Phenylalanine Ammonia-lyase Accumulation Research Articles

Integrated transcriptomic and metabolomic analysis provides insight into the regulation of leaf senescence in rice

Leaf senescence is one of the most precisely modulated developmental process and affects various agronomic traits of rice. Anti-senescence rice varieties are important for breeding application. However, little is known about the mechanisms underlying the metabolic regulatory process of leaf senescence in rice. In this study, we performed transcriptomic and metabolomic analyses of the flag leaves in Yuenong Simiao (YN) and YB, two indica rice cultivars that differ in terms of their leaf senescence. We found 8524 genes/204 metabolites were differentially expressed/accumulated in YN at 30 days after flowering (DAF) compared to 0 DAF, and 8799 genes/205 metabolites were differentially expressed in YB at 30 DAF compared to 0 DAF. Integrative analyses showed that a set of genes and metabolites involved in flavonoid pathway were significantly enriched. We identified that relative accumulation of PHENYLALANINE AMMONIA-LYASE (PAL), CINNAMATE 4-HYDROXYLASE (C4H), 4-COUMAROYL-COA LIGASE (4CL), CHALCONE SYNTHASE (CHS) and CHALCONE ISOMERASE (CHI) in YN30/0 was higher than that in YB30/0. Three flavonoid derivatives, including phloretin, luteolin and eriodictyol, showed lower abundances in YB than in YN at 30 DAF. We further revealed a MYB transcription factor, which is encoded by OsR498G0101613100 gene, could suppress the expression of CHI and CHS. Our results suggested a comprehensive analysis of leaf senescence in a view of transcriptome and metabolome and would contribute to exploring the molecular mechanism of leaf senescence in rice.

Discovery of Novel Isothiazole, 1,2,3-Thiadiazole, and Thiazole-Based Cinnamamides as Fungicidal Candidates.

A series of isothiazole, 1,2,3-thiadiazole, and thiazole-based cinnamamide morpholine derivatives were rationally designed, synthesized, characterized, and evaluated for their fungicidal activities. Bioassay indicated that a combination of 3,4-dichloroisothiazole active substructures with cinnamamide morpholine lead to significant improvement of in vivo antifungal activities of the target compounds; among them, compound 5a exhibited good fungicidal activity against Pseudoperonspera cubensis in vivo with an inhibition rate of 100% at 100 μg/mL. A field experiment indicated that the difference of efficacy between 5a (75.9%) and dimethomorph (77.1%) at 37.5 g ai/667 m2 was not significant; and 5a also exhibited good activity against Botrytis cinerea by triggering accumulation of PAL and NPR1 defense-related gene expression and the defense associated enzyme phenylalanine ammonia-lyase (PAL) expression on cucumber, rather than direct inhibition. These findings strongly supported that 3,4-dichloroisothiazole containing cinnamamide morpholine 5a not only showed good fungicidal activity against P. cubensis but also exhibited plant innate immunity stimulation activity as a promising fungicide candidate with both fungicidal activity and systemic acquired resistance.

Integration of anti-penetrant tricyclazole, signaling molecule salicylic acid and root associated Pseudomonas fluorescens enhances suppression of Bipolaris sorokiniana in bread wheat (Triticum aestivum L.)

Salicylic acid (SA) and root associated Pseudomonas fluorescens are known inducers of systemic resistance and influence plant defense responses including reprogramming of cellular mechanisms in response to biotic stress. However, integration of SA, tricyclazole and bioagent P. fluorescens in the integrated disease management practices is not clearly understood. With this rationale the present study was carried out to understand the effects of integration of an anti-penetrant tricyclazole, signaling molecule SA and P. fluorescens UBSPF-10 in modulation of defense cascades and enhancing suppression of Bipolaris sorokiniana in bread wheat (Triticum aestivum L.). Results showed that exogenous application of SA (100 mg L−1) alone and in combination with tricyclazole (750 mg L−1) and P. fluorescens UBSPF-10 significantly increased activation and accumulation of phenylalanine ammonia lyase (PAL), peroxidase, β 1,3-glucanase and chitinase activities in wheat leaves challenged with B. sorokiniana compared to individually treated and control plants. The influence of combined applications of SA, tricyclazole and P. fluorescens UBSPF-10 on lignin content in plant leaves was investigated and it was observed that the combined treatment resulted significant increase in the acid-soluble lignin content in wheat possibly leading to enhanced suppression of disease progression and severity. Furthermore, correlation analysis clearly indicated that enhanced accumulation of lignin in plant cells significantly reduces pathogen infection and invasion of tissue leading to reduction in disease development compared to other treatments. Therefore, it is concluded that combined application of SA, tricyclazole and P. fluorescens UBSPF-10 played an important role in suppression of B. sorokiniana development in wheat plants in a cooperative manner and thus, could be used in the integrated disease management practices of wheat.

Modulation of auxin and cytokinin responses by early steps of the phenylpropanoid pathway

BackgroundThe phenylpropanoid pathway is responsible for the synthesis of numerous compounds important for plant growth and responses to the environment. In the first committed step of phenylpropanoid biosynthesis, the enzyme phenylalanine ammonia-lyase (PAL) deaminates L-phenylalanine into trans-cinnamic acid that is then converted into p-coumaric acid by cinnamate-4-hydroxylase (C4H). Recent studies showed that the Kelch repeat F-box (KFB) protein family of ubiquitin ligases control phenylpropanoid biosynthesis by promoting the proteolysis of PAL. However, this ubiquitin ligase family, alternatively named Kiss Me Deadly (KMD), was also implicated in cytokinin signaling as it was shown to promote the degradation of type-B ARRs, including the key response activator ARR1. Considering that ubiquitin ligases typically have narrow target specificity, this dual targeting of structurally and functionally unrelated proteins appeared unusual.ResultsHere we show that the KFBs indeed target PAL but not ARR1. Moreover, we show that changes in early phenylpropanoid biosynthesis alter cytokinin sensitivity – as reported earlier - but that the previously documented cytokinin growth response changes are primarily the result of altered auxin signaling. We found that reduced PAL accumulation decreased, whereas the loss of C4H function increased the strength of the auxin response. The combined loss of function of both enzymes led to a decrease in auxin sensitivity, indicating that metabolic events upstream of C4H control auxin sensitivity. This auxin/phenylpropanoid interaction impacts both shoot and root development and revealed an auxin-dependent stimulatory effect of trans-cinnamic acid feeding on leaf expansion and thus biomass accumulation.ConclusionsCollectively, our results show that auxin-regulated plant growth is fine-tuned by early steps in phenylpropanoid biosynthesis and suggest that metabolites accumulating upstream of the C4H step impact the auxin response mechanism.

Plant growth-promoting rhizobacteria mediate induced systemic resistance in rice against bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae

Seven Bacillus plant growth-promoting rhizobacteria spp. were evaluated for growth promotion and induced systemic resistance in rice against Xanthomonas oryzae pv. oryzae (Xoo). The identities of colonies of X. oryzae pv. oryzae grown on mXOS and PSA medium were confirmed by PCR employing specific primers TXTF and TXT4R. Among the seven strains tested as fresh suspensions, talc and sodium alginate formulations under laboratory and green house conditions, maximum germination of 86% was recorded after seed treatments with fresh suspension of Bacillus subtilis GBO3 followed by 85% germination treated with Bacillus pumilus SE34 in comparison to only 71% germination in the untreated controls. Similarly, the maximum vigor index of 1374 was obtained by seed treatment with fresh suspensions of B. subtilis strain GBO3 followed by treatments with strain SE34 with vigor index of 1323 in contrast to an index of only 834 observed in untreated controls. Among the treatments, seed treatments with fresh suspension of seven strains resulted in better germination and vigor assessments than talc based or sodium alginate formulations. Seed treatments with fresh suspension of strain SE34 gave 71% protection, followed by B. subtilis GBO3 and B. pumilus T4 with 58% and 52% protection, respectively, compared to the untreated controls. Seed treatments with talc based formulation of SE34 gave 66% protection, while GBO3 and T4 resulted in 52% and 50% protection, respectively, with similar formulation. Seed treatment with talc and sodium alginate formulations of strain SE34 gave 58% protection followed by GBO3 with 40% protection. Seed treatment with fresh suspensions of strains SE34 and GBO3 followed by challenge inoculations with Xoo increased accumulation of phenylalanine ammonia lyase, peroxidase and polyphenol oxidase compared to untreated control seedlings. Thus, the results of the present study suggest that the PGPR strains used as fresh suspensions and powdered formulations may have commercial potential in plant growth promotion and in management of rice bacterial leaf blight disease.

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

The compartmentation of phenolic compounds in mature leaves of Phyllanthus tenellus and their induction by copper sulphate were analysed at histological and subcellular levels. Light and electron microscopy studies demonstrated that the vacuoles of spongy cells were the main sites of phenolic accumulation. Spraying plants with copper sulphate induced punctated lesions formed by groups of necrotic cells which accumulated brownish substances. Histochemical tests and fluorescence microscopy analysis of the sprayed leaves indicated that the phenolic compounds increased in spongy cells within the lesions. Ultrastructural analyses showed that 3h after elicitation, the organelles of the cells within the lesion started to collapse and the content of phenolic substances increased in the vacuole of spongy cells. Antibody against phenylalanine ammonia-lyase (PAL) from parsley cross-reacted with the crude extract of P. tenellus leaves. Two isoforms, one of 65kD and the other of 66kD, were identified. Immunocytochemical studies showed that PAL was synthesized in the palisade and spongy cells, mainly in the cytoplasm and chloroplasts. The phytotoxicity of Cu2+ions induced the accumulation of PAL in sub-cellular compartments of palisade cells. PAL accumulation started to increase 3h after elicitation and reached a maximum after 6h, decreasing 12h post-induction. The increase of PAL was more evident in cells within the necrotic punctated regions than in surrounding cells. Since the vacuole of palisade cells did not accumulate phenolic compounds, the in situ studies suggested that the end products of PAL synthesis play a role in palisade cell wall reinforcement or might accumulate in other tissues. The symptoms induced by copper sulphate suggest that this abiotic elicitor may be a useful tool in the understanding of the regulation of biosynthetic phenolic pathways inP. tenellus .

Suppression of Phenylalanine Ammonia-Lyase mRNA Accumulation by Suppressors from Phytophthora infestans.

The transient accumulation of phenylalanine ammonia-lyase (PAL) transcript was markedly induced in aged potato tuber slices 1 to 2hr after inoculation with an incompatible race of Phytophthora infestans, whereas the transcript in compatible interaction accumulated to lesser extent. A dramatic increase of PAL transcript accumulation occurred within 30min after treatment with hyphal wall components (HWC) of the pathogen, but the HWC-elicitor-stimulated increase was greatly suppressed by treatment with a suppressor from the compatible race. In contrast, treatment with HWC-elicitor in the presence of the suppressor from the incompatible race caused an acceleration of the accumulation of PAL mRNA compared with that with HWC-elicitor alone. These results for PAL transcript paralleled those based on the changes in PAL enzyme activity. Elicitation and suppression of induction of PAL transcript accumulation and PAL activity were discussed in relation to the race-cultivar specificity in potato late blight.

Spatial and temporal accumulation of defense gene transcripts in bean (Phaseolus vulgaris) leaves in relation to bacteria-induced hypersensitive cell death.

Primary leaves of 7- to 9-day-old Red Mexican bean plants were inoculated with virulent or avirulent isolates of Pseudomonas syringae pv. phaseolicola, or saprophytic P. fluorescens either by vacuum infiltration of the whole leaf lamina, or by syringe-inoculation of selected leaf panels. In the incompatible combination, resistance was associated with a hypersensitive response (HR). Syringe-inoculated leaves were sampled in three zones: zone 1, the inoculated leaf area; zone 2, the surrounding 0.5-0.7 cm of leaf tissue; and zone 3, the remainder of the leaf. Northern blots of RNA from zones 1, 2, and 3 were probed with bean cDNAs for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chitinase (CHT), and lipoxygenase (LOX). Accumulation of PAL, CHS, and CHT transcripts was more rapid and generally of greater magnitude in the incompatible than in the compatible interaction and, in both cases, was observed essentially only in zone 1 tissues. Similarly, antibacterial phytoalexins were only detected in zone 1 from the incompatible interaction. Young primary leaves have a background level of LOX transcripts, which declines as leaves age. This decline was accelerated over the first 12 hr postinoculation (hpi) with avirulent bacteria, whereas a weak transient induction, peaking at 5-6 hpi, was observed in the compatible interaction. A subsequent, strong accumulation of LOX transcripts was seen in both the compatible and incompatible interactions outside the inoculation site starting about 14 hpi. LOX transcripts did not accumulate at the inoculation site itself in the incompatible interaction compared to a relatively strong induction in the compatible interaction. Interestingly, inoculation of leaves with cells of the saprophyte P. fluorescens also induced the accumulation of transcripts for CHS, CHT, and LOX, but generally to a lesser degree than in the incompatible interaction. No HR occurred and no macroscopic cell damage was apparent in leaves inoculated with P. fluorescens. However, at the microscopic level individual, trypan blue-stained, necrotic plant cells were visible. In spite of this and the accumulation of CHS transcripts, no phytoalexin accumulation was found up to 48 hr after inoculation. The spatial and temporal relationship of the hypersensitive reaction to defense gene transcript and phytoalexin accumulation is discussed.

Cellular localization of nonhost resistance reactions of parsley (Petroselinum crispum) to fungal infection

The response of parsley seedlings (Petroselinum crispum) inoculated with zoospores of the soybean-pathogenic fungus, Phytophthora megasperma f. sp. glycinea, ranged from "immunity" to "physiological susceptibility" depending on the post-inoculation environmental conditions. Typical nonhost resistance reactions, hypersensitive cell death and the formation of small local lesions, occurred under high relative humidity and 16 h illumination per day. Localized biochemical reactions were monitored using fluorescence microscopy combined with histochemical and immunohistochemical methods. The rapid accumulation of furanocoumarin phytoalexins, wall-bound phenolics and callose was detected around infection sites. Indirect antibody staining of frozen tissue sections demonstrated the local accumulation of phenylalanine ammonia-lyase, a key enzyme of general phenylpropanoid metabolism, and S-adenosyl-L-methionine: bergaptol O-methyltransferase, a specific enzyme of the furanocoumarin pathway. The results indicate that phenylpropanoid derivatives are synthesized de novo at infection sites.

Phenylalanine Ammonia-Lyase and the Phenylalanine Ammonia-Lyase Inactivating System: Effects of Light, Temperature and Mineral Deficiencies

In constant light, whole apples (cv. Red Spy) that received alternating 6/25°C treatment accumulated more phenylalanine ammonia-lyase (PAL) and anthocyanin than those receiving constant 25° throughout the course of experiment. The stimulatory effect of low temperature (6°) on the activity of PAL and synthesis of anthocyanin was also observed in the alternating light and dark experiment when low temperature was applied in the dark period. In constant light, the activity of phenylalanine ammonia-lyase inactivating system (PAL-IS) was lower in the apples that had received alternating 6/25° treatment than that in apples that had received constant 25°. When the apples were incubated in constant darkness, no accumulation of PAL and anthocyanin occurred but the level of PAL-IS increased steadily throughout the course of incubation. Nitrogen and potassium deficiency increased PAL accumulation and decreased accumulation of PAL-IS in the leaves of greenhouse-grown apple trees. PAL activity was found to be negatively correlated with activity of PAL-IS. It is therefore concluded that low temperature reduces the level of PAL-IS and increases the accumulation of PAL which in turn increase anthocyanin accumulation in the skin of whole apples, and that nitrogen and potassium deficiencies reduce the level of PAL-IS and hence increase the accumulation of PAL in apple leaves.

Relationships and Interactions between Phenylalanine Ammonia-lyase, Phenylalanine Ammonia-lyase Inactivating System, and Anthocyanin in Apples1

Abstract Low temperature (6°C) in light stimulated the accumulation of phenylalanine ammonia-lyase (PAL) and anthocyanin and reduced the level of phenylalanine ammonia-lyase inactivating system (PALS-IS) in the skin of whole apples. Without light, anthocyanin synthesis did not take place in the skin of whole apples, and the level of PAL was found to be very low. In skin disks held in the dark, however, PAL activity increased but no anthocyanin was synthesized. In both the light and dark conditions, PAL-IS in the skin of whole apples was about 3.5 times higher than that in the apple skin disks after 44 hours of incubation at 18°. In the skin of whole apples, cycloheximide (10, 25, and 50 μm), chloramphenicol (50, 100, and 200 μm), and puromycin (10 and 25 μm) increased the accumulation of PAL and anthocyanin and reduced the level of PAL-IS. In the apple skin disks, cycloheximide (0.1, 1.0, 5.0, and 10.0 μm) and chloramphenicol (10.0 and 50.0 μm) inhibited the accumulation of PAL and anthocyanin to varying degrees. Puromycin had little effect on the accumulation of PAL but strongly inhibited the synthesis of anthocyanin. None of the antibiotics, at the concentrations tested, had any effect on the accumulation of PAL-IS in apple skin disks.