Phenylalanine Ammonia-lyase Levels Research Articles

Biochemical responses associated with bacterial blight of rice (Oryza sativa)

Biochemical response of susceptible cultivar TN 1 and resistant near isogenic lines IRBB 13 and IRBB 21 of rice to Xanthomonas oryzae pv. oryzae (Xoo) was studied at different stages of infection. The activities of the defence related enzymes peroxidase (PO), phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) were found to be significantly higher in the resistant isogenic lines, IRBB 13 and IRBB 21 than in the susceptible cultivar TN 1. Total phenols, ortho-dihydroxyphenols and flavonols content also exhibited a similar trend as that of the enzymes. After bacterial infiltration a significant increase in the levels of total phenols, o-dihydroxyphenols, flavonols, PO, PAL and TAL was observed. However, this increase was comparatively rapid and higher in resistant near isogenic lines when compared with the susceptible line. Total sugars, reducing sugars and starch content were significantly higher in the leaves of the susceptible variety TN 1 than in the resistant near isogenic lines IRBB 13 and IRBB 21.

Differences in Responding Sensitivity to Ethephon among Different Maize Genotypes

Lodging is one of the key limiting factors causing serious yield decline in maize cultivation, the plant growth regulator ethephon and its complex formulation are applied to improve quality and lodging-resistance ability of stalks, which is an effective way to prevent stalk lodging. The responses in plant-type, physiological and biochemical characteristics of maize hybrid Nongda 108, Ludan 981 and their corresponding parents Xu 178, Huang-C, Qi 319, and lx9801 varieties, were studied in field condition, with 200 mg L 1 ethephon foliage spray at jointing stage. The results showed that ethephon significantly inhibited the elongation of the first to sixth basal internodes for Nongda 108 and Ludan 981, the plant height and the ear height of Nongda 108 and Ludan 981 were significantly reduced. Ethephon remarkably increased the activities of phenylalanine ammonia lyase (PAL) and indole acetic acid oxidase (IAAO) and content of abscisic acid (ABA) in elongating internodes, and decreased contents of auxin (IAA) and gibberellin (GA4). The genetic traits were analyzed, the plant height and the ear height of Xu 178, Huang-C, Qi 319, and lx9801 were significantly reduced after ethephon treating, and the length of basal internodes was shortened significantly. The PAL and IAAO activities in elongating internodes were significantly increased after ethephon treating. The endogenous IAA content of elongating internodes was significantly decreased, ABA content was significantly increased, but GA4/ABA ratio was significantly decreased. It is indicated that the response differences to ethephon among different hybrids may be attributed to the different sensitivities of the parents lines, showing corresponding performance in plant height, ear height, PAL, IAAO activities and endogenous hormone levels.

MOLECULAR AND METABOLIC ANALYSES IN DEVELOPING OLIVE FRUIT IN RELATION TO DIFFERENT WATER REGIMES

Despite the global economic importance of olive (Olea europaea L.), little is known about the molecular and metabolic changes during fruit development and the modulation of quality-related metabolic pathways during drought stress. In this work, we report the expression pattern of genes involved in important pathways of secondary metabolism (polyphenols, terpenoids) during fruit development in rainfed or fully irrigated olive plants. Phenolic compounds represent a complex mixture in both olive fruits and oil. The interest on these compounds is due to their demonstrated anti-atherogenic and anti-cancerogenic effects providing to the olive oil important nutraceutical properties. Several parameters affect olive fruit phenolic content and these include genotype, pedo-climatic conditions, agronomic techniques (e.g. irrigation) and the develop¬mental stage of the drupe. To gain insight into the transcriptional regulation of phenolic pathways we analysed the pattern of expression of important genes during olive fruit development. Transcript levels of phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), and dihydroflavonol reductase (DFR) genes showed different expression patterns during fruit development. CHS and DFR showed stronger expression in the skin than in the pulp. PAL expression was strong before pit hardening and decreased thereafter. CHS expression peaked at pit hardening and at veraison stage, whereas DFR was only strongly expressed at fruit ripening, suggesting a crucial role for these genes in the regulation of flavonoid biosynthesis in the last developmental stages of olive fruit. Water stress enhanced the expressions of PAL and CHS at pit hardening and of DFR in the skin at ripening. Of the terpenoids, amyrin synthase (AS) showed a decreasing expression trend throughout development, whereas lupeol synthase (LUS) was highly expressed in the skin of ripe fruit. Induction of LUS gene expression was observed at the early developmental stages in fruit of water-stressed plants. To gain insight into the metabolic changes in the fruit under different water regimes in the field, we initiated, in a high-throughput approach, analysis of the mesocarp of ripe fruits by GC-TOF mass spectrometry, and differences in metabolite composition were determined.

Phenol Metabolism in the Leaves of the Olive Tree (Olea europaea L.) cv. Picual, Verdial, Arbequina, and Frantoio during Ripening

The kinetic behavior and protein-expression level of phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO) have been determined in the leaves of the olive tree (Olea europaea L.) of cv. Picual, Verdial, Arbequina, and Frantoio during fruit ripening. Moreover, the concentration of total phenolic compounds, oleuropein, hydroxytyrosol, and tyrosol has been also determined. This study was carried out in 20-year-old olive trees grown in Jaén (Spain). The concentration of total and specific phenols showed a specific pattern in each cultivar. Frantoio showed the highest phenol concentration followed by Arbequina, Picual, and Verdial. A coordinated response between PAL, PPO, and the concentration of total phenols in the four cultivars was found. Also, specific changes were shown over the course of ripening, indicating a regulation of PAL, PPO, and phenol concentration in the olive-tree leaves during fruit ripening.

Rapid suppression of defence enzymes and compounds by sheath blight (Rhizoctonia solani) and sheath rot (Sarocladium oryzae) toxins in rice cell suspension cultures

To understand the suppression mechanisms against disease resistance in rice, we took advantage of the fact that suspension cultured cells exhibit many of the defence responses that are characteristic of intact tissues. In this study we constitutively measured the Rhizoctonia solani and Sarocladium oryzae toxins, induced and suppressed levels of phenylalanine ammonia lyase, peroxidase, superoxide dismutase, phenols, catalase, β-1,3-glucanase and chitinase in rice suspension cultured cells. The addition of Rhizoctonia solani and Sarocladium oryzae toxins separately in suspension cultured cells shows the suppression of defence enzymes and compounds at 24 h and 48 h respectively except SOD. The rice cultivar IR50 delays the disease suppression effect when compared to the other cultivars viz., Pusa Basmati and Co 43. The PR proteins (namely β-1,3-glucanase and chitinase) activities in rice suspension cultured cells were reduced during 48 h and 72 h after the addition of Rhizoctonia solani toxin, whereas the activities were suppressed only after 72 h when inoculated with Sarocladium oryzae toxin. Selective suppression of these defence enzymes and compounds by Rhizoctonia solani and Sarocladium oryzae toxin shows that toxins play a major role during pathogenesis in rice cells.

Differential colonization and defence responses of resistant and susceptible melon lines infected by Fusarium oxysporum race 1·2

A green fluorescent protein (GFP)‐expressing strain of Fusarium oxysporum f.sp. melonis race 1·2 (FOM 1·2‐GFP) was used to visualize infection of a susceptible melon cultivar, Ein Dor (ED). At 1–2 days post‐inoculation (d.p.i.), the fungus grew on the root epidermis and adhered to epidermal cell borders. By day 4, the mycelium crossed the cortex and endodermis through narrow pores in cell walls and reached xylem vessels, where it sporulated and produced secondary hyphae that grew upwards. Colonization dynamics of ED seedlings were compared with those of a genetically resistant line, BIZ. FOM 1·2‐GFP colonized the resistant plant’s vascular system, but the incidence of seedling infection was lower than in ED, suggesting stronger defence responses in BIZ expressed at the pre‐xylem stage of infection. Infection of the vascular system of BIZ was slower: at 11 d.p.i., FOM 1·2‐GFP only colonized the lower hypocotyl sections of BIZ, whilst the upper hypocotyls of ED were already infected, indicating that BIZ also restricted FOM 1·2 movement in the xylem. The expression patterns of three defence genes were compared between the resistant and susceptible genotypes using real‐time PCR. Transcript levels of phenylalanine ammonia lyase (PAL), chitinase (CHI) and hydroperoxide lyase (HPL) were induced to a greater extent in BIZ than in ED. A constitutive two‐ to fourfold difference between BIZ and ED in the basal levels of all three transcripts was also apparent. Both the constitutive and inducible defence responses could contribute to reduced vascular colonization of the resistant genotype.

Glycosylation-mediated phenylpropanoid partitioning in Populus tremuloides cell cultures

BackgroundPhenylpropanoid-derived phenolic glycosides (PGs) and condensed tannins (CTs) comprise large, multi-purpose non-structural carbon sinks in Populus. A negative correlation between PG and CT concentrations has been observed in several studies. However, the molecular mechanism underlying the relationship is not known.ResultsPopulus cell cultures produce CTs but not PGs under normal conditions. Feeding salicyl alcohol resulted in accumulation of salicins, the simplest PG, in the cells, but not higher-order PGs. Salicin accrual reflected the stimulation of a glycosylation response which altered a number of metabolic activities. We utilized this suspension cell feeding system as a model for analyzing the possible role of glycosylation in regulating the metabolic competition between PG formation, CT synthesis and growth. Cells accumulated salicins in a dose-dependent manner following salicyl alcohol feeding. Higher feeding levels led to a decrease in cellular CT concentrations (at 5 or 10 mM), and a negative effect on cell growth (at 10 mM). The competition between salicin and CT formation was reciprocal, and depended on the metabolic status of the cells. We analyzed gene expression changes between controls and cells fed with 5 mM salicyl alcohol for 48 hr, a time point when salicin accumulation was near maximum and CT synthesis was reduced, with no effect on growth. Several stress-responsive genes were up-regulated, suggestive of a general stress response in the fed cells. Salicyl alcohol feeding also induced expression of genes associated with sucrose catabolism, glycolysis and the Krebs cycle. Transcript levels of phenylalanine ammonia lyase and most of the flavonoid pathway genes were reduced, consistent with down-regulated CT synthesis.ConclusionsExogenous salicyl alcohol was readily glycosylated in Populus cell cultures, a process that altered sugar utilization and phenolic partitioning in the cells. Using this system, we identified candidate genes for glycosyltransferases that may mediate the glycosylation, and for transporters that mediate the subcellular compartmentalization of sugars and phenolic glycosides. The suspension cells appear to represent a facile system for dissecting the regulation of phenolic carbon partitioning, and in turn, its effects on growth in Populus.

Analysing scots pine defence‐related transcripts and fungal DNA levels in seedlings single‐ or dual‐inoculated with endophytic and pathogenic Rhizoctonia species

SummaryFungal DNA and induction of host defence‐related transcripts were monitored by real‐time PCR in young Scots pine seedlings inoculated with pathogenic uninucleate (UNR) and endophytic binucleate (BNR) Rhizoctonia species. The UNR (teleomorph Ceratobasidium bicorne) causes root dieback in conifer seedlings following invasion of the vascular cylinder via root apex and destroying apical meristems whilst the BNR, representing anastomosis group AG‐I of genus Ceratobasidium, is primarily restricted to the cortex in basal root regions. In the experiment 1 the fungi were simultaneously inoculated on roots, while in experiment 2, BNR was pre‐inoculated 168 h before inoculation with UNR. Nucleic acids were extracted from infected roots at intervals up to 192 h post‐infection (hpi), and the genomic DNA levels of the host and fungi and the transcript levels of a house‐keeping gene (glyceraldehyde‐3‐phosphate dehydrogenase) and nine putative defence genes were quantified. In simultaneous inoculation UNR was more competitive than BNR whereas pre‐inoculation of BNR suppressed but did not completely prevent root colonization by UNR. Stilbene synthase (STS) transcription was significantly up‐regulated in single‐inoculations with both fungi and in dual inoculation in both experiments. Maximum STS transcript levels were observed in roots single‐inoculated with UNR; the peak level at 48 hpi in experiment 2 was significantly higher than in seedlings single‐inoculated with BNR or co‐inoculated with both fungi, the latter two treatments showing relatively similar STS transcript levels. Similarly, transcript levels of phenylalanine ammonia lyase at 48 hpi in experiment 2 were significantly higher in roots single‐inoculated with UNR compared with BNR or in UNR+BNR co‐inoculations. The other seven putative defence genes monitored did not show any clear‐cut up‐regulation following fungal inoculation. We conclude that BNR suppresses UNR in Scots pine roots via direct competition for infection sites, since the studied transcripts showed no evidence of BNR induced resistance against UNR.

Phenylalanine Ammonia-Lyase, Polyphenol Oxidase, and Phenol Concentration in Fruits ofOlea europaeaL. cv. Picual, Verdial, Arbequina, and Frantoio during Ripening

The kinetics and protein-expression level of phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO) in fruits of olive trees (Olea europaea) cv. Picual, Verdial, Arbequina, and Frantoio have been studied in relation to the concentration of total phenolic compounds, oleuropein, hydroxytyrosol, and tyrosol during fruit ripening. Frantoio was the variety that showed the highest total phenol concentration, the highest PAL activity, the lowest PPO activity, and the lowest protein levels. In contrast, Verdial was the variety that showed the lowest total phenol concentration, the least PAL activity, the greatest PPO activity, and the highest protein levels. Arbequina and Picual showed intermediate levels. These results suggest the existence of a coordinated response between PAL, PPO, and the concentration of total phenols over ripening in the four varieties. The concentration of total and specific phenols differed between varieties and specifically changed over ripening.

Enzyme induction as a possible mechanism for latex-mediated insect resistance in romaine lettuce.

Plant latex is a known storehouse of various secondary metabolites with demonstrated negative impact on insect fitness. A romaine lettuce cultivar, "Valmaine", possesses a high level of latex-mediated resistance against the banded cucumber beetle, Diabrotica balteata LeConte (Coleoptera: Chrysomelidae), compared to a closely related cultivar "Tall Guzmaine". Latex from damaged Valmaine plants was much more deterrent to adult D. balteata feeding than latex from undamaged plants when applied to the surface of artificial diet under choice conditions; no such difference was found in choice tests with latex from damaged and undamaged Tall Guzmaine plants. The intensities of whiteness and browning were significantly higher in Valmaine latex than in Tall Guzmaine latex. The activities of three enzymes (phenylalanine ammonia lyase, polyphenol oxidase, and peroxidase) significantly increased over time in latex from damaged Valmaine plants (i.e., 1, 3, and 6 days after feeding initiation), but they remained the same in Tall Guzmaine latex. The constitutive levels of phenylalanine ammonia lyase and polyphenol oxidase also were significantly higher in Valmaine latex than in Tall Guzmaine latex. These studies suggest that Valmaine latex chemistry may change after plant damage due to increased activity of inducible enzymes and that inducible resistance appears to act synergistically with constitutive resistance against D. balteata.

Time course study on accumulation of cell wall-bound phenolics and activities of defense enzymes in tomato roots in relation to Fusarium wilt

Major cell wall-bound phenolic compounds were detected and identified in roots of tomato at different stages of growth. Alkaline hydrolysis of the cell wall material of the root tissues yielded ferulic acid as the major bulk of the phenolic compounds. Other phenolic compounds identified were 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin and 4-coumaric acid. All the six phenolic acids were higher in very early stage of plant growth. Ferulic acid, 4-hydroxybenzoic acid and 4-coumaric acid exhibited a decreasing trend up to 60 days and then the content of these phenolic acids increased somewhat steadily towards the later stage of growth. Total phenolics, phenylalanine ammonia-lyase (PAL) activity and peroxidase (POD) activity were in tandem match with the occurrence pattern of the phenolic acids. Ferulic acid showed highest antifungal activity against tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici. The results of this study may be interpreted to seek an explanation for high susceptibility of tomato plants at flowering stage to Fusarium wilt. It may also be concluded that greater amounts of ferulic acid in combination with other phenolics and higher level of PAL and POD activities after 60 days of growth may have a role in imparting resistance against Fusarium wilt at a late stage of plant growth.

Expression of PAL and HSPs in fresh-cut banana fruit

Wounding of plants is a widespread stress in nature. Physical damage frequently occurs during harvesting, handling and marketing of fresh produce. Phenylalanine ammonia-lyase (PAL) is a key enzyme involved in plant wound related phenylpropanoid metabolism while heat shock proteins (HSPs) are involved in abiotic, including wounding, and biotic stress responses in plants. However, little is known about postharvest expression at gene and protein levels of PAL and HSPs in wounding (fresh-cut) fruits. In the present study, expression patterns of PAL and HSPs in fresh-cut and intact banana fruit pulp were characterized by Northern and Western blot analyses. Cuting enhanced Ma- PAL2 mRNA accumulation, new PAL protein synthesis, and PAL activity; thereby resulting in accumulation of total phenolics. HSP70 protein and Ma-HSP70-2 transcript were constitutively expressed and were not affected by cutting. However, Ma-HSP70-1 transcript levels increased in fresh-cut pulp tissue at 2–18 h after cutting. Ma-sHSP-1-CI, Ma-sHSP-3-CII, Ma-HSP90, and Ma-HSP101 transcripts also accumulated in fresh-cut tissues at 2–18 h after cuting, but Ma-sHSP-2-CI did not accumulate. Accumulation of sHSP (HSP17.6), HSP90 and HSP101 proteins exhibited the same pattern of expression as their mRNA. There was no evidence of a possible suppressive effect of HSPs synthesis on PAL protein accumulation. Thus, PAL, sHSP, HSP90 and HSP101 were regulated by cutting both at translational and transcriptional levels while HSP70 ( Ma-HSP70-1) was regulated by cutting only at transcription level.

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.

Comparative histochemical analyses of oxidative burst and cell wall reinforcement in compatible and incompatible melon–powdery mildew ( Podosphaera fusca) interactions

The spatial-temporal expression patterns of oxidative burst and cell wall reinforcement were analyzed in leaves of resistant and susceptible melon (Cucumis melo L.) cultivars in response to Podosphaera fusca (Fr.) Braun & Shishkoff, the main causal agent of powdery mildew in cucurbits. Extensive development of powdery mildew mycelia and a progressive increase in haustorial count were recorded in the susceptible cultivar after 4d, while in the resistant cultivar powdery mildew failed to grow and small brownish and necrotic leaf areas were frequently observed. Rapid generation of the reactive oxygen intermediates hydrogen peroxide and superoxide radicals 4h after pathogen challenge, but before the fungal haustoria formation, stood upstream in the cascade of events induced during these interactions. This oxidative burst was followed by the accumulation of strengthening polymers of callose and lignin at the cell wall of attacked resistant plant cells. Interestingly, the transcriptional levels of phenylalanine ammonia-lyase (PAL), an important enzyme for phenylpropanoid metabolism, did not significantly change throughout the experiments. Although these physiological changes were observed in both cultivars, their faster kinetics and amplitude in the resistant line compared to the susceptible cultivar governed the differential visual response of these cultivars against P. fusca. These findings, along with data obtained in previous studies, have provided the bases for an integrated model in which the spatial-temporal response patterns of these resistance mechanisms have been arranged, which may ultimately lead to successful protection of melon plants against P. fusca.

The effects of enhanced methionine synthesis on amino acid and anthocyanin content of potato tubers

BackgroundPotato is a staple food in the diet of the world's population and also being used as animal feed. Compared to other crops, however, potato tubers are relatively poor in the essential amino acid, methionine. Our aim was to increase the methionine content of tubers by co-expressing a gene involved in methionine synthesis with a gene encoding a methionine-rich storage protein in potato plants.ResultsIn higher plants, cystathionine γ-synthase (CgS) is the first enzyme specific to methionine biosynthesis. We attempted to increase the methionine content of tubers by expressing the deleted form of the Arabidopsis CgS (CgSΔ90), which is not regulated by methionine, in potato plants. To increase the incorporation of free methionine into a storage protein the CgSΔ90 was co-transformed with the methionine-rich 15-kD β-zein. Results demonstrated a 2- to 6-fold increase in the free methionine content and in the methionine content of the zein-containing protein fraction of the transgenic tubers. In addition, in line with higher methionine content, the amounts of soluble isoleucine and serine were also increased. However, all of the lines with high level of CgSΔ90 expression were phenotypically abnormal showing severe growth retardation, changes in leaf architecture and 40- to 60% reduction in tuber yield. Furthermore, the colour of the transgenic tubers was altered due to the reduced amounts of anthocyanin pigments. The mRNA levels of phenylalanine ammonia-lyase (PAL), the enzyme catalysing the first step of anthocyanin synthesis, were decreased.ConclusionEctopic expression of CgSΔ90 increases the methionine content of tubers, however, results in phenotypic aberrations in potato. Co-expression of the 15-kD β-zein with CgSΔ90 results in elevation of protein-bound methionine content of tubers, but can not overcome the phenotypical changes caused by CgSΔ90 and can not significantly improve the nutritional value of tubers. The level of PAL mRNA and consequently the amount of anthocyanin pigments are reduced in the CgSΔ90 transgenic tubers suggesting that methionine synthesis and production of anthocyanins is linked.

Improving postharvest quality of mango ‘Haden’ by UV-C treatment

It is well known that UV-C irradiation increases the shelf life of different fruits. However, the biochemical changes associated with these responses are not well understood in tropical fruits. The objective of this work was to study the effects of UV-C treatment on the biochemistry and quality of mango fruit. Mangos cv ‘Haden’ were exposed to UV-C irradiation energy levels of 2.46 and 4.93 kJ m −2 and then stored for 18 days at 25 °C. Total phenols (TP), total flavonoids (TF), enzymatic activity of phenylalanine ammonia-lyase (PAL) and lipoxygenase (LOX) were measured at 0, 0.5, 1, 8, 16, and 24 h and thereafter every 3 days. Decay and overall appearance (OA) were evaluated every 2 days with a hedonic scale. UV-C treatment maintained better OA, lower decay percentage and increased shelf life of fruit. These benefits correlated possitively with higher levels of TP, TF, LOX and PAL. We conclude that UV-C treatment can be a good alternative to increase the shelf life in optimal conditions of mango ‘Haden’.

Quantification of host and pathogen DNA and RNA transcripts in the interaction of Norway spruce with Heterobasidion parviporum

Colonization levels of the pathogenic fungus Heterobasidion parviporum in tissue cultures and bark lesions of 33-year-old Norway spruce trees was monitored using quantitative PCR. Expression of selected host and fungal transcripts was also measured in the same material using quantitative reverse transcriptase (RT)-PCR. The host genes phenylalanine ammonia lyase, peroxidase, glutathione-S-transferase, germin-like protein and class IV chitinase were all induced in tissue cultures in response to infection. Higher transcript levels of phenylalanine ammonia lyase and peroxidase were detected in the more resistant clone 589 than in the less resistant clone 409. Expression of a class IV chitinase was higher in clone 409 than in 589. Several of the fungal genes assayed were differentially expressed during colonization, including putative glutathione-S-transferases, laccase, cellulase, cytochrome P450 and superoxide dismutase genes. The transcriptional responses suggest an important role for the antioxidant systems of both organisms. The changes in the levels of all the examined host and pathogen transcripts were similar between tissue cultures and bark over their overlapping colonization range. This suggests that tissue cultures can be used to select candidate host defense and fungal pathogenicity genes operative in tissues composed of primarily living cells, such as phloem, in the Norway spruce/ H. parviporum pathosystem.

High-level Expression of Phenylalanine Ammonia-lyase in Lactococcus lactis via Synthesized Sequence Based on Bias Codons

To construct a safer and more efficient gene engineering Lactococcus lactis for expressing phenylalanine ammonia lyase (PAL) which will benefit PKU therapy. A PAL cDNA of Parsly and a synthesized PAL sequence based on Lactococcus lactis bias codons ( pal art) were recombined into two Lactococcus lactis NICE systems. The activities of the expressed PALs were detected, and the effect of Lactococcus lactis bias codons on the expression of exterior protein was analyzed. The results showed that the expression level of PAL was increased by using bias codons in the two Lactococcus lactis NICE systems, through which several safer and more efficient strains of the gene engineering Lactococcus lactis were obtained.

Expression of Genes Involved in Proanthocyanidin Biosynthesis during Fruit Development in a Chinese Pollination-constant, Nonastringent (PCNA) Persimmon, `Luo Tian Tian Shi'

Expression patterns of the genes involved in condensed tannin (CT) biosynthesis during fruit development was investigated in a Chinese pollination-constant, nonastringent (PCNA) persimmon (Diospyros kaki Thunb.) `Luo Tian Tian Shi'. The transcript levels of phenylalanine ammonia-lyase (PAL) and dihydroflavonol reductase (DFR) in `Luo Tian Tian Shi' were detected at high levels throughout the fruit growth. Chalcone synthase (CHS) and flavonol 3-hydroxylase (F3H) also continued to be transcribed during fruit growth, although their levels decreased earlier than PAL and DFR. In contrast, expression levels of these genes declined into undetectable levels at an early stage of fruit development in Japanese PCNA persimmon. In addition, anthocyanidin reductase (ANR), which encodes a key enzyme of the proanthocyanidin biosynthesis, was transcribed at high levels in `Luo Tian Tian Shi' during fruit growth, but not in Japanese PCNA persimmon. By contrast, the expression of D. kaki serine carboxypeptidase-like protein 1 (DkSCPL1) that was obtained from suppression subtractive hybridization (SSH) analysis between artificially astringency-removed fruit and astringent fruit in a different experiment, declined earlier than the other flavonoid biosynthesis genes in `Luo Tian Tian Shi', coincident with the termination of the tannin cell development. In the F1 progeny of the cross between `Luo Tian Tian Shi' and Japanese PCNA `Taishu', similar expression patterns were obtained among segregated PCNA and astringent offspring. These results indicate that Chinese PCNA is different from Japanese PCNA in expression of the genes involved in CT biosynthesis. In conclusion, we clarified that expression of the genes (PAL to ANR, but not SCPL) involved in flavonoid biosynthesis was continuous in the Chinese PCNA cultivar, despite the termination of tannin cell development.

Effect of plant growth promoting rhizobacteria on bacterial canker of tomato

Use of plant growth promoting rhizobacteria in managing bacterial canker disease of tomato was studied in the present work. Tomato seeds were treated with PGPR strains viz., Bacillus pumilus INR7, Bacillus pumilus SE34, Bacillus pumilus T4, Bacillus subtilis GBO3, Bacillus amyloliquefaciens IN937a and Brevibacillus brevis IPC11 were subjected for seed germination and seedling vigor. Among the PGPR strains tested, only three strains (IN937a, GBO3 and IPC11) which showed enhancement in the seed quality parameters like seed germination and seedling vigor, were further subjected for estimation of one of the defence-related enzymes, Phenylalanine Ammonia Lyase (PAL) with total phenol contents. The same three strains were recorded for maximum disease protection under greenhouse conditions. The level of PAL and total phenol contents increased significantly upon the PGPR treatment. The rate of reduction in the bacterial canker disease incidence was directly proportional to the amount of increased level of PAL and total phenol content. The possible uses of these PGPR strains in effective management of bacterial canker of tomato were discussed in the present work.