Genetic and biochemical analysis of PadR-padC promoter interactions during the phenolic acid stress response in Bacillus subtilis 168

Bacillus subtilis 168 is resistant to phenolic acids by expression of an inducible enzyme, the phenolic acid decarboxylase (PadC), that decarboxylates these acids into less toxic vinyl derivatives. In the phenolic acid stress response (PASR), the repressor of padC, PadR, is inactivated by these acids. Inactivation of PadR is followed by a strong expression of padC. To elucidate the functional interaction between PadR and the padC promoter, we performed (i) footprinting assays to identify the region protected by PadR, (ii) electrophoretic mobility shift assays (EMSAs) with a modified padC promoter protected region to determine the interacting sequences, and (iii) random mutagenesis of padR to identify amino acid residues essential for the function of PadR. We identified an important consensus dyad sequence called IR1-2 (ATGT-8N-ACAT) overlapping a second dyad element (GTGT-8N-ACAT) that we named dIR1-2bis. The entire dIR1-2bis/IR1-2 sequence permits binding of two PadR dimers in EMSAs, which may be observed for bacteria grown under noninduced conditions where the padC promoter is completely repressed. Three groups of modified PadRs giving a PASR phenotype were characterized in vivo. The DNA sequences of certain mutant padR alleles indicate that important residues are all located in the region containing the coiled-coil leucine zipper domain that is involved in dimerization. These substitutions reduce the affinity of PadR binding to the padC promoter. Of particular interest are residue L128, located at the center of the putative coiled-coil leucine zipper domain, and residue E97, which is conserved among all PadRs.     

Effect of phenolic acids on anthocyanin content in maize roots

Supply of 0.01 to 5.0 mM salicylic, caffeic and gallic acids, either during imbibition of seeds for 24 to 48 h or during seedling growth increased anthocyanin production in maize (Zea mays L. cv. Ganga safed-2) roots. While tyrosine had no effect, phenylalanine either in the presence or absence of the phenolic acids increased anthocyanin content. Glucose in a concentration range of 1 to 20 mM and shikimic acid in 0.01 to 5.0 mM range also increased pigment level, which was higher in the presence of salicylic acid than in it.s absence. The experiments demonstrate the possibility of some indirect effects of salicylic acid and other phenolic acids on anthocyanin synthesis.     

Potent Inhibitory Effects of Some Phenolic Acids on Lactoperoxidase

Lactoperoxidase (LPO) plays a key role in immune response against pathogens. In this study, we examined the effects of some phenolic acids on LPO. For this purpose, bovine milk LPO was purified 380.85‐fold with a specific activity of 26.66 EU/mg and overall yield of 73.33% by using Amberlite CG‐50 H⁺ resin and CNBr‐activated Sepharose‐4B‐l ‐tyrosine‐sulfanilamide affinity chromatography. After purification, the in vitro effects of phenolic acids (tannic acid, 3,4‐dihydroxybenzoic acid, 3,5‐ dihydroxybenzoic acid, chlorogenic acid, sinapic acid, 4‐hydroxybenzoic acid, vanillic acid, salicylic acid, and 3‐hydroxybenzoic acid) were investigated on LPO. These phenolic acids showed potent inhibitory effect on LPO. K_i values for these phenolic acids were found as 0.0129 nM, 0.132 μM, 0.225 μM, 0.286 μM, 0.333 μM, 2.33 μM, 10.82 μM, 0.076 mM, and 0.405 mM, respectively. Sinapic acid and 4‐hydroxybenzoic acid exhibited noncompetitive inhibition; 3,4‐dihydroxybenzoic acid showed uncompetitive inhibition, and other phenolic acids showed competitive inhibition.     

Effect of the engineered indole pathway on accumulation of phenolic compounds in Catharanthus roseus hairy roots

Catharanthus roseus has been well-known to contain indole alkaloids effective for treatment of diverse cancers. We examined the intracellular accumulation profiles of phenolic compounds in response to ectopic overexpression of tryptophan feedback-resistant anthranilate synthase holoenzyme (ASalphabeta) in C. roseus hairy roots. Among 13 phenolic compounds measured, 6 phenolic compounds were detected in late exponential phase ASalphabeta hairy roots. Uninduced and induced ASalphabeta hairy roots accumulated up to 1.2 and 4.5 mg/g DW over a 72-h period, respectively. Upon induction, in parallel with a rapid increase in tryptophan in the first 48 h, accumulation of phenolic compounds tended to increase to a maximum level (4.5 mg/g DW) at 48 h, after which phenolic levels decreased back to the uninduced level by 72 h. Naringin was a predominant form that comprised about 72% and 36% of the total content of phenolic compounds in the uninduced and induced lines, respectively. Upon induction, accumulation of catechin drastically increased with the highest level (3.6 mg/g) occurring at 48 h, whereas that of all others except for salicylic acid showed no statistical difference. Catechin is a final product of the flavonoid pathway, and thus metabolic flux into this pathway is transiently increased by overexpression of AS. Like catechin, salicylic acid is very sensitive to induction as it began to increase to 5-fold within 4 h of induction, but unlike catechin, no significant accumulation of salicylic acid was noted after 4 h of induction. The results suggest differential regulation of this particular biosynthesis branch within the phenolic pathway.     

Combined effect of 24-epibrassinolide and salicylic acid mitigates lead (Pb) toxicity by modulating various metabolites in <Emphasis Type="BoldItalic">Brassica juncea</Emphasis> L. seedlings

The present study demonstrated the combined effect of 24-epibrassinolide and salicylic acid against lead (Pb, 0.25, 0.50, and 0.75 mM) toxicity in Brassica juncea seedlings. Various parameters including water status, metal uptake, total water- and lipid-soluble antioxidants, metal chelator content (total thiols, protein-bound thiols, and non-protein-bound thiols), phenolic compounds (flavonoids, anthocyanins, and polyphenols), and organic acids were studied in 10-day-old seedlings. Dry matter content and the heavy metal tolerance index were reduced by 42.24 and 52.3%, respectively, in response to Pb treatment. Metal uptake, metal-chelating compounds, phenolic compounds, and organic acids were increased in Pb-treated seedlings as compared to control plants. The treatment of Pb-stressed seedlings with combination of EBL and SA resulted in enhancement of heavy metal tolerance index by 40.07%, water content by 1.84%, and relative water content by 23.45%. The total water- and lipid-soluble antioxidants were enhanced by 21.01 and 2.21%, respectively. In contrast, a significant decline in dry weight, metal uptake, thiol, and polyphenol contents was observed following the application of 24-epibrassinolide and salicylic acid. These observations indicate that Pb treatment has an adverse effect on B. juncea seedlings. However, co-application of 24-epibrassinolide and salicylic acid mitigates the negative effects of Pb, by lowering Pb metal uptake and enhancing the heavy metal tolerance index, water content, relative water content, antioxidative capacities, phenolic content, and organic acid levels.     

Non-Involvement of the Human Monocarboxylic Acid Transporter 1 (MCT1) in the Transport of Phenolic Acid

Phenolic acids such asp-coumaric acid and microbial metabolites of poorly absorbed polyphenols are absorbed by the monocarboxylic acid transporter (MCT)-mediated transport system which is identical to the fluorescein/H⁺ cotransport system. We focus here on the physiological impact of MCT-mediated absorption and distribution. We examined whether MCT1, the best-characterized isoform found in almost all tissues, is involved in this MCT-mediated transport system. The induction of MCT1 expression in Caco-2 cells by a treatment with sodium butyrate (NaBut) did not increase the fluorescein permeability. Moreover, the transfection of Caco-2 cells with an expression vector encoding MCT1 caused no increase in either the permeability or uptake of fluorescein. Furthermore, in the MCT1-expressing oocytes, no increase ofp-coumaric acid uptake was apparent, whereas the uptake of salicylic acid, a substrate of MCT1, nearly doubled. Our data therefore establish that MCT1 was not involved in the MCT-mediated transport of phenolic acids.     

Transgenic ipt tobacco overproducing cytokinins overaccumulates phenolic compounds during in vitro growth.

We present evidence that overproduction of endogenous cytokinins (CK) caused stress response in non-rooting Pssu-ipt transgenic tobacco (Nicotiana tabacum L.) grown in vitro. It was demonstrated by overaccumulation of phenolic compounds, synthesis of pathogenesis related proteins (PR proteins), and increase in peroxidase (POD) activities. Immunolocalization of zeatin and also PR-1b protein on leaf cryo-sections proved their accumulation in all mesophyll cells of transgenic tobacco contrary to control non-transgenic plants. Intensive blue autofluorescence of phenolic compounds induced by UV in cross-sections of leaf midrib showed enhanced contents of phenolics in transgenic tobacco compared with controls, nevertheless, no significant difference between both plant types was found in leaf total lignin content. Transgenic plantlets exhibited higher peroxidase activities of both soluble and ionically bound fractions compared with controls. HPLC analysis of phenolic acids confirmed the increase in all phenolic acids in transgenic tobacco except for salicylic acid (SA). The effect of high phenolic content on rooting of transgenic tobacco is discussed.     

Inducers of glycinebetaine synthesis in barley.

Glycinebetaine is an osmoprotectant accumulated by barley (Hordeum vulgare) plants in response to high levels of NaCl, drought, and cold stress. Using barley seedlings in hydroponic culture, we characterized additional inducers of glycinebetaine accumulation. These included other inorganic salts (KCl, MgCl(2), LiCl, and Na(2)SO(4)), oxidants (H(2)O(2) and cumene hydroperoxide), and organic compounds (abscisic acid, polymixin B, n-butanol, salicylic acid, and aspirin). Stress symptoms brought on by high NaCl and other inducers, and not necessarily correlated with glycinebetaine accumulation, include wilting, loss of chlorophyll, and increase in thiobarbituric acid reacting substances. For NaCl, Ca(2+) ions at 10 to 20 mM decrease these stress symptoms without diminishing, or even increasing, glycinebetaine induction. Abscisic acid induces glycinebetaine accumulation without causing any of the stress symptoms. NaCl, KCl, and H(2)O(2) (but not other inducers) induce glycinebetaine at concentrations below those needed for the other stress symptoms. Mg(2+) at 10 to 20 mM induces both stress symptoms and glycinebetaine, but only at low (0.2 mM) Ca(2+). Although illumination is needed for optimal induction, a significant increase in the leaf glycinebetaine level is found in complete darkness, also.     

Antibiotic potential of plant growth promoting rhizobacteria (PGPR) against Sclerotium rolfsii

High performance liquid chromatographic (HPLC) analysis of culture filtrates of plant growth promoting rhizobacteria (PGPR) and medium of inhibitory zone of interaction of Sclerotium rolfsii with PGPR, viz. Pseudomonas aeruginosa, Pseudomonas fluorescens 4, Pseudomonas fluorescens 4 (new) and Pseudomonas sp. varied from sample to sample. In all the culture filtrates of PGPRs, P. aeruginosa had nine phenolic acids in which ferulic acid (14.52 μg/ml) was maximum followed by other phenolic acids. However, the culture filtrates of P. fluorescens 4 had six phenolic acids with maximum ferulic acid (20.54 μg/ml) followed by indole acetic acid (IAA), caffeic, salicylic, o-coumeric acid and cinnamic acids. However, P. fluorescens 4 culture filtrate had seven phenolic acids in which salicylic acid was maximum (18.03 μg) followed by IAA, caffeic, vanillic, ferulic, o-coumeric and cinnamic acids. Pseudomonas sp. also showed eight phenolic acids where caffeic acid (2.75 μg) was maximum followed by trace amounts of ferulic, salicylic, IAA, vanillic, cinnamic, o-coumeric and tannic acids. The analysis of antibiosis zone of PGPRs showed fairly rich phenolic acids. A total of nine phenolic acids were detected in which caffeic acid was maximum (29.14 μg/g) followed by gallic (17.64 μg/g) and vanillic (3.52 μg/g) acids but others were in traces. In P. aeruginosa, antibiosis zone had seven phenolic acids where IAA was maximum (3.48 μg/g) followed by o-coumeric acid (2.08 μg/g), others were in traces. The medium of antibiosis zone of P. fluorescens 4 and P. fluorescens 4 new had eight phenolic acids in which IAA was maximum with other phenolic acids in traces.     

The induction of systemic resistance in barley to powdery mildew infection using salicylates and various phenolic acids

Treatment of the first leaves of barley seedlings with either 5, 10, 15 or 20 mM salicylic acid, sodium salicylate or acetylsalicylic acid resulted in significant reductions in powdery mildew infection on the upper, second leaves. In general, the greatest reduction in mildew infection on the second leaves was obtained by spraying the first leaves with a 15 mM concentration of the compounds. Although the largest reduction in mildew infection of the upper leaves was obtained when the compounds were applied to the first leaves 1–2 days before inoculation, very substantial reductions in infection were still obtained if the first leaves were treated 12 days before inoculation. The three compounds had little direct effect on mildew infection. When ¹⁴C-salicylic acid was fed to first leaves of barley seedlings, uptake was rapid and increased with time. Within 6 h, 0.2% of the salicylic acid appeared in the second leaf and by 24 h after feeding, this had increased to 1.4% (1.1 μmol salicylic acid g^-1 fresh wt). The application of various phenolic acids to first leaves also led to reductions in mildew infection on the second leaves. In particular, treatment of the first leaves with 1 mM vanillic acid, isovanillic acid or syringic acid, reduced mildew infection of the second leaves by 81–87%.     

四种植物酚类物质对舞毒蛾生长发育及繁殖的影响

【目的】为明确植物酚类次生物质对舞毒蛾Lymantria dispar (L.) 的影响,并为应用其防治害虫奠定基础。【方法】本研究选用4种酚酸（水杨酸、丁香酸、绿原酸和水解单宁酸）分别加入人工饲料中饲养舞毒蛾2龄幼虫,调查其对幼虫生长发育及繁殖的影响。【结果】不同酚酸对舞毒蛾的影响差异显著。单宁酸处理第12天时幼虫死亡率达到22.2%,显著高于对照（取食不添加任何酚酸的人工饲料）死亡率（3.3%）（P＜0.01）及其他酚酸处理时的死亡率。到第22天单宁酸及绿原酸使幼虫死亡率迅速上升,至第34天时死亡率达到100%;二者处理的幼虫虫体瘦小,发育历期显著延长,不能正常蜕皮,到4龄期时全部死亡。取食含丁香酸或水杨酸饲料的幼虫可幸存至蛹和成虫,雌性蛹重较对照显著增加,但产卵量和卵受精率均显著降低。其中,取食含丁香酸饲料的成虫产卵量和卵受精率分别比对照减少近90粒和降低约35%,雌性成虫比例下降。【结论】结果说明不同酚酸在害虫体内的积累会对其生长发育及繁殖产生不同程度的影响。     

Effect of plant growth-promoting Rhizobacteria and culture filtrate of Sclerotium rolfsii on phenolic and salicylic acid contents in chickpea (Cicer arietinum)

Two plant growth-promoting rhizobacteria (PGPR), viz., Pseudomonas fluorescens strain Pf4 and P. aeruginosa strain Pag, protected chickpea ( Cicer arietinum) plants from Sclerotium rolfsii infection when applied singly or in combination as seed treatment. Pag gave the best protection to the seedlings, applied either singly (mortality 16%) or in combination with Pf4 (mortality 17%) compared with 44% and 24% mortality in control and Pf4 treatment, respectively. The two PGPR strains induced the synthesis of specific phenolic acids, salicylic acid (SA), as well as total phenolics at different growth stages of chickpea seedlings with varied amount. The maximum amount of total phenolics was recorded in all the aerial parts of 4-week-old plants. Gallic, ferulic, chlorogenic, and cinnamic acids were the major phenolic acids detected in high-performance liquid chromatography (HPLC) analysis. Induction of such phenolic acids in the seedlings was observed up to 6 weeks in comparison with control. Salicylic acid (SA) was induced frequently during the first 3 weeks of growth only. Between the two strains, Pag was more effective in inducing phenolic acid synthesis applied either singly or in combination with strain Pf4 during the entire 6 weeks of growth of chickpea. In the presence of a culture filtrate of S. rolfsii, the two Pseudomonas strains induced more phenolic acids in treated than in non-treated and control plants. The occurrence of salicylic acid was frequent in the first 24 h, but infrequent at 48 and 96 h. Foliar spray of Pseudomonas strains also enhanced the phenolic acid content as well as total phenolics within 24 h of application. Gallic, chlorogenic, and cinnamic acids were consistently discerned in the treated leaves, whereas SA was absent even up to 96 h of application. Resistance in chickpea plants by Pseudomonas strains through induction of phenolic compounds as well as induced systemic resistance via SA-dependent pathway was evident.     

Involvement of autolysin in cellular lysis of Bacillus subtilis induced by short- and medium-chain fatty acids.

The addition of saturated C6, C8, C10, and C12 fatty acids appeared to lyse actively growing cells of Bacillus subtilis 168, as judged by a decrease in the optical density of the culture. Of these fatty acids, dodecanoic acid was the most effective, with 50% lysis occurring in about 30 min at a concentration of 0.5 mM. These conditions also decreased the amount of peptidoglycan estimated by the incorporated radioactivity of N-acetyl-D-[1-14C]glucosamine. At concentrations above 1 mM, however, bacterial lysis was not extensive. Dodecanoic acid did not affect autolysis of the cell wall. The lytic action of dodecanoic acid was greatly diminished in cells in which protein synthesis was inhibited and in an autolytic enzyme-deficient mutant. The results suggest that fatty acid-induced lysis of B. subtilis 168 is due to the induction of autolysis by an autolytic enzyme rather than massive solubilization of the cell membrane by the detergent-like action of the fatty acids.     

Clone-specific responses in leaf phenolics of willows exposed to enhanced UVB radiation and drought stress

The effects of enhanced UVB radiation and drought stress on willow secondary phenolics were studied using the leaves of 8‐week‐old micropropagated plantlets from interspecific hybrids (Salix myrsinites L. ×S. myrsinifolia Salisb.) and pure species (S. myrsinifolia). The plantlets were subjected for 4 weeks to two levels of UVB radiation (ambient, enhanced) and two levels of watering (well‐watered, drought‐stressed) according to a 2 × 2 factorial design. Enhanced UVB radiation increased the total concentration of flavonoids and phenolic acids in all plantlets, while the total concentration of salicylates remained unaffected. Drought stress reduced the total concentration of salicylates and phenolic acids in S. myrsinifolia plantlets, while in hybrids only phenolic acids were affected. The response of phenolic acids to enhanced UVB in drought‐stressed plantlets was different from that in well‐watered ones, indicating that drought stress limited the accumulation of phenolic acids under enhanced UVB radiation. Flavonoids increased in response to enhanced UVB radiation in drought‐stressed plantlets, although drought caused serious physiological stress on growth. There were significant differences between hybrid and S. myrsinifolia plantlets with respect to the composition of phenolics and between families and clones with respect to their concentration. In addition, the response of salicylates, flavonoids and phenolic acids to enhanced UVB and drought stress was clone‐specific, which may indicate that climatic changes will alter the genetic composition of northern forests.     

盐胁迫下水杨酸及其衍生物对小黑麦幼苗生理特性的影响

盐胁迫下,适当浓度的两种酚酸均能降低幼苗叶片电导率和丙二醛(MDA)含量,增加根系活力。在相同处理浓度下．5-磺基水杨酸提高幼苗生理特性的效果比水杨酸好。     

Studies on exudate-depleted sclerotial development in Sclerotium rolfsii and the effect of oxalic acid,sclerotial exudate,and culture filtrate on phenolic acid induction in chickpea (Cicer arietinum)

Exudate depletion from developing sclerotia of Sclerotium rolfsii Sacc. in culture caused reduced size and weight of sclerotia. Germination of exudate-depleted sclerotia was delayed on Cyperus rotundus rhizome meal agar medium when compared with that of control sclerotia. The exudate-depleted sclerotia caused infection in chickpea (Cicer arietinum) plants in a glasshouse. Different temperatures and incubation periods had no effect on the germination ability of the exudate-depleted sclerotia. Oxalic acid, sclerotial exudate, and culture filtrate of S. rolfsii induced the synthesis of phenolic acids, including gallic, ferulic, chlorogenic, and cinnamic acids, as well as salicylic acid, in treated chickpea leaves. Gallic acid content was increased in treated leaves compared with the untreated controls. Maximum induction of gallic acid was seen in both leaves treated with oxalic acid followed by exudate and leaves treated with culture filtrate. Cinnamic and salicylic acids were not induced in exudate-treated leaves. Ethyl acetate fractionation indicated that the sclerotial exudates consisted of gallic, oxalic, ferulic, chlorogenic, and cinnamic acids, whereas the culture filtrate consisted of gallic, oxalic, and cinnamic acids along with many other unidentified compounds.     

Salicylic acid induces amelioration of chromium toxicity and affects antioxidant enzyme activity in Sorghum bicolor L.

Aim: Chromium (Cr(VI)) would inflict serious morphological, metabolic, and physiological anomalies in plants ranging from chlorosis of shoot to lipid peroxidation and protein degradation. Cr(VI) toxicity is often associated with oxidative stress, caused by the excessive formation of reactive oxygen species (ROS). In response, plants are equipped with a repertoire of mechanisms to counteract heavy metal (HM) toxicity. Salicylic acid (SA) plays a key role in the signal transduction pathways of various stress responses, demonstrating the protective effect of SA against abiotic stress factors. So, the present investigation was carried out to study the amelioration of pernicious effects of different concentration of Cr(VI) (0.0, 2.0, and 4.0?mg Cr(VI) kg^?1 soil in the form of potassium dichromate) by treatments of salicylic acid solution viz. pretreatment and foliar spray via antioxidative enzymes and their metabolites.

Results: With different treatments of salicylic acid solution, the reinstatement from ill effects of Cr(VI) toxicity was contemplated but the most conspicuous effect was observed when salicylic acid solution was supplied through the foliar spray (0.50?mM). This was accompanied with an increase in ascorbate peroxidase activity and hydrogen peroxide content and decrease in peroxidase activity and ascorbic acid content.

Significance of the study: This study suggests that salicylic acid when applied through pre-treatment of seeds or through a foliar spray can be used to ameliorate the toxic effects of chromium (VI). Salicylic acid has the great potential for reducing the toxicity of heavy metals without negatively impacting the growth of the plants.