Changes in electron transport,superoxide dismutase and ascorbate peroxidase isoenzymes in chloroplasts and mitochondria of cucumber leaves as influenced by chilling

In order to clarify the relationship between chill-induced disturbance in photosynthetic, respiratory electron transport and the metabolism of reactive oxygen species (ROS), leaf gas exchange, chlorophyll fluorescence quenching, respiration, and activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) were investigated in chloroplasts and mitochondria of cucumber (Cucumis sativus) leaves subjected to a chill (8 °C) for 4 d. Chilling decreased net photosynthetic rate (P _N) and quantum efficiency of photosystem 2 (Φ_PS2), but increased the ratio of Φ_PS2 to the quantum efficiency of CO₂ fixation (ΦCO₂) and non-photochemical quenching (NPQ) in cucumber leaves. While chilling inhibited the activity of cytochrome respiration pathway, it induced an increase of alternative respiration pathway activity and the reduction level of Q-pool. Chilling also significantly increased O₂ ^• production rate, H₂O₂ content, and SOD and APX activities in chloroplasts and mitochondria. There was a more significant increase in SOD and APX activities in chloroplasts than in mitochondria with the increase of membrane-bound Fe-SOD and tAPX in chloroplasts being more significant than other isoenzymes. Taken together, chilling inhibited P _N and cytochrome respiratory pathway but enhanced the photosynthetic electron flux to O₂ and over-reduction of respiratory electron transport chain, resulting in ROS accumulation in cucumber leaves. Meanwhile, chilling resulted in an enhancement of the protective mechanisms such as thermal dissipation, alternative respiratory pathway, and ROS-scavenging mechanisms (SODs and APXs) in chloroplasts and mitochondria.     

Photosynthetic electron transport in tobacco leaves infected with tobacco mosaic virus

Tobacco ( Nicotiana tabacum L. cv. Samsun) plants inoculated with different strains of tobacco mosaic virus (TMV) inducing mosaic symptoms of widely varying severity were studied with in vivo chlorophyll fluorescence. This method was used to deduce photosynthetic electron transport efficiency in relation to symptom expression. The quantum yields of photosystem II (PS II) electron transport rate were significantly diminished in virus strains inducing loss of chlorophyll. The reduction in young mosaic-diseased leaves appeared to be due in part to a reduction in the fraction of open reaction centers, whereas in older leaves exhibiting less pronounced symptoms the reduction was mainly caused by a reduced efficiency of capture of excitation energy of open PS II reaction centers. Upon infection with any of the five virus strains PS II seemed to be irreversibly damaged in the inoculated leaves and the ones directly above, indicative of a possible increased susceptibility to photoinhibition in these leaves (Somersalo and Krause 1989) even when no symptoms were apparent. Symptom expression did not appear to be related to the influence of the virus on PS II activity, because the severest effects occurred in the inoculated leaves, which either remained symptomless or developed slight yellowing only. This study demonstrates the usefulness or modulated chlorophyll fluorescence measurements for the investigation of plant-virus interactions. It is particularly important when visual symptoms are absent.     

Effect of cucumber mosaic virus infection on morphology,yield and phenolic contents of tomato

Ten tomato genotypes were screened for their resistance against cucumber mosaic virus (CMV) and its vector Myzus persicae under natural infection in field, using aphids M. persicae under net-house and mechanical inoculation under greenhouse. Large differences were observed among genotypes for infection percentage (IP) and severity index (SI) among the testing methods used. All genotypes showing tolerance to CMV in the field or through aphid inoculation, however, become susceptible and highly susceptible after mechanical inoculation. All the test genotypes also showed susceptibility to the aphid M. persicae population. Plants inoculated with CMV showed substantial decrease in yield and yield-contributing parameters which varied with cultivars that probably depended upon its genetic make up. All the test genotypes exhibited 0.97–30.19% decrease in plant height, 11.47–52.65% decrease in root length, 46.56–95.56% decrease in fresh plant weight, 65.78–92.84% decrease in root fresh weight, 19.97–87.65% decrease in the dry weight of plants, 75.63–95.43% decrease in dry root weight, 69.51–95.65% reduction in the number of fruits and 89.04–99.89% decrease in yield per plants. After 15 days of inoculation, the quantitative analysis using double beam spectrophotometer showed an increase in total phenolics in CMV-inoculated plants as compared to un-inoculated plants among genotypes. Similarly the thin layer chromatography (TLC) on silica gel G indicated that the number of phenolic compounds was increased in most of the inoculated genotypes while in others they were either decreased or remained same.     

Soluble pathogenesis-related proteins ofCapsicum annuum leaves induced by cucumber mosaic virus infection

Eight major pathogenesis related (PR) proteins were found in a soluble extract of cucumber mosaic virus (CMV) infectedCapsicum annuum leaves. None of them was present in the soluble extract of sham-inoculated controls. The proteins were detected by two-dimensional polyacrylamide gel electrophoresis using (2D-PAGE)PhastSystem, native-PAGE in the first dimension and SDS-PAGE in the second. Two major acidic PR-proteins were identified on the basis of their relative molecular mass (M_r), PR1 of 15.3 kD and PR2 of 29 kD. Six proteins were basic and were identified as PRla of 15.9 kD, PRlb of 15.0 kD, PRlc of 15.9 kD, PR2 of 27.0 kD, PR3 of 36.3 kD and PR4 of 48.8 kD.     

The effect of Botrytis cinerea infection on the antioxidant profile of mitochondria from tomato leaves

Infection of tomato leaves with the necrotrophic fungus Botrytis cinerea resulted in substantial changes in enzymatic and non-enzymatic components of the ascorbate-glutathione cycle as well as in superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione transferase (GST), and l-galactono-gamma-lactone dehydrogenase (GLDH) activities. In the initial phase of the 5 d experiment CuZn SOD was the most rapidly induced isoform (up to 209% of control), whereas later on its activity increase was not concomitant with the constant total SOD enhancement. Starting from the second day B. cinerea infection diminished the mitochondrial antioxidant capacity by decreasing activities of ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) as well as declining ascorbate and glutathione contents. This was accompanied by dehydroascorbate (DHA) and oxidized glutathione (GSSG) accumulation that resulted in ascorbate and glutathione redox ratios decreases. The strongest redox ratio decline of 29% for ascorbate and of 34% for glutathione was found on the 3rd and 2nd days, respectively. Glutathione reductase (GR) induction (185% of control 2 d after inoculation) was insufficient to overcome the decreased antioxidant potential of glutathione. Changes in the ascorbate pool size were closely related to the activity of l-galactono-gamma-lactone dehydrogenase (GLDH). The activities of two glutathione-dependent enzymes: GSH-Px and GST were increased from day 1 to day 4. These results demonstrated that in B. cinerea-tomato interaction mitochondria could be one of the main targets for infection-induced oxidative stress.     

The effect of Benlate and cytokinins on the content of tobacco mosaic virus in tomato leaf disks and cucumber mosaic virus in cucumber cotyledon disks and seedlings

Tomato leaf disks were inoculated with tobacco mosaic virus (TMV) and floated for 7 days on solutions of kinetin and benzyladenine in the range 20-0-002 mg/1. Virus content was reduced at the higher and increased at the lower concentrations. Benlate and benomyl showed a peak of cytokinin activity in the Amaranthus betacyanin bioassay equivalent to c. 0–002 fig/l kinetin. At concentrations above 25 and 100 mg a.i./l for Benlate and benomyl respectively, both compounds increased the TMV content of tomato leaf disks. Cucumber mosaic virus (CMV) content in cucumber cotyledon disks was increased by Benlate and benomyl treatment (50–100 mg/1). Applied as a soil drench (50–500 mg a.i./l) when the plants were inoculated, Benlate increased the CMV content of infected seedlings. The number of starch-iodide lesions (a measure of susceptibility) was unaltered in cotyledons treated with Benlate 7 days before or immediately after inoculation. Infectivity of crude infective cucumber sap was unaffected by benomyl incorporation, whereas Benlate reduced infectivity at higher concentrations (1000–5000 mg/1). Under the experimental conditions described, Benlate, benomyl, benzyladenine and kinetin had no effect on the chlorophyll content of tomato leaf disks, and intact seedlings.     

Replication footprint analysis of cucumber mosaic virus electroporated into tomato protoplasts

Total RNA extracted from cucumber mosaic virus (CMV) strains WT, with its associated satellite CARNA 5 (CMV-associated RNA 5), was successfully electroporated into isolated tomato protoplasts. At various time intervals samples were extracted for total nucleic acids and analyzed by semidenaturing polyacrylamide gel electrophoresis (PAGE). Sequence-specific hybridization probes were used for the detection of viral and satellite RNAs following Northern transfer. The resulting PAGE patterns and/or autoradiographs depict the proportional presence of viral and satellite RNAs in the extracts over time and have been referred to as "replication footprint profiles" (RFPs) of specific CMV/CARNA 5 combinations. The effective isolation and infection of tomato protoplasts, combined with the ability to follow virus/satellite titers during the infection by RFP analysis, yield results similar to those of infected plants and reduces experiments of 21 or more days in whole plants to less than 72 h in protoplasts.     

Electroporation-mediated infection of tobacco leaf protoplasts with tobacco mosaic virus RNA and cucumber mosaic virus RNA

Conditions were established for the introduction of both tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) RNAs into tobacco mesophyll protoplasts by electroporation. The proportion of infected protoplasts was quantified by staining with viral coat protein-specific antibodies conjugated to fluorescein isothiocyanate. Approximately 30–40% of the protoplasts survived electroporation. Under optimal conditions, up to 75% of these were infected with TMV-RNA. Successful infection was demonstrated in 19 out of 20 experiments. Optimal infection was achieved with several direct current pulses of 90 sec at a field strength of 5 to 10 kV/cm. Changing the position of the protoplasts within the chamber between electric pulses was essential for achievement of high rates of infection. Optimal viral RNA concentration was about 10 g/ml in a solution of 0.5 M mannitol without buffer salts.     

Seed-borne cucumber mosaic virus infection of subterranean clover in Western Australia

In Western Australia, infection with cucumber mosaic virus (CMV) was widespread in all three subspecies of subterranean clover (Trifolium subterraneum) growing in plots belonging to the Australian National Subterranean Clover Improvement Programme. Seed-borne CMV was detected in seed harvested in 1984–1986 of 18/25 cultivars from two collections of registered cultivars; seed transmission rates ranged up to 8.8%. Seed samples from CMV-inoculated plants of 11 cultivars transmitted the virus to 0.5–8.7% of seedlings. Seed transmission rates greater than 5% were obtained only with cvs Enfield, Green Range and Nangeela. CMV was not detected in seed harvested in 1975–1981 from one of the registered cultivar collections, in 17 commercial seed stocks from 1986 or in a survey of subterranean clover pastures.
Symptoms in subterranean clover naturally infected with CMV included mottle, leaflet downcurling and dwarfing but severity varied with cultivar and selection. CMV isolates from different sources varied in virulence when inoculated to subterranean clover; two (both from subterranean clover) were severe, two moderate and three (including one from subterranean clover) mild. In pot tests, CMV decreased herbage production and root growth (dry wts) of cv. Green Range by 49% and 59% respectively. In spaced-plants growing in plots, CMV decreased herbage production and root growth of cvs Green Range and Northam by 59–630 and seed production of cv. Green Range by 45%. In rows sown with infected seed, aphid spread increased infection levels to 75% in cv. Green Range and 44% in cv. Esperance and losses in herbage production of 42% and 29% respectively were recorded.
CMV isolated from subterranean clover included isolates from both serogroups.     

Evolutionary relationship of alfalfa mosaic virus with cucumber mosaic virus and brome mosaic virus

The amino acid sequences of the non-structural protein (molecular weight 35,000; 3a protein) from three plant viruses — cucumber mosaic, brome mosaic and alfalfa mosaic have been systematically compared using the partial genomic sequences for these three viruses already available. The 3a protein of cucumber mosaic virus has an amino acid sequence homology of 33.7% with the corresponding protein of brome mosaic virus. A similar protein from alfalfa mosaic virus has a homology of 18.2% and 14.2% with the protein from brome mosaic virus and cucumber mosaic virus, respectively. These results suggest that the three plant viruses are evolutionarily related, although, the evolutionary distance between alfalfa mosaic virus and cucumber mosaic virus or brome mosaic virus is much larger than the corresponding distance between the latter two viruses.     

Inhibition site of the electron transport system in lettuce chloroplasts by fumigation of leaves with SO2

In chloroplasts isolated from SO₂-fumigated leaves at 2.0 ppm,electron flow from water to 2,6-dichloroindophenol (DCIP) wasinhibited, but the electron flow from reduced DCIP to methylviologen was not affected. Neither diphenylcarbazide nor MnCl₂could restore the activity of the DCIP-Hill reaction of SO₂-injuredchloroplasts. Electron flows, from water to ferricyanide orto silicomolybdic acid, were inhibited in a degree similar tothat of the DCIP-Hill reaction. The rate of carotenoid photobleaching in the presence of carbonylcyanide-m-chlorophenylhydrazone was suppressed and paralleledthe inhibition of the DCIP-Hill reaction. In SO₂-injured chloroplasts, the variable part of the fluorescencetransient was diminished, and the fluorescence yield loweredby SO₂ was increased with 3-(3', 4'-dichlorophenyl)-l, l-dimethylurea(DCMU) or more pronouncedly by incubating the sample with sodiumdithionite. However, the yield could not recover to the levelfound in non-fumigated chloroplasts. With SO₂ fumigation, thetime required to reach steady-state level of fluorescence becamelonger in the absence of DCMU, but was not altered in the presenceof DCMU. The pool size of the primary electron acceptors decreasedwith SO₂ fumigation. We concluded that SO₂ inactivated the primaryelectron donor or the reaction center itself. The mode of SO₂action in the electron transport chain is discussed. (Received October 20, 1979; )     

Effects of copper on photosynthetic electron transport systems in spinach chloroplasts

The effects of copper on photosynthetic electron transfer systemsin isolated spinach chloroplasts were studied. Two differentinhibitions were observed. First, copper markedly inhibitedferredoxin-catalyzed reactions such as NADP⁺ photoreduction.The concentration required for 50% inhibition was about 2 µMof cupric sulfate. However, electron flow from reduced 2,6-dichloroindophenol(DCIP) to methyl viologen was not affected. The dissociationconstant between ferredoxin and ferredoxin-NADP⁺ reductase wasunchanged in the presence of 2.5 µM of cupric sulfate.In enzymic reaction systems, the ferredoxin-dependent electronflow from NADPH to cytochrome c was also strongly inhibitedin the presence of cupric sulfate, while DCIP reduction withNADPH as the electron donor was not affected. Second, DCIP photoreductionwas weakly blocked by copper and the lost activity could notbe recovered by adding 1,5-diphenylcarbazide (DPC). It can be concluded that copper directly interacted with ferredoxincausing inhibition of ferredoxin-dependent reactions. Further,copper caused weak inactivation between the oxidizing side ofthe reaction center of photosystem II and the electron donatingsite of DPC. (Received August 8, 1977; )     

Alfalfa mosaic and cucumber mosaic virus infection in chickpea and lentil: incidence and seed transmission

Samples collected in 1994 and 1995 from commercial crops of chickpeas and lentils growing in the agricultural region of south-west Western Australia were tested for infection with alfalfa mosaic (AMV) and cucumber mosaic (CMV) viruses, and for members of the family Potyviridae using enzyme-linked immunosorbent assay (ELISA). In 1994 no virus was detected in the 21 chickpea crops tested but in 1995, out of 42 crops, AMV was found in two and CMV in seven. With lentils, AMV and/or CMV was found in three out of 14 crops in 1994 and 4 out of 13 in 1995, both viruses being detected in two crops in each year. Similar tests on samples from chickpea and lentil crops and plots growing at experimental sites, revealed more frequent infection with both viruses. No potyvirus infection was found in chickpeas or lentils in agricultural areas either in commercial crops or at experimental sites. However, bean yellow mosaic virus (BYMV) was detected along with AMV and CMV in irrigated plots of chickpeas and lentils at a site in Perth. When samples of seed from infected crops or plots of chickpeas and lentils were germinated and leaves or roots of seedlings tested for virus infection by ELISA, AMV and CMV were found to be seed-borne in both while BYMV was seed-borne in lentils. The rates of transmission found through seed of chickpea to seedlings were 0.1–1% with AMV and 0.1–2% with CMV. Seed transmission rates with lentil were 0.1–5% for AMV, 0.1–1% for CMV and 0.8% for BYMV. Individual seed samples of lentil and chickpea sometimes contained both AMV and CMV. With both species, infection with AMV and CMV was sometimes found in commercial seed stocks or seed stocks from multiplication crops of advanced selections nearing release as new cultivars. Seed-borne virus infection has important practical implications, as virus sources can be re-introduced every year to chickpea and lentil crops or plots through sowing infected seed stocks leading to spread of infection by aphid vectors, losses in grain yield and further contamination of seed stocks.     

Effects of purine nucleotides on photosynthetic electron transport in isolated chloroplasts

The effects of guanylates and inosinates (and adenylates) on phosphorylation, ferricyanide reduction, and light-induced H⁺ uptake in spinach chloroplasts were studied. GDP, GTP, IDP, and ITP (but not GMP and IMP) stimulated the light-induced H⁺ uptake and partially inhibited ferricyanide reduction. Phosphate, arsenate, and phlorizin increased the extent of inhibition by these nucleotides and decreased the values of their apparent dissociation constants for the inhibition process. In the presence of phosphate (or arsenate), restoration of ferricyanide reduction from the level inhibited by guanylates and inosinates was observed as phosphorylation (or arsenylation) proceeded. These results suggest that phosphorylation of GDP and IDP as well as ADP takes place after two steps of nucleotide binding to the chloroplast coupling factor 1. The apparent dissociation constants of GDP and IDP for these two binding steps were estimated to be about 34 and 38 µM for the first and 110 and 160 µM for the second step, respectively (at pH 8.3, 15°C). Above pH 9, the ratio (P/e) of the extent of phosphorylation to the increment of electron transport from the basal level measured in the presence of [ATP + Pi] or [ADP + Pi + phlorizin], became increasingly large. When the electron transport level inhibited by dicyclohexylcarbodiimide was taken to be the basal activity, the P/e ratio remained almost constant ( 1) from pH 7.0 up to 10.     

Biological properties of pseudorecombinant and recombinant strains created with cucumber mosaic virus and tomato aspermy virus.

Cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) are closely related cucumoviruses. We have made pseudorecombinant viruses in which the RNAs 3 of these two viruses have been exchanged and recombinant viruses containing chimeric RNA 3 molecules, in which the coat proteins and the 3'-end regions of CMV and TAV have been exchanged, giving rise to recombinants designated RT3 and TR3. The replication properties and the cell-to-cell and long-distance movement patterns of these pseudorecombinant and recombinant viruses were examined in different hosts. All the viruses were able to replicate and accumulate RNA 4 in protoplasts. The pseudorecombinants and the R1R2RT3 recombinant infected tobacco systemically, but the R1R2TR3 recombinant was not detectable, even in the inoculated leaves. Comparison of the abilities of the viruses to replicate in protoplasts and intact cucumber plants suggests that cell-to-cell movement factors are also encoded by RNAs 1 and/or 2. Major determinants of symptom severity in Nicotiana glutinosa are localized on the 3' part of RNA 3, and in Nicotiana benthamiana, more severe symptoms were observed with the T1T2R3 strain than with the others tested.     

Interaction between cucumber green mottle mosaic virus MP and CP promotes virus systemic infection

The movement protein (MP) and coat protein (CP) of tobamoviruses play critical roles in viral cell-to-cell and long-distance movement, respectively. Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus. The functions of CGMMV MP and CP during viral infection remain largely unclear. Here, we show that CGMMV MP can interact with CP in vivo, and the amino acids at positions 79–128 in MP are vital for the MP–CP interaction. To confirm this finding, we mutated five conserved residues within the residue 79–128 region and six other conserved residues flanking this region, followed by in vivo interaction assays. The results showed that the conserved threonine residue at the position 107 in MP (MP^T107) is important for the MP–CP interaction. Substitution of T107 with alanine (MP^T107A) delayed CGMMV systemic infection in Nicotiana benthamiana plants, but increased CGMMV local accumulation. Substitutions of another 10 conserved residues, not responsible for the MP–CP interaction, with alanine inhibited or abolished CGMMV systemic infection, suggesting that these 10 conserved residues are possibly required for the MP movement function through a CP-independent manner. Moreover, two movement function-associated point mutants (MP^F17A and MP^D97A) failed to cause systemic infection in plants without impacting on the MP–CP interaction. Furthermore, we have found that co-expression of CGMMV MP and CP increased CP accumulation independent of the interaction. MP and CP interaction inhibits the salicylic acid-associated defence response at an early infection stage. Taken together, we propose that the suppression of host antiviral defence through the MP–CP interaction facilitates virus systemic infection.     

Interaction of cucumber mosaic virus and potato virus y with tobacco mosaic virus

A study was performed on the interaction of cucumber mosaic virus (CMV) of potato virus Y (PVY) with tobacco mosaic virus (TMV). Interference was evaluated using tobacco plantsNicotiana tabacum cv. Java responding to CMV and PVY with a systemic infection and to TMV with local necrotic lesions. The decrease in TMV — induced lesion number gave evidence of a decrease in susceptibility caused by the previous infection with CMV or PVY, the decrease of lesion enlargement demonstrated a decreased TMV reproduction in the plants previously infected with CMV or PVY. The interference concerned was incomplete, as evaluated from reproduction of the challenging TMV and from the decrease in susceptibility of the host to TMV brought about by the first infection with CMV or PVY.     

Inheritance and genetic mapping of cucumber mosaic virus resistance introgressed from Lycopersicon chilense into tomato

Cucumber mosaic virus (CMV) infects a wide variety of crop plants and in tomato (Lycopersicon esculentum Mill.) causes significant economic losses in many growing regions, particularly the Mediterranean. The objective of the present study was to identify the number and map locations of genes controlling resistance to CMV in breeding lines (BC₁–inbreds) derived from the related wild species L. chilense. These lines also carried the gene Tm-2 ^a for resistance to ToMV, which facilitated the interpretation of disease symptoms. The segregation for CMV resistance in the BC₂F₁ and BC₂F₂ generations, following mechanical inoculation with subgroup-I isolates, was consistent with expectations for a single dominant gene, for which the symbol Cmr (cucumber mosaic resistance) was given. Resistant and susceptible BC₁-inbreds were analyzed with RFLP and isozyme markers to identify genomic regions introgressed from L. chilense. The only L. chilense-specific markers found were on chromosome 12; some resistant lines contained a single introgression comprising the entire short arm and part of the long arm of this chromosome, while others contained a recombinant derivative of this introgression. The chromosome 12 markers were significantly associated with CMV resistance in both qualitative and quantitative models of inheritance. The qualitative analysis, however, demonstrated that CMV resistance was not expressed as a reliable monogenic character, suggesting a lack of penetrance, significant environmental effects, or the existence of additional (undetected) resistance factors. In the quantitative analysis, the marker interval TG68 – CT79 showed the most significant association with CMV resistance. No association between CMV resistance and the Tm-2 ^a gene was observed. These breeding lines are potentially useful sources of CMV resistance for tomato improvement, in which context knowledge of the map location of Cmr should accelerate introgression by marker-assisted selection. Received: 9 August 1999 / Accepted: 22 December 1999