Ethylene insensitivity impairs resistance to soilborne pathogens in tobacco and Arabidopsis thaliana

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.     

Influence of salicylic acid on rubisco and rubisco activase in tobacco plant grown under sodium chloride in vitro

The present study was designed to evaluate the influence of salicylic acid (SA) on the growth of salt stress (sodium chloride) induced in tobacco plants. In addition, quantification of rubisco and rubisco activase contents of the plants was also determined in treatments with the control, 10⁻⁴ mM SA, 50 mM NaCl, 100 mM NaCl, 150 mM NaCl, SA + 50 mM NaCl, SA + 100 mM NaCl and SA + 150 mM NaCl, respectively after in vitro culture for 5 weeks. The growth of the tobacco plant decreased in 50 mM and 100 mM NaCl when not treated with SA. However, the growth was accelerated by SA, and the growth retardation caused by NaCl was improved by SA. The content of rubisco was improved by SA only in plants treated with 50 mM NaCl, and the activity of rubisco was increased by SA resulting in the decreased effect of NaCl, but only in 50 mM NaCl treated plants. The content of rubisco activase decreased due to NaCl, and SA did not improve the effect caused by NaCl. The activity of rubisco activase was increased by SA resulting in decreased activity caused by NaCl, but increased effect by SA was not recovered to the level of NaCl untreated plants. The activity of rubisco and rubisco activase, which decreased due to denaturing agents, did not demonstrate significant improvement when compared to the control.     

Influence of exogenous application of glutathione on rubisco and rubisco activase in heavy metal-stressed tobacco plant grown in vitro

The effect of glutathione on the influences of heavy metals affecting rubisco and rubisco activase was studied in tobacco plants grown in vitro where the shoot explants of the tobacco plant cultured on MS medium under aseptic conditions and two explants were placed in the control, 0.1 mM GSH, 1 mM GSH, 0.2 mM Cd, 0.2 mM Cu, 0.2 mM Zn, and a mixture of Cd and GSH, Cu and GSH, Zn and GSH, respectively. The effect of GSH on the growth of the tobacco plant was minimal, but the heavy metals clearly retarded its growth. GSH recovered the growth retarded by heavy metals, and the concentration of GSH required to recover the growth differed depending on the heavy metals. The content of chlorophyll in the plant increased through GSH and Zn, and decreased through Cd and Cu. The chlorophyll content which decreased due to Cd and Cu was recovered by GSH, and the content which increased due to Zn was decreased by 1 mM GSH. The content of rubisco decreased due to GSH and heavy metals, and the content which decreased due to heavy metals was recovered by GSH, and when GSH was treated with Zn, the increased rate was maximum compared to other heavy metals. The activity of rubisco was increased due to GSH and heavy metals, and the activity increased by Cd and Zn decreased through GSH. In the case of Cu, the activity of GSH increased even more. There was no effect of GSH on the influences of heavy metals on the content and activity of rubisco activase. The activity of rubisco decreased by thiourea among six denaturing agents, and increased by l-cysteine, and in most cases the activity level was recorded as high. The activity of rubisco activase all decreased as a result of six denaturing agents, and the effect caused by EDTA and guanidine-HCl was the greatest, while the effect caused by l-cysteine and urea was minimal.     

Ethylene insensitivity modulates ozone-induced cell death in birch

We have used genotypic variation in birch (Betula pendula Roth) to investigate the roles of ozone (O(3))-induced ethylene (ET), jasmonic acid, and salicylic acid in the regulation of tissue tolerance to O(3). Of these hormones, ET evolution correlated best with O(3)-induced cell death. Disruption of ET perception by transformation of birch with the dominant negative mutant allele etr1-1 of the Arabidopsis ET receptor gene ETR1 or blocking of ET perception with 1-methylcyclopropene reduced but did not completely prevent the O(3)-induced cell death, when inhibition of ET biosynthesis with aminooxyacetic acid completely abolished O(3) lesion formation. This suggests the presence of an ET-signaling-independent but ET biosynthesis-dependent component in the ET-mediated stimulation of cell death in O(3)-exposed birch. Functional ET signaling was required for the O(3) induction of the gene encoding beta-cyanoalanine synthase, which catalyzes detoxification of the cyanide formed during ET biosynthesis. The results suggest that functional ET signaling is required to protect birch from the O(3)-induced cell death and that a decrease in ET sensitivity together with a simultaneous, high ET biosynthesis can potentially cause cell death through a deficient detoxification of cyanide.     

Inheritance of insensitivity to tobacco rattle virus in potatoes

The sensitivity of 20 genotypes from each of 13 progenies of parental potato clones was assessed using recently developed glasshouse testing procedures. Significant heritable differences between the progenies with respect to their degree of sensitivity to TRV were observed. Significant general and specific combining abilities were also demonstrated. The results are discussed in relation to modes of inheritance of this trait. The results indicate that though a major gene for TRV insensitivity may be operating this does not explain all the observed variation and it is suggested that there is also a system inherited in a polygenic manner that can confer good levels of insensitivity to TRV.     

Winter down-regulation of intrinsic photosynthetic capacity coupled with up-regulation of Elip-like proteins and persistent energy dissipation in a subalpine forest

Overwintering, sun-exposed and photosynthetically inactive evergreens require powerful photoprotection. The goal of this study was to seasonally characterize photosynthesis and key proteins/components involved in electron transport and photoprotection. Maximal photosystem II (PSII) efficiency and photosynthetic capacity, amounts of zeaxanthin (Z), antheraxanthin (A), pheophytin and proteins (oxygen-evolving 33 kDa protein (OEC), PSII core protein D1 and subunit S (PsbS) protein, and members of the early light-inducible protein (Elip) family) were assessed in five conifer species at high altitude and in ponderosa pine (Pinus ponderosa) at moderate altitude during summer and winter. Relative to summer, winter down-regulation of photosynthetic capacity and loss of PSII efficiency at the high-altitude sites were paralleled by decreases in OEC, D1, and pheophytin; massive nocturnal retention of (Z + A) and up-regulation of two to four proteins cross-reactive with anti-Elip antibodies; and no change in PsbS amount. By contrast, ponderosa pine at moderate altitude exhibited no down-regulation of photosynthetic capacity, smaller depressions in PSII efficiency, and less up-regulation of Elip family members. These results support a function for members of the Elip family in the acclimation of sun-exposed needles that down-regulate photosynthesis during winter. A possible role in sustained photoprotection is considered.     

Stomatal conductance does not correlate with photosynthetic capacity in transgenic tobacco with reduced amounts of Rubisco

High-resolution imaging of chlorophyll a fluorescence from intact tobacco leaves was used to compare the quantum yield of PSII electron transport in the chloroplasts of guard cells with that in the underlying mesophyll cells. Transgenic tobacco plants with reduced amounts of Rubisco (anti-Rubisco plants) were compared with wild-type tobacco plants. The quantum yield of PSII in both guard cells and underlying mesophyll cells was less in anti-Rubisco plants than in wild-type plants, but closely matched between the two cell types regardless of genotype. CO2 assimilation rates of anti-Rubisco plants were 4.4 micromol m(-2) s(-1) compared with 17.3 micromol m(-2) s(-1) for the wild type, when measured at a photon irradiance of 1000 micromol m(-2) s(-1) and ambient CO2 of 380 micromol mol(-1). Despite the large difference in photosynthetic capacity between the anti-Rubisco and wild-type plants, there was no discernible difference in the rate of stomatal opening, steady-state stomatal conductance or response of stomatal conductance to ambient CO2 concentration. These data demonstrate clearly that the commonly observed correlation between photosynthetic capacity and stomatal conductance can be disrupted in the long term by manipulation of photosynthetic capacity via antisense RNA technology. It was concluded that stomatal conductance is not directly determined by the photosynthetic capacity of guard cells or the leaf mesophyll.     

Reduced sedoheptulose-1,7-bisphosphatase levels in transgenic tobacco lead to decreased photosynthetic capacity and altered carbohydrate accumulation

Transgenic tobacco (Nicotiana tabacum L. cv. Samsun) plants with reduced levels of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase; EC 3.1.3.37) were produced using an antisense construct in which the expression of a tobacco SBPase cDNA clone was driven by the cauliflower mosaic virus (CaMV) promoter. The reduction in SBPase protein levels observed in the primary transformants correlated with the presence of the antisense construct and lower levels of the endogenous SBPase mRNA. No changes in the amounts of other Calvin cycle enzymes were detected using Western blot analysis. The SBPase antisense plants with less than 20% of wild-type SBPase activity were observed to display a range of phenotypes, including chlorosis and reduced growth rates. Measurements of photosynthesis, using both light-dosage response and CO₂ response curves, of T1 plants revealed a reduction in carbon assimilation rates, which was apparent in plants retaining 57% of wild-type SBPase activity. Reductions were also observed in the quantum efficiency of photosystem II. This decrease in photosynthetic capacity was reflected in a reduction in the carbohydrate content of leaves. Analysis of carbohydrate status in fully expanded source leaves showed a shift in carbon allocation away from starch, whilst sucrose levels were maintained in all but the most severely affected plants. Plants with less than 15% of wild-type SBPase activity were found to contain less than 5% of wild-type starch levels. The results of this preliminary analysis indicate that SBPase activity may limit the rate of carbon assimilation. Received: 23 February 1997 / Accepted: 2 May 1997     

Photoperiod and photosynthetic capacity in Lotus scoparius

Abstract Leaves of Lotus scoparius Nutt., a Mediterranean-climate shrub, exhibited higher photosynthetic capacities when grown under winter (10/14 h, day/night) than summer (14/10 h) photo-periods, even though total daily photon irradiance was higher under summer photoperiods. Photosynthetic dependence on natural variations in photo-period, such that activity was higher under photo-periods associated with expected precipitation, may be a more dependable environmental parameter than total irradiance in temperate habitats with winter-spring precipitation patterns and where seasonal cloudiness may cause total daily irradiance levels to be highly variable.     

Ethylene insensitivity impedes a subset of responses to phosphorus deficiency in tomato and petunia

The role of ethylene in growth and developmental responses to low phosphorus stress was evaluated using ethylene-insensitive 'Never-ripe' (Nr) tomato and etr1 petunia plants. Low phosphorus increased adventitious root formation in 'Pearson' (wild-type) tomato plants, but not in Nr, supporting a role for ethylene in adventitious root development and showing that ethylene is important for this aspect of phosphorus response. Low phosphorus reduced ethylene production by adventitious roots of both genotypes, suggesting that ethylene perception--not production--regulates carbon allocation to adventitious roots at the expense of other roots under low phosphorus stress. With the exception of its effect on adventitious rooting, Nr had positive effects on growth and biomass accumulation in tomato whereas etr1 tended to have negative effects on petunia. This was particularly evident during the recovery from transplanting, when the effective quantum yield of photosystem II of etr1 petunia grown with low phosphorus was significantly lower than 'Mitchell Diploid', suggesting that etr1 petunia plants may undergo more severe post-transplant stress at low phosphorus availability. Our results demonstrate that ethylene mediates adventitious root formation in response to phosphorus stress and plays an important role for quick recovery of plants exposed to multiple environmental stresses, i.e. transplanting and low phosphorus.     

A new rubisco-like protein coexists with a photosynthetic rubisco in the planktonic cyanobacteria Microcystis

Two genes encoding proteins related to large subunits of Rubisco were identified in the genome of the planktonic cyanobacterium Microcystis aeruginosa PCC 7806 that forms water blooms worldwide. The rbcL(I) gene belongs to the form I subfamily typically encountered in cyanobacteria, green algae, and land plants. The second and newly discovered gene is of the form IV subfamily and widespread in the Microcystis genus. In M. aeruginosa PCC 7806 cells, the expression of both rbcL(I) and rbcL(IV) is sulfur-dependent. The purified recombinant RbcL(IV) overexpressed in Escherichia coli cells did not display CO(2) fixation activity but catalyzed enolization of 2,3-diketo-5-methylthiopentyl-1-phosphate, and the rbcL(IV) gene rescued a Bacillus subtilis MtnW-deficient mutant. Therefore, the Microcystis RbcL(IV) protein functions both in vitro and in vivo and might be involved in a methionine salvage pathway. Despite variations in the amino acid sequences, RbcL(IV) shares structural similarities with all members of the Rubisco superfamily. Invariant amino acids within the catalytic site may thus represent the minimal set for enolization, whereas variations, especially located in loop 6, may account for the limitation of the catalytic reaction to enolization. Even at low protein concentrations in vitro, the recombinant RbcL(IV) assembles spontaneously into dimers, the minimal unit required for Rubisco forms I-III activity. The discovery of the coexistence of RbcL(I) and RbcL(IV) in cyanobacteria, the ancestors of chloroplasts, enlightens episodes of the chaotic evolutionary history of the Rubiscos, a protein family of major importance for life on Earth.     

Ethylene insensitivity conferred by the Green-ripe and Never-ripe 2 ripening mutants of tomato

The ripening of a fleshy fruit represents the summation of an array of biochemical processes that are regulated by interactions between developmental programs and environmental inputs. Analysis of tomato (Solanum lycopersicum) mutants and inhibitor studies indicate that ethylene is necessary for full development of the ripening program of climacteric fruit such as tomato, yet ethylene alone is not sufficient. This suggests that an interaction between ethylene and nonethylene (or developmental) pathways mediates ripening. In this study, we have examined the physiological basis for ripening inhibition of the dominant Green-ripe (Gr) and Never-ripe 2 (Nr-2) mutants of tomato. Our data suggest that this inhibition is due to ethylene insensitivity in mutant fruit. Further investigation of ethylene responses in Gr and Nr-2 plants also revealed weak ethylene insensitivity during floral senescence and abscission and, during inhibition of root elongation, a phenotype associated with the triple response. However, ethylene-induced inhibition of hypocotyl elongation and petiole epinasty are normal in Gr and Nr-2, suggesting that these loci regulate a subset of ethylene responses. We have mapped both dominant mutations to a 2-cM overlapping region of the long arm of chromosome 1 of tomato, a region not previously linked to any known ethylene signaling loci. The phenotypic similarity and overlapping map location of these mutations suggest Gr and Nr-2 may be allelic and may possibly encode a novel component of the ethylene response pathway.     

Ethylene regulates the timing of anther dehiscence in tobacco

We investigated the involvement of ethylene signaling in the development of the reproductive structures in tobacco ( Nicotiana tabacum L.) by studying flowers that were insensitive to ethylene. Ethylene-insensitivity was generated either by expression of the mutant etr1-1 ethylene-receptor allele from Arabidopsis thaliana or by treatment with the ethylene-perception inhibitor 1-methylcyclopropene (MCP). Development of ovaries and ovules was unaffected by ethylene-insensitivity. Anther development was also unaffected, but the final event of dehiscence was delayed and was no longer synchronous with flower opening. We showed that in these anthers degeneration of the stomium cells and dehydration were delayed. In addition, we found that MCP-treatment of detached flowers and isolated, almost mature anthers delayed dehiscence whereas ethylene-treatment accelerated dehiscence. This indicated that ethylene has a direct effect on a process that takes place in the anthers just before dehiscence. Because a similar function has been described for jasmonic acid in Arabidopsis, we suggest that ethylene acts similarly to or perhaps even in concurrence with jasmonic acid as a signaling molecule controlling the processes that lead to anther dehiscence in tobacco.     

Transpiration-induced changes in the photosynthetic capacity of leaves

High transpiration rates were found to affect the photosynthetic capacity of Xanthium strumarium L. leaves in a manner analagous to that of low soil water potential. The effect was also looked for and found in Gossypium hirsutum L., Agathis robusta (C. Moore ex Muell.) Bailey, Eucalyptus microcarpa Maiden, Larrea divaricata Cav., the wilty flacca tomato mutant (Lycopersicon esculentum (L.) Mill.) and Scrophularia desertorum (Munz) Shaw. Two methods were used to distinguish between effects on stomatal conductance, which can lower assimilation by reducing CO₂ availability, and effects on the photosynthetic capacity of the mesophyll. First, the response of assimilation to intercellular CO₂ pressure (C _i) was compared under conditions of high and low transpiration. Second, in addition to estimating C _i using the usual Ohm's law analogy, C _i was measured directly using the closed-loop technique of T.D. Sharkey, K. Imai, G.D. Farquhar and I.R. Cowan (1982, Plant Physiol, 60, 657–659). Transpiration stress responses of Xanthium strumarium were compared with soil drought effects. Both stresses reduced photosynthesis at high C _i but not at low C _i; transpiration stress increased the quantum requirement of photosynthesis. Transpiration stress could be induced in small sections of leaves. Total transpiration from the plant did not influence the photosynthetic capacity of a leaf kept under constant conditions, indicating that water deficits develop over small areas within the leaf. The effect of high transpiration on photosynthesis was reversed approximately half-way by returning the plants to low-transpiration conditions. This reversal occurred as fast as measurements could be made (5 min), but little further recovery was observed in subsequent hours.Abbreviations and symbols A photosynthetic CO₂ assimilation rate - C _a ambient CO₂ partial pressure - C _i partial pressure of CO₂ inside the leaf - VPD leaf-to-air water-vapor pressure difference This research was begun while the author was a Postdoctoral Research Fellow at the Australian National University, Canberra