporphyrin IX at the rate of 1–3 pmol/h per mg protein. Maximum activity in these membrane preparations was dependent upon added EDTA, GSH, ATP and MgCl₂. Activity was totally dependent upon added ATP, probably as the species MgATP^2? and there was also a requirement for Mg²⁺ in addition to that used to form the MgATP^2? complex.     

The Reciprocal Transfer of Radiocarbon between a Developing Tiller and its Parent Shoot in Vegetative Plants of Lolium multiflorum Lam.

The transfer of ¹⁴C-labelled assimilates between a tiller andits parent shoot was examined in young plants of Lolium multiflorumLam. Radiocarbon was exported freely from an expanded laminato sinks within the shoot axis from which it originated andto the root system. Lesser amounts of radiocarbon were exportedto the other shoot. It is suggested that the reciprocal exchangeof radiocarbon between tiller and main shoot occurred principallyvia a direct pathway through stem tissues rather than via apathway involving the roots.     

Studies on soluble RNA binding indoleacetic acid in etiolated mung bean hypocotyl sections

Nucleic acid was extracted by the SLS-phenol method from Phaseolusaureus hypocotyl treated with IAA-2-¹⁴C. Radioactivity in thenucleic acid fraction was found at the positions of sRNA andrRNA on an MAK column. IAA-¹⁴C was released from the radioactivecompound(s) in the sRNA fraction, by alkaline hydrolysis, butnot by ethanol extraction, or by dialysis to 2 M NaCl, 8 M urea,and 0.1 M EDTA. When the radioactive compound at the positionof sRNA on an MAK column was further re-chromatographed on aDEAE-cellulose column and on a BD-cellulose column, it was alwayslocalized only in a settled part of the fraction of each column.From this fraction IAA-¹⁴C was released by alkaline hydrolysis.Also, IAA-¹⁴C was released from the radioactive compound insRNA fraction, by RNase digestion, but not by pronase treatment.Results of these experiments suggest the existence of some kindsof sRNA binding IAA. The genesis of this sRNA binding IAA-¹⁴Cwas observed within 30 min after the supply of IAA-¹⁴C, andthe sRNA became saturated with IAA-¹⁴C about 2 hr after thebeginning of incubation. The behavior of sRNA binding IAA, representedby sRNA binding IAA-¹⁴C, may have a role in IAA induced growthof mung bean hypocotyl sections. (Received July 6, 1971; )