Effects of ethylene and auxin on polyamine levels in suspension-cultured tobacco cells

The effects of ethylene and auxin on polyamine levels were studied in suspension-cultured cells of tobacco ( Nicotiana tabacum . L). Treatment of 4-day-cultured cells with ethylene increased the levels of spermidine and spermine. The activities of arginine decarboxylase (ADC; EC 4.1.1.19), ornithine decarboxylase (ODC: EC 4.1.1.17), and S -adenosylmethionine decarboxylase (SAMDC: EC 4.1.1.50) rapidly increased between 3 and 12 h. An auxin, indole-3-acetic acid (IAA), increased polyamine levels and activities of ADC, ODC and SAMDC. The spermine level continued to increase significantly during a 24-h incubation with IAA. The increases in polyamine accumulation induced by ethylene were partially offset by an inhibitor of ethylene action, 2,5-norbornadiene. It is suggested that the auxin-induced polyamine accumulation occurred directly, without metabolic competition between ethylene and polyamine biosynthesis, and indirectly, through auxin-induced ethylene formation.     

Role of ammonium accumulation in bacteria-induced hypersensitive and compatible reactions of tobacco and cotton plants

Leaves of 12-week-old tobacco plants (Nicotiana tabacum L. cv. Samsun NN) were infiltrated with suspensions of Pseudomonas syringae pv, pisi (DSM 50291) to induce hypersensitive reaction (HR). Cotyledons of 2-week-old cotton plants (Gossypium hirsutum L. cv. Acala 442 and Coker BR) were infiltrated with Xanthomonas campestris pv. malvacearum (race 10) to induce the disease. In tobacco, HR-related increases in NH⁺₄ levels started within 2 h after infection and continued up to the time of tissue decay. Increase of NH⁺₄ and especially K⁺ efflux were detected in intercellular washing fluids (IWF). Antibiotics stopped and later reverted NH⁺₄ production and K⁺ efflux, but only if applied within 2 h after infection. When 10 mM NH⁺₄ was injected into leaves, it was rapidly consumed from the IWF, and also, although more slowly, within the leaf cells. The concomitant K⁺ efflux was strong but delayed, and most of the K⁺ was reabsorbed after 2 h. Bacterial cell multiplication in HR stopped before the appearance of HR symptoms and cell necrosis. In the compatible reaction in cotton cotyledons, both NH₊⁴accumulation and K⁺ efflux proceeded much more slowly than in the HR with tobacco, and bacteria continued to multiply until general cell necrosis occurred. The compatible reaction developed faster in constant darkness than in a light/dark rhythm. Bacterial enzymes produced NH⁺₄, mainly from proteins of host cells, in both light and darkness. Continuous light delayed the main peak of both NH⁺₄ production and K⁺ efflux. High CO₂ concentration inhibited both processes, thus indicating that photorespiration plays a role in enhancing the release of free ammonium during bacterial pathogenesis. This is supported by shifts in the pattern of amino acids. The results demonstrate the accelerating and aggravating effect of ammonium in pathogenesis and HR, though ammonium is not the primary agent.     

Changes in endogenous cyanide and 1-aminocyclopropane-1-carboxylic acid levels during the hypersensitive response of tobacco mosaic virus-infected tobacco leaves

Leaves of Nicotiana tabacum L. cv. Xanthi necroticum plants form local necrotic lesions at the site of infection by tobacco mosaic virus. During the first seven days post-inoculation, endogenous levels of 1-aminocyclopropane-1-carboxylic acid (ACC) and N-malonyl-ACC increased in the lesion area. The time course of ACC accumulation coincided with an increase in the endogenous cyanide level which began within two days after inoculation. Concomitantly, the activity of -cyanoalanine synthase, the main HCN detoxifying enzyme, decreased. Likewise, treatment of leaf discs of uninfected plants with ACC led to cyanide accumulation. Exogenously applied KCN caused necrotic spots on tobacco leaves very similar to the whitish centers of virus-induced local lesions. Possible implications of cyanide in cell death during TMV-induced lesion development are discussed.     

Cucumber mosaic virus movement protein affects sugar metabolism and transport in tobacco and melon plants

In addition to its influence on plasmodesmal function, tobacco mosaic virus movement protein (TMV‐MP) causes an alteration in carbon metabolism in source leaves and in resource partitioning among the various plant organs. The present study was aimed at characterizing the influence of cucumber mosaic virus (CMV)‐MP on carbohydrate metabolism and transport in both tobacco and melon plants. Transgenic tobacco plants expressing the CMV‐MP had reduced levels of soluble sugars and starch in their source leaves and a significantly reduced root‐to‐shoot ratio in comparison with control plants. A novel virus‐vector system was employed to express the CMV‐coat protein (CP), the CMV‐MP or the TMV‐MP in melon plants. This set of experiments indicated that the viral MPs cause a significant elevation in the proportion of sucrose in the phloem sap collected from petioles of source leaves, whereas this sugar was at very low levels or even absent from the sap of control melon plants. The mode by which the CMV‐MP exerts its effect on phloem‐sap sugar composition is discussed in terms of possible alterations in the mechanism of phloem loading.     

A glycine-rich RNA-binding protein gene is differentially expressed during acute hypersensitive response following Tobacco Mosaic Virus infection in tobacco

During efforts for cloning disease resistance-responsive genes, a cDNA encoding a putative Nicotiana glutinosa glycine-rich RNA binding protein (ngRBP) was isolated from TMV induced cDNA library. Northern blot hybridization revealed that ngRBP gene is negatively regulated during early hours of TMV induced acute hypersensitive response (HR). Under greenhouse conditions induced expression of ngRBP gene was observed after 24 h following TMV infection. Salicylic acid and copper also induced ngRBP mRNA expression. Our findings are suggestive of some possible role for ngRBP in plant-pathogen interaction.     

Markers for hypersensitive response and senescence show distinct patterns of expression

Controlled cellular suicide is an important process that can be observed in various organs during plant development. From the generation of proper sexual organs in monoecious plants to the hypersensitive response (HR) that occurs during incompatible pathogen interactions, programmed cell death (PCD) can be readily observed. Although several biochemical and morphological parameters have been described for various types of cell death in plants, the relationships existing between those different types of PCD events remain unclear. In this work, we set out to examine if two early molecular markers of HR cell death (HIN1 and HSR203J) as well as a senescence marker (SAG12) are coordinately induced during these processes. Our result indicates that although there is evidence of some cross-talk between both cell death pathways, spatial and temporal characteristics of activation for these markers during hypersensitive response and senescence are distinct. These observations indicate that these markers are relatively specific for different cell death programs. Interestingly, they also revealed that a senescence-like process seems to be triggered at the periphery of the HR necrotic lesion. This suggests that cells committed to die during the HR might release a signal able to induce senescence in the neighboring cells. This phenomenon could correspond to the establishment of a second barrier against pathogens. Lastly, we used those cell death markers to better characterize cell death induced by copper and we showed that this abiotic induced cell death presents similarities with HR cell death.     

De novo root formation in thin cell layers of tobacco: changes in free and bound polyamines

Thin cell layers excised from tobacco ( Nicotiana tabacum L. cv. Samsun) stem internodes, with an appropriate exogenous hormonal balance, were able to form a greater number of roots, and in a larger percentage of the explants (93%) than when they were excised from pedicels (40%). The developmental sequence of root formation and explant growth were followed by histological analysis. Free and bound [trichloroacetic acid (TCA)-soluble and -insoluble] putrescine and spermidine increased in the explants, particularly when root meristemoids appeared. These meristemoids originated in the superficial (day 6 in culture) or deep (days 10–11) layers and inside the newly formed callus (day 25). At those times, TCA-soluble and, to a lesser extent, TCA-insoluble bound putrescine predominated over the other polyamines. Spermine was always present in trace amounts. Polyamines decreased again when root and callus formation was completed (day 30). The involvement of these three classes of polyamines (free, TCA-soluble and -insoluble) in morphogenic processes is discussed.     

Determination of ornithine, putrescine, N-methylputrescine and N-methylpyrroline pools in tobacco tissue by high-performance liquid chromatography

A procedure for the determination of metabolites of the biochemical pathway ornithine to N-methyl-δ¹-pyrrolinium salt (N-methylpyrroline) is described. Plant tissue was extracted with 0.5 M HCl and the extract purified on C₁₈-cartridges. Ornithine was reacted with o -phthaldialdehyde, putrescine and N-methylputrescine with dansyl chloride and the products were separated by reversed-phase high-performance liquid chromatography (HPLC). N-methylpyrroline was determined by cation-exchange HPLC without derivatization. The metabolites in the roots of tobacco ( Nicotiana ) species with different nicotine-producing capacities were determined. Furthermore, the specific activities of the enzymes ornithine decarboxylase (EC 4.1.1.17), putrescine N-methyltransferase (EC 2.1.1.53) and N-methylputrescine oxidase were determined. Both the metabolite pools and the enzyme activities were correlated with the different nicotine-producing capacities of the different tobacco species.     

Growth kinetics, polyamine pattern and biosynthesis in hairy root lines of Nicotiana tabacum

The possibility of a relation between the expression of root inducing (Ri) T-DNA genes of Agrobacterium rhizogenes and changes in polyamine metabolism has been explored in fast-growing tobacco hairy roots. Transformed root cultures have been established on hormone-fee medium; they came from transgenic plants of Nicotiana tabacum L. cv. Xanthi with different altered phenotypes, designated transformed (T) and supertransformed (T'). T and especially T' roots developed more rapidly both by elongation and lateral branching, and showed a higher growth rate than the untransformed control. After 3 weeks in culture, normal roots showed a very reduced meristematic zone, and flow cytometric analysis indicated that 2C nuclei were predominant in the apical parts in contrast to T and T' roots, in which endopolyploidisation also appeared. Putrescine, spermidine and traces of spermine were present in all the samples, both in free and in conjugated forms. Putrescine was the major polyamine detected in controls and in transformed roots. At the time of excision, the polyamine levels were similar in normal, T and T' roots. Significant differences were found during the progression of growth, particularly in the TCA-insoluble fraction in which polyamines varied differently according to the type of roots, increasing considerably in T roots on day 8, then decreasing. The lower polyamine contents found in growing transformed roots were concomitant to low arginine (EC 4.1.1.19) and ornithine (EC 4.1.1.17) decarboxylase activities. It is suggested that polyamine levels and related enzyme activities are linked to growth kinetics rather than being a consequence of foreign gene expression.     

Involvement of NtERF3 in the cell death signalling pathway mediated by SIPK/WIPK and WRKY1 in tobacco plants

We previously reported that one of the ethylene response factors (ERFs), NtERF3, and other members of the subgroup VIII‐a ERFs of the AP2/ERF family exhibit cell death‐inducing ability in tobacco leaves. In this study, we focused on the involvement of NtERF3 in a cell death signalling pathway in tobacco plants, particularly downstream of NtSIPK/NtWIPK and NtWRKY1, which are mitogen‐activated protein kinases and a phosphorylation substrate of NtSIPK, respectively. An ERF‐associated amphiphilic repression (EAR) motif‐deficient NtERF3b mutant (NtERF3bΔEAR) that lacked cell death‐inducing ability suppressed the induction of cell death caused by NtERF3a. The transient co‐expression of NtERF3bΔEAR suppressed the hypersensitive reaction (HR)‐like cell death induced by NtSIPK and NtWRKY1. The induction of cell death by NtSIPK and NtWRKY1 was also inhibited in transgenic plants expressing NtERF3bΔEAR. Analysis of gene expression, ethylene production and cell death symptoms in salicylic acid‐deficient tobacco plants suggested the existence of some feedback regulation in the HR cell death signalling pathway mediated by SIPK/WIPK and WRKY1. Overall, these results suggest that NtERF3 functions downstream of NtSIPK/NtWIPK and NtWRKY1 in a cell death signalling pathway, with some feedback regulation.     

Over-expression of a scopoletin glucosyltransferase in Nicotiana tabacum leads to precocious lesion formation during the hypersensitive response to tobacco mosaic virus but does not affect virus resistance

Nicotiana tabacum Togt encodes a scopoletin glucosyltransferase (UDPglucose:scopoletin O -beta-D-glucosyltrans- ferase, EC 2.4.1.128) known to act in vitro on many different substrates including the 6-methoxy-7-hydroxy- coumarin scopoletin. This phenolic compound accumulates in vast amounts, essentially in its glucosylated form scopolin, in tobacco during the hypersensitive response (HR) to tobacco mosaic virus (TMV). To identify the physiological role of this pathogen-inducible UDP-Glc glucosyltransferase (UGT), we generated TOGT over-expressing transgenic plants. Although no endogenous scopoletin or scopolin could be detected before infection, the accumulation of both the aglycone and the glucoside was found to be 2-fold higher in transgenic plants after inoculation with TMV than in wild-type plants. Scopoletin UGT activity in plants over-expressing Togt was significantly higher during the HR than in control plants. This up-regulated activity was associated with a strong increase of the bright blue fluorescence surrounding the HR-necrotic lesions under UV light, which is known to correlate with scopoletin and scopolin abundance. Necrosis appeared sooner in transgenic plants and lesions developed faster, suggesting an accelerated HR. Unexpectedly, the viral content in each lesion was not significantly different in transgenic and in wild-type plants. These results are discussed in relation to the role of TOGT as the major UDP-Glc: scopoletin glucosyltransferase and to the importance of scopoletin accumulation during the HR.     

Alteration in carbon partitioning induced by the movement protein of tobacco mosaic virus originates in the mesophyll and is independent of change in the plasmodesmal size exclusion limit

The influence of the 30 kDa movement protein of tobacco mosaic virus (TMV-MP) on carbon partitioning in trans-genie tobacco plants (Nicotiana tabacum cv. Xanthi) expressing the TMV-MP was investigated. Using reciprocal grafting of transgenic tobacco plants expressing this movement protein and vector control plants, as well as transgenic tobacco plants expressing the TMV-MP in phloem cells only, we showed that the interactive site involved in carbon allocation to roots is localized to the mesophyll tissue. Biomass partitioning experiments conducted on transgenic plants, in which various deletion mutant forms of the TMV-MP (two of which included deletions in the domain responsible for increasing the size exclusion limit) were expressed, revealed that the TMV-MP exerts its influence on carbon allocation via a mechanism that is completely independent of the TMV-MP-induced increase in the plasmodesmal size exclusion limit. Furthermore, small N- and C-terminal deletions in the MP revealed the complexity of the interactions likely to be involved between the MP and an endogenous regulatory mechanism. We propose that the TMV-MP interferes with an endogenous signal transduction pathway that involves macromolecular trafficking through plasmodesmata to regulate biomass partitioning between the source and various sink tissues.     

Two amino acids on 2a polymerase of Cucumber mosaic virus co-determine hypersensitive response on legumes

Thehypersensitiveresponse(HR)isoneofmostextensivelystudiedresistantreactionsdur-ingtheincompatibleinteractionbetweenplantandpathogen.Inthisprocess,plantinitiatesdiversedefensesystemssuchasdepositionofligninandcalloseinthecellwall,productionofphytoalex-ins,expressionofpathogenesis-relatedproteins(PRprotein)andactivationofprogrammedcelldeath(PCD),whichresultinlimitationofthepathogenwithintheinitialinfectionsites[1,2].TheHRinducedbybacteria,fungiandvirusesusuallyactivatesasystemicacquiredresis…     

Ornithine decarboxylase activity and the hypersensitive reaction to tobacco mosaic virus in Nicotiana tabacum

The activity of ornithine decarboxylase (ODC) is increased 20 fold in leaves of Nicotiana tabacum cv. Xanthi n.c. following infection with tobacco mosaic virus at 20°. The activity reaches its maximum when localized necrotic lesions appear. There is little or no increase in plants kept at 32° when infection is systemic. However, if the infected plants are transferred to 20°, a marked and rapid increase in ODC activity occurs in the upper leaves, which collapse seven to nine hours after the transfer. ODC activity therefore parallels the activity of phenylalanine ammonia lyase during the hypersensitive reaction. Tyrosine decarboxylase was found to be activated in the same conditions. By contrast no increase in arginine decarboxylase activity could be detected. Temperature has a much greater effect on the polyamine and tyramine content of Xanthi n.c. leaves than does infection with TMV.     

Free and conjugated polyamines during de novo floral and vegetative bud formation in thin cell layers of tobacco

The concentrations of three classes of polyamines, trichloroacetic acid-soluble (free), TCA-soluble conjugated (to small molecules) and TCA-insoluble conjugated (to macromolecules), was examined during de novo floral and vegetative bud formation in thin cell layers of Nicotiana tabacum L. cv. Samsun. Explants (consisting of 5–6 layers of epidermal, subepidermal and parenchyma cells) were excised either from floral pedicels or from stem internodes at the unripe fruit stage and cultured on the same medium. In the former, the first de novo formed flower buds appeared on day 8 of culture, while in the latter the first vegetative domes appeared on day 10. In both cases the number of floral and vegetative buds increased up to day 12 and 15, respectively. Changes in dry weight were determined throughout the culture period. Free and conjugated putrescine titer increased 5–60 times in both types of culture and in the three classes of polyamines examined; spermidine content also increased, while spermine, when present, did not show significant changes. TCA-soluble conjugated polyamines were most abundant, being about 2-fold the TCA-insoluble conjugated ones and 10-fold the free ones. The major increment in putrescine and spermidine content occurred in stem internode explants developing vegetative buds. In pedicel explants the maximum putrescine level was reached before or on day 8 in culture (emergence of the first flower buds with calyx initials), while in stem internode explants the maximum level was reached on day 12, at the emergence of the first vegetative buds with leaf primordia. While spermidine prevailed on day 0, putrescine was the most abundant polyamine during both differentiation processes. The putrescine content rapidly increased immediately after the onset of culture. Thus conjugated polyamines, especially putrescine, and not only the free ones, seem to be involved in both the reproductive and vegetative phases of tobacco growth and development.