Phenylalanine Ammonia-lyase Transcripts Research Articles

Melatonin and calcium modulate the production of rosmarinic acid, luteolin, and apigenin in Dracocephalum kotschyi under salinity stress

Melatonin (Mel) and calcium (Ca2+) have a regulatory role in the induction of specialized metabolites production and defensive responses against stresses. Therefore, in this study, the effects of Mel and Ca2+ and the possible relationship between them in the increase of the production of phenolic compounds in Dracocephalum kotschyi Boiss. under both control and salinity stress conditions were investigated. The results showed that 75 mM NaCl reduced shoot dry biomass but elevated H2O2 content, electrolyte leakage (EL) level, total phenolic and flavonoid contents (TPC and TFC), and DPPH scavenging capacity. Salinity stress also upregulated gene expression of phenylalanine ammonia-lyase (PAL) and rosmarinic acid synthase (RAS), as well as the activities of PAL and tyrosine ammonia-lyase (TAL) enzymes. Pre-treatment of the plants with CaCl2 and Mel affected these attributes in a dose-dependent manner. Application of 5 mM Ca2+ and 100 μM Mel improved shoot dry biomass and reduced the level of EL and H2O2 content but enhanced TPC and TFC, DPPH scavenging capacity, PAL and TAL activities, PAL and RAS transcripts, and content of rosmarinic acid (RA), luteolin flavone (LF) and apigenin flavone (AF) under salinity stress. Pre-treatment of D. kotschyi with lanthanum chloride (LaCl3) as a plasma membrane channel blocker, ethylene glycol tetra-acetic acid (EGTA) as a Ca2+ chelator and trifluoperazine (TFP) as a calmodulin (CaM) antagonist, impaired Mel effects on the above attributes under salinity stress. In contrast, pre-treatment with p–chlorophenylalanine (p-CPA), as an inhibitor of Mel biosynthesis, did not impair the impacts of Ca2+ on the production of phenolic compounds in salt-exposed plants. These results suggested that the effect of Mel on the induction of phenolic compounds production requires the influx of extracellular Ca2+ into the cells and is dependent on Ca2+/CaM signaling.

Dynamics of antioxidant properties, phenolic compounds, and transcriptional expression of key enzymes for the phenylpropanoid pathway in leaves of field‐grown winter wheat with different nitrogen fertilization schemes

AbstractWheat is a field crop with high relevance for food production worldwide. An adequate supply with nitrogen (N) is crucial for the grain yield and grain quality of wheat. However, little is known about the influence of N fertilization on phenolic compounds, such as phenolic acids and flavonoids, in the leaves of wheat (Triticum aestivum L. ssp. aestivum cv. Genius) under field conditions. Therefore, a field experiment was set up with winter wheat grown under different N fertilization regimes to investigate the influence of N supply on antioxidant properties in general as well as on distinct phenolic acids and flavonoids in the leaves of wheat plants. In the present study, total phenolic concentration and antioxidant capacity were negatively influenced by higher dosages of N fertilizer. Antioxidant properties were mainly determined by chlorogenic acid and homoorientin, a phenolic acid and a flavonoid, respectively. The production of these phenolic compounds was not limited by the gene transcription of phenylalanine ammonia lyase or cinnamate 4‐hydroxylse, the key enzymes of the phenylpropanoid pathway, since the transcription of these enzymes was not significantly influenced by N fertilization.

In silico characterization and transcriptional modulation of phenylalanine ammonia lyase (PAL) by abiotic stresses in the medicinal orchid Vanda coerulea Griff. ex Lindl.

Phenylalanine ammonia lyase (PAL) is the first enzyme of phenylpropanoid pathway. In the present study, a full-length PAL transcript from Vanda coerulea Griff. ex Lindl. (Family: Orchidaceae) was isolated and characterized. It was found that complete PAL transcript of V. coerulea (VcPAL; Gene Bank no. MG745168) contained 2175 bp with the open reading frame (ORF) of 2112 bp, encoding 703 amino acid residues. The multiple sequence alignment showed that VcPAL protein had 81% identity with that of the orchid, Bromheadia finlaysoniana. Phylogenetic analysis also disclosed that VcPAL shared the same evolutionary relationship with PAL proteins of other orchid species and to be closely related to that of other angiosperm species as well. The three-dimensional structure of VcPAL was found to be homo-tetrameric in nature consisting of four identical subunits with a molecular mass of 75 kDa per subunit. In silico characterization revealed the deduced protein to be a stable protein, comprising three major functional domains as reported in PAL proteins of other species. The transcription profiling of VcPAL exhibited the highest expression level to be present in the in vitro - raised leaf and root samples as compared to that of the ex vitro plant. The differential expression of VcPAL transcript was observed to be up-regulated by different types of abiotic stresses like wounding, cold, UV-B, salinity, and down-regulated by dark treatment. The study also exhibited that the VcPAL enzyme activity was directly proportional to the gene expression after the tissues were subjected to salinity and wounding stresses wherein a 1.7- fold increase in the enzyme activity was recorded in the leaf tissues exposed to salinity stress. A positive correlation could be found between the enzyme activity and the accumulation of phenylpropanoids such as total phenolic and flavonoid contents with R2 = 0.85 and 0.842 respectively.

Expression profile of phenylalanine ammonia-lyase (PAL) and phenolic content during early stages of graft development in bud grafted Hevea brasiliensis

Abstract Phenylalanine ammonia lyase (PAL) is a key enzyme in the phenylpropanoid pathway responsible for the biosynthesis of many secondary metabolites, such as anthocyanins, flavanols, and lignins. This study investigated the effect of grafting on vegetative growth, PAL expression and phenolic content of RRIM 600 and RRIT 251 bud grafted on different rubber rootstocks: for homografts (RRIM 600/RRIM 600 and RRIT 251/RRIT 251) and heterografts (RRIM 600/RRIM 623, RRIM 600/Clone#1, RRIM 600/Clone#2) (RRIT 251/RRIM 623, RRIT 251/Clone#1, RRIT 251/Clone#2) at 0, 7, 14, 21 and 28 days after bud grafting. For the RRIM 600 scion, the rootstock that provided the highest growth rate was Clone#1 while RRIT 251 showed uniform growth on different graft combinations. The results indicate that grafting alters PAL gene expression. PAL transcripts at higher levels during the early stages of grafting development especially 7 days after grafting. However, no significant differences were detected in the phenolic content at different post-grafting times. These findings suggest that the expression of the PAL gene is related to grafting compatibility in grafted H. brasiliensis.

Manganese Phosphite in Coffee Defence against Hemileia vastatrix, the Coffee Rust Fungus: Biochemical and Molecular Analyses

AbstractPhosphites (Phi) are inorganic salts of phosphorous acid that have been used as resistance elicitors to activate mechanisms underlying plant defence. This study assessed the effectiveness of manganese phosphite (MnPhi) for the management of rust (Hemileia vastatrix) on coffee seedlings. The effect of this compound on the induction of resistance was also measured by the expression of defence‐related genes such as POX (peroxidase), CAT (catalase), GLU (β‐1.3‐glucanase) and PAL (phenylalanine ammonia‐lyase) and by the activity of defence enzymes such as ascorbate peroxidase (APX), superoxide dismutase (SOD) and polyphenol oxidase (PPO). Foliar sprayings of MnPhi on coffee seedlings provided 63% control of rust severity. This compound induced defence responses in seedlings with increased transcription of genes POX, CAT, GLU and PAL in non‐inoculated plants and increased activity of APX, SOD and PPO enzymes in plants inoculated with H. vastatrix and in non‐inoculated plants. It is suggested that MnPhi can induce resistance in coffee seedlings.

A kinetic model for flavonoid production in tea cell culture.

As one of the strategies for efficient production of a metabolite from cell cultures, a kinetic model is very useful tool to predict productivity under various culture conditions. In this study, we propose a kinetic model for flavonoid production in tea cell culture based on the cell life cycle and expression of PAL, the gene encoding phenylalanine ammonia-lyase (PAL)-the key enzyme in flavonoid biosynthesis. The flavonoid production rate was considered to be related to the amount of active PAL. Synthesis of PAL was modelled based on a general gene expression/translation mechanism, including the transcription of DNA encoding PAL into mRNA and the translation of PAL mRNA into the PAL protein. The transcription of DNA was assumed to be promoted at high light intensity and suppressed by a feedback regulatory mechanism at high flavonoid concentrations. In the model, mRNA and PAL were considered to self-decompose and to be lost by cell rupture. The model constants were estimated by fitting the experimental results obtained from tea cell cultures under various light intensities. The model accurately described the kinetic behaviors of dry and fresh cell concentrations, glucose concentration, cell viability, PAL specific activity, and flavonoid content under a wide range of light intensities. The model simulated flavonoid productivity per medium under various culture conditions. Therefore, this model will be useful to predict optimum culture conditions for maximum flavonoid productivity in cultured tea cells.

Correlation of nitric oxide produced by an inducible nitric oxide synthase-like protein with enhanced expression of the phenylpropanoid pathway in Inonotus obliquus cocultured with Phellinus morii.

Fungal interspecific interactions enhance biosynthesis of phenylpropanoid metabolites (PM), and production of nitric oxide (NO) is known to be involved in this process. However, it remains unknown which signaling pathway(s) or regulator(s) mediate fungal PM biosynthesis. In this study, we cocultured two white-rot fungi, Inonotus obliquus and Phellinus morii, to examine NO production, expression of the genes involved in phenylpropanoid metabolism and accumulation of phenylpropanoid-derived polyphenols by I. obliquus. Coculture of the two fungi caused an enhanced NO biosynthesis followed by increased transcription of the genes encoding phenylalanine ammonia lyase (PAL) and 4-coumarate CoA ligase (4CL), as well as an upregulated biosynthesis of styrylpyrone polyphenols in I. obliquus. Addition of the NO synthase (NOS) selective inhibitor aminoguanidine (AG) inhibited NO production by more than 90% followed by cease in transcription of PAL and 4Cl. Treatment of guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one did not affect NO production but suppressed transcription of PAL and 4CL and reduced accumulation of total phenolic constituents. Genome-wide analysis of I. obliquus revealed two genes encoding a constitutive and an inducible NOS-like protein, respectively (cNOSL and iNOSL). Coculture of the two fungi did not increase the expression of the cNOSL gene but triggered expression of the iNOSL gene. Cloned iNOSL from Escherichia coli shows higher activity in transferring L-arginine to NO, and this activity is lost upon AG addition. Thus, iNOSL is more responsible for NO production in I. obliquus and may act as an important regulator governing PM production during fungal interspecific interactions.

Metabolic shift from withasteroid formation to phenylpropanoid accumulation in cryptogein-cotransformed hairy roots of Withania somnifera (L.) Dunal.

Cotransformed hairy roots containing a gene that encodes a fungal elicitor protein, β-cryptogein, were established in Withania somnifera, a medicinal plant widely used in Indian systems of medicine. To find out whether β-cryptogein protein endogenously elicits the pathway of withasteroid biosynthesis, withaferin A and withanolide A contents along with transcript accumulation of farnesyl pyrophosphate (FPP) synthase, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), and sterol glycosyltransferase (SGT) were analyzed in both cryptogein-cotransformed and normal hairy roots of W. somnifera. It was observed that the withaferin A and withanolide A contents were drastically higher in normal hairy roots than cryptogein-cotransformed ones. Similar trends were also observed on the levels of transcript accumulation. Subsequently, the enzyme activity of phenylalanine ammonia lyase (PAL), one of the key enzymes of phenylpropanoid pathway, was measured in both cryptogein-cotransformed and normal hairy roots of W. somnifera along with the levels of PAL transcript accumulation. Upliftment of PAL activity was observed in cryptogein-cotransformed hairy roots as compared to the normal ones, and the PAL expression also reflected a similar trend, i.e., enhanced expression in the cryptogein-cotransformed lines. Upliftment of wall-bound ferulic acid accumulation was also observed in the cryptogein-cotransformed lines, as compared to normal hairy root lines. Thus, the outcome of the above studies suggests a metabolic shift from withanolide accumulation to phenylpropanoid biosynthesis in cryptogein-cotransformed hairy roots of W. somnifera.

Expression of genes related to flavonoid and stilbene synthesis as affected by signaling chemicals and Botrytis cinerea in grapevines

Recent studies have shown that the expression of genes related to the synthesis of flavonoids, such as the phenylalanineammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and stilbene synthase (STS) genes were induced in response to different signaling molecules and Botrytis cinerea inoculation in grapevine leaves. Therefore, in the present study, the nucleotide and deduced amino acid sequences of STS genes from cultivars Campbell Early and Kyoho were compared. The deduced amino acid sequences of VlKSTS12, VlKSTS11, VICESTS13, and VlKSTS1 showed 100 % homology to VlCESTS12, VlCESTS11, VICESTS24, and VlKSTS13, respectively, in Campbell Early and Kyoho. In addition, the maximum transcription was observed 6 h after chemical treatments. In Campbell Early, the transcription of PAL, CHS, and CHI was higher in leaves treated with ethephon than in those treated with hydrogen peroxide, methyl jasmonate, and salicylic acid. The PAL, CHS, and CHI genes were more induced in Campbell Early than in Kyoho. The mRNA content of STS genes started to increase at 6 h and peaked at 48 h after the treatments. In Kyoho leaves, the expression of STSs was highly up-regulated at 1 h and peaked at 6 h after the treatments. The expression of the STS genes was induced in both the cultivars in leaves inoculated with B. cinerea. STS11 and STS12 showed differential expression patterns from STS1, STS24, and STS13 in Campbell Early leaves inoculated with B. cinerea.

A Myb transcription factor regulates genes of the phenylalanine pathway in maritime pine

During the life cycles of conifer trees, such as maritime pine (Pinus pinaster Ait.), large quantities of carbon skeletons are irreversibly immobilized in the wood. In energetic terms this is an expensive process, in which carbon from photosynthesis is channelled through the shikimate pathway for the biosynthesis of phenylpropanoids. This crucial metabolic pathway is finely regulated, primarily through transcriptional control, and because phenylalanine is the precursor for phenylpropanoid biosynthesis, the precise regulation of phenylalanine synthesis and use should occur simultaneously. The promoters of three genes encoding the enzymes prephenate aminotransferase (PAT), phenylalanine ammonia lyase (PAL) and glutamine synthetase (GS1b) contain AC elements involved in the transcriptional activation mediated by R2R3-Myb factors. We have examined the capacity of the R2R3-Myb transcription factors Myb1, Myb4 and Myb8 to co-regulate the expression of PAT, PAL and GS1b. Only Myb8 was able to activate the transcription of the three genes. Moreover, the expression of this transcription factor is higher in lignified tissues, in which a high demand for phenylpropanoids exits. In a gain-of-function experiment, we have shown that Myb8 can specifically bind a well-conserved eight-nucleotide-long AC-II element in the promoter regions of PAT, PAL and GS1b, thereby activating their expression. Our results show that Myb8 regulates the expression of these genes involved in phenylalanine metabolism, which is required for channelling photosynthetic carbon to promote wood formation. The co-localization of PAT, PAL, GS1b and MYB8 transcripts in vascular cells further supports this conclusion.

Accumulation of selected phenolics and expression of PAL genes in carrots differing in their susceptibility to carrot fly (Psila rosae F.)

AbstractThree research problems were addressed in this work: an extent of root damage caused by carrot fly (Psila rosae) in a collection of carrot breeding accessions as well as correlation between resistance to this pest and accumulation of certain phenolics and mRNAs of the genes coding for phenylalanine ammonia-lyase (PAL). Differences in susceptibility between the studied accessions were statistically significant. The most resistant were lines 7262A and DC 96367. Moreover, no correlation was observed between accumulation of the specific metabolites and the extent of the root damage. However, proportion of certain phenolics exhibited positive correlation with the resistant phenotype. This was shown for the ratios of luteolin 7-O-glucoside to kaempferol 3-O-glucoside and methyluteolin 7-O-glucoside to kaempferol 3-O-glucoside. Susceptibility of roots to larval damage also correlated with semi-quantitatively estimated accumulation of PAL1 and PAL3 mRNAs in leaves. Both PAL genes were expressed at the highest level in line DC 96367 and less strongly in line 7262A. Distinctly lower accumulation of the PAL transcripts was observed for the susceptible varieties: Dolanka and Karotan.

Effects of irrigation on fruit ripening behavior and metabolic changes in olive

Olive (Olea europaea, cv Leccino) fruits grown under different water regimes were analyzed by metabolomics and specific transcript accumulation analyses. The fruit from non-irrigated (rain-fed) and irrigated trees cultivated under field conditions, with a seasonal water amount equivalent to the calculated crop evapotranspiration (ETc) was compared in the last developmental phase and, in particular, at commercial harvest. Metabolomics (GC–MS) analysis identified several hundred metabolites in ripe mesocarp, 46 of which showed significantly different contents in the rain-fed and irrigated samples. Some compounds involved in primary metabolism (carbohydrates, amino acids, organic acids) and secondary metabolism (squalene, simple phenols) appeared to be more abundant when irrigation was performed. Higher levels of total polyphenol were observed in the rain-fed fruit, which at ripening showed an increase in anthocyanin concentration. These data indicate that ripening in olives is affected by irrigation. In addition, expression analyses of three key polyphenol biosynthetic genes (phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), dihydroflavonol reductase (DFR)) and two genes involved in triterpenoid metabolism (β-amyrin synthase and cycloartenol synthase) were also performed. The analyzed genes showed different expression patterns throughout ripening, and the resulting PAL, DFR and β-amyrin synthase transcript accumulation was found to be affected by the different water regimes at specific stages of fruit development.

Activation of phenylpropanoid pathway in legume plants exposed to heavy metals. Part I. Effects of cadmium and lead on phenylalanine ammonia-lyase gene expression, enzyme activity and lignin content.

Species-specific changes in expression of phenylalanine ammonia-lyase (PAL) and lignin content were detected in roots of soybean (Glycine max L.) and lupine (Lupinus luteus L.) seedlings treated with different concentrations of cadmium (Cd(2+), 0-25 mg/l) or lead (Pb(2+), 0-350 mg/l). The stimulatory effect of both metals was observed in mRNA coding for PAL in soybean. In the case of lupine, changes of PAL mRNA level were dependent on the metal used: Cd(2+) caused a decrease, whereas Pb(2+) an increase of PAL transcript level. The activity of PAL was enhanced in both plant species at higher metal concentrations (15-25 mg/l of Cd(2+) or 150-350 mg/l of Pb(2+)); however it was not directly correlated with PAL mRNA. This suggests a transcriptional and posttranscriptional control of PAL expression under heavy metals stress. In soybean, Cd(2+) or Pb(2+) treatment increased lignin content, while in lupine the effect was opposite. The decreased lignin accumulation in lupine roots in response to heavy metals, despite an increased PAL activity, suggests that the activated phenylpropanoid pathway was involved in the synthesis of secondary metabolites other than lignin.

4-Coumarate:CoA ligase family members from elicitor-treated Sorbus aucuparia cell cultures

Sorbus aucuparia cell cultures accumulate biphenyl and dibenzofuran phytoalexins in response to elicitor treatment. These polyketide derivatives arise from the starter substrate benzoyl-CoA, the biosynthesis of which is largely unresolved. Two CoA ligases involved are cinnamate:CoA ligase and benzoate:CoA ligase, which were assumed to be related in S. aucuparia to the ubiquitous 4-coumarate:CoA ligase (4CL). cDNAs encoding three distinct 4CLs from elicitor-treated S. aucuparia cell cultures were isolated using RT-PCR and RACE techniques and functionally expressed in Escherichia coli as His 6-tagged proteins (Sa4CL2 and Sa4CL3) or GST-fusion protein (Sa4CL1). All three isoenzymes preferred 4-coumaric acid over cinnamic acid in spectrophotometric assays and failed to utilize benzoic acid in radioisotopic assays. After elicitor treatment of S. aucuparia cell cultures, the transcript levels of all three Sa4CLs increased but were significantly lower than the maximum expression rates of the phenylalanine ammonia-lyase ( PAL) and biphenyl synthase 1 ( BIS1) genes. The substrate specificities and the expression profiles indicate that the three 4CL isoenzymes are not involved in benzoyl-CoA biosynthesis in S. aucuparia cell cultures. Sa4CL3 and PAL transcripts also accumulated in response to light treatment. Phylogenetically, Sa4CL1 and Sa4CL2 belong to the class I cluster and Sa4CL3 groups in the class II cluster. Sa4CL3 contains a 49-amino acid N-terminal extension, which includes a chloroplast sorting signal.

Age-induced loss of wound-healing ability in potato tubers is partly regulated by ABA

Wounding of potato (Solanum tuberosum L.) tubers induces the development of a suberized closing layer and wound periderm that resists desiccation and microbial invasion. Wound-healing ability declines with tuber age (storage period). The mechanism of loss in healing capacity with age is not known; however, upregulation of superoxide production, increased ABA biosynthesis and phenylalanine ammonia lyase (PAL) activity in response to wounding are processes critical to the development of a suberized closing layer and wound periderm. Therefore, the role of ABA in modulating the age-induced loss of wound-healing ability of tubers was examined. Non-wounded older tubers had 86% less ABA (dry matter basis) than younger tubers. PAL transcript increased in younger tubers within 24h of wounding, but transcription was delayed by 5days in older tubers. Wound-induced PAL activity increased more rapidly in younger than older tubers. ABA treatment increased PAL expression and activity in tissue from both ages of tubers and restored the 24h transcription time line in older tubers. Moreover, ABA treatment of wounded older tubers enhanced their resistance to water vapor loss following a 6-day wound-healing period. Wound-induced accumulation of suberin poly(phenolic(s)) (SPP) and suberin poly(aliphatic(s)) (SPA) was measurably slower in older versus younger tubers. ABA treatment hastened SPP accumulation in older tubers to match that in younger tubers, but only enhanced SPA accumulations over the initial 4days of healing. Age-induced loss of wound-healing ability is thus partly due to reduced ability to accumulate ABA and modulate the production of SPP through PAL in response to wounding and to dysfunction in the downstream signaling events that couple SPA biosynthesis and/or deposition to ABA. ABA treatment partly restored the healing ability of older tubers by enhancing the accumulation of SPP without restoring wound-induced superoxide forming ability to the level of younger tubers. The coupling of phenolic monomers into the poly(phenolic) domain of suberin was therefore not limited by the diminished wound-induced superoxide production of older tubers.

Riboflavin induces resistance in rice against Rhizoctonia solani via jasmonate-mediated priming of phenylpropanoid pathway

Vitamins are plant growth regulators and activators of defense responses against pathogens. The cytomolecular mechanisms involved in the induction of resistance by chemicals especially vitamins on monocotyledonous plants are largely unknown. Here, we show that riboflavin, which acts as a defense activator in rice against economically important sheath blight caused by Rhizoctonia solani, primed the expression of lipoxygenase ( LOX) as a key gene in octadecanoid pathway, and enhanced lignification. Exogenous jasmonic acid (JA) application on rice induces resistance against R. solani in a manner similar to riboflavin. Application of jasmonate-deficient rice mutant hebiba and using a LOX inhibitor revealed the main role of octadecanoid pathway in riboflavin-induced resistance (IR). In riboflavin-treated inoculated plants, upregulation of phenylalanine ammonia-lyase ( PAL) expression, as a major marker of phenylpropanoid pathway, was detected downstream of LOX upregulation. Co-application of riboflavin and 5, 8, 11, 14-eicosatetraynoic acid (ETYA) on rice leaves revealed no upregulation of PAL and no priming in lignification. Furthermore, lower levels of PAL transcripts and lignin were detected in hebiba compared with control. These findings indicate the role of octadecanoid pathway in the induction of phenylpropanoid metabolism leading to lignification as a novel mechanism of riboflavin-IR in Oryza sativa–R. solani pathosystem.

Acibenzolar-S-methyl-induced alteration of defence gene expression and enzyme activity in cotton infected with Fusarium oxysporum f. sp. vasinfectum

The effect of acibenzolar-S-methyl (ASM) applied in the form of Bion ® as a seed soak or a foliar spray, on transcript accumulation of six defence related genes, and activities of four defence related enzymes, was assessed after cotton was inoculated with Fusarium oxysporum f. sp. vasinfectum ( Fov). Significant upregulation of chitinase class I, peroxidase, and β-1, 3-glucanase transcripts occurred in the Bion ® seed soak treatment with Fov inoculation compared to all other treatments. Bion ® alone did not significantly increase transcripts compared with water control treatments in the majority of cases. Phenylalanine ammonia lyase transcript in Bion ® treated plants following fungal inoculation was greater than in the respective Bion ® alone treatments, and osmotin-like protein transcript was significantly greater in inoculated plants sprayed with Bion ® than in all other treatments. Transcripts of 4-coumarate-CoA ligase-like protein were generally lower than for all other genes, and significant upregulation compared with water control occurred in extracts from Fov inoculated plants receiving no Bion ® treatment, and Fov inoculated plants cultivated from Bion ® treated seed. The greatest increases in chitinase, β-1, 3-glucanase and peroxidase activity occurred in root and shoot tissue from Bion ® seed treated material inoculated with Fov. Bion ® treatment alone did not consistently enhance enzyme activities above those measured in water treated controls. Polyphenol oxidase activities were enhanced by various Bion ® or Fov treatments at 3 days after inoculation only.

Targeted gene analysis in Ulmus americana and U. pumila tissues

Summary Steady-state gene expression was compared between Dutch elm disease (DED)-susceptible Ulmus americana and DED-resistant U. pumila callus, leaf midrib, root and inner bark tissues. Stress-related cDNAs including phenylalanine ammonia-lyase (PAL), chitinase (CHT) and polygalacturonase-inhibiting protein (PGIP) were isolated and compared following RT-PCR of elm tissues. Complete CHT and partial PAL and PGIP cDNA transcripts were identified, each displaying sequence variation between elm species. These transcripts were Dig-labelled and subsequently used for northern analyses of the elm tissues. Midrib and root tissue displayed highest steady-state gene expression compared with inner bark and callus tissues. A modified nucleic acid isolation technique was necessary for downstream RNA analyses. Lithium chloride and polyvinylpyrrolidone were critical for efficient removal of polysaccharides and phenolics associated with some of the elm tissues. Steady-state gene expression is discussed in relation to the tissues investigated. The use of tissues other than in vitro callus culture more closely represents the tissues associated with the elm’s vascular response to DED.

Rice Allelopathy Induced by Methyl Jasmonate and Methyl Salicylate

Methyl jasmonate (MeJA) and methyl salicylate (MeSA) are important signaling molecules that induce plant defense against insect herbivores and microbial pathogens. We tested the hypothesis that allelopathy is an inducible defense mechanism, and that the JA and SA signaling pathways may activate allelochemicals release. Exogenous application of MeJA and MeSA to rice (Oryza sativa L.) enhanced rice allelopathic potential and led to accumulation of phenolics, an increase in enzymatic activities, and gene transcription of phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H), two key enzymes in the phenylpropanoid pathway. Aqueous extracts of the leaves of rice IAC165, a putative allelopathic variety, treated with MeSA (5 mM) or MeJA (0.05 mM), showed increased inhibitory effects (25 and 21%, respectively) on root growth of barnyardgrass (Echinochloa crus-galli L.), and increased inhibitory effects (18 and 23%, respectively) on shoot growth. Aqueous extracts from leaves of Huajingxian 1 rice, a putative nonallelopathic variety treated with MeJA and MeSA, caused 63 and 24% inhibition of root growth in barnyardgrass seedlings. The root exudates of both IAC165 and Huajingxian 1 plants treated with MeJA and MeSA for 48 hr also showed significant increases in their inhibitory effects on root growth of barnyardgrass seedlings. At the four-leaf stage, levels of 3,4-hydroxybenzoic acid, vanillic acid, coumaric acid, and ferulic acid that accumulated in the leaves were 5.3-, 31.3-, 2.2-, and 1.7-fold higher in response to MeJA exposure, and 3.3-, 13.1-, 2.0-, and 2.2-fold higher in response to MeSA. Treatments of MeSA and MeJA enhanced the PAL activity in the rice leaves up to 52.3 and 80.1%, respectively, whereas C4H activity was increased by 40.2 and 67%. Gene transcription of PAL and C4H in rice leaves significantly increased after the plants were subjected to treatment with MeJA and MeSA. These results suggest that allelopathy may be an active defense mechanism, and that plant signaling compounds are potentially valuable in its regulation.

Nitric oxide promotes the wound-healing response of potato leaflets

Nitric oxide (NO) is an essential regulatory molecule in several developmental and (patho) physiological processes. In this work, it is demonstrated that NO participates in the wound-healing response of potato leaves. The experimental approaches showed that the deposition of the cell-wall glucan callose was induced by the application of the NO donor sodium nitroprusside (SNP), and such induction was additive to the wound-induced callose production. Additionally, the expression of wound-related genes as phenylalanine ammonia-lyase ( PAL) and extensin showed an accumulation of their transcript levels by SNP treatment. Moreover, the SNP-mediated increase of the PAL transcript level was additive to the induction mediated by wounding. These results indicate that increased levels of NO might potentiate the healing responses in plants leading to a rapid restoration of the damaged tissue.