Polyamine Metabolism and Osmotic Stress : I. Relation to Protoplast Viability

Cereal leaves subjected to the osmotica routinely used for protoplast isolation show a rapid increase in arginine decarboxylase activity, a massive accumulation of putrescine, and slow conversion of putrescine to the higher polyamines, spermidine, and spermine (HE Flores, AW Galston 1984 Plant Physiol 75: 102). Mesophyll protoplasts from these leaves, which have a high putrescine:polyamine ratio, do not undergo sustained division. By contrast, in Nicotiana, Capsicum, Datura, Trigonella, andVigna, dicot genera that readily regenerate plants from mesophyll protoplasts, the response of leaves to osmotic stress is opposite to that in cereals. Putrescine titer as well as arginine and ornithine decarboxylase activities decline in these osmotically stressed dicot leaves, while spermidine and spermine titers increase. Thus, the putrescine:polyamine ratio in Vigna protoplasts, which divide readily, is 4-fold lower than in oat protoplasts, which divide poorly. We suggest that this differing response of polyamine metabolism to osmotic stress may account in part for the failure of cereal mesophyll protoplasts to develop readily in vitro.     

Age-Dependent Decrease of Nucleoside Pools in Cereal Leaves

The amounts of nucleosides, the nucleobase adenine, and theadenine-containing nucleotides were determined in leaves ofbarley and wheat using recently developed methods of extractpurification and HPLC analysis. There were large amounts ofnucleosides in the flag leaves but markedly lower amounts inthe older leaves. Their role as nucleotide precursors in graindevelopment or transport vehicles in leaf senescence is discussed.The adenine nucleotide concentration slightly increased withleaf age but the energy charge values were rather constant.Thus the energy charge cannot serve as monitor of leaf age orleaf senescence. (Received April 20, 1987; Accepted October 12, 1987)     

Analysis of Free Amino Acid Pools in Fungal Mycelia

Free amino acid pools derived from three different types of fungal mycelia have been analyzed by the method of Heathcote and Haworth by using thinlayer chromatography. The preliminary extraction was carried out with boiling water and interfering proteins; peptides and salts were first removed by means of an ion-retardation resin. As far as determined, the results obtained represent the first quantitative analysis of fungal amino acid pools.     

The Relation between Accumulation of Abscisic Acid and Proline in Detached Rice Leaves

The relation between abscisic acid (ABA) and proline accumulation was investigated in detached rice (Oryza sativa L.) leaves. In darkness, proline content increased about 2-, 2,5- and 6-fold after 24, 48 and 72 h. ABA content reached maximum after 48 h. In the light, proline content remained almost unchanged until 48 h and subsequently increased slightly. ABA content in the light was lower than in darkness, but the maximum was also after 48 h. During 12-h exposure to decreased air humidity, proline content gradually increased, but ABA content increased about 25-fold after 4 h and declined thereafter. Exogenous application of ABA resulted in an increase in proline content in detached rice leaves under both light and darkness.     

Changes in Amino Acid Content of Excised Leaves During Incubation. III. Role of Sugar in the Accumulation of Proline in Wilted Leaves

A previously reported accumulation of proline in wilted turnip leaves has been observed in 6 additional species representing 5 different families. The results of experiments on the conditions affecting the behavior of proline in wilted leaves were interpreted to mean that the supply and metabolism of available carbohydrate was essential for proline accumulation.     

Nitrate Accumulation and its Relation to Leaf Elongation in Spinach Leaves

The leaf elongation rate (LER) of spinach leaves during theday was twice that during the night when grown at a photon fluxdensity of 145 µmol m^–2 s^–1. All leaves showedthe same LER-pattern over 24 h. Due to low turgor, LER was lowin the afternoon and in the first hours of the night until wateruptake restored full turgor. Osmotic potential remained constantdue to increased nitrate uptake and starch degradation in thisperiod. LER increased to high rates in the second part of thenight and in the morning. The lower rate in the dark comparedto the light was not caused by the lower night temperatures,as increased photon flux density during growth resulted in equalrates in the light and the dark. Increased relative humiditydecreased LER and afternoon rates were most sensitive to waterstress. A ‘low light’ night period did not changeLER-pattern during the night or on the following day. We concludethat nitrate is not an obligatory osmoticum during the nightand can be exchanged for organic osmotica without decreasingLER. During the night the turgor is first restored by increasingwater uptake, nitrate uptake and starch degradation. This resultedin increased leaf fresh weight in this period. Thereafter, elongationincreased by simultaneous uptake of nitrate and water. Nitrateconcentration was, therefore, constant in the older leaves.In the younger leaves nitrate concentration increased to replacesoluble carbohydrates. The vacuoles of the old leaves were filledwith nitrate before those of the young leaves. Key words: Spinacia oleracea L., nitrate accumulation, osmotic potential, organic acids     

Accumulation of delta-Aminolevulinic Acid and Its Relation to Chlorophyll Synthesis and Development of Plastid Structure in Greening Leaves

Levulinic acid inhibited the greening of etiolated maize (Zea mays) and bean (Phaseolus vulgaris) leaves and caused accumulation of δ-aminolevulinic acid (ALA). ALA accumulation in maize was equivalent to the decrease in chlorophyll, over a wide range of experimental conditions. It was saturated at low light intensities and was not limited by the supply of substrates during the early hours of greening. During 20 hours in light, levulinic acid had little effect on the structural development of thylakoids in bundle sheath chloroplasts but significantly reduced the number and size of thylakoids in grana of mesophyll chloroplasts. Recrystallization of prolamellar bodies and their reformation was inhibited. Mitochondria appeared not to be affected.     

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.     

Polyamine Titre in Relation to Chill-Sensitivity in Phaseolus sp.

Guye, M G., Vigh, L. and Wilson, J. M. 1986. Polyamine titrein relation to chill-sensitivity in Phaseolus sp.—J. exp.Bot. 37: 1036–1043. Endogenous levels of the polyamines putrescine, spermidine andspermine were quantified in the primary leaves of five cultivarsof bean (Phaseolus sp.) differing in their ‘wilting response’to a chilling exposure of 5 ?C for 24 h. Levels of polyamines prior to chilling treatment did not appearto be correlated with chill-tolerance as levels in the non-chilledcontrols were highest in cultivars of medium chill-sensitivity.Plants grown under a vapour saturation deficit (VSD) of 8?4gm^–3 day/6?1 g m^–3 night exhibited a mild hardeningas compared to plants grown under a VSD of 5?7 gm^–3 day/4?1gm^–3 night, as the former showed less wilting on chilling.Hardening at high VSD had the effect of slightly lowering theputrescine content of non-chilled tissue but total polyaminecontent remained unchanged. However, on chilling, the largestrelative increase in polyamine levels, in particular that ofputrescine, occurred in hardened plants. There was also a significantrelative increase in putrescine titre in response to chillingin non-hardened genotypes of high chill-tolerance, whereas morechill-sensitive genotypes remained unchanged or slightly declinedin putrescine content on chilling. Relative changes in putrescine content rather than absolutelevels appears to be correlated with chill-tolerance. Theseresults are discussed in view of present knowledge on the adaptivesignificance of stress-induced changes in polyamines, especiallywith regard to membrane stability Key words: Chilling, polyamines, Phaseolus sp.     

枣树叶中氨基酸的含量

通过对枣树叶中氨基酸进行测定证实,两个品种之间在氨基酸总量上无显著性差异,氨基酸含量最高的是谷氨酸、最低的是蛋氨酸。     

Regional Amino Acid Distribution in Relation to Function in Insulin Hypoglycaemia

The amino acids glutamate, aspartate, gamma-aminobutyric acid (GABA), and glutamine were measured as their dansyl derivatives in whole brain and specific brain regions by a sensitive double-labelling technique at various times during the development of hypoglycaemic encephalopathy. Hypoglycaemia was induced by administration of insulin (100 i.u./kg) to 24-h fasted rats. No significant changes in glutamate, GABA, or glutamine were detected in whole brain at any time up to and including the onset of hypoglycaemic convulsions. In cerebral cortex, however, GABA levels were reduced to 65% or normal prior to the appearance of neurological symptoms of hypoglycaemia. Onset of symptoms (severe catalepsy and loss of righting reflex, but before the onset of convulsions) was accompanied by marked decreases of glutamate and glutamine in striatum and hippocampus. These regions, in addition to cerebral cortex, show the greatest vulnerability to hypoglycaemic insult, according to previous anatomical studies. Aspartate levels were significantly increased (p less than 0.01) in the cerebral cortex of convulsing animals, confirming a previous report. No changes were detectable in any of the amino acids studied in medulla-pons at any time during the progression of hypoglycaemia. Cerebral cortex and striatum showed a selective net loss of amino acids (2.2 and 3.5 mumol/g. respectively) prior to the onset of insulin-hypoglycaemic convulsions.     

Factors Affecting Polyamine Accumulation in Barley (Hordeum vulgare L.) Leaf Sections during Osmotic Stress

Turner, L. B. and Stewart, G. R. 1988. Factors affecting polyamineaccumulation in barley (Hordeum vulgare L.) leaf sections duringosmotic stress.-J. exp. Bot. 39: 311–316. Polyamine concentrations in peeled leaf sections of Hordeumvulgare were unaffected by decreases in leaf water potentialif osmotic adjustment took place and leaf turgor was maintained.Putrescine accumulation occurred concomitantly with a decreasein leaf turgor. Increases in the order of 3-to 4-fold were observed.An apparently greater putrescine accumulation (7-fold) occurredwhen leaf sections were osmotically stressed in the presenceof exogenous phosphate ions. This was the result of water lossfrom the tissue and the large decline which occurred in theputrescine levels of control tissue sections incubated withphosphate ions. Putrescine accumulation was at its maximum after4 h osmotic stress. In contrast, proline accumulation took placebetween 4 h and 24 h after the imposition of osmotic stress. Key words: Hordeum vulgare, osmotic stress, polyamine     

Adenovirus Proteins II. N-Terminal Amino Acid Analysis

Adenovirus types 2, 4, 5, 6, 18, 21 and 27, were analyzed for N-terminal amino acids by use of (35)S-labeled phenylisothiocyanate of high specific radioactivity. Two free N-terminal amino acids, alanine and glycine, were found in these viruses in the molar ratio (alanine-glycine) of 2.5:1, with the exception of type 2 where the ratio was 3.6:1 and type 5 where the ratio was 5.5:1 Adenovirus type 2 was disrupted by acetone treatment, and two protein fractions were obtained after sucrose gradient centrifugation. One of these fractions, which was associated with the viral deoxyribonucleic acid and comprised approximately 18% of the total protein of the virus, was greatly enriched with respect to N-terminal alanine and glycine.     

Polyamine Metabolism and Osmotic Stress : II. Improvement of Oat Protoplasts by an Inhibitor of Arginine Decarboxylase

We have attempted to improve the viability of cereal mesophyll protoplasts by pretreatment of leaves with dl-α-difluoromethylarginine (DFMA), a specific `suicide' inhibitor of the enzyme (arginine decarboxylase) responsible for their osmotically induced putrescine accumulation. Leaf pretreatment with DFMA before a 6 hour osmotic shock caused a 45% decrease of putrescine and a 2-fold increase of spermine titer. After 136 hours of osmotic stress, putrescine titer in DFMA-pretreated leaves increased by only 50%, but spermidine and spermine titers increased dramatically by 3.2- and 6-fold, respectively. These increases in higher polyamines could account for the reduced chlorophyll loss and enhanced ability of pretreated leaves to incorporate tritiated thymidine, uridine, and leucine into macromolecules. Pretreatment with DFMA significantly improved the overall viability of the protoplasts isolated from these leaves. The results support the view that the osmotically induced rise in putrescine and blockage of its conversion to higher polyamines may contribute to the lack of sustained cell division in cereal mesophyll protoplasts, although other undefined factors must also play a major role.     

Relationship between Stress-Induced ABA and Proline Accumulations and ABA-Induced Proline Accumulation in Excised Barley Leaves

When excised second leaves from 2-week-old barley (Hordeum vulgare var Larker) plants were incubated in a wilted condition, abscisic acid (ABA) levels increased to 0.6 nanomole per gram fresh weight at 4 hours then declined to about 0.3 nanomole per gram fresh weight and remained at that level until rehydrated. Proline levels began to increase at about 4 hours and continued to increase as long as the ABA levels were 0.3 nanomole per gram fresh weight or greater. Upon rehydration, proline levels declined when the ABA levels fell below 0.3 nanomole per gram fresh weight.

Proline accumulation was induced in turgid barley leaves by ABA addition. When the amount of ABA added to leaves was varied, it was observed that a level of 0.3 nanomole ABA per gram fresh weight for a period of about 2 hours was required before proline accumulation was induced. However, the rate of proline accumulation was slower in ABA-treated leaves than in wilted leaves at comparable ABA levels. Thus, the threshold level of ABA for proline accumulation appeared to be similar for wilted leaves where ABA increased endogenously and for turgid leaves where ABA was added exogenously. However, the rate of proline accumulation was more dependent on ABA levels in turgid leaves to which ABA was added exogenously than in wilted leaves.

Salt-induced proline accumulation was not preceded by increases in ABA levels comparable to those observed in wilted leaves. Levels of less than 0.2 nanomole ABA per gram fresh weight were measured 1 hour after exposure to salt and they declined rapidly to the control level by 3 hours. Proline accumulation commenced at about 9 hours. Thus, ABA accumulation did not appear to be involved in salt-induced proline accumulation.

     

Senescence in Detached Oat Leaves I. Changes in Free Amino Acid Levels

Changes in the levels of free amino acids have been measuredduring light and dark senescence of oat leaf segments. Leaveswere aged either on water, 5 ppm kinetin or 30 ppm abscisicacid. The patterns with which levels of individual amino acidschange differ a great deal in leaves senescing either in darkor light, signifying that different mechanisms may regulateoat leaf senescence in light and dark. Levels of serine andmost of the other amino acids that increase substantially duringdark senescence of oat leaves change parallel to mitochondrialrespiration. Kinetin depresses the rise in amino acids justas it does with respiration in the dark. The synthesis of serineproteases does not seem to be limited by the availability ofendogenous serine. The levels of glutamine increase dramaticallyin leaves kept in light (ca. 2,200% of initial value within7 days) but only a little in the dark, which may reflect a possiblerole of photorespiration during the senescence of oat leavesin the light. Abscisic acid enhances the release of amino acidsmore strongly in the light than dark. The senescence promotingeffect of abscisic acid in the light seems to bring about changesin amino acid levels similar to those that appear in leavessenescing on water in the dark. ¹ Present address: C.F. Kettering Research Laboratory, 150 EastSouth College Street, Yellow Springs, Ohio 45387, U.S.A. (Received June 24, 1981; Accepted October 30, 1981)     

Osmotic Stress in Coleoptiles and Primary Leaves of Wheat. II. Main Phosphorylated Fractions

Seedlings of wheat (Triticum durum, cv. Balcarceno-INTA) werewater-stressed in darkness with 20% polyethylene glycol (PEG)6000 or 0.3 M mannitol added to the root medium. At differenttimes and up to a total of 36 h of treatment the coleoptilesand primary leaves were cut and analysed for acid soluble-P,lipid-P, protein-P, alkali-stable organic-P, and nucleic acid-P.All the phosphorylated fractions were expressed on 100 mg oftotal coleoptile and primary leaves dry wt. Acid soluble-P (14.6µmol) accounted for most of the phosphorus. The lipid-Pfraction remained at an unchanged level (6.2 µmol/100mg dry wt) in control or in mildly osmotic stressed shoots ofseedlings over the period of treatment. The protein-P representedonly 2.4% (0.8 µmol) of the total phosphorus found incoleoptiles and primary leaves. The alkali-stable organic-Pand nucleic acid-P fractions represented 22.2% (7.2µ mol)and 11.1% (3.6 µmol) of the total phosphorus content ofshoots, respectively. All the phosphorylated fractions, exceptingthe lipid-P fraction, underwent a significant (P < 0.025)fall over periods of up to 36 h of wheat seedlings growth indarkness. A common characteristic found in all the phosphorylatedfractions was the fact that there were not significant differencesbetween control and 20% PEG 6000 or 0.3 M mannitol treated seedlingsduring 36 h of treatment and growth in darkness. However, whenseedlings were pulse-labelled with 32P during imbibition, someeffects of the osmotic stressants on several fractions couldbe seen. Specific radioactivity fell in acid soluble-P of controlshoots, but increased in 20% PEG 6000 and in 0.3 M mannitoltreated seedlings. Radioactive phosphorus was not found in thealkali-stable organic-P fraction. Lipid-P, nucleic acid-P, andprotein-P fractions increased their specific activities during36 h of shoot growth in control plants. Such 20% PEG 6000 and0.3 M mannitol restricted this increase during the same periodof time, the former being more active. Possible implicationsof phospholipid and phosphoprotein turnover in relation to waterstress are discussed.