Inhibitor Of Phenylalanine Ammonia-lyase Research Articles

Overexpression of GmPAL Genes Enhances Soybean Resistance Against Heterodera glycines.

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

Enhancing Protoplast Isolation and Early Cell Division from Cannabis sativa Callus Cultures via Phenylpropanoid Inhibition.

De novo regeneration of Cannabis sativa L. (cannabis) using tissue culture techniques remains unreliable and infrequent. Conventional methods for the regeneration and transformation of cannabis have not achieved the reliability and replicability that need to be integrated into research and breeding programs. Protoplast systems are effective for gene expression studies and transformation and genome-editing technologies and open the possibility of somatic hybridization to create interspecific hybrids. To date, leaf-derived protoplasts have been isolated for transient gene expression studies, but protoplast-to-plant regeneration has not been reported. The present study aims to evaluate the efficacy of using a callus culture system as an abundant tissue source for protoplast isolation and lays the groundwork for a protoplast-to-plant regeneration system. Using hypocotyl-derived callus cultures, which are known to have relatively greater regenerative potential, the efficacy of protoplast isolation and initial cell division were assessed. In this study, the effect of 2-aminoindane-2-phosphonic acid (AIP), a competitive inhibitor of phenylalanine ammonia lyase (PAL), in callus culture media and the effect of subculture frequency on protoplast yield were assessed. This study found that inclusion of AIP at 1 mM resulted in a 334% increase in protoplast yield compared with AIP-free medium, representing the first known use of AIP in cannabis tissue culture. Inclusion of AIP led to a 28% decrease in total soluble phenolics and 52% decrease in tissue browning compared with the control medium. Lastly, a two-phase culture system for protoplast regeneration was tested. At a concentration of 2.0 × 105 protoplasts per mL, cell wall reconstitution and cell division were observed, providing one of the first know reports of cell division from cannabis protoplasts and setting the stage for the future development of a protoplast-to-plant regeneration system.

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

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

The use of L-phenylalanine ammonia lyase inhibitors in plant ecophysiological studies

Phenylalanine ammonia lyase (PAL) is a key enzyme controlling the biosynthesis of phenolic compounds in plants. PAL catalyzes ammonia elimination from L-phenylalanine in a reaction that yields cinnamic acid, a precursor of a large group of phenylpropanoid compounds. Phenylpropanoids and their derivatives play an important role in regulating plant resistance mechanisms under environmental stresses. By reducing the level of phenolic compounds, PAL inhibitors can induce changes in plant metabolism. This paper presents the current state of knowledge on the use of PAL inhibitors in plant biology, and draws attention to the possibilities of using PAL inhibitors in agriculture in the context of the witnessed climate changes which increase the frequency and intensity of some disasters such as droughts, floods and storms. By reducing the level of phenolic compounds, PAL inhibitors can induce changes in plant metabolism. This paper presents the current state of knowledge on the use of PAL inhibitors in plant biology, and draws attention to the possibilities of using PAL inhibitors in agriculture in the context of the witnessed climate changes.

Elucidation of lignin and polysaccharide linkages in wheat straw by 2H/13C isotopic tracer

To elucidate chemical linkages between lignin and polysaccharides, the aqueous mixed solutions of coniferin-[α-13C], syringin-[α-13C], D-glucose-[6-2H], and phenylalanine ammonia-lyase inhibitor were injected into a living wheat stalk. Internode tissues with high abundance of 2H-13C were collected. The milled wood lignin, lignin-carbohydrate complex (LCC), and residual LCC (R-LCC) with enrichment of 2H-13C were isolated. The 13C and 2H abundances showed that the lignin and polysaccharides of internode tissues were labeled by 13C and 2H, respectively. Analysis with carbon-13 nuclear magnetic resonance (13C-NMR) showed that ketal and benzyl ether bonds were formed between α-C of lignin and carbohydrates. The R-LCC and LCC were further treated with enzymes to obtain enzymatic degraded R-LCC (ED-R-LCC) and enzymatic degraded LCC (ED-LCC). 13C-NMR spectra of ED-LCC showed that the α-C of lignin side chain was combined with 6-C of carbohydrates by ether, ester, and ketal linkages. 1H-NMR differential spectra of ED-LCCs revealed an LC linkage of benzyl ether bond. Glucan-lignin (En-R-GL) and xylan-lignin (En-R-XL) complexes were separated from ED-R-LCC by ionic liquid. A part of lignin α-C was linked to cellulose 6-C by benzyl ether and α-ketal linkages. 13C-NMR spectra of En-R-XL showed there were α-benzyl ether and α-ketal bonds between lignin and xylan.

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.

Elucidation of the Structure of Lignin-Carbohydrate Complexes in Ginkgo CW-DHP by 13C-2H Dual Isotope Tracer.

To elucidate the chemical linkages between lignin and carbohydrates in ginkgo cell walls, 13C-2H-enriched cell wall-dehydrogenation polymers (CW-DHP) were selectively prepared with cambial tissue from Ginkgo biloba L. by feeding D-glucose-[6-2H2], coniferin-[α-13C], and phenylalanine ammonia-lyase (PAL) inhibitor. The abundant detection of 13C and 2H confirmed that D-glucose-[6-2H2] and coniferin-[α-13C] were involved in the normal metabolism of ginkgo cambial cells that had been effectively labelled with dual isotopes. In the ginkgo CW-DHP, ketal and ether linkages were formed between the C-α of lignin side chains and carbohydrates, as revealed by solid state CP/MAS 13C-NMR differential spectroscopy. Furthermore, the DMSO/TBAH ionic liquids system was used to fractionate the ball-milled CW-DHP into three lignin-carbohydrate complex (LCC) fractions: glucan–lignin complex (GL), glucomannan–lignin complex (GML), and xylan–lignin complex (XL). The XRD determination indicated that the cellulose type I of the GL was converted into cellulose type II during the separation process. The molecular weight was in the order of Ac-GL > Ac-GML > XL. The 13C-NMR and 1H-NMR differential spectroscopy of 13C-2H-enriched GL fraction indicated that lignin was linked with cellulose C-6 by benzyl ether linkages. It was also found that there were benzyl ether linkages between the lignin side chain C-α and glucomannan C-6 in the 13C-2H-enriched GML fraction. The formation of ketal linkages between the C-α of lignin and xylan was confirmed in the 13C-2H-enriched XL fraction.

Assessment of the Role of PAL in Lignin Accumulation in Wheat (Tríticum aestívum L.) at the Early Stage of Ontogenesis.

The current study evaluates the role of phenylalanine ammonia-lyase (PAL) and the associated metabolic complex in the accumulation of lignin in common wheat plants (Tríticum aestívum L.) at the early stages of ontogenesis. The data analysis was performed using plant samples that had reached Phases 4 and 5 on the Feekes scale—these phases are characterized by a transition to the formation of axial (stem) structures in cereal plants. We have shown that the substrate stimulation of PAL with key substrates, such as L-phenylalanine and L-tyrosine, leads to a significant increase in lignin by an average of 20% in experimental plants compared to control plants. In addition, the presence of these compounds in the nutrient medium led to an increase in the number of gene transcripts associated with lignin synthesis (PAL6, C4H1, 4CL1, C3H1). Inhibition was the main tool of the study. Potential competitive inhibitors of PAL were used: the optical isomer of L-phenylalanine—D-phenylalanine—and the hydroxylamine equivalent of phenylalanine—O-Benzylhydroxylamine. As a result, plants incubated on a medium supplemented with O-Benzylhydroxylamine were characterized by reduced PAL activity (almost one third). The lignin content of the cell wall in plants treated with O-Benzylhydroxylamine was almost halved. In contrast, D-phenylalanine did not lead to significant changes in the lignin-associated metabolic complex, and its effect was similar to that of specific substrates.

Induced Resistance by Ascorbate Oxidation Involves Potentiating of the Phenylpropanoid Pathway and Improved Rice Tolerance to Parasitic Nematodes.

Anticipating an increased ecological awareness, scientists have been exploring new strategies to reduce the use of chemical pesticides to control pests and diseases. Triggering the intrinsic plant defense system is one of the promising strategies to reduce yield loss by pathogenic organisms, such as nematodes. Ascorbate oxidase (AO) enzyme plays an important role in plant defense by regulating the apoplastic ascorbate/dehydroascorbate (DHA) ratio via the ascorbate oxidation process. Ascorbate oxidation is known to induce systemic resistance in rice against parasitic root-knot nematodes (RKN). Here, we sought to evaluate if AO- or DHA-induced resistance (IR) against RKN M. graminicola involves activation of the phenylpropanoid pathway and whether this IR phenotype has potential effects on growth of rice seedlings under stressed and unstressed conditions. Our results show that AO/DHA-IR against these parasitic nematodes is dependent on activation of phenylalanine ammonia lyase (PAL). However, application of reduced ascorbic acid (AA) did not induce this response. Gene expression analysis via qRT-PCR showed that OsPAL2 and OsPAL4 are highly expressed in AO/DHA-sprayed nematode-infected roots and PAL-activity measurements confirmed that AO/DHA spraying triggers the plants for primed activation of this enzyme upon nematode infection. AO/DHA-IR is not effective in plants sprayed with a chemical PAL inhibitor confirming that AO/DHA-induced resistance is dependent on PAL activity. Improved plant growth and low nematode infection in AO/DHA-sprayed plants was found to be correlated with an increase in shoot chlorophyll fluorescence (Fv/Fm), chlorophyll index (ChlIdx), and modified anthocyanin reflection index which were proven to be good above-ground parameters for nematode infestation. A detailed growth analysis confirmed the improved growth of AO/DHA-treated plants under nematode-infected conditions. Taken together, our results indicate that ascorbate oxidation enhances the phenylpropanoid-based response to nematode infection and leads to a tolerance phenotype in treated rice plants.

Uptake and phytotoxicity of lead are affected by nitrate nutrition and phenolic metabolism

Complex environmental interactions may modify toxicity of metals. We studied responses of the common medicinal plant chamomile to lead (Pb) toxicity under nitrate sufficient (+N) or deficient (–N) conditions involving also inhibitor of the key phenolic enzyme phenylalanine ammonia-lyase (2-aminoindane-2-phosphonic acid, AIP). Data indicate that N deficiency depleted nitrogenous but elevated phenolic metabolites or peroxidase activity and enhanced ROS (but not nitric oxide) formation evoked by Pb or AIP. Pb stimulated individual phenolic acids in the shoots but had no impact on total soluble phenols or flavonols where the impacts of AIP and N nutrition were more evident. Pb rather affected glutathione while N deficiency ascorbic and malic acids and the involvement of AIP in these changes was visible. PCA analyses showed clear separation with respect to N deficiency or AIP application. Besides, application of AIP stimulated accumulation of Pb in shoots and in absorbed root fraction while N deficiency suppressed Pb amount in shoots and stimulated in roots. Our data, to our knowledge for the first time, confirm that uptake of metals is not only a simple function of its presence but that mineral nutrition and state of the secondary metabolism have crucial impact on resulting responses.

Enhancement of polyphenolic metabolism as an adaptive response of lettuce (Lactuca sativa) roots to aluminum stress

Polyphenols, pivotal secondary metabolites, are involved in plant adaption to abiotic stresses. Here, we investigated the role and metabolism profile of polyphenols under aluminum (Al) stress in different lettuce genotypes grown in 0.5 mM CaCl2 solution with AlCl3 (pH = 4.5). The complementary use of high-resolution mass spectrometry and quantitative biochemical approaches allowed the characterization of total and unique phenols, as well as their roles in Al tolerance. By comparing the most tolerant and sensitive genotype, 8 polyphenols, including 4 phenolic acids, 2 flavonoids, 1 xanthone and 1 unknown compound, were identified in the roots of the tolerant genotype. The total phenolic and flavonoid contents significantly increased in the tolerant genotype under Al stress. Seedlings with more phenolic accumulation usually performed greater Al tolerance. Meanwhile, principal enzymes related to phenolic biosynthesis significantly increased in roots of the tolerance genotype after Al treatment, with phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase, and 4-coumarate coenzyme A ligase increased by 16, 18 and 30%, respectively. The elevated total phenolics were significantly suppressed by AIP, a highly specific PAL inhibitor. Consequently, the antioxidant capacity was inhibited, leading to lettuce sensitivity to Al stress. These results clearly suggested the enhancement of unique polyphenolic biosynthesis as an adaptive strategy of lettuce to Al stress by protecting plants from oxidative stress.

Improving callus regeneration of Miscanthus × giganteus J.M.Greef, Deuter ex Hodk., Renvoize ‘M161’ callus by inhibition of the phenylpropanoid biosynthetic pathway

In previous studies, the regeneration rates of Miscanthus × giganteus J.M.Greef, Deuter ex Hodk., Renvoize from callus tissue cultured on semi-solid media significantly declined after 4 mo of culture, which presents problems with germplasm conservation and use as an alternative propagation system. Due to the species’ lignocellulosic nature, it was hypothesized that the accumulation of phenolic compounds in the callus may be responsible for inhibiting regeneration. The current study aimed to optimize regeneration of M. × giganteus callus by culturing the callus tissue in the presence of 2-aminoindan-2-phosphonic acid (AIP), a competitive inhibitor of phenylalanine ammonia lyase (PAL), to reduce the biosynthesis of phenolics. Embryogenic callus was cultured on media supplemented with 9.0- or 11.3-μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0-, 1-, 10-, 100-, or 1000-μM AIP. Every 28 d for 7 mo, the callus tissue was visually classified based on morphology and regeneration rate. Over the duration of the study, regeneration of shoots was consistently highest in callus cultured on 11.3-μM 2,4-D supplemented with 10- and 100-μM AIP (13–58.3%), and in vitro plantlet development from callus cultured on all concentrations of AIP demonstrated tillering and rooting. Total soluble phenolic content of the callus decreased in a dose-dependent manner from 2242.34-μg g−1 dry weight in the control to 1569.71-μg g−1 dry weight in AIP-treated callus. These data indicate that inhibiting PAL in M. × giganteus cultures increased the percentage of calluses exhibiting regeneration over time.

UV-B shock induces photoprotective flavonoids but not antioxidant activity in Antarctic Colobanthus quitensis (Kunth) Bartl

Colobanthus quitensis, the only dicot that inhabits Antarctica, is subjected to different abiotic factors such as high UV-B levels in spring-summer. Several authors described the flavonoid biosynthesis as crucial to tolerating UV-B enriched environments. The goal of this work is the study of the relationship between biosynthesis of flavonoids, resistance to high UV-B levels and the maintenance of redox homeostasis. With this objective, C. quitensis plantlets were subjected to low, medium and high levels of UV-B radiation, in the presence and absence of aminooxiacetic acid (AOA), a phenylalanine ammonia-lyase (PAL) inhibitor with the aim to determine the role of flavonoids in the capacity of C. quitensis to survive to UV-B. The results showed that plants subjected to increasing levels of UV-B, in the absence of the inhibitor, did not present oxidative damage; on the other hand, plants that were treated by the inhibitor showed oxidative stress symptoms and photosynthetical damage. Oxidative damage was correlated with flavonoids content; at the molecular level, these results showed an increase in transcript levels related to the phenylpropanoid pathway enzymes and their activity, as well as the expression of the UVR8−COP1-HY5 transduction system. Moreover, in AOA treated plants, the results show significant damage and plant death. Our results suggest that the survival mechanism showed by C. quitensis to UV-B radiation is mediated by biosynthesis of flavonoids.

Influence of media supplements on inhibition of oxidative browning and bacterial endophytes of Camellia sinensis var. sinensis.

Explant oxidative browning and necrosis of Camellia sinensis var. sinensis is a severe problem in tissue culture, often associated with the exuded phenolic compounds and microbial contamination from the explants. In this study, 2-aminoindane-2-phosphonic acid (AIP), an inhibitor of the polyphenol production-required enzyme phenylalanine ammonia lyase (PAL), and different antibiotics were tested to control tea explant necrosis and browning. These compounds were supplemented in the regular plant growth medium together with 6-benzylaminopurine and thidiazuron at different concentrations. Our data indicated that application of 2µM of AIP was able to effectively inhibit callus browning, significantly reduce EGC abundance, and greatly improve callus induction and growth. Moreover, the use of 150mg/L of timentin and 30mg/L gentamycin resulted in an effective elimination of the surface and endophytic microbes associated with explants of C. sinensis var. sinensis. Our study revealed that the inhibition of PAL using AIP combined with the two tested antibiotics could open up new doors to control oxidative tissue browning and endophyte contamination in tissue culture for tea genetic manipulation.

Substituted phosphonic analogues of phenylglycine as inhibitors of phenylalanine ammonia lyase from potatoes

A series of phosphonic acid analogues of phenylglycine variously substituted in phenyl ring have been synthesized and evaluated for their inhibitory activity towards potato l-phenylalanine ammonia lyase. Most of the compounds appeared to act as moderate (micromolar) inhibitors of the enzyme. Analysis of their binding performed using molecular modeling have shown that they might be bound either in active site of the enzyme or in the non-physiologic site. The latter one is located in adjoining deep site nearby the to the entrance channel for substrate into active site.

Modulating role of ROS in re-establishing desiccation tolerance in germinating seeds of Caragana korshinskii Kom.

In close agreement with visible germination, orthodox seeds lose desiccation tolerance (DT). This trait can be regained under osmotic stress, but the mechanisms are poorly understood. In this study, germinating seeds of Caragana korshinskii Kom. were investigated, focusing on the potential modulating roles of reactive oxygen species (ROS) in the re-establishment of DT. Germinating seeds with 2 mm long radicles can be rendered tolerant to desiccation by incubation in a polyethylene glycol (PEG) solution (-1.7 MPa). Upon PEG incubation, ROS accumulation was detected in the radicles tip by nitroblue tetrazolium chloride staining and further confirmed by confocal microscopy. The PEG-induced re-establishment of DT was repressed when ROS scavengers were added to the PEG solution. Moreover, ROS act downstream of abscisic acid (ABA) to modulate PEG-mediated re-establishment of DT and serve as a new inducer to re-establish DT. Transcriptomic analysis revealed that re-establishment of DT by ROS involves the up-regulation of key genes in the phenylpropanoid-flavonoid pathway, and total flavonoid content and key enzyme activity increased after ROS treatment. Furthermore, DT was repressed by an inhibitor of phenylalanine ammonia lyase. Our data suggest that ROS play a key role in the re-establishment of DT by regulating stress-related genes and the phenylpropanoid-flavonoid pathway.

1-Aminobenzocyclobutene-1-phosphonic Acid and Related Compounds as Inhibitors of Phenylalanine Ammonia-Lyase.

Five new geminal aminocycloalkanephosphonic acids (4 - 8) containing both an aromatic ring and a cycloalkane ring were synthesized and evaluated as potential inhibitors of buckwheat phenylalanine ammonia-lyase (PAL). Within the set of compounds which are related to 2-aminoindane-2-phosphonic acid (AIP, 3), a known powerful inhibitor of PAL, racemic 1-aminobenzocyclobutene-1-phosphonic acid (4), was six times weaker than AIP as an in vitro inhibitor of buckwheat PAL, but six times stronger than AIP as an in vivo inhibitor of phenylalanine-derived anthocyanin synthesis in buckwheat.

Secondary metabolic responses to treatment of 2-aminoindan-2-phosphonic acid in cell lines from Arnebia euchroma

The accumulation of rosmarinic acid, acetylshikonin, deoxyshikonin, β, β'-dimethylacrylshikonin and isovalerylshikonin was investigated in cell suspension cultures of Arnebia euchroma (Royle) Johnst under the influence of 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia lyase (PAL), and of the effector methyl jasmonate (MeJA). The results showed that methyl jasmonate promoted the accumulation of rosmarinic acid and shikonin derivatives. Conversely, 2-aminoindan-2-phosphonic acid suppressed the formation of rosmarinic acid, which indicated that AIP, indeed, was able to inhibit the phenylpropanoid pathway in A. euchroma. Meanwhile, the content of total shinkonins and other four kinds of shikonin derivatives, though varied in degrees, was also inhibited. And the inhibition was dose-dependent and time-dependent. Acetylshikonin responsed most rapidly to the treatment of AIP, the content reduced after 24 h of treatment and decreased to only half of those untreated control 48 h after teratment. β, β'-Dimethylacrylshikonin, difffer from acetylshikonin, responded much slowly to the treatment, inhibition could only be observed 96 h later. These suggest that phenylpropanoid pathway plays an important role in the shikoninsbiosynthesis, and this study provides a reference for the further research in metabolic regulation of producing shikonins by cell culture technology and biosynthesis pathways of shikonin derivatives. Still, shikonins biosynthesis pathways is complicated, the exact dose- and time-effect relationship of AIP and interaction between AIP and other effectors like MeJA need further research.

Methyl jasmonate and α-aminooxi-β-phenyl propionic acid alter phenylalanine ammonia-lyase enzymatic activity to affect the longevity and floral scent of cut tuberose

Longevity is a determining factor of the marketability of cut flowers, and is therefore a key target in commercial floriculture. Floral scent is another important factor in the popularity of cut flowers. The phenylpropanoid pathway is one of the key pathways in floral scent production. The first step of this pathway is catalyzed by the enzyme phenylalanine ammonia-lyase (PAL). Here, we evaluated the pre-harvest foliar application of a PAL promoter (methyl jasmonate [MeJA]) and a PAL inhibitor (α-aminooxi-β-phenyl propionic acid [AOPP]) on the physiological parameters, floral scent, and longevity of cut tuberose flowers (Polianthes tuberosa L.). Relative water content (RWC) was significantly affected by foliar application of treatments. The highest and lowest RWCs were observed in plants treated with AOPP (3 mM) and the control, respectively. Catalase (CAT) enzyme activity was not significantly affected by treatments, while peroxidase (POD) activity was significantly affected. Foliar application significantly affected stem water uptake and the 3 mM AOPP resulted in the highest stem water uptake. A GC-MS analysis of floral scent volatiles detected 37 different compounds, with the most abundant being methyl benzoate, benzyl benzoate, pentacosane, and methyl salicylate. The pre-harvest foliar application of MeJA increased the total scent of tuberose, as well as increasing the relative abundances of methyl benzoate and methyl salicylate and reducing the benzyl benzoate and pentacosane content. In contrast, the foliar application of AOPP decreased the total floral scent; while the relative amount of pentacosane increased, the methyl benzoate and methyl salicylate contents decreased. Both treatments extended the longevity of the tuberose flowers; however, the flowers of the AOPP-treated plants lasted longer than those treated with MeJA which could be due the different effects of these treatments on RWC, antioxidant enzymes activity, and/or stem water uptake. These results indicate that PAL plays an important role in the generation of floral scent and the extension of longevity in cut tuberose.

In vitro plant regeneration and alleviation of physiological disorders in Scadoxus puniceus

The frequent overharvesting of slow-growing bulbs has led to a severe reduction in wild plant populations. In vitro propagation techniques have been useful in reducing the stress placed on wild plant populations. The techniques involved in in vitro propagation of the Amaryllidaceae are often challenging. The current study evaluated the role of phloroglucinol (PG), gallic acid (GCA; an inhibitor of phenylalanine ammonia lyase) and photoperiod on the development of bulblets, reduction of phenolic exudation and hyperhydricity in Scadoxus puniceus grown in vitro. The combination of PG and naphthaleneacetic acid (NAA) significantly increased the number of bulblets that developed on leaf explants. GCA (1, 5 and 10μM) and 5μM benzyladenine (BA) significantly improved the number of bulblets produced on leaf explants and plant development. The longest shoots (66.0±9.03mm) were produced at 10μM GCA. In addition, red pigmentation characteristic of phenolic exudation in the media were reduced with treatments of PG and GCA as well as a 24h dark photoperiod. Hyperhydricity was alleviated with the application of meta-topolins. The shoots of regenerated bulb halves developed to a length of 180mm under a 24h dark photoperiod. The current study is the first to describe an in vitro propagation protocol for S. puniceus by limiting the effect of phenolic exudation thus providing a better culture condition and it provides a better conservation strategy for this over-utilised species.