Characterisation, expression and functional analysis of PAL gene family in Cephalotaxus hainanensis.

Phenylalanine ammonia lyase (PAL) is the first committed step in the formation of phenylpropanoids, and catalyses the deamination of L-phenylalanine (L-Phe) to yield cinnamic acid. While PALs are common in plants, PAL genes involved in alkaloid biosynthesis in Cephalotaxus hainanensis have never been described. To obtain better knowledge of PAL genes and their number and function involved in Cephalotaxus alkaloid biosynthesis four PAL genes were screened and cloned. In vitro enzymatic analysis showed that all four PAL recombinant proteins could convert L-Phe to product trans-cinnamic acid, and showed strict substrate specificity. Moreover, the expression profiles of four ChPALs were analysed using qRT-PCR, which showed that they had higher transcript levels in roots and stems, and that different ChPALs displayed different response sensitivities and change patterns in response to stimuli. Several metabolic compounds were measured in stimulated leaves using UPLC-MS, and indicating the concentration of Cephalotaxus alkaloids and cinnamic acid in leaves subjected to different conditions. These concentrations increased significantly after treatment with 100 mM NaCl, 100 mM mannitol, 100 µM SA and 10 µM ABA. The expression levels of four PAL genes showed indications of upregulation after treatment. These results supply an important foundation for further research on candidate genes involved in the biosynthesis of Cephalotaxus alkaloids.

[Roles of phenylalanine ammonia-lyase in low temperature tolerance in cucumber seedlings].

To reveal the roles of phenylalanine ammonia-lyase (PAL) in low temperature tolerance in cucumber seedlings, a specific PAL inhibitor (AOPP) was sprayed to the seedlings, and then the stress tolerance was determined. The results suggested that both gene expression and enzymatic activity of PAL in cucumber leaves were induced under low temperature. The seedlings pretreated with AOPP showed lower PAL activity and less accumulation of phenolics and flavonoids. Low temperature caused damages in cucumber seedlings, and pretreatment of AOPP aggravated these damages. Compared to the control, the seedlings pretreated with AOPP showed significantly higher relative electrolyte leakage and MDA production, lower maximum photochemical efficiency of PSII (Fv/Fm) but higher photo-chemical quenching coefficient Y(NO), and reduced expression of low temperature-responsive genes (PR1-la, COR47, P5CS and HSP70). In cucumber seedlings, low temperature stress induced the accumulation of H2O2, increased the contents of ascobate (AsA) but decreased the contents of dehydroascobate (DHA), and thus reduced the value of AsA: DHA. In the AOPP-pretreated seedlings, the activities of antioxidant enzymes (CAT and APX) were significantly repressed, and excess H2O2 accumulated. The value of AsA: DHA was also lower than the control. Furthermore, co-application of H2O2 scavenger alleviated the low temperature-induced damages in the AOPP-pretreated seedlings, while coapplication of a CAT inhibitor made the seedlings more sensitive to low temperature stress. These results indicated that under low temperature stress, the enhanced activities of PAL could increase the biosynthesis of phenylpropanoid compounds and activate the cellular antioxidant enzymes, which could scavenge the excess ROS and maintain the cellular redox status, and thereby reduce the photo- and oxidative damages caused by low temperature stress.

Medium salt strength induced changes in growth, physiology and secondary metabolite content in adventitious roots of Morinda citrifolia: the role of antioxidant enzymes and phenylalanine ammonia lyase.

In an attempt to improve growth and secondary metabolite production, and to understand the possible mechanism involved in relation to the changes in physiology and activities of antioxidant enzymes, we cultured Morinda citrifolia adventitious roots in different strength (0.25, 0.50, 0.75, 1.0, 1.5 and 2.0) of Murashige and Skoog (MS) medium supplemented with 5 mg l(-1) indole butyric acid and 30 g l(-1) sucrose. Quarter-strength MS medium was proven suitable for the production of both root biomass and secondary metabolites [anthraquinone (AQ), phenolics and flavonoids]. With the increasing salt strength, root growth and AQ accumulation decreased significantly. Higher (1.5 and 2 MS) salt strength provoked osmotic stress resulted in more than twofold free proline accumulation than lower salt strength treated roots and induced free radical scavenging activity. Phenylalanine ammonia lyase activity showed a positive correlation in relation to salt strength that leads to an increase in phenol biosynthesis in expense of AQ formation. The elevated catalase (CAT), guaiacol peroxidase (G-POD) and superoxide dismutase activities and decreased ascorbate peroxidase (APX) activities were observed in roots treated with 2.0 MS. On the other hand, APX activity was strongly activated along with considerable increase in CAT activity at 0.25 MS treated culture. However, the joint functions of CAT, G-POD and APX at 0.25 MS treated cultures were efficient to eliminate the potential danger of hydrogen peroxide (H(2)O(2)) as evidenced from low H(2)O(2) accumulation and low level of lipid peroxidation.

Immunohistochemical localization of enzymes that catalyze the long sequential pathways of lignin biosynthesis during differentiation of secondary xylem tissues of hybrid aspen (Populus sieboldii x Populus grandidentata).

We have investigated the spatial localization of enzymes that catalyze the sequential pathways of lignin biosynthesis in developing secondary xylem tissues of hybrid aspen (Populus sieboldii Miq. x Populus grandidentata Michx.) using immunohistochemical techniques. The enzymes phenylalanine ammonia-lyase, caffeic acid 3-O-methyltransferase and 4-coumarate:CoA ligase in the common phenylpropanoid pathway, cinnamyl-alcohol dehydrogenase (CAD) and peroxidase in the specific lignin pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) in the shikimate pathway and glutamine synthetase (GS) in the nitrogen reassimilation system were abundantly localized in the 6th to 9th wood fibers away from cambium; these wood fibers are likely undergoing the most intense lignification. Only weak immunolabeling of enzymes involved in the general phenylpropanoid and specific lignin pathways was detected in the cells near the cambium; lignification of these cells has likely been initiated after primary cell wall formation. In contrast, distinct localization of DAHPS and GS was observed around the cambium, which may be involved not only in lignin biosynthesis, but also in amino acid and protein synthesis, which are essential for cell survival. Our observations suggest that co-localization of enzymes related to the sequential shikimate, general phenylpropanoid and specific lignin branch pathways and to the nitrogen recycling system is associated with cell wall lignification of wood fibers during secondary xylem development.

Characterization of the phenylalanine ammonia-lyase gene (SlPAL5) from tomato (Solanum lycopersicum L.).

Phylogenetic analysis based on the deduced amino acid sequence of phenylalanine ammonia-lyase gene (SlPAL5) cDNA from tomato (Solanum lycopersicum L.) revealed high sequence similarity to PAL genes in Nicotiana tabacum (92%), Ipomoea nil (87%), Manihot esculenta (84%), and Catharanthus roseus (84%). The SlPAL5 gene exists as multiple copies in the tomato plant, and its transcription was strongly expressed in old leaves and flowers. From 5 days post-anthesis to the onset of ripening, SlPAL5 expression decreased gradually but was maintained at a comparatively high level; SlPAL5 transcript expression was very low at the mature-green stage. SlPAL5 expression was significantly induced in response to NaCl, mannitol, and cold treatment; SlPAL5 expression decreased gradually after treatment with abscisic acid and H(2)O(2); SlPAL5 transcript decreased after exposure to methyl viologen for 3 h and increased after 6 h and maintained a stable expression level until 24 h, suggesting that the SlPAL5 gene may function in the response to abiotic stress.

High levels of linkage disequilibrium and associations with forage quality at a phenylalanine ammonia-lyase locus in European maize (Zea mays L.) inbreds.

Forage quality of maize is influenced by both the content and structure of lignin in the cell wall. Phenylalanine Ammonia-Lyase (PAL) catalyzes the first step in lignin biosynthesis in plants; the deamination of L-phenylalanine to cinnamic acid. Successive enzymatic steps lead to the formation of three monolignols, constituting the complex structure of lignin. We have cloned and sequenced a PAL genomic sequence from 32 maize inbred lines currently employed in forage maize breeding programs in Europe. Low nucleotide diversity and excessive linkage disequilibrium (LD) was identified at this PAL locus, possibly reflecting selective constrains resulting from PAL being the first enzyme in the monolignol, and other, pathways. While the association analysis was affected by extended LD and population structure, several individual polymorphisms were associated with neutral detergent fiber (not considering population structure) and a single polymorphism was associated with in vitro digestibility of organic matter (considering population structure).

Activation of a mitogen-activated protein kinase pathway is involved in disease resistance in tobacco.

Hypersensitive response (HR), a form of programmed cell death, is frequently associated with plant disease resistance. It has been proposed that mitogen-activated protein kinase (MAPK) cascades regulate HR cell death based on pharmacological studies by using kinase inhibitors. However, direct evidence is lacking. Here, we demonstrate that NtMEK2, a MAPK kinase, is upstream of salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two tobacco MAPKs that are activated by various pathogens or pathogen-derived elicitors. Expression of a constitutively active mutant of NtMEK2 induces HR-like cell death in tobacco, which is preceded by the activation of endogenous SIPK and WIPK. In addition, NtMEK2-SIPK/WIPK cascade appears to control the expression of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and l-phenylalanine ammonia lyase (PAL), two defense genes encoding key enzymes in the phytoalexin and salicylic acid biosynthesis pathways. These results demonstrate that a plant MAPK cascade controls multiple defense responses against pathogen invasion.

Inverse relationship between systemic resistance of plants to microorganisms and to insect herbivory.

Pre-inoculation of plants with a pathogen that induces necrosis leads to the development of systemic acquired resistance (SAR) to subsequent pathogen attack [1]. The phenylpropanoid-derived compound salicylic acid (SA) is necessary for the full expression of both local resistance and SAR [2] [3]. A separate signaling pathway involving jasmonic acid (JA) is involved in systemic responses to wounding and insect herbivory [4] [5]. There is evidence both supporting and opposing the idea of cross-protection against microbial pathogens and insect herbivores [6] [7]. This is a controversial area because pharmacological experiments point to negative cross-talk between responses to systemic pathogens and responses to wounding [8] [9] [10], although this has not been demonstrated functionally in vivo. Here, we report that reducing phenylpropanoid biosynthesis by silencing the expression of phenylalanine ammonialyase (PAL) reduces SAR to tobacco mosaic virus (TMV), whereas overexpression of PAL enhances SAR. Tobacco plants with reduced SAR exhibited more effective grazing-induced systemic resistance to larvae of Heliothis virescens, but larval resistance was reduced in plants with elevated phenylpropanoid levels. Furthermore, genetic modification of components involved in phenylpropanoid synthesis revealed an inverse relationship between SA and JA levels. These results demonstrate phenylpropanoid-mediated cross-talk in vivo between microbially induced and herbivore-induced pathways of systemic resistance.

Differential regulation of phenylalanine ammonia-lyase genes during plant development and by environmental cues.

Phenylalanine ammonia-lyase (PAL) catalyzes the first reaction in the biosynthesis from phenylalanine of a wide variety of phenylpropanoid natural products including lignin, flavonoid pigments, and phytoalexins. In bean (Phaseolus vulgaris L.), PAL is encoded by a family of three genes. We show here by RNase protection with gene-specific probes that these genes are expressed differentially during development and in response to different environmental cues. While all three genes are expressed at high levels in roots, only PAL1 and PAL2 are expressed in shoots and only PAL1 is expressed in leaves. Strikingly, PAL2 is expressed at very high levels in petals, where PAL1 is only very weakly expressed and PAL3 is not expressed. All three genes are induced by mechanical wounding of hypocotyls, but fungal infection only activates PAL1 and PAL3. Illumination of etiolated hypocotyls activates PAL1 and PAL2 but not PAL3. Corresponding differential patterns of synthesis of specific PAL polypeptide isoforms were observed by two-dimensional gel electrophoretic analysis of in vitro translation products encoded by RNA isolated from hypocotyls stimulated by light, wounding, or infection. The specific isoforms encoded by transcripts of the three PAL genes were identified by inhibition of synthesis in vitro with gene-specific anti-sense transcripts followed by comparative two-dimensional gel electrophoretic analysis of the pattern of translation products. These data indicate that selective expression of PAL genes encoding functional variants is governed by a complex set of regulatory networks for developmental and environmental control of phenylpropanoid biosynthesis.