Benzothiadiazole and l-2-oxothiazolidine-4-carboxylic acid reduce the severity of Sharka symptoms in pea leaves: effect on antioxidative metabolism at the subcellular level

The effect of treatment with benzothiadiazole (BTH) or l -2-oxothiazolidine-4-carboxylic acid (OTC), and their interaction with Plum pox virus (PPV) infection, on antioxidative metabolism of pea plants was studied at the subcellular level. PPV infection produced a 20% reduction in plant growth. Pre-treatment of pea plants with OTC or BTH afforded partial protection against PPV infection, measured as the percentage of leaves showing symptoms, but neither BTH nor OTC significantly reduced the virus content. PPV infection caused oxidative stress, as monitored by increases in lipid peroxidation and protein oxidation in soluble and chloroplastic fractions. In leaves of non-infected plants, OTC increased the content of reduced glutathione (GSH) and total glutathione; accordingly, an increase in the redox state of glutathione was observed. An increase in oxidized glutathione (GSSG) was found in symptomatic leaves from infected plants. A similar increase in GSSG was also observed in asymptomatic leaves from infected, untreated plants. However, no changes in GSSG occurred in asymptomatic leaves from infected plants treated with BTH and OTC and, accordingly, a higher redox state of GSH was recorded in those leaves, which could have had a role in the reduction of symptoms, as observed in asymptomatic leaves from infected plants treated with BTH or OTC. Treatment with BTH or OTC had some effect on antioxidant enzymes in soluble and chloroplastic fractions from infected pea leaves. An increase in antioxidative mechanisms, such as GSH-related enzymes (DHAR, GR and G6PDH), as well as APX and POX, at the subcellular level was observed, which could play a role in reducing the severity of cellular damage induced by Sharka in pea leaves.     

Oxidative stress induced by long-term plum pox virus infection in peach (Prunus persica)

In this study, the effect of long-term plum pox virus (PPV) infection on the response of certain antioxidant enzymes at the subcellular level was studied in peach plants ( Prunus persica (L.) Batch) (cv. GF305), which are characterized by great susceptibility to the virus. In infected plants, a decrease in the efficiency of excitation energy capture by PSII ( F _v'/ F _m') was observed, which was accompanied by a decrease in non-photochemical quenching (NPQ). p -Hydroxy-mercury benzoic acid (pHMB)-insensitive ascorbate peroxidase (APX) activity (class III peroxidase) was detected in both chloroplast and soluble fractions. In soluble fractions from inoculated peaches, a significant increase in pHMB-sensitive APX activity and a significant decrease in superoxide dismutase (SOD) activity were observed. These changes were correlated with the observations in isolated chloroplasts, where an increase in both pHMB-sensitive and pHMB-insensitive APX activities was observed, whereas significant decreases in SOD, monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) activities were produced. According to these results, as a consequence of PPV infection, an oxidative stress, indicated by an increase in lipid peroxidation and protein oxidation, was produced in peach leaves, which was monitored by the diaminobenzidine (DAB) peroxidase-coupled H₂O₂ probe. PPV infection produced an alteration in chloroplast ultrastructure, giving rise to dilated thylakoid membranes. PPV-infected peach leaves showed a decreased amount of starch in chloroplasts from palisade parenchyma, as well as an increase in the number and size of plastoglobuli, in relation to control plants. The results suggest that long-term PPV infection produces an oxidative stress, and that an antioxidative metabolism imbalance may be related to the progress of PPV infection and symptoms in peach plants.     

Long-term plum pox virus infection produces an oxidative stress in a susceptible apricot, Prunus armeniaca, cultivar but not in a resistant cultivar

The effect of plum pox virus (PPV) infection on the response of some antioxidant enzymes was studied in two apricot cultivars, which behaved differently against PPV infection: cultivar Real Fino (susceptible) and cultivar Stark Early Orange (cv. SEO, resistant). In the susceptible cultivar, PPV produced a decrease in Φ _PSII, F '_v/ F '_m and Q _p. PPV infection produced a drop in p -hydroxy mercury benzoic acid (pHMB)-sensitive ascorbate peroxidase, dehydroascorbate reductase and peroxidase in the soluble fraction from susceptible plants, whereas in the resistant apricot cultivar, pHMB-insensitive ascorbate peroxidase, monodehydroascorbate reductase, glutathione reductase and superoxide dismutase increased. However, catalase decreased in the soluble fractions from both infected cultivars. Long-term PPV infection also produced a decrease in the chloroplastic ascorbate–glutathione cycle enzymes only in the susceptible plants. As a consequence of PPV infection, an oxidative stress, indicated by an increase in lipid peroxidation and in protein oxidation, was produced only in the leaves from the susceptible cultivar which was also monitored by the diaminobenzidine peroxidase-coupled H₂O₂ probe. The loss of Φ _PSII, indicative of activated oxygen species production, and the decrease in the levels of antioxidant enzymes in chloroplasts from susceptible plants could be responsible for the chlorosis symptoms observed. The results suggest that the higher antioxidant capacity showed by cv. SEO could be a consequence of a systemic acquired resistance induced by PPV penetration in stem tissue at the graft site and could be related, among other factors, to their resistance to PPV.     

Chloroplast protection in plum pox virus‐infected peach plants by L‐2‐oxo‐4‐thiazolidine‐carboxylic acid treatments: effect in the proteome

Sharka, a disease caused by plum pox virus (PPV), has a significant economic impact on fruit tree production. In this work, we analysed the effect of (2,1,3)‐benzothiadiazole (BTH) and L‐2‐oxo‐4‐thiazolidine‐carboxylic acid (OTC) on plant growth and virus content. OTC reduced sharka symptom, stimulated plant growth and alleviated PPV‐induced oxidative stress, indicated by a lack of changes in some oxidative stress parameters. PPV infection reduced chloroplast electron transport efficiency. However, in the presence of BTH or OTC, no changes in the chlorophyll fluorescence parameters were observed. PPV produced an alteration in chloroplast ultrastructure, giving rise to a decrease in starch contents that was less dramatic in OTC‐treated plants. Furthermore, PPV reduced the abundance of proteins associated with photosynthesis, carbohydrate and amino acid metabolism and photorespiration. These changes did not take place in OTC‐treated plants, and increases in the expression of proteins related with the aforementioned processes, including ADP‐glucose pyrophosphorylase, were produced, which correlated with the lower decrease in starch contents observed in PPV‐infected plants treated with OTC. The results suggested that OTC treatment provides protection to the photosynthetic machinery and/or the chloroplast metabolism in PPV‐infected peaches. Thus, OTC could have practical implications in agriculture in improving the vigour of different plant species as well as in immunizing plants against pathogens.     

Targeting of proteins to the outer envelope membrane uses a different pathway than transport into chloroplasts.

The chloroplastic envelope is composed of two membranes, inner and outer, each with a distinct set of polypeptides. Like proteins in other chloroplastic compartments, most envelope proteins are synthesized in the cytosol and post-translationally imported into chloroplasts. Considerable knowledge has been obtained concerning protein import proteins. We isolated a cDNA clone from pea that encodes a 14-kilodalton outer envelope membrane protein. The precursor form of this protein does not possess a cleavable transit peptide and its import into isolated chloroplasts does not require either ATP or a thermolysin-sensitive component on the chloroplastic surface. These findings, together with similar observations made with a spinach chloroplastic outer membrane protein, led us to propose that proteins destined for the outer membrane of the chloroplastic envelope follow an import pathway distinct from that followed by proteins destined for other chloroplastic compartments.     

Experimental Transmission of Plum Pox Virus (PPV) to Prunus Mahaleb and Prunus avium

Sharka caused by plum pox virus (PPV) is a disease spread in France since 1970, and causing severe damages essentially on apricot but also on plums and peach. Cherry is generally considered as not infected by PPV. Experimental transmissions by chip budding or aphids allowed to show that 3 isolates of PPV can multiply inside three cherry rootstocks (P. Mahaleb cv.‘SL 64′, P. avium cv.‘F 12-1′, and P. avium*P. pseudocerasus cv. ‘Colt') (Tables 1 and 2). But generally, the virus remained localized to the infection site and disappeared quickly (Table 3). Typical symptoms of chlorotic ringspot or vein clearing are also limited to the leaves probed by the aphids. The fact that no translocation was detected is discussed.     

Contrasting effect of dark-chilling on chloroplast structure and arrangement of chlorophyll-protein complexes in pea and tomato: plants with a different susceptibility to non-freezing temperature

The effect of dark-chilling and subsequent photoactivation on chloroplast structure and arrangements of chlorophyll–protein complexes in thylakoid membranes was studied in chilling-tolerant (CT) pea and in chilling-sensitive (CS) tomato. Dark-chilling did not influence chlorophyll content and Chl a/b ratio in thylakoids of both species. A decline of Chl a fluorescence intensity and an increase of the ratio of fluorescence intensities of PSI and PSII at 120 K was observed after dark-chilling in thylakoids isolated from tomato, but not from pea leaves. Chilling of pea leaves induced an increase of the relative contribution of LHCII and PSII fluorescence. A substantial decrease of the LHCII/PSII fluorescence accompanied by an increase of that from LHCI/PSI was observed in thylakoids from chilled tomato leaves; both were attenuated by photoactivation. Chlorophyll fluorescence of bright grana discs in chloroplasts from dark-chilled leaves, detected by confocal laser scanning microscopy, was more condensed in pea but significantly dispersed in tomato, compared with control samples. The chloroplast images from transmission-electron microscopy revealed that dark-chilling induced an increase of the degree of grana stacking only in pea chloroplasts. Analyses of O-J-D-I-P fluorescence induction curves in leaves of CS tomato before and after recovery from chilling indicate changes in electron transport rates at acceptor- and donor side of PS II and an increase in antenna size. In CT pea leaves these effects were absent, except for a small but irreversible effect on PSII activity and antenna size. Thus, the differences in chloroplast structure between CS and CT plants, induced by dark-chilling are a consequence of different thylakoid supercomplexes rearrangements. Dedicated to Prof. Zbigniew Kaniuga on the 25th anniversary of his initiation of studies on chilling-induced stress in plants.     

Targeting of the turnip yellow mosaic virus 66K replication protein to the chloroplast envelope is mediated by the 140K protein

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two replication proteins, 140K and 66K, both being required for its RNA genome replication. The 140K protein contains domains indicative of methyltransferase, proteinase, and NTPase/helicase, and the 66K protein encompasses the RNA-dependent RNA polymerase domain. During viral infection, the 66K protein localizes to virus-induced chloroplastic membrane vesicles, which are closely associated with TYMV RNA replication. To investigate the determinants of its subcellular localization, the 66K protein was expressed in plant protoplasts from separate plasmids. Green fluorescent protein (GFP) fusion and immunofluorescence experiments demonstrated that the 66K protein displayed a cytoplasmic distribution when expressed individually but that it was relocated to the chloroplast periphery under conditions in which viral replication occurred. The 66K protein produced from an expression vector was functional in viral replication since it could transcomplement a defective replication template. Targeting of the 66K protein to the chloroplast envelope in the course of the viral infection appeared to be solely dependent on the expression of the 140K protein. Analysis of the subcellular localization of the 140K protein fused to GFP demonstrated that it is targeted to the chloroplast envelope in the absence of other viral factors and that it induces the clumping of the chloroplasts, one of the typical cytological effects of TYMV infection. These results suggests that the 140K protein is a key organizer of the assembly of the TYMV replication complexes and a major determinant for their chloroplastic localization and retention.     

The apoplastic antioxidant system in Prunus: response to long-term plum pox virus infection

This work describes, for the first time, the changes taking place in the antioxidative system of the leaf apoplast in response to plum pox virus (PPV) in different Prunus species showing different susceptibilities to PPV. The presence of p-hydroxymercuribenzoic acid (pHMB)-sensitive ascorbate peroxidase (APX) (class I APX) and pHMB-insensitive APX (class III APX), superoxide dismutase (SOD), peroxidase (POX), NADH-POX, and polyphenoloxidase (PPO) was described in the apoplast from both peach and apricot leaves. PPV infection produced different changes in the antioxidant system of the leaf apoplast from the Prunus species, depending on their susceptibility to the virus. In leaves of the very susceptible peach cultivar GF305, PPV brought about an increase in class I APX, POX, NADH-POX, and PPO activities. In the susceptible apricot cultivar Real Fino, PPV infection produced a decrease in apoplastic POX and SOD activities, whereas a strong increase in PPO was observed. However, in the resistant apricot cultivar Stark Early Orange, a rise in class I APX as well as a strong increase in POX and SOD activities was noticed in the apoplastic compartment. Long-term PPV infection produced an oxidative stress in the apoplastic space from apricot and peach plants, as observed by the increase in H2O2 contents in this compartment. However, this increase was much higher in the PPV-susceptible plants than in the resistant apricot cultivar. Only in the PPV-susceptible apricot and peach plants was the increase in apoplastic H2O2 levels accompanied by an increase in electrolyte leakage. No changes in the electrolyte leakage were observed in the PPV-inoculated resistant apricot leaves, although a 42% increase in the apoplastic H2O2 levels was produced. Two-dimensional electrophoresis analyses revealed that the majority of the polypeptides in the apoplastic fluid had isoelectric points in the range of pI 4-6. The identification of proteins using MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) and peptide mass fingerprinting analyses showed the induction of a thaumatin-like protein as well as the decrease of mandelonitrile lyase in peach apoplast due to PPV infection. However, most of the selected polypeptides showed no homology with known proteins. This fact emphasizes that, at least in Prunus, most of the functions of the apoplastic space remain unknown. It is concluded that long-term PPV infection produced an oxidative stress in the leaf apoplast, contributing to the deleterious effects produced by PPV infection in leaves of inoculated, susceptible Prunus plants.     

Age-related changes in oxidative stress markers and abscisic acid levels in a drought-tolerant shrub, Cistus clusii grown under Mediterranean field conditions

Compared with our knowledge of senescence in annuals and biennials, little is known about age-related changes in perennials. To get new insights into the mechanisms underlying aging in perennials, we measured oxidative stress markers in leaves and organelles, together with abscisic acid levels in leaves of 2- and 7-year-old Cistus clusii dunal plants grown under Mediterranean field conditions. Recently emerged leaves, which either appeared during autumn or spring, were compared to evaluate the effects of environmental constraints on oxidative stress and abscisic acid accumulation as plants aged. Plant aging led to an enhanced oxidation of α-tocopherol and ascorbate, increased lipid peroxidation and reduced PSII efficiency in leaves during the more stressful conditions of spring and summer, but not during autumn. Analyses of lipid peroxidation in organelles isolated from the same leaves revealed that oxidative stress occurred both in chloroplasts and mitochondria. Although both plant groups showed similar leaf water and nitrogen contents throughout the study, abscisic acid levels were markedly higher (up to 75%) in 7-year-old plants compared to 2-year-old plants throughout the study. It is concluded that (a) meristematic tissues of C. clusii maintain the capacity to make new leaves with no symptoms of oxidative stress for several years, unless these leaves are exposed to environmental constraints, (b) leaves of oldest plants show higher oxidative stress than those of young plants when exposed to adverse climatic conditions, thus supporting the idea that the oxidative stress associated with aging is due at least partly to extrinsic factors, (c) at the subcellular level, age-induced oxidative stress occurs both in chloroplasts and mitochondria, and (d) even in the absence of environmental stress, newly emerged leaves accumulate higher amounts of ABA as plants age.     

Plant aging increases oxidative stress in chloroplasts

Aging has received considerable attention in biomedicine, but little is known about the regulatory mechanisms responsible for the aging not associated with senescence in plants. This study provides new insights into the relationship between oxidative stress and plant aging, and points out chloroplasts as one of the target organelles of age-associated oxidative stress in plants. We simultaneously analyzed lipid oxidation, photosynthesis, chlorophyll content, de-epoxidation state of the xanthophyll cycle, and levels of chloroplastic antioxidant defenses such as beta-carotene and alpha-tocopherol in leaves of the same age in 1-, 3- and 7-year-old Cistus clusii Dunal plants growing under Mediterranean field conditions. Enhanced formation of malondialdehyde in leaves (2.7-fold) and chloroplasts (2.8-fold), decreased photosynthetic activity (25%), and lower chlorophyll (ca. 20%) and chloroplastic antioxidant defense levels (ca. 25%-85%) were observed in 7-year-old plants, when compared with 1- and 3-year-old plants. The differences observed, which were associated with plant aging, were only noticeable in mature nonsenescing plants (7-year-old plants). No differences were observed between pre-reproductive (1-year-old plants) and young plants (3-year-old plants). This study shows that from a certain age, oxidative stress increases progressively in chloroplasts as plants age, whereas photosynthesis is reduced. The results indicate that the oxidative stress associated with the aging in plants accumulates progressively in chloroplasts, and that the contribution of oxidative stress to aging increases as plants age.     

Differential turnover of the photosystem II reaction centre D1 protein in mesophyll and bundle sheath chloroplasts of maize

Photoinhibition is caused by an imbalance between the rates of the damage and repair cycle of photosystem II D1 protein in thylakoid membranes. The PSII repair processes include (i) disassembly of damaged PSII-LHCII supercomplexes and PSII core dimers into monomers, (ii) migration of the PSII monomers to the stroma regions of thylakoid membranes, (iii) dephosphorylation of the CP43, D1 and D2 subunits, (iv) degradation of damaged D1 protein, and (v) co-translational insertion of the newly synthesized D1 polypeptide and reassembly of functional PSII complex. Here, we studied the D1 turnover cycle in maize mesophyll and bundle sheath chloroplasts using a protein synthesis inhibitor, lincomycin. In both types of maize chloroplasts, PSII was found as the PSII-LHCII supercomplex, dimer and monomer. The PSII core and the LHCII proteins were phosphorylated in both types of chloroplasts in a light-dependent manner. The rate constants for photoinhibition measured for lincomycin-treated leaves were comparable to those reported for C3 plants, suggesting that the kinetics of the PSII photodamage is similar in C3 and C4 species. During the photoinhibitory treatment the D1 protein was dephosphorylated in both types of chloroplasts but it was rapidly degraded only in the bundle sheath chloroplasts. In mesophyll chloroplasts, PSII monomers accumulated and little degradation of D1 protein was observed. We postulate that the low content of the Deg1 enzyme observed in mesophyll chloroplasts isolated from moderate light grown maize may retard the D1 repair processes in this type of plastids.     

Diagnosis of the Earliest Strain-Specific Interactions between Tobacco Mosaic Virus and Chloroplasts of Tobacco Leaves in Vivo by Means of Chlorophyll Fluorescence Imaging

Fluorescence imaging was used to diagnose early stages of the strain-specific interactions between tobacco mosaic virus (strain PV230) and chloroplasts following infection of tobacco leaves (Nicotiana tabacum cv Xanthi). The earliest indication of interaction in tissues that ultimately become chlorotic was a reduction in chlorophyll fluorescence, and there was little fluorescence quenching compared with adjacent healthy tissues. Subsequently, fluorescence increased but remained unquenched. In the late stages fluorescence declined again in chlorotic regions as the chloroticmosaic symptoms developed. These in vivo data showing altered fluorescence yields confirm strain-specific interaction of virus coat protein with photosystem II (PSII) components in vitro, leading to photoinhibition and photooxidation of chlorophyll in infected cells and the development of visible chlorotic-mosaic symptoms. Although mechanisms leading to the low, unquenched fluorescence condition are not known, the intermediate high, unquenched fluorescence condition is consistent with impaired PSII electron transport as measured in vitro. Fluorescence lesions appear more rapidly and develop more extensively in high light, consistent with the faster and larger extent of symptom formation in high-light-grown leaves than in low-light-grown leaves.     

Characterization of photosystem II in transgenic tobacco plants with decreased iron superoxide dismutase

Iron superoxide dismutases (FeSODs) play an important role in preventing the oxidative damage associated with photosynthesis. To investigate the mechanisms of FeSOD in protection against photooxidative stress, we obtained transgenic tobacco (Nicotiana tabacum) plants with severely decreased FeSOD by using a gene encoding tobacco chloroplastic FeSOD for the RNAi construct. Transgenic plants were highly sensitive to photooxidative stress and accumulated increased levels of O??? under normal light conditions. Spectroscopic analysis and electron transport measurements showed that PSII activity was significantly reduced in transgenic plants. Flash-induced fluorescence relaxation and thermoluminescence measurements revealed that there was a slow electron transfer between Q(A) and Q(B) and decreased redox potential of Q(B) in transgenic plants, whereas the donor side function of PSII was not affected. Immunoblot and blue native gel analyses showed that PSII protein accumulation was also decreased in transgenic plants. PSII photodamage and D1 protein degradation under high light treatment was increased in transgenic plants, whereas the PSII repair was not affected, indicating that the stability of the PSII complex was decreased in transgenic plants. The results in this study suggest that FeSOD plays an important role in maintaining PSII function by stabilizing PSII complexes in tobacco plants.     

Immunogold Localization and Quantification of Cellular and Subcellular Abscisic Acid, Prior to and During Drought Stress

An Immunogold labeling procedure and experimental data are presented, which demonstrate that antibodies produced against a bovine serum albumin-abscisic acid conjugate can be used both to characterize the cellular and subcellular localization of abscislc acid (ABA), and to permit quantitative comparisons of this hormone in the subcellular compartments prior to and at times of drought stress. At the control leaf water potential (approximately -0.45 MPa), a quantitatively similar positive labeling pattern was observed in the chloroplasts and apoplast. A twofold drought stress-induced increase in the apoplastic ABA concentration was observed in the drought stressed leaf tissue (i.e., at a leaf water potential of approximately -1.55 MPa), while the ABA concentration in the chloroplasts did not differ from that of the controls. Three histochemical controls and the physiological observations validated the specificity of the procedure. Based on the labeling patterns we observed and literature cited, the validity of the hypothesis that drought stress induces a release of chloroplastic ABA is questioned. We interpreted our results as providing indirect evidence for a drought stress-induced root source origin for the increased apoplastic ABA concentrations.     

Scavenger Enzyme Activities in Subcellular Fractions of White Clover (Trifolium repens L.) under PEG-induced Water Stress

Scavenger enzyme activities in subcellular fractions under polyethylene glycol (PEG)-induced water stress in white clover (Trifolium repens L.) were studied. Water stress decreased ascorbic acid (AA) content and catalase (CAT) activity and increased the contents of hydrogen peroxide (H₂O₂), thiobarbituric acid reactive substances (TBARS) (measure of lipid peroxidation), and activities of superoxide dismutase (SOD), its various isozymes, ascorbate peroxidase (APOX), and glutathione reductase (GR) in cellular cytosol, chloroplasts, mitochondria, and peroxisomes of Trifolium repens leaves. In both the PEG-treated plants and the control, chloroplastic fractions showed the highest total SOD, APOX, and GR activities, followed by mitochondrial fractions in the case of total SOD and GR activities, whereas cytosolic fractions had the second greatest APOX activity. However, CAT activity was the highest in peroxisomes, followed by the cytosol, mitochondria, and chloroplasts in decreasing order. Although Mn-SOD activity was highest in mitochondrial fractions, residual activity was also observed in cytosolic fractions. Cu/Zn-SOD and Fe-SOD were observed in all subcellular fractions; however, the activities were the highest in chloroplastic fractions for both isoforms. Total Cu/Zn-SOD activity, the sum of activities observed in all fractions, was higher than other SOD isoforms. These results suggest that cytosolic and chloroplastic APOX, chloroplastic and mitochondrial GR, mitochondrial Mn-SOD, cytosolic and chloroplastic Cu/Zn-SOD, and chloroplastic Fe-SOD are the major scavenger enzymes, whereas cellular CAT may play a minor role in scavenging of O₂⁻ and H₂O₂ produced under PEG-induced water stress in Trifolium repens.     

Photosynthetic alterations of pea leaves infected systemically by pea enation mosaic virus: A coordinated decrease in efficiencies of CO2 assimilation and photosystem II photochemistry

We have investigated photosynthetic changes of fully expanded pea leaves infected systemically by pea enation mosaic virus (PEMV) that often attacks legumes particularly in northern temperate regions. A typical compatible virus–host interaction was monitored during 40 post-inoculation days (dpi). An initial PEMV-induced decrease in photosynthetic CO₂ assimilation was detected at 15 dpi, when the virus appeared in the measured leaves. This decrease was not induced by stomata closure and corresponded with a decrease in the efficiency of photosystem II photochemistry (Φ_PSII). Despite of a slight impairment of oxygen evolution at this stage, PSII function was not primarily responsible for the decrease in Φ_PSII. Chlorophyll fluorescence imaging revealed that Φ_PSII started to decrease from the leaf tip to the base. More pronounced symptoms of PEMV disease appeared at later stages, when a typical mosaic and enations appeared in the infected leaves and oxidative damage of cell membranes was detected. From 30 dpi, a degradation of photosynthetic pigments accelerated, stomata were closing and corresponding pronounced decline in CO₂ assimilation was observed. A concomitant photoprotective responses, i.e. an increase in non-photochemical quenching and accumulation of de-epoxidized xanthophylls, were also detected. Interestingly, alternative electron sinks in chloroplasts were not stimulated by PEMV infection, which is in contradiction to earlier reports dealing with virus-induced plant stresses. The presented results show that the PEMV-induced alterations in mature pea leaves accelerated leaf senescence during which a decrease in Φ_PSII took place in coordinated manner with an inhibition of CO₂ assimilation.     

A novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts

Most chloroplast and mitochondrial precursor proteins are targeted specifically to either chloroplasts or mitochondria. However, there is a group of proteins that are dual targeted to both organelles. We have developed a novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The mitochondrial precursor of alternative oxidase, AOX was specifically targeted only to mitochondria. The chloroplastic precursor of small subunit of pea ribulose bisphosphate carboxylase/oxygenase, Rubisco, was mistargeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual import system. The dual targeted glutathione reductase GR precursor was targeted to both mitochondria and chloroplasts in both systems. The GR pre-sequence could support import of the mature Rubisco protein into mitochondria and chloroplasts in the single import system but only into chloroplasts in the dual import system. Although the GR pre-sequence could support import of the mature portion of the mitochondrial FAd subunit of the ATP synthase into mitochondria and chloroplasts, mature AOX protein was only imported into mitochondria under the control of the GR pre-sequence in both systems. These results show that the novel dual import system is superior to the single import system as it abolishes mistargeting of chloroplast precursors into pea mitochondria observed in a single organelle import system. The results clearly show that although the GR pre-sequence has dual targeting ability, this ability is dependent on the nature of the mature protein.     

Two chloroplastic protein translocation components,Tic110 and Toc75, are conserved in different plastid types from multiple plant species

Most chloroplastic proteins are nuclear-encoded and must be transported into the organelle post-translationally. Proteinaceous components in the outer and inner envelope membranes of chloroplasts responsible for this import process were originally identified from pea seedlings. We sought to determine whether these proteins are conserved among different plant species other than pea and among different plastid types. We analyzed plant EST databases and found the presence of homologues to pea chloroplastic protein translocation components, Tic110 and Toc75, in both monocot and dicot species. Because these clones were obtained from various tissues, their presence in different types of plastids is proposed. Protein extracts were prepared from several plant species and from different plant tissues, and then probed with antisera raised against pea Tic110 and Toc75. The results support the idea that translocation components originally found in pea chloroplasts are conserved among different plant species and are present in various plastid types.     

Isolation and characterization of a cDNA clone encoding the major hsp70 of the pea chloroplastic stroma

The 70-kD heat shock proteins (hsp70s) are a group of ubiquitous, highly conserved molecular chaperones that have been implicated in a variety of processes, ranging from DNA replication to protein folding and transport. To learn more about the evolution and possible functions of higher plant chloroplastic hsp70s, we isolated a cDNA clone encoding the major stromal hsp70 of pea chloroplasts, which we term CSS1 (Chloroplastic Stress Seventy). This cDNA clone encodes a 75,490-D protein that is very closely related to an hsp70 from the cyanobacterium, Synechocystis. CSS1 is nuclear encoded and synthesized as a higher molecular mass precursor with a chloroplastic transit peptide approximately 65 amino acids long. CSS1 mRNA was detected in RNA samples from leaves and roots of pea (Pisum sativum) plants grown at 18°C but increased 9- and 6-fold, respectively, after brief exposure of the plants to elevated temperature. We discuss the possible role(s) of CSS1 in chloroplastic protein transport and other processes.