Osmotic Stress as a Pregrowth Procedure for Cryopreservation 2. Water Relations and Metabolic State of Sycamore and Soybean Cell Suspensions

Addition of 6 per cent mannitol or sorbitol to liquid culturemedium decreased the water potential (_w) by –0.93 MPa( 382 ± 7 mOsm kg^–1 water). Sycamore cells grownto exponential phase in such media exhibited increased levelsof total and soluble protein and respiratory activity, but decreasedamounts of free proline. Soybean cells showed increased respiratoryactivity and free proline levels, but total protein levels remainedunaffected. Soluble protein levels were reduced under sorbitol-inducedstress. In both species osmotic stress had little effect oncell dry weight. Water relation studies indicate that sycamore cells are capableof much greater osmotic adjustment than soybean cells, and thatmannitol uptake does not contribute significantly to that adjustment. Acer pseudoplatanus L., sycamore, Glycine max L. var. Biloxi, soybean, suspension culture cells, osmotic stress, water relations, metabolism     

Effect of cycocel and furfuryl amino purine on protein metabolism of pearl millet (Pennisetum americanum L.) under simulated drought conditions

Water stress drastically decreased relative water content and total soluble protein whereas it increased free amino acids, proline and protease enzyme in P. americanum. Treatments with 6-furfuryl amino purine (0.50 mM) and cycocel (60 mM) maintained the levels of soluble protein contents but the applications of furfuryl amino purine decreased free amino acids and free proline under stress conditions.     

Iso-osmotic effect of NaCl and PEG on growth, cations and free proline accumulation in callus tissue of two indica rice (Oryza sativa L.) genotypes

One-month old calli of two indica rice genotypes, i.e., Basmati-370 and Basmati-Kashmir were subjected to two iso-osmotic concentrations (−0.57 MPa and −0.74 MPa) created with 50 and 100 mol m⁻³ NaCl or 10 and 18% solutions of PEG-8000. Both genotypes tolerated only low levels of stress and showed severe growth suppression at −0.74 MPa. The degree of stress tolerance of both genotypes was greater for PEG induced stress than for NaCl induced stress. The relative growth rate of callus was reduced under both stresses, however, the reverse was true for callus dry weight. Sodium (Na⁺) content of the callus tissue was increased only under NaCl induced stress. Salt induced stress reduced K⁺ and Ca²⁺ contents, but the PEG induced stress increased them. Higher levels of stress increased the proline content many folds with more increase being under PEG stress than that under NaCl. Water and osmotic potentials of the callus tissue decreased, whereas turgor potential increased under both abiotic stresses. Overall, Basmati-370 was more tolerant to both NaCl and PEG induced stresses than Basmati-Kashmir, because of less reduction in growth and more dry weight. Moreover, Basmati-370 accumulated higher amounts of cations, free proline, and maintained maximum turgor as compared to Basmati-Kashmir. In conclusion, at cellular level, mechanism of NaCl induced osmotic stress tolerance was found to be associated with more ionic accumulation of inorganic solutes and that of PEG induced osmotic stress tolerance with the accumulation of free proline, as an important osmolyte in the cytosol.     

Regulation of Aspartokinase Isoenzyme Levels in Cultured Cells of Vinca rosea

The levels of aspartokinase isoenzymes were followed as a functionof days after transfer of V. rosea cells to fresh medium. Whencells were subcultured in a 7-day cycle, both isoenzymes showedpeaks at early, but not identical, stages of cell proliferation.Levels of the intracellular amino acids lysine, threonine, isoleucineand methionine decreased as the cellular level of protein increased.As soon as the increase in protein ceased, the amino acid levelbegan to increase. The stationary cells accumulated large amountsof free amino acids. When late stationary cells were used asthe inoculum, growth was slow and, as expected, it took longerbefore the depletion of endogenous free amino acids and thedevelopment of aspartokinase isoenzymes was significantly retarded.These results are further evidence that the syntheses of aspartokinaseisoenzymes are under repression-derepression control in higherplants as they are in bacterial systems. (Received April 22, 1981; Accepted August 31, 1981)     

Effect of Salt Stress on Viability, Germination and Endogenous Levels of Some Metabolites and Ions in Maize (Zea mays L.) Pollen

Maize plants, subjected to 0, 80, 120 and 160 meq l^–1salinity using NaCl, showed adverse effects on viability, germinationand tube growth of pollen, besides enhancing the bursting ofpollen. The endogenous levels of various metabolites in pollenwere also affected. Pollen grains from salinized plants hadmore soluble carbohydrates, free amino acids, especially proline,phenols and DNA and less starch, protein and RNA compared tothe non-saline controls. Salinity also resulted in the accumulationof ions such as Na⁺, K⁺ and Cl^– while it caused a reductionin the boron content of pollen. These metabolic disturbancespossibly lead to decreased viability, germination and tube growthof pollen thereby resulting into a reduction in reproductivecapacity of the plants under salt stress. Zea mays L., maize, pollen, viability, germination, salt stress     

Changes in the Contents of Free and Protein Amino Acids in Tobacco Seeds and Placentae during Maturation

Changes in the levels of protein and free amino acids in theseeds and placentae of Nicotiana tabacum were studied duringseed development. Seed maturation was completed 24 days afteranthesis. During maturation, protein rapidly accumulated inthe seeds between the 6th and 18th day, along with an appreciablecompositional change in the protein amino acids as the proportionsof glutamic acid and arginine increased. The amount of freeamino acids in the seeds gradually decreased throughout maturation.The major free amino acid on the 6th day after anthesis wasglutamine, which then drastically decreased between the 6thand 12th day with increases of glutamic acid, proline, arginineand alanine. The latter amino acids decreased thereafter untilthe 24th day. On the other hand, the amount and composition of the proteinsin the placentae did not change significantly throughout seedmaturation. In the early stage of development, the major freeamino acids in the placentae were glutamine, asparagine andglutamic acid, while in the later stage asparagine was mostabundant. (Received March 12, 1982; Accepted August 16, 1982)     

Amino Acid Content of Leaves in Mycorrhizal and Non-mycorrhizal Citrus Rootstocks

Nitrogen status was examined in leaves of sour orange and roughlemon citrus rootstocks grown in a low phosphorus sand inoculatedwith Glomus etunicatus, in sand amended with superphosphateat a rate of 2240 kg ha^–1, and in a sand control leftuntreated. Sour orange was 3.1- and 3.5-fold taller and roughlemon was 1.8- and 2.0-fold taller than the controls in theinoculated and phosphorus treatments, respectively. In the controls,leaf N was up to 2.5-fold higher than in the other treatments.Both total and free amino acids accumulated in leaves of bothrootstocks to higher levels in the control than in the othertreatments. Most total amino acids in the control were lowerthan in the treatments, with the exceptions of arginine (upto 12-fold increase), proline (up to 1.8-fold increase), lysine,and free ammonia. Twenty-two free amino acids, urea, and ammoniawere detected. Both rootstocks grown in control sand had significantincreases in citrulline, ornithine, lysine, histidine, arginineand ammonia. Levels of total and most free amino acids in theinoculated and phosphorus treatments were similar to one another.It is suggested that mineral deficiency caused by the absenceof G. etunicatus causes a reorganization of N-metabolism witha shift to a greater synthesis of ornithine cycle intermediates. Citrus aurantium L., Citrus limon (L.) Burm, amino acid content, citrus rootstocks, mycorrhiza, nitrogen metabolism     

The Extent and Pattern of Osmotic Adjustment in White Clover (Trifolium repens L.) During the Development of Water Stress

White clover plants were subjected to water stress followingthe cessation of watering. As a water deficit developed, waterand osmotic potentials were measured in stolon tips, leavesfrom the stolon tip and leaves from the plant crown. Pressurepotentials were calculated. Pressure potential was maintainedin stolon tips even when water potential fell to around –2·0MPa. In contrast, pressure potential in leaves fell rapidlyas water stress developed. Total amino acid and potassium levels were largely unaffectedin both stolon tips and leaves. Water-soluble carbohydratesand proline accumulated during water stress. The increase inproline level in leaves did not follow the same pattern as thatin stolon tips, although toward the end of the water stressperiod the level had increased by a similar extent in both partsof the plant. Additionally, pressure potential and osmotic potentialappeared to be significantly related to proline content in stolontips. No such relationship was found for leaves. The role ofproline in osmotic adjustment is discussed. Trifolium repens L. cv. Olwen, white clover, water stress, osmotic adjustment, proline     

Respiration and Carbohydrate Accumulation in the Water-Stressed Wheat Apex

The effect of water stress on the respiration of the immaturefloral apex of wheat was studied in a controlled environmentand related to changes in water relations, growth, protein synthesis,and solute accumulation. Apex respiration measured in vitropolarographically showed no wounding response and was cyanide-and malonate-sensitive. It decreased with each decrease in apexwater potential _a reaching 40% of the non-stress control rateat –5·0 MPa, irrespective of whether the waterstress was induced by droughting in vivo or non-permeating osmoticain vitro. Apex respiration was not quantitatively related toturgor potential. During drought stress there was a conservation of ethanol-insolubledry matter and water in the apex while ethanol-soluble carbohydratesand amino acids accumulated. The calculated daily import ofsoluble carbohydrate into the apex during the whole droughtstress period remained nearly constant despite falling waterpotential. Respiration of the apex during a drought period wasnot limited by the suistrate supply within the apex.     

Sagebrush (Artemisia tridentata Nutt.) roots maintain nutrient uptake capacity under water stress

Phosphorus and nitrogen uptake capacities were assessed during36–58 d drying cycles to determine whether the abilityof sagebrush (Artemisia tridentata Nutt.) to absorb these nutrientschanged as the roots were subjected to increasing levels ofwater stress. Water was withheld from mature plants in large(6 I) containers and the uptake capacity of excised roots insolution was determined as soil water potentials decreased from–0.03 MPa to –5.0 MPa. Phosphorus uptake rates of excised roots at given substrateconcentrations increased as preharvest soil water potentialsdecreased to –5.0 MPa. V_max and K_m also increased as soilwater potentials declined. Declining soil water potentials depressednitrogen uptake at set substrate concentrations, but uptakecapacity, calculated as the sum V_max for both NH⁺₄+NO^–₃,did not change significantly with drying. The sum V_max correlatedwith root nitrogen concentration. Root uptake capacity for nitrogen and phosphorus was extremelystable under severe water stress in this aridland shrub. Maintenanceof uptake capacity, coupled with a previously demonstrated abilityto conduct hydraulic lift, may enable A. tridentata better tomaintain nitrogen and phosphorus uptake as soil water availabilitydeclines. These mechanisms may be important in the ability ofA. tridentata to maintain growth, complete reproduction, andgain an advantage against competitors late in the season whenthe soil layers with higher nutrient availability are dry. Key words: Kinetics, nitrogen, phosphorus, roots, water stress     

Accumulation of some nitrogen compounds in response to salt stress and their relationships with salt tolerance in rice (Oryza sativa L.) seedlings

The salt-induced accumulation of some nitrogen compounds (free amino acids, ammonium and urea) in shoots of eight rice cultivars differing in salt tolerance was investigated. Salt treatment (100 mM, 6 days) significantly increased the proline content of shoots but this appeared to be a reaction to stress damage and not associated with salt tolerance, because proline contents were higher in the more sensitive cultivars. Besides proline, some other free amino acids also accumulated leading to a significant increase in the total amino acid content of the stressed seedlings. High levels of free ammonium also accumulated under conditions of stress; this was highly correlated with the accumulation of Na⁺ in the shoots and negatively correlated with salt tolerance. The accumulation of ammonium was positively correlated with the accumulation of many free amino acids, and also associated with the production of urea in the stressed seedlings. Results from the present investigations suggest that an increase in the concentration of some free amino acids including proline, may be a result of the reassimilation of the stress-induced ammonium. A high capacity to assimilate ammonium may be an important factor in alleviating the consequence of stress because ammonium can be toxic at high concentrations.     

Composition and Distribution of Free Amino Acids in Flatpea (Lathyrus sylvestris L.) as Influenced by Water Deficit and Plant Age

Drought-stressed flatpea (Lathyrus sylvestris L.) plants from8 to 22 weeks old were analysed for nitrogen, soluble proteinand free amino acids. An increase in nitrogen and free aminoacid concentrations and a decrease in soluble protein levelwere observed in roots of plants up to 16 weeks old. The cumulativeconcentration of free amino acids increased with drought stress.Tissue concentrations of 2, 4-diaminobutyric acid (1.6–2.6%of the dry weight) were highest in leaves. Levels increasedsteadily, nearly doubling, in leaves and stems between weeks10 and 16. Levels in drought-stressed leaves were, on average,11.9% higher than those of controls. Estimated concentrationsof a mixture of 4-aminobutyric acid and an unknown amino acidwere highest in stems, increased in this tissue with age andtended to increase in stems and leaves and decrease in rootsin response to water deficit. Levels of the mixture of homoserineand another unidentified amino acid were not influenced by ageor water status of the plants. Root concentrations of asparagine,arginine, glutamine, aspartate, and another prominent, unidentifiedamino acid increased with plant age and reached a peak at thetime of flowering (14 to 18 weeks). Only the concentration ofthe unknown compound was elevated following drought stress.Concentrations of valine, isoleucine, leucine, phenylalanine,and methionine also increased during this period and were elevatedin drought-stressed plants. Proline levels increased with plantage and drought stress, but proline accounted for only about10% of the total free amino acids in the drought-stressed plants. Key words: 2, 4-Diaminobutyric acid, drought, flatpea     

Salt Stress Control of Intracellular Solutes in Streptomycetes Indigenous to Saline Soils

Actinomycetes were isolated from a number of saline and saline-sodic California soils. From these isolates, two species of Streptomyces (S. griseus and S. californicus) were selected to assess their physiological response to salinity. NaCl was more inhibitory to growth rates and specific growth yields than were equivalent concentrations of KCl. Intracellular concentrations of the free amino acid pool increased in response to salt stress. Whereas the neutral free amino acids proline, glutamine, and alanine accumulated as salinity increased, concentrations of the acidic free amino acids glutamate and aspartate were reduced. Accumulation of free amino acids by streptomycetes under salt stress suggests a response typical of procaryotes, although the specific amino acids involved differ from those associated with other gram-positive bacteria. Above a salinity threshold of about 0.75 M (−3.8 MPa), there was little further intracellular accumulation of free amino acids, whereas accumulation of K⁺ salts sharply increased.     

Osmotic Adjustment in Lolium perenne; Its Heritability and the Nature of Solute Accumulation

Plants derived from Lolium perenne L. cv. ‘Melle’were selected on the basis of extreme high or low lamina solutepotential (_s) and pair-crossed to produce divergent G₂ lines.The high and low lines had mean lamina _s values significantlydifferent from each other and from ‘Melle’, andshowed an enhanced range of phenotypic expression of _s duringdrought. Both mature lamina _s and meristem _s values of droughtedplants were highly heritable. Extreme G₂ genotypes were selectedand clonally replicated for further study. During drought mean ₂ values fell from –1·21 to–1·80 MPa. Fructans of large molecular weight,and total free amino acids, especially proline, all accumulatedwhen estimated on a dry-matter (DM) or plant-water (PW) basis.Oligosaccharide content was largely unchanged. Minerals declinedwhen estimated on a DM basis but accumulated on a PW basis becausehydration (g water in turgid tissue per g DM) declined morerapidly. In comparison with genotypes having high constitutive _s, low-_sgenotypes (a) were larger, had faster leaf extension rate, hadfewer tillers, and were proportionally more affected by drought,(b) showed greater osmotic adjustment, (c) contained and accumulatedmuch more fructan (but not oligosaccharides), and amino acids,especially proline, and (d) accumulated more mineral ions ona PW basis, but less on a DM basis. The relatively high repeatabilitiesfor organic solutes in particular show that further divergentselection for individual solutes would not be difficult. Solutes accumulated, probably because they were not consumedin growth. There was no evidence of ‘competition’between growth and osmotic adjustment for metabolites, or thatplants which accumulated more solutes were better able to recoverwhen water deficits were relieved. Perennial ryegrass, drought, genetic variation, carbohydrate, amino acids, proline, mineral uptake, Lolium perenne L. cv. ‘Melle’     

CHANGES OF AMINO ACID COMPOSITION OF CHLORELLA CELLS DURING THEIR LIFE CYCLE

The cells of Chlorella ellipsoidea were grown synchronously,and at different stages of their life cycle, the cells wereanalysed for their contents in amino acids existing in freeforms as well as in the fractions of bulk protein and peptides.Throughout the algal life cycle, the content of bulk protein(per unit dry weight of cells) remained relatively constant,being about 20 to 40 times those of peptides and free aminoacids. The amino acid composition of the protein fraction alsoremained fairly constant, the predominant amino acids beingalanine, glutamic acid, glycine and leucine. The contents inthe bulk peptides increased appreciably during the periods ofgrowth and "ripening" (light period), and decreased markedlyduring the periods of "post-ripening" and cellular division(dark period). Similar modes of change in content were alsoobserved in most of the individual amino acids contained inthe peptide fraction. The most abundant component in the peptidefraction was arginine followed by glutamic acid, glycine andcyst(e)ine. Rather irregular was the mode of change of the levelsof individual free amino acids, although, as a whole, theirbehavior was similar to that of bulk peptides, increasing duringthe light period and decreasing during the dark period. Themost predominant free amino acids were glutamic acid and alaninefollowed by proline. Experimental evidence showed that the processes of formationof free amino acids and peptides are for the most part lightdependent, while the synthesis of protein, which is thoughtto be effected using as building blocks mostly free amino acids—formeddirectly or indirectly from early photosynthates or derivedfrom pre-formed peptides—is essentially a light-independentprocess. Peptides, as a whole, seem to have significance asreservoirs of building blocks for the syntheses in the darkof protein and other nitrogenous cellular substances. The synthesisof protein in the dark takes place not only by consuming thefree amino acids and peptides that have been accumulated duringthe light period, but also by assimilating the exogenous nitrogensource (nitrate). The distribution of individual amino acidsin the three main fractions mentioned above as it changed duringthe course of algal cell cycle was followed in detail, and theresults obtained were discussed in relation to various relevantdata reported by other workers. (Received June 29, 1964; )     

Sugar, Amino Acid and Inorganic Contents in Rice Phloem Sap

Rice phloem sap was obtained through severed stylets of brownplanthoppers and its chemical composition was determined. Sucrose,the only sugar detected, was present at 17–25% (w/v) inthe sap. Amino acids were present at a total of 3–8% (w/v)and were found to be mostly in a free form. The amount of boundamino acids was estimated to be small, if any were present.Among the free amino acids, asparagine (17–33% on a molarbasis), glutamate (6–14%), serine (10–13%), glutamine(7–15%), threonine (5–6%) and valine (6–7%)were dominant, while cystine and methionine (0–0.2%) werepresent in minor amounts and -aminobutyric acid was not detected.The sap had a slightly alkaline pH (ca. 8.0). The inorganicconstituents detected by electron probe x-ray microanalyzerwere Na, S, P and K, with the K content being the highest. Theosmotic pressure was estimated to be 13–15 atm. The aminoacid composition of the plant parts was determined and the differencesin the ease of phloem loading among amino acids were compared. (Received October 5, 1981; Accepted December 17, 1981)     

Changes in Proline Content Accompanying the Uptake of Zinc and Copper by Lemna minor

Lemna minor L. grown in Hoagland solution containing zinc (10ppm) or copper (5 ppm) for 4 d accumulated high levels of thesemetals. Zinc and Cu accumulation in the test plants was accompaniedby a specific pattern of change in proline content. The lattershowed a steep rise during early stages (peak at 12-24 h) followedby a gradual decline until 96 h of treatment. In a dose-responsestudy, lower metal concentrations induced a sharp rise in prolinelevel with a maximum value at 5 ppm, which declined when theconcentration was further enhanced. There was a correspondencebetween the level of proline and total free amino acids in metaltreated plants. The possibility of proline involvement in tolerancemechanisms to heavy metals has been discussed.Copyright 1993,1999 Academic Press Heavy metals, zinc, copper, proline, total free amino acids, Lemna minor     

Sources of proline-nitrogen in water-stressed soybean (Glycine max). II. Fate of 15N-labelled protein

The absorption of nitrate, protein metabolism and the source of nitrogen for proline synthesis were studied in soybean ( Glycine max L. cv. Akisengoku) with ¹⁵N tracer technique under water stress conditions. The absorption of nitrate was sensitive to water stress and the flow of nitrate into the leaves completely ceased under severe stress conditions. Net protein loss from the water-stressed leaves was attributable to both a decrease in synthetic activity and a stimulation of protein degradation. Proline and asparagine accumulated extensively in the severely water-stressed plant tissues, especially in the younger green leaves. Fifty four % of the loss of leaf protein-¹⁵N during the stress period was balanced by a gain in ¹⁵N in the free amino acids, 41% being found in proline and asparagine. The increase in ¹⁵N content of the free proline was 3 times greater than the decrease in ¹⁵N content of the protein-bound proline in the leaf. The results indicate that the accumulation of proline in response to water stress was caused by enhanced synthesis and that the nitrogen source for this proline is the leaf protein. The possible association of these findings with stress tolerance is discussed.     

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.