Role of Calcium on Phenolic Compounds and Enzymes Related to Lignification in Soybean (Glycine max L.) Root Growth

Changes in soluble and cell wall bound peroxidases activities, phenylalanine ammonia-lyase activity and phenolic compounds and lignin contents in roots of calcium-treated soybean (Glycine max (L.) Merr.) seedlings and their relationships with root growth were investigated. Three-day-old soybean seedlings were cultivated in nutrient solution with or without 0.025–5.0 mM calcium for 24 h. In general, length and fresh and dry weights of roots increased, while activities of enzymes (soluble and cell-wall peroxidases and phenylalanine ammonia-lyase) and phenolic compounds and lignin contents decreased against calcium concentrations. In the absence of calcium, phenylalanine ammonia-lyase and peroxidases activities increased by accumulating phenolic compounds and lignin due to restricted growth of roots. Enhanced calcium supply reduced the production of phenolic compounds and lignification due to low phenylalanine ammonia-lyase and peroxidases activities, reinforcing the essential role of calcium to improve the soybean root growth.     

Allocation of proteolytic activity in the seedling of Cynara cardunculus L. in the initial growth stages

Cynara cardunculus L. seeds were germinated in vitro under environmentally controlled conditions. Seeds showed a 60% germination rate, and three growth stages were established based on the seedling mean relative growth rate (RGR). Root, stem and cotyledons were compared in these stages with respect to the emergence of total proteases and cardosin activity and its allocation in the seedling. In growth stage I (1st-5th post-germinative days), seedlings grew very slowly. Total proteases and cardosins were already active at the onset of seedlings in the stem. Total soluble protein remained constant in cardoon seedlings during stage I, and the content of all free amino acids (aa) but proline (Pro) was equally allocated on the 1st post-germinative day. In growth stage II (5th-10th post-germinative days), seedlings grew intensively and exhibited fully developed cotyledons. A pronounced increase in the content of all free aa up to the middle of growth stage II in both stems and roots was observed. In addition, the allocation of the total proteolytic activity and cardosins followed a gradient from the root to the seedling shoot. However, the whole seedling soluble protein remained constant up to the 7th day in and tended to peak on the 10th post-germinative day, being allocated mainly to the seedling stem. In growth stage III (10th-15th post-germinative days), cardoon seedlings exhibited the lowest mean RGR and the highest R/S growth ratio. An intensive degradation of total soluble protein present in the whole seedling except for cotyledons (ca. 5-fold) was observed. Nevertheless, in growth stage III, both the gradients exhibited by total proteases and cardosins activities between the root and the seedling shoot were enhanced, as were contents of all aa except Pro, exhibiting the highest levels in cotyledons on the 15th post-germinative day.     

Changes in trigonelline (N-methylnicotinic acid) content and nicotinic acid metabolism during germination of mungbean (Phaseolus aureus) seeds

Changes in trigonelline content and in biosynthetic activity were determined in the cotyledons and embryonic axes of etiolated mungbean (Phaseolus aureus) seedlings during germination. Accumulation of trigonelline (c. 240 nmol per pair of cotyledons) was observed in the cotyledons of dry seeds; trigonelline content decreased 2 d after imbibition. Trigonelline content in the embryonic axes increased with seedling growth and reached a peak (c. 380 nmol per embryonic axis) at day 5. Trigonelline content did not change significantly during the differentiation of hypocotyls, and the concentration was greatest in the apical 5 mm. Nicotinic acid and nicotinamide were better precursors for pyridine nucleotide synthesis than quinolinic acid, but no great differences were found in the synthesis of trigonelline from these three precursors. Trigonelline synthesis was always higher in embryonic axes than in cotyledons. Activity of quinolinate phosphoribosyltransferase (EC 2.4.2.19), nicotinate phosphoribosyltransferase (EC 2.4.2.11), and nicotinamidase (EC 3.5.1.19) was found in cotyledons and embryonic axes, but no nicotinamide phosphoribosyltransferase (EC 2.4.2.12) activity was detected. It follows that quinolinic acid and nicotinic acid were directly converted to nicotinic acid mononucleotide by the respective phosphoribosyltransferases, but nicotinamide appeared to be converted to nicotinic acid mononucleotide after conversion to nicotinic acid. Trigonelline synthase (nicotinate N-methyltransferase, EC 2.1.1.7) activity increased in the embryonic axes, but decreased in cotyledons during germination. [14C]Nicotinic acid and trigonelline absorbed by the cotyledons were transported to the embryonic axes during germination. Trigonelline had no effect on the growth of seedlings, but nicotinic acid and nicotinamide significantly inhibited the growth of roots. Based on these findings, the role of trigonelline synthesis in mungbean seedlings is discussed.     

The Effect of L-Phenylalanine on Phenylalanine Ammonia-lyase and Cell Division in Artichoke Callus Culture

5 x 10^–5 M L-phenylalanine overcame the inhibitory effectof white light on cell division in artichoke callus culturesand increased extractable phenylalanine ammonia-lyase (PAL)activity compared to cultures grown in the presence of 5 x 10^–4M phenylalanine The lower concentration of the amino acid alsoenhanced rates of uptake and incorporation of ¹⁴C labelled phenylalaninethroughout G₁ and S. Differences between the two concentrationswere greatest during S with a 4-fold increase in uptake anda 3-fold increase in incorporation It is suggested thereforethat the capacity of 5 x10^–5 M phenylalanine to offsetthe light effect is due to an indirect stimulatory effect onamino acid and protein metabolism Increased levels of extractablePAL activity would then be reflected by this general stimulationof protein synthesis. Helianthus tuberosus L, Jerusalem artichoke, callus culture, cell division, phenylalanine ammonia-lyase     

Control of phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase in gherkin tissues

Blue light mediates a transient increase in the extractable activity of phenylalanine ammonia-lyase from both cotyledons and hypocotyls of etiolated gherkin seedlings, but concurrent changes in extractable cinnamic acid 4-hydroxylase activity only occur in cotyledons. Excision, followed by incubation in the dark, also results in stimulation of the lyase activity in both tissues, but the hydroxylase activity is only stimulated in cotyledons, again concurrently with the lyase. Stimulated levels of hydroxycinnamic acid esters are, however, only formed following light treatment, indicating the presence of another light-sensitive step in their biosynthesis. Treatment of gherkin tissues with 2-aminooxyacetic acid or α-aminooxy-β-phenylpropionic acid inhibits phenylalanine ammonia-lyase activity in situ, reduces the accumulation of hydroxycinnamic acid esters and presumably reduces the endogenous cinnamic acid pool. This treatment increases extractable lyase activity and delays its peak in activity. In cotyledons, these changes in the lyase are associated with concurrent and similar changes in extractable hydroxylase activity, whilst in hypocotyls the hydroxylase is relatively unaffacted. The increase in phenylalanine ammonia-lyase activity following excision of cotyledons and hypocotyls is prevented by cinnamic acid; in cotyledons the hydroxylase is similarly affected, but after a longer lag. Thus whilst cinnamic acid can modify the extractable activity of the lyase, it cannot itself mediate changes in the extractable activity of the hydroxylase.     

Phenylalanine ammonia lyase and cinnamic acid hydroxylases as assembled consecutive enzymes on microsomal membranes of cucumber cotyledons: Cooperation and subcellular distribution

1. Cooperation between phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and cinnamic acid hydroxylases was investigated using microsomal fractions from cotyledons of cucumber (Cucumis sativus L.). The interpretations were based on experiments which demonstrate a limited exchange between the pool of cinnamic acid formed by the membrane-bound phenylalanine ammonia-lyase and the cinnamic acid pool external to the enzyme-membrane system. 2. The extent of cooperation between the microsomal enzymes was proved to be influenced by treatment of the cotyledons with light. On exposure to UV-light, which is known to enhance greatly the soluble phenylalanine ammonia-lyase activity in cell cultures, differential effects on the levels of microsomal and soluble phenylalanine ammonia-lyase, and of cinnamic acid hydroxylases, were observed. The time course of the enzyme activities and their cooperation in vitro after treatment of the cotyledons with light were studied. 3. The extent of cooperation in vitro was found to vary depending on the concentration of L-phenylalanine. 4. Homogenates obtained from etiolated cotyledons of Cucumis sativus in the absence of Mg²⁺ were fractionated by sucrose density gradient centrifugation and examined for phenylalanine ammonia-lyase, cinnamic acid o-hydroxylase, cinnamic acid o-hydroxylase, and several marker enzymes. Ammonia-lyase activity was highest in fractions with 25% sucrose, in which primarily smooth endoplasmic reticulum is localized. Hydroxylase activities co-occur with phenylalanine ammonia-lyase in these fractions (density=1.100 g/cm³), and also in fractions at higher densities (d=1.12–1.13 and 1.15 g/cm³).Abbreviations PAL L-phenylalanine ammonia-lyase - Tris tris-(hydroxymethyl)aminomethane - EDTA ethylenediamine tetraacetic acid - ATPase ATP phosphohydrolase     

Differential response of cultured parsley cells to elicitors from two non-pathogenic strains of fungi. 2. Effects on enzyme activities

Parsley cell cultures produce linear furanocoumarins and the linear benzodipyrandione, graveolone, in response to treatment with an elicitor from either Phytophthora megasperma or Alternaria carthami. Activities of enzymes involved in general phenylpropanoid metabolism, phenylalanine ammonia-lyase and 4-coumarate: CoA ligase, as well as of an enzyme involved specifically in furanocoumarin biosynthesis, dimethylallyl diphosphate: umbelliferone dimethylallyltransferase, were monitored over several days after treatment with A. carthami elicitor. In addition, the activities of chalcone synthase, an enzyme involved in flavonoid formation, and of glucose-6-phosphate: NADP 1-oxidoreductase were also monitored. The lyase and the ligase activities increased steadily for 48 h and the dimethylallyltransferase activity for 54 h, while the synthase activity was not altered and the oxidoreductase activity decreased gradually. In some experiments, phenylalanine ammonia-lyase activity reached a maximum value of 250 mukat/kg, twice the maximal activity observed previously in parsley cells after treatment with either ultraviolet light or an elicitor preparation from P. megasperma. In crude extracts, phenylalanine ammonia-lyase activity was shown to be inhibited by unidentified small-molecular-weight compounds which were formed in proportion to the elicitor treatment. While phenylalanine ammonia-lyase and dimethylallyl diphosphate: umbelliferone dimethylallyltransferase are known to be required for furanocoumarin biosynthesis, the involvement of 4-coumarate: CoA ligase is as yet unclear. The concomitant increase and decrease of the ligase activity with the activities of the lyase and the dimethylallyltransferase, as well as its similar response to elicitor concentrations, suggest that CoA esters of cinnamic acids play a role in the biosynthesis of furanocoumarins.     

Developmental Effects of Sandoz 6706 on Activities of Enzymes of Phenolic and General Metabolism in Barley Shoots Grown in the Dark or under Low or High Intensity Light

The activities of three enzymes of phenolic biosynthesis and six of general metabolism were studied at 24-hour intervals between the 3rd and 8th day after planting in barley shoots treated with the chlorosis-inducing herbicide Sandoz 6706 and grown in the dark or under high or low intensity light. The herbicide had no effect on fresh weight or soluble protein (per shoot) in plants grown in the dark or under low intensity light, but slightly decreased these parameters in plants grown for more than 5 days under high intensity light. In dark-grown seedlings the herbicide had no detectable effects on plastid ultrastructure or on the activity of malate dehydrogenase, cytochrome c oxidase, NADP-cytochrome c reductase, triose phosphate isomerase, peroxidase, catalase, shikimate dehydrogenase, phenylalanine ammonia-lyase, or chalcone-flavanone isomerase. Under low intensity light, Sandoz 6706-treated plants developed plastids with single thylakoids extending across the organelle, and the activity of all enzymes examined was increased to varying degrees. When the herbicide-treated plants were grown under high intensity light, plastid lamellar organization was severely disrupted. Activities of shikimate dehydrogenase and chalcone-flavanone isomerase were markedly enhanced, phenylalanine ammonia-lyase activity slightly promoted, and catalase activity severely inhibited. The other enzymes were not appreciably affected by Sandoz 6706 under high intensity light. It is concluded that the changes in plastid ultrastructure and enzyme activities of the herbicide-treated plants are largely secondary photomorphogenetic or photooxidative responses in the carotenoid-free plants in which chlorophylls accumulate in reduced amounts (low intensity light) or are completely absent (high intensity light).     

The effect of phenylalanine on biosynthesis of protoberberine alkaloids in the cell culture of low meadow-rue

The addition of 7 mM phenylalanine to the nutrient medium for low meadow-rue (Thalictrum minus L.) cell culturing on the 7th or 8th day doubled berberine secretion into medium. Simultaneously, the content of phenolic compounds increased in the cells and medium. Investigation of phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) activities showed that exogenous Phe activated PAL by 35% and inactivated TAL by 20%. When the crude extract was separated on DEAE-Sephacel column, two proteins were isolated. One of them displayed both PAL and TAL activities, whereas another protein displayed only PAL activity. This activity disappeared after cell culturing longer than 20 days and also under the effect of Phe at a concentration reducing alkaloid biosynthesis. Phe addition to medium also increased the content of protein in both the cells and culture medium. The proportion of low-molecular proteins in the medium increased. Testing antimicrobial activity of the medium showed that it was determined by berberine and to a lesser degree by palmatine. Protein fraction also demonstrated antimicrobial activity. An improved antimicrobial activity after Phe adding to medium resulted from alkaloid and protein accumulation. The conclusion was made that one of the mechanisms of Phe action was the control of alkaloid biosynthesis with the involvement of the enzyme system of the early steps of the phenylpropanoid pathway, which, in its turn, is one of the stages in stress-induced plant response to pathogen action.     

Light Control of Anthocyanin Biosynthesis in Zea Seedlings

Evidence for involvement of two non-photosynthetic pigments in photoinduction of anthocyanin biosynthesis in the roots and mesocotyls of Zea mays L. seedlings is presented. Short (5 min), low energy (4.5 × 10³ J m^?2) fluences of red light neither induced anthocyanin synthesis nor enhanced phenylalanine ammonia-lyase activity in dark-grown maize seedlings. Little anthocyanin synthesis and no enhancement of phenylalanine ammonia-lyase activity was induced by continuous far-red light. Continuous white or blue light induced both anthocyanin synthesis and enhanced phenylalanine ammonia-lyase activity. These results show that phytochrome alone cannot induce anthocyanin synthesis in maize seedlings. However, a strong phytochrome mediation of white light induced pigment synthesis was demonstrated. This effect was not demonstrable with white light enhanced phenylalanine ammonia-lyase activity, indicating that phytochrome controls another step in anthocyanin biosynthesis.     

Effect of GA3, kinetin and indole acetic acid on carbohydrate metabolism in chickpea seedlings germinating under water stress

Enhanced amylase activity was observed during a 7-day-growth period in the cotyledons of PEG imposed water stressed chickpea seedlings grown in the presence of GA₃ and kinetin, when compared with stressed seedlings. During the first 5 days of seedling growth, the seedlings growing under water deficit conditions as well as those growing in the presence of PGRs had a higher amylase activity in shoots than that of control seedlings. Neither GA₃ nor kinetin increased the amylase activity of roots whereas IAA reduced root amylase activity. Activity of acid and alkaline invertases was maximum in shoots and at a minimum in cotyledons. Compared with alkaline invertase, acid invertase activity was higher in all the tissues. The reduced acid and alkaline invertase activities in shoots of stressed seedlings were enhanced by GA₃ and kinetin. Roots of stressed seedlings had higher alkaline invertase activity and GA₃ and IAA helped in bringing the level near to those in the controls. GA₃ and kinetin increased the sucrose synthase (SS) and sucrose phosphate synthase (SPS) activities in cotyledons of stressed seedlings, whereas they brought the elevated level of SPS of stressed roots to near normal level. The higher level of reducing sugars in the shoots of GA₃ and kinetin treated stressed seedlings could be due to the high acid invertase activity observed in the shoots, and the high level of bound fructose in the cotyledons of stressed seedlings could be due to the high activity of SPS in this tissue.     

Amino-acid biosynthesis in the cotyledons of Sinapis alba L. in darkness and far-red light studied by deuterium labelling and mass spectrometry

The incorporation of deuterium from deuterium oxide into the free amino acids of the cotyledons of Sinapis alba L. was studied by gas chromatography-mass spectrometry and was similar, both qualitatively and quantitatively, after incubation of the seedlings in darkness or far-red light. The results support studies which show that phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is synthesised de novo, rather than activated, in response to far-red light.Abbreviations GC-MS Gas chromatography-mass spectrometry - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - HFB n-propyl heptafluorobutyryl n-propyl     

Photoregulation of Nicotinamide Adenine Dinucleotide Kinase Activity in Cell-free Extracts

This article gives evidence that NAD kinase activity is controlled by the action of phytochrome. The NADP level rapidly increased in the cotyledons of seedlings of Pharbitis nil strain Violet (a short day plant), when the inductive dark for flowering was interrupted with a 5-minute illumination of red light. Illumination with far red light immediately after illumination with red light counteracted partly the effect of the latter.     

STUDIES ON THE SOLUBLE CARBOHYDRATES AND CARBOHYDRATE PRECURSORS IN GERMINATING SOYBEAN SEED

The total soluble carbohydrate fraction of the cotyledons and embryo axis of germinating soybean seedlings declined rapidly during the first 3 days of germination. This depletion began earlier in the embryo axis than in the cotyledon. The total carbohydrate content of the cotyledons of plants grown in light and plants grown in dark was approximately the same for the first 7 days of germination. Between day 9 and 13 the total carbohydrate content of the cotyledons of soybean seedlings grown in dark was higher than that of plants grown in light. The reducing sugar content of light-grown soybean cotyledons increased approximately 5-fold during the first 9 days of germination, whereas that of dark-grown soybean cotyledons increased more slowly during this interval. Reducing sugars in the embryo increased during the early stages of germination until they approximately equalled the total carbohydrate. Between day 4 and 13, oil was depleted more rapidly in the cotyledons of seedlings grown in light than those grown in the dark. The reserve carbohydrates of soybean embryos and cotyledons consisted primarily of low molecular weight oligosaccharides, particularly sucrose, stachyose, and raffinose. These compounds decreased rapidly during germination. The isocitritase activity in the cotyledons of germinating soybean seeds increased rapidly for the first 6 days of germination and then decreased for the next 7 days. The isocitritase activity of plants grown in the dark was higher than that of the plants grown in light at all stages of development, particularly between day 7 and 11.     

Activities of enzymes of polyphenol metabolism inPhaseolus aureus seedlings germinated in the presence of 2-Chloroethylphosphonic acid

The plant growth regulator 2-ohloroethylphosphonic acid inhibited the elongation of growth inPhaseolus aureus seedlings. In comparison to the control, the polyphenol oxidase and peroxidase activity of treated seedlings was low up to 24 and 48 h of germination, respectively and that of phenylalanine ammonia-lyase and tyrosine ammonia-lyase was slightly less at 120 h and that of α- and β-glucosidases were less at 48 and 72 h, respectively. At other stages of germination, it greatly stimulated the activities of these enzymes. Part of Ph. D. dissertation submitted by Y. K. Arora to Punjab Agricultural University, Ludhiana, India     

Adenosylhomocysteinase and adenosine nucleosidase activities in Lupinus luteus cotyledons during seed formation and germination

The activities of adenosylhomocysteinase (EC 3.3.1.1) and adenosine nucleosidase (EC 3.2.2.7) were assayed in extracts from yellow lupin (Lupinus luteus L.) cotyledons at different stages of seed formation and seedling development. Adenosylhomocysteinase activity was demonstrated in all the cotyledon extracts examined. Its lowest level was found in the dry seeds and the highest, in 4-day-old seedling cotyledons. Extracts from the cotyledons of maturating seeds, dry seeds, and seedlings up to the second day of growth exhibited no adenosine nucleosidase activity. Adenosine nucleosidase activity appeared in the cotyledons of 2-day-old seedlings and its highest level was reached in 4-to 5-day-old seedlings. There is no inhibitor of adenosine nucleosidase in the maturating and dry yellow lupin seeds. No activator of a possible zymogen form of adenosine nucleosidase from maturating or dry seeds occurs in the growing seedlings.     

Phytochrome-induced flavonoid biosynthesis in mustard (Sinapis alba L.) cotyledons. Enzymic control and differential regulation of anthocyanin and quercetin formation

Phytochrome-induced increases in enzyme activities for phenylalanine ammonia-lyase (EC 4.3.1.5) and chalcone isomerase (EC 5.5.1.6), and in amounts of the related end products, anthocyanin and the flavonol, quercetin, were measured in cotyledons of mustard (Sinapis alba L.). There was no correlation between the activities of these enzymes and the rate of anthocyanin accumulation; however, some correlation was found with the quercetin accumulation rate. Since anthocyanin and flavonol accumulation is spatially separated in mustard (flavonols in the upper epidermis, anthocyanin in the lower epidermis), it was possible to measure anthocyanin-associated phenylalanine ammonia-lyase independently. This activity correlated well with the accumulation rate for anthocyanin during the first few hours after induction. The phytochrome effect on anthocyanin formation differed from that on quercetin formation: anthocyanin was strongly induced by continuous far-red light and by both continuous red light and red light pulses, whereas quercetin was only effectively induced by continuous far-red light.Abbreviations CHI chalcone isomerase - PAL phenylalanine ammonia-lyase     

Circadian Rhythmicity in the Activities of Phenylalanine Ammonia-Lyase from Lemna perpusilla and Spirodela polyrhiza

The oscillations in phenylalanine ammonia-lyase activity from Spirodela polyrhiza and phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities from Lemna perpusilla displayed a circadian rhythm under continuous light. Rhythmicity in enzymic activity could not be detected in continuous darkness since under this condition phenylalanine ammonia-lyase activity remains at a fairly constantly low level. Results from our studies of the oscillatory pattern of the respective activities of phenylalanine and tyrosine ammonia-lyase support their “inseparability.”     

On the Mechanism of the Changes in Phenylalanine Ammonia-lyase Activity Induced by Ultraviolet and Blue Light in Gherkin Hypocotyls

Irradiation with ultraviolet light causes in the hypocotyl of dark-grown gherkin seedlings the partial conversion of trans-hydroxycinnamic acids to the cis-isomers. The trans-hydroxycinnamic acids inhibit the development of phenylalanine ammonia-lyase activity, and the transformation of these compounds to the much less inhibitory cis-isomers forms a ready explanation for the increase in phenylalanine ammonia-lyase activity in the hypocotyl of gherkin seedlings irradiated with ultraviolet light. Arguments are advanced that the increase in phenylalanine ammonia-lyase activity caused by irradiation with blue light is also (at least in part) initiated by trans-cis isomerisation of the hydroxycinnamic acids.