PAL2 Gene Research Articles

Trichoderma gamsiiT6085, a biocontrol agent of Fusarium head blight, modulates biocontrol‐relevant defence genes expression in wheat

AbstractTo enhance the framework of the mechanisms of action used by Trichoderma gamsii T6085 for the control of Fusarium head blight (FHB), this work investigated its ability to modulate the expression of defence‐related genes of wheat (Triticum aestivum ‘Apogee’) in response to endophytic colonization of plant tissues. Changes in relative expression of pal1, pr1, pgip2 and lox1 genes were assessed over time in wheat roots, in spikes colonized by T6085 alone and both T6085 and Fusarium graminearum, and in leaves from wheat seedlings root‐inoculated with T6085. Results indicate the ability of T6085 to induce local and systemic defence responses in wheat plants in the presence of one of the causal agents of FHB. There was a general significant up‐regulation of the plant defence‐related genes analysed, especially in the first days after the application of T6085. According to these results, modulation of plant defence genes could be included within the arsenal of mechanisms used by T6085 when applied to wheat, an additional feature of interest in the management of FHB. To evaluate the effect of the plant genotype on the ability of T6085 to endophytically colonize roots, root colonization was assessed on four cultivars of T. aestivum and two cultivars of T. durum. Data showed that roots of only two T. aestivum cultivars were endophytically colonized by T6085, similar to cv. Apogee used here as control, thus demonstrating an effect of the host genotype on the endophytic ability of T6085.

Expression of Mn-sod, PAL1, aos1 and HPL genes in soybean plants overexpressing the NmDef02 defensin.

Plant defensins are a potential tool in crop improvement programs through biotechnology. Their antifungal action makes them attractive molecules for the production of transgenic plants. Information is currently lacking on what happens to the expression of defense genes in transgenic plants that overexpress a defensin. Here we show the relative expression of four defense-related genes: Mn-sod, PAL1, aos1 and HPL evaluated in two transgenic soybean events (Def1 and Def17) constitutively expressing the NmDef02 defensin gene from Nicotiana megalosiphon. The expression of these defense genes showed a differential profile in the transgenic events, with the increased expression of the aos1 gene and the repression of the Mn-sod gene in both events, when compared to the non-transgenic control. Furthermore, the expression of the PAL1 gene only increased in the Def17 event. The results indicate that although there were some changes in the expression of defense genes in transgenic plants overexpressing the defensin NmDef02; the morphoagronomic parameters evaluated were similar to the non-transgenic control. Understanding the molecular changes that occur in these transgenic plants could be of interest in the short, medium and long term.

Biosynthesis of capsaicinoids in pungent peppers under salinity stress.

The synthesis of capsaicinoids occurs in the placenta of the fruits of pungent peppers. However, the mechanism of capsaicinoids' biosynthesis in pungent peppers under salinity stress conditions is unknown. The Habanero and Maras genotypes, the hottest peppers in the world, were chosen as plant material for this study, and they were grown under normal and salinity (5 dS m-1 ) conditions. The results showed that salinity stress harmed plant growth but increased the capsaicin content by 35.11% and 37.00%, as well as the dihydrocapsaicin content by 30.82% and 72.89% in the fruits of the Maras and Habanero genotypes, respectively, at 30 days after planting. The expression analysis of key genes in capsaicinoids biosynthesis revealed that the PAL1, pAMT, KAS, and PUN1 genes were overexpressed in the vegetative and reproductive organs of pungent peppers under normal conditions. However, under salinity stress, overexpression of PAL1, pAMT, and PUN1 genes was identified in the roots of both genotypes, which was accompanied by an increase in capsaicin and dihydrocapsaicin content. The findings showed that salinity stress caused an enhancement in the capsaicin and dihydrocapsaicin contents in the roots, leaves, and fruits of pungent peppers. Nonetheless, it was found that the production of capsaicinoids is generally not restricted to the fruits of pungent peppers.

Salinity alleviator bacteria in rice (Oryza sativa L.), their colonization efficacy, and synergism with melatonin.

In this study, rhizospheric and endophytic bacteria were tested for the alleviation of salinity stress in rice. Endophytic isolates were taken from previous studies based on their salt stress-alleviating traits. The rhizospheric bacteria were isolated from rice and screened based on salt tolerance and plant growth-promoting traits. Molecular identification indicated the presence of class Gammaproteobacteria, Bacillota, and Actinomycetia. Two-two most potential isolates each from rhizospheric and endophytic bacteria were selected for in planta trials. Results showed that microbial inoculation significantly improved germination and seedling vigor under elevated salinity. The confocal scanning laser microscopy showed higher bacterial colonization in inoculated rice roots than in control. Based on this experiment, rhizospheric bacteria Brevibacterium frigoritolerans W19 and endophytic Bacillus safensis BTL5 were selected for pot trial along with a growth-inducing compound melatonin 20 ppm. Inoculation of these two bacteria improved the levels of chlorophyll, proline, phenylalanine ammonia-lyase, catalase, superoxide dismutase, polyphenol oxidase, root-shoot length, and dry weight under elevated salt concentration. The gene expression studies showed modulation of SOD1, CATa, NHX1, and PAL1 genes by the bacterial strains and melatonin application. The inoculation was found to have additive effects with 20 ppm melatonin. This enhancement in dry matter accumulation, compatible solute production, and oxidative stress regulation could help plants in mitigating the ill effects of high salinity. Exploring such a combination of microbes and inducer molecules could be potentially useful in developing stress-alleviating bioformulations.

Analysis of flavonoid metabolism during fruit development of Lycium chinense

Lycium chinense is an important medicinal plant in the northwest of China. Flavonoids are the major pharmacological components of L. chinense fruits. However, flavonoid metabolism during fruit development of L. chinense remains to be studied. Here, we analyzed the change of flavonoid contents, enzyme activity, and gene expression during fruit development of L. chinense. We found that flavonoids, anthocyanins, and catechins are the most important components of L. chinense fruits. Flavonoid content was increased with fruit development and was high at the late developmental stage. PAL, CHS, and F3H enzymes played a significant role in flavonoid accumulation in fruits. Transcriptomic analysis showed that anthocyanin pathway, flavonol pathway, flavonoid biosynthesis, and phenylpropanoid synthesis pathway were the major pathways involved in flavonoid metabolism in L. chinense. Gene expression analysis indicated that PAL1 and CHS2 genes were critical for flavonoid metabolism in L. chinense fruits. These discoveries help us understand the dynamic changes in flavonoids during fruit development and enhance the use of L. chinense fruits.

A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana

BackgroundCRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS).ResultsIn this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programmable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold and 245-fold for the endogenous N. benthamiana DFR and PAL2 genes, respectively, with negligible expression in the absence of the trigger.ConclusionsThe tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.

Exploring Two Streptomyces Species to Control Rhizoctonia solani in Tomato

Streptomyces species are effective biocontrol agents toward many plant pathogens. These microorganisms are well known for producing secondary metabolites, promoting plant growth and inducing plant defense mechanisms. In this study, the ability of tomato root-colonizing Streptomyces strains to trigger the resistance against Rhizoctonia solani (J.G. Kühn) AG4 was investigated. For this goal, we evaluated the pattern of LOXB and PAL1 genes expression changes upon pathogen inoculation in primed tomato plants. The results revealed that Streptomyces globisporous (Krasil’nikov) strain F8 and S. praecox (Millard and Burr) strain R7 were able to enhance the expression of lipoxygenase and phenylalanine ammonia lyase in tomato plants. This finding suggests that Streptomyces strains F8 and R7 may trigger jasmonic acid and phenyl propanoid signaling pathways in plants, therefore, resulting an induced defense status in tomatoes against R. solani. Biochemical characterization of these Streptomyces strains showed that they were strong producers of siderophores. S. praecox strain R7 produced siderophores of hyderoxamate and catechol types and S. globisporous strain F8 produced a phenolic siderophore. Moreover, they also produced protease while only the S. praecox strain R7 was able to produce amylase. Taken together, these results indicate that S. globisporous strain F8 and S. praecox strain R7 promote plant growth and reduces disease and hence are suitable for future in depth and field studies with the aim to attain appropriate biocontrol agents to protect tomatoes against R. solani.

Sequencing and phylogenetic analysis of phenylalanine ammonia lyase (pal) and chalcone synthase (chs) genes in some Iranian endemic species of Apiaceae

The Apiaceae family includes many valuable medicinal species. The Phenyl propanoid pathway is a source of many of critical secondary metabolites. Hence, the identification of involved genes will improve a better understanding of their medicinal properties. The purpose of this research was to obtain a genomic DNA sequence of two important genes including phenylalanine ammonia lyase 2 (pal2) and chalcone synthase (chs) in some Iranian endemic species of Apiaceae viz. Dorema aucheri, Ferula assa-foetida, Prangos ferulaceae, Kelussia odoratissima Mozaff., Ferulago angulata, Heracleum persicum, and Ducrosia anethifolia. For this purpose, conserved regions of each gene were sequenced. Multiple alignments revealed that studied sequences shared a high degree of similarity based on genes pal2 (78%) and chs (66.9%). The results from the neighbor-joining analysis produced different topologies with moderately to high support. The genera of Ferula and Dorema were the closest, while Ferulago and Ducrosia possessed a greater genetic distance to each other based on the pal2 gene. Moreover, the two genera (Ferulago and Prangos) were grouped based on genes pal2 and chs. The NJ analysis revealed that the four Apiaceae genera did not form a monophyletic group based on the chs gene, which showed a higher level of diversity in the chs family alongside the expansion of Apiaceae. Partial-length genomic DNA sequence of these genes helped realize their evolutionary relationships and led to a better understanding of antioxidant and medicinal properties.

LED light mediates phenolic accumulation and enhances antioxidant activity in Melissa officinalis L. under drought stress condition.

The popularity of lemon balm in conventional medicine is suggested by the existence of two groups of compounds, namely essential oil and phenylpropanoids pathway derivatives. One of the promising approaches to improve tolerance to drought stress inducedoxidative damage in seedlings grownin greenhouses and plant growth chambers is replacing the traditional and high-cost light technologies by recently developed light emitting diodes (LED). In this experiment, we analyzed the role of various LED lights including red (R), blue (B), red (70%) + blue (30%) (RB), and white (W) as well as normalgreenhouse light(as control) to stimulate defense mechanisms against drought stress in two genotypes of Melissa officinalis L. The present study demonstrates that pre-treatment with LEDs with high-intensity output for 4weeks alleviated harmful effects of drought stress in the two genotypes. Under drought stress, RB LED pre-treated plantlets of the two genotypes exhibited the highest relative growth index of shoot and root and total phenolic and anthocyanin content compared to those irradiated with other LEDs and greenhouse lights. The highest amount of malondialdehyde level was detected in R LED exposed plants. In response to drought, LED-exposed plants especially RB light-irradiated plants of the two genotypes maintained significantly higher antioxidant and phenylalanine ammonia-lyase (PAL) enzyme activities and higherexpression level of the PAL1 and 4CL-1 genes compared to those irradiated with greenhouse light. We concluded that RB LED light provides a better growthcondition and resistance to drought stress for the two genotypes of lemon balm by the highest antioxidant activity and the least amount of damage to the cell membranes. Our data suggest that LED light pre-treatments as moderate stress activate antioxidant systems, enhance the scavenging of ROS and induce drought stress tolerance in the two genotypes of lemon balm plants.

Expression patterns of two pal genes of Pleurotus ostreatus across developmental stages and under heat stress

BackgroundPhenylalanine ammonia-lyase (PAL, EC 4.3.1.24) is the first key enzyme in the phenylpropanoid pathway. The pal gene has been widely studied in plants and participates in plant growth, development and defense systems. However, in Pleurotus ostreatus, the biological functions of pal during organismal development and exposure to abiotic stress have not been reported.ResultsIn this study, we cloned and characterized the pal1 (2232 bp) and pal2 (2244 bp) genes from the basidiomycete P. ostreatus CCMSSC 00389. The pal1 and pal2 genes are interrupted by 6 and 10 introns, respectively, and encode proteins of 743 and 747 amino acids, respectively. Furthermore, prokaryotic expression experiments showed that PAL enzymes catalyzed the conversion of L-phenylalanine to trans-cinnamic acid. The function of pal1 and pal2 was determined by constructing overexpression (OE) and RNA interference (RNAi) strains. The results showed that the two pal genes had similar expression patterns during different developmental stages. The expression of pal genes was higher in the reproductive growth stage than in the vegetative growth stage. And the interference of pal1 and pal2 delayed the formation of primordia. The results of heat stress assays showed that the RNAi-pal1 strains had enhanced mycelial tolerance to high temperature, while the RNAi-pal2 strains had enhanced mycelial resistance to H2O2.ConclusionsThese results indicate that two pal genes may play a similar role in the development of P. ostreatus fruiting bodies, but may alleviate stress through different regulatory pathways under heat stress.

Chitosan enhances rosmarinic acid production in shoot cultures of Melissa officinalis L. through the induction of methyl jasmonate

BackgroundChitosan is a polycationic polysaccharide derived from chitin that has been recognized as an effective elicitor in the production of secondary metabolites of many medicinal plants. In this study, the effect of abiotic elicitor (chitosan) at various concentrations on rosmarinic acid (RA) and total phenolic accumulation in shoot cultures of lemon balm was investigated.ResultsTreatment of shoots by chitosan led to a noticeable induction of phenylalanine ammonia-lyase (PAL), catalase (CAT), guaiacol peroxidase (GPX) and lipoxygenase (LOX) activities. Besides, the expression of PAL1, TAT and RAS genes and accumulation of RA and phenolic compound increased in chitosan-treated lemon balm shoots. Chitosan treatment also increased H2O2 accumulation and the expression of RBOH, an essential gene implicated in ROS production. Also, the up-regulation of the OPR gene by exogenous chitosan was associated with the induction of endogenous JA determined by GC-MASS.ConclusionThe present study showed that the induced production of rosmarinic acid by chitosan involves the trigger of defense-related enzymes, up-regulated expression of TAT and RAS genes, and stimulation of JA biosynthesis.

Characterisation of the willow phenylalanine ammonia-lyase (PAL) gene family reveals expression differences compared with poplar

Willow is an important biomass crop for the bioenergy industry, and therefore optimal growth with minimal effects of biotic and abiotic stress is essential. The phenylpropanoid pathway is responsible for the biosynthesis of not only lignin but also of flavonoids, condensed tannins, benzenoids and phenolic glycosides which all have a role in protecting the plant against biotic and abiotic stress. All products of the phenylpropanoid pathway are important for the healthy growth of short rotation cropping species such as willow. However, the phenylpropanoid pathway in willow remains largely uncharacterised. In the current study we identified and characterised five willow phenylalanine ammonia-lyase (PAL) genes, which encode enzymes that catalyse the deamination of l-phenylalanine to form trans-cinnamic acid, the entry point into the phenylpropanoid pathway. Willow PAL1, PAL2, PAL3 and PAL4 genes were orthologous to the poplar genes. However no orthologue of PAL5 appears to be present in willow. Moreover, two tandemly repeated PAL2 orthologues were identified in a single contig. Willow PALs show similar sub-cellular localisation to the poplar genes. However, the enzyme kinetics and gene expression of the willow PAL genes differed slightly, with willow PAL2 being more widely expressed than its poplar orthologues implying a wider role for PALs in the production of flavonoids, condensed tannins, benzenoids, and phenolic glycosides, in willow.

Wound response in orange as a resistance mechanism against Penicillium digitatum (pathogen) and P. expansum (non-host pathogen)

Penicillium digitatum is the most devastating postharvest pathogen of citrus. In addition, Penicillium expansum is the main pathogen of pome fruit, although recent studies have demonstrated its ability to infect oranges under some conditions. In this study, we evaluated wound response in ‘Valencia’ oranges harvested at three different maturity stages and the effect of wound response on the establishment of both pathogens when fruit were stored at two different temperatures (20 and 4°C). The effect of wounding and pathogen inoculation on lignin content, was also quantified. Lastly, the expression of several phenylpropanoid pathway-related genes was also analyzed by semi-quantitative RT-PCR. Results indicated that, in general, P. digitatum exhibited lower decay incidence and severity as time between wounding and inoculation increased. Decay incidence and severity were higher in fruit from the over-mature harvest than in fruit from immature and commercial harvests. P. expansum was able to infect fruit at 20°C but lesions were small compared to lesion size of fruit stored at 4°C. Lignin content in wounded fruit (control) and in samples wounded and inoculated with P. expansum was highest in fruit from the immature harvest at 7d post-wounding and inoculation. Wounded fruit had higher expression of pal1, comt1 and pox1 genes at 48h than at 24h. However, samples inoculated with P. digitatum showed lower expression at 48h than at 24h. Our results indicated that maturity and storage temperature play an important role in orange wound response.

Molecular and analysis of a phenylalanine ammonia-lyase gene (LrPAL2) from Lycoris radiata

Phenylalanine ammonia-lyase (PAL), the first enzyme of phenylpropanoid biosynthesis, participates in the biosynthesis of flavonoids, lignins, stilbenes and many other compounds. In this study, we cloned a 2,326 bp full-length PAL2 gene from Lycoris radiata by using degenerate oligonucleotide primer PCR (DOP-PCR) and the rapid amplification of cDNA ends method. The cDNA contains a 2,124 bp coding region encoding 707 amino acids. The LrPAL2 shares about 77.0 % nucleic acid identity and 83 % amino acid identity with LrPAL1. Furthermore, genome sequence analysis demonstrated that LrPAL2 gene contains one intron and two exons. The 5' flanking sequence of LrPAL2 was also cloned by self-formed adaptor PCR (SEFA-PCR), and a group of putative cis-acting elements such as TATA box, CAAT box, G box, TC-rich repeats, CGTCA motif and TCA-element were identified. The LrPAL2 was detected in all tissues examined, with high abundance in bulbs at leaf sprouting stage and in petals at blooming stage. Besides, LrPAL2 drastically responded to MJ, SNP and UV, moderately responded to GA and SA, and a little increased under wounding. Comparison of LrPAL2 expression and LrPAL1 expression demonstrated that LrPAL2 can be more significantly induced than LrPAL1 under the above treatments, and LrPAL2 transcripts accumulated prominently at blooming stage, especially in petals, while LrPAL1 transcripts did not accumulated significantly at blooming stage. All these results suggested that LrPAL2 might play distinct roles in different branches of the phenylpropanoid pathway.

Differential Expression of Potato Defence Genes Associated with the Salicylic Acid Defence Signalling Pathway in Response to Weakly and Highly Aggressive Isolates of Verticillium dahliae

AbstractVerticillium wilt is a vascular disease affecting hundreds of important dicotyledonous crops worldwide. Its main causal agent in potato is Verticillium dahliae Kleb. A differential potato‐V. dahliae system consisting of two cultivars of potato (susceptible; S and moderately resistant; MR) and two V. dahliae isolates (weakly, WA and highly aggressive, HA), was used to evaluate the expression of five defence‐related genes, PAL1, PAL2, PR‐1, PR‐2 and PR‐5. These genes were selected because they are in general associated with the salicylic acid defence signalling pathway. Expression levels of these genes were assessed in potato roots and leaves at 0, 4 and 21 h (hpi), and 3, 7 and 14 days postinoculation (dpi). In the roots, the expression of PAL1, PR‐1 and PR‐2 in the MR was higher than in the susceptible cultivar in response to inoculation with either one of the tested V. dahliae isolates. PAL2 gene expression increased gradually starting at 21 hpi in the MR cultivar as opposed to the susceptible one. Higher expression of PR‐1 was detected at 7 dpi in roots. In the leaves, both PAL1 and PAL2 genes showed higher expression in the MR cultivar relative to the susceptible one. Interestingly, the expression of PR‐2 was slightly higher in the susceptible cultivar. Combined data analyses revealed that PAL1, PAL2, PR‐1 and PR‐2 genes are regulated at the transcriptional level in response to infection by V. dahliae. These results indicate that the salicylic acid pathway is also involved in potato defence against V. dahliae and add to the data gathered to elucidate the signalling mechanisms in this host–pathogen interaction.

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.

Quantitative RT‐PCR Expression Analysis of Lipodepsipeptides Synthetase and Defence‐related Genes in Orange Fruit in Response to Antagonist–pathogen Interaction

AbstractStrains of Pseudomonas syringae are effective in controlling postharvest diseases of citrus fruits, and antagonistic activity has been correlated with in vitro production of lipodepsipeptides. Additionally, biocontrol agents can induce a range of defence mechanisms of resistance in citrus tissue that result in a broad spectrum of metabolic modifications, such as systemic acquired resistance, induced systemic resistance and production of reactive oxygen species. The aim of this study was to evaluate the expression of syringomycin (syrB1) and syringopeptin (sypA) synthetase genes from P. syringae pv. syringae biocontrol strains in vitro on different culture media and in vivo on citrus fruits (Citrus sinensis cv. Tarocco) during the interaction with Penicillium digitatum by quantitative RT‐PCR. Similarly, gene transcript levels of chitinase (CHI1), allene oxide synthase (AOS), glutathione peroxidase (GPX1) and phenylalanine ammonia‐lyase (PAL1) were measured. SyrB1 and sypA genes were more actively expressed when antagonistic Pseudomonas strains were grown on orange peel broth as compared to NB and PDB. Penicillium digitatum resulted to be strongly stimulatory only to syrB1 expression, thus suggesting that syrB1 gene could be involved in biocontrol activity. QRT‐PCR showed that both P. s. pv. syringae and P. digitatum strains increase CHI1 transcription in inoculated flavedo tissues relative to the untreated control. Interestingly, CHI1 transcription was markedly induced by co‐inoculation of P. s. pv syringae and P. digitatum strains. Pseudomonas syringae pv. syringae, alone or co‐inoculated with P. digitatum, was weakly effective in enhancing GPX1, AOS and PAL1 gene expression, whereas P. digitatum alone strongly enhanced GPX1, AOS and PAL1 expression. Moreover, we assume that CHI1 gene is most likely part of the molecular mechanisms involved in pathogen defence responses in citrus fruit. This is the first example of monitoring the expression of syrB1‐sypA and CHI1, GPX1, AOS and PAL1 genes in interactions between P. s. pv. syringae biocontrol agents and P. digitatum in vitro and in vivo.

Inhibitory mechanisms induced by the endophytic bacterium MGY2 in controlling anthracnose of papaya

An endophytic bacterium named Pseudomonas putida MGY2 was isolated from papaya fruit. The effect of MGY2 on reducing anthracnose caused by Colletotrichum gloeosporioides infection in harvested papaya fruit and the possible mechanisms involved were investigated. Fruit treated with MGY2 showed a significant lower disease index, disease incidence, and lesion diameter than the control. MGY2 reduced the decrease in firmness and delayed the peak of ethylene production of harvested papaya fruit stored at 25 °C. MGY2 also significantly enhanced the activities of phenylalanine ammonia-lyase (PAL), catalase (CAT), and peroxidase (POD) and increased the phenolic content. The expression levels of PAL1, CAT1, and POD genes were obviously elevated in MGY2-treated fruit. These results suggested that papaya fruit is capable of responding to the endophyte P. putida MGY2, which could activate defensive enzymes and genes and thereby induce host disease resistance.

Control of phenylalanine ammonia-lyase gene promoters from pea by UV radiation

The gene fusion system was used to study UV light-control of PS PAL1 and PS PAL2 genes encoding phenylalanine ammonia-lyase of pea. The induction of pea PAL promoters was analysed in transgenic tobacco plants. Binary plasmids (derivatives of pBI101.2 vector) containing 5′ regulatory fragments of PS PAL1 and PS PAL2 linked to reporter genes (GUS,LUC) were constructed. The analyses were performed with the use of single constructs (containing one variant of PS PAL promoter and one reporter gene) and dual constructs (containing both PS PAL1 and PS PAL2 promoters connected with different reporter genes). The use of dual constructs enabled the evaluation of both PS PAL promoters activity in the same plant. The analyses of in vitro grown plants have shown that both PAL promoters are strongly induced in leaves subjected to UV radiation. In some cases, the UV-stimulated expression exceeded the exposed areas. This phenomenon was observed more often in the leaves of plants containing the PS PAL1::GUS than PS PAL2::GUS construct. Removal of boxes 2, 4, 5 from PS PAL1 promoter and deletion of its 5′ end region (-339 to -1394) decreases the level of gene expression but does not eliminate its responsiveness to UV.

Changes in phenylpropanoid metabolites associated with homology‐dependent silencing of phenylalanine ammonia‐lyase and its somatic reversion in tobacco

Gene silencing of phenylalanine ammonia‐lyase (PAL) in transgenic tobacco (Nicotiana tabacumL.) is manifested phenotypically by reduced growth, altered leaf morphology, and reduced levels of phenylpropanoid compounds. Here we report the rare event of somatic reversion from silencing to PAL over‐expression in tobacco harboring the beanPAL2gene. This phenomenon allows the comparison of the effects of PAL under‐ and over‐expression in the same plant. A comparison of gene silenced and revertant tissues reveals striking differences in lignin content and monomer composition, and both qualitative and quantitative differences in soluble and cell wall bound phenylpropanoid compounds in the recovered sector.