Expression Of PAL Research Articles

The bHLH Rac Immunity1 (RAI1) Is Activated by OsRac1 via OsMAPK3 and OsMAPK6 in Rice Immunity

The Rac/Rop GTPase OsRac1 plays an essential role in rice immunity. However, the regulatory genes acting downstream of OsRac1 are largely unknown. We focused on the RAI1 gene, which is up-regulated in suspension cells expressing a constitutively active form of OsRac1. RAI1 encodes a putative basic helix-loop-helix transcription factor. A microarray analysis of cells transformed with an inducible RAI1 construct showed increased expression of PAL1 and OsWRKY19 genes after induction, suggesting that these genes are regulated by RAI1. This was confirmed using RAI1 T-DNA activation-tagged and RNA interference lines. The PAL1 and OsWRKY19 genes were also up-regulated by sphingolipid and chitin elicitors, and the RAI1 activation-tagged plants had increased resistance to a rice blast fungus. These results indicated that RAI1 is involved in defense responses in rice. RAI1 interacted with OsMAPK3 and OsMAPK6 proteins in vivo and in vitro. Also, RAI1 was phosphorylated by OsMAPK3/6 and OsMKK4-dd in vitro. Overexpression of OsMAPK6 and/or OsMAPK3 together with OsMKK4-dd increased PAL1 and OsWRKY19 expression in rice protoplasts. Therefore, the regulation of PAL1 and OsWRKY19 expression by RAI1 could be controlled via an OsMKK4-OsMAPK3/6 cascade. Co-immunoprecipitation assays indicated that OsMAPK3 and OsRac1 occur in the same complex as OsMAPK6. Taken together, our results indicate that RAI1 could be regulated by OsRac1 through an OsMAPK3/6 cascade. In this study, we have identified RAI1 as the first transcription factor acting downstream of OsRac1. This work will help us to understand the immune system regulated by OsRac1 in rice and its orthologs in other plant species.

REF4 and RFR1, Subunits of the Transcriptional Coregulatory Complex Mediator, Are Required for Phenylpropanoid Homeostasis in Arabidopsis

The plant phenylpropanoid pathway produces an array of metabolites that impact human health and the utility of feed and fiber crops. We previously characterized several Arabidopsis thaliana mutants with dominant mutations in REDUCED EPIDERMAL FLUORESCENCE 4 (REF4) that cause dwarfing and decreased accumulation of phenylpropanoids. In contrast, ref4 null plants are of normal stature and have no apparent defect in phenylpropanoid biosynthesis. Here we show that disruption of both REF4 and its paralog, REF4-RELATED 1 (RFR1), results in enhanced expression of multiple phenylpropanoid biosynthetic genes, as well as increased accumulation of numerous downstream products. We also show that the dominant ref4-3 mutant protein interferes with the ability of the PAP1/MYB75 transcription factor to induce the expression of PAL1 and drive anthocyanin accumulation. Consistent with our experimental results, both REF4 and RFR1 have been shown to physically associate with the conserved transcriptional coregulatory complex, Mediator, which transduces information from cis-acting DNA elements to RNA polymerase II at the core promoter. Taken together, our data provide critical genetic support for a functional role of REF4 and RFR1 in the Mediator complex, and for Mediator in the maintenance of phenylpropanoid homeostasis. Finally, we show that wild-type RFR1 substantially mitigates the phenotype of the dominant ref4-3 mutant, suggesting that REF4 and RFR1 may compete with one another for common binding partners or for occupancy in Mediator. Determining the functions of diverse Mediator subunits is essential to understand eukaryotic gene regulation, and to facilitate rational manipulation of plant metabolic pathways to better suit human needs.

Influence of Human Lactoferrin Expression on Iron Homeostasis, Flavonoids, and Antioxidants in Transgenic Tobacco

This study was aimed at to check the influence of human lactoferrin (hLF) expression on iron homeostasis, flavonoids, and antioxidants in transgenic tobacco. Transgenic tobacco expressing hLF cDNA under the control of a CaMV 35S promoter was produced. The iron content as well as chlorophyll content of transgenic tobacco was lower compared to mock and untransformed wild plants. Interestingly, hLF transgenic tobacco showed higher level of transcript expression for genes related to iron content regulation like iron transporter and metal transporter. While expression of genes related to iron storage such as ferritin 1 and ferritin 2 was downregulated. The transcript expression of genes encoding antioxidant enzymes such as glutathione reductase, glutathione-S-transferase, ascorbate peroxidase, and catalase was downregulated in hLF transgenic tobacco compared to controls. Further, the transcript expression of two important genes encoding dihydroflavonol reductase (DFR) and phenylalanine ammonia lyase regulatory enzymes of flavonoid biosynthesis pathway was analyzed. The expression of DFR was found to be downregulated, while PAL expression was upregulated in hLF transgenic tobacco compared to mock and untransformed wild plant. Total phenolics, flavonoids, and proanthocyanidins contents were found to be higher in hLF transgenic tobacco than the mock and untransformed wild plant. Results suggest that hLF expression in transgenic tobacco leads to iron deficiency, downregulation of antioxidant enzymes, and increase in total flavonoids.

Expression of flavonoid biosynthesis genes vis-à-vis rutin content variation in different growth stages of Fagopyrum species

Buckwheat is one of the field crops with the highest concentration of rutin, an important flavonoid of medicinal value. Two species of buckwheat, Fagopyrum esculentum and Fagopyrum tataricum, are the major sources of rutin. Seeds of latter contain 40–50× higher rutin compared to the former. The physiological and molecular bases of rutin content variation between Fagopyrum species are not known. The current study investigated the differences in rutin content in seeds and in other tissues and growth stages of two Fagopyrum species, and also correlated those differences with the expression of flavonoid pathway genes. The analysis of rutin content dynamics at different growth stages, S1–S9 (from seed germination to mature seed formation) of Fagopyrum species revealed that rutin content was higher during seedling stages of F. tataricum (3.5 to 4.6-fold) compared to F. esculentum and then increased exponentially from stages S3 to S6 (different leaf maturing stages and inflorescence) of F. esculentum, whereas it fluctuated in F. tataricum. The rutin content was highest in the inflorescence stage (S6) of both species, with a relatively higher biosynthesis and accumulation during post-flowering stages of F. tataricum compared to F. esculentum. The expression of flavonoid pathway genes, through qRT-PCR, in different growth stages vis-à-vis rutin content variation showed differential expression for four genes, PAL, CHS, CHI and FLS with the amounts of transcripts relatively higher in F. tataricum compared to F. esculentum, thereby, correlating these genes with the biosynthesis and accumulation of rutin. The expression of PAL was highest, 7.69 and 8.96-fold in Stages 2 (seedling stage) and 9 (fully developed seeds) of F. tataricum compared to F. esculentum, respectively. The expression of the CHS gene correlated with the rutin content because it was highest in the flowers (S6) and fully developed seeds (S9) of both Fagopyrum species, with relatively higher transcript amounts (2.13 and 3.19-fold, respectively) in F. tataricum (IC-329457) compared to F. esculentum (IC-540858). This study provides useful information on molecular and physiological dynamics of rutin biosynthesis and accumulation in Fagopyrum species and the correlation of expression of flavonoid biosynthesis genes with the rutin content can be useful in planning for genetic improvement.

Effects of PEG stress on flavonoids accumulation and related gene expression in suspension of Scutellaria baicalensis

To study the effects of PEG stress on baicalin, baicalein accumulation induced by an increased concentration of PEG solution and the related genes' expression in suspension of Scutellaria baicalensis. The content of baicalin, baicalein in suspension of S. baicalensis was determined by HPLC. The related genes' expression was analyzed by semi-quantitative PCR. The content of proline in suspension of S. baicalensis was promoted by PEG treatment. Ten percent PEG treatment promoted the expression of PAL and the content of baicalein in experimental material via a drought stress. 20% PEG solution treatment promoted the expression of UBGAT. At the same time, the increased activity of APX inhibited the progress of eliminating reactive oxygen by baicalein, which induced the transformation from baicalein to baicalin. Active ingredient in suspension of S. baicalensis was promoted significantly via a stress of light concentration of PEG solution.

Effects of solar UV-A and UV-B radiation on gene expression and phenolic accumulation in Betula pendula leaves

Ultraviolet (UV) radiation is an important environmental factor for plant communities; however, plant responses to solar UV are not fully understood. Here, we report differential effects of solar UV-A and UV-B radiation on the expression of flavonoid pathway genes and phenolic accumulation in leaves of Betula pendula Roth (silver birch) seedlings grown outdoors. Plants were exposed for 30 days to six UV treatments created using three types of plastic film. Epidermal flavonoids measured in vivo decreased when UV-B was excluded. In addition, the concentrations of six flavonoids determined by high-performance liquid chromatography-mass spectrometry declined linearly with UV-B exclusion, and transcripts of PAL and HYH measured by quantitative real-time polymerase chain reaction were expressed at lower levels. UV-A linearly regulated the accumulation of quercetin-3-galactoside and quercetin-3-arabinopyranoside and had a quadratic effect on HYH expression. Furthermore, there were strong positive correlations between PAL expression and accumulation of four flavonols under the UV treatments. Our findings in silver birch contribute to a more detailed understanding of plant responses to solar UV radiation at both molecular and metabolite levels.

Systemic induced effects of mechanical wounding on the chemical defense of Bt corn (Zea mays)

Aims The major objective was to explore the difference in the systematic induced effects of mechanical wounding on Bt corn and conventional corn after the first leaf of corn seedlings was mechanically wounded. Methods Two Bt corns,'5422Bt1' (Bt11) and '5422CBCL' (Mon810),as well as their conventional corn '5422' were used to detect changes in the content of direct defense chemical DIMBOA and expression of its genes (Bx1,Bx6 and Bx9),content of direct defense chemical total phenol and expression of its gene (PAL),direct defense protein genes (MPI and PR-2a),and the indirect defense chemical volatile genes (FPS and TPS) in the non-wounded parts (second leaf and roots) by employing chemical analysis and gene expression analysis methods after the first leaf of corn seedlings was mechanically wounded for 6 h. Important findings Mechanical wounding systematically induced the expression of PAL and TPS in the second leaf and PAL and FPS in the roots of conventional corn '5422' as compared with the healthy plants,which significantly increased the DIMBOA content in the second leaf but reduced the DIMBOA content and its gene expression of Bx6 and Bx9 in the roots. In addition,mechanical wounding systematically induced the gene expression of Bx6,PAL,PR-2a and TPS in the second leaf and Bx6,Bx9,PAL,MPI,PR-2a and TPS in the roots of Bt corn '5422Bt1',resulting in increased DIMBOA content of the second leaf. In terms of '5422CBCL',mechanical wounding systematically induced the gene expression of Bx9,PAL,PR-2a and TPS in the second leaf and the gene expression of Bx6,PAL,MPI and TPS in the roots,which remarkably increased the total phenol content in the roots. The systematic induced effect of mechanical wounding on the chemical defense response is more obvious in the two Bt corns as compared with the conventional corn '5422',suggesting that there is a synergistic relationship between the Bt gene introduction and chemical defense response during the systematic induced defense processes inflicted by mechanical wounding.

Effect of Fusarium oxysporum endophyte inoculation on the activities of phenylpropanoid pathway enzymes and Radopholus similis numbers in susceptible and tolerant East African Highland bananas

AbstractPhenylpropanoid pathway enzymes are involved in plant defence pathways leading to lignification, synthesis of secondary metabolites, such as salicylic acid and phytoalexins, wound healing and the oxidative burst. Endophytic non-pathogenic Fusarium oxysporum isolate V5w2 has been reported to show antagonistic effects against Radopholus similis in the screenhouse. An experiment was designed to study root biochemical changes (PAL, POX and PPO activities) and R. similis numbers in susceptible and tolerant bananas following inoculation with isolate V5w2. Constitutive expression of PAL and PPO were similar between the susceptible and tolerant cultivars, while constitutive POX activity was higher in the tolerant cultivar. PAL activity was suppressed in both cultivars 7 days after endophyte inoculation (7 dai), but was significantly up-regulated in the susceptible cv. Nabusa at 30 days post nematode challenge (dpnc) in endophyte-inoculated plants. In the tolerant cultivar, PAL activity was up-regulated in R. similis-challenged plants at 7 and 30 dpnc, irrespective of endophyte inoculation. POX and PPO were transiently up-regulated in cv. Nabusa 7 dai, exceeding levels observed in non-inoculated plants of the same cultivar. POX activity was up-regulated at 7 dpnc in endophyte-inoculated cv. Nabusa plants challenged with R. similis. In the tolerant cultivar, POX and PPO activities were similarly up-regulated in R. similis-challenged plants at 7 dpnc, irrespective of endophyte inoculation. The findings of this study implicate PAL, POX and PPO in banana defence against the root-burrowing nematode R. similis. Our findings further demonstrate the ability of endophytic F. oxysporum isolate V5w2 directly to induce POX and prime PAL in the susceptible cv. Nabusa for greater up-regulation following R. similis challenge.

TheMtSNF4b subunit of the sucrose non-fermenting-related kinase complex connects after-ripening and constitutive defense responses in seeds ofMedicago truncatula

Dormant seeds are capable of remaining alive in the hydrated state for extended periods of time without losing vigor, until environmental cues or after-ripening result in the release of dormancy. Here, we investigated the possible role of the regulatory subunit of the sucrose non-fermenting-related kinase complex, MtSNF4b, in dormancy of Medicago truncatula seeds. Expression of MtSNF4b and its involvement in a high-molecular-weight complex are found in dormant seeds, whereas imbibition of fully after-ripened, non-dormant seeds leads to dissociation of the complex. MtSNF4b is capable of complementing the yeast Delta snf4 mutant and of interacting with the MtSnRK1 alpha-subunit in a double hybrid system. Transcriptome analyses on freshly harvested and after-ripened RNAi Mtsnf4b and wild-type embryos implicate MtSNF4b in the defense response in hydrated dormant embryonic tissues, affecting the expression of genes encoding enzymes of flavonoid and phenylpropanoid metabolism, WRKY transcription factors and pathogenesis-related proteins. Silencing MtSNF4b also increased the speed of after-ripening during dry storage, an effect that appears to be related to a change in base water potential. No significant difference in ABA content or sensitivity was detected between mutant and wild-type seeds. Pharmacological studies using hexoses and sugar analogs revealed that mannose restored germination behavior and expression of the genes PAL, CHR and IFR in RNAi Mtsnf4b seeds towards that of the wild-type, suggesting that MtSNF4b might act upstream of sugar-sensing pathways. Overall, the results suggest that MtSNF4b participates in regulation of a constitutively activated defense response in hydrated, dormant seeds.

Cloning and Induction of Phenylalanine Ammonia-lyase Gene from Salvia miltiorrhiza and Its Effect on Hydrophilic Phenolic Acids Levels

Aim To Clone phenylalanine ammonia-lyase gene (designated as Smpal) from Salvia miltiorrhiza and to study its expression, induction and effect on hydrophilic phenolic acids levels in S. miltiorrhiza. Methods Smpal was cloned and well characterized from S. miltiorrhiza, using Rapid Amplification of cDNA ends (RACE) technology. The expression of Smpal could be induced by MeJA, SA and GA3. The metabolite content was determined by LC-MS. Results The full-length of Smpal cDNA was 2354 bp in size and contained an ORF of 2133 bp encoding 711 amino-acids, a 5′ untranslated region (UTR) of 21 bp, and a 3′ UTR of 171 bp. RT-PCR analysis revealed that the Smpal expressed constitutively in all test plant organs with the highest expression level in root, followed by stem and leaves. Conclusion The deduced Smpal shared an overall identity of 62.4%-93% with other known pals. Southern hybridization analyses suggested that Smpal belonged to a multigene family. The fluctuation of RA and LAB content was directly correlated with pal expression.

Timing and concentration effects on the defense response of Zea mays seedlings after application of jasmonic acid to leaves.

Aims Our objectives were to investigate temporal dynamics of the contents of direct defense chemicals (DIMBOA and total phenolics) and the expression levels of their corresponding key genes (Bx1, Bx9 and PAL), direct defense protein-related genes (PR-1, PR-2a and MPI), and indirect defense chemical volatile-related genes (FPS and TPS) in leaves and root systems of Zea mays seedlings under the application of exogenous jasmonic acid (JA) on leaf surface. Methods Leaves of Z. mays cultivar Gaoyou 115 were treated with JA with concentrations of 1, 2.5, 5 and 10 mmol·L–1. We examined contents of the defense chemicals and expression dynamics of their synthesis-mediated genes, direct defense protein-related genes and indirect defense chemical volatile-related genes in both leaves and roots from 3 to 48 h after the application. Important findings Exogenous JA application resulted in significant timing and concentration effects on the chemical defense response. Gene expression of Bx9 and PAL in the leaves could be induced 3 to 6 h after treatment, resulting in an apparent increase in the content of DIMBOA and total phenolics. The increase was positively correlated with JA concentrations, but the inductive effect gradually decreased afterward. Gene expression of PR-2a and MPI in the leaves was also dramatically induced by JA up to 24 and 48 h, respectively. High JA concentrations induced the expression of FPS gene, whereas low JA concentrations had inducible effects on the expression of TPS after 3–6 h. In addition, JA treatment to leaves could indirectly induce the defense response in roots. Most parameters measured in the indirect induction mainly occurred in the later phase (after 24–48 h) of the treatment. For example, JA systematically increased the contents of DIMBOA and total phenolics in the roots and enhanced the expression of PR-2a, MPI, FPS and TPS genes in the roots 48 h after the treatment, showing a tendency of positive relationship with JA concentrations. These findings provided evidence that the indirect induction by JA application to Z. mays leaves on roots was not as strong as on leaves. Leaves started the defense response earlier than roots, and the induction of defense response both in leaves and roots was increased with increasing JA concentrations.

Pythium infection activates conserved plant defense responses in mosses

The moss Physcomitrella patens (P. patens) is a useful model to study abiotic stress responses since it is highly tolerant to drought, salt and osmotic stress. However, very little is known about the defense mechanisms activated in this moss after pathogen assault. In this study, we show that P. patens activated multiple and similar responses against Pythium irregulare and Pythium debaryanum, including the reinforcement of the cell wall, induction of the defense genes CHS, LOX and PAL, and accumulation of the signaling molecules jasmonic acid (JA) and its precursor 12-oxo-phytodienoic acid (OPDA). However, theses responses were not sufficient and infection could not be prevented leading to hyphae colonization of moss tissues and plant decay. Pythium infection induced reactive oxygen species production and caused cell death of moss tissues. Taken together, these data indicate that Pythium infection activates in P. patens common responses to those previously characterized in flowering plants. Microscopic analysis also revealed intracellular relocation of chloroplasts in Pythium-infected tissues toward the infection site. In addition, OPDA, JA and its methyl ester methyl jasmonate induced the expression of PAL. Our results show for the first time JA and OPDA accumulation in a moss and suggest that this defense pathway is functional and has been maintained during the evolution of plants.

Low responsiveness of grapevine flowers and berries at fruit set to UV-C irradiation

In grapevine, stimulation of defence responses was evidenced in response to various types of abiotic stresses in both leaves and berries, as revealed by the increasing expression of genes encoding defence-related proteins or the stimulation of their corresponding activities. However, the capability of inflorescences to respond to abiotic stresses has never been investigated. Therefore, plant defence reactions in response to UV-C irradiation were followed in inflorescences and young clusters focusing on both bunchstems (peduncle and pedicels) and developing flowers/berries from separated floral buds stage [Biologische Bundesanstalt, Bundessortenamt and CHemical industry (BBCH) stage 57] to groat-sized berries stage (BBCH 73). For this purpose, the expression of various genes coding for pathogenesis-related (PR) proteins (class I and III chitinases, Chi1b and CH3; beta-1,3-glucanase, GLUC), an enzyme of the phenylpropanoid pathway (phenylalanine ammonia-lyase, PAL), and stilbene synthase (STS) was analysed in parallel with variations of chitinase activity and the accumulation of the phytoalexin resveratrol. Multiple defence responses were induced in bunchstems of both inflorescences and clusters following UV-C treatment. First, expression of genes encoding PR proteins was stimulated and chitinase activity was enhanced. Secondly, PAL and STS expression increased in association with resveratrol accumulation. Amazingly, none of the tested defence processes was induced in grapevine flowers following UV-C exposure, whatever the stage analysed. Similarly, in berries at fruit set, induction of gene expression was weak and neither an increase in chitinase activity nor resveratrol synthesis was noticed. However, in groat-sized berries, responsiveness to UV-C increased, as revealed by the induction of CH3, PAL, and STS expression, together with resveratrol accumulation. The differential responsiveness between bunchstems, flowers, and berries is discussed.

Nitric Oxide Regulates Shikonin Formation in Suspension-Cultured Onosma paniculatum Cells

Endogenously occurring nitric oxide (NO) is involved in the regulation of shikonin formation in Onosma paniculatum cells. NO generated after cells were inoculated into shikonin production medium reached the highest level after 2 d of culture, which was 16 times that at the beginning of the experiment, and maintained a high level for 6 d. A nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine (L-NNA), and a nitrate reductase (NR) inhibitor, sodium azide (SoA), consistent with their inhibition of NO biosynthesis, decreased shikonin formation significantly. This reduction could be alleviated or even abolished by exogenous NO supplied by sodium nitroprusside (SNP), suggesting that the inhibition of NO biosynthesis resulted in decreased shikonin formation. However, when endogenous NO biosynthesis was up-regulated by the elicitor from Rhizoctonia cerealis, shikonin production was enhanced further, showing a dependence on the elicitor-induced NO burst. Real-time PCR analysis showed that NO could significantly up-regulate the expression of PAL, PGT and HMGR, which encode key enzymes involved in shikonin biosynthesis. These results demonstrated that NO plays a critical role in shikonin formation in O. paniculatum cells.

Inhibition of wound-induced ethylene does not prevent red discoloration in fresh-cut endive (Cichorium intybus L.)

Consumer demand for lightly processed ready-to-eat fruits and vegetables increases consistently as convenience and easiness of preparation become decision factors in food purchases. However, red discoloration of cut leafy vegetables limit storage life and the flexibility of offering specific products. This is the case of fresh-cut endive (Cichorium intybus L.), that turn red quickly and thus have a limited shelf life either alone or in mixed vegetables products. Heat shock was shown to block red discoloration in cut endive, through significantly reducing the level of phenylalanine ammonia-lyase (PAL1) transcripts and stimulating peak levels of heat shock protein (HSP90) transcript accumulation shortly after cut. With the aim of evaluating to what extent the effect of heat shock was related to the blockage of wound-induced ethylene, we applied [S]-trans-2-amino-4-(2-aminoethoxy)-3-butenoic acid hydrochloride (AVG) to block ethylene synthesis and 1-methylcyclopropene (1-MCP) to block the response to ethylene in fresh-cut endive, and analysed wound induced ethylene emission as well as HSP90 and PAL expression patterns as compared to samples treated by heat shock (46 °C/120 s). Ethylene was measured continuously during 48 h with a laser-based ethylene detector, and real-time quantitative PCR (qPCR) was performed for PAL1 and PAL2 as well as for degenerated HSP90 primers. Red discoloration limited the shelf life of 1-MCP and AVG treated endives to 48 h, whereas samples treated with heat shock maintained good quality for 8 days. Wound-induced ethylene was blocked by AVG, inhibited by 1-MCP but not by heat shock, which was shown to delay ethylene emission by at least 8 h after cutting, after which the rates increased to higher levels than in the control and took longer to decrease. It was concluded that ethylene is not the main factor in the processes that lead to tissue discoloration in fresh-cut endive.

An early Ca2+ influx is a prerequisite to thaxtomin A-induced cell death in Arabidopsis thaliana cells

The pathogenicity of various Streptomyces scabies isolates involved in potato scab disease was correlated with the production of thaxtomin A. Since calcium is known as an essential second messenger associated with pathogen-induced plant responses and cell death, it was investigated whether thaxtomin A could induce a Ca2+ influx related to cell death and to other putative plant responses using Arabidopsis thaliana suspension cells, which is a convenient model to study plant-microbe interactions. A. thaliana cells were treated with micromolar concentrations of thaxtomin A. Cell death was quantified and ion flux variations were analysed from electrophysiological measurements with the apoaequorin Ca2+ reporter protein and by external pH measurement. Involvement of anion and calcium channels in signal transduction leading to programmed cell death was determined by using specific inhibitors. These data suggest that this toxin induces a rapid Ca2+ influx and cell death in A. thaliana cell suspensions. Moreover, these data provide strong evidence that the Ca2+ influx induced by thaxtomin A is necessary to achieve this cell death and is a prerequisite to early thaxtomin A-induced responses: anion current increase, alkalization of the external medium, and the expression of PAL1 coding for a key enzyme of the phenylpropanoid pathway.

Production of l-phenylalanine from trans-cinnamic acids by high-level expression of phenylalanine ammonia lyase gene from Rhodosporidium toruloides in Escherichia coli

A combined promoter expression vector pBV–PAL for high-level expression of phenylalanine ammonia lyase gene of Rhodosporidium toruloides was constructed. Pal gene was cloned and inserted into the region between SalI and PstI restriction sites of expression vector pBV220 (containing P L P R promoter) to obtain recombinant expression vector pBV220–PAL. The tac promoter obtained from the plasmid pKtac was inserted into the expression vector pBV220–PAL to construct expression vector pBV–PAL. The recombinant plasmid pBV220–PAL and pBV–PAL were introduced into Escherichia coli JM109 by transformation. The result showed that the transformant E. coli JM109 (pBV–PAL) gave a much higher PAL activity than that transformant E. coli JM109 (pBV220–PAL). Recombinant PAL expression level of the transformant JM109 (pBV–PAL) was about 9.6% of total cellular protein, specific enzyme activity was 2.3-fold higher than that of the transformant JM109 (pBV220–PAL), reached 35 U/g (dry cells weight, DCW). PAL specific activity of 123 U/g (DCW) could be achieved in a 5-l fermentor. 80.5% conversion rate of trans-cinnamic acid to l-phenylalanine and 5.12 g/l l-phenylalanine were obtained after 3 h bioconversion using the transformant JM109 (pBV–PAL). The recombinant strain JM109 containing the combined promoter expression vector pBV–PAL was shown to be effective and practical to product l-phenylalanine.

Salicylic acid activates phenylalanine ammonia-lyase in grape berry in response to high temperature stress

Our previous work has indicated that salicylic acid (SA) is involved in the development of pea plant thermotolerance and induces the gene expression of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), a key enzyme in phenylpropanoid metabolism of grape berry. However, the relationship between SA and PAL during high temperature stress remains obscure. The present experiment, using the technique of in vivo incubation of the grape berry (Vitis vinifera L. cv. Cabernet Sauvignon) tissue in the SA-contained medium, the effects of exogenous SA on the gene expression of PAL and the accumulation of polyphenols during high temperature stress were investigated. The results showed that SA could induce the accumulation of PAL mRNA and the synthesis of new PAL protein, and increase the activity under high temperature stress. A significant accumulation of phenolics was also observed in the SA-treated berries. But, the activation of PAL by SA could be blocked by the pretreatments of berry tissues with the protein synthesis inhibitor cycloheximide, and mRNA transcription inhibitor, actinomycin D, respectively. It is thus speculated that SA may induce the activation of PAL and the accumulation of pheonlics leading to the development of thermotolerance.

Defense-related gene expression in susceptible and tolerant bananas ( Musa spp.) following inoculation with non-pathogenic Fusarium oxysporum endophytes and challenge with Radopholus similis

Radopholus similis is a major pest of East African highland cooking bananas ( Musa spp.) in Uganda. Non-pathogenic Fusarium oxysporum endophytes, isolated from bananas in farmers’ fields, have shown potential to reduce R. similis numbers in tissue culture banana. The mechanism through which endophytes confer resistance to nematodes has previously been demonstrated to involve induced resistance. In this study, the expression of eight defense-related genes in banana was investigated using quantitative real-time reverse transcription PCR. Plants of susceptible (cv. Nabusa, genomic group AAA-EA) and tolerant (cv. Kayinja, genomic group ABB) banana cultivars were inoculated with endophytic F. oxysporum strain V5w2. Gene expression levels were analysed following endophyte inoculation and nematode challenge. Endophyte colonization of roots of the tolerant cultivar induced transient expression of POX and suppressed expression of PR-3, lectin, PAE, PAL and PIR7A. Catalase and PR-1 activities were up-regulated in the tolerant cultivar 33 days after endophyte colonization of roots, but their expressions were further up-regulated following nematode challenge. Apart from POX and lectin, the other genes analysed were not responsive to endophyte colonization or R. similis challenge in the susceptible cv. Nabusa. This is the first report of endophyte-induced defense-related gene expression in banana.

Introduction of sense constructs of cinnamate 4-hydroxylase (CYP73A24) in transgenic tomato plants shows opposite effects on flux into stem lignin and fruit flavonoids

Understanding regulation of phenolic metabolism underpins attempts to engineer plants for diverse properties such as increased levels of antioxidant flavonoids for dietary improvements or reduction of lignin for improvements to fibre resources for industrial use. Previous attempts to alter phenolic metabolism at the level of the second enzyme of the pathway, cinnamate 4-hydroxylase have employed antisense expression of heterologous sequences in tobacco. The present study describes the consequences of homologous sense expression of tomato CYP73A24 on the lignin content of stems and the flavonoid content of fruits. An extensive number of lines were produced and displayed four developmental variants besides a normal phenotype. These aberrant phenotypes were classified as dwarf plants, plants with distorted (curly) leaves, plants with long internodes and plants with thickened waxy leaves. Nevertheless, some of the lines showed the desired increase in the level of rutin and naringenin in fruit in a normal phenotype background. However this could not be correlated directly to increased levels of PAL and C4H expression as other lines showed less accumulation, although all lines tested showed increases in leaf chlorogenic acid which is typical of Solanaceous plants when engineered in the phenylpropanoid pathway. Almost all transgenic lines analysed showed a considerable reduction in stem lignin and in the lines that were specifically examined, this was correlated with partial sense suppression of C4H. Although not the primary purpose of the study, these reductions in lignin were amongst the greatest seen in plants modified for lignin by manipulation of structural genes. The lignin showed higher syringyl to coniferyl monomeric content contrary to that previously seen in tobacco engineered for downregulation of cinnamate 4-hydroxylase. These outcomes are consistent with placing CYP73A24 more in the lignin pathway and having a role in flux control, while more complex regulatory processes are likely to be involved in flavonoid and chlorogenic acid accumulation.