Regulation of Sulfate Assimilation by Nitrogen Nutrition in the Duckweed Lemna minor L

The effect of nitrate and ammonium on the extractable activity of two enzymes of assimilatory sulfate reduction, ATP sulfurylase (EC 2.7.7.4) and adenosine 5′-phosphosulfate sulfotransferase (APSSTase), was examined in Lemna minor L. cultivated under steady state conditions. Nitrate reductase (EC 1.6.6.1) was measured for comparison. Low nitrate concentrations (0.2 and 0.04 millimolar) caused a decrease in the specific activity of all three enzymes measured. Twenty-four hours after transfer to medium without a nitrogen source, the specific activity of APSSTase and nitrate reductase was at less than 30% of the original level, whereas ATP sulfurylase was still at about 80%. NH₄⁺ added to the nutrient solution caused a 50 to 100% increase in the specific activity of APSSTase within 24 hours, followed by a slow decrease. After 72 hours with NH₄⁺, the specific activity was still 25% higher than originally. During the same period, the extractable protein increased by 30% on a fresh weight basis, and total protein by 55 to 60%. Nitrate reductase activity decreased to less than 5%. After omission of NH₄⁺ from the nutrient solution extractable APSSTase activity rapidly decreased to the level of cultures with NO₃⁻ as a nitrogen source. Using [³⁵S]SO₄²⁻ as a sulfur source, an increased incorporation of label into the protein fraction could be detected when NH₄⁺ was added to the nutrient solution. This indicated that more sulfate was assimilated and used for protein synthesis. The higher extractable activity of APSSTase with NH₄⁺ may be a regulatory mechanism involved in the formation of sufficient sulfur amino acids during a period of increased protein synthesis.     

Regulation of Sulfate Assimilation in Plants: 7. Cysteine Inactivation of Adenosine 5'-Phosphosulfate Sulfotransferase in Lemna minor L

When 0.5 mm cysteine is added to cultures of Lemna minor L. growing with sulfate as the sole sulfur source, there is a rapid 80% loss of extractable adenosine 5′-phosphosulfate sulfotransferase. This loss is accompanied by an inhibition of sulfate uptake; however, lack of sulfate is not responsible for the decreasing adenosine 5′-phosphosulfate sulfotransferase activity.     

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.     

Regulation of root growth by gibberellin in Lemna minor

Hormonal control of root growth was studied in Lemna minor. Although addition of gibberellic acid (GA3) to the culture medium did not promote the root growth, a gibberellin biosynthesis inhibitor, uniconazole P (Un-P), significantly inhibited root growth. Both length and diameter of roots in Un-P-treated plants were significantly smaller than those in control plants, mainly caused by inhibition of cell division. In epidermal cells, the length was slightly decreased and the width increased by Un-P treatment, indicating inhibition of elongation growth. GA3 completely nullified the inhibition caused by Un-P. Transverse cortical microtubules (CMTs) of epidermal cells in the elongation zone were significantly fragmented by treatment with Un-P, but not by that in the presence of GA3. The cellulose microfibril array in the Un-P-treated cells was more random and more oblique than that in the control cells. These results suggested that root growth in L. minor is regulated by endogenous gibberellin.     

Nitrogen Metabolism of Lemna minor. II. Enzymes of Nitrate Assimilation and Some Aspects of Their Regulation

In L. minor grown in sterile culture, the primary enzymes of nitrate assimilation, nitrate reductase (NR), nitrite reductase (NiR) and glutamate dehydrogenase (GDH) change in response to nitrogen source. NR and NiR levels are low when grown on amino acids (hydrolyzed casein) or ammonia; both enzymes are rapidly induced on addition of nitrate, while addition of nitrite induces NiR only. Ammonia represses the nitrate induced synthesis of both NR and NiR.NADH dependent GDH activity is low when grown on amino acids and high when grown on nitrate or ammonia, but the activities of NADPH dependent GDH and Alanine dehydro-genase (AIDH) are much less affected by nitrogen source. NADH-GDH and AIDH are induced by ammonia, and it is suggested that these enzymes are involved in primary nitrogen assimilation.     

Regulation of elongation growth by gibberellin in root segments of Lemna minor

Hormonal control of elongation growth was analyzed in segments excised from the elongation zone of Lemna roots. Exogenous GA3 did not promote the segment elongation but rather inhibited it. Uniconazole-P, a gibberellin biosynthesis inhibitor, significantly inhibited the segment elongation, and the inhibitory effect was completely nullified by GA3. In the epidermis, cell elongation was inhibited, but lateral cell expansion was not affected by uniconazole-P. Orientation of cortical microtubules of epidermal cells was disturbed by treatment with uniconazole-P for 12 h, and the disorganization of cortical microtubules was ameliorated by GA3. These findings suggested that disorganization of cortical microtubules induced inhibition of elongation growth of root. However, stabilization of cortical microtubules by taxol, a microtubule-stabilizing agent, did not affect the inhibition of segment elongation by uniconazole-P. These results suggested that endogenous gibberellin controls the elongation growth of root by regulating cell elongation.     

Aspartokinase in Lemna minor L: Studies on the in Vivo and in Vitro Regulation of the Enzyme

Aspartokinase has been isolated from wheat germ and a preliminary survey made of its properties in a partially purified extract. The enzyme has an absolute requirement for ATP and a divalent metal ion. The phosphate donor can be either ATP or GTP, but other nucleotides are ineffective. Both magnesium and manganese will activate the enzyme, whereas calcium shows a trace amount of activity. The enzyme has a Km of 16.7 mm for aspartate, 1.2 mm for ATP, and 3.3 mm for MgCl₂. Lysine inhibits the reaction at fairly low concentrations, and threonine inhibits at high concentrations. Other amino acids which are derived from aspartate (methionine, homoserine, threonine, and isoleucine) have little effect. When lysine and threonine are added together, they show a concerted inhibition of the reaction. The enzyme is also stabilized against heat inactivation by lysine and threonine together but not by either when added separately. It is suggested that aspartokinase from plants is a regulatory enzyme and exhibits a concerted feedback mechanism.     

Sulfate Uptake and Its Regulation in Lemna paucicostata Hegelm. 6746

The steady state concentrations of S-containing compounds formed in Lemna paucicostata Hegelm. 6746 in response to variations in source and concentrations of sulfur were measured. Neither growth rates nor protein accumulation were markedly affected by the various growth conditions. Ignoring complications due to possible compartmentation, the results are consistent with internal pools of both SO(4) (2-) and cyst(e)ine (or products of their metabolism), but not methionine, being effectors of regulation of high affinity SO(4) (2-) uptake. As SO(4) (2-) in the growth medium was increased to 10 mm, down-regulation of high affinity SO(4) (2-) uptake was more than compensated for by unregulated uptake via the "non-saturating" uptake system. Tissue inorganic SO(4) (2-) accumulated but formation of reduced sulfur remained constant. Some conversion of l-cystine sulfur to SO(4) (2-) occurred. Presence of l-cystine in the medium (a) down-regulated high affinity SO(4) (2-) uptake and (b) decreased the rate of SO(4) (2-) organification. The net results were decreased (7 mum l-cystine) or normal (14 mum l-cystine) total tissue SO(4) (2-) and dose-dependent accumulation of soluble cyst(e)ine and glutathione, but not of soluble methionine. l-Methionine was not metabolized to cyst(e)ine or its products. Presence of l-methionine in the medium led to increased total tissue sulfur, accounted for almost wholly by manyfold increases in soluble methionine, AdoMet, and S-methylmethionine sulfonium. Soluble cyst(e)ine increased slightly.     

Purification and properties of hydroxypyruvate reductase from Lemna minor L

• 1.1. Hydroxypyruvate reductase has been purified 193-fold from Lemna minor L. by affinity chromatography on Blue Sepharose.
• 2.2. The enzyme has activity over a broad pH range (optimum pH 6), a K_m hydroxypyruvate of 59 μ M and K_m NADH of 12μM.
• 3.3. Crude extracts of Lemna exhibit substrate inhibition of activity above 1 mM hydroxypyruvate, a property which is lost on purification.
• 4.4. Oxaloacetate inhibits purified preparations of the enzyme and a possible role for such regulation in vivo is 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.     

Regulation of Sulfate Assimilation in Plants : XIII. Assimilatory Sulfate Reduction during Ontogenesis of Primary Leaves of Phaseolus vulgaris L

The correlation between the extractable activities of three key enzymes of assimilatory sulfate reduction and the in vivo incorporation of ³⁵SO₄²⁻ into amino acids, proteins, and sulfolipids was investigated from greening to senescence in primary leaves of beans (Phaseolus vulgaris L.). The total extractable activity of ATP sulfurylase (EC 2.7.7.4) and of adenosine 5′-phosphosulfate sulfotransferase reached a maximum in the leaves of approximately 7- and 11-day-old seedlings, respectively. During senescence, there was a decrease in both enzyme activities. After approximately 17 days, no appreciable activities remained. In contrast, total O-acetyl-l-serine sulfhydrylase (EC 4.3.99.8) activity decreased to only approximately 50% of the maximal value during the same period. The in vivo incorporation of ³⁵SO₄²⁻ into amino acid and protein fractions showed a time-course similar to that of the total extractable adenosine 5′-phosphosulfate sulfotransferase activity. Both cysteine and sulfate markedly decreased during senescence. The total extractable activity of ribulosebisphosphate carboxylase (EC 4.1.1.39) was maximal in the primary leaves of 13-day-old seedlings, and approximately 40% of this value was still detectable after 17 days. Taken together with results from the literature, these results show that assimilatory sulfate reduction in primary leaves of P. vulgaris L. stops before CO₂ and nitrate assimilation.     

Exchange Properties of Isolated Cell Walls of Lemna minor L

From our theoretical treatment which is an extension of the classical Donnan theory, we have estimated the rational selectivity coefficients of the carboxylic groups of the walls during exchanges of divalent ions against monovalent ones (i.e. calcium and potassium or calcium and sodium ions). These coefficients express the interactions between the different ions, those between the counter-ions and the ionized groups of the wall, and the influence of water. These quantitative values are consistent with the great affinity of the carboxylic groups for the calcium ions. They vary with the experimental conditions, showing a purely physicochemical mechanism of “regulation” of the exchanges in the cells walls of Lemna minor L.     

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     

Isolation of polysaccharides from callus culture of Lemna minor L

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

Genetic transformation of duckweed Lemna gibba and Lemna minor

Summary We developed efficient genetic transformation protocols for two species of duckweed, Lemna gibba (G3) and Lemna minor (8627 and 8744), using Agrobacterium-mediated gene transfer. Partially differentiated nodules were co-cultivated with Agrobacterium tumefaciens harboring a binary vector containing β-glucuronidase and nptII expression cassettes. Transformed cells were selected and allowed to grow into nodules in the presence of kanamycin. Transgenic duckweed fronds were regenerated from selected nodules. We demonstrated that transgenic duckweed could be regenerated within 3 mo. after Agrobacterium-mediated transformation of nodules. Furthermore, we developed a method for transforming L. minor 8627 in 6 wk. These transformation protocols will facilitate genetic engineering of duckweed, ideal plants for bioremediation and large-scale industrial production of biomass and recombinant proteins.