Localization of Pathogenesis-Related Proteins in the Epidermis and Intercellular Spaces of Tobacco Leaves after Their Induction by Potassium Salicylate or Tobacco Mosaic Virus Infection

The localization of pathogenesis-related (PR) proteins inducedin tobacco leaves by treatment with potassium salicylate ora hypersensitive response to tobacco mosaic virus (TMV) infectionwas studied using immunochemical methods. Total PR protein levelsincreased with time after these treatments. The proportion ofPR proteins in the intercellular spaces to the total contentin the leaf discs rapidly rose in the later stage of the treatmentto about 75% on the 9th day after salicylate treatment and tomore than 80% on the 6th to 9th day after TMV inoculation. After5 days of salicylate treatment, the amounts of PR proteins inthe peeled leaf epidermis were two fold those in the mesophylltissue. Only five percent or less of the total PR proteins inthe epidermal and mesophyll tissues of salicylate-treated leaveswere detected in the isolated epidermal and mesophyll protoplasts.The sugar content in highly purified PR la, lb and lc was lessthan one mole of monosaccharide per mole of each protein. Theseresults show that the PR proteins are non-glycoproteins secretedinto the intercellular spaces. (Received January 16, 1987; Accepted July 14, 1987)     

Auxin-induced ethylene production as related to auxin metabolism in leaf discs of tobacco and sugar beet

Exogenously supplied indole-3-acetic acid (IAA) stimulated ethylene production in tobacco (Nicotiana glauca) leaf discs but not in those of sugar beet (Beta vulgaris L.). The stimulatory effect of IAA in tobacco was relatively small during the first 24 hours of incubation but became greater during the next 24 hours. It was found that leaf discs of these two species metabolized [1-¹⁴C]IAA quite differently. The rate of decarboxylation in sugar beet discs was much higher than in tobacco. The latter contained much less free IAA but a markedly higher level of IAA conjugates. The major conjugate in the sugar beet extracts was indole-3-acetylaspartic acid, whereas tobacco extracts contained mainly three polar IAA conjugates which were not found in the sugar beet extracts. The accumulation of the unidentified conjugates corresponded with the rise of ethylene production in the tobacco leaf discs. Reapplication of all the extracted IAA conjugates resulted in a great stimulation of ethylene production by tobacco leaf discs which was accompanied by decarboxylation of the IAA conjugates. The results suggest that in tobacco IAA-treated leaf discs the IAA conjugates could stimulate ethylene production by a slow release of free IAA. The inability of the exogenously supplied IAA to stimulate ethylene production in the sugar beet leaf discs was not due to a deficiency of free IAA within the tissue but rather to the lack of responsiveness of this tissue to IAA, probably because of an autoinhibitory mechanism existing in the sugar beet leaf discs.     

Some Soybean Cultivars Have Ability to Induce Germination of Sunflower Broomrape

Sunflower broomrape is a noxious parasitic weed which has caused severe damage to crop ecosystems. Trap crops can release a mixture of allelochemicals to induce the germination of sunflower broomrape. We studied the allelopathic effects of soybean on sunflower broomrape. Fourteen common soybean cultivars were grown in pots. Samples were collected from soybean plants and rhizosphere soil at five growth stages (V1, V3, V5, R2, and R4). The allelopathic effects of soybean reached highest at the V3 stage. Methanolic extracts of soybean roots induced higher broomrape germination than methanolic extracts of stems or leaves. The germination rates induced by root extracts (10-fold dilution) were positively correlated with germination rates induced by stem (10-fold dilution) and leaf extracts (10-fold dilution). The broomrape germination rates induced by root extracts were also positively correlated with soybean nodule diameter and dry weight. The results indicated that soybeans could induce sunflower broomrape germination. We conclude that soybean has the potential to be used as a trap crop for sunflower broomrape.     

Virus-induced synthesis of messenger RNAs for precursors of pathogenesis-related proteins in tobacco

Infection of Samsun NN tobacco with tobacco mosaic virus (TMV) induces a number of host-encoded, so-called pathogenesis-related (PR-) proteins, which are found in the intercellular space of the leaf and are associated with induced resistance. By immunoprecipitation of their in vitro translation products we were able to detect the mRNAs corresponding to a number of PR-proteins in TMV-infected tobacco, but not in healthy plants. Analysis by the Northern blot technique using cloned cDNA of PR1-mRNAs as probe showed that the mRNAs for the closely related proteins PR1a, 1b and 1c occur at a low level in healthy tobacco; upon TMV infection this level is increased > 100-fold. The PR1-specific probe did no hybridize to mRNAs corresponding to other PR-proteins. Sequencing of the 5'-terminal region of PR1-mRNAs showed that PR1-proteins are derived from precursors by removal of an N-terminal signal peptide of 30 amino acids.     

Immunochemical Localization of Pathogenesis-Related Proteins Secreted into the Intercellular Spaces of Salicylate-Treated Tobacco Leaves

In tobacco leaves, pathogenesis-related (PR) 1 proteins areabundantly induced by hypersensitive reaction to the infectionwith tobacco mosaic virus (TMV) and by treatment with salicylicacid, and are secreted into the intercellular spaces. To studythe distribution of PR 1 proteins outside of the cells, theimmunogold technique was used with anti-PR 1 antibody. Whensections of salicylate-treated tobacco leaf were reacted withantibody against PR 1a and then with protein A-gold complex,most of the gold label was localized in the intercellular spacesin the region between cells, and a little label was found inthe cytoplasmic matrix and in small electron-dense granulesinside the cells. When salicylate-treated leaves were incubatedwith polygalacturonase and/or cellulase to liberate protoplastsor single cells from the tissue, most of PR 1 proteins weresolubilized far before complete liberation of single cells,suggesting their localization in free spaces or a region susceptableto maceration, such as the secondary cell wall. (Received May 30, 1988; Accepted June 27, 1988)     

WATER RELATIONS OF WATER-STRESSED,SPLIT-ROOT C4 (SORGHUM BICOLOR; POACEAE) AND C3 (HELIANTHUS ANNUUS; ASTERACEAE) PLANTS

Sorghum [Sorghum bicolor (L.) Moench] and sunflower (Helianthus annuus L.) were grown in a greenhouse with roots divided between sand irrigated with nutrient solution (–0.097 MPa) or nutrient solution containing polyethylene glycol (PEG) (–0.570 MPa) to compare the effect of unequal root zone stress on plant water relations of a C₄ (sorghum) and a C₃ (sunflower) plant. Roots also were divided between two pots of sand irrigated only with nutrient solution (controls) or only with PEG in nutrient solution. In addition to plant water-status measurements, photosynthetic rate, growth (height, root, and shoot dry weights), and evolution of ethylene (a gaseous hormone indicative of stress) were measured. Under all three split-root treatments, sunflower had a lower leaf water potential and produced more ethylene than sorghum. Sunflower was able to survive the PEG stress if half of its root system was under nonstressed conditions. Sunflower with half its root system irrigated with PEG usually had values of leaf water potential, osmotic potential, stomatal resistance, transpiration rate, photosynthetic rate, ethylene evolution, height, and dry weights that were close to those of the control plants. Sunflower with all roots exposed to PEG was wilted severely. Sorghum was little affected by PEG stress applied either to half or all the root system. Growth of sorghum was the same under all treatments. Apparently because stomata of sorghum were more closed in the partial stress test than those of sunflower, sorghum conserved water and had a higher leaf water potential, which might have permitted growth with stress.     

Bemnisia tabaci feeding induces pathogenesis-related proteins in cassava (Manihot esculenta Crantz)

Cassava (Manihot esculenta Cranzts) plants fed upon by whitefly Bemisia tabaci showed increased levels of pathogenesis-related (PR) proteins, such as beta-1, 3-glucanase, peroxidase and chitinase activities, as compared to uninfested plants. The enzymes increased in specific activities from 2 to 7 fold and protein content in leaf extracts decreased in whitefly-infested plants, compared to uninfested plants. Among the three PR proteins, B. tabaci feeding induced significantly higher beta-1, 3-glucanase activities, when compared with other two PR proteins. Study also discussed the possible application of PR proteins in whitefly control program.     

Weed control in sunflower (helianthus annuus L.) with post-emergent herbicides

Sunflower is the most important oil crop in Hungary, is the base of the production of cooking oil and moreover takes an important part in production of margarine too. Extracted sunflower groats as a secondary product origining from the mentioned procedure can be used in forage successfully. The amount of harvested sunflower reaches the 20-25% of the EU's yield. The sowing area approaches 500 thousand hectares. The essential condition of successful crop production is the perfect weed control. Sowing areas are infected with monocotyledon and dicotyledonous weeds too. Annual dicotyledonous weeds are the most troublesome. The worst species is the Ambrosia artemisiifolia L. Many other weed species as Abutilon theophrasti MEDIC., Datura stramonium L. and Xanthium strumarium L. can cause serious damages. In our model experiments we examined the herbicide sensibility of two commercial sunflower cultivars as "Iregi szürke csikos", "Marica II" and a sulfonylurea-urea tolerant new hybrid "PR63E82". The experiment was set up under greenhouse conditions with the use of four important weed and different post-emergent herbicide as Modown 4F (bifenox), Pledge 50 WP (flumioxazin) and Granstar 75 DF (tribenuron-methyl). We applied normal and double doses too. Sunflower was cultivated to 4-6 leaf stage. Post-emergent herbicides were sprayed out when weeds were in 2, 2-4 and 4-6 leaf stage. Weed killer and phytotoxic effects of post-emergent herbicides were examined. We declared that development of weeds had significally effect on the effectiveness of different herbicides.     

Sunflower (Helianthus annuus) long‐chain acyl‐coenzyme A synthetases expressed at high levels in developing seeds

Long chain fatty acid synthetases (LACSs) activate the fatty acid chains produced by plastidial de novo biosynthesis to generate acyl‐CoA derivatives, important intermediates in lipid metabolism. Oilseeds, like sunflower, accumulate high levels of triacylglycerols (TAGs) in their seeds to nourish the embryo during germination. This requires that sunflower seed endosperm supports very active glycerolipid synthesis during development. Sunflower seed plastids produce large amounts of fatty acids, which must be activated through the action of LACSs, in order to be incorporated into TAGs. We cloned two different LACS genes from developing sunflower endosperm, HaLACS1 and HaLACS2, which displayed sequence homology with Arabidopsis LACS9 and LACS8 genes, respectively. These genes were expressed at high levels in developing seeds and exhibited distinct subcellular distributions. We generated constructs in which these proteins were fused to green fluorescent protein and performed transient expression experiments in tobacco cells. The HaLACS1 protein associated with the external envelope of tobacco chloroplasts, whereas HaLACS2 was strongly bound to the endoplasmic reticulum. Finally, both proteins were overexpressed in Escherichia coli and recovered as active enzymes in the bacterial membranes. Both enzymes displayed similar substrate specificities, with a very high preference for oleic acid and weaker activity toward stearic acid. On the basis of our findings, we discuss the role of these enzymes in sunflower oil synthesis.     

Spermine Is a Salicylate-Independent Endogenous Inducer for Both Tobacco Acidic Pathogenesis-Related Proteins and Resistance against Tobacco Mosaic Virus Infection

Intercellular spaces are often the first sites invaded by pathogens. In the spaces of tobacco mosaic virus (TMV)-infected and necrotic lesion-forming tobacco (Nicotiana tabacum L.) leaves, we found that an inducer for acidic pathogenesis-related (PR) proteins was accumulated. The induction activity was recovered in gel-filtrated fractions of low molecular mass with a basic nature, into which authentic spermine (Spm) was eluted. We quantified polyamines in the intercellular spaces of the necrotic lesion-forming leaves and found 20-fold higher levels of free Spm than in healthy leaves. Among several polyamines tested, exogenously supplied Spm induced acidic PR-1 gene expression. Immunoblot analysis showed that Spm treatment increased not only acidic PR-1 but also acidic PR-2, PR-3, and PR-5 protein accumulation. Treatment of healthy tobacco leaves with salicylic acid (SA) caused no significant increase in the level of endogenous Spm, and Spm did not increase the level of endogenous SA, suggesting that induction of acidic PR proteins by Spm is independent of SA. The size of TMV-induced local lesions was reduced by Spm treatment. These results indicate that Spm accumulates outside of cells after lesion formation and induces both acidic PR proteins and resistance against TMV via a SA-independent signaling pathway.     

Antifungal activity of chitin-binding PR-4 type proteins from barley grain and stressed leaf.

Antifungal activity in vitro has been associated with barley leaf and grain proteins which are homologous with pathogenesis related proteins of type 4 (PR-4) from tobacco and tomato and with C terminal domains of potato win and Hevea hevein precursor proteins. One protein (pI approximately 9.3, M(r) approximately 13.7 kDa) from barley grain and two very similar proteins from leaves infected with Erysiphe graminis were isolated by chitin affinity chromatography, but none of the proteins showed chitinase activity in vitro. The leaf proteins were increased several fold in response to either Erysiphe infection or NiCl2 infiltration and accumulated extracellularly. The three barley proteins were found to inhibit growth of Trichoderma harzianum in microtiter plate assays using approximately 10 micrograms/ml concentrations and in lower concentrations in a synergistic way when mixed either with barley chitinase C (a PR-3 type protein) or with barley protein R (a PR-5 type protein). Structurally similar proteins were detected in wheat, rye and oats grain extracts.     

Leaf catalase mRNA and catalase-protein levels in a high-catalase tobacco mutant with o(2)-resistant photosynthesis

Experiments were conducted with a tobacco (Nicotiana tabacum) mutant with 40 to 50% greater catalase activity than wild type that is associated with a novel form of O₂-resistant photosynthesis. The apparent K_m for H₂O₂ was the same in mutant and wild-type leaf extracts. Tobacco RNAs were hybridized with Nicotiana sylvestris catalase cDNA, and a threefold greater steady-state level of catalase mRNA was found in mutant leaves. Steady-state levels of ribulose-1,5-bisphosphate carboxylase small subunit mRNA were similar in mutant and wild type. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially purified catalase showed that the protein concentration in the band corresponding to catalase was higher in the mutant than in the wild type. Separation of leaf catalase proteins by isoelectric focusing revealed the presence of five major bands and one minor band of activity. The distribution of the catalase activity among these forms was similar in mutant and wild type, although the total activity was higher in the mutant in all five major bands. The results indicate that the enhanced catalase activity in mutant leaves is caused by an increase in synthesis of catalase protein and that this trait is mediated at the nucleic acid level.     

Effect of glycidate, an inhibitor of glycolate synthesis in leaves, on the activity of some enzymes of the glycolate pathway

Under conditions where glycolate synthesis was inhibited at least 50% in tobacco (Nicotiana tabacum L.) leaf discs treated with glycidate (2,3-epoxypropionate), the ribulose diphosphate carboxylase activity in extracts and the inhibition of the activity by 100% oxygen were unaffected by the glycidate treatment. [1-¹⁴C]Glycidate was readily taken into leaf discs and was bound to leaf proteins, but the binding occurred preferentially with proteins of molecular weight lower than ribulose diphosphate carboxylase. Glycidate added to the isolated enzyme did not inhibit ribulose diphosphate carboxylase activity or affect its inhibition by 100% O₂. Thus, glycidate did not inhibit glycolate synthesis by a direct effect on ribulose diphosphate carboxylase/oxygenase.     

The biosynthesis of sulfoquinovosyldiacylglycerol: studies with groundnut (Arachis hypogaea) leaves

The biosynthetic pathway of sulfoquinovosyldiacylglycerol (SQDG) was investigated using groundnut (Arachis hypogaea) leaf discs and 35S-labeled precursors. [35S]SO4(2-) was actively taken up by the leaf discs and rapidly incorporated into SQDG. After 2 h, 1.5% of the [35S]SO4(2-) added to the incubation medium was taken up, of which 28% was incorporated into SQDG. The methanol-water phases of the lipid extracts of the leaf discs were analyzed for the 35S-labeled intermediates. Up to 2 h of incubation, cysteic acid, 3-sulfopyruvate, 3-sulfolactate, 3-sulfolactaldehyde, and sulfoquinovose (SQ) which have been proposed as intermediates [Davies et al. (1966) Biochem. J. 98, 369-373] were not labeled. Only a negligible amount of radioactivity was observed in these compounds after incubation for 4 h and more. Addition of sodium molybdate inhibited the uptake of [35S]SO4(2-) as well as its incorporation into SQDG by the leaf discs, suggesting that 3'-phosphoadenosine-5'-phosphosulfate may be involved in the biosynthesis of SQDG. Addition of unlabeled cysteic acid to the incubation medium enhanced the uptake of [35S]SO4(2-) but did not affect its incorporation into SQDG. 35S-labeled cysteic acid was taken up by the leaf discs and metabolized to sulfoacetic acid but not incorporated into SQ or SQDG. These results show that cysteic acid is not an intermediate in SQDG biosynthesis. [35S]SQ was taken up by the leaf discs and incorporated into SQDG in a time-dependent manner. [35S]Sulfoquinovosylglycerol was also taken up by the leaf discs but not incorporated into SQDG. It is concluded that SQDG is not biosynthesized by the proposed sulfoglycolytic pathway in higher plants. Though [35S]SQ was converted to SQDG, the rates are much lower compared to [35S]SO4(2-) incorporation, which suggests that a more direct pathway involving sulfonation of a lipid precursor may exist in higher plants.     

Biological and molecular comparison between localized and systemic acquired resistance induced in tobacco by a Phytophthora megasperma glycoprotein elicitin

We have compared localized (LAR) and systemic (SAR) acquired resistance induced in tobacco by a hypersensitive response (HR) inducing Phytophthora megasperma glycoprotein elicitin. Three different zones were taken into account: LAR, SAR_T and SAR_S. The LAR zone was 5–10 mm wide and surrounded the HR lesion. SAR_T was the tissue of the elicitor-treated leaf immediately beyond the LAR zone. The systemic leaf was called SAR_S. Glycoprotein-treated plants showed enhanced resistance to challenge infection by tobacco mosaic virus (TMV). Disease resistance was similar in SAR_T and SAR_S, and higher in LAR. The expression pattern, in glycoprotein-treated plants, of acidic and basic PR1, PR2, PR3 and PR5 proteins and of O-methyltransferases (OMT), enzymes of the phenylpropanoid pathway, was similar to that in TMV-infected plants. OMT was stimulated in LAR but not in SAR_T and SAR_S. The four classes of acidic and basic PR proteins accumulated strongly in LAR. Reduced amounts of acidic PR1, PR2, PR3 and only minute amounts of basic PR2 and PR3 accumulated in SAR_T and SAR_S. In glycoprotein-treated plants, expression of the acidic and basic PR proteins in LAR and SAR of transgenic NahG and ETR tobacco plants and in LAR of plants treated with inhibitors of salicylic acid accumulation and of ethylene biosynthesis indicated a salicylic acid-dependent signalling pathway for acidic isoform activation and an ethylene-dependent signalling pathway for basic isoform activation.     

Synthesis and localization of pathogenesis-related proteins in tobacco.

The PR1 family of pathogenesis-related proteins from tobacco (Nicotiana tabacum L.) leaves is induced by a variety of pathogenic and chemical agents and is associated with resistance to tobacco mosaic virus. The majority of the PR1 proteins did not copurify with mesophyll protoplasts (the major cell type of the leaf) isolated from tobacco mosaic virus-infected N. tabacum cv. Xanthi-nc leaves. However, these isolated protoplasts were capable of synthesizing and selectively secreting the PR1 proteins. Using monoclonal antibodies for immunofluorescence microscopy, we localized these proteins to the extracellular spaces predominantly in regions adjacent to viral lesions as well as in xylem elements of infected leaves.     

Effect of Ethephon on Protein Degradation and the Accumulation of ;Pathogenesis-Related' (PR) Proteins in Tomato Leaf Discs

The effect of ethephon (2-chloroetylphosphonic acid) on the degradation of proteins and on the induction of Lycopersicon esculentum pathogenesis-related (PR) proteins was studied in tomato leaf discs. The rate of ribulose, -1,5-bisphosphate carboxylase/oxygenase (Rubisco) degradation was maximal in discs after 48 hours of incubation with 1 millimolar ethephon, leading to complete disappearance of Rubisco after 96 hours. This effect was correlated with an increase in PR protein synthesis and the induction of the previously reported alkaline proteolytic enzyme PR-P69 (P Vera, V Conejero [1988] Plant Physiol 87: 58-63). In vivo pulse-chase experiments demonstrated that ethephon not only affected Rubisco content but that of many other ³⁵S-labeled proteins as well, indicating that ethylene activates a general and nonspecific mechanism of protein degradation. This effect was partially inhibited in vivo by the action of pCMB, a selective inhibitor of cysteine-proteinases such as P69. These data reinforce the hypothesis that P69 and perhaps other PR proteins are involved in the mechanism of accelerated protein degradation activated by ethylene.     

The Association of "Pathogenesis-related" Proteins with Viral Induced Necrosis in Nicotiana sylvestris

The association of “pathogenesis-related” (PR) proteins with protection from superinfection, systemic acquired resistance and production of localized necrotic lesions was examined with a system using tobacco mosaic virus (TMV) and Nicotiana sylvestris. Leaves of N. sylvestris with a mosaic from earlier inoculation with a systemically infecting strain of TMV (TMV-C) and control plants were challenged with a necrotizing strain of TMV (TMV-P), RNA of TMV-P and turnip mosaic virus (TuMV). TMV-P virions produced localized necrotic lesions only in the dark green areas of the mosaic of TMV-C infected plants. Both RNA of TMV-P and TuMV produced localized necrotic lesions in both light green and dark green areas of the mosaic of TMV-C infected plants. All three challenge inocula produced localized necrotic lesions in previously uninoculated plants. Six days after challenge inoculation proteins were extracted from separated dark green and light green mosaic leaf tissue, and leaf material from control plants. Proteins were separated by electrophoresis in a 5 % polyacrylamide spacer gel and 10 % polyacrylamide running gel. PR proteins were found in tissue where localized necrotic lesions were produced as a result of challenge inoculation, but not in tissue that was not superinfected. PR proteins were not found in light green or dark green mosaic leaf tissue as a result of TMV-C inoculation. No PR proteins were evident in protein extracts from light green tissue challenged with TMV-P, although PR proteins were produced in dark green tissue, where necrosis occurred, from the same leaves. Systemic acquired resistance (reduction in size of lesions formed by a challenge inoculation) to TuMV or RNA of TMV-P and PR protein concentration was measured at various times in light green areas of mosaic leaves where dark green areas of the mosaic leaves had been inoculated with TMV-P. No quantitative or temporal relationship between the onset of resistance and PR protein production was found. It is concluded that PR proteins are a result of pathogen induced necrosis and not significantly involved in the mechanism(s) of viral induced resistance.