The Potassium Relations of Lemna minor L.: II. THE MECHANISM OF POTASSIUM UPTAKE

The experiments described in this paper concern the nature ofthe K⁺-influx mechanism in Lemna minor L. It is establishedthat K⁺ influx is ‘site restricted’ and that theaffinity of the system involved is in close agreement with thatof the well-documented System I mechanisms that are found inmany plants (Epstein, 1972). Experiments with ATP and CCCP suggestthat K⁺ influx is an active process, that the influx machineryresides at the plasmalemma and that this machinery containsan ATPase activity. Membrane vesicles isolated from the plantsabsorb K⁺(Rb⁺) with an affinity comparable to that of the systeminvolved in physiological K⁺ influx.     

Amino Acid Metabolism of Lemna minor L. : I. Responses to Methionine Sulfoximine

When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with [¹⁵N]H₄⁺ prior to supplying the inhibitor. Analyses of the ¹⁵N abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [¹⁵N]H₄⁺, are ¹⁴N enriched and are not apparently derived from ¹⁵N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in vivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.     

The Regulation of Nitrite Reductase Level in Lemna minor L.

The regulation of nitrite reductase in Lemna minor has beenstudied. The evidence indicates that in nitrate-fed plants nitrateitself is the inducer of nitrite reductase. The enzyme is subjectto end-product repression by ammonia and various amino acids.Nitrate reductase is also repressed by a similar range of compounds.Most of the repressors tested are more effective when nitraterather than nitrite is supplied as the inducer. The effectsof cyclo-heximide, D-threo-chloramphenicol and lincomycin onthe induction by nitrate and nitrite suggest that both enzymesare synthesized on cytoplasmic ribosomes. The mechanism of repressionby ammonia and amino acids is discussed.     

The control of glutamine synthetase level in Lemna minor L.

Summary The specific activity of glutamine synthetase (E.C. 6.3.1.2) of Lemna minor L. is markedly reduced when either ammonium ions or glutamine are present in the growth medium. Combinations of 5 mM ammonia and 5 mM glutamic acid or 5 mM ammonia and 5 mM glutamine as nitrogen source, lead to a 4–5 fold reduction of the maximum activity measurable on 5 mM -aminobutyric acid. Analyses of the soluble pool of nitrogen indicate that the reduction in enzyme level is associated with an increase in the pool of glutamine. There is an inverse correlation between the apparent rate of synthesis of glutamine synthetase and the intracellular concentration of glutamine, and this relationship suggests that the glutamine synthetase of Lemna minor is subject to end product repression by the endogenous pool of glutamine.     

Phytoremediation potential of Lemna minor L. for heavy metals

Phytoremediation potential of L. minor for cadmium (Cd), copper (Cu), lead (Pb), and nickel (Ni) from two different types of effluent in raw form was evaluated in a glass house experiment using hydroponic studies for a period of 31 days. Heavy metals concentration in water and plant sample was analyzed at 3, 10, 17, 24, and 31 day. Removal efficiency, metal uptake and bio-concentration factor were also calculated. Effluents were initially analyzed for physical, chemical and microbiological parameters and results indicated that municipal effluent (ME) was highly contaminated in terms of nutrient and organic load than sewage mixed industrial effluent (SMIE). Results confirmed the accumulation of heavy metals within plant and subsequent decrease in the effluents. Removal efficiency was greater than 80% for all metals and maximum removal was observed for nickel (99%) from SMIE. Accumulation and uptake of lead in dry biomass was significantly higher than other metals. Bio-concentration factors were less than 1000 and maximum BCFs were found for copper (558) and lead (523.1) indicated that plant is a moderate accumulator of both metals. Overall, L. minor showed better performance from SMIE and was more effective in extracting lead than other metals.     

Amino Acid Metabolism of Lemna minor L. : II. Responses to Chlorsulfuron

Chlorsulfuron, an inhibitor of acetolactate synthase (EC 4.1.3.18) (TB Ray 1984 Plant Physiol 75: 827-831), markedly inhibited the growth of Lemna minor at concentrations of 10⁻⁸ molar and above, but had no inhibitory effects on growth at 10⁻⁹ molar. At growth inhibitory concentrations, chlorsulfuron caused a pronounced increase in total free amino acid levels within 24 hours. Valine, leucine, and isoleucine, however, became smaller percentages of the total free amino acid pool as the concentration of chlorsulfuron was increased. At concentrations of chlorsulfuron of 10⁻⁸ molar and above, a new amino acid was accumulated in the free pool. This amino acid was identified as α-amino-n-butyrate by chemical ionization and electron impact gas chromatography-mass spectrometry. The amount of α-amino-n-butyrate increased from undetectable levels in untreated plants, to as high as 840 nanomoles per gram fresh weight (2.44% of the total free pool) in plants treated with 10⁻⁴ molar chlorsulfuron for 24 hours. The accumulation of this amino acid was completely inhibited by methionine sulfoximine. Chlorsulfuron did not inhibit the methionine sulfoximine induced accumulations of valine, leucine, and isoleucine, supporting the idea that the accumulation of the branched-chain amino acids in methionine sulfoximine treated plants is the result of protein turnover rather than enhanced synthesis. Protein turnover may be primarily responsible for the failure to achieve complete depletion of valine, leucine, and isoleucine even at concentrations of chlorsulfuron some 10⁴ times greater than that required to inhibit growth. Tracer studies with ¹⁵N demonstrate that chlorsulfuron inhibits the incorporation of ¹⁵N into valine, leucine, and isoleucine. The α-amino-n-butyrate accumulated in the presence of chlorsulfuron and [¹⁵N]H₄⁺ was heavily labeled with ¹⁵N at early time points and appeared to be derived by transamination from a rapidly labeled amino acid such as glutamate or alanine. We propose that chlorsulfuron inhibition of acetolactate synthase may lead to accumulation of 2-oxobutyrate in the isoleucine branch of the pathway, and transamination of 2-oxobutyrate to α-amino-n-butyrate by a constitutive transaminase utilizing either glutamate or alanine as α-amino-N donors.     

Amino Acid Metabolism of Lemna minor L. : III. Responses to Aminooxyacetate

Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by amino-oxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of α-amino-n-butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate.     

Influence of Nitrogen Availability on Aminotransferases in Lemna minor L.

Protein contents and glutamate: glyoxylate, serine: glyoxylate,alanine: glyoxylate and glutamate: pyruvate aminotransferaseactivities per gram fresh weight declined sharply when Lemnaminor L., previously grown on nitrate medium, was starved ofnitrogen. Nitrogen replenishment after 5 d caused complete recoveryof these parameters with higher values in ammonium-fed thannitrate-fed plants 7 d after transfer of plants from nitrogen-freemedium. Glutamate: glyoxylate and alanine: glyoxylate aminotransferasespecific activities (based on total extracted protein) showedlittle change with nitrogen availability. Serine: glyoxylateaminotransferase increased slowly during nitrogen starvationand decreased following nitrogen replenishment whether withammonium or nitrate. After 1 d of nitrogen starvation the specificactivity of glutamate: pyruvate aminotransferase declined; itincreased following nitrogen replenishment and ammonium gaverise to agreater activity than nitrate. The results are discussed in relation to the differences instability of the various enzymes relative to the overall proteinturnover rate. Key words: Aminotransferases, Nitrogen source, Photorespiration     

Some characteristics of nitrate reductase induction in Lemna minor L.

Summary Low levels of nitrate reductase can be detected in plants of Lemna minor grown on some organic nitrogen sources. Nitrogen-starvation does not lead to a derepression of nitrate reductase activity. Nitrate ions are necessary for the development of maximum enzyme activity and the maintenance of high enzyme levels. Nitrogen-starvation of ammonia-grown plants increases the subsequent rate of nitrate-mediated induction. It is suggested that ammonium ions, either directly or indirectly modulate the rate of nitrate reductase induction. The pattern of control regulating nitrate reductase levels in Lemna is contrasted with that in some species of algae.     

Purification and properties of glycollate oxidase from Lemna minor L.

• 1.1. Glycollate oxidase has been purified to apparent homogeneity from Lemna minor L. grown on medium containing 7mM NO⁻₃.
• 2.2. The enzyme is a highly basic protein with a sub-unit molecular weight of 42,000 and a holoprotein molecular weight of 250,000.
• 3.3. The Lemna enzyme is a flavoprotein with a broad specificity for straight chain α-hydroxy acids, the preferred substrate being glycollate.
• 4.4. It is also competitively inhibited by oxalate and phenyllactate.
• 5.5. A comparison is drawn between the physical properties of glycollate oxidase from a number of higher plants and the degree of sub-unit aggregation in the resulting protomers.

     

The control of aldolase in Lemna minor L. in relation to nitrogen deficiency

The loss of activity of aldolase which occurs when Lemna is deprived of nitrogen is shown to be due to the accumulation of a specific inhibitor of aldolase. The inhibitor has been purified 600-fold and has the properties of a low molecular weight protein. The inhibitor is not a proteolytic enzyme and the kinetics of the interaction between aldolase and the inhibitor are reported. The possible physiolgocal significance of the inhibition of aldolase is briefly discussed.     

Titration of Isolated Cell Walls of Lemna minor L

A theoretical model has been built to bypass the equation of titration of the cell wall. This equation, which is an extension of the Henderson-Hasselbach equation, underlines the importance of the exchange constant, the ionic strength as well as the rate of neutralization. The model is restricted to the case when the ionization degree is equal to the neutralization degree. The shape of the titration curve is shown to be strongly dependent on the valency of the base used.     

Isolation of Polysaccharides from the Callus Culture of Lemna minor L.

Two fractions that included acid arabinogalactan and pectin were extracted from the callus culture of duckweed plants (Lemna minorL.) with water and ammonium oxalate. Residues of galactose and arabinose (ratio, (2.0–2.5) : 1) were the major constituents of acid arabinogalactan. The pectin fraction contained primarily residues of glycuronic acids, galactose, and arabinose. The percentages of arabinogalactan and pectin were similar. The yield of polysaccharide fractions did not depend on the method used for their isolation. Extraction with water, treatment of the biomass with aqueous formalin and dilute hydrochloric acid, and extraction with aqueous ammonium oxalate allowed us to obtain the pectin polysaccharide with the highest purity.