Phenylalanine Ammonia-lyase Levels Research Articles

Modifications in gene expression and phenolic compounds content by methyl jasmonate and fungal elicitors in Ficus carica. Cv. Siah hairy root cultures

BackgroundOne of the most effective strategies to increase phytochemicals production in plant cultures is elicitation. In the present study, we studied the effect of abiotic and biotic elicitors on the growth, key biosynthetic genes expression, antioxidant capacity, and phenolic compounds content in Rhizobium (Agrobacterium) rhizogenes-induced hairy roots cultures of Ficus carica cv. Siah.MethodsThe elicitors included methyl jasmonate (MeJA) as abiotic elicitor, culture filtrate and cell extract of fungus Piriformospora indica as biotic elicitors were prepared to use. The cultures of F. carica hairy roots were exposed to elicitores at different time points. After elicitation treatments, hairy roots were collected, and evaluated for growth index, total phenolic (TPC) and flavonoids (TFC) content, antioxidant activity (2,2-diphenyl-1-picrylhydrazyl, DPPH and ferric ion reducing antioxidant power, FRAP assays), expression level of key phenolic/flavonoid biosynthesis genes, and high-performance liquid chromatography (HPLC) analysis of some main phenolic compounds in comparison to control.ResultsElicitation positively or negatively affected the growth, content of phenolic/flavonoid compounds and DPPH and FRAP antioxidant activities of hairy roots cultures in depending of elicitor concentration and exposure time. The maximum expression level of chalcone synthase (CHS: 55.1), flavonoid 3′-hydroxylase (F3’H: 34.33) genes and transcription factors MYB3 (32.22), Basic helix-loop-helix (bHLH: 45.73) was induced by MeJA elicitation, whereas the maximum expression level of phenylalanine ammonia-lyase (PAL: 26.72) and UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT: 27.57) genes was obtained after P. indica culture filtrate elicitation. The P. indica elicitation also caused greatest increase in the content of gallic acid (5848 µg/g), caffeic acid (508.2 µg/g), rutin (43.5 µg/g), quercetin (341 µg/g), and apigenin (1167 µg/g) phenolic compounds.ConclusionsThis study support that elicitation of F. carica cv. Siah hairy roots can be considered as an effective biotechnological method for improved phenolic/flavonoid compounds production, and of course this approach requires further research.

Exogenous melatonin prolongs raspberry postharvest life quality by increasing some antioxidant and enzyme activity and phytochemical contents

There is a growing trend towards enhancing the post-harvest shelf life and maintaining the nutritional quality of horticultural products using eco-friendly methods. Raspberries are valued for their diverse array of phenolic compounds, which are key contributors to their health-promoting properties. However, raspberries are prone to a relatively short post-harvest lifespan. The present study aimed to investigate the effect of exogenous melatonin (MEL; 0, 0.001, 0.01, and 0.1 mM) on decay control and shelf-life extension. The results demonstrated that MEL treatment significantly reduced the fruit decay rate (P ≤ 0.01). Based on the findings, MEL treatment significantly increased titratable acidity (TA), total phenolics content (TPC), total flavonoid content (TFC), and total anthocyanin content (TAC). Furthermore, the MEL-treated samples showed increased levels of rutin and quercetin content, as well as antioxidant activity as measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reduction activity potential (FRAP). Additionally, the samples exhibited higher levels of phenylalanine ammonia-lyase (PAL) and catalase (CAT) enzymes compared to the control samples. Moreover, the levels of pH, total soluble solids (TSS), and IC50 were decreased in the MEL-treated samples (P ≤ 0.01). The highest amount of TA (0.619 g/100 ml juice), rutin (16.722 µg/ml juice) and quercetin (1.467 µg/ml juice), and PAL activity (225.696 nm/g FW/min) was observed at 0.001 mM treatment, while, the highest amount of TAC (227.235 mg Cy-g/100 ml juice) at a concentration of 0.01 mM and CAT (0.696 u/g FW) and TAL activities (9.553 nm/100 g FW) at a concentration of 0.1 mM were obtained. Considering the lack of significant differences in the effects of melatonin concentrations and the low dose of 0.001 mM, this concentration is recommended for further research. The hierarchical cluster analysis (HCA) and principal component analysis (PCA) divided the treatments into three groups based on their characteristics. Based on the Pearson correlation between TPC, TFC, TAC, and TAA, a positive correlation was observed with antioxidant (DPPH and FRAP) and enzyme (PAL and CAT) activities. The results of this study have identified melatonin as an eco-friendly compound that enhances the shelf life of raspberry fruits by improving phenolic compounds, as well as antioxidant and enzyme activities.

Elicitation of salicylic acid and methyl jasmonate provides molecular and physiological evidence for potato susceptibility to infection by Erwinia carotovora subsp. carotovora

Among the range of severe plant diseases, bacterial soft rot caused by Erwinia carotovora is a significant threat to crops. This study aimed to examine the varying response patterns of distinct potato cultivars to the influence of E. carotovora. Furthermore, it seeks to highlight the potential role of salicylic acid (SA) and methyl jasmonate (MeJA) in stimulating the antioxidant defence system. We collected eight bacterial isolates from diseased and rotted tubers which were morphologically and physiologically identified as E. carotovora subsp. carotovora. We conducted a greenhouse experiment to analyse the antioxidant responses of three different potato cultivars (Diamont, Kara, and Karros) at various time intervals (2, 4, 6, 8, 12, and 24 h) after bacterial infection (hpi). We assessed the extent of disease damage by applying a foliar spray of 0.9 mM salicylic acid (SA) and 70 μM methyl jasmonate (MeJA). Inoculating with Ecc led to an increase in total phenolic levels, as well as the activities and gene expression of phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POX) as time progressed. Additionally, the application of SA and MeJA resulted in a further increase relative to the diseased treatments. The Karros cultivar, unlike the Diamont and Kara cultivars, demonstrated the highest expression levels of PAL, PPO and POX through inoculation, reflecting its higher levels of activity and resistance. Furthermore, the genetic response of potato cultivars to infection at 0 hpi varied depending on their susceptibility. The examination of the rate of PAL activity upregulation following SA or MeJA stimulation clarifies the cultivars' susceptibility over time. In conclusion, the study identified E. carotovora subsp. carotovora as the most virulent isolate causing soft rot disease in potato tubers. It further revealed that the Karros cultivar displayed superior resistance with high activities and gene expression of PAL, PPO and POX, while the cv. Diamont exhibited sensitivity. Additionally, foliar exposure to SA and MeJA induced antioxidant responses, enhancing the potato plants' resistance against Ecc pathogenesis and overall plant defence.

The efficacy of chemical inducers and fungicides in controlling tomato root rot disease caused by Rhizoctoniasolani

Chitosan is an environmentally friendly natural substance that is used in crop disease management as an alternative to chemical pesticides. A significant issue restricting output in Egypt is root rot, which is a disease, caused by Rhizoctonia solani. Therefore, a greenhouse experiment was conducted to assess the effects of R. solani on 60-day-old tomato plants under fungal infection and to determine the antifungal activity of chitosan and Rizolax T fungicide against the pathogenic fungus. The findings demonstrated that 4 g/L of chitosan seed application completely obstructed the radial mycelial growth of R. solani and decreased the disease severity. Pathogenic infection significantly decreased morphological characteristics and total chlorophyll but significantly increased carotenoid, total thiol, non-protein thiol, protein thiol, antioxidant enzymes, oxidative stress, total phenolic, total flavonoid, and isoflavone compared to healthy plants. Tomato plants treated with chitosan exhibited lower rates of oxidative stress, but higher levels of all previously mentioned parameters compared to untreated infected plants. The number and molecular mass of protein banding patterns varied in all treated tomato plants as compared to the healthy control. There are 42 bands in the treatments, and their polymorphism rate is 69.55%. Moreover, the number and density of α- and β-esterase, and peroxidase isozymes in treated tomato plants exhibited varied responses. Moreover, in treated and control plants, chitosan treatment raised the expression levels of phenylalanine ammonia-lyase, pathogenesis-related protein-1, β-1,3-glucanases and chitinase. In conclusions, chitosan reduces R. solani infection by controlling the biochemical and molecular mechanisms in tomato plants during infection.

Transcriptomic-Proteomic Analysis Revealed the Regulatory Mechanism of Peanut in Response to Fusarium oxysporum.

Peanut Fusarium rot, which is widely observed in the main peanut-producing areas in China, has become a significant factor that has limited the yield and quality in recent years. It is highly urgent and significant to clarify the regulatory mechanism of peanuts in response to Fusarium oxysporum. In this study, transcriptome and proteome profiling were combined to provide new insights into the molecular mechanisms of peanut stems after F. oxysporums infection. A total of 3746 differentially expressed genes (DEGs) and 305 differentially expressed proteins (DEPs) were screened. The upregulated DEGs and DEPs were primarily enriched in flavonoid biosynthesis, circadian rhythm-plant, and plant-pathogen interaction pathways. Then, qRT-PCR analysis revealed that the expression levels of phenylalanine ammonia-lyase (PAL), chalcone isomerase (CHI), and cinnamic acid-4-hydroxylase (C4H) genes increased after F. oxysporums infection. Moreover, the expressions of these genes varied in different peanut tissues. All the results revealed that many metabolic pathways in peanut were activated by improving key gene expressions and the contents of key enzymes, which play critical roles in preventing fungi infection. Importantly, this research provides the foundation of biological and chemical analysis for peanut disease resistance mechanisms.

Postharvest caffeic acid dipping enhances disease resistance and storage capacity of ‘Zaosu’ pear fruit via regulating phenylpropane metabolism

Caffeic acid, a naturally occurring and safe phenolic compound, acts as a precursor in lignin synthesis within the phenylpropane metabolism pathway. The aims of this study was to evaluate the impacts of postharvest caffeic acid immersion on Alternaria rot and postharvest quality of ‘Zaosu’ pear fruit, as well as elucidate the underlying mechanisms involved. Results showed that postharvest caffeic acid dipping effectively inhibited spore germination and colony diameter of Alternaria alternata, as well as lesion diameter in A. alternata-inoculated pears. Caffeic acid treatment also delayed the increase of weight loss and decline in flesh firmness, increased total soluble solids content, and reduced surface yellowing of pears. Additionally, caffeic acid treatment enhanced the enzymatic activities and gene transcription levels of phenylalanine ammonialyase, 4-coumarate/coenzyme A ligase, cinnamate 4-hydroxylase, peroxidase, cinnamyl alcohol dehydrogenase, and polyphenol oxidase in pears. Meanwhile, the transcription levels of p-coumarate-3-hydroxylase, caffeic acid O-methyltransferase, cinnamoyl CoA reductase, caffeoyl shikimate esterase, hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase, caffeoyl CoA O-methyltransferase, ferulic acid 5-hydroxylase, and laccase genes were also up-regulated by caffeic acid treatment in pears. Furthermore, caffeic acid dipping enhanced phenylalanine, flavonoids, caffeic acid, total phenolic compounds, and lignin levels in the fruit. Therefore, caffeic acid exhibits promising potential for enhancing resistance against A. alternata and maintaining the storage capacity of pears via inducing phenylpropane metabolism.

Exogenously applied abscisic acid accelerates wound healing of Chinese yam tubers (Dioscorea opposita) through promoting suberin polyphenolics biosynthesis

Water loss, nutrient elution and pathogen attacks caused by mechanical wounding are the primary sources of postharvest loss of fruit and vegetables. Continuously increasing information has unraveled that abscisic acid (ABA) can accelerate wound healing of fruit and vegetables via stimulating suberin polyphenolics (SPP) biosynthesis, whereas the effect of ABA in wound healing of Chinese yam tubers remains unclear. The treatment of ABA and fluridone (FLD, an inhibitor of ABA biosynthesis) on postharvest wound healing of yam tubers was investigated in this study. The results showed that ABA treatment facilitated wound healing process of yam tubers, representing by the reduced weight loss and relative electrolyte leakage (REL). ABA enhanced the expression levels of phenylalanine ammonialyase (PAL), cinnamic acid 4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL), as well as the corresponding enzyme contents and activities. Correspondingly, the contents of major monomers such as ferulic acid, caffeic acid and p-coumaric acid also increased. Simultaneously, ABA could improve the expression of peroxidase (POD), the enzyme content and activity, as well as hydrogen peroxide (H2O2) production, which might contribute to the polymerization of SPP and lignin. Inversely, inhibition of ABA production by FLD application displayed an opposite effect to ABA treatment. These results demonstrate that application of ABA stimulated wound healing of Chinese yam tubers via improving the gene expressions of relevant enzymes, their contents and activities, so that maintain high quality of postharvest yam tubers.

Red light induces salicylic acid accumulation by activating CaHY5 to enhance pepper resistance against Phytophthora capsici.

Pepper (Capsicum annuum L.) is frequently challenged by various pathogens, among which Phytophthora capsici is the most devastating to pepper production. Red light signal acts as a positive induction of plant resistance against multiple pathogens. However, little is known about how the red light signal affects pepper resistance to P. capsici infection (PCI). Here, we report that red light regulates salicylic acid (SA) accumulation by activating elongated hypocotyl5 (CaHY5), a basic leucine zipper (bZIP) transcription factor, thereby decreasing pepper susceptibility to PCI. Exogenous SA treatment reduced pepper susceptibility to PCI, while silencing of CaPHYB (a red light photoreceptor) increased its susceptibility. PCI significantly induced CaHY5 expression, and silencing of CaHY5 reduced SA accumulation, accompanied by decreases in the expression levels of phenylalanine ammonia-lyase 3 (CaPAL3), CaPAL7, pathogenesis-related 1 (CaPR1), and CaPR1L, which finally resulted in higher susceptibility of pepper to PCI. Moreover, CaHY5 was found to activate the expression of CaPAL3 and CaPAL7, which are essential for SA biosynthesis, by directly binding to their promoters. Further analysis revealed that exogenous SA treatment could restore the resistance of CaHY5-silenced pepper plants to PCI. Collectively, this study reveals a critical mechanism through which red light induces SA accumulation by regulating CaHY5-mediated CaPAL3 and CaPAL7 expression, leading to enhanced resistance to PCI. Moreover, red light-induced CaHY5 regulates pepper resistance to PCI, which may have implications for PCI control in protected vegetable production.

Exogenous methyl jasmonate combined with Ca2+ promote resveratrol biosynthesis and stabilize sprout growth for the production of resveratrol-rich peanut sprouts

Promoting resveratrol accumulation in plants and utilizing resveratrol-rich plants as raw materials for the development of functional foods is a promising development direction. The effects of methyl jasmonate (MeJA), in combination with CaCl2 and Ca2+ inhibitors, on physiological metabolism and resveratrol enrichment of peanut sprouts were investigated. MeJA combined with CaCl2 increased Ca2+ content, calmodulin content, and Ca2+- adenosine triphosphatase activity, as well as upregulated calcium-binding proteinase expression levels. Treatment with MeJA plus CaCl2 significantly increased peroxidase and superoxide dismutase activities and antioxidant capacities, significantly decreased the content of malondialdehyde and hydrogen peroxide, which resulted in a significantly increased in sprout length and fresh weight, and alleviated the inhibition of sprout growth. MeJA plus CaCl2 significantly increased the activities of phenylalanine ammonia-lyase and 4-coumarate coenzyme A ligase and upregulated the expression levels of phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, and resveratrol synthase, thus significantly increasing resveratrol content. However, MeJA combined with Ca2+ antagonists reversed these effects. These results indicate that MeJA interacts with Ca2+ to promote resveratrol synthesis in peanut sprouts and to improve sprout stress tolerances.

Adaptive response of flavonoids in Robinia pseudoacacia L. affected by the contamination of cadmium and elevated CO2 to arbuscular mycorrhizal symbiosis

As a key component in non-enzyme resistance system, flavonoids play a crucial role in the plant growth and defenses, which are significantly affected by biotic and abiotic factors such as fungi, bacteria, viruses, heavy metals, and atmospheric CO2. Arbuscular mycorrhizal fungi (AMF) play an important role in enhancing plant tolerance to adverse environments, which can significantly affect the synthesis of flavonoids by forming mycorrhizal symbionts with plant roots. However, few studies explored the combined effects of AMF, elevated CO2, and heavy metals on flavonoids in plants. Here, we investigated the adaptive response of flavonoids accumulation in Robinia pseudoacacia L. seedlings affected by the contamination of cadmium (Cd) and elevated CO2 to arbuscular mycorrhizal symbiosis. The results showed that G. mosseae decreased (p < 0.05) Cd content in leaves by 62.2% under elevated CO2. Moreover, G. mosseae colonization led to significant decreases in robinin, quercetin, kaempferol and acacetin by 17.4%, 11.1%, 15.5% and 23.1% under elevated CO2 + Cd, respectively. Additionally, G. mosseae down-regulated (p < 0.05) expression levels of phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) genes under elevated CO2 + Cd, and CHS and uridine diphosphate flavonoid glucosyltransferase (UFGT) activities decreased (p < 0.05). Quercetin, kaempferol and acacetin showed positive (p < 0.05) correlation with PAL and CHS genes expression and PAL, CHS, and UFGT activities. Cadmium, C/N ratio, carotenoids, leaf biomass, total chlorophyll, P, and starch in leaves and G. mosseae colonization rate in roots influenced (p < 0.05) flavonoids content. Overall, G. mosseae reduced flavonoids synthesis by down-regulating gene expression levels and activities of key enzymes under elevated CO2 + Cd. The results improved our understanding of the regulation of AMF on non-enzymatic resistance of plants grown in heavy metal-contaminated soils under increasing atmospheric CO2 scenarios.

Pre-harvest application of proline, methionine, and melatonin improves shelf-life and maintains nutritional quality of Brassica oleracea florets during cold storage

The reduction in shelf-life and nutritional value of cauliflower florets are the most vital problems during cold storage. This research was performed to explore the impact of the pre-harvest foliar implementation of proline (PR), methionine (MT), and melatonin (ML), at a rate of 25 mg.L−1, on shelf-life and active composites of cauliflower florets (cv. ‘Arasya’) stored at 10 °C for 14 d. The obtained results exhibited that florets from treated plants with PR, MT, and ML were lower in water loss, electrolyte leakage, browning index, and titratable acidity (TA) compared with untreated plants, at the end of storage. Furthermore, these pre-harvest treatments significantly slow down the loss of total sugar content, reducing sugar, total soluble solid (TSS), glucosinolates concentration (Gly), total phenols (TP), vitamin C (VC), and antioxidant activity (DPPH) than control samples. After 14 days of storage, the application of PR, MT, and ML significantly improved the activity levels of phenylalanine ammonia-lyase (PAL), and catalase (CAT) as well as declined the polyphenol oxidase (PPO), peroxidase (POD) activates in the florets during the cold storage. Therefore, the pre-harvest application of PR, MT, and ML might be promising substances to keep the nutritional quality of cauliflower forests and reduce the browning index during cold storage.

Effects of Exogenous Nitric Oxide Treatment on Grape Berries Against Botrytis cinerea and Alternaria alternata Related Enzymes and Metabolites.

Postharvest losses of grape berries caused by the pathogenic fungi Botrytis cinerea and Alternaria alternata have been widely reported, and nitric oxide (NO) as a plant signaling molecule to control postharvest diseases has recently become an active research topic. This study aimed to investigate the regulatory effect of NO on the interaction between grape berries and fungi. During interactions between grape berries and pathogenic fungi, treatment with 10 mM sodium nitroprusside (SNP, an NO donor) delayed the decline of the physiological quality of the grape berries and had positive effects on the weight loss rate, firmness, and respiration intensity. SNP treatment increased the activities of superoxide dismutase (SOD) and polyphenol oxidase (PPO) and inhibited the activities of peroxidase (POD) and catalase (CAT) of grape berries during the resistance to fungal pathogen infection. In addition, the increase in browning degree and the accumulation of hydrogen peroxide were inhibited by SNP treatment. In the phenylpropane metabolic pathway, the activities of phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumaric acid coenzyme A ligase (4CL) were increased during the activation of grape berries during the resistance to pathogen infection by SNP, and the intermediate metabolites lignin, flavonoids, and total phenols were accumulated. In addition, SNP treatment had a regulatory effect on the gene expression levels of SOD, POD, PPO, PAL, and 4CL. These results suggested that SNP treatment was effective for the preservation and disease reduction of grape berries.

Oligoalginates from Padina pavonica obtained by chemical and mechanical fractionation differentially induce resistance of date palm vitroplants to biotic and abiotic stresses

Alginate and/or oligoalginates are known as plant defense stimulators that become a novel alternative to agrochemicals. The elicitor effects of alginate and oligoalginates derived from the brown algae Padina pavoniva on the date palm vitroplants was tested. Firstly, alginate (SAPP) was extracted and the structure was characterized by ATR-FTIR, GC/MS and 1H/13C NMR. The M/G ratio was 0.8 indicating that G units were abundant in the alginate polymer. Secondly, alginates depolymerization was performed using ball milling as mechanical depolymerization (OABM) and acid hydrolysis as chemical fractionation (G + M, G and M). The expression level of four plant defense marker genes PAL, SOD, PR1 and DHN in date palm vitroplants after 48 h treatments with alginate (SAPP), dried algae powder treated by ball milling (PPBM), and oligoalginates were investigated. The four genes were up-regulated differentially, according to the elicitor treatment. The highest transcription levels of PAL (phenylalanine ammonia-lyase), PR1 (pathogenesis-related protein 1), SOD (superoxide dismutase) and DHN (dehydrin) (~11, 7, 5 and 4-fold respectively more than obtained in control vitroplants) were observed in the vitroplants treated with OABM followed by oligoalginates obtained by chemical hydrolysis. The lowest transcription levels were observed in both SAPP and PPPM treated plants. OABM treated vitroplants showed increased resistance to salt, drought and fungal contamination. These findings open up promising prospects for a new depolymerization method of alginate as a more efficient tool to induce a defense response in plants against abiotic and biotic stresses.

In vitro application of proline in potato tubers under newly emerging bacteria Lelliottia amnigena infection

Biotic stress deleteriously affects growth, development, and productivity in plants. Proline (Pro) plays a significant role in enhancing plant resistance to pathogen infection. However, its effects on reducing Lelliottia amnigena-induced oxidative stress in potato tubers remain unknown. The present study aims to evaluate the in vitro Pro treatment in potato tubers exposed to a newly emerging bacterium, L. amnigena. Sterilized healthy potato tubers were inoculated with 0.3 mL of L. amnigena suspension (3.69 × 107 CFU mL−1) 24 h before Pro (5.0 mM) application. The L. amnigena treatment significantly increased the contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2) in the potato tubers by 80.6 and 85.6%, respectively, compared to the control. Application of proline (Pro) decreased MDA and H2O2 contents by 53.6 and 55.9%, respectively, compared to the control. Application of Pro to L. amnigena-stressed potato tubers increased the activities of NADPH oxidase (NOX), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), 4-coumaryl-CoA ligase (4CL) and cinnamate-4-hydroxylase (C4H) C4H by 94.2, 96.3, 97.3, 97.1, 96.6, 79.3, 96.4, 93.6, and 96.2%, respectively, compared to the control. In comparison to the control, the genes PAL, SOD, CAT, POD, and NOX were significantly increased in the Pro-treated tubers at 5.0 mM concentration. Tubers treated with Pro + L. amnigena increased the transcript levels of PAL, SOD, CAT, POD, and NOX by 2.3, 2.2, 2.3, 2.5, and 2.8-fold respectively, compared to the control. Our findings suggested that pretreatment of tubers with Pro might reduce lipid peroxidation and oxidative stress by enhancing enzymatic antioxidant activity and gene expression.

GmWAK1, Novel Wall-Associated Protein Kinase, Positively Regulates Response of Soybean to Phytophthora sojae Infection

Phytophthora root rot is a destructive soybean disease worldwide, which is caused by the oomycete pathogen Phytophthora sojae (P. sojae). Wall-associated protein kinase (WAK) genes, a family of the receptor-like protein kinase (RLK) genes, play important roles in the plant signaling pathways that regulate stress responses and pathogen resistance. In our study, we found a putative Glycine max wall-associated protein kinase, GmWAK1, which we identified by soybean GmLHP1 RNA-sequencing. The expression of GmWAK1 was significantly increased by P. sojae and salicylic acid (SA). Overexpression of GmWAK1 in soybean significantly improved resistance to P. sojae, and the levels of phenylalanine ammonia-lyase (PAL), SA, and SA-biosynthesis-related genes were markedly higher than in the wild-type (WT) soybean. The activities of enzymatic superoxide dismutase (SOD) and peroxidase (POD) antioxidants in GmWAK1-overexpressing (OE) plants were significantly higher than those in in WT plants treated with P. sojae; reactive oxygen species (ROS) and hydrogen peroxide (H2O2) accumulation was considerably lower in GmWAK1-OE after P. sojae infection. GmWAK1 interacted with annexin-like protein RJ, GmANNRJ4, which improved resistance to P. sojae and increased intracellular free-calcium accumulation. In GmANNRJ4-OE transgenic soybean, the calmodulin-dependent kinase gene GmMPK6 and several pathogenesis-related (PR) genes were constitutively activated. Collectively, these results indicated that GmWAK1 interacts with GmANNRJ4, and GmWAK1 plays a positive role in soybean resistance to P. sojae via a process that might be dependent on SA and involved in alleviating damage caused by oxidative stress.

Salicylic acid biosynthesis is not from phenylalanine in Arabidopsis.

The phytohormone salicylic acid (SA) regulates biotic and abiotic stress responses in plants. Two distinct biosynthetic pathways for SA have been well documented in plants: the isochorismate (IC) pathway in the chloroplast and the phenylalanine ammonia-lyase (PAL) pathway in the cytosol. However, there has been no solid evidence that the PAL pathway contributes to SA biosynthesis. Here, we report that feeding Arabidopsis thaliana with Ring-13 C-labeled phenylalanine (13 C6 -Phe) resulted in incorporation of the 13 C label not into SA, but into its isomer 4-hydroxybenzoic acid (4-HBA) instead. We obtained similar results when feeding 13 C6 -Phe to the SA-deficient ics1 ics2 mutant and the SA-hyperaccumulating mutant s3h s5h. Notably, we detected 13 C6 -SA when 13 C6 -benzoic acid (BA) was provided, suggesting that SA can be synthesized from BA. Furthermore, despite the substantial accumulation of SA upon pathogen infection, we did not observe incorporation of 13 C label from Phe into SA. We also did not detect 13 C6 -SA in PAL-overexpressing lines in the kfb01 kfb02 kfb39 kfb50 background after being fed 13 C6 -Phe, although endogenous PAL levels were dramatically increased. Based on these combined results, we propose that SA biosynthesis is not from Phe in Arabidopsis. These results have important implications for our understanding of the SA biosynthetic pathway in land plants.

The long non-coding RNA GhlncRNA149.1 improves cotton defense response to aphid damage as a positive regulator

Long non-coding RNAs (lncRNAs) play crucial roles in various plant biological regulatory processes and defense responses to all sorts of biotic and abiotic stresses. However, there are limited studies on the function and mechanism of lncRNAs in plant defense responses to phytophagous insects. In this research, a lncRNA GhlncRNA149.1 was identified from upland cotton (Gossypium hirsutum L.). Depending on the position relationship between lncRNA and mRNA, we identified CC-NBS-LRR family gene GhA01G0129 as the target gene of GhlncRNA149.1. Expression levels of GhlncRNA149.1 and its target gene GhA01G0129 were significantly down-regulated in cotton cotyledons upon cotton aphid infestation. The expression level of GhlncRNA149.1 was significantly down-regulated after the induction of methyl jasmonate and salicylic acid, whereas expression level of its target gene GhA01G0129 was significantly up-regulated. Overexpression of GhlncRNA149.1 in cotton cotyledons enhanced the tolerance of cotton plants to cotton aphid feeding. Expectedly, silencing GhlncRNA149.1 increased susceptibility of cotton plants to cotton aphids. The expression level of GhA01G0129, the target gene of GhlncRNA149.1, was significantly up-regulated in cotton plants overexpressing GhlncRNA149.1, and significantly down-regulated in cotton plants silencing GhlncRNA149.1. The activity and expression levels of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were up-regulated in cotton plants transiently overexpressing GhlncRNA149.1, while opposite results were obtained in cotton plants silencing GhlncRNA149.1. In addition, similar results were obtained in the expression levels of phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), enhanced disease susceptibility 1 (EDS1) and non-expression of pathogenesis-related gene 1 (NPR1) in cotton plants transiently overexpressing GhlncRNA149.1 and silencing GhlncRNA149.1. These results collectively suggest that GhlncRNA149.1 improve the plant defense response to aphid attack, and thus has potential for enforcing cotton aphid prevention and control.

Arbuscular mycorrhizal fungi induce flavonoid synthesis for mitigating oxidative damage of trifoliate orange under water stress

Arbuscular mycorrhizal (AM) fungi can increase the tolerance of host plants to drought, whereas the intrinsic mechanisms are not still fully elucidated. Flavonoids are a kind of antioxidants to scavenge reactive oxygen species (ROS), whereas whether and how flavonoids are involved in increased drought tolerance of host plants by AM fungi is not known. Here, Funneliformis mosseae , was inoculated into potted trifoliate orange seedlings, which were subjected to adequate water and water stress conditions. The AM effect on plant growth, concentrations of flavonoid substances and total flavonoids, and activities and expression levels of enzymes related to flavonoids synthesis (clnnamate 4-dydroxylate (C4H), phenylalanine ammonialyase (PAL), chalcone isomerase (CHI), and 4-coumaroyl-CoA ligase (4CL)) in roots was analyzed, along with changes in scavenging activity of ROS by root flavonoid extracts. Compared with non-AM control, F . mosseae inoculation distinctly improved plant growth performance and increased phenylalanine, glycitin, luteolin-7- O -glucoside, and total flavonoid concentrations, coupled with the decrease in p -coumaric acid, regardless of soil water regimes. Activities of C4H, CHI, 4CL, and PAL and expression levels of PtPAL1 and Pt4CL were induced by F . mosseae inoculation under water stress. AM plants exhibited higher scavenging activity of hydroxyl radical and superoxide (O 2 •− ) by root flavonoid extracts under water stress, along with lower levels of O 2 •− and hydrogen peroxide and the degree of membrance lipid peroxidation, compared with non-AM plants. It is concluded that AM fungi accelerated flavonoids synthesis of trifoliate orange for mitigating oxidative damage under water stress. • AMF increased phenylalanine, glycitin, luteolin-7- O -glucoside, and total flavonoid concentrations, regardless of soil moisture regimes. • C4H, CHI, 4CL, and PAL activities and PtPAL1 and Pt4CL expressions were induced by AMF under water stress. • AMF plants exhibited higher scavenging activity of ROS by root flavonoid extracts under water stress. • AM fungi accelerated flavonoids synthesis for mitigating oxidative damage under water stress.

Identification of vanillic acid and its new amide derivative from Hyoscyamus niger and their modes of action in controlling tobacco mosaic virus

Plant viruses represented by tobacco mosaic virus (TMV) are hard to control due to the characteristics of wide hosts and difficulties to be inactivated in vitro . The discovery of candidates to damage the virus directly, and elicitors to induce plant resistances are important strategies against TMV. This study aims to identify new candidates from the title plants as biopesticides in controlling TMV. Herein, vanillic acid ( 6 ) and its new amide derivative, nigeroate ( 1 ), and seven known ones were discovered from the seeds of Hyoscyamus niger L. Compound 1 , elucidated by spectral methods, was synthesized by two steps for industrial application in agriculture as a new elicitor. Compared with ningnanmycin, compound 1 appeared higher curative efficacies against TMV with the inhibitory rates of 58.00% and 69.14% at 250 and 500 μg/mL, respectively, while compound 6 displayed higher inactivation and protective efficacies with the inhibitory rates of 47.30–79.71% and 43.28–63.87% at 125–500 μg/mL, respectively. Compound 6 could destroy TMV particles into small fragments dramatically by decreasing the transcription level of TMV-CP gene, while compound 1 just broke the TMV particles slightly, and was no influence on TMV-CP gene. Interestingly, compound 1 induced potent hypersensitive response (HR), and systemic acquired resistance (SAR) activities (53.82% at 500 μg/mL) in tobaccos. The accumulation of H 2 O 2 by activating SOD (superoxide dismutase), CAT (catalase), as well as POD (peroxidase) was the mechanism of compound 1 induced HR. The SAR induced by compound 1 was referred to upregulate SA (salicylic acid) by activating PAL (phenylalanine ammonia-lyase) pathway, ICS (isochorismate synthase) pathway, as well as PR (pathogenesis-related) proteins due to the increasement of PAL enzyme, and PAL , ICS1 , NPR1 , PR1 , and PR2 transcription levels. Thus, vanillic acid ( 6 ) and its new amide derivative ( 1 ) could be developed as antiviral agents and plant immune elicitors for biopesticides in controlling TMV, respectively. • Vanillic acid and its new amide derivative, nigeroate, were identified from Hyoscyamus niger . • Tobacco mosaic virus coat protein is the target for vanillic acid exerting antiviral activity. • Nigeroate was synthesized by two steps for industrial application as an elicitor. • Accumulation of H 2 O 2 and salicylic acid contributed to nigeroate’s systemic resistance. • Defense enzymes, pathogenesis-related, and salicylic acid biosynthesis-related genes manipulated nigeroate’s resistance.

Identification of Key Genes during Ethylene-Induced Adventitious Root Development in Cucumber (Cucumis sativus L.).

Ethylene (ETH), as a key plant hormone, plays critical roles in various processes of plant growth and development. ETH has been reported to induce adventitious rooting. Moreover, our previous studies have shown that exogenous ETH may induce plant adventitious root development in cucumber (Cucumis sativus L.). However, the key genes involved in this process are still unclear. To explore the key genes in ETH-induced adventitious root development, we employed a transcriptome technique and revealed 1415 differentially expressed genes (DEGs), with 687 DEGs up-regulated and 728 DEGs down-regulated. Using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, we further identified critical pathways that were involved in ETH-induced adventitious root development, including carbon metabolism (starch and sucrose metabolism, glycolysis/gluconeogenesis, citrate cycle (TCA cycle), oxidative phosphorylation, fatty acid biosynthesis, and fatty acid degradation), secondary metabolism (phenylalanine metabolism and flavonoid biosynthesis) and plant hormone signal transduction. In carbon metabolism, ETH reduced the content of sucrose, glucose, starch, the activity of sucrose synthase (SS), sucrose-phosphate synthase (SPS) and hexokinase (HK), and the expressions of CsHK2, pyruvate kinase2 (CsPK2), and CsCYP86A1, whereas it enhanced the expressions of β-amylase 1 (CsBAM1) and β-amylase 3 (CsBAM3). In secondary metabolism, the transcript levels of phenylalanine ammonia-lyase (CsPAL) and flavonoid 3'-monooxygenase (CsF3'M) were negatively regulated, and that of primary-amine oxidase (CsPAO) was positively regulated by ETH. Additionally, the indole-3-acetic acid (IAA) content and the expressions of auxin and ETH signaling transduction-related genes (auxin transporter-like protein 5 (CsLAX5), CsGH3.17, CsSUAR50, and CsERS) were suppressed, whereas the abscisic acid (ABA) content and the expressions of ABA and BR signaling transduction-related genes (CsPYL1, CsPYL5, CsPYL8, BRI1-associated kinase 1 (CsBAK1), and CsXTH3) were promoted by ETH. Furthermore, the mRNA levels of these genes were confirmed by real-time PCR (RT-qPCR). These results indicate that genes related to carbon metabolism, secondary metabolite biosynthesis, and plant hormone signaling transduction are involved in ETH-induced adventitious root development. This work identified the key pathways and genes in ETH-induced adventitious rooting in cucumber, which may provide new insights into ETH-induced adventitious root development and will be useful for investigating the molecular roles of key genes in this process in further studies.