Identification of tomato F-box proteins functioning in phenylpropanoid metabolism.

Phenylpropanoids, a class of specialized metabolites, play crucial roles in plant growth and stress adaptation and include diverse phenolic compounds such as flavonoids. Phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) are essential enzymes functioning at the entry points of general phenylpropanoid biosynthesis and flavonoid biosynthesis, respectively. In Arabidopsis, PAL and CHS are turned over through ubiquitination-dependent proteasomal degradation. Specific kelch domain-containing F-Box (KFB) proteins as components of ubiquitin E3 ligase directly interact with PAL or CHS, leading to polyubiquitinated PAL and CHS, which in turn influences phenylpropanoid and flavonoid production. Although phenylpropanoids are vital for tomato nutritional value and stress responses, the post-translational regulation of PAL and CHS in tomato remains unknown. We identified 31 putative KFB-encoding genes in the tomato genome. Our homology analysis and phylogenetic study predicted four PAL-interacting SlKFBs, while SlKFB18 was identified as the sole candidate for the CHS-interacting KFB. Consistent with their homolog function, the predicted four PAL-interacting SlKFBs function in PAL degradation. Surprisingly, SlKFB18 did not interact with tomato CHS and the overexpression or knocking out of SlKFB18 did not affect phenylpropanoid contents in tomato transgenic lines, suggesting its irreverence with flavonoid metabolism. Our study successfully discovered the post-translational regulatory machinery of PALs in tomato while highlighting the limitation of relying solely on a homology-based approach to predict interacting partners of F-box proteins.

Expression and function analysis of phenylalanine ammonia-lyase genes involved in Bamboo lignin biosynthesis.

Bamboo, renowned as the fastest-growing plant globally, matures within an astonishingly short period of 40-50 days from shoots, reaching heights of 10-20 meters. Moreover, it can be harvested for various uses within 3-5 years. Bamboo exhibits exceptional mechanical properties, characterized by high hardness and flexibility, largely attributed to its lignin content. Phenylalanine ammonia-lyase (PAL) catalyzes the crucial initial step in lignin biosynthesis, but its precise role in bamboo lignification processes remains elusive. Thus, elucidating the functions of PAL genes in bamboo lignification processes is imperative for understanding its rapid growth and mechanical strength. Here, we systematically identified and classified PAL genes in Moso bamboo, ensuring nomenclature consistency across prior studies. Subsequently, we evaluated PAL gene expression profiles using publicly available transcriptome data. The downregulation of PePALs expression in Moso bamboo through in planta gene editing resulted in a decrease in PAL activity and a subsequent reduction in lignin content. In contrast, overexpression of PePAL led to enhanced PAL activity and an increase in lignin content. These findings highlight the critical role of PAL in the lignin biosynthesis process of Moso bamboo, which will help to unravel the mechanism underpinning bamboo's rapid growth and mechanical strength, with a specific emphasis on elucidating the functions of PAL genes.

Gene Expression Profiling and Qualitative Characteristics in Delaying Flesh Softening of Avocado Fruits.

In this research, qualitative characteristics were studied under different post-harvest treatments in Hass and Fuerte cultivars of avocado (Persea americana) fruits. The post-harvest treatments performed in fruits of these cultivars comprised Ethrel application and plastic film (membrane) covering. The measurements of qualitative characteristics were related to color; flesh consistency; measurements of titratable acidity, total soluble solids, percentage of total phenolic contents, and ascorbic peroxidase activity; and the real-time (quantitative) polymerase chain reaction (qPCR) of gene expression and enzyme activities of phenylalanine ammonia-lyase (PAL) and beta-galactosidase (ß-gal). The experiments found that the application of plastic film has excellent results in retaining qualitative characteristics and enzyme activities via maintaining firmness in higher levels. The plastic film covering appeared to delay ripening without the use of chemicals and, therefore, it has the potential to extend the duration of the post-harvest life of the avocado fruit. Variations between the two cultivars were found in the measurements of total soluble solids (Fuerte cultivar showed an increase of 22%, whereas Hass cultivar showed an increase of 120% in Brix values) and total phenolic contents (Fuerte cultivar showed a decrease of 16% and Hass cultivar showed an increase of 29%). It is worth noting that PAL's activity increased significantly (over 44%), as compared to other treatments, and ß-galactosidase's activity decreased, as compared to other treatments. In conclusion, plastic film covering results in a decrease in the activity of ß-galactosidase, as shown by the reaction of hydrolysis (enzyme activity) but also from the expression of the related genes.

Combined analysis of transcriptomics with metabolomics provides insights into the resistance mechanism in winter jujube using L-Methionine.

Black rots lead to great economic losses in winter jujube industry. The objective of this research was to delve into the underlying mechanisms of enhanced resistance of winter jujube fruit to black rot by L-Methionine (Met) treatment. The findings revealed that the application of Met significantly curtailed lesion diameter and decay incidence in winter jujube fruit. The peroxidase (POD) activity in the Met-treated jujubes was 3.06-fold that in the control jujubes after 4 d of treatment. By day 8, the activities of phenylalanine ammonia-lyase (PAL), chitinase (CHI) and ß-1,3-glucanase (GLU) in the Met-treated jujubes had surged to their zenith, being 1.39, 1.22, and 1.52 times in the control group, respectively. At the end of storage, the flavonoid and total phenol content remained 1.58 and 1.06 times than that of the control group. Based on metabolomics and transcriptomics analysis, Met treatment upregulated 6 key differentially expressed metabolites (DEMs) (succinic acid, trans-ferulic acid, salicylic acid, delphinium pigments, (S)-abscisic acid, and hesperidin-7-neohesperidin), 12 key differentially expressed genes (DEGs) (PAL, CYP73A, COMT, 4CL, CAD, POD, UGT72E, ANS, CHS, IAA, TCH4 and PR1), which were involved in phenylpropanoid biosynthesis pathway, flavonoid biosynthesis pathway and plant hormone signal transduction pathway. Further analysis revealed that the most of the enzymes, DEMs and DEGs in this study were associated with both antioxidant and disease resistance. Consequently, Met treatment enhanced disease resistance of winter jujube fruit by elevating antioxidant capacity and triggering defense response. This study might provide theoretical support for utilizing Met in the management and prevention of post-harvest black rot in winter jujube.

Effect of ozone treatment on phenylpropanoid metabolism in harvested cantaloupes.

Phenylpropanoid metabolism plays an important role in cantaloupe ripening and senescence, but the mechanism of ozone regulation on phenylpropanoid metabolism remains unclear. This study investigated how ozone treatment modulates the levels of secondary metabolites associated with phenylpropanoid metabolism, the related enzyme activities, and gene expression in cantaloupe. Treating cantaloupes with 15 mg/m3 of ozone after precooling can help maintain postharvest hardness. This treatment also enhances the production and accumulation of secondary metabolites, such as total phenols, flavonoids, and lignin. These metabolites are essential components of the phenylpropanoid metabolic pathway, activating enzymes like phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4CL, chalcone synthase, and chalcone isomerase. The results of the transcriptional expression patterns showed that differential gene expression related to phenylpropanoid metabolism in the peel of ozone-treated cantaloupes was primarily observed during the middle and late storage stages. In contrast, the pulp exhibited significant differential gene expression mainly during the early storage stage. Furthermore, it was observed that the level of gene expression in the peel was generally higher than that in the pulp. The correlation between the relative amount of gene changes in cantaloupe, activity of selected enzymes, and concentration of secondary metabolites could be accompanied by positive regulation of the phenylpropanoid metabolic pathway. Therefore, ozone stress induction positively enhances the biosynthesis of flavonoids in cantaloupes, leading to an increased accumulation of secondary metabolites. Additionally, it also improves the postharvest storage quality of cantaloupes.

Isolation and Characterization of Phenylalanine Ammonia Lyase (PAL) Genes in Ferula pseudalliacea: Insights into the Phenylpropanoid Pathway.

Phenylalanine ammonia lyase (PAL) is a key enzyme regulating the biosynthesis of the compounds of the phenylpropanoid pathway. This study aimed to isolate and characterize PAL genes from Ferula pseudalliacea Rech.f. (Apiales: Apiaceae) to better understand the regulation of metabolite production. Three PAL gene isoforms (FpPAL1-3) were identified and cloned using the 3'-RACE technique and confirmed by sequencing. Bioinformatics analysis revealed important structural features, such as phosphorylation sites, physicochemical properties, and evolutionary relationships. Expression analysis by qPCR demonstrated the differential transcription profiles of each FpPAL isoform across roots, stems, leaves, flowers, and seeds. FpPAL1 showed the highest expression in stems, FpPAL2 in roots and flowers, and FpPAL3 in flowers. The presence of three isoforms of PAL in F. pseudalliacea, along with the diversity of PAL genes and their tissue-specific expression profiles, suggests that complex modes of regulation exist for phenylpropanoid biosynthesis in this important medicinal plant. The predicted interaction network revealed associations with key metabolic pathways, emphasizing the multifaceted roles of these PAL genes. In silico biochemical analyses revealed the hydrophilicity of the FpPAL isozyme; however, further analysis of substrate specificity and enzyme kinetics can clarify the specific role of each FpPAL isozyme. These comprehensive results increase the understanding of PAL genes in F. pseudalliacea, helping to characterize their contributions to secondary metabolite biosynthesis.

Offsetting pb induced oxidative stress in Vicia faba plants by foliar spray of chitosan through adjustment of morpho-biochemical and molecular indices.

In the course of their life, plants face a multitude of environmental anomaly that affects their growth and production. In recent decades, lead (Pb) gained an increasing attention as it is among the most significant contaminants in the environment. Therefore, in this study the effects of Pb concentrations (0, 50 and 100 ppm) on Vicia faba plants and attempts to alleviate this stress using chitosan (Chs; 0 and 0.1%) were performed. The results validated that with increasing Pb concentrations, a decline in growth, pigments and protein contents was observed. In the same time, a significant upsurge in the stress markers, both malondialdehyde (MDA) and H2O2, was observed under Pb stress. Nonetheless, foliar spraying with Chs improves the faba bean growth, pigment fractions, protein, carbohydrates, reduces MDA and H2O2 contents and decreases Pb concentrations under Pb stress. Pb mitigation effects by Chs are probably related with the activity of antioxidant enzymes, phenylalanine ammonia lyase (PAL) and proline. The application of Chs enhanced the activities of peroxidase, catalase and PAL by 25.77, 17.71 and 20.07%, respectively at 100 ppm Pb compared to their control. Plant genomic material exhibits significant molecular polymorphism, with an average polymorphism of 91.66% across all primers. To assess the genetic distance created among treatments, the dendrogram was constructed and the results of the similarity index ranged from 0.75 to 0.95, indicating genetic divergence. Our research offers a thorough comprehension of the role of Chs in lessening the oxidative stress, which will encourage the use of Chs in agricultural plant protection.

Root suberization in the response mechanism of melon to autotoxicity.

Continuous cropping obstacles poses significant challenges for melon cultivation, with autotoxicity being a primary inducer. Suberization of cells or tissues is a vital mechanism for plant stress response. Our study aimed to elucidate the potential mechanism of root suberization in melon's response to autotoxicity. Cinnamic acid was used to simulate autotoxicity. Results showed that autotoxicity worsened the root morphology and activity of seedlings. Significant reductions were observed in root length, diameter, surface area, volume and fork number compared to the control in the later stage of treatment, with a decrease ranging from 20% to 50%. The decrease in root activity ranged from 16.74% to 29.31%. Root suberization intensified, and peripheral suberin deposition became more prominent. Autotoxicity inhibited phenylalanineammonia-lyase activity, the decrease was 50% at 16 h. The effect of autotoxicity on cinnamylalcohol dehydrogenase and cinnamate 4-hydroxylase activity showed an initial increase followed by inhibition, resulting in reductions of 34.23% and 44.84% at 24 h, respectively. The peroxidase activity only significantly increased at 24 h, with an increase of 372%. Sixty-three differentially expressed genes (DEGs) associated with root suberization were identified, with KCS, HCT, and CYP family showing the highest gene abundance. GO annotated DEGs into nine categories, mainly related to binding and catalytic activity. DEGs were enriched in 27 KEGG pathways, particularly those involved in keratin, corkene, and wax biosynthesis. Seven proteins, including C4H, were centrally positioned within the protein interaction network. These findings provide insights for improving stress resistance in melons and breeding stress-tolerant varieties.

Physiological and molecular mechanism of Populus pseudo-cathayana × Populus deltoides response to Hyphantria cunea.

Populus pseudo-cathayana × Populus deltoides is a crucial artificial forest tree species in Northeast China. The presence of the fall webworm (Hyphantria cunea) poses a significant threat to these poplar trees, causing substantial economic and ecological damage. This study conducted an insect-feeding experiment with fall webworm on P. pseudo-cathayana × P. deltoides, examining poplar's physiological indicators, transcriptome, and metabolome under different lengths of feeding times. Results revealed significant differences in phenylalanine ammonia-lyase activity, total phenolic content, and flavonoids at different feeding durations. Transcriptomic analysis identified numerous differentially expressed genes, including AP2/ERF, MYB, and WRKY transcription factor families exhibiting the highest expression variations. Differential metabolite analysis highlighted flavonoids and phenolic acid compounds of poplar's leaves as the most abundant in our insect-feeding experiment. Enrichment analysis revealed significant enrichment in the plant hormone signal transduction and flavonoid biosynthetic pathways. The contents of jasmonic acid and jasmonoyl-L-isoleucine increased with prolonged fall webworm feeding. Furthermore, the accumulation of dihydrokaempferol, catechin, kaempferol, and naringenin in the flavonoid biosynthesis pathway varied significantly among different samples, suggesting their crucial role in response to pest infestation. These findings provide novel insights into how poplar responds to fall webworm infestation.

Unveiling phenylpropanoid regulation: the role of DzMYB activator and repressor in durian (Durio zibethinus) fruit.

KEY MESSAGE: DzMYB2 functions as an MYB activator, while DzMYB3 acts as an MYB repressor. They bind to promoters, interact with DzbHLH1, and influence phenolic contents, revealing their roles in phenylpropanoid regulation in durian pulps. Durian fruit has a high nutritional value attributed to its enriched bioactive compounds, including phenolics, carotenoids, and vitamins. While various transcription factors (TFs) regulate phenylpropanoid biosynthesis, MYB (v-myb avian myeloblastosis viral oncogene homolog) TFs have emerged as pivotal players in regulating key genes within this pathway. This study aimed to identify additional candidate MYB TFs from the transcriptome database of the Monthong cultivar at five developmental/postharvest ripening stages. Candidate transcriptional activators were discerned among MYBs upregulated during the ripe stage based on the positive correlation observed between flavonoid biosynthetic genes and flavonoid contents in ripe durian pulps. Conversely, MYBs downregulated during the ripe stage were considered candidate repressors. This study focused on a candidate MYB activator (DzMYB2) and a candidate MYB repressor (DzMYB3) for functional characterization. LC-MS/MS analysis using Nicotiana benthamiana leaves transiently expressing DzMYB2 revealed increased phenolic compound contents compared with those in leaves expressing green fluorescence protein controls, while those transiently expressing DzMYB3 showed decreased phenolic compound contents. Furthermore, it was demonstrated that DzMYB2 controls phenylpropanoid biosynthesis in durian by regulating the promoters of various biosynthetic genes, including phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR). Meanwhile, DzMYB3 regulates the promoters of PAL, 4-coumaroyl-CoA ligase (4CL), CHS, and CHI, resulting in the activation and repression of gene expression. Moreover, it was discovered that DzMYB2 and DzMYB3 could bind to another TF, DzbHLH1, in the regulation of flavonoid biosynthesis. These findings enhance our understanding of the pivotal role of MYB proteins in regulating the phenylpropanoid pathway in durian pulps.

The Transcription Factor MiMYB8 Suppresses Peel Coloration in Postharvest 'Guifei' Mango in Response to High Concentration of Exogenous Ethylene by Negatively Modulating MiPAL1.

Anthocyanin accumulation is regulated by specific genes during fruit ripening. Currently, peel coloration of mango fruit in response to exogenous ethylene and the underlying molecular mechanism remain largely unknown. The role of MiMYB8 on suppressing peel coloration in postharvest 'Guifei' mango was investigated by physiology detection, RNA-seq, qRT-PCR, bioinformatics analysis, yeast one-hybrid, dual-luciferase reporter assay, and transient overexpression. Results showed that compared with the control, low concentration of exogenous ethylene (ETH, 500 mg·L-1) significantly promoted peel coloration of mango fruit (cv. Guifei). However, a higher concentration of ETH (1000 mg·L-1) suppressed color transformation, which is associated with higher chlorophyll content, lower a* value, anthocyanin content, and phenylalanine ammonia-lyase (PAL) activity of mango fruit. M. indica myeloblastosis8 MiMYB8 and MiPAL1 were differentially expressed during storage. MiMYB8 was highly similar to those found in other plant species related to anthocyanin biosynthesis and was located in the nucleus. MiMYB8 suppressed the transcription of MiPAL1 by binding directly to its promoter. Transient overexpression of MiMYB8 in tobacco leaves and mango fruit inhibited anthocyanin accumulation by decreasing PAL activity and down-regulating the gene expression. Our observations suggest that MiMYB8 may act as repressor of anthocyanin synthesis by negatively modulating the MiPAL gene during ripening of mango fruit, which provides us with a theoretical basis for the scientific use of exogenous ethylene in practice.

Genome-Wide Identification and Expression Profile Analysis of the Phenylalanine Ammonia-Lyase Gene Family in Hevea brasiliensis.

The majority of the world's natural rubber comes from the rubber tree (Hevea brasiliensis). As a key enzyme for synthesizing phenylpropanoid compounds, phenylalanine ammonia-lyase (PAL) has a critical role in plant satisfactory growth and environmental adaptation. To clarify the characteristics of rubber tree PAL family genes, a genome-wide characterization of rubber tree PALs was conducted in this study. Eight PAL genes (HbPAL1-HbPAL8), which spread over chromosomes 3, 7, 8, 10, 12, 13, 14, 16, and 18, were found to be present in the genome of H. brasiliensis. Phylogenetic analysis classified HbPALs into groups I and II, and the group I HbPALs (HbPAL1-HbPAL6) displayed similar conserved motif compositions and gene architectures. Tissue expression patterns of HbPALs quantified by quantitative real-time PCR (qPCR) proved that distinct HbPALs exhibited varying tissue expression patterns. The HbPAL promoters contained a plethora of cis-acting elements that responded to hormones and stress, and the qPCR analysis demonstrated that abiotic stressors like cold, drought, salt, and H2O2-induced oxidative stress, as well as hormones like salicylic acid, abscisic acid, ethylene, and methyl jasmonate, controlled the expression of HbPALs. The majority of HbPALs were also regulated by powdery mildew, anthracnose, and Corynespora leaf fall disease infection. In addition, HbPAL1, HbPAL4, and HbPAL7 were significantly up-regulated in the bark of tapping panel dryness rubber trees relative to that of healthy trees. Our results provide a thorough comprehension of the characteristics of HbPAL genes and set the groundwork for further investigation of the biological functions of HbPALs in rubber trees.

Engineered Phenylalanine Ammonia-Lyases for the Enantioselective Synthesis of Aspartic Acid Derivatives.

Biocatalytic hydroamination of alkenes is an efficient and selective method to synthesize natural and unnatural amino acids. Phenylalanine ammonia-lyases (PALs) have been previously engineered to access a range of substituted phenylalanines and heteroarylalanines, but their substrate scope remains limited, typically including only arylacrylic acids. Moreover, the enantioselectivity in the hydroamination of electron-deficient substrates is often poor. Here, we report the structure-based engineering of PAL from Planctomyces brasiliensis (PbPAL), enabling preparative-scale enantioselective hydroaminations of previously inaccessible yet synthetically useful substrates, such as amide- and ester-containing fumaric acid derivatives. Through the elucidation of cryo-electron microscopy (cryo-EM) PbPAL structure and screening of the structure-based mutagenesis library, we identified the key active site residue L205 as pivotal for dramatically enhancing the enantioselectivity of hydroamination reactions involving electron-deficient substrates. Our engineered PALs demonstrated exclusive α-regioselectivity, high enantioselectivity, and broad substrate scope. The potential utility of the developed biocatalysts was further demonstrated by a preparative-scale hydroamination yielding tert-butyl protected l-aspartic acid, widely used as intermediate in peptide solid-phase synthesis.

Identification and characterization of PAL genes involved in the regulation of stem development in Saccharum spontaneum L.

BACKGROUND: Saccharum spontaneum L. is a closely related species of sugarcane and has become an important genetic component of modern sugarcane cultivars. Stem development is one of the important factors for affecting the yield, while the molecular mechanism of stem development remains poorly understanding in S. spontaneum. Phenylalanine ammonia-lyase (PAL) is a vital component of both primary and secondary metabolism, contributing significantly to plant growth, development and stress defense. However, the current knowledge about PAL genes in S. spontaneum is still limited. Thus, identification and characterization of the PAL genes by transcriptome analysis will provide a theoretical basis for further investigation of the function of PAL gene in sugarcane. RESULTS: In this study, 42 of PAL genes were identified, including 26 SsPAL genes from S. spontaneum, 8 ShPAL genes from sugarcane cultivar R570, and 8 SbPAL genes from sorghum. Phylogenetic analysis showed that SsPAL genes were divided into three groups, potentially influenced by long-term natural selection. Notably, 20 SsPAL genes were existed on chromosomes 4 and 5, indicating that they are highly conserved in S. spontaneum. This conservation is likely a result of the prevalence of whole-genome replications within this gene family. The upstream sequence of PAL genes were found to contain conserved cis-acting elements such as G-box and SP1, GT1-motif and CAT-box, which collectively regulate the growth and development of S. spontaneum. Furthermore, quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that SsPAL genes of stem had a significantly upregulated than that of leaves, suggesting that they may promote the stem growth and development, particularly in the + 6 stem (The sixth cane stalk from the top to down) during the growth stage. CONCLUSIONS: The results of this study revealed the molecular characteristics of SsPAL genes and indicated that they may play a vital role in stem growth and development of S. spontaneum. Altogether, our findings will promote the understanding of the molecular mechanism of S. spontaneum stem development, and also contribute to the sugarcane genetic improving.

Engineered Zea mays phenylalanine ammonia-lyase for improve the catalytic efficiency of biosynthesis trans-cinnamic acid and p-coumaric acid.

Phenylalanine ammonia-lyase (PAL) plays a pivotal role in the biosynthesis of phenylalanine. PAL from Zea mays (ZmPAL2) exhibits a bi-function of direct deamination of L-phenylalanine (L-Phe) or L-tyrosine(-L-Tyr) to form trans-cinnamic acid or p-coumaric acid. trans-Cinnamic acid and p-coumaric acid are mainly used in flavors and fragrances, food additives, pharmaceutical and other fields. Here, the Activity of ZmPAL2 toward L-Phe or L-Tyr was improved by using semi-rational and rational designs. The catalytic efficiency (kcat/Km) of mutant PT10 (V258I/I459V/Q484N) against L-Phe was 30.8 µM-1 s-1, a 4.5-fold increase compared to the parent, and the catalytic efficiency of mutant PA1 (F135H/I459L) to L-tyrosine exhibited 8.6 µM-1 s-1, which was 1.6-fold of the parent. The yield of trans-cinnamic acid in PT10 reached 30.75 g/L with a conversion rate of 98%. Meanwhile, PA1 converted L-Tyr to yield 3.12 g/L of p-coumaric acid with a conversion rate of 95%. Suggesting these two engineered ZmPAL2 to be valuable biocatalysts for the synthesis of trans-cinnamic acid and p-coumaric acid. In addition, MD simulations revealed that the underlying mechanisms of the increased catalytic efficiency of both mutant PT10 and PA1 are attributed to the substrate remaining stable within the pocket and closer to the catalytically active site. This also provides a new perspective on engineered PAL.

Genome-Wide Characterization of the Phenylalanine Ammonia-Lyase Gene Family and Their Potential Roles in Response to Aspergillus flavus L. Infection in Cultivated Peanut (Arachis hypogaea L.).

Phenylalanine ammonia-lyase (PAL) is an essential enzyme in the phenylpropanoid pathway, in which numerous aromatic intermediate metabolites play significant roles in plant growth, adaptation, and disease resistance. Cultivated peanuts are highly susceptible to Aspergillus flavus L. infection. Although PAL genes have been characterized in various major crops, no systematic studies have been conducted in cultivated peanuts, especially in response to A. flavus infection. In the present study, a systematic genome-wide analysis was conducted to identify PAL genes in the Arachis hypogaea L. genome. Ten AhPAL genes were distributed unevenly on nine A. hypogaea chromosomes. Based on phylogenetic analysis, the AhPAL proteins were classified into three groups. Structural and conserved motif analysis of PAL genes in A. hypogaea revealed that all peanut PAL genes contained one intron and ten motifs in the conserved domains. Furthermore, synteny analysis indicated that the ten AhPAL genes could be categorized into five pairs and that each AhPAL gene had a homologous gene in the wild-type peanut. Cis-element analysis revealed that the promoter region of the AhPAL gene family was rich in stress- and hormone-related elements. Expression analysis indicated that genes from Group I (AhPAL1 and AhPAL2), which had large number of ABRE, WUN, and ARE elements in the promoter, played a strong role in response to A. flavus stress.

Overexpression of GmPAL Genes Enhances Soybean Resistance Against Heterodera glycines.

Soybean cyst nematode (Heterodera glycines, soybean cyst nematode [SCN]) disease adversely affects the yield of soybean and leads to billions of dollars in losses every year. To control the disease, it is necessary to study the resistance genes of the plant and their mechanisms. Isoflavonoids are secondary metabolites of the phenylalanine pathway, and they are synthesized in soybean. They are essential in plant response to biotic and abiotic stresses. In this study, we reported that phenylalanine ammonia-lyase (PAL) genes GmPALs involved in isoflavonoid biosynthesis, can positively regulate soybean resistance to SCN. Our previous study demonstrated that the expression of GmPAL genes in the resistant cultivar Huipizhi (HPZ) heidou are strongly induced by SCN. PAL is the rate-limiting enzyme that catalyzes the first step of phenylpropanoid metabolism, and it responds to biotic or abiotic stresses. Here, we demonstrate that the resistance of soybeans against SCN is suppressed by PAL inhibitor l-α-(aminooxy)-ß-phenylpropionic acid (L-AOPP) treatment. Overexpression of eight GmPAL genes caused diapause of nematodes in transgenic roots. In a petiole-feeding bioassay, we identified that two isoflavones, daidzein and genistein, could enhance resistance against SCN and suppress nematode development. This study thus reveals GmPAL-mediated resistance against SCN, information that has good application potential. The role of isoflavones in soybean resistance provides new information for the control of SCN. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Comparative analyses reveal a phenylalanine ammonia lyase dependent and salicylic acid mediated host resistance in Zingiber zerumbet against the necrotrophic soft rot pathogen Pythium myriotylum.

Little is known about the molecular basis of host defense in resistant wild species Zingiber zerumbet (L.) Smith against the soil-borne, necrotrophic oomycete pathogen Pythium myriotylum Drechsler, which causes the devastating soft rot disease in the spice crop ginger (Zingiber officinale Roscoe). We investigated the pattern of host defense between Z. zerumbet and ginger in response to P. myriotylum inoculation. Analysis of gene expression microarray data revealed enrichment of phenylpropanoid biosynthetic genes, particularly lignin biosynthesis genes, in pathogen-inoculated Z. zerumbet compared to ginger. RT-qPCR analysis showed the robust activation of phenylpropanoid biosynthesis genes in Z. zerumbet, including the core genes PAL, C4H, 4CL, and the monolignol biosynthesis and polymerization genes such as CCR, CAD, C3H, CCoAOMT, F5H, COMT, and LAC. Additionally, Z. zerumbet exhibited the accumulation of the phenolic acids including p-coumaric acid, sinapic acid, and ferulic acid that are characteristic of the cell walls of commelinoid monocots like Zingiberaceae and are involved in cell wall strengthening by cross linking with lignin. Z. zerumbet also had higher total lignin and total phenolics content compared to pathogen-inoculated ginger. Phloroglucinol staining revealed the enhanced fortification of cell walls in Z. zerumbet, specifically in xylem vessels and surrounding cells. The trypan blue staining indicated inhibition of pathogen growth in Z. zerumbet at the first leaf whorl, while ginger showed complete colonization of the pith within 36 h post inoculation (hpi). Accumulation of salicylic acid (SA) and induction of SA regulator NPR1 and the signaling marker PR1 were observed in Z. zerumbet. Silencing of PAL in Z. zerumbet through VIGS suppressed downstream genes, leading to reduced phenylpropanoid accumulation and SA level, resulting in the susceptibility of plants to P. myriotylum. These findings highlight the essential role of PAL-dependent mechanisms in resistance against P. myriotylum in Z. zerumbet. Moreover, our results suggest an unconventional role for SA in mediating host resistance against a necrotroph. Targeting the phenylpropanoid pathway could be a promising strategy for the effective management of P. myriotylum in ginger.

The phenylalanine ammonia-lyase inhibitor AIP induces rice defence against the root-knot nematode Meloidogyne graminicola.

The phenylalanine ammonia-lyase (PAL) enzyme catalyses the conversion of l-phenylalanine to trans-cinnamic acid. This conversion is the first step in phenylpropanoid biosynthesis in plants. The phenylpropanoid pathway produces diverse plant metabolites that play essential roles in various processes, including structural support and defence. Previous studies have shown that mutation of the PAL genes enhances disease susceptibility. Here, we investigated the functions of the rice PAL genes using 2-aminoindan-2-phosphonic acid (AIP), a strong competitive inhibitor of PAL enzymes. We show that the application of AIP can significantly reduce the PAL activity of rice crude protein extracts in vitro. However, when AIP was applied to intact rice plants, it reduced infection of the root-knot nematode Meloidogyne graminicola. RNA-seq showed that AIP treatment resulted in a rapid but transient upregulation of defence-related genes in roots. Moreover, targeted metabolomics demonstrated higher levels of jasmonates and antimicrobial flavonoids and diterpenoids accumulating after AIP treatment. Furthermore, chemical inhibition of the jasmonate pathway abolished the effect of AIP on nematode infection. Our results show that disturbance of the phenylpropanoid pathway by the PAL inhibitor AIP induces defence in rice against M. graminicola by activating jasmonate-mediated defence.

Biochemical Pathways of Salicylic Acid Derived from l-Phenylalanine in Plants with Different Basal SA Levels.

As a plant hormone, salicylic acid (SA) has diverse regulatory roles in plant growth and stress resistance. Although SA is widely found in plants, there is substantial variation in basal SA among species. Tea plant is an economically important crop containing high contents of SA whose synthesis pathway remains unidentified. The phenylalanine ammonia-lyase (PAL) pathway is responsible for basal SA synthesis in plants. In this study, isotopic tracing and enzymatic assay experiments were used to verify the SA synthesis pathway in tea plants and evaluate the variation in phenylalanine-derived SA formation among 11 plant species with different levels of SA. The results indicated that SA could be synthesized via PAL in tea plants and conversion efficiency from benzoic acid to SA might account for variation in basal SA among plant species. This research lays the foundation for an improved understanding of the molecular regulatory mechanism for SA biosynthesis.