Peroxidase activity and lignification in soybean root growth-inhibition by juglone

The changes in activities of soluble and cell wall-bound peroxidases and lignin contents in juglone-stressed soybean (Glycine max) seedlings and their relationships with root growth were investigated. Soybean seedlings (3-d-old) were cultivated in nutrient solution supplemented with 0.5 to 25 μM juglone for 24 h. Length and dry mass of roots decreased after 5 to 25 μM juglone treatments. Low juglone concentrations (≤ 1 μM) increased soluble peroxidase activity, while high concentrations (≥ 10 μM) inhibited activities of soluble and cell wall-bound peroxidases. Juglone (≤ 1 μM) did not affect lignin content but highly increased lignification after 5 to 25 μM treatments. Results indicate that lignification may be an important step in root growth reduction of juglone-stressed soybean.     

Peroxidase and phenylalanine ammonia-lyase activities,phenolic acid contents,and allelochemicals-inhibited root growth of soybean

The influence of the allelochemicals ferulic (FA) and vanillic (VA) acids on peroxidase (POD, EC 1.11.1.7) and phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activities and their relationships with phenolic acid (PhAs) contents and root growth of soybean (Glycine max (L.) Merr.) were examined. Three-day-old seedlings were cultivated in nutrient solution containing FA or VA (0.1 to 1 mM) for 48 h. Both compounds (at 0.5 and 1 mM) decreased root length (RL), fresh weight (FW) and dry weight (DW) and increased PhAs contents. At 0.5 and 1 mM, FA increased soluble POD activity (18% and 47%, respectively) and cell wall (CW)-bound POD activity (61% and 34%), while VA increased soluble POD activity (33% and 17%) but did not affect CW-bound POD activity. At 1 mM, FA increased (82%) while VA reduced (32%) PAL activities. The results are discussed on the basis of the role of these compounds on phenylpropanoid metabolism and root growth and suggest that the effects caused on POD and PAL activities are some of the many mechanisms by which allelochemicals influence plant growth.     

Nitric oxide affecting root growth,lignification and related enzymes in soybean seedlings

This study analyzed the involvement of nitric oxide (NO) in the root lignification of soybean seedlings. To this end, changes in root cell viability; phenylalanine ammonia-lyase (PAL) and soluble and cell wall bound peroxidase (POD) activities and lignin and hydrogen peroxide (H₂O₂) contents of soybean roots treated with the NO-donor sodium nitroprusside (SNP) and its relationships with root growth were evaluated. Seedlings were cultivated in a nutrient solution supplemented with 5 to 1,000 μM SNP for 24 h. At an extremely low concentration (5 μM), SNP induced root growth and increased lignification and activities of related enzymes (PAL and cell wall-bound POD). At a high concentration (1,000 μM), SNP reduced root growth and lignification (PAL activity and H₂O₂ and lignin contents) and caused a loss of cell viability. Application of potassium ferrocyanide (an analog of SNP that cannot release NO) and PTIO (2-phenyl-4,4,5,5,-tetramethylimidazoleline-1-oxyl-3-oxide, a scavenger of NO) revealed that the inhibitory/stimulatory effects on root lignification may be due to NO itself. These results indicate that NO, depending on its concentration, may act as a stress factor, due to its toxic action, or as a signal molecule, inducing soybean root growth and lignification.     

Phenolics and the activities of phenylalanine ammonia-lyase, phenol-β-glucosyltransferase and β-glucosidase in cucumber roots as affected by phenolic allelochemicals

Seven-day-old seedlings of cucumber (Cucumis sativus L.) cv. Wisconsin were treated with 0.1 mM solutions of cinnamic acid (ferulic and p-coumaric acids) and benzoic acid (p-hydroxybenzoic and vanillic acids) derivatives as stressors. The content of free and glucosylated soluble phenols and the activity of phenylalanine ammonia-lyase (E.C.4.3.1.5), phenol-β-glucosyltransferase (E.C.2.4.1.35.), and β-glucosidase (E.C.3.2.1.21.) in seedling roots as well as their length and fresh weight were examined. Changes in glucosylated phenolic content and phenol-β-glucosyltranspherase activity were observed under the influence of all phenolics applied. Treatment with ferulic and p-coumaric acids stimulated the increase of phenylalanine ammonia-lyase and β-glucosidase activity and slightly inhibited cucumber root growth.     

The effects of dopamine on root growth and enzyme activity in soybean seedlings

In the present study, we investigated the effects of dopamine, an allelochemical exuded from the velvetbean (Mucuna pruriens L DC. var utilis), on the growth and cell viability of soybean (Glycine max L. Merrill) roots. We analyzed the effects of dopamine on superoxide dismutase, phenylalanine ammonia-lyase and cell wall-bound peroxidase activities as well as its effects on lignin contents in the roots. Three-day-old seedlings were cultivated in half-strength Hoagland nutrient solution (pH 6.0), without or with 0.25 to 1.0 mM dopamine, in a growth chamber (25°C, 12L:12D photoperiod, irradiance of 280 μmol m⁻² s⁻¹) for 24 h. In general, the length, fresh weight and dry weight of roots, cell viability, PAL and POD activities decreased, while SOD activities increased after dopamine treatment. The content of lignin was not altered. The data demonstrate the susceptibility of soybean to dopamine and reinforce the role of this catecholamine as a strong allelochemical. The results also suggest that dopamine-induced inhibition in soybean roots is not related to the production of lignin, but may be related to damage caused by reactive oxygen species.     

Plant hormone interaction and phenolic metabolism in the regulation of russet spotting in iceberg lettuce

Russet spotting (RS) is a physiological disorder induced in iceberg lettuce (Lactuca sativa L.) by exposure to parts per million levels of ethylene at 5 ± 2°C. Ethylene induced phenylalanine ammonia-lyase and ionically bound peroxidase activities that correlated with development of RS symptoms. The ethylene-treated tissue had significantly higher lignin content than air control tissue with lignification localized in walls of RS-affected cells. Ethylene also caused the accumulation of the flavonoids (+)catechin and (−)epicatechin and the chlorogenic acid derivatives 3-caffeoyl-quinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid. These soluble phenolic compounds were readily oxidized to brown substances by polyphenol oxidase isolated from RS tissue. Ethylene substantially increased ionically bound indole-3-acetic acid (IAA) oxidase activity, while IAA application greatly reduced ethylene-induced phenylalanine ammonia-lyase, peroxidase, and IAA oxidase activities, soluble phenolic content, and RS development.     

Changes of phenolic metabolism and oxidative status in nitrogen-deficient Matricaria chamomilla plants

The effects of nitrogen deficiency on selected physiological attributes, phenylalanine ammonia-lyase (PAL, EC. 4.3.1.5) activity, phenolic contents, peroxidase (EC. 1.11.1.7) and catalase (EC. 1.11.1.6) activities, lipid peroxidation status and H₂O₂ accumulation were studied in N-deficient Matricaria chamomilla (L.) over 12 days. N deficiency enhanced root growth and inhibited shoot growth. Chlorophyll composition and F _v/F _m were not affected by N stress, but nitrogen and soluble proteins decreased in both the rosettes and the roots. PAL activity, expressed per mg protein, was enhanced in N-deficient rosettes and tended to decrease by the end of the experiment, while in the roots PAL activity was maintained. Total phenolic contents increased in both rosettes and roots. Peroxidase and catalase activities in N-deficient rosettes tended to decrease by the end of the experiment, while in the roots they increased on the 12th day of deficiency. Furthermore, lipid peroxidation status increased in N-deficient roots on the 12th day, indicating that antioxidative protection was insufficient to scavenge reactive oxygen species being generated. Surprisingly, H₂O₂ content was even lower in N-deficient roots by the end of the experiment, while in the leaves increased. This observation in correlation to lipid peroxidation and H₂O₂ degradation is discussed. The importance of PAL activity and phenolic metabolites in combination with antioxidative enzymes for plant protection against oxidative stress and the significance of PAL activity for the mobilization of N availability in N-deficient tissue are also discussed in view of existing information.     

Soybean root growth inhibition and lignification induced by p-coumaric acid

The effects of 0.25–2 mM p-coumaric acid, a phenylpropanoid metabolite with recognized allelopathic properties, were tested on root growth, cell viability, phenylalanine ammonia-lyase (PAL) activities, soluble and cell wall-bound peroxidase (POD) activities, hydrogen peroxide (H₂O₂) level and lignin content and its monomeric composition in soybean (Glycine max (L.) Merr.) roots. At ≥0.25 mM, exogenously supplied p-coumaric acid induced premature cessation of root growth, increased POD activity and lignin content and decreased the H₂O₂ content. At ≥0.5 mM, the allelochemical decreased the cell viability and PAL activity. When applied jointly with PIP (an inhibitor of the cinnamate 4-hydroxylase, C4H), 1 mM p-coumaric acid increased lignin content. In contrast, the application of MDCA (an inhibitor of the 4-coumarate:CoA ligase, 4CL) with p-coumaric acid did not increase lignin content. The lignin monomeric composition of p-coumaric acid-exposed roots revealed a significant increase of p-hydroxyphenyl (H) and guaiacyl (G) units. Taken together, these results suggest that p-coumaric acid's mode of action is entry via the phenylpropanoid pathway, resulting in an increase of H and G lignin monomers that solidify the cell wall and restrict soybean root growth.     

Exogenous caffeic acid inhibits the growth and enhances the lignification of the roots of soybean (Glycine max)

The allelopathic effect of caffeic acid was tested on root growth, phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities, hydrogen peroxide (H₂O₂) accumulation, lignin content and monomeric composition of soybean (Glycine max) roots. We found that exogenously applied caffeic acid inhibited root growth, decreased the PAL activity and H₂O₂ content and increased the soluble and cell wall-bound POD activities. The p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) monomers and total lignin (H + G + S) increased in the caffeic acid-exposed roots. When applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), caffeic acid equalized the inhibitory effect of PIP, whereas the application of methylene dioxocinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL) plus caffeic acid decreased lignin production. These results indicate that exogenously applied caffeic acid can be channeled into the phenylpropanoid pathway via the 4CL reaction, resulting in an increase of lignin monomers that solidify the cell wall and inhibit root growth.     

The effects of l-DOPA on root growth, lignification and enzyme activity in soybean seedlings

In the present study, we investigated the effects of l-DOPA (l-3,4-dihydroxyphenylalanine), an allelochemical exuded from the velvetbean (Mucuna pruriens L DC. var. utilis), on the growth and cell viability of soybean (Glycine max L. Merrill) roots. We analyzed the effects of l-DOPA on phenylalanine ammonia lyase (PAL), cinnamyl-alcohol dehydrogenase (CAD) and cell wall-bound peroxidase (POD) activities as well as its effects on phenylalanine, tyrosine and lignin contents in the roots. 3-day-old seedlings were cultivated in half-strength Hoagland nutrient solution (pH 6.0), with or without 0.5?mM l-DOPA, in a growth chamber at 25?°C for 6, 12, 18 or 24?h with a day/night regime of 1:1, and a photon flux density of 280???mol?m^?2 s^?1. In general, the length, fresh weight and dry weight of the roots decreased followed by a significant loss of cell viability. Phenylalanine, tyrosine and lignin contents as well as PAL, CAD and cell wall-bound POD activities increased after l-DOPA treatment. These results reinforce the susceptibility of soybean to l-DOPA, which increases the enzyme activity in the phenylpropanoid pathway and, therefore, provides precursors for the polymerization of lignin. In brief, these findings suggest that the inhibition of soybean root growth induced by exogenously applied l-DOPA may be due to excessive production of lignin in the cell wall.     

Degradation of aromatic compounds through the β‐ketoadipate pathway is required for pathogenicity of the tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici

Plant roots react to pathogen attack by the activation of general and systemic resistance, including the lignification of cell walls and increased release of phenolic compounds in root exudate. Some fungi have the capacity to degrade lignin using ligninolytic extracellular peroxidases and laccases. Aromatic lignin breakdown products are further catabolized via the β‐ketoadipate pathway. In this study, we investigated the role of 3‐carboxy‐cis,cis‐muconate lactonizing enzyme (CMLE), an enzyme of the β‐ketoadipate pathway, in the pathogenicity of Fusarium oxysporum f. sp. lycopersici towards its host, tomato. As expected, the cmle deletion mutant cannot catabolize phenolic compounds known to be degraded via the β‐ketoadipate pathway. In addition, the mutant is impaired in root invasion and is nonpathogenic, even though it shows normal superficial root colonization. We hypothesize that the β‐ketoadipate pathway in plant‐pathogenic, soil‐borne fungi is necessary to degrade phenolic compounds in root exudate and/or inside roots in order to establish disease.     

Physiological and biochemical events leading to vitrification of plants cultured in vitro

Water content, peroxidase activity and isoperoxidases, phenylalanine ammonia-lyase activity and phenolic content were comparatively analyzed in tissues of normal and vitreous plants cultured in vitro. The release of ethylene in flask atmospheres by normal and vitrifying plants was also measured. On the basis of the results, it is hypothesized that vitrification results from a burst of ethylene controlled by the peroxidase-IAA-oxidase system. An initiating stress (e.g. excess of cytokinins or of NH₄⁺ ions) would mediate the enhancement of the activity of soluble and membrane-bound peroxidases through a rapid modification of the phenolic level. The excess of ethylene in the atmosphere of stressed plants would retroinhibit its own biosynthesis and as a consequence decrease the activities of PAL and acidic peroxidases, thus hindering lignification processes. A parallel decrease in cellulose synthesis due to a diverted conversion of sugars to amino acids is expected (from data in the literature). Deficiency of both cellulose and lignin would allow more water uptake due to reduced wall pressure and bring about the hyperhydric malformations.     

Time course study on accumulation of cell wall-bound phenolics and activities of defense enzymes in tomato roots in relation to <Emphasis Type="Italic">Fusarium</Emphasis> wilt

Major cell wall-bound phenolic compounds were detected and identified in roots of tomato at different stages of growth. Alkaline hydrolysis of the cell wall material of the root tissues yielded ferulic acid as the major bulk of the phenolic compounds. Other phenolic compounds identified were 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin and 4-coumaric acid. All the six phenolic acids were higher in very early stage of plant growth. Ferulic acid, 4-hydroxybenzoic acid and 4-coumaric acid exhibited a decreasing trend up to 60 days and then the content of these phenolic acids increased somewhat steadily towards the later stage of growth. Total phenolics, phenylalanine ammonia-lyase (PAL) activity and peroxidase (POD) activity were in tandem match with the occurrence pattern of the phenolic acids. Ferulic acid showed highest antifungal activity against tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici. The results of this study may be interpreted to seek an explanation for high susceptibility of tomato plants at flowering stage to Fusarium wilt. It may also be concluded that greater amounts of ferulic acid in combination with other phenolics and higher level of PAL and POD activities after 60 days of growth may have a role in imparting resistance against Fusarium wilt at a late stage of plant growth.     

Induced defence responses and protective effects on tomato against Xanthomonas vesicatoria by an aqueous extract from Solanum lycocarpum infected with Crinipellis perniciosa

This study investigated the induced defence responses and protective effects on susceptible tomato (Lycopersicon esculentum Mill.) against Xanthomonas vesicatoria (Doidge) by a heat-treated aqueous extract (VLA) from dry necrotic tissue of ‘Lobeira’ (Solanum lycocarpum St. Hil.) branches infected with the fungus Crinipellis perniciosa (Stahel) compared with acibenzolar-S-methyl (ASM), a commercial inducer of resistance. Plantlets were sprayed with VLA and ASM and challenged 4 days later with a virulent strain of X. vesicatoria, under greenhouse conditions. The disease severity, fresh weight of shoots, the activities of phenol peroxidase (POX), polyphenol oxidase (PPO), chitinase (CHI), phenylalanine ammonia-lyase (PAL), lignin deposition, and soluble phenolic contents were evaluated in the leaf tissues. Reduction of the bacterial spot severity was observed in plantlets treated with VLA which conferred 63% of the ASM protection. This protective effect and lesion reduction promoted by VLA were probably associated particularly with POX and PAL activities, lignin deposition on leaf tissues and, to a less extent, CHI activity.     

Herbicide effects on phenolic metabolism in maize (Zea mays L.)and soybean (Glycine max L.) seedling

The activities of phenylalanine ammonia lyase [PAL; EC 4.3.1.5 [EC] ]and chalcone isomerase [Cl; EC 5.5.1.5 [EC] ] as well as the contentsof anthocyanin and total soluble hydroxyphenolic compounds wereinvestigated in maize (Zea mays L.) and soybean (Glycine maxL.) seedlings 120 h after treatment with the field dose of fiveherbicides from different groups (trifluralin, fluometuron,atrazine, alachlor, and rimsulfuron) having varied modes ofaction. The fresh weight of both species was greatly decreasedby trifluralin followed by fluometuron and atrazine. The dryweight was, in general, only slightly decreased by all the herbicideswith the largest response with trifluralin. On the other hand,the activities of PAL and Cl were greatly enhanced in both speciesby alachlor and rimsulfuron, but decreased by trifluralin. Fluometuroninduced decreases in PAL activity of maize only and decreasedCl activity of maize and soybean seedlings. Moreover, hydroxyphenoliccompounds were increased in both species by alachlor and rimsulfuronand decreased by trifluralin and atrazine. Similarly, anthocyanincontent was increased in both seedlings by alachlor and rimsulfuron,but decreased by trifluralin and fluometuron, whereas atrazinedecreased the anthocyanin content in maize only. The presentresults indicate that stress is maintained by the differentherbicides and confirm the controlling action of PAL and Clon the production of anthocyanin and phenolic compounds duringthe induced state of stress. In addition, dry weight reductionappeared to coincide with the changes in the parameters of secondarymetabolism, suggesting a regulatory role of secondary metabolismon seedling growth. Key words: Herbicides, phenylalanine ammonia lyase, chalcone isomerase, anthocyanin, hydroxyphenolics     

Alterations in phenylpropanoid content in soybean roots during low temperature acclimation

L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity, growth and phenolic acid content during low temperature acclimation in soybean (Glycine max. (L.) Merr.) roots were investigated. Elongation of soybean roots was inhibited after the transfer of 3-d-old seedlings grown at 25 to 10 °C. Extractable PAL activity as well as the total amount of phenolics increased 24 h after plant transfer to low temperature. The high pressure liquid chromatography analyses revealed the presence of six phenolic acids in soybean roots: p-hydroxybenzoic, vanillic, syringic, anisic, p-coumaric and ferulic. Analyses of different fractions of phenolic acids showed that during 24 h of low temperature exposure, an increase in the relative level of ester-bound-soluble phenolic acids occurred. The highest increase in this fraction was observed for ferulic acid (26 %). At the same time, a decrease in phenolic glycosides took place. The amount of phenolic acids released after alkaline treatment of the cell wall material was strongly inhibited (3-fold), which may suggest an alteration of the physical properties of the wall in acclimation to low temperature. The possible role of phenolics in acclimation to low temperature in roots is discussed.     

Ferulic acid impairs rhizogenesis and root growth,and alters associated biochemical changes in mung bean (Vigna radiata) hypocotyls

The present study investigated the effect of ferulic acid (FA; 0–1000 µM) on early growth, and rhizogenesis in mung bean (Vigna radiata) hypocotyls and associated biochemical changes. FA severely affected the radicle elongation and number of secondary roots after 72 h. The root and shoot length, number and length of secondary roots, and seedling dry weight of one-week-old seedlings of mung bean were decreased by 64%. The rooting potential (percent rooting, number and length of adventitious roots) of mung bean hypocotyls under in vitro conditions was significantly inhibited in response to 1–100 µM FA. At 1000 µM there was complete cessation of rooting. FA caused a reduction in the contents of water-soluble proteins and endogenous total phenolics, whereas the activities of proteases, peroxidases, and polyphenol peroxidases increased. The study concludes that FA inhibits root growth and development, and in vitro rooting process in mung bean by interfering with biochemical processes that are crucial for root formation.     

Effect of a strong cold storage induced desiccation on metabolic solutes,stock quality and regrowth,in seedlings of two oak species

Bare-root seedlings of pedunculate oak (Quercus robur L.) and northern red oak (Quercus rubra L.) were lifted in January and stored at 1.8°C, at 82% relative humidity, until their fresh weight declined by 33%. Root growth potential (RGP), fine root electrolyte leakage (REL), fine root water content (RWC), shoot tip water content (SWC), starch and metabolic solute contents in root and shoot, were measured just after lifting and after treatment. Survival of treated seedlings was also assessed in a field trial. RWC, SWC, REL, RGP were dramatically affected by desiccation during cold storage. In both species, root soluble carbohydrate level, inositol level and isocitrate level increased, whereas root starch level and shoot soluble carbohydrate level decreased. In northern red oak, treated seedlings had higher root contents of soluble carbohydrates, inositol and proline than in pedunculate oak. Moreover, treatment induced proline accumulation only in northern red oak roots. These differences could explain why field survival of treated seedlings was significantly better in northern red oak than in pedunculate oak.