Iodus 40, salicylic acid, heptanoyl salicylic acid and trehalose exhibit different efficacies and defence targets during a wheat/powdery mildew interaction

Prophylactic efficacies of Iodus 40 and salicylic acid (SA) against wheat powdery mildew caused by Blumeria graminis f. sp. tritici have been shown and compared with those of heptanoyl salicylic acid (HSA) and trehalose. Plantlets treated once exhibited 55%, 50%, 95%, and 38% protection levels, respectively. Two sprayings increased these levels up to 60%, 65%, 100%, and 60%, respectively. Biological effects of these resistance inducers on reactive oxygen species (ROS) metabolism and lipid peroxidation were also investigated. We found clear differences in the extent and the type of induced responses, with HSA exhibiting both the most numerous and the highest effects. HSA and SA induced a 5.5-fold increase of whole cell DAB staining due to hydrogen peroxide accumulation, whereas Iodus 40 and trehalose increased staining intensity at the penetration sites only. However, these effects were not correlated with any modification of catalase (CAT), oxalate oxidase (OXO) or lipoxygenase (LOX) activities, except for HSA which decreased CAT in non-inoculated conditions and increased LOX in infectious conditions. HSA also induced an increase in the rate of lipid peroxidation, whereas Iodus 40 induced a decrease. The effects of the inducers on germinating conidia and wheat epidermal cells responding to fungal penetration were also investigated. Papilla-linked autofluorescence was affected by SA and Iodus 40 whereas germination was slightly altered by Iodus 40. The newly described protective efficacies and the partial, distinct and non-overlapping activities of these inducers on the wheat/powdery mildew interaction are discussed.     

Changes in lipid composition of Blumeria graminis f.sp. tritici conidia produced on wheat leaves treated with heptanoyl salicylic acid

Treatment of wheat leaves with heptanoyl salicylic acid (HS) and trehalose at concentrations of 0.1 and 15 g l(-1), prior to fungal inoculation, resulted in 40% and 60% protection, respectively, against powdery mildew. The total lipid composition of Blumeria graminis f.sp. tritici (Bgt) conidia, the causal agent of wheat powdery mildew, was compared when produced on wheat leaves, respectively, untreated and treated with the two elicitors, HS and trehalose. An obvious effect was observed on lipid composition (sterol and fatty acid (FA)) of Bgt conidia produced on wheat leaves treated with HS. A total of 16 FA (C12-C24 saturated and unsaturated) as well as unusual methoxylated Fatty Acids (mFA) (3-methoxydocosanoic and 3-methoxytetracosanoic acids) were detected in the conidia. Medium chain FA were predominant in HS treated conidia (64.65%) while long chain fatty acids constituted the major compounds in untreated conidia (62%). The long chain/medium chain FA ratio decreased from 1.8 in the conidia produced on untreated leaves to 0.5 in the conidia obtained from HS treated leaves. When comparing the sterol composition of Bgt conidia produced on leaves treated with HS versus conidia obtained from untreated ones, very important changes within the two major classes can be seen. In particular, 24-methylsterols, e.g., 24-methylenecholesterol and 24-methylcholesta-7,24-dien were reduced by about 82% whereas 24-ethylsterols, e.g., 24-ethylcholesterol and 24-ethylcholesta-5,22-dienol were increased by about 85%. The 24-methylsterols/24-ethylsterols ratio was reduced by ninefold in the conidia produced from HS treated leaves.     

Chemical-induced resistance against powdery mildew in barley: the effects of chitosan and benzothiadiazole

Chitosan (CHT), a deacetylated chitin derivative, and benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a non toxic synthetic functional analogue of salicylic acid, were applied as foliar spray to barley plants (Hordeum vulgare L.), to compare their effectiveness in inducing resistance against Blumeria graminis f. sp. hordei and to investigate the underlying defence response. After an induction phase of 3 days (IP, time elapsed between treatment and fungal inoculation) both compounds reduced significantly the infection on the primary leaf, namely of 55.5% for CHT and of 68.9% for BTH, showing the induction of a good level of local resistance (LAR). A 5-day IP further reduced the infected areas in BTH treated plants (−77.2%) but not in CHT treated ones (−47.1%). Furthermore, both CHT and BTH also induced SAR, being the infection in the second non treated leaves reduced of 57% and 76.2%, respectively, as evaluated at 10-day IP. Both BTH and CHT induced oxidative burst and phenolic compound deposition in treated leaves, creating an hostile environment that slowed down the fungal spreading by impairing haustorium development. However, the greater efficacy of BTH was possibly due to: i) a greater reinforcement of papilla; ii) a higher level and the more homogeneous diffusion of H₂O₂ in the treated leaf tissues and iii) an induced hypersensitive-like response in many penetrated cells.     

Molecular cloning of two novel peroxidases and their response to salt stress and salicylic acid in the living fossil Ginkgo biloba

Background and Aims

Peroxidase isoenzymes play diverse roles in plant physiology, such as lignification and defence against pathogens. The actions and regulation of many peroxidases are not known with much accuracy. A number of studies have reported direct involvement of peroxidase isoenzymes in the oxidation of monolignols, which constitutes the last step in the lignin biosynthesis pathway. However, most of the available data concern only peroxidases and lignins from angiosperms. This study describes the molecular cloning of two novel peroxidases from the ‘living fossil’ Ginkgo biloba and their regulation by salt stress and salicylic acid.

Methods

Suspension cell cultures were used to purify peroxidases and to obtain the cDNAs. Treatments with salicylic acid and sodium chloride were performed and peroxidase activity and gene expression were monitored.

Key Results

A novel peroxidase was purified, which preferentially used p-hydroxycinnamyl alcohols as substrates and was able to form dehydrogenation polymers in vitro from coniferyl and sinapyl alcohols. Two peroxidase full-length cDNAs, GbPrx09 and GbPrx10, were cloned. Both peroxidases showed high similarity to other basic peroxidases with a putative role in cell wall lignification. Both GbPrx09 and GbPrx10 were expressed in leaves and stems of the plant. Sodium chloride enhanced the gene expression of GbPrx09 but repressed GbPrx10, whereas salicylic acid strongly repressed both GbPrx09 and GbPrx10.

Conclusions

Taken together, the data suggest the participation of GbPrx09 and GbPrx10 in the developmental lignification programme of the cell wall. Both peroxidases possess the structural characteristics necessary for sinapyl alcohol oxidation. Moreover, GbPrx09 is also involved in lignification induced by salt stress, while salicylic acid-mediated lignification is not a result of GbPrx09 and GbPrx10 enzymatic activity.     

Stress enhances the gene expression and enzyme activity of phenylalanine ammonia-lyase and the endogenous content of salicylic acid to induce flowering in pharbitis

The involvement of salicylic acid (SA) in the regulation of stress-induced flowering in the short-day plant pharbitis (also called Japanese morning glory) Ipomoea nil (formerly Pharbitis nil) was studied. Pharbitis cv. Violet was induced to flower when grown in 1/100-strength mineral nutrient solution under non-inductive long-day conditions. All fully expanded true leaves were removed from seedlings, leaving only the cotyledons, and flowering was induced under poor-nutrition stress conditions. This indicates that cotyledons can play a role in the regulation of poor-nutrition stress-induced flowering. The expression of the pharbitis homolog of PHENYLALANINE AMMONIA-LYASE, the enzyme activity of phenylalanine ammonia-lyase (PAL; E.C. 4.3.1.5) and the content of SA in the cotyledons were all up-regulated by the stress treatment. The Violet was also induced to flower by low-temperature stress, DNA demethylation and short-day treatment. Low-temperature stress enhanced PAL activity, whereas non-stress factors such as DNA demethylation and short-day treatment decreased the activity. The PAL enzyme activity was also examined in another cultivar, Tendan, obtaining similar results to Violet. The exogenously applied SA did not induce flowering under non-stress conditions but did promote flowering under weak stress conditions in both cultivars. These results suggest that stress-induced flowering in pharbitis is induced, at least partly, by SA, and the synthesis of SA is promoted by PAL.     

Use of blue-green and chlorophyll fluorescence measurements for differentiation between nitrogen deficiency and pathogen infection in winter wheat

In recent years, several sensor-based approaches have been established to early detect single plant stresses, but the challenge of discriminating between simultaneously occurring stressors still remains. Earlier studies on wheat plants strongly affected by pathogens and nitrogen deficiency indicated that chlorophyll fluorescence might be suited to distinguish between the two stressors. Nevertheless, there is lack of information on the pre-symptomatic detection of synchronized occurrence of slight N-deficiency and the early stages of pathogen infection. The usefulness of the blue, green, and yellow fluorescence signals in this context has not yet been explored. We hypothesized that differentiation between wheat plants’ physiological reaction due to N-deficiency and leaf rust (Puccinia triticina) as well as N-deficiency and powdery mildew (Blumeria graminis f. sp. tritici) might be accomplished by means of UV laser-induced fluorescence spectral measurements between 370 and 620 nm in addition to chlorophyll fluorescence (640-800 nm). Plants were provided with either a normal or a modified Hoagland nutrient solution in order to induce a slight N deficit. Pathogen inoculation was carried out on the second fully developed leaf. Four experimental groups were evaluated: (a) N-full-supply [N+]; (b) N-deficiency [N−]; (c) N-full-supply + pathogen [N+/LR] or [N+/PM]; (d) N-deficiency + pathogen [N−/LR] or [N−/PM]. The results revealed that, in addition to the amplitude ratio of R/FR fluorescence, B/G fluorescence also facilitated reliable and robust discrimination among the four experimental groups. The discrimination among the experimental groups was accomplished as early as one and two days after inoculation for powdery mildew and leaf rust infection, respectively. During the 3 days evaluation period, the differences among the treatment groups became more evident. Moreover, several other amplitude ratios and half-bandwidth ratios proved to be suited to early detect fungal infection, irrespective of the nitrogen status of the plant.     

Transcription of the Hsp30, Hsp70, and Hsp90 heat shock protein genes is modulated by the PalA protein in response to acid pH-sensing in the fungus <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>

Heat shock proteins are molecular chaperones linked to a myriad of physiological functions in both prokaryotes and eukaryotes. In this study, we show that the Aspergillus nidulans hsp30 (ANID_03555.1), hsp70 (ANID_05129.1), and hsp90 (ANID_08269.1) genes are preferentially expressed in an acidic milieu, whose expression is dependent on the palA ⁺ background under optimal temperature for fungal growth. Heat shock induction of these three hsp genes showed different patterns in response to extracellular pH changes in the palA⁺ background. However, their accumulation upon heating for 2 h was almost unaffected by ambient pH changes in the palA ⁻ background. The PalA protein is a member of a conserved signaling cascade that is involved in the pH-mediated regulation of gene expression. Moreover, we identified several genes whose expression at pH 5.0 is also dependent on the palA ⁺ background. These results reveal novel aspects of the heat- and pH-sensing networks of A. nidulans.     

Effect of pentoxifylline on arachidonic acid metabolism,neutral lipid synthesis and accumulation during induction of the lipocyte phenotype by retinol in murine hepatic stellate cell

In liver fibrosis, the quiescent hepatic stellate cells (HSC) are activated to proliferate and express the activated myofibroblast phenotype, losing fat droplets and the stored vitamin A, and depositing more extracellular matrix. Therapeutic strategies for liver fibrosis are focused on HSC. Pentoxifylline (PTF), an analog of the methylxanthine, prevents the biochemical and histological changes associated with animal liver fibrosis. The aim of the present study was to investigate the phenotypic change of myofibroblasts into quiescent lipocytes by PTF and/or retinol, using a permanent cell line GRX that represents murine HSC. We studied the action of both drugs on the synthesis of neutral lipids, activity of phospholipase A₂ (PLA₂), release of arachidonic acid (AA) and prostaglandins synthesis. Accumulation and synthesis of neutral lipids was dependent upon association of retinol with PTF. PTF (0.5 mg/mL) alone did not induce lipid accumulation and synthesis, but in cells induced by physiologic concentration of retinol (1–2.5 M), it increased the quantity of stored lipids. Retinol and PTF (5 M and 0.1 mg/mL, respectively) had a synergistic effect on neutral lipid synthesis and accumulation. In higher PTF concentrations (0.5 and 0.7 mg/ml), the synthesis was stimulated but accumulation decreased. Membrane-associated PLA₂ activity decreased after PTF treatment, which increased the AA release 8 fold, and significantly increased the production of PGE₂, but not of PGF₂. However, when in presence of retinol, we observed a slightly higher increase in PGE₂ and PGF₂ production. In conclusion, PTF treatment generated an excess of free AA. We propose that retinol counteracts the action of PTF on the AA release and PGs production, even though both drugs stimulated the lipocyte induction in the HSC.