Role of Chitinase and Chitin Oligosaccharides in Lignification Response of Cultured Carrot Cells Treated with Mycelial Walls

Chitinase activity was induced in cultured carrot cells by incubationwith mycelial walls of a fungus, Chaetomium globosum. Both intra-and extracellular chitinases were resolved into four componentsby gel filtration chromatography. The extracellular enzymesliberated soluble oligosaccharides of different sizes from insolublechitin, suggesting that these carrot chitinases are endo-hydrolases.The solubilized chitinase digests obtained from insoluble mycelialwalls of C. globosum and chitin were fractionated by gel filtrationchromatography, and the elicitor activity of each fraction forthe accumulation of phenolic acids in cultured carrot cellswas determined. In both solubilized fragments of fungal wallsand of chitin, elicitor-active oligosaccharides were distributedin many fractions, however, potent activity for inducing phenolicacid synthesis was observed in the high molecular weight fractions. (Received October 5, 1987; Accepted February 12, 1988)     

Induction of Phenylalanine Ammonia-Lyase Activation and Isoflavone Glucoside Accumulation in Suspension-Cultured Cells of Red Bean, Vigna angularis, by Phytoalexin Elicitors, Vanadate, and Elevation of Medium pH

Treatment of suspension-cultured cells of red bean, Vigna angularis,with nigeran resulted in an accumulation of isoflavone glucosides,such as daidzein 7-O-ß-D-glucoside, daidzein 7,4'-di-O-ß-D-glucoside,and 2'-hydroxydaidzein 7,4'-di-O-ß-D-glucoside, whichwas accompanied by a transient increase in the activity of phenylalanineanimonia-lyase (PAL). Similar effects were also seen with otherphytoalexin elicitors, such as RNase A and cell wall componentsof Phytophthora megasperma var. sojae. Interestingly, the accumulation of isoflavone glucosides andthe transient increase in PAL activity were induced also byvanadate, a specific inhibitor of plasma membrane adenosinetriphosphatase. K₃PO₄ showed similar effects, but this was ascribedto the elevation of medium pH caused by adding this basic salt.In fact, merely raising the pH of the medium was found to besufficient for the induction of PAL activity. Experiments usinginhibitors showed that the induction depends on RNA and proteinsyntheses. The results are discussed in relation to the possiblemechanism of action of phytoalexin elicitors. ¹ Present address: Laboratory of Biochemistry, Faculty of Agriculture,Nagoya University, Furocho, Chikusa, Nagoya 464, Japan.     

Effects of Glyphosate on the Shikimate Pathway and Regulation of Phenylalanine Ammonia-lyase in Cryptomeria and Perilla Cell Suspension Cultures

Treatment of Cryptomeria and Perilla cell suspension cultureswith glyphosate resulted in a marked suppression of the formationof flavans and caffeic acid derivatives, respectively, whileit caused only a slight decline in the cell growth. In contrastwith 3-deoxy-D-arabino-heptulosonate (DAHP) synthase-Mn isozyme,DAHP synthase-Co isozyme from Cryptomeria and Perilla cellswas much more sensitive to inhibition by glyphosate. The additionof 1 to 2 mM glyphosate caused an accumulation of shikimateand quinate and a reduction of L-phenylalanine in both cellcultures. The inhibition of phenylalanine ammonia-lyase (PAL)activity by glyphosate was reversed by exogenously suppliedL-phenylalanine to near the control level. Cycloheximide andactinomycin D nullified the recovery by exogenous L-phenylalanineon PAL activity. L-Phenylalanine itself promoted PAL activityto some extent. No recovery of PAL activity in L--aminooxy-ß-phenylpropionate(L-AOPP)-treated cell cultures could be observed by the additionof L-phenylalanine. Therefore, L-AOPP seems to inhibit the formationof PAL, though it has been considered a competitive inhibitor. ³Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai 980, Japan. (Received October 28, 1985; Accepted March 13, 1986)     

Effect of Ethylene on the Phenylalanine Ammonia-lyase Activity of Carrot Tissues

Ethylene stimulated the activity of l -phenylalanine ammonia-lyase (PAL) in carrot (Daucus carota L.) tissues. It is also known to induce the formation of isochlorogenic acid. However, the induction patterns were different for isochlorogenic acid and PAL activity. Ethylene action seems to be indirect, as it did not affect the PAL activity of cell-free extracts.     

Differential regulation of phenylalanine ammonia-lyase genes during anthocyanin synthesis and by transfer effect in carrot cell suspension cultures

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) catalyses the first step in the phenylpropanoid pathway and is induced during differentiation and by various stimuli. In carrot ( Daucus carota L. cv. Kurodagasun) suspension culture cells, PAL is slowly induced during anthocyanin synthesis which occurs in a medium lacking 2,4-dichlo-rophenoxyacetic acid and is also induced rapidly and transiently by transferring and diluting cells to fresh medium. Analyses of nucleotide sequences derived from PAL cDNAs revealed that the PAL mRNAs induced by transfer were transcribed from different carrot PAL genes than the PAL mRNAs induced during anthocyanin synthesis. Northern blotting using probes derived from 3'non-coding regions for PAL cDNAs confirmed that different PAL genes were induced during anthocyanin synthesis and after transfer. Induction of different PAL genes occurs in response to differences in the induction trigger.     

Correlation between Phenylalanine Ammonia Lyase Activity and Phenolic Biosynthesis in p-Fluorophenylalanine-sensitive and -resistant Tobacco and Carrot Tissue Cultures

Phenylalanine ammonia lyase (PAL) activity was measured in p-fluorophenylalanine (PFP)-sensitive and -resistant tobacco (Nicotiana tabacum L.) and carrot (Daucus carota L.) cell lines which are known to oversynthesize phenylalanine. A correlation between phenolic levels and PAL activities was detected. The phenylalanine analog-resistant and -sensitive carrot cells showed no differences in the accumulation of phenolic compounds and PAL activities. The PFP-resistant tobacco cells, however, had 10 times higher levels of phenolics and also 10 to 20 times higher PAL activities than the PFP-sensitive line. The PAL activity in the resistant tobacco line increased dramatically after inoculation of the cells into fresh medium. Conditions affecting this increase were characterized.     

The Effects of 2,5-Norbornadiene on the Induction of the Activity of 1-Aminocyclopropane-l-carboxylate Synthase and of Phenylalanine Ammonia-lyase in Wounded Mesocarp Tissue of Cucurbita maxima

Activities of both 1-aminocyclopropane-l-carboxylate (ACC) synthaseand phenylalanine ammonia-lyase (PAL) were rapidly induced inexcised mesocarp discs of Cucurbita maxima Duch. The increasein activity of ACC synthase preceded that of PAL. 2,5-Norbornadiene(NBD), an inhibitor of the action of ethylene [Sisler and Yang(1984) Phytochemistry 12: 2765-2768.], enhanced the level ofactivity of ACC synthase in excised mesocarp disc and overcamethe suppression by exogenous ethylene. NBD, by contrast, suppressedthe level of PAL activity induced in the wounded tissue. Theseresults suggest that endogenous ethylene produced in the woundedmesocarp tissue suppresses the induction of ACC synthase butpromotes the induction of PAL. (Received March 9, 1989; Accepted June 14, 1989)     

Light-induced Synthesis of Anthocyanin in Carrot Cells in Suspension: II EFFECTS OF LIGHT AND 2, 4-D ON INDUCTION AND REDUCTION OF ENZYME ACTIVITIES RELATED TO ANTHOCYANIN SYNTHESIS

Time-courses of light-induced activities of enzymes relatingto anthocyanin formation were studied. Phenylalanine ammonia-lyase(PAL), 4-cumarate CoA ligase (4CL) and cinnamate-4-hydroxylase(C4H) (group 1 enzymes) and chalcone synthase (CHS) and chalcone-flavanoneisomerase (CHFI) (group 2 enzymes), were studied in carrot suspensioncells which were irradiated 5 d after transfer to a 2,4-dichlorophenoxyaceticacid (2,4-D)-free medium in the dark. Time-courses of group1 enzymes showed two peaks (fast and slow) with the slow peakincreasing almost parallel to anthocyanin accumulation. Time-coursesof group 2 enzymes showed one peak corresponding to the slowpeak of group 1. From the inhibitor experiment, the fast peakalso corresponded to the activity of the newly synthesized enzyme.From the initial phase of the time-courses, enzymes belongingto group 1 always induced more rapidly than those of group 2,and their induction was co-operative. However, once anthocyanin synthesis was induced by light, neitheraddition of 2,4-D nor transfer to darkness could prohibit anthocyaninsynthesis completely. Addition of 2,4-D in the dark completelysuppressed anthocyanin synthesis within 1 d and the activityof CHS also disappeared within 1 d. These results are explainedby a previous hypothesis (Takeda, 1988) that 2,4-D induces thestate change of cells. Key words: Anthocyanin, co-ordinate induction, Daucus carota, 2,4-dichlorophenoxyacetic acid, light-triggered     

Compartmentation of Phenolic Compounds and Phenylalanine Ammonia-Lyase in Leaves of Phyllanthus tenellus Roxb. and their Induction by Copper Sulphate

The compartmentation of phenolic compounds in mature leavesof Phyllanthus tenellus and their induction by copper sulphatewere analysed at histological and subcellular levels. Lightand electron microscopy studies demonstrated that the vacuolesof spongy cells were the main sites of phenolic accumulation.Spraying plants with copper sulphate induced punctated lesionsformed by groups of necrotic cells which accumulated brownishsubstances. Histochemical tests and fluorescence microscopyanalysis of the sprayed leaves indicated that the phenolic compoundsincreased in spongy cells within the lesions. Ultrastructuralanalyses showed that 3 h after elicitation, the organelles ofthe cells within the lesion started to collapse and the contentof phenolic substances increased in the vacuole of spongy cells.Antibody against phenylalanine ammonia-lyase (PAL) from parsleycross-reacted with the crude extract of P. tenellus leaves.Two isoforms, one of 65 kD and the other of 66 kD, were identified.Immunocytochemical studies showed that PAL was synthesized inthe palisade and spongy cells, mainly in the cytoplasm and chloroplasts.The phytotoxicity of Cu²⁺ions induced the accumulation of PALin sub-cellular compartments of palisade cells. PAL accumulationstarted to increase 3 h after elicitation and reached a maximumafter 6 h, decreasing 12 h post-induction. The increase of PALwas more evident in cells within the necrotic punctated regionsthan in surrounding cells. Since the vacuole of palisade cellsdid not accumulate phenolic compounds, the in situ studies suggestedthat the end products of PAL synthesis play a role in palisadecell wall reinforcement or might accumulate in other tissues.The symptoms induced by copper sulphate suggest that this abioticelicitor may be a useful tool in the understanding of the regulationof biosynthetic phenolic pathways inP. tenellus . Copyright2000 Annals of Botany Company Cell death, copper sulphate, heavy metal, immunolabelling, phenolic compounds, phenylalanine ammonia-lyase,Phyllanthus , transmission electron microscopy, ultrastructure     

Induction of chitinase and {beta}-1,3-glucanase activity in sunflower suspension cells in response to an elicitor from Phytophthora megasperma f. sp. glycinea (Pmg). Evidence for regulation by ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC)

In heterotrophic cell suspensions of sunflower (Helianthus annuusL. cv. Spanners Allzweck) the effect of Pmg elicitor, a fungalelicitor preparation from Phytophthora megasperma f. sp. glycinea,on the induction of chitinase and ß-1,3-glucanaseactivity was studied in relation to changes in ethylene biosynthesis.Dose-response experiments with Pmg elicitor showed that theonset of the induction of intracellular chitinase and ß-1,3-glucanaseactivity coincided or followed a transient rise in ethyleneand particularly endogenous 1-aminocyclopropane-1-carboxylicacid (ACC) levels within 5 h of application. Treatment with5 µg ml^–1 elicitor stimulated ethylene and ACC levels1.6-fold and 4-fold, relative to control, respectively. Themolar ratio of ACC to ethylene changed from approximately 3:1in controls to 9:1 in treated cells. During further incubation,ethylene formation and, to a lesser degree, ACC levels declinedand the ACC/ethylene ratio increased to 56:1 in elicitor-treatedcells. On a protein basis, the activities of ß-1,3-glucanaseand chitinase increased approximately 5-fold and 8-fold, respectively,48 h after elicitor application. Additional treatment with theACC synthesis inhibitor aminoethoxyvinyiglycine (AVG) decreasedelicitor-induced enzyme activities and the levels of both ethyleneand ACC. Elicitor effects on chitinase and ß-1,3-glucanaseactivities could be fully restored when ACC was additionallyapplied. Concomitantly, the ACC/ ethylene ratio increased. Neithertreatments with ACC alone, which simultaneously increased internalACC and ethylene levels, nor treatments with AVG alone, whichsimultaneously reduced ACC and ethylene levels, could generallystimulate chitinase or ß-1,3-glucanase activitiesin the cells. It is suggested that ACC functions as a promotingfactor in the induction of chitinase and ß-1,3-glucanaseactivity triggered by Pmg elicitor and appears to reverse aninhibiting influence of ethylene. Key words: 1-Aminocyclopropane-1-carboxylic acid, chitinase, ß-1,3-glucanase, ethylene, Helianthus cellsuspension cultures, Phytophthora megasperma-elicitor     

An Endogenous Suppressor of the Defense Response in Pisum sativum

Healthy pea plants contain a substance, tentatively called "endogenoussuppressor", which specifically suppresses the accumulationof pisatin in pea plants that is induced by treatment with CuCl₂or an elicitor from Mycosphaerella pinodes. This suppressorelicits the accumulation of phytoalexins in other legumes, suchas kidney bean, soybean and cowpea. The endogenous suppressorfunctions to delay the accumulation of pisatin, the activationof phenylalanine ammonialyase (PAL) and the accumulation ofmRNAs for PAL and chalcone synthase induced by the elicitorfrom M. pinodes. The substance specifically induces susceptibilityto nonpathogens, such as Mycosphaerella ligulicola and M. melonis,in pea out of four species of legume tested, but the effectis not cultivar-specific. Thus, the endogenous suppressor inhealthy pea plants suppresses a series of self-defense reactionsand induces susceptibility in pea plants in a species-specificmanner, being similar to the exogenous fungal suppressor fromthe pea pathogen, M.pinodes. (Received February 19, 1992; Accepted May 11, 1992)     

A Vesicular Arbuscular Mycorrhizal Fungus (Glomus intraradix) Induces a Defense Response in Alfalfa Roots

Flavonoid accumulation and activities of phenylalanine ammonia-lyase (PAL), chalcone isomerase (CHI), and chitinase were followed during early colonization of alfalfa roots (Medicago sativa L. cv Gilboa) by vesicular arbuscular (VA) fungi (Glomus intraradix). Formononetin was the only flavonoid detected that showed a consistent increase in the inoculated roots. This increase depended only on the presence of the fungus in the plant rhizosphere; no colonization of the root tissue was required. CHI and chitinase activities increased in inoculated roots prior to colonization, whereas the increase in PAL activity coincided with colonization. After reaching a maximum, activities of all enzymes declined to below those of uninoculated roots. PAL inactivation was not caused by a soluble inhibitor. Our results indicate that VA fungi initiate a host defense response in alfalfa roots, which is subsequently suppressed.     

Kinetics of Phenylalanine Ammonia-Lyase and the Effect of L-{alpha}-aminooxy-{beta}-phenylpropionic Acid on Enzyme Activity and Radicle Growth in Germinating Lettuce Seeds

The effects of different concentrations of L--aminooxy-ß-phenyIpropionicacid (AOPP), an analog of L-phenylalanine, on the activity ofphenylalanine ammonia-lyase (PAL, EC 4.3.1.5 [EC] ) and the growthof radicles in 24 h old germinating lettuce (Lactuca salivaL.) seeds were investigated. AOPP causes a significant inhibitionof PAL activity in the seeds (85% inhibition at 10⁴ M). It alsocauses a stimulation of radicle growth at that concentration.The results show that the inhibition of PAL by AOPP may be dueto an irreversible binding of the inhibitor to the enzyme leadingto its inactivation. AOPP also inhibits ethylene biosynthesisin germinating lettuce seeds which could probably explain thestimulation of radicle growth in these seeds. The enzyme shows typical Michaelis-Menten kinetics. The K_m forL-phenylalanine is 4.2 x 10⁵ M. The enzyme does not show anytyrosine ammonia-lyase activity. Various substrate analogs suchas D-phenylalanine, p-fluorophenylalanine, ß-phenyllacticacid, tryptophan and the product of the enzyme reaction, trans-cinnamicacid, inhibit the enzyme competitively. A number of intermediatesand endproducts of the phenylpropanpid pathway, except chlorogenicacid, do not show any inhibition. ¹Scientific contribution number 1423 from the New HampshireAgricultural Experiment Station. (Received May 9, 1986; Accepted September 8, 1986)     

Responses of cultured parsley cells to elicitors from phytopathogenic fungi : timing and dose dependency of elicitor-induced reactions

Cultured parsley cells (Petroselinum crispum) responded to treatment with heat-released soluble cell-wall fragments (elicitors) from several different phytopathogenic fungi by forming coumarin derivatives (phytoalexins). This response was preceded in all cases by large but transient increases in the activities of two enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL). The activities of two hydrolytic enzymes, chitinase and 1,3-β-glucanase, also increased strongly in elicitor-treated cells, whereas the activities of three enzymes participating in primary metabolism were affected differently by the elicitor treatment. Glucose-6-phosphate dehydrogenase increased, phosphofructokinase remained almost constant, and pyrophosphate:fructose-6-phosphate phosphotransferase declined sharply in activity. Different amounts of cell-wall preparations from various phytopathogenic fungi were required for maximum elicitor activity. While three oomycetes (Phytophthora spp.) yielded the most active elicitors studied (maximum coumarin accumulation at concentrations of about 10 microgram per milliliter), cell-wall preparations from an ascomycete and three deuteromycetes gave comparable results only at 10 to 100 times higher concentrations. Optimal induction of PAL, 4CL, and chitinase with Phytophthora elicitor required only about 1 microgram per milliliter, whereas 1,3-β-glucanase induction showed a dose dependence similar to that observed for coumarins. The elicitor concentration had pronounced effects not only on the extent, but also on the timing of all induced reactions.     

Activity profile of defence-related enzymes in rice genotypes (Oryza sativa L.) against rice blast (Magnaporthe oryzae)

The control and infected leaf samples of blast resistant and susceptible rice genotypes were evaluated for activities of defence-related enzymes viz., total phenol content, chitinase, phenylalanine ammonia lyase (PAL), β-glycosidase, antioxidative enzymes, superoxide dismutase, peroxidase and ascorbate peroxidase. The level of total phenol content and the activity profile of chitinase, PAL and β-glycosidase significantly increased in both blast-resistant and susceptible rice genotypes with comparatively higher level induction Tetep, NLR-20104 and Swarnadhan the blast-resistant genotypes. The antioxidative enzymes were comparatively higher in the leaf samples of blast-resistant genotypes recording highest increase in NLR-20104 and KJT-5. The constitutive levels of total phenols and activity of defence-related and antioxidative enzymes in the control leaf samples differed among the genotypes and were even higher in the two blast susceptible genotypes (EK-70 and Chimansal). However, the level of induction as evident from the activity profile differences between control and infected leaf samples suggests higher level of induction was more which is indicative of the induced defence response. The genotype recording maximum induction of defence-related and antioxidative enzymes activity could be useful criteria in screening for blast resistant genotype in rice.     

Regulation of phenylalanine ammonia-lyase genes in carrot suspension cultured cells

Induction of anthocyanin synthesis occurs during metabolic differentiation in carrot suspension cultured cells grown in medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), and is closely correlated with embryogenesis. Anthocyanin synthesis may also be induced by light-irradiation under different culture conditions. The phenylalanine ammonia-lyase (PAL) gene (TRN-PAL), which was transiently induced by the transfer effect, was also rapidly induced after light-irradiation. However, TRN-PAL was not involved in anthocyanin synthesis. A second PAL gene, ANT-PAL, was involved in anthocyanin synthesis. ANT-PAL was induced during metabolic differentiation in medium lacking 2,4-D parallel with the induction of chalcone synthase (CHS). PAL genes in the carrot genome are expressed differentially depending on the nature of the environmental stimulus, e.g. transfer effect and light, and other parameters which also affect anthocyanin synthesis.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - Luc firefly luciferase - PAL phenylalanine ammonia-lyase - UV ultraviolet