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Systemically induced resistance is a promising strategy to control plant diseases, as it affects numerous pathogens. However, since induced resistance reduces one or both growth and activity of plant pathogens, the indigenous microflora may also be affected by an enhanced defensive state of the plant. The aim of this study was to elucidate how much the bacterial rhizosphere microflora of Arabidopsis is affected by induced systemic resistance (ISR) or systemic acquired resistance (SAR). Therefore, the bacterial microflora of wild-type plants and plants affected in their defense signaling was compared. Additionally, ISR was induced by application of methyl jasmonate and SAR by treatment with salicylic acid or benzothiadiazole. As a comparative model, we also used wild type and ethylene-insensitive tobacco. Some of the Arabidopsis genotypes affected in defense signaling showed altered numbers of culturable bacteria in their rhizospheres; however, effects were dependent on soil type. Effects of plant genotype on rhizosphere bacterial community structure could not be related to plant defense because chemical activation of ISR or SAR had no significant effects on density and structure of the rhizosphere bacterial community. These findings support the notion that control of plant diseases by elicitation of systemic resistance will not significantly affect the resident soil bacterial microflora.

Rhizobacteria–induced systemic resistance (ISR) and pathogen-induced systemic acquired resistance (SAR) have a broad, yet partly distinct, range of effectiveness against pathogenic microorganisms. Here, we investigated the effectiveness of ISR and SAR in Arabidopsis against the tissue-chewing insects Pieris rapae and Spodoptera exigua. Resistance against insects consists of direct defense, such as the production of toxins and feeding deterrents and indirect defense such as the production of plant volatiles that attract carnivorous enemies of the herbivores. Wind-tunnel experiments revealed that ISR and SAR did not affect herbivore-induced attraction of the parasitic wasp Cotesia rubecula (indirect defense). By contrast, ISR and SAR significantly reduced growth and development of the generalist herbivore S. exigua, although not that of the specialist P. rapae. This enhanced direct defense against S. exigua was associated with potentiated expression of the defense-related genes PDF1.2 and HEL. Expression profiling using a dedicated cDNA microarray revealed four additional, differentially primed genes in microbially induced S. exigua-challenged plants, three of which encode a lipid-transfer protein. Together, these results indicate that microbially induced plants are differentially primed for enhanced insect-responsive gene expression that is associated with increased direct defense against the generalist S. exigua but not against the specialist P. rapae.

Ran, L. X., van Loon, L. C., and Bakker, P. A. H. M. 2005. No role for bacterially produced salicylic acid in rhizobacterial induction of systemic resistance in Arabidopsis. Phytopathology 95:1349-1355. The role of bacterially produced salicylic acid (SA) in the induction of systemic resistance in plants by rhizobacteria is far from clear. The strong SA producer Pseudomonas fluorescens WCS374r induces resistance in radish but not in Arabidopsis thaliana, whereas application of SA leads to induction of resistance in both plant species. In this study, we compared P. fluorescens WCS374r with three other SA-producing fluorescent Pseudomonas strains, P. fluorescens WCS417r and CHA0r, and P. aeruginosa 7NSK2 for their abilities to produce SA under different growth conditions and to induce systemic resistance in A. thaliana against bacterial speck, caused by P. syringae pv. tomato. All strains produced SA in vitro, varying from 5 fg cell –1 for WCS417r to >25 fg cell –1 for WCS374r. Addition of 200 µM FeCl3 to standard succinate medium abolished SA production in all strains. Whereas the incubation temperature did not affect SA production by WCS417r and 7NSK2, strains WCS374r and CHA0r produced more SA when grown at 33 instead of 28°C. WCS417r, CHA0r, and 7NSK2 induced systemic resistance apparently associated with their ability to produce SA, but WCS374r did not. Conversely, a mutant of 7NSK2 unable to produce SA still triggered induced systemic resistance (ISR). The possible involvement of SA in the induction of resistance was evaluated using SA-nonaccumulating transgenic NahG plants. Strains WCS417r, CHA0r, and 7NSK2 induced resistance in NahG Arabidopsis. Also, WCS374r, when grown at 33 or 36°C, triggered ISR in these plants, but not in ethylene-insensitive ein2 or in non-plant pathogenesis-related protein-expressing npr1 mutant plants, irrespective of the growth temperature of the bacteria. These results demonstrate that, whereas WCS374r can be manipulated to trigger ISR in Arabidopsis, SA is not the primary determinant for the induction of systemic resistance against bacterial speck disease by this bacterium. Also, for the other SAproducing strains used in this study, bacterial determinants other than SA must be responsible for inducing resistance.

The ability of selected strains of fluorescent Pseudomonas spp. to cause induced systemic resistance (ISR) in Eucalyptus urophylla against bacterial wilt caused by Ralstonia solanacearum was investigated. Four of the five strains used can produce salicylic acid (SA) in vitro and, therefore, chemical SA, that is known to induce resistance in many plant species, was used as a reference treatment. Whereas a soil drench with SA did induce systemic resistance in E. urophylla, infiltration of SA into leaves did not. None of the fluorescent Pseudomonas spp. strains caused ISR against bacterial wilt when applied to the soil, but two strains, P. putida WCS358r and P. fluorescens WCS374r triggered ISR when infiltrated into two lower leaves 3–7 days before challenge inoculation. A mutant of strain WCS358r defective in the biosynthesis of the fluorescent siderophore pseudobactin, did not cause ISR, while the purified siderophore of WCS358r did, suggesting that pseudobactin358 is the ISR determinant of WCS358. A siderophore-minus mutant of WCS374r induced the same level of disease resistance as its parental strain, but the purified siderophore induced resistance as well, indicating that both the siderophore and another, unknown, inducing determinant(s) of WCS374r can trigger ISR in Eucalyptus. A possible role of WCS374r-produced SA remains uncertain. Transformation of a siderophore-minus mutant of WCS358 with the SA biosynthetic gene cluster from WCS374 did not enable this transformant to cause ISR in E. urophylla.

Transgenic tobacco plants (Tetr) expressing the mutant etr1-1 gene from Arabidopsis thaliana are insensitive to ethylene and develop symptoms of wilting and stem rot when grown in nonautoclaved soil. Several isolates of Fusarium, Thielaviopsis, and Pythium were recovered from stems of diseased Tetr plants. Inoculation with each of these isolates of 6-week-old plants growing in autoclaved soil caused disease in Tetr plants but not in nontransformed plants. Also, when 2-week-old seedlings were used, nontransformed tobacco appeared nonsusceptible to the Fusarium isolates, whereas Tetr seedlings did develop disease. Tetr seedlings were not susceptible to several nonhost Fusarium isolates. In contrast to results with Fusarium isolates, inoculation of 2-week-old seedlings with a Thielaviopsis isolate resulted in equal symptom development of nontransformed and Tetr tobacco. In order to explore the potential range of pathogens to which Tetr tobacco plants display enhanced susceptibility, the pathogenicity of several root and leaf pathogens was tested. Tetr plants were more susceptible to the necrotrophic fungi Botrytis cinerea and Cercospora nicotianae and the bacterium Erwinia carotovora, but only marginally more to the bacterium Ralstonia solanacearum. In contrast, the biotrophic fungus Oidium neolycopersici, the oomycete Peronospora tabacina, and Tobacco mosaic virus caused similar or less severe symptoms on Tetr plants than on nontransformed plants. Total peroxidase activity of Tetr plants was lower than that of nontransformed plants, suggesting a role for peroxidases in resistance against necrotrophic microorganisms. A comparable range of pathogens was examined on Arabidopsis and its ethylene-insensitive mutants etr1-1 and ein2-1. With the exception of one Fusarium isolate, ethylene insensitivity increased susceptibility of Arabidopsis plants to a similar spectrum of necrotizing pathogens as in tobacco. Thus, both ethylene-insensitive tobacco and Arabidopsis plants appear to be impaired in their resistance to necrotrophic pathogens.

Transgenic ethylene-insensitive tobacco (Tetr) plants spontaneously develop symptoms of wilting and stem necrosis when grown in nonautoclaved soil. Fusarium oxysporum, F. solani, Thielaviopsis basicola, Rhizopus stolonifer, and two Pythium spp. were isolated from these diseased Tetr plants and demonstrated to be causal agents of the disease symptoms. Pathogenicity of the two Pythium isolates and four additional Pythium spp. was tested on ethylene-insensitive tobacco and Arabidopsis seedlings. In both plant species, ethylene insensitivity enhanced susceptibility to the Pythium spp., as evidenced by both a higher disease index and a higher percentage of diseased plants. Based on the use of a DNA probe specific for Pythium spp., Tetr plants exhibited more pathogen growth in stem and leaf tissue than similarly diseased control plants. These results demonstrate that ethylene signaling is required for resistance to different root pathogens and contributes to limiting growth and systemic spread of the pathogen.

Selected strains of rhizosphere bacteria reduce disease by activating a resistance mechanism in the plant named rhizobacteria-mediated induced systemic resistance (ISR). Rhizobacteria-mediated ISR resembles pathogeninduced systemic acquired resistance (SAR) in that both types of induced resistance render uninfected plant parts more resistant towards a broad spectrum of plant pathogens. Some rhizobacteria trigger the salicylic acid (SA)dependent SAR pathway by producing SA at the root surface. In other cases, rhizobacteria trigger a different signalling pathway that does not require SA. The existence of a SA-independent ISR pathway has been demonstrated in Arabidopsis thaliana. In contrast to pathogen-induced SAR, ISR induced by Pseudomonas fluorescensWCS417r is independent of SA accumulation and pathogenesis-related (PR) gene activation but, instead, requires responsiveness to the plant hormones jasmonic acid (JA) and ethylene. Mutant analyses showed that ISR follows a novel signalling pathway in which components from the JA and ethylene response are successively engaged to trigger a defensive state that, like SAR, is controlled by the regulatory factor NPR1. Interestingly, simultaneous activation of both the JA/ethylene-dependent ISR pathway and the SA-dependent SAR pathway results in an enhanced level of protection. Thus combining both types of induced resistance provides an attractive tool for the improvement of disease control. This review focuses on the current status of our research on triggering, signalling, and expression of rhizobacteria-mediated ISR in Arabidopsis.

▪ Abstract Nonpathogenic rhizobacteria can induce a systemic resistance in plants that is phenotypically similar to pathogen-induced systemic acquired resistance (SAR). Rhizobacteria-mediated induced systemic resistance (ISR) has been demonstrated against fungi, bacteria, and viruses in Arabidopsis, bean, carnation, cucumber, radish, tobacco, and tomato under conditions in which the inducing bacteria and the challenging pathogen remained spatially separated. Bacterial strains differ in their ability to induce resistance in different plant species, and plants show variation in the expression of ISR upon induction by specific bacterial strains. Bacterial determinants of ISR include lipopolysaccharides, siderophores, and salicylic acid (SA). Whereas some of the rhizobacteria induce resistance through the SA-dependent SAR pathway, others do not and require jasmonic acid and ethylene perception by the plant for ISR to develop. No consistent host plant alterations are associated with the induced state, but upon challenge inoculation, resistance responses are accelerated and enhanced. ISR is effective under field conditions and offers a natural mechanism for biological control of plant disease.

Conditions for UV-induced cross-linking of abscisic acid (ABA) through its enone chromophore to binding proteins were evaluated. The effects of a UV-light band between 260 and 530 nm on both unconjugated and protein-conjugated ABA, as well as on anti-ABA antibodies as models of ABA-binding proteins were determined. UV irradiation caused both isomerization and photolysis of ABA, but increasing the lower irradiation boundary to 345 nm strongly reduced photolysis and largely prevented isomerization. When conjugated to alkaline phosphatase (AP), ABA remained stable when using either a 320 or a 345 nm filter. At these wavelengths both binding of ABA to antibodies as well as AP enzymatic activity were maintained. UV-induced cross-linking of monoclonal anti-ABA antibodies to immobilized ABA was analysed by immunoassays. Optimal cross-linking was achieved after a 5 min irradiation period at 0°, using a long pass, cut-on filter to quench wavelengths below 290 nm. This cross-linking faithfully reflected cognate binding activity.

The rate of senescence and the two-dimensional pattern of soluble proteins from detached oat leaves senescing in either darkness or light were analyzed, and compared to those of leaves in which senescence was delayed by application of the cytokinin benzyladenine or enhanced through the action of abscisic acid.

Publisher Summary This chapter provides an overview of the processes and mechanisms. It has become clear that plants have evolved sophisticated mechanisms to deal with environmental challenges. To ward off attack by different types of organisms, they possess a surveillance system that recognizes common microbial components, such as bacterial lipopolysaccharides and flagellin, or fungal constituents comprising ergosterol and chitin. Plants flourish in almost all natural environments, even though they are surrounded by potentially harmful organisms. Because of their sessile nature, they have to cope effectively with these threats. During evolution they have developed effective mechanisms to counteract microbial invasion and animal attack. Apart from morphological adaptations, they rely on inducible defenses that are activated in response to infection or attack and limit proliferation of, and tissue colonization by the attacker.

To protect themselves from disease, plants have evolved sophisticated defense mechanisms in which the signal molecules salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) often play crucial role. Elucidation of signaling pathways controlling disease resistance is a major objective in research on plant-pathogen interactions. The capacity of a plant to develop a broad spectrum, systemic acquired resistance (SAR) after primary infection with a necrotizing pathogen is well known and its signal transduction pathway extensively studied. Plants of which the roots have been colonized by specific strains of nonpathogenic fluorescent Pseudomonas spp. develop a phenotypically similar form of protection that is called rhizobacteria-mediated induced systemic resistance( ISR). In contrast to pathogen induced SAR, which is regulated by SA, rhizobacteria mediated ISR is controlled by a signaling pathway in which ET and JA play key roles.In the past decade, the model plant species Arabidopsis thaliana was explored to study the molecular basis of rhizobacteria mediated ISR. Here we review the current knowledge of the signal transduction steps involved in the ISR pathway that leads from recognition of the rhizobacteria in the roots to systemic expression of broad-spectrum disease resistance in above-ground foliar tissues.

Plant growth-promoting rhizobacteria can suppress diseases through antagonism between the bacteria and soil-borne pathogens, as well as by inducing a systemic resistance in the plant against both root and foliar pathogens. The generally non-specific character of induced resistance constitutes an increase in the level of basal resistance to several pathogens simultaneously, which is of benefit under natural conditions where multiple pathogens may be present. Specific Pseudomonas strains induce systemic resistance in e.g. carnation, cucumber, radish, tobacco and Arabidopsis, as evidenced by an enhanced defensive capacity upon challenge inoculation. Although some bacterial strains are equally effective in inducing resistance in different plant species, others show specificity, indicating specific recognition between bacteria and plants at the root surface. In carnation, radish and Arabidopsis, the O-antigenic side chain of the bacterial outer membrane lipopolysaccharide acts as an inducing determinant, but other bacterial traits are also involved. Pseudobactin siderophores have been implicated in the induction of resistance in tobacco and Arabidopsis, and another siderophore, pseudomonine, may explain induction of resistance associated with salicylic acid (SA) in radish. Although SA induces phenotypically similar systemic acquired resistance (SAR), it is not necessary for the systemic resistance induced by most rhizobacterial strains. Instead, rhizobacteria-mediated induced systemic resistance (ISR) is dependent on jasmonic acid (JA) and ethylene signaling in the plant. Upon challenge inoculation of induced Arabidopsis plants with a pathogen, leaves expressing SAR exhibit a primed expression of SA-, but not JA/ethylene-responsive defense-related genes, whereas leaves expressing ISR are primed to express JA/ethylene-, but not SA-responsive genes. Combination of ISR and SAR can increase protection against pathogens that are resisted through both pathways, as well as extend protection to a broader spectrum of pathogens than ISR or SAR alone.

Induced resistance is the phenomenon in which a plant, once appropriately stimulated, exhibits an enhanced resistance upon “challenge” inoculation with a pathogen. Induced resistance can be localized as well as systemic, and can be induced by limited pathogen infection, avirulent pathogens, certain non-pathogenic bacteria, and certain chemicals. Systemic induced resistance has been particularly well studied in tobacco, cucumber and Arabidopsis. Under the influence of the inducing stimulus, a mobile signal is generated and transported to other parts of the plant where it enhances the mechanisms normally functioning to limit infection, growth, multiplication and spread of fungi, bacteria and viruses. In the systemic acquired resistance (SAR) induced by pathogens causing hypersensitive necrosis, resistance mechanisms to challenge inoculation become operative earlier or operate at a higher level than in non-induced plants. The expression of SAR is dependent on the accumulation of salicylic acid (SA) and associated with the induction of pathogenesis-related proteins (PRs) with anti-pathogen activities. In contrast, rhizobacterially-mediated induced systemic resistance (ISR) does not require SA, can occur without production of PRs, and is dependent on ethylene and jasmonic acid (JA) signalling. Mutants in the signal-transduction pathways of SAR and ISR have been identified in Arabidopsis which point to both pathways converging at the final step, while additional antimicrobial peptides are independently regulated. A different type of systemic induced resistance, involving production of proteinase inhibitors against herbivores, also requires JA. Synthesis of JA is initiated on perception of the Polypeptide systemin, which is released on wounding of tomato and potato and acts as the mobile signal. These various types of systemic induced resistance confer an enhanced defensive capacity on plants against a broad spectrum of attackers and offer great potential for exploitation in crop protection.

Download : Download full-size image Various rhizobacterial strains suppress soil-borne plant diseases by competition with the pathogen for nutrients, siderophore-mediated competition for iron, antibiosis, or production of lytic enzymes. Some strains can also enhance the defensive capacity of plants by mechanisms that are either dependent on the production of salicylic acid and associated with the accumulation of pathogenesis-related proteins in the plant (systemic acquired resistance), or that are not but require responsiveness of the plant to jasmonate and ethylene (induced systemic resistance). To exploit the mechanisms involved in disease suppression by rhizobacteria, the genes responsible are being cloned. Combinations of rhizobacterial strains or genetic modification of Pseudomonas bacteria are used to improve biological control of economically important plant diseases. Risk assessment studies have been initiated with regard to the introduction of such genetically-modified microorganisms in the field.

In incompatible host-pathogen interactions, damage caused by the pathogen remains restricted as a result of the plant's defensive response. Most effective is the hypersensitive reaction, in which the cells around the infection site rapidly necrose. This response is associated with a coordinated and integrated set of metabolic alterations that are instrumental in impeding further pathogen ingress, as well as in enhancing the capacity of the host to limit subsequent infection by different types of pathogens [27, 77]. Altered ion fluxes across the plant cell membrane, generation of active oxygen species, changes in the phosphorylation state of regulatory proteins and transcriptional activation of plant defense systems culminate in cell death at the site of infection, local accumulation of phytoalexins and cell wall rigidification as a result of callose, lignin and suberin deposition [31, 89]. In addition, various novel proteins are induced which are collectively referred to as ``pathogenesis-related proteins '' (PRs). These PRs, defined as proteins coded for by the host plant but induced specifically in pathological or related situations [4, 81], do not only accumulate locally in the infected leaf, but are also induced systemically, associated with the development of systemic acquired resistance (SAR) against further infection by fungi, bacteria and viruses. Induction of PRs has been found in many plant species belonging to various families [78], suggestive of a general role for these proteins in adaptation to biotic stress conditions. SAR, likewise, is a generally occurring phenomenon, that engenders an enhancement of the defensive capacity of plants in response to necrotizing infections [70]. Since some of the tobacco PRs were identified as chitinases [45] and b-1,3-glucanases [38] with potential antifungal activity, it has often been suggested that the collective set of PRs may be effective in inhibiting pathogen growth, multiplication and/or spread, and be responsible for the state of SAR [42, 65]. Originally, five main classes of PRs (PR-1-5) were characterized by both biochemical and molecular-biological techniques in tobacco [9, 80]. Thereupon, in 1994 a unifying nomenclature for PRs was proposed based on their grouping into families sharing amino acid sequences, serological relationship, and/or enzymatic or biological activity. By then 11 families (PR-1--11) were recognized and classified for tobacco and tomato [81] (cf. Table 1). Criteria used for the inclusion of new families of PRs were that (i) protein(s) must be induced by a pathogen in tissues that do not normally express the protein(s), and (ii) induced expression must have been shown to occur in at least two different plant-pathogen combinations, or expression in a single plant-pathogen combination must have been confirmed independently in diaerent laboratories.

Ethylene is involved in many physiological and developmental processes in plants, as well as in the responses of plants to abiotic and biotic stresses [1, 2]. In hypersensitively reacting tobacco, tobacco mosaic virus (TMV) infection results in increased ethylene synthesis [3]. Associated with TMV infection is the production of acidic and basic pathogenesis-related (PR)-proteins several of which have antifungal activity [4, 5, 6]. Associated with the production of PR-proteins, the plant becomes more resistant to subsequent pathogen attack, a phenomenon known as systemic acquired resistance (SAR) [7, 8]. Under the influence of the primary infection, a signal moves through the plant and induces the resistant state.

In contrast to wild-type seeds of Arabidopsis thaliana and to seeds deficient in (aba) or insensitive to (abi3) abscisic acid (ABA), maturing seeds of recombinant (aba,abi3) plants fail to desiccate, remain green, and lose viability upon drying. These double-mutant seeds acquire only low levels of the major storage proteins and are deficient in several low mol wt polypeptides, both soluble and bound, and some of which are heat stable. A major heat-stable glycoprotein of more than 100 kilodaltons behaves similarly; during seed development, it shows a decrease in size associated with the abi3 mutation. In seeds of the double mutant from 14 to 20 days after pollination, the low amounts of various maturation-specific proteins disappear and many higher mol wt proteins similar to those occurring during germination are induced, but no visible germination is apparent. It appears that in the aba,abi3 double mutant seed development is not completed and the program for seed germination is initiated prematurely in the absence of substances protective against dehydration. Seeds may be made desiccation tolerant by watering the plants with the ABA analog LAB 173711 or by imbibition of isolated immature seeds, 11 to 15 days after pollination, with ABA and sucrose. Whereas sucrose stimulates germination and may protect dehydration-sensitive structures from desiccation damage, ABA inhibits precocious germination and is required to complete the program for seed maturation and the associated development of desiccation tolerance.

Summary Of the three molecular forms of petunia (Petunia hybrida) peroxidase a, the form PRXa 1.1 (37.5 kDa) was retarded on columns of concanavalin A-Sepharose, whereas forms PRXa 1.2 and 1.3 (37.0 and 36.5 kDa, respectively) were not. Chemical deglycosylation of the two fractions yielded only poorly resolved products of 33–35 kDa. Enzymatic deglycosylation with c-mannosidase converted PRXa 1.1 and 1.2 to immunoreactive products migrating in polyacrylamide gels at the position of PRXa 1.3. The presence of xylose, arabinose, fucose, mannose, glucose, and galactose was indicated by gas- chromatographic analysis. Lectin affinity blotting using lectins conjugated to horseradish peroxidase suggested that PRXa contains small complex-type N- glycosyl carbohydrate chains. These results indicate that the heterogeneity of PRXa is caused by differences in the terminal a-linked mannose residues. Some ce-mannosidase activity was present in the apoplast of leaves, which is consistent with the suggestion that it may be responsible for the conversion of PRXa 1.1 into PRXa 1.2 and 1.3 during leaf development.

Plant defenses against pathogens and insects are regulated differentially by cross-communicating signaling pathways in which salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) play key roles. To understand how plants integrate pathogen- and insect-induced signals into specific defense responses, we monitored the dynamics of SA, JA, and ET signaling in Arabidopsis after attack by a set of microbial pathogens and herbivorous insects with different modes of attack. Arabidopsis plants were exposed to a pathogenic leaf bacterium (Pseudomonas syringae pv. tomato), a pathogenic leaf fungus (Alternaria brassicicola), tissue-chewing caterpillars (Pieris rapae), cell-content-feeding thrips (Frankliniella occidentalis), or phloem-feeding aphids (Myzus persicae). Monitoring the signal signature in each plant-attacker combination showed that the kinetics of SA, JA, and ET production varies greatly in both quantity and timing. Analysis of global gene expression profiles demonstrated that the signal signature characteristic of each Arabidopsis-attacker combination is orchestrated into a surprisingly complex set of transcriptional alterations in which, in all cases, stress-related genes are overrepresented. Comparison of the transcript profiles revealed that consistent changes induced by pathogens and insects with very different modes of attack can show considerable overlap. Of all consistent changes induced by A. brassicicola, Pieris rapae, and F. occidentalis, more than 50% also were induced consistently by P. syringae. Notably, although these four attackers all stimulated JA biosynthesis, the majority of the changes in JA-responsive gene expression were attacker specific. All together, our study shows that SA, JA, and ET play a primary role in the orchestration of the plant’s defense response, but other regulatory mechanisms, such as pathway cross-talk or additional attacker-induced signals, eventually shape the highly complex attacker-specific defense response.

The induction of a hypersensitive reaction in ‘Samsun NN’ tobacco by tobacco mosaic virus (TMV) at 20°C leads to the development of both localized and systemic acquired resistance, and is associated with the appearance of pathogenesis-related proteins (PR's) and large increases in peroxidase activity and ethylene production. Salicylic acid (SA) induced a similar resistance in treated plant parts and occasionally also in untreated upper leaves of plants of which three lower leaves had been injected. SA also induced the same four PR's, but these were confined to the treated leaves. Thus, the connection between the presence of PR's and the reduction of TMV multiplication and spread may not be direct. In contrast to TMV, SA did not stimulate ethylene production and hardly increased peroxidase activity. Induction of acquired resistance and PR's by SA developed equally well at 20°C and at 32°C. However, pricking leaves with needles moistened with the ethylene-releasing compound ethephon mimicked TMV infection in inducing acquired resistance and PR's in both treated and untreated leaves at 20°C, but not at 32°C. Ethephon increased peroxidase activity at both temperatures, but only at 20°C dit it induce changes in both the anodic and the cathodic isoenzymes that were similar to those induced as a result of TMV infection. SA induced PR's and reduced TMV multiplication in ‘Samsun’ tobacco, and inhibited virus spread in ‘Samsun NN’ at 32°C. These observation indicate that neither the induction of PR's, nor the development of acquired resistance is temperature-sensitive. On the other hand, the effects of ethephon are temperature-sensitive in the same way as the hypersensitive response to TMV. It can thus be hypothesized that ethylene, produced naturally during the hypersensitive reaction of tobacco to TMV, leads to the temperature-sensitive synthesis or release of a presumably benzoic acid-type compound that functions as the natural inducer of PR's and acquired resistance. Although vanillic acid has been shown to accumulate in hypersensitively reacting tobacco leaves, it produced none of the effects of SA, and thus cannot be the natural inducer. Inductie van een overgevoeligheidsreactie in ‘Samsun NN’-tabak door tabaksmozaiekvirus (TMV) bij 20°C leidt tot de ontwikkeling van een verworven resistentie die zowel lokaal als systemisch werkzaam is, en gaat samen met het verschijnen van ‘pathogenesis-related proteins’ (PR's) en sterke toename in de activitieit van peroxidase en de produktie van ethyleen. Salicylzuur (SA) induceerde een vergelijkbare resistentie in behandelde plantedelen en af en toe ook in niet behandelde bovenbladeren van planten waarvan drie onderbladeren waren ingespoten. SA induceerde ook dezelfde vier PR's, maar deze waren beperkt tot de behandelde bladeren. Er bestaat dus geen directe samenhang tussen de aanwezigheid van PR's en de remming van de vermeerdering en uitbreiding van TMV in de plant. In tegenstelling tot TMV stimuleerde SA de ethyleenproduktie niet en verhoogde het de peroxidaseactiviteit nauwelijks. Inductie van verworven resistentie en PR's door SA trad even goed op bij 32°C als bij 20°C. Net als infectie met TMV leidde aanprikken van bladeren met naalden die gedoopt waren in een oplossing van ethefon — waaruit in het blad ethyleen vrijkomt — echter tot inductie van verworven resistentie en PR's in zowel behandelde als onbehandelde bladeren bij 20°C, maar niet bij 32°C. Ethefon verhoogde de peroxidaseactiviteit bij beide temperaturen, maar alleen bij 20°C induceerde het veranderingen in zowel de anodische als de kathodische isoenzymen die vergelijkbaar waren met die welke geinduceerd werden als gevolg van infectie met TMV. SA induceerde PR's en verminderde de vermenigvuldiging van TMV in ‘Samsun’ tabak, en remde de uitbreiding van het virus in ‘Samsun NN’ bij 32°C. Deze waarnemingen tonen dat noch de inductie van PR's, noch de ontwikkeling van verworven resistentie een temperatuurgevoelig proces is. Daarentegen zijn de effecten van ethefon op dezelfde wijze temperatuurgevoelig als de overgevoeligheidsreactie op TMV. Men kan daarom veronderstellen dat ethyleen, dat op natuurlijke wijze geproduceerd wordt tijdens de overgevoeligheidsreactie van tabak op TMV, aanleiding geeft tot een temperatuurgevoelig proces, namelijk de synthese of het vrijkomen van een verbinding, vermoedelijk een benzoezuurderivaat, dat fungeert als de natuurlijke inductor van PR's en verworven resistentie. Hoewel is aangetoond dat vanillinezuur zich ophoopt in overgevoelig reagerende tabaksbladeren, veroorzaakte deze verbinding geen enkel van de effecten van SA. Vanillinezuur kan dus niet de natuurlijke inductor zijn.

In Samsun NN tobacco reacting hypersensitively to tobacco mosaic virus (TMV), the natural auxin indoleacetic acid (IAA) decreased lesion enlargement in a concentration-dependent manner when sprayed on young, expanding leaves between 40 h before and 40 h after inoculation, and again between 60 and 100 h after inoculation. A maximum decrease in lesion size of 30% was routinely obtained by application of 10−4 m IAA within 12 h of inoculation. The synthetic auxin, a-naphthylacetic acid, likewise reduced lesion size, whereas the less active natural auxin, phenylacetic acid, was effective at higher concentrations only. The auxin-like regulator, 2,4-dichlorophenoxyacetic acid, was atypical in that it reduced lesion size at low, but promoted it at higher concentrations. The non-auxin analogues, β-naphthyl-acetic aced and benzoic acid, were only marginally active. In non-inoculated plants, IAA increased peroxidase activity similarly to TMV infection, but application of the hormone to TMV-inoculated plants counteracted the virus-induced increase in enzyme activity, mainly through partial suppression of the quantitative changes in peroxidase isoenzymes characteristic of the infection. The latter effect is related to the decrease in necrotizing tissue resulting from the reduction in lesion size. Since the effect of the auxin on peroxidase activity was prolonged and systemic, whereas the effect on lesion size was not, the two effects are not directly related. The ability to restrict lesion spread in response to auxin was gradually lost during leaf development or after detachment. It could not be restored by kinetin. However, kinetin selectively inhibited the increase in lesion enlargement due to detachment. Like in mature, fully grown and in old, senescing leaves, auxin did not reduce lesion size in young, systemic resistant leaves further, possibly because of lack of an additional factor. Similar results can be obtained by treatment with ethephon, suggesting that the action of auxin is mediated by auxin-induced ethylene. IAA also inhibited TMV multiplication in young, inoculated leaves of the systemically reacting tobacco cultivar Samsun. Hence, the limiting action of exogenous auxin on lesion spread may depend both on a direct inhibitory effect on virus multiplication in young, hypersensitively reacting leaves, and on a stimulation of factors involved in virus localization.

Pricking Samsum NN tobacco leaves with needles moistened with the ethylene-releasing compound 2-chloroethylphosphonic acid (ethephon) gave rise to necrotic spots resembling virus-induced local lesions. Treatment with ethephon resulted in changes in protein constitution and peroxidase isoenzyme patterns qualitatively and quantitatively similar to those induced by tobacco mosaic virus (TMV). Moreover, a systemic resistance was induced comparable to the result of virus infection. Abundant local ethylene synthesis during TMV infection thus seems to be a causative factor in redirecting plant metabolism during the hypersensitive reaction.

Infection of Nicotiana tabacum var. “Samsun” and “Samsun NN” with tobacco mosaic virus “Wageningen” U 1 (TMV W U 1) at 30 °C leads to systemic mosaic symptoms in both varieties. Electrophoretic patterns of soluble leaf proteins in polyacrylamide gels showed identical changes for both varieties and were the same as those in the combination TMV W U 1-Samsun at 20 °C and similar to those in the combination TMV W U 1-Samsun EN, in which identical symptoms are induced. Four new protein components (I to IV) accumulating at 20 °C in Samsun NN plants showing local lesions did not increase in amount after transfer of these plants to 30 °C; bands I, II and IV were considerably reduced, whereas band III appeared unaffected. When Samsun plants were infected with the Holmes' ribgrass strain of TMV (TMV HR) at 20 °C, both free TMV HR coat protein and relatively small amounts of the new components I to IV were apparent, proving that these components cannot be products of the N gene. The formation of small local lesions on the variety Samsun EN upon infection with TMV HR resulted in induction of the new components I to IV in apparently trace amounts, whereas quantitative changes seemed to be extremely limited. Inoculation of Samsun NN plants with TMV HR led to symptoms under greenhouse conditions only. In this combination, systemic acquired resistance was expressed when traces of protein components I to IV were present. In general, the changes in soluble proteins were connected with the type of symptoms—either mosaic or localized necrosis—produced, rather than the genetic make-up of the host plant variety.

Summary Using 2-naphthylamides of nineteen amino acids as substrates, five aminopeptidases and one arginine-specific endopeptidase were identified in the first leaf of oats (Avena sativa L. cv. Victory) by activity staining after electrophoretic separation of the soluble proteins on polyacrylamide gels. The main amino-peptidase showed preference towards alanine, glycine and leucine. Two aminopeptidases were specific for arginine and lysine and one acted as an iminopeptidase specific for proline. The fifth was most active with methionine but also liberated some other N-terminal amino acids. All aminopeptidases were active at pHs 7.5, 6.0 and 4.5. The activity of the main aminopeptidase strongly decreased in the course of leaf development. The arginine-specific endopeptidase was active at pHs 4.5 and 7.5 and was inhibited at pH 7.5 by o-phenanthroline, suggesting it to be a metalloenzyme. Its activity also decreased during leaf development. During storage of protein extracts in the cold, changes in the electrophoretic protein profile, indicative of protein degradation, were evident. Under such conditions, some of the arginine-specific endopeptidase was associated with the protein band containing ribulose bisphosphate carboxylase, suggestive of a possible role in protein breakdown during leaf senescence. The aminopeptidases may be involved in the rapid degradation of peptides generated by endopeptidase action.

Summary When detached oat leaves were aged in darkness, loss of protein and chlorophyll was similar whether leaf segments were placed horizontally in Petri dishes on wet filter paper, or held vertically in plastic holders with their bases in water. In horizontally placed leaves, the activities of the two proteases tended to increase but in leaves kept vertically protease activities decreased as in attached leaves. In light, both chlorophyll and protein loss occurred at about half the rate observed in darkness. However, in vertically held leaves the acidic protease started to increase by day 2, whereas the neutral protease first declined but increased later. These increases were not mimicked by incubation on sucrose in darkness and neither could they be attributed to a greater accumulation of free amino acids in the light. Kinetin and, to a lesser extent zeatin, maintained chlorophyll and protein levels and counteracted both the increases and decreases in protease activities observed during ageing in light or darkness, respectively. Since accelerated loss of protein in darkness was not accompanied by increases in protease activities, these observations are at variance with those of Martin and Thimann (Plant Physiol. 49: 64, 1972) and suggest that additional synthesis of the major proteases is not a prerequisite for protein breakdown during senescence.

Summary The role of methionine as the precursor of ethylene in healthy and tobacco mosaic virus-infected tobacco leaves was studied by comparison of the specific radioactivities of methionine and ethylene after labelling leaves with L-[U-14C] methionine by petiolar uptake. The specific activity of the ethylene produced was far less than expected on the basis of the methionine pool size within the leaves and the amount of label applied. This was caused, on the one hand, by an irreversible binding of the applied methionine to the cell wall fraction and, on the other hand, its rapid metabolization. The rise in ethylene production due to virus infection was associated with a corresponding decrease in specific activity, suggesting a second precursor or another (unlabeled) methionine pool to be used for virus-induced ethylene production. However, virus-induced ethylene production occured only in the interveinal leaf parts where the specific radioactivity of methionine was (relatively) low. Hence, the decrease in the specific radioactivity of the ethylene resulted from the nonhomogeneous distribution of the applied 14C-labeled methionine within the leaf rather than from another precusor.

Summary By using extraction at pH 4.5, followed by gel filtration and anion-exchange chromatography, the acidic and neutral proteases present in oat (Avena sativa L. cv. Victory) leaves were purified up to 87 and 22 times, respectively. The enzymes were only marginally separated, however, indicating that they have similar molecular weights and are rather similarly charged between pHs 6 and 9. Both enzymes appeared to be endopeptidases. The neutral activity was decreased by inhibitors of metalloproteinases, whereas the acidic activity was not. However, both were inhibited to varying extens by cysteine- or serine-type protease inhibitors. Inhibition of enzyme activity by polyamines could not be ascertained. Contrary to a previous report, serine did not activate the enzymes. These discrepancies may be due to anomalies encountered in the ninhydrin staining used for determination of the enzyme activity.

Endogenous ethylene production of tobacco leaves was similar in light and in darkness. However, the rate of conversion of exogenously applied l-aminocyclopropane-l-carboxylic acid (ACC) to ethylene was reversibly inhibited by light. Virus-stimulated ethylene production, during the hypersensitive reaction of tobacco leaves to tobacco mosaic virus, was likewise inhibited by light. Under such circumstances ethylene production is limited at the level of the conversion of ACC to ethylene. Inhibition of the increase in ACC-stimulated ethylene production by cycloheximide and 2-(4-methyl-2,6-dinitroanilino)-N-methyl-propionamide after shifting leaf discs from light to darkness indicated that de novo protein synthsis was involved. Regulation of ACC-dependent ethylene production by reversible oxidation/reduction of essential SH groups, as suggested by Gepstein and Thimann (1980, Planta 149, 196-199) could be excluded. Instead, regulation of the ACC-converting enzyme at the level of both synthesis/degradation and activation/inactivation is suggested. Phytochrome was not involved in light inhibition, but low intensities of either red or blue light decreased the rate of ACC conversion. Dichlorophenyldimethylurea counteracted the inhibitory effect of light, indicating that (part of) the photosynthetic system is involved in the light inhibition. The ethylene production of Pharbitis cotyledons grown in darkness or light, either in the presence of absence of the inhibitor of carotenoid synthesis, SAN 9789 (norflurazon), supported this view.

Specific localization of polyphenoloxidases after electrophoresis on polyacrylamide gels is described. The method consists of using caffeic acid as the substrate and m -phenylenediamine as a coupling agent at pH 5·5. Isoenzyme patterns of tobacco polyphenoloxidases showed six bands which may be considered to represent true isozymic forms of the enzyme. These bands occupied positions on the gel different from those of tobacco peroxidase isoenzymes developed with guaiacol. Apparent identity of polyphenoloxidases and peroxidases revealed with other substrates and differentiation of these enzymes are discussed.

Different conditions for the extraction of soluble proteins from tobacco leaves were evaluated. Best results were obtained by mixing and homogenizing freshly harvested leaf material with a Tris-HCl buffer containing 0·5 M sucrose, 0·1 per cent ascorbic acid and 0·1 per cent cysteine-HCl at pH 8·0. The extract was fractionated by centrifugation, further purified by gel filtration and concentrated by ammonium sulphate precipatation. The resulting solution showed an u.v. spectrum characteristic of protein, and was separated by electrophoresis on polyacrylamide gels of different concentrations into about thirty protein zones. No differences were detected in the electrophoretic patterns of purified extracts from the varieties Samsun and Samsun NN.

Zusammenfassung In naturlicherweise alternden Gerstenblattern nehmen der Proteingehalt und die Aktivitat RNS-hydrolysierender Enzyme schneller ab als in abgeschnittenen Blattern. Verabreichung einer physiologischen Konzentration Indolessigsaure an die abgeschnittenen Blatter verringert diese Unterschiede. Mittels Polyacrylamidgelelektrophorese wurden mindestens 7 Polyuridylsaure-hydrolysie-rende Enzymbander in den Blattextrakten nachgewiesen. Die Anderungen in der RNase-Aktivitat infolge naturliches oder kunstliches Altern, mit oder ohne IES, zeigten sich durch ungefahr proportionelle Anderungen aller Bander. Zugabe von IES in vitro aber, an zentrifugierten Extrakten, hemmte teilweise die RNase-Aktivitat, insbesondere bei zwei Bandern. Die physiologische Bedeutung dieser Wirkungen von IES in vivo und in vitro werden diskutiert.

Staining with amido black or light green in 5% trichloroacetic acid (TCA) instead of 7% acetic acid results in improved resolution of soluble leaf protein patterns developed by means of polyacrylamide gel electrophoresis. Destaining time is considerably shortened or even eliminated by using this modification. The protein patterns are qualitatively identical, but quantitatively markedly different from those stained with Coomassie blue in 12.5% TCA. Amino black used as a stain in disc gel electrophoresis shows linearity between staining intensity and amount of protein in the range 0–40 μg. This is not the case for the Coomassie blue stain which does not impart enough contrast in regions of the gel crowded with bands. Evidence is presented that the anomalous protein patterns, due to the use of Coomassie blue, result from clogging of the gels caused by protein precipitation. On the other hand, preferential affinity of Coomassie blue towards new protein components in tobacco plants infected with tobacco mosaic virus (TMV), indicates that the use of this dye may be helpful in searching for small qualitative differences.

Non-pathogenic soilborne microorganisms can promote plant growth, as well as suppress diseases. Plant growth promotion is taken to result from improved nutrient acquisition or hormonal stimulation. Disease suppression can occur through microbial antagonism or induction of resistance in the plant. Several rhizobacterial strains have been shown to act as plant growth-promoting bacteria through both stimulation of growth and induced systemic resistance (ISR), but it is not clear in how far both mechanisms are connected. Induced resistance is manifested as a reduction of the number of diseased plants or in disease severity upon subsequent infection by a pathogen. Such reduced disease susceptibility can be local or systemic, result from developmental or environmental factors and depend on multiple mechanisms. The spectrum of diseases to which PGPR-elicited ISR confers enhanced resistance overlaps partly with that of pathogen-induced systemic acquired resistance (SAR). Both ISR and SAR represent a state of enhanced basal resistance of the plant that depends on the signalling compounds jasmonic acid and salicylic acid, respectively, and pathogens are differentially sensitive to the resistances activated by each of these signalling pathways. Root-colonizing Pseudomonas bacteria have been shown to alter plant gene expression in roots and leaves to different extents, indicative of recognition of one or more bacterial determinants by specific plant receptors. Conversely, plants can alter root exudation and secrete compounds that interfere with quorum sensing (QS) regulation in the bacteria. Such two-way signalling resembles the interaction of root-nodulating Rhizobia with legumes and between mycorrhizal fungi and roots of the majority of plant species. Although ISR-eliciting rhizobacteria can induce typical early defence-related responses in cell suspensions, in plants they do not necessarily activate defence-related gene expression. Instead, they appear to act through priming of effective resistance mechanisms, as reflected by earlier and stronger defence reactions once infection occurs.

Ethylene production was stimulated severalfold during the hypersensitive reaction of Samsun NN tobacco to tobacco mosaic virus (TMV). Exogenous methionine or S -adenosylmethionine (SAM) did not increase ethylene evolution from healthy or TMV-infected leaf discs, although both precursors were directly available for ethylene production. This indicates that ethylene production is not controlled at the level of methionine concentration or availability, nor at the level of SAM production or concentration. In contrast, 1-aminocyclopropane-1-carboxylic acid (ACC) stimulated ethylene production considerably. Thus, ethylene production is primarily limited at the level of ACC production. The regulation of ethylene production during the hypersensitive reaction to TMV was further studied by determining the time course of the concentrations of methionine, SAM, and ACC, as well as the course of their in vivo conversion rates. Endogenous concentrations of methionine and SAM remained unaffected until late in infection. On the contrary, the peak in ethylene production near the time of local lesion development was preceded by a large increase in ACC production. As a result of this increase, ACC accumulated in the leaf tissue. Only after local lesions became visible, the capacity to convert ACC into ethylene increased severalfold, associated with a sharp decrease in ACC content and a large increase in ethylene production. Ethylene production in tobacco leaves reacting hypersensitively to TMV is thus regulated at the level of both the production of ACC and its conversion to ethylene.

During the hypersensitive reaction of Samsun NN tobacco to tobacco mosaic virus (TMV) the inoculated leaves synthesize large quantities of ethylene. At the same time, 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), a conjugate of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) accumulates. Smaller amounts of MACC are formed concomitant with ethylene synthesis during the normal development of tobacco leaves. The conjugate appears neither to be hydrolysed to liberate ACC, nor to be transported to other plant parts. Its accumulation thus reflects the history of the operation of the pathway of ethylene synthesis in the leaf. In floating leaf discs exogenously applied ACC was converted only slowly to both ethylene and MACC. More ethylene and less MACC were produced in darkness than in light, suggesting that environmental conditions may influence the ratio at which ACC in converted to either ethylene or MACC.

Ten pH-3 soluble, low-molecular-weight pathogenesis-related proteins (PRs) were found to accumulate in leaves of tobacco cv. Samsun NN reacting hypersensitively to tobacco mosaic virus. Besides the previously characterized PRs 1a, 1b, 1c and 2, these proteins were provisionally designated N, O, P, Q, R, and S in order of decreasing electrophoretic mobility in native polyacrylamide gels. Two-dimensional gel electrophoresis indicated that the PRs consist of single polypeptides, except for R, which is composed of two components with slightly different molecular weights. Estimated molecular weights in SDS-containing gels were: PRs 1a and 1b 17 kD, 1c 16.5 kD, 2 31 kD, N 33 kD, O 35 kD, P 27 kD, Q 28 kD, R 13 and 15 kD, and S 25 kD. However, based on their elution from gel filtration columns and relative moblities in native gels of different acrylamide concentrations, P and Q appeared to have molecular weights similar to those of the PR 1 group. Upon chromatofocusing no additional components were resolved. The PRs were eluted between pH 7 and 4; except for R, their pIs, as judged from isoelectric focusing, appeared to lie in the range from pH 4 to 5.2. In the presence of 6 M urea PR 1a was split into two components, one of which was strongly retarded on gels, as were P and Q. None of the PRs was detected when gels were stained for glycoproteins.By combinations of gel filtration, DEAE-cellulose chromatography, and chromatofocusing, PRs 1a, 1b, 1c, 2 and N were purified, their amino acid compositions determined, and antisera raised against each of these components. By Western blotting, antisera against either PR 1a, 1b, or 1c reacted with each of the components of the PR 1 group, as well as with PR S. Similarly, the antisera against either PR 2 or N reacted with both 2 and N, as well as with O and R. On the basis of major similarities in molecular weight characteristics, amino acid compositions, and serological relationships, it is proposed to classify tobacco PRs into five groups: 1: PRs 1a, 1b, and 1c; 2: 2a (formerly 2), 2b (N), and 2c (O); 3: 3a (P), and 3b (Q); 4: 4a and 4b (the two components of R); and 5: PR 5 (S).

Summary From different Nicotiana species ( N. sylvestris Spegaz. et Comes, N. glutinosa L., and N. tabacum L. cultivars) which differ in their sensitivity towards tobacco mosaic virus (TMV), chromatin-associated protein profiles were compared by two-dimensional polyacrylamide gel electrophoresis. After removal of DNA from dissociated chromatin preparations, the remaining chromatin-associated proteins were extracted with phenol and precipitated with ethanol. The sensitive silver staining of the gel enabled visualization of up to 350 different polypeptides. Comparative analysis showed that over 90% of the polypeptides were similar in all Nicotiana species but discrete differences provided unique patterns for each of the species and cultivars examined. However, no specific polypeptide spot could be associated with the presence of the gene N , which governs hypersensitivity towards TMV. Nevertheless, the method presented here allows study of the chromatin proteins involved in the regulation of gene expression during growth and development, as well as analyses of the influence of environmental factors on genetic regulatory elements.

Preparations of pathogenesis-related (b) proteins (PRs) from different Nicotiana species, tomato,Gynura aurantiaca, bean, and cowpea were compared to each other and to bean chitinase and a constitutive apple agglutinin by electrophoresis in polyacrylamide gels both in the absence and in the presence of SDS, and by serological double diffusion analysis using antisera against tobacco PRs and bean chitinase. PRs from different plant genera displayed a similar but not identical range of relative mobilities in both native and SDS gels, whereas bean chitinase and apple agglutinin were clearly different. None of the antisera reacted with any of the PR preparations from plant genera other than the one from which the antigen(s) had been derived. Whilst PRs within the genus Nicotiana are serologically related and can be identical, PRs from different plant genera seem to be sufficiently different to be considered as genus-specific.

The effects of temperature, light and leaf age on tobacco mosaic virus (TMV)-induced ethylene production and on virus localization in Samsun NN tobacco in relation to the hypersensitive reaction were investigated. Lesion size increased with increasing temperature up to 28 °C and in continuous light at intensities above 750 lx, but decreased with leaf age. The enhanced localization of the virus in darkness and in old leaves was associated with a sharp peak in ethylene production near the time of lesion appearance, whereas in continuous light or in young leaves lesion expansion continued and virus-induced ethylene production was delayed. Hence, the early burst in ethylene production appears to be associated with the virus-localizing reaction. Temperature-shift experiments indicated that at 20 °C the hypersensitive reaction is initiated between 15 and 18 h after inoculation. Activity of the temperature-sensitive N gene is required for at least 6 h during the 24 h period after inoculation to induce development of necrotic local lesions, although an increased capacity to produce ethylene develops within 3 h. Since, after induction at 20 °C, both the increase in ethylene production and the development of necrotic local lesions take place at 30 °C, the N gene appears to be required only for the initiation of the hypersensitive reaction with other factors determining its continued development.

The regulation of ethylene biosynthesis was studied in Samsun and Samsun NN tobacco (Nicotiana tabacum L.), infected with tobacco mosaic virus (TMV) strains W U1 or Holmes' Ribgrass (HR), or with tobacco necrosis virus (TNV). Stimulation of ethylene production upon infection was not primarily determined by the genetic constitution of the host plant, nor by the infecting virus, but appeared to be related to symptom expression and virus localization. A hypersensitive reaction (Samsun NN infected with TMV W U1 (20 °C) or TNV, and Samsun infected with TMV HR or TNV), was accompanied by a sharp rise in the production, and accumulation of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), followed by rapid evolution of ethylene when the capacity to convert ACC to ethylene had increased. None of these changes occurred in systemically reacting plants (TMV W U1-infected Samsun NN at 30 °C or Samsun at 20 °C). These observations indicate that the increase in ethylene production during a hypersensitive reaction depends primarily on an increase in tissue production of ACC. Both the increase in ACC production and its accumulation were restricted to the cells surrounding the necrotic areas. Consequently, virus-stimulated ethylene production also occurred locally at the site where the virus is actively localized. After primary infection of hypersensitively reacting plants, the capacity for converting ACC to ethylene was increased systemically. Challenge inoculation did not lead to an accumulation of ACC because it was converted immediately to ethylene. This rapid local ethylene production may be responsible for limiting lesion enlargement in tobacco leaves exhibiting systemic acquired resistance. Ethylene production commenced 2 h after shifting TMV W U1-infected Samsun NN from 30 to 20 °G, 6 h before necrosis developed. Thus, the increase in ethylene production is not determined by the onset of necrosis, but by a much earlier event occurring in the plant-virus interaction at 20 °C.

This paper represents attempts to observe alterations in the pattern of chromatin protein phosphorylation in Lemna and barley ( Hordeum vulgare ). As judged by in vitro labeling the phosphorylation profile is substantially altered during germination. This may not be the result of specific tissue differentiation, however, because phosphorylation does not differ markedly between the embryonic root and shoot. Treatment of nuclei from germinating embryos with low concentrations of sodium or potassium chloride produced phosphorylation patterns similar but not identical to those found in nuclei from ungerminated embryos. Treatment of Lemna with abscisic acid in vivo causes substantial alterations in the labeling of three protein bands and part of this may be duplicated by labeling isolated nuclei from treated tissue with γ 32 P-ATP. Some effects of light/dark transition on Lemna chromatin protein phosphorylation are also described.

The distribution and subcellular localization of the two major proteases present in oat (Avena sativa L. cv Victory) leaves was investigated. Both the acidic protease, active at pH 4.5, and the neutral protease, active at pH 7.5, are soluble enzymes; a few percent of the enzyme activity was ionically bound or loosely associated with organellar structures sedimenting at 1000g. On the average, 16% of the acidic protease could be washed out of the intercellular space of the leaf. Since isolated protoplasts contained correspondingly lower activities as compared to crude leaf extracts, part of the acidic activity is associated with cell walls. No neutral protease activity was recovered in intercellular washing fluid. Of the activities present in protoplasts, the acidic protease was localized in the vacuole, whereas the neutral protease was not. The localization of the acidic protease in vacuoles did not change during leaf development up to an advanced stage of senescence, when more than 50% of the leaf protein had been degraded. These observations indicate that protein degradation during leaf senescence is not due to a redistribution of acidic protease activity from the vacuole to the cytoplasm.

Tobacco (Nicotiana tabacum L.) leaves were found to contain an extracellular proteinase that endoproteolytically cleaves tobacco pathogenesis-related (PR) proteins. This proteinase was partially purified from tobacco leaves and characterized as an aspartyl proteinase with a pH optimum around pH 3 and a molecular mass of 36,000 to 40,000 daltons. In vitro, the enzyme cleaved purified tobacco and tomato PR proteins into discrete fragments. The characteristics of this proteinase were similar to pepsin and identical to those displayed by a previously described tomato 37-kilodalton aspartyl proteinase active against tomato PR proteins (I Rodrigo, P Vera, V Conejero [1989] Eur J Biochem 184: 663-669), suggesting that these extracellular proteases could play a role in a conserved mechanism for PR protein turnover in plants.