Characterization, high-resolution mapping and differential expression of three homologous PAL genes in Coffea canephora Pierre (Rubiaceae)

Phenylalanine ammonia lyase (PAL) is the first entry enzyme of the phenylpropanoid pathway producing phenolics, widespread constituents of plant foods and beverages, including chlorogenic acids, polyphenols found at remarkably high levels in the coffee bean and long recognized as powerful antioxidants. To date, whereas PAL is generally encoded by a small gene family, only one gene has been characterized in Coffea canephora (CcPAL1), an economically important species of cultivated coffee. In this study, a molecular- and bioinformatic-based search for CcPAL1 paralogues resulted successfully in identifying two additional genes, CcPAL2 and CcPAL3, presenting similar genomic structures and encoding proteins with close sequences. Genetic mapping helped position each gene in three different coffee linkage groups, CcPAL2 in particular, located in a coffee genome linkage group (F) which is syntenic to a region of Tomato Chromosome 9 containing a PAL gene. These results, combined with a phylogenetic study, strongly suggest that CcPAL2 may be the ancestral gene of C. canephora. A quantitative gene expression analysis was also conducted in coffee tissues, showing that all genes are transcriptionally active, but they present distinct expression levels and patterns. We discovered that CcPAL2 transcripts appeared predominantly in flower, fruit pericarp and vegetative/lignifying tissues like roots and branches, whereas CcPAL1 and CcPAL3 were highly expressed in immature fruit. This is the first comprehensive study dedicated to PAL gene family characterization in coffee, allowing us to advance functional studies which are indispensable to learning to decipher what role this family plays in channeling the metabolism of coffee phenylpropanoids

Salicylic acid biosynthesis in plants

Salicylic acid (SA) is an important plant hormone that is best known for mediating host responses upon pathogen infection. Its role in plant defense activation is well established, but its biosynthesis in plants is not fully understood. SA is considered to be derived from two possible pathways; the ICS and PAL pathway, both starting from chorismate. The importance of both pathways for biosynthesis differs between plant species, rendering it hard to make generalizations about SA production that cover the entire plant kingdom. Yet, understanding SA biosynthesis is important to gain insight into how plant pathogen responses function and how pathogens can interfere with them. In this review, we have taken a closer look at how SA is synthesized and the importance of both biosynthesis pathways in different plant species

Phenylalanine ammonia-lyase through evolution: A bioinformatic approach

Phenylalanine ammonia-lyase (PAL) is the first entry enzyme of the phenylpropanoid pathway that converts phenylalanine to cinnamic acid which is the precursor of various secondary metabolites. PAL is recently formulated for Phenylketonuric patients in pegylated forms. Screening a PAL with the highest affinity to the substrate is of great importance for this purpose.  PAL exists in all higher plants and some fungi and few bacteria. Ancestors of land plants have been adopted by evolving metabolic pathways. A multi-gene family encodes PAL by gene duplication events in most plants. In this study, the taxonomic distribution and phylogeny of pal gene found in land plants, fungi and bacteria have been analyzed. It seems that the ancestor of plants acquired a pal gene via horizontal gene transfer in symbioses with bacteria and fungi. Gymnosperms have kept a diverse set of pal genes that arose from gene duplication events. In angiosperms, after the divergence of dicotyledons from monocots, pal genes were duplicated many times. The close paralogues of pal genes in some species indicate expansion of gene families after the divergence in plant pal gene evolution. Interestingly, some of the plant pals clustered by species a way that pals within one species are more closely related to each other than to homologs in the other species which indicates this duplication event occurred more recently.</p

The Ve-mediated resistance response of the tomato to Verticillium dahliae involves H2O2, peroxidase and lignins and drives PAL gene expression

<p>Abstract</p> <p>Background</p> <p><it>Verticillium dahliae </it>is a fungal pathogen that infects a wide range of hosts. The only known genes for resistance to <it>Verticillium </it>in the Solanaceae are found in the tomato (<it>Solanum lycopersicum</it>) <it>Ve </it>locus, formed by two linked genes, <it>Ve1 </it>and <it>Ve2</it>. To characterize the resistance response mediated by the tomato <it>Ve </it>gene, we inoculated two nearly isogenic tomato lines, LA3030 (<it>ve</it>/<it>ve</it>) and LA3038 (<it>Ve</it>/<it>Ve</it>), with <it>V. dahliae</it>.</p> <p>Results</p> <p>We found induction of H₂O₂production in roots of inoculated plants, followed by an increase in peroxidase activity only in roots of inoculated resistant plants. Phenylalanine-ammonia lyase (PAL) activity was also increased in resistant roots 2 hours after inoculation, while induction of PAL activity in susceptible roots was not seen until 48 hours after inoculation. Phenylpropanoid metabolism was also affected, with increases in ferulic acid, <it>p</it>-coumaric acid, vanillin and <it>p</it>-hydroxybenzaldehyde contents in resistant roots after inoculation. Six tomato <it>PAL </it>cDNA sequences (<it>PAL1 </it>- <it>PAL6</it>) were found in the SolGenes tomato EST database. RT-PCR analysis showed that these genes were expressed in all organs of the plant, albeit at different levels. Real-time RT-PCR indicated distinct patterns of expression of the different <it>PAL </it>genes in <it>V. dahliae</it>-inoculated roots. Phylogenetic analysis of 48 partial <it>PAL </it>cDNAs corresponding to 19 plant species grouped angiosperm <it>PAL </it>sequences into four clusters, suggesting functional differences among the six tomato genes, with <it>PAL2 </it>and <it>PAL6 </it>presumably involved in lignification, and the remaining <it>PAL </it>genes implicated in other biological processes.</p> <p>An increase in the synthesis of lignins was found 16 and 28 days after inoculation in both lines; this increase was greater and faster to develop in the resistant line. In both resistant and susceptible inoculated plants, an increase in the ratio of guaiacyl/syringyl units was detected 16 days after inoculation, resulting from the lowered amount of syringyl units in the lignins of inoculated plants.</p> <p>Conclusions</p> <p>The interaction between the tomato and <it>V. dahliae </it>triggered a number of short- and long-term defensive mechanisms. Differences were found between compatible and incompatible interactions, including onset of H₂O₂production and activities of peroxidase and PAL, and phenylpropanoid metabolism and synthesis of lignins.</p

The first step into phenolic metabolism in the hornwort Anthoceros agrestis: molecular and biochemical characterization of two phenylalanine ammonia-lyase isoforms

Two isoforms of phenylalanine ammonia-lyase (PAL) have been isolated as cDNA sequences from the hornwort Anthoceros agrestis. The encoded enzymes convert L-phenylalanine and to lower extents L-tyrosine and L-histidine. Thus, the functional presence of the general phenylpropanoid pathway in one of the earliest land plant groups is established. The hornwort Anthoceros agrestis has an elaborated phenolic metabolism resulting in phenolic compounds, such as rosmarinic acid or megacerotonic acid. The general phenylpropanoid pathway is involved in the biosynthesis of these compounds. Two phenylalanine ammonia-lyase (PAL) genes, AaPAL1 and AaPAL2, have been identified in Anthoceros agrestis and the protein with an N-terminal 6xHis-tag heterologously synthesized in Escherichia coli for a full biochemical characterization. Both PAL proteins accept L-phenylalanine, L-tyrosine as well as L-histidine as substrates, although the activity is explicitly the highest with L-phenylalanine. K$_{m}$ values as well as catalytic efficiencies were determined for phenylalanine (K$_{m}$ AaPAL1 39 µM, AaPAL2 18 µM) and tyrosine (K$_{m}$ AaPAL1 3.3 mM, AaPAL2 3.5 mM). In suspension cultures of Anthoceros agrestis, PAL genes were transcribed in parallel to rosmarinic acid (RA) accumulation and both showed highest abundance in the early growth phase. In a phylogenetic tree, both AaPAL amino acid sequences grouped within a clade with PAL amino acid sequences of diverse origin ranging from non-vascular to vascular plants, while most PALs from eudicots and monocots were mainly found in two other clades. The similarity of the hornwort PAL amino acid sequences to PAL sequences from vascular plants is more than 80% showing a strong conservation within the land plants. With this characterization of PALs from Anthoceros agrestis together with former investigations concerning cinnamic acid 4-hydroxylase and 4-coumaric acid CoA-ligase, the functional presence of the general phenylpropanoid pathway in this hornwort is proven

Effects of single or combined water deficit and aphid attack on tomato volatile organic compound (VOC) emission and plant-plant communication

Plants release a broad spectrum of volatile organic compounds (VOCs). The composition of the released VOC blend is dependent on the physiological status and, consequently, is affected by biotic and abiotic stresses. Stress-related VOCs can be perceived by different organisms, including natural enemies of herbivores and neighboring plants. Here, the responses of tomato plants (emitters) to single or combined abiotic (water stress) and biotic (aphid attack) stresses, and the effect of VOC released by emitters on neighboring unstressed plants (receivers), have been investigated. Emissions of α-pinene and methyl salicylate from plants exposed to single or combined stress, and of camphene from plants exposed to water or combined stress were significantly higher than in unstressed plants. In receivers, only the release of methyl salicylate increased when companion emitters were stressed. The expression of genes related to VOC biosynthesis and plant defense responses was unaffected or declined in water-stressed emitters, and was generally higher in receivers than in emitters. The gene coding for methyl salicylate biosynthesis was particularly active in aphid-attacked emitters and in receivers that were conditioned by the infested emitters. In addition, VOCs emitted by stressed plants induce VOC emission in unstressed receivers, and this increases attraction of parasitic wasps, which may improve protection against aphid attacks under conditions of reduced water availability

Tomato Pathogenesis-related Protein Genes are Expressed in Response to Trialeurodes vaporariorum and Bemisia tabaci Biotype B Feeding

The temporal and spatial expression of tomato wound- and defense-response genes to Bemisia tabaci biotype B (the silverleaf whitefly) and Trialeurodes vaporariorum (the greenhouse whitefly) feeding were characterized. Both species of whiteflies evoked similar changes in tomato gene expression. The levels of RNAs for the methyl jasmonic acid (MeJA)- or ethylene-regulated genes that encode the basic β-1,3-glucanase (GluB), basic chitinase (Chi9), and Pathogenesis-related protein-1 (PR-1) were monitored. GluB and Chi9 RNAs were abundant in infested leaves from the time nymphs initiated feeding (day 5). In addition, GluB RNAs accumulated in apical non-infested leaves. PR-1 RNAs also accumulated after whitefly feeding. In contrast, the ethylene- and salicylic acid (SA)-regulated Chi3 and PR-4 genes had RNAs that accumulated at low levels and GluAC RNAs that were undetectable in whitefly-infested tomato leaves. The changes in Phenylalanine ammonia lyase5 (PAL5) were variable; in some, but not all infestations, PAL5 RNAs increased in response to whitefly feeding. PAL5 RNA levels increased in response to MeJA, ethylene, and abscisic acid, and declined in response to SA. Transcripts from the wound-response genes, leucine aminopeptidase (LapA1) and proteinase inhibitor 2 (pin2), were not detected following whitefly feeding. Furthermore, whitefly infestation of transgenic LapA1:GUS tomato plants showed that whitefly feeding did not activate the LapA1 promoter, although crushing of the leaf lamina increased GUS activity up to 40 fold. These studies indicate that tomato plants perceive B. tabaci and T. vaporariorum in a manner similar to baterical pathogens and distinct from tissue-damaging insects