Inhibitor Of Phenylalanine Ammonia-lyase Research Articles

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.

Activities of some defense proteins associated with age or plant family in larch needles of Larix olgensis and L. kaempferi × L. gmelinii

Defence proteins are a kind of chemical defence compounds. They play a key role in plant restraining biotic and abiotic harm. To illuminate activities of some defense proteins associated with age or plant family of larch, the larch needles were collected from two different families of Korean larch, Larix olgensis and a hybrid larch, L. kaempferi × L. gmelinii, respectively, and then the activities of defence proteins in those were tested using a UV spectrophotometry. The results showed that the activities of protective enzymes at the16-/17- and 19-year-age groups were higher than those at the other age groups in the both larch species. While the activities of polyphenol oxidase (PPO) at the 16-/17-year-age group and phenylalanine ammonia-lyase (PAL) and protease inhibitors at the 19-year-age group were the highest among all age groups. Then, compared with L. olgensis, the hybrid larch had significant effects on the activities of protective enzymes. The effects of plant family on the activities of PAL and chymotrypsin inhibitor were significantly different, and then those on the activities of PPO were not significantly different. The effects of the two families in L. olgensis on the activities of trypsin inhibitor (TI) were significantly different, while those in the hybrid larch on the activities of TI were contrary. To conclusions showed that the different age and plant family significantly affected the activities of defence proteins in the needles of two larch species, and then enhanced the larch resistance to pests. These could play a key function in forestry tree genetic improvement and management in future.

Thiamine-induced priming against root-knot nematode infection in rice involves lignification and hydrogen peroxide generation.

Thiamine (vitamin B1, VB1) can act as a plant defence trigger, or priming agent, leading to a rapid counterattack on pathogen invasion. In this study, the priming effect of thiamine on rice (Oryza sativa cv. Nipponbare) and its activity against root-knot nematode (Meloidogyne graminicola) infection were evaluated. Thiamine treatment and subsequent nematode inoculation activated hydrogen peroxide (H2O2) accumulation and lignin deposition in plant roots, and this correlated with enhanced transcription of OsPAL1 and OsC4H, two genes involved in the phenylpropanoid pathway. The number of nematodes in rice roots was slightly but significantly reduced, and the development of the nematodes was delayed, whereas no direct toxic effects of VB1 on nematode viability and infectivity were observed. The combined application of thiamine with l-2-aminooxy-3-phenylpropionic acid (AOPP), an inhibitor of phenylalanine ammonia-lyase (PAL), significantly hampered the VB1-priming capacity. These findings indicate that thiamine-induced priming in rice involves H2O2 and phenylpropanoid-mediated lignin production, which hampers nematode infection. Further cellular and molecular studies on the mechanism of thiamine-induced defence will be useful for the development of novel nematode control strategies.

Nitric oxide: a signaling molecule which activates cell wall-associated defense of tomato against Rhizoctonia solani

We investigated the role of sodium nitroprusside (SNP), as a NO donor, in activating cell wall-related defense responses of tomato against R. solani. Based on previous knowledge on the cross talk of NO with various pathways and its effect on cell wall components, experiments were carried out to investigate the function of cell wall-related defense responses, octadecanoid and phenylpropanoid pathways (which are associated with cell wall modification) not only in SNP-activated defense but also in basal resistance of tomato to the pathogen. In detached leaves and intact plants, we observed a considerable decrease in disease progress on both partially resistant (CH Falat) and susceptible (Mobil) tomato cultivars treated with SNP. The SNP treatment regulated malondialdehyde (MDA), H2O2 and O2 −levels in plant cells, whereas SNP primed callose depsition, phenolics and lignification, as defense responses related to cell wall modification in the inoculated tomato plants. Priming in the activity and expression of lipoxygenase (LOX) and phenylalanine ammonia-lyase (PAL) as key markers of octadecanoid and phenylpropanoid pathways, respectively, was observed in SNP-treated inoculated plants. Co-application of LOX or PAL inhibitors and SNP completely suppressed SNP-activated defense responses. Using DEA-NONOate, as an independent NO releaser, supported the data obtained by SNP on disease progress and the role of PAL and LOX in NO-related protection of tomato against the pathogen. These findings highlight the role of phenylpropanoid and octadecanoid pathways and involvement of cell wall modifications in NO-associated induction of defense as novel mechanisms of SNP-IR in tomato–R. solani pathosystem.

Identification of lettuce phenylalanine ammonia-lyase inhibitors based on in silico virtual screening followed by in vitro evaluation

Identification of lettuce phenylalanine ammonia-lyase (PAL) inhibitors, and evaluation of activities were performed. The structure of lettuce PAL was predicted based on template-based protein structure prediction followed by molecular dynamic simulation. Molecular docking was conducted with 1,465 ligands against lettuce PAL. Glycyrrhetic acid derivatives, carotenoids, curcuminoids, sesamin, and sesamolin were determined to be potent lettuce PAL inhibitors. Curcumin was selected due to its high solubility in aqueous ethanol. Lettuce slices were soaked in a 0.1 mg/mL (w/v) curcumin solution for 1 h, followed by 24 h storage at 20°C. The accumulated chlorogenic acid content was lower in lettuce treated with curcumin than in a control group. Curcumin is a potent PAL inhibitor to be used as an anti-browning agent. Thus, in silico virtual screening can be applied for identification of biomaterials for food preservation.

Differential deployment of parthenolide and phenylpropanoids in feverfew plants subjected to divalent heavy metals and trans-cinnamic acid

Accumulation of defense metabolites is an adaptive response to heavy metals in plants. However, deployment of multiple classes of defense metabolites under cadmium (Cd) and copper (Cu) toxicity is not understood. Here, the responses of parthenolide and phenylpropanoids were examined. Quantification of parthenolide and phenylpropanoids, and the expression of parthenolide biosynthesis genes were performed in feverfew leaves. Photosynthetic and physiological analysis of feverfew seedling treated with Cd and Cu were used to confirm the relationship between concentration of heavy metals and secondary metabolites accumulation. In addition, we examined the role of trans-Cinnamic acid (CA) as a phenylalanine ammonia-lyase (PAL) inhibitor alone or together with different Cd and Cu concentrations on plant growth and metabolites accumulation. Application of different Cu concentrations alone was more phytotoxic than respective Cd concentrations alone in terms of plant growth, photosynthesis, and oxidative status; however, addition of CA improved these attributes compared with their respective treatments alone. The results also revealed the repressing parthenolide biosynthesis versus phenylpropanoids when plants treated with heavy metals, whereas CA treatment showed the inverse effect. This study provides valuable information into the response of feverfew plants to Cd or Cu alone or together with a PAL inhibitor by linking stress-related physiological responses to changes in metabolites.

Participation of phenylalanine ammonia-lyase (PAL) in increased phenolic compounds in fresh cold stressed walnut (Juglans regia L.) kernels

Mature fresh walnut (Juglans regia L.) kernels showed increased phenolics under cold storage in a previous study. The aim of the present work was to investigate changes in individual soluble phenolic compounds under cold stress in relation to changes in total and specific activity of phenylalanine ammonia-lyase (PAL) as an approach to study the association of particular compounds with phenylpropanoid pathway (PhP). Treatments with inhibitors of phenylalanine ammonia-lyase (PAL), mRNA, RNA and protein synthesis, as well as, activities of the enzymes polyphenol oxidase (PPO) and peroxidase (POD) were included in the experiment. Treated samples were stored at 1°C and 90% RH for 20d. Results revealed increases in specific and total activity of PAL during cold storage. Among phenolics, 2,4-dihydroxybenzoic acid and protocatechuic acid ethyl ester were identified by HPLC–DAD–ESI-MS. Increases in acids 4-hydroxybenzoic, 2,4-dihydroxybenzoic, syringic and vanillic agreed with PAL activity increases, whereas in ellagic were largely independent on PAL. Increases in protocatechuic acid and decreases in protocatechuic acid ethyl ester exhibited no direct relation to PAL.

A possible role of an anthocyanin filter in low-intensity light stress-induced flowering in Perilla frutescens var. crispa

The red-leaved form of Perilla frutescens var. crispa was induced to flower by low-intensity light stress. The leaves of this form are normally red, but turned green under low-intensity light due to anthocyanin depletion in the epidermis. Flowering did not occur when plants were grown under light passed through a red-colored cellophane paper, which has an absorption spectrum similar to that of anthocyanins. High-concentration anthocyanins may play the role of a red-colored optical filter under normal light conditions, and this filter effect may be lost under low-intensity light, causing a change in the wavelength characteristics of the light with which the mesophyll cells are irradiated. This change may induce a photobiological effect leading to flowering. The gene expression and enzyme activity of phenylalanine ammonia-lyase (PAL), the key enzyme for anthocyanin biosynthesis, decreased under low-intensity light. L-2-aminooxy-3-phenylpropionic acid (AOPP), which is widely used as a PAL inhibitor, inhibited low-intensity light stress-induced flowering and increased PAL activity and anthocyanin content. The inhibition of flowering by AOPP in P. frutescens may be through different mechanisms than PAL inhibition.

Endogenous factors regulating poor-nutrition stress-induced flowering in pharbitis: The involvement of metabolic pathways regulated by aminooxyacetic acid

The short-day plant pharbitis (also called Japanese morning glory), Ipomoea nil (formerly Pharbitis nil), was induced to flower by poor-nutrition stress. This stress-induced flowering was inhibited by aminooxyacetic acid (AOA), which is a known inhibitor of phenylalanine ammonia-lyase (PAL) and the synthesis of indole-3-acetic acid (IAA) and 1-aminocycropropane-1-carboxylic acid (ACC) and thus regulates endogenous levels of salicylic acid (SA), IAA and polyamine (PA). Stress treatment increased PAL activity in cotyledons, and AOA suppressed this increase. The observed PAL activity and flowering response correlate positively, indicating that AOA functions as a PAL inhibitor. The inhibition of stress-induced flowering by AOA was also overcome by IAA. An antiauxin, 4-chlorophenoxy isobutyric acid, inhibited stress-induced flowering. Both SA and IAA promoted flowering induced by stress. PA also promoted flowering, and the effective PA was found to be putrescine (Put). These results suggest that all of the pathways leading to the synthesis of SA, IAA and Put are responsive to the flowering inhibition by AOA and that these endogenous factors may be involved in the regulation of stress-induced flowering. However, as none of them induced flowering under non-stress conditions, they may function cooperatively to promote flowering.

Investigating the roles of phenylpropanoids in the growth and development of Zea mays L.

The Poaceae includes some of the most important food, fiber, and bio-fuel crops. While there have been many studies investigating the function of phenylpropanoids in this family, most of our understanding is based on correlative data rather than experimental evidence. The current study was conducted to evaluate the roles of phenylpropanoids in the growth and development of Zea mays and to develop an experimental model for further investigations. Z. mays seedlings were grown in vitro with various concentrations of the competitive phenylalanine ammonia lyase inhibitor, 2-aminoindane-2-phosphonic acid (AIP). Ferulic acid, a downstream biosynthetic product, was added to determine if it could rescue the induced phenotypes. At lower concentrations of AIP, plants exhibited elongated roots and shoots, but at higher concentrations, growth was extremely stunted. At the cellular level, the epidermal cells of roots cultured with AIP exhibited a loss of intercellular adhesion and organization, and their cell walls were more readily degraded by enzymatic digestion. These characteristics were accompanied by significant reductions in primary cell wall autofluorescence, indicating that less ferulic acid and other phenolics were incorporated in the cell wall. The majority of these symptoms could be partially or entirely rescued by ferulic acid, providing further evidence that these differences were due to the inhibition of phenylpropanoid biosynthesis. This study provides experimental evidence supporting and expanding upon hypothesized functions of phenylpropanoids in the growth and development of Z. mays and provides an experimental system for further investigations in the Poaceae and other taxonomic groups.

Stress and salicylic acid induce the expression of PnFT2 in the regulation of the stress-induced flowering of Pharbitis nil

Poor nutrition and low temperature stress treatments induced flowering in the Japanese morning glory Pharbitis nil (synonym Ipomoea nil) cv. Violet. The expression of PnFT2, one of two homologs of the floral pathway integrator gene FLOWERING LOCUS T (FT), was induced by stress, whereas the expression of both PnFT1 and PnFT2 was induced by a short-day treatment. There was no positive correlation between the flowering response and the homolog expression of another floral pathway integrator gene SUPPRESSOR OF OVEREXPRESSION OF CO1 and genes upstream of PnFT, such as CONSTANS. In another cultivar, Tendan, flowering and PnFT2 expression were not induced by poor nutrition stress. Aminooxyacetic acid (AOA), a phenylalanine ammonia-lyase inhibitor, inhibited the flowering and PnFT2 expression induced by poor nutrition stress in Violet. Salicylic acid (SA) eliminated the inhibitory effects of AOA. SA enhanced PnFT2 expression under the poor nutrition stress but not under non-stress conditions. These results suggest that SA induces PnFT2 expression, which in turn induces flowering; SA on its own, however, may not be sufficient for induction.

Reactive oxygen species promote chloroplast dysfunction and salicylic acid accumulation in fumonisin B1-induced cell death

We report a novel regulatory mechanism by which reactive oxygen species (ROS) regulate fumonisin B1 (FB1)-induced cell death. We found that FB1 induction of light-dependent ROS production promoted the degradation of GFP-labeled chloroplast proteins and increased phenylalanine ammonia lyase (PAL) activity, PAL1 gene expression and SA content, while pretreatment with ROS manipulators reversed these trends. Moreover, treatment with H2O2 or 3-amino-1,2,4-triazole increased PAL activity, PAL1 gene expression and SA content. PAL inhibitor significantly blocked FB1-induced lesion formation and SA increase. Our results demonstrate that light-dependent ROS accumulation stimulates the degradation of chloroplastic proteins and up-regulates PAL-mediated SA synthesis, thus promoting FB1-induced light-dependent cell death.

Veratrole Biosynthesis in White Campion

White campion (Silene latifolia) is a dioecious plant that emits 1,2-dimethoxybenzene (veratrole), a potent pollinator attractant to the nocturnal moth Hadena bicruris. Little is known about veratrole biosynthesis, although methylation of 2-methoxyphenol (guaiacol), another volatile emitted from white campion flowers, has been proposed. Here, we explore the biosynthetic route to veratrole. Feeding white campion flowers with [(13)C9]l-phenylalanine increased guaiacol and veratrole emission, and a significant portion of these volatile molecules contained the stable isotope. When white campion flowers were treated with the phenylalanine ammonia lyase inhibitor 2-aminoindan-2-phosphonic acid, guaiacol and veratrole levels were reduced by 50% and 63%, respectively. Feeding with benzoic acid (BA) or salicylic acid (SA) increased veratrole emission 2-fold, while [(2)H5]BA and [(2)H6]SA feeding indicated that the benzene ring of both guaiacol and veratrole is derived from BA via SA. We further report guaiacol O-methyltransferase (GOMT) activity in the flowers of white campion. The enzyme was purified to apparent homogeneity, and the peptide sequence matched that encoded by a recently identified complementary DNA (SlGOMT1) from a white campion flower expressed sequence tag database. Screening of a small population of North American white campion plants for floral volatile emission revealed that not all plants emitted veratrole or possessed GOMT activity, and SlGOMT1 expression was only observed in veratrole emitters. Collectively these data suggest that veratrole is derived by the methylation of guaiacol, which itself originates from phenylalanine via BA and SA, and therefore implies a novel branch point of the general phenylpropanoid pathway.

Root Growth and Enzymes Related to the Lignification of Maize Seedlings Exposed to the Allelochemical L‐DOPA

L-3,4-Dihydroxyphenylalanine (L-DOPA) is a known allelochemical exuded from the roots of velvet bean (Mucuna pruriens L. Fabaceae). In the current work, we analyzed the effects of L-DOPA on the growth, the activities of phenylalanine ammonia-lyase (PAL), tyrosine ammonia-lyase (TAL), and peroxidase (POD), and the contents of phenylalanine, tyrosine, and lignin in maize (Zea mays) roots. Three-day-old seedlings were cultivated in nutrient solution with or without 0.1 to 2.0 mM L-DOPA in a growth chamber (25°C, light/dark photoperiod of 12/12, and photon flux density of 280 μmol m−2 s−1) for 24 h. The results revealed that the growth (length and weight) of the roots, the PAL, TAL, and soluble and cell wall-bound POD activities decreased, while phenylalanine, tyrosine, and lignin contents increased after L-DOPA exposure. Together, these findings showed the susceptibility of maize to L-DOPA. In brief, these results suggest that the inhibition of PAL and TAL can accumulate phenylalanine and tyrosine, which contribute to enhanced lignin deposition in the cell wall followed by a reduction of maize root growth.

Stress-Induced Flowering in Pharbitis—A Review

Many plant species are induced to flower by stress. Stress-induced flowering has been studied mostly in the short-day plant pharbitis (also called Japanese morning glory; Ipomoea nil, formerly Pharbitis nil). In this article, physiological characteristics, the regulation by salicylic acid (SA) and the expression of flowering-related genes in stress-induced flowering in pharbitis are reviewed. Pharbitis flowered under long-days in response to poor nutrition or low temperature. The pharbitis plants induced to flower by stress reached anthesis, fruited and produced fertile seeds. The progeny of the stressed plants developed normally. Grafting experiments indicated that a transmissible flowering stimulus is involved in poor nutrition stress-induced flowering. Aminooxyacetic acid (AOA), a phenylalanine ammonia-lyase (PAL) inhibitor, inhibited the stress-induced flowering, and this inhibition was overcome by SA. Stress induced PAL activity and SA biosynthesis. PnFT2, a pharbitis ortholog of the flowering gene FLOWERING LOCUS T of Arabidopsis thaliana, was expressed when the plants were induced to flower by stress. The overexpression of PnFT2 induced flowering, and PnFT2RNAi inhibited it. AOA inhibited PnFT2 expression induced by stress, and SA eliminated this inhibitory effect. SA enhanced PnFT2 expression under poor nutrition but not under non-stressful conditions. Therefore, stress may induce the production of SA and other unknown factor(s) that may work in combination to induce PnFT2 expression and flowering.

The primary module in Norway spruce defence signalling against H. annosum s.l. seems to be jasmonate-mediated signalling without antagonism of salicylate-mediated signalling

A key tree species for the forest industry in Europe is Norway spruce [Picea abies (L.) Karst.]. One of its major diseases is stem and butt rot caused by Heterobasidion parviporum (Fr.) Niemelä & Korhonen, which causes extensive revenue losses every year. In this study, we investigated the parallel induction of Norway spruce genes presumably associated with salicylic acid- and jasmonic acid/ethylene-mediated signalling pathways previously observed in response to H. parviporum. Relative gene expression levels in bark samples of genes involved in the salicylic acid- and jasmonic acid/ethylene-mediated signalling pathways after wounding and inoculation with either the saprotrophic biocontrol fungus Phlebiopsis gigantea or with H. parviporum were analysed with quantitative PCR at the site of the wound and at two distal locations from the wound/inoculation site to evaluate their roles in the induced defence response to H. parviporum in Norway spruce. Treatment of Norway spruce seedlings with methylsalicylate, methyljasmonate and inhibitors of the jasmonic acid/ethylene signalling pathway, as well as the Phenylalanine ammonia lyase inhibitor 2-aminoindan-2-phosphonic acid were conducted to determine the responsiveness of genes characteristic of the different pathways to different hormonal stimuli. The data suggest that jasmonic acid-mediated signalling plays a central role in the induction of the genes analysed in this study irrespective of their responsiveness to salicylic acid. This may suggest that jasmonic acid-mediated signalling is the prioritized module in the Norway spruce defence signalling network against H. parviporum and that there seems to be no immediate antagonism between the modules in this interaction.

Storage of fresh walnuts (Juglans regia L.) – Low temperature and phenolic compounds

Undried or fresh walnut (Juglans regia L.) kernels are a less widespread product than the dried kernels with no information concerning their storage. Weight loss, respiration, ethylene production, colour, total phenolics (TP) and total antioxidant capacity (TAC) were examined in fresh de-hulled walnuts of cv Franquette with or without extraction from the shell during storage in air at 1°C and 90% RH for 20–40d. The effect of low temperature on TP, TAC and individual phenolic compounds was also investigated during the storage of fresh shelled kernels at 1°C and 8°C for up to 30d. Walnuts were harvested at the commercially mature stage. Respiration, as determined at 20°C, decreased sharply in shelled kernels during the first 20d at 1°C, but gradually in whole walnuts during 40d storage. No ethylene production was detected in samples. Before storage, the TP concentration in fresh kernels was already 1.2-times higher than in kernels dried at 36°C for 24h. Additionally, TP and TAC increased by 1.2- and 1.3-fold, respectively, in fresh kernels stored at 1°C for 20d and remained almost stable thereafter. Shelling increased kernel browning slightly at the lower temperature, but had no effect on TP and TAC values. At 1°C, 4-hydroxybenzoic, protocatechuic, vanillic, ellagic, 2,4-dihydroxybenzoic and syringic acids exhibited major increases within the first 20d and did not change subsequently. At 8°C, no increases in TP, TAC and most phenolic compounds were observed apart from those in 2,4-dihydroxybenzoic and syringic acids. By contrast, protocatechuic acid ethyl ester decreased at both storage temperatures, the decrease being promoted by the higher temperature used. Ellagic acid was the major acid observed in all cases. Treatments with actinomycin D, cordycepin, cycloheximide and 2-aminoindane-2-phosphonic acid (inhibitor of phenylalanine ammonia-lyase or PAL inhibitor) inhibited TP increases at 1°C, indicating the induction of at least the phenylpropanoid pathway in fresh walnuts at 1°C.

Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana)

BackgroundProtoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L.) was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall re-synthesis and cell division.ResultsThis study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L.) leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 μM), an inhibitor of phenylalanine ammonia lyase (PAL), reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (±3.27) in controls to 65.3% (±4.60). Protoplasts isolated from callus grown in 100 μM AIP developed cell walls by day 2, had a division rate of 28.5% (±3.59) by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose.ConclusionsThis study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated protoplasts survived electrofusion and viable heterokaryons were produced. Together, these results provide the first evidence of sustained cell division, callus regeneration, and potential application of somatic cell fusion in American elm, suggesting that this source of protoplasts may be ideal for genetic manipulation of this species. The technological advance made with American elm in this study has potential implications in other woody species for fundamental and applied research which require availability of viable protoplasts.

Salicylic acid is involved in the regulation of starvation stress-induced flowering in Lemna paucicostata

The short-day plant, Lemna paucicostata (synonym Lemna aequinoctialis), was induced to flower when cultured in tap water without any additional nutrition under non-inductive long-day conditions. Flowering occurred in all three of the tested strains, and strain 6746 was the most sensitive to the starvation stress conditions. For each strain, the stress-induced flowering response was weaker than that induced by short-day treatment, and the stress-induced flowering of strain 6746 was completely inhibited by aminooxyacetic acid and l-2-aminooxy-3-phenylpropionic acid, which are inhibitors of phenylalanine ammonia-lyase. Significantly higher amounts of endogenous salicylic acid (SA) were detected in the fronds that flowered under the poor-nutrition conditions than in the vegetative fronds cultured under nutrition conditions, and exogenously applied SA promoted the flowering response. The results indicate that endogenous SA plays a role in the regulation of stress-induced flowering.

Feedback inhibition of the general phenylpropanoid and flavonol biosynthetic pathways upon a compromised flavonol-3-O-glycosylation

Flavonols, phenylalanine-derived secondary metabolites, have protective and regulatory functions in plants. In Arabidopsis thaliana, they are consecutively glycosylated at their 3-OH and 7-OH groups. UGT78D1 and UGT78D2 are the major flavonol 3-O-glycosyltransferases in Arabidopsis leaves. The ugt78d1 ugt78d2 double mutant, which was strongly compromised in the initial 3-O-glycosylation, showed a severe and specific repression of flavonol biosynthesis, retaining only one-third of the wild-type level. This metabolic phenotype was associated with a repressed transcription of several flavonol biosynthetic genes including the committed step chalcone synthase [(CHS) or TRANSPARENT TESTA 4 (TT4)]. Furthermore, the committed step of the upstream, general phenylpropanoid pathway, phenylalanine ammonia-lyase (PAL), was down-regulated in its enzyme activity and in the transcription of the flavonol-related PAL1 and PAL2. However, a complete blocking of flavonoid biosynthesis at CHS released PAL inhibition in a tt4 ugt78d1 ugt78d2 line. PAL activity was even enhanced in the flavonol synthase 1 mutant, which compromises the final formation of flavonol aglycones. The dependence of the PAL feedback inhibition on flavonols was confirmed by chemical complementation of tt4 ugt78d1 ugt78d2 using naringenin, a downstream flavonoid intermediate, which restored the PAL repression. Although aglycones were not analytically detectable, this study provides genetic evidence for a novel, flavonol-dependent feedback inhibition of the flavonol biosynthetic pathway and PAL. It was conditioned by the compromised flavonol-3-O-conjugation and a decrease in flavonol content, yet dependent on a residual, flavonol synthase 1 (FLS1)-related capacity to form flavonol aglycones. Thus, this regulation would not react to a reduced metabolic flux into flavonol biosynthesis, but it might prevent the accumulation of non-glycosylated, toxic flavonols.