Phenylalanine Ammonia-lyase Gene Research Articles

Genome-Wide Characterization and Expression Analysis of CsPALs in Cucumber (Cucumis sativus L.) Reveal Their Potential Roles in Abiotic Stress and Aphid Stress Tolerance

Phenylalanine ammonia lyase (PAL) is a pivotal enzyme in the phenylalanine metabolic pathway in plants and has a crucial role in the plant’s response to environmental stress. Although the PAL family has been widely studied in many plant species, limited is known about its particular role in cucumbers under stress. We investigated the physicochemical properties, gene structure, gene duplication events, conserved motifs, cis-acting elements, protein interaction networks, stress-related transcriptome data, and quantitatively validated key stress-related genes. The main results indicated that 15 PAL genes were grouped into four clades: I, II, and III when arranged in a phylogenetic tree of PAL genes in angiosperms. The analysis of the promoter sequence revealed the presence of multiple cis-acting elements related to hormones and stress responses in the cucumber PAL genes (CsPALs). The analysis of protein interaction networks suggested that CsPAL1 interacts with eight other members of the PAL family through CsELI5 and CsHISNA, and directly interacts with multiple proteins in the 4CL family. Further investigation into the expression patterns of CsPAL genes in different tissues and under various stress treatments (NaCl, Cu2+, Zn2+, PEG6000, aphids) demonstrated significant differential expression of CsPALs across cucumber tissues. In summary, our characterization of the CsPAL family offers valuable insights and provides important clues regarding the molecular mechanisms of CsPALs in managing abiotic and biotic stress interactions in cucumbers.

Genome-wide identification of the phenylalanine ammonia-lyase gene from Epimedium Pubescens Maxim. (Berberidaceae): novel insight into the evolution of the PAL gene family

BackgroundPhenylalanine ammonia-lyase (PAL) serves as a key gateway enzyme, bridging primary metabolism and the phenylpropanoid pathway, and thus playing an indispensable role in flavonoid, anthocyanin and lignin biosynthesis. PAL gene families have been extensively studied across species using public genomes. However, a comprehensive exploration of PAL genes in Epimedium species, especially those involved in prenylated flavonol glycoside, anthocyanin, or lignin biosynthesis, is still lacking. Moreover, an in-depth investigation into PAL gene family evolution is warranted.ResultsSeven PAL genes (EpPAL1-EpPAL7) were identified. EpPAL2 and EpPAL3 exhibit low sequence identity to other EpPALs (ranging from 61.09 to 64.38%) and contain two unique introns, indicating distinct evolutionary origins. They evolve at a rate ~ 10 to ~ 54 times slower compared to EpPAL1 and EpPAL4-7, suggesting strong purifying selection. EpPAL1 evolved independently and is another ancestral gene. EpPAL1 formed EpPAL4 through segmental duplication, which lead to EpPAL5 and EpPAL6 through tandem duplications, and EpPAL7 through transposed duplication, shaping modern EpPALs. Correlation analysis suggests EpPAL1, EpPAL2 and EpPAL3 play important roles in prenylated flavonol glycosides biosynthesis, with EpPAL2 and EpPAL3 strongly correlated with both Epimedin C and total prenylated flavonol glycosides. EpPAL1, EpPAL2 and EpPAL3 may play a role in anthocyanin biosynthesis in leaves. EpPAL2, EpPAL3, EpPAL6, and EpPAL7 might be engaged in anthocyanin production in petals, and EpPAL2 and EpPAL3 might also contribute to anthocyanin synthesis in sepals. Further experiments are needed to confirm these hypotheses. Novel insights into the evolution of PAL gene family suggest that it might have evolved from a monophyletic group in bryophytes to large-scale sequence differentiation in gymnosperms, basal angiosperms, and Magnoliidae. Ancestral gene duplications and vertical inheritance from gymnosperms to angiosperms likely occurred during PAL evolution. Most early-diverging eudicotyledons and monocotyledons have distinct histories, while modern angiosperm PAL gene families share similar patterns and lack distant gene types.ConclusionsEpPAL2 and EpPAL3 may play crucial roles in biosynthesis of prenylated flavonol glycosides and anthocyanins in leaves and flowers. This study provides novel insights into PAL gene family evolution. The findings on PAL genes in E. pubescens will aid in synthetic biology research on prenylated flavonol glycosides production.

Genome wide identification of phenylalanine ammonia-lyase (PAL) gene family in Cucumis sativus (cucumber) against abiotic stress

Phenylalanine ammonia lyase (PAL) is a widely studied enzyme in plant biology due to its role in connecting primary metabolism to secondary phenylpropanoid metabolism, significantly influencing plant growth, development, and stress response. Although PAL genes have been extensively studied in various plant species but their exploration in cucumber has been limited. This study successfully identified 11 CsPAL genes in Cucumis sativus (cucumber). These CsPAL genes were categorized based on their conserved sequences revealing patterns through MEME analysis and multiple sequence alignment. Interestingly, cis-elements related to stress were found in the promoter regions of CsPAL genes, indicating their involvement in responding to abiotic stress. Furthermore, these gene’s promoters contained components associated with light, development and hormone responsiveness. This suggests that they may have roles in hormone developmental processes. MicroRNAs were identified as a key regulators for the CsPAL genes, playing a crucial role in modulating their expression. This discovery underscores the complex regulatory network involved in the plant’s response to various stress conditions. The influence of these microRNAs further highlights the complicated mechanisms that plants use to manage stress. Gene expression patterns were analyzed using RNA-seq data. The significant upregulation of CsPAL9 during HT3h (heat stress for 3 h) and the heightened upregulation of both CsPAL9 and CsPAL7 under HT6h (heat stress for 6 h) in the transcriptome study suggest a potential role for these genes in cucumber’s tolerance to heat stress. This comprehensive investigation aims to enhance our understanding of the PAL gene family’s versatility, offering valuable insights for advancements in cucumber genetics.

Spinach flavonoid-rich extract: Unleashing plant defense mechanisms against cucumber powdery mildew

In response to the escalating demand for organic commodities, the exploration of non-chemical alternatives such as phytochemicals for plant disease management has gained momentum. This investigation focused on assessing the efficacy of a sustainable compound, Spinach Flavonoid-Rich Extract (SFRE), in mitigating cucumber powdery mildew (Podosphaera fusca) under controlled greenhouse conditions, and its potential in eliciting plant defense mechanisms at both biochemical and genetic levels. The application of SFRE resulted in a significant reduction in P. fusca disease severity, with a decrease of approximately 99%. High-Performance Liquid Chromatography (HPLC) analysis identified spinacetin as the dominant flavonoid in SFRE, with a concentration of 81.97 μg per 100 mg of the extract's dry weight. Furthermore, SFRE was found to induce systemic resistance in the infected seedlings, as evidenced by the upregulation of defense-related enzymes, including β-1,3-glucanase, chitinase, peroxidase, phenylalanine ammonia-lyase, and polyphenol oxidase, and the enhanced expression of β-1,3-glucanase, chitinase, and phenylalanine ammonia-lyase genes. In addition, SFRE application led to an accumulation of flavonoid/phenolic compounds and an enhancement in chlorophyll synthesis. A robust correlation was observed between phenolic compounds and the activities of defense enzymes, as well as the expression of the tested genes. This study substantiates that SFRE, as a reservoir of natural compounds, presents an effective and environmentally friendly alternative to chemical fungicides for P. fusca control, and could be incorporated into a comprehensive approach to managing cucumber powdery mildew through integrated disease management strategies.

Deciphering High-Temperature-Induced Lignin Biosynthesis in Wheat through Comprehensive Transcriptome Analysis.

This study systematically investigated the physiological and molecular responses of the wheat mutant 'XC-MU201' under high-temperature stress through comprehensive transcriptome analysis and physiological measurements. RNA sequencing of 21 samples across seven different treatment groups revealed, through Weighted Gene Co-expression Network Analysis (WGCNA), 13 modules among 9071 genes closely related to high-temperature treatments. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed significant enrichment of lignin biosynthesis-related modules under high-temperature conditions, especially at the H-10DAT, H-20DAT, and H-30DAT time points. Experimental results demonstrated a significant increase in lignin content in high-temperature-treated samples, confirmed by tissue staining methods, indicating wheat's adaptation to heat damage through lignin accumulation. The phenylalanine ammonia-lyase gene (TaPAL33) was significantly upregulated under high-temperature stress, peaking at H-30DAT, suggesting its critical role in cellular defense mechanisms. Overexpression of TaPAL33 in the wheat variety 'Xinchun 11' enhanced lignin synthesis but inhibited growth. Subcellular localization of GFP-labeled TaPAL33 in tobacco cells showed its distribution mainly in the cytoplasm and cell membrane. Transgenic wheat exhibited higher PAL enzyme activity, enhanced antioxidant defense, and reduced oxidative damage under high-temperature stress, outperforming wild-type wheat. These results highlight TaPAL33's key role in improving wheat heat tolerance and provide a genetic foundation for future research and applications.

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.

Comprehensive Analysis of the Yield and Leaf Quality of Fresh Tea (Camellia sinensis cv. Jin Xuan) under Different Nitrogen Fertilization Levels.

Reasonable application of nitrogen fertilizer can improve the yield and quality of tea. This study used Jin Xuan as the tested variety and applied nitrogen fertilizer at rates of 0 kg/ha (N0), 150 kg/ha (N150), 300 kg/ha (N300), and 450 kg/ha (N450) in the summer and autumn seasons to analyze the effects of nitrogen application on the quality components and gene expression of tea leaves. The results showed that the N150 treatment significantly increased total polyphenols (TP), total catechins (TC), and caffeine contents, with the most significant increase observed in the content of six monomers of catechins (EGCG, ECG, EGC, GCG, GC, and EC) in the summer. The N300 treatment significantly increased TP and AA contents in the autumn while decreasing TC content. Additionally, the N300 treatment significantly increased caffeine and theanine contents in the autumn. Notably, the N300 treatment significantly increased both summer and autumn tea yields. Multivariate statistical analysis showed that TPs, AAs, TCs, EGC, and caffeine were key factors affecting the quality of Jin Xuan. Furthermore, the N150 treatment upregulated the expression of the phenylalanine ammonia-lyase (PAL) gene, which may increase the accumulation of catechins. In conclusion, it is recommended to apply 150 kg/ha of nitrogen fertilizer in the summer and 300 kg/ha of nitrogen fertilizer in the autumn. This recommendation provides a theoretical basis for improving the quality and yield of tea leaves in summer and autumn.

Chemical induction of DNA demethylation by 5-Azacytidine enhances tomato fruit defense against gray mold through dicer-like protein DCL2c.

Postharvest decay, primarily caused by pathogenic fungi in ripening fruits and fresh vegetables, poses a challenge to agricultural sustainability and results in significant economic losses. The regulation of the fruit ripening by DNA methylation has been well demonstrated, while defense response of fruit underlying epigenetic regulation against postharvest decay remains uncertain. In the present study, treatment of tomato fruits with the DNA methyltransferase inhibitor 5-Azacytidine (5-Aza) notably decreased their susceptibility to gray mold. Following 5-Aza treatment, we observed a substantial increase in activities of chitinase (CHI) and glucanase (GLU) in tomato fruits, as well as an increase in the expression of the dicer-like SlDCL2 gene family. Suppression of SlDCL2c through double-stranded RNA-induced RNA interference (RNAi) resulted in a decrease in the expression of chitinases CHI3, CHI9, Class V chitinase, and endochitinase 4 by 71%, 29%, 55%, 64%, as well as glucanases Cel1, Cel2, and GluB by 19%, 93%, and 87%, respectively. This was accompanied by decreased activities of resistance-related enzymes, including CHI and GLU. The expression levels of genes phenylalanine ammonia-lyase PAL2, peroxidase POD 12, POD P7, CCR1, CYP84A2, and COMT in phenylpropanoid biosynthesis pathway also decreased by 33%, 53%, 18%, 50%, 30%, and 24% in SlDCL2c-RNAi fruit, resulting in decreased activities of PAL and POD. Consequently, the lesion diameter of gray mold in SlDCL2c-RNAi fruit increased by 55% compared to the control group. Overall, the present study indicated that DNA methyltransferase inhibitor 5-Aza reduces susceptibility of tomato fruit to gray mold through regulation of DCL2c-mediated inducible defense response.

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.

Molecular mechanism of delayed development by interfering RNA targeting the phenylalanine ammonia lyase gene (pal1) in Pleurotus ostreatus

The blockage of the development of edible mushrooms will affect the production cycle and yield of fruiting bodies. Phenylalanine ammonia lyase (PAL, EC 4.3.1.5) is an enzyme that catalyses the deamination of phenylalanine to form trans-cinnamic acid. Previous results have shown that a decrease in pal1 gene transcription delays fruiting body development in Pleurotus ostreatus. Herein, we used wild type (WT) and RNA interference (RNAi) strains to study the molecular regulation of pal1 by RNA sequencing and agrobacterium-mediated genetic transformation. Our results showed that interference with the pal1 gene resulted in a decrease in the total PAL enzyme activity and the total phenol content, as well as an increase in the intracellular H2O2 content. RNA-Seq data demonstrated that the significantly enrichment KEGG terms were mainly related to the peroxisome pathway, MAPK signalling pathway-yeast and three other pathways, moreover, the catalase gene cat1 involved in multiple pathways that were enriched above. Exogenous H2O2 significantly enhanced the transcription of the cat1 gene and elevated total CAT enzyme activity. Moreover, the levels of cat1 gene transcription and the total CAT enzyme activity in the RNAi-pal1 strains were gradually closed to those in the WT strain through the removal of H2O2, which indicated that pal1 regulated the expression of cat1 by affecting the intracellular H2O2 content. Finally, the overexpression of the cat1 gene in P. ostreatus caused growth retardation, especially during the process of primordia formation. In conclusion, this study demonstrated that PAL1 affects cat1 gene expression through the signalling molecule H2O2 and regulates the development of P. ostreatus. Our findings have enhanced the understanding of the molecular developmental mechanism of edible mushrooms.

Methyl jasmonate stimulates growth and upregulates the expression of Phenylalanine Ammonia-Lyase (PAL) gene in Gynura pseudochina in vitro micropropagation

Methyl jasmonate stimulates growth and upregulates the expression of Phenylalanine Ammonia-Lyase (PAL) gene in Gynura pseudochina in vitro micropropagation

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.

Integrated transcriptome and metabolome analysis reveals the regulation of phlorizin synthesis in Lithocarpus polystachyus under nitrogen fertilization

BackgroundNitrogen (N) is essential for plant growth and development. In Lithocarpus polystachyus Rehd., a species known for its medicinal and food value, phlorizin is the major bioactive compound with pharmacological activity. Research has revealed a positive correlation between plant nitrogen (N) content and phlorizin synthesis in this species. However, no study has analyzed the effect of N fertilization on phlorizin content and elucidated the molecular mechanisms underlying phlorizin synthesis in L. polystachyus.ResultsA comparison of the L. polystachyus plants grown without (0 mg/plant) and with N fertilization (25, 75, 125, 175, 225, and 275 mg/plant) revealed that 75 mg N/plant fertilization resulted in the greatest seedling height, ground diameter, crown width, and total phlorizin content. Subsequent analysis of the leaves using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) detected 150 metabolites, including 42 flavonoids, that were differentially accumulated between the plants grown without and with 75 mg/plant N fertilization. Transcriptomic analysis of the L. polystachyus plants via RNA sequencing revealed 162 genes involved in flavonoid biosynthesis, among which 53 significantly differed between the N-treated and untreated plants. Fertilization (75 mg N/plant) specifically upregulated the expression of the genes phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), and phlorizin synthase (PGT1) but downregulated the expression of trans-cinnamate 4-monooxygenase (C4H), shikimate O-hydroxycinnamoyltransferase (HCT), and chalcone isomerase (CHI), which are related to phlorizin synthesis. Finally, an integrated analysis of the transcriptome and metabolome revealed that the increase in phlorizin after N fertilization was consistent with the upregulation of phlorizin biosynthetic genes. Quantitative real-time PCR (qRT‒PCR) was used to validate the RNA sequencing data. Thus, our results indicated that N fertilization increased phlorizin metabolism in L. polystachyus by regulating the expression levels of the PAL, PGT1, 5-O-(4-coumaroyl)-D-quinate 3’-monooxygenase (C3’H), C4H, and HCT genes.ConclusionsOur results demonstrated that the addition of 75 mg/plant N to L. polystachyus significantly promoted the accumulation of flavonoids, including phlorizin, and the expression of flavonoid synthesis-related genes. Under these conditions, the genes PAL, 4CL, and PGT1 were positively correlated with phlorizin accumulation, while C4H, CHI, and HCT were negatively correlated with phlorizin accumulation. Therefore, we speculate that PAL, 4CL, and PGT1 participate in the phlorizin pathway under an optimal N environment, regulating phlorizin biosynthesis. These findings provide a basis for improving plant bioactive constituents and serve as a reference for further pharmacological studies.

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

BackgroundSaccharum spontaneumL. 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.ResultsIn 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.ConclusionsThe 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.

Salivary Protein Cyclin-Dependent Kinase-like from Grain Aphid Sitobion avenae Suppresses Wheat Defense Response and Enhances Aphid Adaptation.

Aphids are insect pests that suck phloem sap and introduce salivary proteins into plant tissues through saliva secretion. The effector of salivary proteins plays a key role in the modulation of host plant defense responses and enhancing aphid host adaptation. Based on previous transcriptome sequencing results, a candidate effector cyclin-dependent kinase-like (CDK) was identified from the grain aphid Sitobion avenae. In this study, the function of SaCDK in wheat defense response and the adaptation of S. avenae was investigated. Our results showed that the transient overexpression of SaCDK in tobacco Nicotiana benthamiana suppressed cell death triggered by mouse pro-apoptotic protein-BAX or Phytophthora infestans PAMP-INF1. SaCDK, delivered into wheat cells through a Pseudomonas fluorescens-mediated bacterial type III secretion system, suppressed callose deposition in wheat seedlings, and the overexpression of SaCDK in wheat significantly decreased the expression levels of salicylic acid and jasmonic acid signaling pathway-related genes phenylalanine ammonia lyase (PAL), pathogenesis-related 1 protein (PR1), lipoxygenase (LOX) and Ω-3 fatty acid desaturase (FAD). In addition, aphid bioassay results showed that the survival and fecundity of S. avenae were significantly increased while feeding on the wheat plants carrying SaCDK. Taken together, our findings demonstrate that the salivary protein SaCDK is involved in inhibiting host defense response and improving its host adaptation, which lays the foundation to uncover the mechanism of the interaction of cereal aphids and host plants.

Identification of transcription factor genes responsive to MeJA and characterization of a LaMYC2 transcription factor positively regulates lycorine biosynthesis in Lycoris aurea

Jasmonates (JAs) are among the main phytohormones, regulating plant growth and development, stress responses, and secondary metabolism. As the major regulator of the JA signaling pathway, MYC2 also plays an important role in plant secondary metabolite synthesis and accumulation. In this study, we performed a comparative transcriptome analysis of Lycoris aurea seedlings subjected to methyl jasmonate (MeJA) at different treatment times. A total of 31,193 differentially expressed genes (DEGs) were identified by RNA sequencing. Among them, 732 differentially expressed transcription factors (TFs) comprising 51 TF families were characterized. The most abundant TF family was WRKY proteins (80), followed by AP2/ERF-EFR (67), MYB (59), bHLH (52), and NAC protein (49) families. Subsequently, by calculating the Pearson's correlation coefficient (PCC) between the expression level of TF DEGs and the lycorine contents, 41 potential TF genes (|PCC| >0.8) involved in lycorine accumulation were identified, including 36 positive regulators and 5 negative regulators. Moreover, a MeJA-inducible MYC2 gene (namely LaMYC2) was cloned on the basis of transcriptome sequencing. Bioinformatic analyses revealed that LaMYC2 proteins contain the bHLH-MYC_N domain and bHLH-AtAIB_like motif. LaMYC2 protein is localized in the cell nucleus, and can partly rescue the MYC2 mutant in Arabidopsis thaliana. LaMYC2 protein could interact with most LaJAZs (especially LaJAZ3 and LaJAZ4) identified previously. Transient overexpression of LaMYC2 increased lycorine contents in L. aurea petals, which might be associated with the activation of the transcript levels of tyrosine decarboxylase (TYDC) and phenylalanine ammonia lyase (PAL) genes. By isolating the 887-bp-length promoter fragment upstream of the start codon (ATG) of LaTYDC, we found several different types of E-box motifs (CANNTG) in the promoter of LaTYDC. Further study demonstrated that LaMYC2 was indeed able to bind the E-box (CACATG) present in the LaTYDC promoter, verifying that the pathway genes involved in lycorine biosynthesis could be regulated by LaMYC2, and that LaMYC2 has positive roles in the regulation of lycorine biosynthesis. These findings demonstrate that LaMYC2 is a positive regulator of lycorine biosynthesis and may facilitate further functional research of the LaMYC2 gene, especially its potential regulatory roles in Amaryllidaceae alkaloid accumulation in L. aurea.

Evaluation of Virus-Free Chrysanthemum 'Hangju' Productivity and Response to Virus Reinfection in the Field: Molecular Insights into Virus-Host Interactions.

The shoot apical meristem culture has been used widely to produce virus-free plantlets which have the advantages of strong disease resistance, high yield, and prosperous growth potential. However, this virus-free plant will be naturally reinfected in the field. The physiological and metabolic responses in the reinfected plant are still unknown. The flower of chrysanthemum 'Hangju' is a traditional medicine which is unique to China. In this study, we found that the virus-free 'Hangju' (VFH) was reinfected with chrysanthemum virus B/R in the field. However, the reinfected VFH (RVFH) exhibited an increased yield and medicinal components compared with virus-infected 'Hangju' (VIH). Comparative analysis of transcriptomes was performed to explore the molecular response mechanisms of the RVFH to CVB infection. A total of 6223 differentially expressed genes (DEGs) were identified in the RVFH vs. the VIH. KEGG enrichment and physiological analyses indicated that treatment with the virus-free technology significantly mitigated the plants' lipid and galactose metabolic stress responses in the RVFH. Furthermore, GO enrichment showed that plant viral diseases affected salicylic acid (SA)-related processes in the RVFH. Specifically, we found that phenylalanine ammonia-lyase (PAL) genes played a major role in defense-related SA biosynthesis in 'Hangju'. These findings provided new insights into the molecular mechanisms underlying plant virus-host interactions and have implications for developing strategies to improve plant resistance against viruses.

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.

Phenylalanine Ammonia-Lyase-Mediated Differential Response of Tomato (Solanum lycopersicum L.) Cultivars with Different Stress Tolerance to Treatment with Low-Molecular-Weight Chitosan

The latest research has shown that chitosan acts as a growth stimulator and elicitor in plants, including resistance to biotic and abiotic factors. However, increasing concentrations could possibly make chitosan a source of stress for plants. In this study, we investigated the effect of low-molecular-weight chitosan hydrolysate on the root development of tomato (Solanum lycoperscum) cultivars Red Cherry, Lel, and Tytan. The growth rate change, together with total phenolic content, phenylalanine ammonia-lyase (PAL) activity, and gene expression, were studied in relation to tomatoes. High concentrations of chitosan negatively affect the growth of tomato seedlings and contribute to changes in the tropism of the seedling roots. After the addition of chitosan hydrolysate, the PAL activity and the total phenolic content decreased 24 h later. PAL is a key enzyme in the biosynthesis of many plant stress factors. An analysis of the tomato PAL gene family was carried out. The SlPAL gene expression in the seedlings of cv. Cherry increased 1.5 times after 48 h, while in cv. Lel, the expression stably decreased in the presence of chitosan. The obtained results are supposed to aid our understanding of the mechanisms underlying the effects of chitosan on plant development and further its successful application in agriculture as well as in research on plant stress.

Molecular regulatory mechanisms of staminate strobilus development and dehiscence in Torreya grandis.

Gymnosperms are mostly dioecious, and their staminate strobili undergo a longer developmental period than those of angiosperms. However, the underlying molecular mechanisms remain unclear. This study aimed to identify key genes and pathways involved in staminate strobilus development and dehiscence in Torreya grandis. Through weighted gene co-expression network analysis (WGCNA), we identified fast elongation-related genes enriched in carbon metabolism and auxin signal transduction, whereas dehiscence-related genes were abundant in alpha-linolenic acid metabolism and the phenylpropanoid pathway. Based on WGCNA, we also identified PHYTOCHROME-INTERACTING FACTOR4 (TgPIF4) as a potential regulator for fast elongation of staminate strobilus and 2 WRKY proteins (TgWRKY3 and TgWRKY31) as potential regulators for staminate strobilus dehiscence. Multiple protein-DNA interaction analyses showed that TgPIF4 directly activates the expression of TRANSPORT INHIBITOR RESPONSE2 (TgTIR2) and NADP-MALIC ENZYME (TgNADP-ME). Overexpression of TgPIF4 significantly promoted staminate strobilus elongation by elevating auxin signal transduction and pyruvate content. TgWRKY3 and TgWRKY31 bind to the promoters of the lignin biosynthesis gene PHENYLALANINE AMMONIA-LYASE (TgPAL) and jasmonic acid metabolism gene JASMONATE O-METHYLTRANSFERASE (TgJMT), respectively, and directly activate their transcription. Overexpression of TgWRKY3 and TgWRKY31 in the staminate strobilus led to early dehiscence, accompanied by increased lignin and methyl jasmonate levels, respectively. Collectively, our findings offer a perspective for understanding the growth of staminate strobili in gymnosperms.