Phenylalanine Ammonia-lyase Gene Research Articles

A reference-grade genome assembly for Astragalus mongholicus and insights into the biosynthesis and high accumulation of triterpenoids and flavonoids in its roots

Astragalus membranaceus var. mongholicus (AMM), a member of the Leguminosae, is one of the most important medicinal plants worldwide. The dried roots of AMM have a wide range of pharmacological effects and are a traditional Chinese medicine. Here, we report the first chromosome-level reference genome of AMM, comprising nine pseudochromosomes with a total size of 1.47 Gb and 27 868 protein-encoding genes. Comparative genomic analysis reveals that AMM has not experienced an independent whole-genome duplication (WGD) event after the WGD event shared by the Papilionoideae species. Analysis of long terminal repeat retrotransposons suggests a recent burst of these elements at approximately 0.13 million years ago, which may explain the large size of the AMM genome. Multiple gene families involved in the biosynthesis of triterpenoids and flavonoids were expanded, and our data indicate that tandem duplication has been the main driver for expansion of these families. Among the expanded families, the phenylalanine ammonia-lyase gene family was primarily expressed in the roots of AMM, suggesting their roles in the biosynthesis of phenylpropanoid compounds. The functional versatility of 2,3-oxidosqualene cyclase genes in cluster III may play a critical role in the diversification of triterpenoids in AMM. Our findings provide novel insights into triterpenoid and flavonoid biosynthesis and can facilitate future research on the genetics and medical applications of AMM.

Phylogenomic analysis of phenylalanine ammonia-lyase (PAL) multigene family and their differential expression analysis in wheat (Triticum aestivum L.) suggested their roles during different stress responses.

Phenylalanine ammonia-lyase (PAL) is a key enzyme in the phenylalanine metabolism pathway and plays an important role in plant growth and stress response. It has been widely reported in plants, but less studied in wheat. In this study, 54 PAL genes were identified in the wheat genome. Based on phylogenetic analysis, the 54 TaPAL genes were divided into four groups (I, II, III, and IV). Then, the expression levels of TaPALs under biotic stresses were analyzed by transcriptome data analysis. The results showed that 31 genes were up-regulated and one gene was down-regulated after inoculation with Fusarium graminearum, 11 genes were up-regulated and 14 genes were down-regulated after inoculation with Puccinia striiformis, and 32 up-regulated and three down-regulated genes after inoculation with powdery mildew. The expression patterns of the five TaPALs were further analyzed by qRT-PCR. After inoculation with F. graminearum, the expression levels of five TaPALs were up-regulated. However, the TaPALs (expect TaPAL49) were down-regulated when inoculated with P. striiformis. Finally, the functions of TaPAL32 and TaPAL42 in resistance of wheat to the stripe rust were further analyzed by virus induced gene silencing (VIGS) assays. The results showed that the disease severity of TaPAL32 and TaPAL42 silenced plants was higher than that of control plants at 14 days after inoculation. It indicated that these two genes played a positive role in wheat stripe rust resistance. This study provided new evidence support for the functional study of PAL genes in wheat, and provided potential application value for the breeding of wheat resistant varieties.

Validation of Appropriate Reference Genes for qRT-PCR Normalization in Oat (Avena sativa L.) under UV-B and High-Light Stresses.

Oat is a food and forage crop species widely cultivated worldwide, and it is also an important forage grass in plateau regions of China, where there is a high level of ultraviolet radiation and sunlight. Screening suitable reference genes for oat under UV-B and high-light stresses is a prerequisite for ensuring the accuracy of real-time quantitative PCR (qRT–PCR) data used in plant adaptation research. In this study, eight candidate reference genes (sulfite oxidase, SUOX; victorin binding protein, VBP; actin-encoding, Actin1; protein PSK SIMULATOR 1-like, PSKS1; TATA-binding protein 2-like, TBP2; ubiquitin-conjugating enzyme E2, UBC2; elongation factor 1-alpha, EF1-α; glyceraldehyde-3-phosphate dehydrogenase 1, GAPDH1;) were selected based on previous studies and our oat transcriptome data. The expression stability of these reference genes in oat roots, stems, and leaves under UV-B and high-light stresses was first calculated using three frequently used statistical software (geNorm, NormFinder, and BestKeeper), and then the comprehensive stability of these genes was evaluated using RefFinder. The results showed that the most stably expressed reference genes in the roots, stems, and leaves of oat under UV-B stress were EF1-α, TBP2, and PSKS1, respectively; the most stably expressed reference genes in the roots, stems, and leaves under high-light stress were PSKS1, UBC2, and PSKS1, respectively. PSKS1 was the most stably expressed reference gene in all the samples. The reliability of the selected reference genes was further validated by analysis of the expression of the phenylalanine ammonia-lyase (PAL) gene. This study highlights reference genes for accurate quantitative analysis of gene expression in different tissues of oat under UV-B and high-light stresses.

Volatile Dimethyl Disulfide from Guava Plants Regulate Developmental Performance of Asian Citrus Psyllid through Activation of Defense Responses in Neighboring Orange Plants.

Intercropping with guava (Psidium guajava L.) can assist with the management of Asian citrus psyllid (ACP, Diaphorina citri Kuwayama), the insect vector of the huanglongbing pathogen, in citrus orchards. Sulfur volatiles have a repellent activity and physiological effects, as well as being important components of guava volatiles. In this study, we tested whether the sulfur volatiles emitted by guava plants play a role in plant–plant communications and trigger anti-herbivore activities against ACP in sweet orange plants (Citrus sinensis L. Osbeck). Real-time determination using a proton-transfer-reaction mass spectrometer (PTR-MS) showed that guava plants continuously release methanethiol, dimethyl sulfide (DMS), and dimethyl disulfide (DMDS), and the contents increased rapidly after mechanical damage. The exposure of orange plants to DMDS resulted in the suppression of the developmental performance of ACP. The differential elevation of salicylic acid (SA) levels; the expression of phenylalanine ammonia lyase (PAL), salicylate-O-methyl transferase (SMT), and pathogenesis-related (PR1) genes; the activities of defense-related enzymes PAL, polyphenol oxidase (PPO), and peroxidase (POD); and the total polyphenol content were observed in DMDS-exposed orange plants. The emission of volatiles including myrcene, nonanal, decanal, and methyl salicylate (MeSA) was increased. In addition, phenylpropanoid and flavonoid biosynthesis, and aromatic amino acid (such as phenylalanine, tyrosine, and tryptophan) metabolic pathways were induced. Altogether, our results indicated that DMDS from guava plants can activate defense responses in eavesdropping orange plants and boost their herbivore resistance to ACP, which suggests the possibility of using DMDS as a novel approach for the management of ACP in citrus orchards.

Gene expression pattern and taxane biosynthesis in a cell suspension culture of Taxus baccata L. subjected to light and a phenylalanine ammonia lyase (PAL) inhibitor.

Taxus baccata L. cell culture is a promising commercial method for the production of taxanes with anti-cancer activities. In the present study, a T. baccata cell suspension culture was exposed to white light and 2-aminoindan-2-phosphonic acid (AIP), a phenylalanine ammonia lyase (PAL) inhibitor, and the effects of this treatment on cell growth, PAL activity, total phenol content (TPC), total flavonoid content (TFC), taxane production and the expression of some key taxane biosynthetic genes (DXS, GGPPS, T13OH, BAPT, DBTNBT) as well as the PAL were studied. Light reduced cell growth, whereas AIP slightly improved it. Light increased PAL activity up to 2.7-fold relative to darkness. The highest TPC (24.89mg GAE/g DW) and TFC (66.94mg RUE/g DW) were observed in cultures treated with light and AIP. Light treatment also resulted in the maximum content of total taxanes (154.78μg/g DW), increasing extracellular paclitaxel and cephalomannin (3.3-fold) and intracellular 10-deacetyl paclitaxel (2.5-fold). Light significantly increased the expression level of PAL, DBTNBT, BAPT, and T13αOH genes, whereas it had no effect on the expression of DXS, a gene active at the beginning of the taxane biosynthetic pathway. AIP had no significant effect on the expression of the target genes. In conclusion, the light-induced activation of PAL transcription and altered expression of relevant biosynthetic genes reduced cell growth and increased the content of total phenolic compounds and taxanes. These findings can be applied to improve taxane production in controlled cultures and bioreactors.

Physiological and Molecular Responses of Vitis vinifera cv. Tempranillo Affected by Esca Disease.

Esca is a multi-fungal disease affecting grapevines. The objective of the study was to evaluate the physiological and molecular response of the grapevine cv. Tempranillo to esca disease, carried out in a vineyard under Mediterranean climatic conditions in western Spain. The photosynthetic pigments in the leaves decreased, with a strong decrease in the photosynthetic efficiency. The proline content increased significantly in the early stages of affected leaves, being possibly involved in the maintenance of lipid peroxidation levels in leaves, which do not increase. The phenol, flavonoid, and phenylpropanoid content decreased in esca-affected leaves, as does the total antioxidant capacity (FRAP), while the polyphenol oxidase (PPO) activity suffers a strong increase with the development of the disease. In affected grapes, the lipid peroxidation and the total phenol content decrease, but not the anthocyanin content. The ascorbate pool decreases with the disease and with time. On the other hand, pool GSH + GSSG is lower in affected leaves, but increases with time. These alterations show a clear change in the redox homeostasis. The expression of genes phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), superoxide dismutase (SOD), and chalcone synthase (ChaS1 and ChaS3) become considerably higher in response to esca, being even higher when the infection time increases. The alteration of AsA and GSH levels, phenolic compounds, PPO activity, proline content, and FRAP, together with the increase of the PAL, PPO, SOD, ChaS1, and ChaS3 gene expression, are clearly implicated in the esca response in plants. The expression of these genes, similar to the PPO activity, can be used as markers of state in the development of the disease.

An insight into the mechanisms underpinning the anti-browning effect of Codium tomentosum on fresh-cut apples

This work focuses on understanding the action of a novel seaweed extract with anti-browning functionality in fresh-cut apples. Organic fresh-cut apples were coated by immersion in an aqueous Codium tomentosum seaweed extract (0.5 % w/v), packaged under ambient atmospheric conditions in plastic bags, and stored at 4 ˚C for 15 days. Browning-related enzymatic activities, as well as targeted gene expression related to superficial browning, were monitored immediately after coating and followed at five-day intervals, until a final storage period of 15 days. Gene expression was particularly affected one hour after coating application (day 0), with no expression registered for peroxidase (mdPOD) and phenylalanine ammonia-lyase (mdPAL) genes in the coated samples. A reduction in polyphenol oxidase expression levels was also observed. After 15 days of storage, the coated samples developed lower browning levels and presented distinctly lower activities of polyphenol oxidase and peroxidase - the oxidative enzymes predominantly involved in enzymatic browning. The observed post-coating suppression of mdPAL and mdPOD expression, and reduction in mdPPO expression, suggest that the seaweed C. tomentosum extract delays the activation of these genes, and decreases enzymatic activity, which in turn accounts for the coating’s anti-browning effect.

Cloning, characterization and prokaryotic expression analysis of two phenylalanine ammonia-lyase genes from Peucedanum praeruptorum Dunn

Cloning, characterization and prokaryotic expression analysis of two phenylalanine ammonia-lyase genes from Peucedanum praeruptorum Dunn

Engineering of triterpene metabolism and overexpression of the lignin biosynthesis gene PAL promotes ginsenoside Rg3 accumulation in ginseng plant chassis.

The ginsenoside Rg3 found in Panax species has extensive pharmacological properties, in particular anti-cancer effects. However, its natural yield in Panax plants is limited. Here, we report a multi-modular strategy to improve yields of Rg3 in a Panax ginseng chassis, combining engineering of triterpene metabolism and overexpression of a lignin biosynthesis gene, phenylalanine ammonia lyase (PAL). We first performed semi-rational design and site mutagenesis to improve the enzymatic efficiency of Pq3-O-UGT2, a glycosyltransferase that directly catalyzes the biosynthesis of Rg3 from Rh2 . Next, we used clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing to knock down the branch pathway of protopanaxatriol-type ginsenoside biosynthesis to enhance the metabolic flux of the protopanaxadiol-type ginsenoside Rg3 . Overexpression of PAL accelerated the formation of the xylem structure, significantly improving ginsenoside Rg3 accumulation (to 6.19-fold higher than in the control). We combined overexpression of the ginsenoside aglycon synthetic genes squalene epoxidase, Pq3-O-UGT2, and PAL with CRISPR/Cas9-based knockdown of CYP716A53v2 to improve ginsenoside Rg3 accumulation. Finally, we produced ginsenoside Rg3 at a yield of 83.6 mg/L in a shake flask (7.0 mg/g dry weight, 21.12-fold higher than with wild-type cultures). The high-production system established in this study could be a potential platform to produce the ginsenoside Rg3 commercially for pharmaceutical use.

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

Main conclusionTwo 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. Km values as well as catalytic efficiencies were determined for phenylalanine (Km AaPAL1 39 µM, AaPAL2 18 µM) and tyrosine (Km 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.

Characterization of phenylalanine ammonia-lyase genes facilitating flavonoid biosynthesis from two species of medicinal plant Anoectochilus.

BackgroundAnoectochilus roxburghii and Anoectochilus formosanus, belong to the Anoectochilus genus, have been used for Chinese herbal drugs as well as health food. Phenylalanine ammonia-lyase (PAL), the key enzyme in primary metabolism and phenylpropanoid metabolism, produces secondary metabolites (flavonoids) in plants, which are beneficial for the biosynthesis of phenylpropanoid metabolites.MethodsThe PAL genes were cloned from A. formosanus and A. roxburghii according to our previous transcriptomic analysis. The PALs were introduced into pCAMBIA2300-35S-PAL-eGFP to generate 35S-PAL-eGFP. The constructs were further used for subcellular localization and transgenic Arabidopsis. The expression of AfPAL and ArPAL under precursor substance (L-Phe), NaCl, UV, and red-light were analyzed by real-time quantitative PCR (RT-qPCR).ResultsAfPAL and ArPAL , encoding 2,148 base pairs, were cloned from A. formosanus and A. roxburghii. The subcellular localization showed that the ArPAL and AfPAL were both localized in the nucleus with GPF. Quantitative RT-PCR analysis indicated that the ArPAL and AfPAL genes function in the phenylalanine pathway as well as response to induced conditions. Overexpression of the AfPAL and ArPAL could increase flavonoids and anthocyanin content in the transgenic Arabidopsis.DiscussionThe results suggest that AfPAL and ArPAL play a crucial role in the flavonoid biosynthesis in Anoectochilus. Also, our study provides new insights into the enrichment of secondary metabolites of traditional Chinese medicines A. formosanus and A. roxburghii, which can improve their medicinal active ingredients and be used for drug discovery in plants.

Molecular cloning and characterization of three phenylalanine ammonia-lyase genes from Schisandra chinensis.

Phenylalanine ammonia-lyase (PAL), which catalyzes the conversion from L-phenylalanine to trans-cinnamic acid, is a well-known key enzyme and a connecting step between primary and secondary metabolisms in the phenylpropanoid biosynthetic pathway of plants and microbes. Schisandra chinensis, a woody vine plant belonging to the family of Magnoliaceae, is a rich source of dibenzocyclooctadiene lignans exhibiting potent activity. However, the functional role of PAL in the biosynthesis of lignan is relatively limited, compared with those in lignin and flavonoids biosynthesis. Therefore, it is essential to clone and characterize the PAL genes from this valuable medicinal plant. In this study, molecular cloning and characterization of three PAL genes (ScPAL1-3) from S. chinensis was carried out. ScPALs were cloned using RACE PCR. The sequence analysis of the three ScPALs was carried out to give basic characteristics followed by docking analysis. In order to determine their catalytic activity, recombinant protein was obtained by heterologous expression in pCold-TF vector in Escherichia coli (BL21-DE3), followed by Ni-affinity purification. The catalytic product of the purified recombinant proteins was verified using RP-HPLC through comparing with standard compounds. The optimal temperature, pH value and effects of different metal ions were determined. Vmax, Kcat and Km values were determined under the optimal conditions. The expression of three ScPALs in different tissues was also determined. Our work provided essential information for the function of ScPALs.

PECULIARITIES OF THE GROWTH OF Artemisia tilesii Ledeb. “HAIRY” ROOTS WITH DIFFERENT FOREIGN GENES

Aim. To compare Artemisia tilesii “hairy” root lines with different transferred genes in terms of the relationship between the total content of flavonoids, the levels of antioxidant activity (AOA) and reducing power (RP), as well as the activity of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), rolB and rolC genes. Methods. We compared the root lines Nos. 10 and 16, obtained by transformation with the wild Agrobacterium rhizogenes strain A4, lines Nos. 2 and 4, obtained using A. rhizogenes carrying pSV124 vector with ifn-α2b and nptII genes, as well as the roots of non-transformed plants that were cultivated in vitro. The presence and activity of rolB, rolC, PAL, and CHS genes were determined by PCR and real-time PCR, respectively. The content of flavonoids, AOA and RP were determined by standard tests with AlCl3, DPPH (2,2-diphenyl-1-picrylhydrazyl) and K3[Fe(CN)6] accordingly. Results. The content of flavonoids in most of the lines was higher than in the control, and correlated with AOA and RP. Roots No. 10 were characterized by the fastest growth, which coincided with higher activity of rolB and rolC genes. The activities of PAL and CHS in “hairy” roots were lower than those in non-transformed ones. Conclusions. Root lines carrying only rolB and rolC and lines with additional ifn-α2b and nptII genes had similar ranges of flavonoids concentration, AOA and RP levels that exceeded those in the control. The dependence of the root growth rate, and lack of the dependence of the flavonoids content with the activity of the rol genes were demonstrated. PAL activity inversely correlated with flavonoids content in all experimental lines, which may be the result of overproduction of compounds in transgenic roots.

The different deletion mutation in the phenylalanine ammonia-lyase (PAL) gene affects the peel color of mango (Mangifera indica L.)

The different deletion mutation in the phenylalanine ammonia-lyase (PAL) gene affects the peel color of mango (Mangifera indica L.)

Genome-Wide Analysis and Expression Profiling of the Phenylalanine Ammonia-Lyase Gene Family in Solanum tuberosum.

Phenylalanine ammonia-lyase is one of the most widely studied enzymes in the plant kingdom. It is a crucial pathway from primary metabolism to significant secondary phenylpropanoid metabolism in plants, and plays an essential role in plant growth, development, and stress defense. Although PAL has been studied in many actual plants, only one report has been reported on potato, one of the five primary staple foods in the world. In this study, 14 StPAL genes were identified in potato for the first time using a genome-wide bioinformatics analysis, and the expression patterns of these genes were further investigated using qRT-PCR. The results showed that the expressions of StPAL1, StPAL6, StPAL8, StPAL12, and StPAL13 were significantly up-regulated under drought and high temperature stress, indicating that they may be involved in the stress defense of potato against high temperature and drought. The expressions of StPAL1, StPAL2, and StPAL6 were significantly up-regulated after MeJa hormone treatment, indicating that these genes are involved in potato chemical defense mechanisms. These three stresses significantly inhibited the expression of StPAL7, StPAL10, and StPAL11, again proving that PAL is a multifunctional gene family, which may give plants resistance to multiple and different stresses. In the future, people may improve critical agronomic traits of crops by introducing other PAL genes. This study aims to deepen the understanding of the versatility of the PAL gene family and provide a valuable reference for further genetic improvement of the potato.

Enhancing salinity stress tolerance and phenylalanine ammonia lyase gene activity using osmolytes in Moringa seedling production

The salinity of irrigation water is one of the major abiotic stresses that have a variable reaction to soil structure, which has detrimental impacts on seedling development and quality. The osmolytes can help lessen these adverse effects on the establishment of Moringa oleifera seedlings. Therefore, this study was conducted in Egypt (30″53′30, 87″ N and 30″41′29, 77″ E) using polyethylene bags during May – July 2020 and 2021. Three factors in a factorial split –split plot experiment were arranged in randomized complete block design. The main plots were soil structure; sand:clay 2:1 (V/V) and sand 100 %, whereas the subplot treatments were salinity levels; tap water as a control 0.5, 1.5, 4, and 6 dS m−1, and sub–subplot osmolytes; tap water as a control, and glycine betaine, mannitol, proline, and sorbitol all at 0.2 g L−1 as a soil drench. This study aimed to evaluate the effects of osmolytes and soil structure on growth parameters and phenylalanine ammonia lyase (PAL) gene expression in Moringa seedlings. The study concluded that Moringa seedlings in sand:clay performed significantly (P ˂ 0.05) better than those in the sand. With increasing salinity levels, osmolytes enhanced all seedling growth parameters and PAL gene expression compared with the control in the two soil structures. The order of tolerance to salinity stress was sorbitol ˃ proline ˃ mannitol ˃ glycine betaine ˃ control. Moringa seedlings produced in sand:clay treated with 0.2 g L−1 sorbitol and salinity at 4 or 6 dS m−1 were superior in most growth parameters and PAL gene expression.

Mushrooms Do Produce Flavonoids: Metabolite Profiling and Transcriptome Analysis of Flavonoid Synthesis in the Medicinal Mushroom Sanghuangporus baumii

Mushrooms produce a large number of medicinal bioactive metabolites with antioxidant, anticancer, antiaging, and other biological activities. However, whether they produce flavonoids and, if so, how they synthesize them remains a matter of some debate. In the present study, we combined flavonoid-targeted metabolomics and transcriptome analysis to explore the flavonoid synthesis in the medicinal mushroom Sanghuangporus baumii. The S. baumii synthesized 81 flavonoids on a chemically defined medium. The multiple classes of flavonoids present were consistent with the biosynthetic routes in plants. However, paradoxically, most of the genes that encode enzymes involved in the flavonoid biosynthetic pathway are missing from S. baumii. Only four genes related to flavonoid synthesis were found in S. baumii, among which phenylalanine ammonia lyase gene (PAL) is a key gene regulating flavonoid synthesis, and overexpression of SbPAL increases the accumulation of flavonoids. These results suggest that the flavonoid synthesis pathway in S. baumii is different from that in known plants, and the missing genes may be replaced by genes from the same superfamilies but are only distantly related. Thus, this study provides a novel method to produce flavonoids by metabolic engineering using mushrooms.

Unveiling Molecular Mechanisms of Nitric Oxide-Induced Low-Temperature Tolerance in Cucumber by Transcriptome Profiling.

Cucumber (Cucumis sativus L.) is one of the most popular cultivated vegetable crops but it is intrinsically sensitive to cold stress due to its thermophilic nature. To explore the molecular mechanism of plant response to low temperature (LT) and the mitigation effect of exogenous nitric oxide (NO) on LT stress in cucumber, transcriptome changes in cucumber leaves were compared. The results showed that LT stress regulated the transcript level of genes related to the cell cycle, photosynthesis, flavonoid accumulation, lignin synthesis, active gibberellin (GA), phenylalanine metabolism, phytohormone ethylene and salicylic acid (SA) signaling in cucumber seedlings. Exogenous NO improved the LT tolerance of cucumber as reflected by increased maximum photochemical efficiency (Fv/Fm) and decreased chilling damage index (CI), electrolyte leakage and malondialdehyde (MDA) content, and altered transcript levels of genes related to phenylalanine metabolism, lignin synthesis, plant hormone (SA and ethylene) signal transduction, and cell cycle. In addition, we found four differentially expressed transcription factors (MYB63, WRKY21, HD-ZIP, and b-ZIP) and their target genes such as the light-harvesting complex I chlorophyll a/b binding protein 1 gene (LHCA1), light-harvesting complex II chlorophyll a/b binding protein 1, 3, and 5 genes (LHCB1, LHCB3, and LHCB5), chalcone synthase gene (CSH), ethylene-insensitive protein 3 gene (EIN3), peroxidase, phenylalanine ammonia-lyase gene (PAL), DNA replication licensing factor gene (MCM5 and MCM6), gibberellin 3 beta-dioxygenase gene (GA3ox), and regulatory protein gene (NPRI), which are potentially associated with plant responses to NO and LT stress. Notably, HD-ZIP and b-ZIP specifically responded to exogenous NO under LT stress. Taken together, these results demonstrate that cucumber seedlings respond to LT stress and exogenous NO by modulating the transcription of some key transcription factors and their downstream genes, thereby regulating photosynthesis, lignin synthesis, plant hormone signal transduction, phenylalanine metabolism, cell cycle, and GA synthesis. Our study unveiled potential molecular mechanisms of plant response to LT stress and indicated the possibility of NO application in cucumber production under LT stress, particularly in winter and early spring.

In vivo Antiphytoviral Activity of Essential Oils and Hydrosols From Origanum vulgare, Thymus vulgaris, and Rosmarinus officinalis to Control Zucchini Yellow Mosaic Virus and Tomato Leaf Curl New Delhi Virus in Cucurbita pepo L.

In the last decades, the interest in biological activity of natural compounds has been growing. In plant protection, essential oils have been reported to exhibit antiviral, antimycotic, and antiparasitic activities, and are regarded as promising for the formulation of safe antimicrobial agents. Attention has also been focused on hydrosols, the by-products of hydro-distillation of essential oils. Their production is easy, fast, and cheap, and they seem to arise less concern for human health than essential oils. Plant viruses represent a major concern for agricultural crops since no treatment compound is available for virus control. This work was aimed at evaluating the antiphytoviral effectiveness of treatments with three essential oils and corresponding hydrosols extracted from Origanum vulgare, Thymus vulgaris, and Rosmarinus officinalis on Cucurbita pepo plants infected by zucchini yellow mosaic virus or tomato leaf curl New Delhi virus. Treatments were applied either concurrently or after virus inoculation to ascertain an inhibition or curative activity, respectively. Symptoms were observed and samplings were performed weekly. Virus titer and expression levels of phenylalanine ammonia lyase gene (PAL) were measured on treated and untreated infected plants by real-time PCR. PAL gene plays an important role in plant defense response as it is involved in tolerance/resistance to phytopathogens. Results indicated that treatments were effective against tomato leaf curl New Delhi virus whether applied simultaneously with the inoculation or after. A major inhibition was observed with O. vulgare essential oil and hydrosol, resulting in 10–4-fold decrease of virus titer 3 weeks after treatment. Curative activity gave maximum results with all three essential oils and T. vulgaris and R. officinalis hydrosols, recording from 10–2-fold decrease to virus not detected 4 weeks after treatment. An induction of PAL gene expression was recorded at 12 d.p.i. and then was restored to the levels of untreated control. This allows to hypothesize an early plant defense response to virus infection, possibly boosted by treatments. Plant extracts’ composition was characterized by gas chromatography-mass spectrometry. Phenols were largely main components of O. vulgare and T. vulgaris extracts (carvacrol and thymol, respectively), while extracts from R. officinalis were based on monoterpene hydrocarbons (essential oil) and oxygenated monoterpenes (hydrosol).

Candidate marker genes and enzymes for selection of potato with resistance to early blight, caused by Alternaria alternata

Early blight, caused by Alternaria alternata (Fr.) Keissler is a serious disease of potato and other cultivated Solanum species. The molecular components defining defense responses to A. alternata in potato are limited. Host transcript accumulation after A. alternata inoculation of six potato genotypes (10/33/R1, 3/33/R2 and 21/33/R2, resistant to the pathogen, and 8707/106, 8703/804 and 8707/112, susceptible) was examined to develop understanding of mechanisms of their responses to A. alternata genotypes. The marker genes PR-2, ChtA, PR-5, PR1-b, PIN2, ERF3, PAL and LOX, activity of catalase (CAT), superoxide dismutase (SOD), peroxidase (POX), polyphenol oxidase (PPOs) and phenylalanine ammonia-lyase (PAL), as well as biomass growth parameters, were analysed. Expression of PR-2, ChtA, PR-5, PR1-b and PAL genes was greatly increased in the inoculated resistant genotypes compared to the susceptible genotypes and un-inoculated controls. Transcription levels of PIN2, ERF3 and LOX genes were decreased in resistant inoculated plants. Simultaneously, activities of POX, SOD and PPOs were greatly increased in the inoculated resistant host genotypes, compared to the susceptible and non-inoculated controls. CAT activity in genotype 21/33/R2 and PAL activity in resistant genotypes 21/33/R2 and 10/33/R1 increased in the susceptible and non-inoculated. Host growth parameters of inoculated plants decreased compared to un-inoculated controls. Knowledge of changes in gene expression levels and enzyme production in defense processes in infected potato plants can inform future studies to identify the defense mechanisms, and assist generation of potato cultivars resistant to early blight.