The effect of inoculation with <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> on the contents of cytoplasmic protein and free amino acids in the roots of pea seedlings

The changes in the contents of protein and free amino acids in pea plants inoculated with Rhizobium leguminosarum were studied taking into account the susceptibility of roots to root nodule bacteria. The content of cytoplasmic protein during infection increased in the actively growing root zone (0–5 mm) and decreased in the root zones susceptible to rhizobia (5–20 mm from the root tip). The quantitative composition of free amino acids changed essentially upon inoculation of pea seedlings with R. leguminosarum.     

Salinity Stress and the Content of Proline in Roots of Pisum sativum and Tamarix tetragyna

Amino acid composition of the free amino acid pool and the TCA-insolubleprotein fraction were investigated in root tips of pea and Tamarixtetragyna plants grown at various levels of NaCl salinity. Salinitystress induced an increase of proline content, mainly in thefree amino acid pool in both plants, and of proline or hydroxyprolinecontent in the protein. Externally-supplied proline was absorbedand incorporated into protein, by pea roots, more effectivelythan by Tamarix roots. Salinity stress, apparently, stimulatedthe metabolism of externally-supplied labelled proline. Pearoots have a very large pool of free glutamic acid; however,70 per cent of the ¹⁴C from externally-supplied ¹⁴C-U-glutamicacid was released as CO₂. Very small amounts of it were incorporatedinto protein. No measurable amount of radioactivity could bedetected in any one of the individual amino acids, either ofprotein hydrolysate or the free amino acid pool. Proline very effectively counteracted the inhibitory effectof NaCl on pea seed germination and root growth. A similar effectbut to a lesser degree was achieved with phenylalanine and asparticacid. The feasibility of proline being a cytoplasmic osmoticumis discussed.     

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic‐active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane‐localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element‐companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.     

Synthesis and Interconversion of Amino Acids in Developing Cotyledons of Pea (Pisum sativum L.)

Freshly isolated cotyledons from 10-day developing pea (Pisum sativum) seeds were fed radiolabeled precursors for 5 hours, and the specific radioactivity of the free and total protein amino acids was determined using a dansylation procedure. When the seven most abundant amino acids in phloem exudate of pea fruits (asparagine, serine, glutamine, homoserine, alanine, aspartate, glycine) were fed singly, their carbon was distributed widely among the aliphatic amino acids, proline and tryptophan; sporadic labeling of tyrosine and histidine also occurred. Feeding of glucose led to relatively greater labeling of aromatic amino acids including phenylalanine. The data support the involvement of known plant pathways in these interconversions. Labeling patterns were consistent with participation of the cyanoalanine pathway in the conversion of serine to homoserine, and with the synthesis of histidine from adenosine. All of the labeled amino acids were incorporated into protein.     

The Effect of Darkness on the Leghemoglobin Content and Amino Acid Levels in the Root Nodules of Pea Plants

The dependence of the nitrogen fixing system in the root nodules of pea plants (Pisum sativum) L. cv. Torsdag II) on light induced reactions was studied. The pots of the inoculated pea plants, after the nolules had fixed nitrogen for a fornight, were transferred to a dark room. The control plants were kept under normal lighting conditions. The decay of leghemoglobin was measured after photosynthesis had ceased. In the dark the red nodules turned green in three days, when about half of the haem had been broken down. The plants in normal lighting conditions had maintained the red nodules. The appearence of leghemoglobin and bacteroids was simultaneouos. In normal lighting conditions the number of bacteroids was about 1.6 × 10⁸ per g fresh nodules. The appearance of leghemoglobin and bacteroids was simultaneous. In normal lighting conditons the number of bacteroids was bout 1.6 × 10⁸ per g fresh nodules. At the same time as the nodules turned green in the dark most of the bacteroids disappeared and the number of rod-shaped bacteria increased. After five days int the dark thenumber of bacteria of the green nodules was 2.2 × 10⁸ per g fresh nodules. A large increase of of bacteria in the nodules is one of the results after the termination of effective symbiosis. Quantitative estimations were made with an automatic amino acid analysator of the amino acid composition in the root nodules of pea plants grown in the light and of pea plants grown in the dark. Altogether 27 amino acids and amides and 3 unknown ninhydrin positive compounds were found in the free amino acid fraction. In the red N-fixing nodules asparagine, the amide of aspartic acid, was the most prominent (more than 50 per cent of the total amino acid fraction), indicating the energy charge of the nitrogen fixation. 5 days in the dark affected the proportions of the amino acids as follows. Asparagine, homoserine, γ-aminobutyric acid and ethanolamine were decreased and the most of the others increased. In the hydrolysate of the non-soluble protein fraction 25 amino acids could be detected. The proportions of the amino acids in the root nodules of light-grown and dark-grown pea plants were very similar. Hydroxyproline and α, γ-diaminopimelic acid (DAP) were found in these fraction. Most of the DAP was contained in the peptide fraction. Also hydroxyproline was found to a small extent. It was assumed that the amino acids in this fraction were derived from the peptides of both plant cells and rhizobia.     

Dynamics of amino acids in pea stem sections during root formation and its inhibition by kinetin and ethionine

An investigation was conducted to study the interrelation of free amino acid metabolism and root formation in etiolated pea stem sections as dependent on time and on inhibition of root formation by kinetin and ethionine. The rise in the level of aspartic acid and increase in the rate of conversion of¹⁴C-labeled glucose to free amino acids were found to be characteristic features of the formation of foci of meristematic cells in pericyclo region. The formation of roots was reflected, in general, much more in the rate of conversion of labeled glucose to free amino acids than in the levels of corresponding amino acids. The total amount of free amino acids was not significantly changed during incubation of stem sections in a solution of kinetin (5×10^?5 m). A rapid fall in their level was recorded in the next 24 hours. The incorporation of¹⁴C from glucose into a precursor of lignin, phenylalanine, was completely inhibited by kinetin which stimulated simultanously the growth of adjacent buds. Stimulation of secondary xylem formation, which appeared later, was accompanied by the resumption of¹⁴C-incorporation into phenylalanine. Inhibition of root formation by ethionine resulted in the rapid fall of the level of most amino acids and in a significant decrease in the rate of incorporation of¹⁴C from glucose into amino acids. A decreasing level of ethionine in tissues during cultivation of ethionine-treated stem sections was accompanied by a gradual rise in the individual amino acids and in the rate of conversion of glucose into free amino acids.     

Dormancy in Seeds of Charlock (Sinapis arvensis L.): Early Effects of Gibberellic Acid on the Synthesis of Amino Acids and Proteins

Charlock (Sinapis arvensis L.) seeds were imbibed with 10 mm GA(3) for 24 hours at 0 C. After equilibration at 25 C, a 5-fold increase in radioactivity in the amino acids labeled from 2-(14)C-acetate was observed within 2 hours. The total amount of amino acids was reduced to half, and the specific radioactivity increased approximately 10-fold, indicating a diversion of metabolites for amino acid and protein synthesis in GA(3)-treated seeds. The rate of incorporation of l-(14) C-leucine into protein was doubled. Autoradiographs showed that enhancement of protein synthesis was localized in the shoot and root meristems, the developing vascular tissues, and in the endosperm cells inside the testa.     

Determination of specific radioactivity of plant amino acids using dansylation

A sensitive procedure is described for measuring the absolute specific activity of all the protein amino acids, both free and in total protein, as well as of some nonprotein amino acids. It is illustrated with developing pea cotyledons. Particular attention is paid to optimization of tlc loading and resolution, protection of cysteine, and stabilization of fluorescence of the eluted derivatives.     

Intervarietal Differences in the Amino Acid Composition of Pea Roots as Related to their Response to Salinity

The salt tolerance of three cultivars of Pisum sativum L. asdefined by root growth on saline substrate was in the orderDan > Alaska > Laxton Progress. The total content of freeamino acids decreased in Alaska, but not in Laxton Progress,with increasing salinity, while the content of the free basicamino acids increased with salinity. In both cultivars salinityinduced accumulation of free proline, but not enough to maintainthe osmotic adaptation of the cytoplasm. Proline accumulationcould not account for the greater tolerance of the Alaska cultivar.Both cultivars contain large amounts of glutamic acid and homoserine.The amino acid composition of protein of the Alaska cultivaris affected much more by salinity than is that of Laxton Progress.In Alaska salinity induces an increase in aspartic and glutamicacids, threonine, serine, leucine and isoleucine. The totalcontent of basic amino acids decreases with salinity. The proteinof Laxton Progress is very rich in leucine and its amount slightlydecreases with salinity. In the most tolerant cultivar, Dan,there is more proline in the protein than in the other two cultivarsand its content of leucine increases with salinity. The onlyfeatures which show some correlation with salinity toleranceare proline content (Dan > Alaska > Laxton Progress) andan increase in leucine content in the protein, with increasingsalinity. Pisum sativum L, pea, amino acid composition of roots, salinity     

Modulation of the enzymatic efficiency of ferredoxin-NADP(H) reductase by the amino acid volume around the catalytic site

Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs) are ubiquitous flavoenzymes that deliver NADPH or low-potential one-electron donors (ferredoxin, flavodoxin, adrenodoxin) to redox-based metabolic reactions in plastids, mitochondria and bacteria. Plastidic FNRs are quite efficient reductases. In contrast, FNRs from organisms possessing a heterotrophic metabolism or anoxygenic photosynthesis display turnover numbers 20- to 100-fold lower than those of their plastidic and cyanobacterial counterparts. Several structural features of these enzymes have yet to be explained. The residue Y308 in pea FNR is stacked nearly parallel to the re-face of the flavin and is highly conserved amongst members of the family. By computing the relative free energy for the lumiflavin-phenol pair at different angles with the relative position found for Y308 in pea FNR, it can be concluded that this amino acid is constrained against the isoalloxazine. This effect is probably caused by amino acids C266 and L268, which face the other side of this tyrosine. Simple and double FNR mutants of these amino acids were obtained and characterized. It was observed that a decrease or increase in the amino acid volume resulted in a decrease in the catalytic efficiency of the enzyme without altering the protein structure. Our results provide experimental evidence that the volume of these amino acids participates in the fine-tuning of the catalytic efficiency of the enzyme.     

Effect of substrate salinity on the ability for protein synthesis in pea roots

The effect of salinity on incorporation of amino acids into root tip protein is apparently of dual nature: in presence of salts the uptake is depressed and the normal metabolic pathways are disturbed. If the roots were grown at high salt concentration, uptake and incorporation are affected even if they are carried out in the absence of salt. NaCl and Na₂SO₄ affect uptake, incorporation, and metabolism of ¹⁴C leucine in different ways. There are also preliminary indications that in pea roots grown at different types of salinity, different proteins may be synthesized. Kinetin was found to inhibit incorporation of amino acids into non stressed and Na₂SO₄ stressed roots, but promotes uptake and incorporation of amino acids into protein in NaCl stressed tissue. It seems that there are some pronounced differences between the effects of NaCl and Na₂SO₄ salinities on the metabolism of pea root tissue.     

Developmental Changes in the Free Amino Acid Pool and Total Protein Amino Acids of Pea Cotyledons (Pisum sativum L.)

Changes in the levels of twenty-two free amino acids and in the amino acid composition of the total protein were measured throughout the development of cotyledons of a dwarf garden pea, Pisum sativum cv Greenfeast, grown in a constant environment. A sensitive double-isotope dansylation technique was used. Fresh weight, dry weight, and protein content were also followed. Twenty of the amino acids showed synchronous changes in levels, giving a developmental pattern containing four peaks; major peaks occurred very early and very late in development. The amino acid composition of the total protein, which was always very different from that of the free amino acid pool, showed early changes to one consistent with the final storage protein composition of the seed. These changes included a 50% drop in methionine content and a 70% rise in cysteine. While the maximum free methionine level occurred early in development, that of cysteine was late.     

Temperature impact on localization of "free" phenolic compounds in the root tissues and deformation of root hairs in pea seedlings inoculated by Rhizobium

The study was focused on localization of "free" phenolic compounds in pea Pisum sativum L. seedling roots grown at 22 and 8 degrees C 24 h after their inoculation with Rhizobium leguminosarum bv. viceae bacteria. A comparison of phenolic compound distribution along the root in root tissues, and results of observation of root hair development on the root surface, response of root hairs to inoculation, manifesting itself in various deformation degree (bends, twists, ect.) enabled us to reveal differences between roots grown at different temperatures. These differences are basically referred to a sector localized 0-5 mm away from the root tip containing meristematic and extending cells. A distribution of phenolic compounds in sectors with root hairs responding to inoculation by various degrees of contortion practically did not depend on the temperature of plant growth. The evidence provided in the course of this work enabled us to suggest that inhibition of pea root infection at low temperature is caused by decelerated growth processes characteristic of both the root itself and root hairs, as well by a slow increase in the root hair zone.     

Synthesis of proteins and nucleic acids by the pea aphid Acyrthosiphon pisum (aphididae) in the absence of a full complement of dietary amino acids

Protein, nucleic acids, and nucleotide syntheses were studied in pea aphids, Acyrthosiphon pisum (Harris), by feeding them labeled ¹⁴C-amino acids and [5-³H]-orotic acid in sucrose. It was demonstrated that in the absence of dietary essential amino acids, aphids were capable of synthesizing nucleic acids, nucleotides, and proteins when provided with a single dietary amino acid in sucrose. It is suggested that other required amino acids were possibly supplied by the symbionts present in the pea aphid and/or were obtained from the amino acid pool in the hemolymph or glucose, one of the end products of sucrose digestion. Of the various amino acids tested, synthesis of measurable amounts of protein or other compounds occurred when alanine, aspartic acid, glutamic acid, glycine, proline, or serine were provided, but no synthesis occurred with cysteine.     

The nature of protein differentiation in the growing pea root

Abstract Sections of the growing root of pea (Pisum sativum) have been microdissected into stele, cortex and epidermis. Using labelled amino acids, two dimensional separations employing non-equilibrium pH gel electrophoresis (NEPHGE) and isoelectric focusing (IEF), and silver staining, the complexity of protein differences between the cortex and the stele has been assessed. Analyses commenced as cells in these two tissues appear in the meristem (0.7—1 mm from the tip) and continued up to 30 mm from the tip as they subsequently mature. From the earliest stages at which the cortex and stele can be distinguished and dissected apart the protein patterns differ substantially. However these tissue differences, involving over one third of the detected protein species, are almost all quantitative. Very few qualitative (i.e. tissue specific) proteins were detected. Many proteins also show quantitative stage-specific variation, detected using successively older root segments. In vitro translation studies involving isolated mRNA showed only a very limited stage-specific variation in translated proteins. This supports the notion that translational controls may contribute significantly to the development of these two tissue types.     

Amino acids in exudates of healthy and fungus-affected pea roots

Summary Amino acids in exudates of uninoculated pea roots were compared quantitatively and qualitatively with exudates of roots inoculated with Gliocladium catenulatum. This fungus has the potential of causing severe root necrosis. Twenty-one amino acids were found in exudates of healthy roots and apparently some of these were utilized by the fungus. A relatively high concentration of ammonia was detected in exudates of inoculated pea roots, indicating an intense deamination by the fungus. No other imbalance in amino acids was found which could be related to known toxic effects of amino acids on plant tissues.     

Short term physiological implications of NBPT application on the N metabolism of Pisum sativum and Spinacea oleracea

The application of urease inhibitors in conjunction with urea fertilizers as a means of reducing N loss due to ammonia volatilization requires an in-depth study of the physiological effects of these inhibitors on plants. The aim of this study was to determine how the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) affects N metabolism in pea and spinach. Plants were cultivated in pure hydroponic culture with urea as the sole N source. After 2 weeks of growth for pea, and 3 weeks for spinach, half of the plants received NBPT in their nutrient solution. Urease activity, urea and ammonium content, free amino acid composition and soluble protein were determined in leaves and roots at days 0, 1, 2, 4, 7 and 9, and the NBPT content in these tissues was determined 48 h after inhibitor application. The results suggest that the effects of NBPT on spinach and pea urease activity differ, with pea being most affected by this treatment, and that the NBPT absorbed by the plant caused a clear inhibition of the urease activity in pea leaf and roots. The high urea concentration observed in leaves was associated with the development of necrotic leaf margins, and was further evidence of NBPT inhibition in these plants. A decrease in the ammonium content in roots, where N assimilation mainly takes place, was also observed. Consequently, total amino acid contents were drastically reduced upon NBPT treatment, indicating a strong alteration of the N metabolism. Furthermore, the amino acid profile showed that amidic amino acids were major components of the reduced pool of amino acids. In contrast, NBPT was absorbed to a much lesser degree by spinach plants than pea plants (35% less) and did not produce a clear inhibition of urease activity in this species.     

Nitrate Assimilation in Higher Plants with Special Reference to the Cocklebur (Xanthium pennsylvanicum Wallr.)

A soluble NADH-dependent nitrate reductase is described forthe shoot system of Xanthium. Young leaves and immature stemtissues contain high levels of the enzyme. They are relativelyrich in free amino acids and amides but store little free nitrate.The specific activity of the enzyme is lower in fully expandedleaves, although these leaves exhibit higher rates of fixationof carbon in photosynthesis than do younger leaves. Neithernitrate nor free amino acids accumulate in the mesophyll ofthe leaf. Older parts of the stem axis accumulate large amountsof soluble nitrogen, almost entirely as free nitrate. Reservesof nitrate in the shoot and root are rapidly depleted if nitrateis removed from the external medium. Nitrate reductase is apparently absent from roots of Xanthium.This finding is supported by analyses of bleeding sap from nitrate-fedplants which show that 95 per cent of the nitrogen exportedfrom roots is present as free nitrate. However, roots are capableof synthesizing and exporting large amounts of amino nitrogenif supplied with reduced nitrogen such as urea or ammonium. A scheme is presented summarizing the main features of the metabolismof nitrate in Xanthium and this is compared with the situationin nitrate-fed plants of the field pea (Pisum arvense L.), aspecies previously shown to be capable of reducing nitrate inits root system.     

Changes in the Soluble Protein and Free Amino Acid Content of Chill-Sensitive and Chill-Resistant Plants During Chilling and Hardening Treatments

The effects of low temperature (5 °C and 12°C) and droughttreatments on leaf soluble protein content and free amino acidcontent have been investigated in four species, which were rankedaccording to chilling-sensitivity: pea (chill-resistant), mungbean (highly chill-sensitive), and tomato and french bean (intermediatechilling-sensitivity). Drought treatment caused a 30–40% decrease in proteinlevels, and in all but the mung bean, a 100–200% increasein free amino acid concentration. Four days chilling at 5°C,85% r.h. caused leaf water content to decrease by almost 50%in the mung bean, but by only approximately 6–7% in theother three species. During this treatment the leaf solubleprotein content decreased in all four species although the decreasewas greatest and most rapid in the mung bean, commencing with8 h of chilling (coinciding closely with the onset of waterloss), and decreasing by over 80% after 4 d. In the chill-sensitivespecies (but not in the pea) the decrease in protein contentwas accompanied by an increase in free amino acid content. However,on a mgg^–1 dry wt. basis, this increase was insufficientto account for all the protein lost. When plants of each specieswere chilled at 5°C, 100% r.h., water loss was greatly reducedor prevented and there was no significant decrease in leaf solubleprotein. It is concluded that the protein decrease which occurredat 5°C, 85% r.h., was a response to water loss and not thedirect result of low temperature. However, chilling at 100%r.h. did cause an increase in free amino acid content of thechill-sensitive species, suggesting that this was a direct responseto low temperature. Although drought treatment caused a 6–20 fold increasein free proline content in the leaves of the four species examined,chilling (5°C) and chill-hardening (12°C) caused littlechange in free proline content, indicating that the accumulationof this ‘protective’ amino acid is unlikely to contributeto the effectiveness of the chill-hardening treatment. Key words: Low Temperature, Drought, Leaf soluble protein.content, Amino acids     

Free amino acids are important for the retention of protein and non-protein meals by the midgut of Aedes aegypti females

There is a relationship between the normal progress of digestion and the retention or elimination of the proteins ingested with the meal by Aedes aegyti females. The addition of soybean trypsin inhibitor (STI) to a protein meal prevented digestion and resulted in a rapid elimination of the undigested proteins. The addition of a mix of free amino acids to a protein meal together with STI resulted in a significant increase in the retention of the undigested proteins during the first 10-15 hrs after feeding. The effect of the free amino acids on the retention of the proteins was concentration-dependent between 250 microg/ml and 5 mg/ml. Free amino acids were also important for the retention of non-protein meals. When females were fed a meal containing FITC-dextran (20 kD), most of this compound was eliminated into the feces by 10 hrs; the addition of free amino acid resulted in a significant increase in the retention of the FITC-dextran by the midgut during the first 15 hrs after feeding. The presence of free amino acids in the midgut lumen seems to be an important signal used by the mosquito to regulate the retention of the meal.