Influence of natural substances of phenolic character and diethyldithiocarbamate on the metabolism of L-tryptophan in cabbage,maize and pea

The effect of phenolic substances isolated earlier from cabbage, maize and pea on L-tryptophan-3-¹⁴G (L-Try-¹⁴C) metabolism in those plants was investigated. For the sake of comparison the effect of diethyldithiocarbamate (DIECA) on cabbage was also observed. A phenolic substance of unknown structure isolated from cabbage was utilised in a 0.5 mg/ml concentration, p-coumarie acid (PCA) from maize in 0.7 mg/ml concentration and quercetin-glucosyl-coumarate (QGC) found in pea in 8 mg/ml concentration were used. The chosen concentrations were on the limit of their inhibitory effect on the growth of the respective plant apical segments. The effect of DIECA was investigated at 0.1 mg/ml concentration. Using non-labelled L-Try as substrate (5 × 10^?3 M) biological tests have shown that QGC and DIECA have a weaker but distinct effect on the increase in auxin level (+ 17% and + 15%, respectively). All phenolic compounds, as well as DIECA increase the intake of L-Try-¹⁴C from the incubation solutions. Phenolic substances decrease the conversion of L-Try-¹⁴C to its metabolites. The changes were studied after a 20 hour period of L-Try-¹⁴C metabolism. On chromatograms changes in the proportion of L-Try-¹⁴C metabolites took place, especially in the areas of substances of complex character from which IAA splits off easily,i.e. in the areas of 3-indolylacetylglucose and 3-indolylaeetamide. The radioactivity of these areas is generally decreased (e.g. the decrease of radioactivity in the 3-indolylacetylglucose area under the influence of the phenolic substance ofBrassica plants is ?7,4%, under the influence of PCA in maize it is —8.9% and under the influence of QGC in pea it is ?17.1% DIECA also decreased the radioactivity of this zone, by ?10,5%. In cabbage a large part of L-Try-¹⁴C was transformed to glucobrassicin; its biosynthesis increases in the presence of the phenolic substance of cabbage by +3%, in the presence of DIECA by +27%. The results are discussed in a working hypothesis based on the key formation of IAA, accompanied by its oxidation and detoxication.     

Biogenesis of indole compounds from D- and L-tryptophan in segments of etiolated seedlings of cabbage,maize and pea

The metabolism of D-and L-tryptophan-3-¹⁴C (Try-3-¹⁴C) was studied and compared for three different plant species, cabbage, maize and pea. Apical segments of the seedlings were incubated for 6 hours in solutions of L- or D-Try-3-¹⁴C (1·5 μc/ml) with the addition of chloramphenicol (10^?4g/ml) and then allowed to stand for another 20 hours in moist chambers. The methanolic extract of the tissues was analyzed radiochromatographically and by paper electrophoresis in combination with biological tests. Chloramphenicol in a concentration of 10^?4 g/ml had little influence on the growth of the segments, though the antibiotic slightly decreased the uptake of L-Try, it did not prevent the formation of IAA from L-Try. In the segments of cabbage the following metabolites were formed from L-Try-3-¹⁴C (accounting for 52% of the activity of the chromatographically separated extract): glucobrassicin (26·0%), neoglucobrassicin (3·6%), a spot corresponding according to its Rf to 3-indolylacetamide (IAAmide—10·9%), β-glucoside of 3-indolylacetic acid (IAGluc—3·3%) and traces of 3-indolylacetonitrile (IAN), IAA and indole-3-carboxylic acid (total 5%). In maize segments L-Try-3-¹⁴C (53·0%) was transformed to several unidentified hydrophilic substances, one of them possessing auxin activity (total amount 6·9%), IAGlue (9·3%) accompanied by a small amount of tryptamine, a spot corresponding according to its Rf to IAAmide (16·5%), IAA and another unidentified hydrophobic substance (4·1%). In pea segments L-Try-3-¹⁴C (66·7%) gave a zone corresponding according to its Rf to IAAmide (20·0%), a substance similar to IAGluc (10·5%) and also hydrophobic substances (3·1%) containing traces of IAA, which could be demonstrated only by bioassay. D-Try is metabolised in the three plants by the virtually exclusive formation of malonyltryptophan.     

Effects of phenolic substances on metabolism of exogenous indole-3-acetic acid in maize stems

Four-day-old stem segments of Zea mays L. cv. Seneca 60 were treated sequentially with phenolic substances and indole-3-acetic [2-¹⁴C] acid ([2-¹⁴C]IAA). Formation of bound IAA was rapid, but a pretreatment with p-coumaric acid, ferulic acid or 4-methylumbelliferone decreased the level of bound IAA. The decrease is not likely related to the effect of the phenolics on enzymic oxidation of IAA, since the level of free IAA was not limiting and the activity of ferulic acid in enzymic oxidation of IAA is different from that of p-coumaric acid and 4-methylum-belliferone. Apparently these compounds inhibited the formation of bound IAA and consequently caused an accumulation of free IAA. In contrast, caffeic acid, protocatechuic acid and 2,3-dihydro-2, 2-dimethyl-7-benzofuranol had little effect. After the uptake of IAA there was a slow but steady incorporation of the radioactivity into the 80% ethanol-insoluble, 1 M NaOH-soluble fraction. Phenolic substances also affected this process. The compounds which are cofactors of IAA-oxidase increased the incorporation while those which are inhibitors of IAA-oxidase decreased the incorporation. Results suggest that the phenolics also affected the enzymic oxidation of IAA in vivo in the same way as in vitro.     

Effects of natural and synthetic neuroactive substances on the growth and feeding of cabbage looper, Trichoplusia ni

In this study we investigated the effects of two naturally occurring beta-carboline alkaloids and two synthetic tricyclic antidepressants on the growth and food consumption of fifth instar larvae of the cabbage looper, Trichoplusia ni Hübner (Lepidoptera: Noctuidae). In artificial diets at high concentrations (3,000 ppm), harmane, amitriptyline, and imipramine reduce growth and feeding; harmane reduced feeding consistently at a lower concentration (200 ppm). In animals other than insects, beta-carboline alkaloids inhibit monoamine oxidase (MAO) activity and thus affect rates of disposition of serotonin and other monoamine neurotransmitters. Because brain serotonin levels are associated with variation in rates of carbohydrate and protein intake in insects, the effects of beta-carboline alkaloid ingestion on dietary self-selection behavior were examined. Choosing between diets lacking carbohydrate but containing protein and diets lacking protein but containing carbohydrate, larvae consumed a greater proportion of diet containing protein but lacking carbohydrate in the presence of harmane than in its absence. These results are consistent with beta-carboline alkaloid-mediated persistence of serotonin in the brain due to MAO inhibition. Alternatively, these results could reflect alkaloid-mediated peripheral inhibition of sucrose taste receptors influencing ingestive behaviors. That beta-carboline alkaloid ingestion is associated with changes in feeding behavior is consistent with a possible defensive role for these compounds in plant foliage.     

Growth-correlative effect of the root on the apical part of the epicotyl in pea seedlings regarding the IAA content and L-tryptophan aminotransferase and L-tryptophan dehydrogenase activities

In five-day-old etiolated pea seedlings an increase in IAA content and in L-tryptophan aminotransferase (TAT) and L-tryptophan dehydrogenase (TDH) activities in the apical part of the epicotyl was found 12 h after root excision, which was followed by a decrease up to 48 h. In ten-day-old seedlings a continuous decrease in IAA, TAT and TDH levels in the apical part of the epicotyl was recorded up to 48 h after root excision. This indicates a growth-correlative effect of the root becoming evident in dependence on plant ontogenesis in changes of IAA content and activities of the above enzymes catalysing IAA synthesis.     

Influence of arbuscular mycorrhizae on the metabolism of maize under drought stress

A greenhouse experiment was carried out to investigate the influence of the arbuscular mycorrhizal (AM) fungus (Glomus intraradices Schenck & Smith) on metabolic changes in tropical maize (Zea mays L.) under drought. Two cultivars, Tuxpeno sequia CO (drought sensitive) and C8 (drought resistant), were subjected for 3 weeks to water stress following tasselling (75–95 days after sowing). Fully expanded 7th or 8th leaves were sampled and assessed for levels of chlorophyll, sugars, proteins, and amino acids. Chlorophyll content was not altered either by water stress or the presence of mycorrhizae. Mycorrhizal plants (M+) had higher total and reducing sugars than nonmycorrhizal plants (M-) at the end of 3 weeks of the drought cycle. An increase in protein content was observed with drought stress in M + plants of the cultivar C0. Most of the amino acids showed a linear increase during the period of water stress in M+ and M- plants for both cultivars. Total amino acids increased by 40.6% and 43.7% in M- plants of C0 and C8, respectively. With the presence of AM fungus, amino acid levels increased by only 10.7% and 19.2% of leaf dry mass in C0 and C8, respectively. Alanine, asparagine, glutamine, and glycine accounted for 70% of the amino acid pool. Under drought, AM inoculation enabled the plants to retain considerable amounts of sugars and proteins, especially in the drought-sensitive cultivar C0. This may be of physiological importance in helping the plant to withstand moderate drought.     

Cell wall remodeling under salt stress: Insights into changes in polysaccharides,feruloylation, lignification,and phenolic metabolism in maize

Although cell wall polymers play important roles in the tolerance of plants to abiotic stress, the effects of salinity on cell wall composition and metabolism in grasses remain largely unexplored. Here, we conducted an in-depth study of changes in cell wall composition and phenolic metabolism induced upon salinity in maize seedlings and plants. Cell wall characterization revealed that salt stress modulated the deposition of cellulose, matrix polysaccharides and lignin in seedling roots, plant roots and stems. The extraction and analysis of arabinoxylans by size-exclusion chromatography, 2D-NMR spectroscopy and carbohydrate gel electrophoresis showed a reduction of arabinoxylan content in salt-stressed roots. Saponification and mild acid hydrolysis revealed that salinity also reduced the feruloylation of arabinoxylans in roots of seedlings and plants. Determination of lignin content and composition by nitrobenzene oxidation and 2D-NMR confirmed the increased incorporation of syringyl units in lignin of maize roots. Salt stress also induced the expression of genes and the activity of enzymes enrolled in phenylpropanoid biosynthesis. The UHPLC–MS-based metabolite profiling confirmed the modulation of phenolic profiling by salinity and the accumulation of ferulate and its derivatives 3- and 4-O-feruloyl quinate. In conclusion, we present a model for explaining cell wall remodeling in response to salinity.     

Studies on plant growth-regulating substances: The metabolism of indole-3-acetonitrile and related compounds in wheat and pea tissues

The plant growth-regulating activity of a number of new indole derivatives is reported. It is shown that indole-3-acetonitrile (IAN) is converted to indole-3-carboxylic acid by metabolism within wheat and pea tissues, and the mechanism of this a-oxidation reaction has been studied. The relevant indole compounds were synthesized and their metabolism investigated by T.L.C. techniques. N -Methylindole-3-acetonitrile was also shown to be degraded by a-oxidation in wheat and pea tissues and this was separately investigated. While no definite conclusions can be drawn, the evidence indicates that conversion of indole- and Af-methylindole-3-acetonitriles to the corresponding indole-3-aldehyde-cyanohydrins can occur. These compounds then become metabolized to the aldehydes and then to the respective indole-3-carboxylic acids. Indole- and A⁷-methylindole-3-glyoxylic acids do not appear to be involved in the a-oxidation reaction to any significant extent. Relevant studies on the metabolism of indole-3-acetaldehyde-cyanohydrin are also described.     

Purification and characterization of ferritins from maize, pea, and soya bean seeds. Distribution in various pea organs

Ferritins from maize, pea, and soya bean seeds were purified. They contain two polypeptides of 28 and 26.5 kDa. The molecular weight of native pea seed ferritin has been estimated to be 540,000. Pea and maize seed ferritins were compared by reverse phase high performance liquid chromatography, amino acid composition, and two-dimensional gel electrophoresis. They are very similar, although four isoforms of the 28-kDa polypeptide from the pea were observed in contrast to a unique polypeptide in maize. No isoforms of the 26.5-kDa polypeptide were detected. Rabbit antibodies were produced in response to pea seed ferritin. It was shown by Western blot analysis that ferritins of the three plants analyzed share immunological determinants. However, horse spleen ferritin was not recognized by the phytoferritin antibodies. Antibodies were also used to demonstrate that ferritins are not uniformly distributed in different pea organs from 30-day-old iron-unloaded plants. The protein was more abundant in flowers than in fruits and roots, and was not detected in leaves.     

Effect of BCAA intake during endurance exercises on fatigue substances,muscle damage substances,and energy metabolism substances

The increase rate of utilization of branched-chain amino acids (BCAA) by muscle is reduced to its plasma concentration during prolonged exercise leading to glycogen. BCAA supplementation would reduce the serum activities of intramuscular enzymes associated with muscle damage. To examine the effects of BCAA administration on fatigue substances (serotonin, ammonia and lactate), muscle damage substances (CK and LDH) and energy metabolism substances (FFA and glucose) after endurance exercise. Subjects (n = 26, college-aged males) were randomly divided into an experimental (n = 13, EXP) and a placebo (n = 13, CON) group. Subjects both EXP and CON performed a bout of cycle training (70% VO₂max intensity) to exhaustion. Subject in the EXP were administrated BCAA (78ml/kg·w) prior to the bout of cycle exercise. Fatigue substances, muscle damage substances and energy metabolism substances were measured before ingesting BCAAs and placebos, 10 min before exercise, 30 min into exercise, immediately after exercise, and 30 min after exercise. Data were analyzed by two-way repeated measure ANCOVA, correlation and statistical significance was set at p < 0.05. The following results were obtained from this study; 1. In the change of fatigue substances : Serotonin in the EXP tended to decreased at the 10 min before exercise, 30 min into exercise, post exercise, and recovery 30 min. Serotonin in the CON was significantly greater than the EXP at the10 min before exercise and recovery 30. Ammonia in the EXP was increased at the 10 min before exercise, 30 min into exercise, and post exercise, but significantly decreased at the recovery 30min (p < 0.05). Ammonia in the CON was significantly lower than the EXP at the 10 min before exercise, 30 min into exercise, and post exercise (p < 0.05). Lactate in the EXP was significantly increased at the 30 min into exercise and significantly decreased at the post exercise and recovery 30 min. Lactate in the CON was significantly lower than the EXP at the post exercise (p < 0.05). 2. In the change of muscle damage substances : CK in the EXP was decreased at the 10 min before exercise and increased at the 30 min into exercise and then decreased at the post exercise and recovery 30 min. CK in the CON was greater than the EXP. LDH in the EXP was decreased at the 10 min before exercise and increased at the 30 min into exercise and then decreased at the post exercise and recovery 30 min. LDH in the CON was higher than the EXP. 3. In the change of energy metabolism substances :Glucose in the EXP tended to decrease at the 10 min before exercise, 30 min into exercise, post exercise and recovery 30 min. Glucose in the CON was significantly greater than the EXP at the recovery 30 min (p < .05). FFA in both EXP and CON was increased at the post exercise and recovery 30 min. % increase for FFA in the EXP was greater than the CON at the post exercise and recovery 30 min. 4. The relationship of the fatigue substances, muscle damage substances and energy metabolism substances after endurance exercise indicated strongly a positive relationship between LDH and ammonia and a negative relationship between LDH and FFA in the EXP. Also, there were a strong negative relationship between glucose and FFA and a positive relationship between glucose and serotonin in the EXP. There was a strong positive relationship between CK and LDH and a strong negative relationship between FFA and glucose in the CON. These results indicate that supplementary BCAA decreased serum concentrations of the intramuscular enzymes as CK and LDH following exhaustive exercise. This observation suggests that BCAA supplementation may reduce the muscle damage associated with endurance exercise.     

Ocean acidification and the loss of phenolic substances in marine plants

Rising atmospheric CO(2) often triggers the production of plant phenolics, including many that serve as herbivore deterrents, digestion reducers, antimicrobials, or ultraviolet sunscreens. Such responses are predicted by popular models of plant defense, especially resource availability models which link carbon availability to phenolic biosynthesis. CO(2) availability is also increasing in the oceans, where anthropogenic emissions cause ocean acidification, decreasing seawater pH and shifting the carbonate system towards further CO(2) enrichment. Such conditions tend to increase seagrass productivity but may also increase rates of grazing on these marine plants. Here we show that high CO(2) / low pH conditions of OA decrease, rather than increase, concentrations of phenolic protective substances in seagrasses and eurysaline marine plants. We observed a loss of simple and polymeric phenolics in the seagrass Cymodocea nodosa near a volcanic CO(2) vent on the Island of Vulcano, Italy, where pH values decreased from 8.1 to 7.3 and pCO(2) concentrations increased ten-fold. We observed similar responses in two estuarine species, Ruppia maritima and Potamogeton perfoliatus, in in situ Free-Ocean-Carbon-Enrichment experiments conducted in tributaries of the Chesapeake Bay, USA. These responses are strikingly different than those exhibited by terrestrial plants. The loss of phenolic substances may explain the higher-than-usual rates of grazing observed near undersea CO(2) vents and suggests that ocean acidification may alter coastal carbon fluxes by affecting rates of decomposition, grazing, and disease. Our observations temper recent predictions that seagrasses would necessarily be "winners" in a high CO(2) world.