The Arabidopsis SAFEGUARD1 suppresses singlet oxygen-induced stress responses by protecting grana margins.

Singlet oxygen (1O2), the major reactive oxygen species (ROS) produced in chloroplasts, has been demonstrated recently to be a highly versatile signal that induces various stress responses. In the fluorescent (flu) mutant, its release causes seedling lethality and inhibits mature plant growth. However, these drastic phenotypes are suppressed when EXECUTER1 (EX1) is absent in the flu ex1 double mutant. We identified SAFEGUARD1 (SAFE1) in a screen of ethyl methanesulfonate (EMS) mutagenized flu ex1 plants for suppressor mutants with a flu-like phenotype. In flu ex1 safe1, all 1O2-induced responses, including transcriptional rewiring of nuclear gene expression, return to levels, such as, or even higher than, those in flu Without SAFE1, grana margins (GMs) of chloroplast thylakoids (Thys) are specifically damaged upon 1O2 generation and associate with plastoglobules (PGs). SAFE1 is localized in the chloroplast stroma, and release of 1O2 induces SAFE1 degradation via chloroplast-originated vesicles. Our paper demonstrates that flu-produced 1O2 triggers an EX1-independent signaling pathway and proves that SAFE1 suppresses this signaling pathway by protecting GMs.

Arabidopsis FAR-RED ELONGATED HYPOCOTYL3 negatively regulates carbon starvation responses.

Light is one of the most important environmental factors that affects various cellular processes in plant growth and development; it is also crucial for the metabolism of carbohydrates as it provides the energy source for photosynthesis. Under extended darkness conditions, carbon starvation responses are triggered by depletion of stored energy. Although light rapidly inhibits starvation responses, the molecular mechanisms by which light signalling affects this process remain largely unknown. In this study, we showed that the Arabidopsis thaliana light signalling protein FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and its homolog FAR-RED IMPAIRED RESPONSE1 (FAR1) are essential for plant survival after extended darkness treatment at both seedling and adult stages. Transmission electron microscopy analyses revealed that disruption of both FHY3 and FAR1 resulted in destruction of chloroplast envelopes and thylakoid membranes under extended darkness conditions. Furthermore, treatment with sucrose, but not glucose, completely rescued carbon starvation-induced cell death in the rosette leaves and arrested early seedling establishment in the fhy3 far1 plants. We thus concluded that the light signalling proteins FHY3 and FAR1 negatively regulate carbon starvation responses in Arabidopsis.

WHITE STRIPE LEAF8, encoding a deoxyribonucleoside kinase, is involved in chloroplast development in rice.

KEY MESSAGE: WSL8 encoding a deoxyribonucleoside kinase (dNK) that catalyzes the first step in the salvage pathway of nucleotide synthesis plays an important role in early chloroplast development in rice. The chloroplast is an organelle that converts light energy into chemical energy; therefore, the normal differentiation and development of chloroplast are pivotal for plant survival. Deoxyribonucleoside kinases (dNKs) play an important role in the salvage pathway of nucleotides. However, the relationship between dNKs and chloroplast development remains elusive. Here, we identified a white stripe leaf 8 (wsl8) mutant that exhibited a white stripe leaf phenotype at seedling stage (before the four-leaf stage). The mutant showed a significantly lower chlorophyll content and defective chloroplast morphology, whereas higher reactive oxygen species than the wild type. As the leaf developed, the chlorotic mutant plants gradually turned green, accompanied by the restoration in chlorophyll accumulation and chloroplast ultrastructure. Map-based cloning revealed that WSL8 encodes a dNK on chromosome 5. Compared with the wild type, a C-to-G single base substitution occurred in the wsl8 mutant, which caused a missense mutation (Leu 349 Val) and significantly reduced dNK enzyme activity. A subcellular localization experiment showed the WSL8 protein was targeted in the chloroplast and its transcripts were expressed in various tissues, with more abundance in young leaves and nodes. Ribosome and RNA-sequencing analysis indicated that some components and genes related to ribosome biosynthesis were down-regulated in the mutant. An exogenous feeding experiment suggested that the WSL8 performed the enzymic activity of thymidine kinase, especially functioning in the salvage synthesis of thymidine monophosphate. Our results highlight that the salvage pathway mediated by the dNK is essential for early chloroplast development in rice.

Selective Elimination of Membrane-Damaged Chloroplasts via Microautophagy.

Plant chloroplasts constantly accumulate damage caused by visible wavelengths of light during photosynthesis. Our previous study revealed that entire photodamaged chloroplasts are subjected to vacuolar digestion through an autophagy process termed chlorophagy; however, how this process is induced and executed remained poorly understood. In this study, we monitored intracellular induction of chlorophagy in Arabidopsis (Arabidopsis thaliana) leaves and found that mesophyll cells damaged by high visible light displayed abnormal chloroplasts with a swollen shape and 2.5 times the volume of normal chloroplasts. In wild-type plants, the activation of chlorophagy decreased the number of swollen chloroplasts. In the autophagy-deficient autophagy mutants, the swollen chloroplasts persisted, and dysfunctional chloroplasts that had lost chlorophyll fluorescence accumulated in the cytoplasm. Chloroplast swelling and subsequent induction of chlorophagy were suppressed by the application of exogenous mannitol to increase the osmotic pressure outside chloroplasts or by overexpression of VESICLE INDUCING PROTEIN IN PLASTID1, which maintains chloroplast envelope integrity. Microscopic observations of autophagy-related membranes showed that swollen chloroplasts were partly surrounded by autophagosomal structures and were engulfed directly by the tonoplast, as in microautophagy. Our results indicate that an elevation in osmotic potential inside the chloroplast due to high visible light-derived envelope damage results in chloroplast swelling and serves as an induction factor for chlorophagy, and this process mobilizes entire chloroplasts via tonoplast-mediated sequestering to avoid the cytosolic accumulation of dysfunctional chloroplasts.

Molecular and Ultrastructural Mechanisms Underlying Yellow Dwarf Symptom Formation in Wheat after Infection of Barley Yellow Dwarf Virus.

Wheat (Tritium aestivum L.) production is essential for global food security. Infection of barley yellow dwarf virus-GAV (BYDV-GAV) results in wheat showing leaf yellowing and plant dwarfism symptom. To explore the molecular and ultrastructural mechanisms underlying yellow dwarf symptom formation in BYDV-GAV-infected wheat, we investigated the chloroplast ultrastructure via transmission electron microscopy (TEM), examined the contents of the virus, H2O2, and chlorophyll in Zhong8601, and studied the comparative transcriptome through microarray analyses in the susceptible wheat line Zhong8601 after virus infection. TEM images indicated that chloroplasts in BYDV-GAV-infected Zhong8601 leaf cells were fragmentized. Where thylakoids were not well developed, starch granules and plastoglobules were rare. Compared with mock-inoculated Zhong8601, chlorophyll content was markedly reduced, but the virus and H2O2 contents were significantly higher in BYDV-GAV-infected Zhong8601. The transcriptomic analyses revealed that chlorophyll biosynthesis and chloroplast related transcripts, encoding chlorophyll a/b binding protein, glucose-6-phosphate/phosphate translocator 2, and glutamyl-tRNA reductase 1, were down-regulated in BYDV-GAV-infected Zhong8601. Some phytohormone signaling-related transcripts, including abscisic acid (ABA) signaling factors (phospholipase D alpha 1 and calcineurin B-like protein 9) and nine ethylene response factors, were up-regulated. Additionally, reactive oxygen species (ROS)-related genes were transcriptionally regulated in BYDV-GAV infected Zhong8601, including three up-regulated transcripts encoding germin-like proteins (promoting ROS accumulation) and four down-regulated transcripts encoding peroxides (scavenging ROS). These results clearly suggest that the yellow dwarf symptom formation is mainly attributed to reduced chlorophyll content and fragmentized chloroplasts caused by down-regulation of the chlorophyll and chloroplast biosynthesis related genes, ROS excessive accumulation, and precisely transcriptional regulation of the above-mentioned ABA and ethylene signaling- and ROS-related genes in susceptible wheat infected by BYDV-GAV.

Effects of imidazolium chloride ionic liquids and their toxicity to Scenedesmus obliquus.

The low volatility of ionic liquids effectively eliminates a major pathway for environmental release and contamination; however, the good solubility, low degree of environmental degradation and biodegradation of ILs may pose a potential threat to the aquatic environment. The growth inhibition of the green alga Scenedesmus obliquus by five 1-alkyl-3-methylimidazolium chloride ionic liquids (ILs) ([Cnmim]Cl, n=6, 8, 10, 12, 16) was investigated, and the effect on cellular membrane permeability and the ultrastructural morphology by ILs ([Cnmim]Cl, n=8, 12, 16) were studied. The results showed that the growth inhibition rate increased with increasing IL concentration and increasing alkyl chain lengths. The relative toxicity was determined to be [C6mim]Cl<[C8mim]Cl<[C10mim]Cl<[C12mim]Cl<[C16mim]Cl. The algae were most sensitive to imidazolium chloride ILs at 48 h according to the results from the growth inhibition rate and cellular membrane permeability tests. The ultrastructural morphology showed that the ILs had negative effects on the cellular morphology and structure of the algae. The cell wall of treated algae became wavy and separated from the cell membrane. Chloroplast grana lamellae became obscure and loose, osmiophilic material was deposited in the chloroplast, and mitochondria and their cristae swelled. Additionally, electron-dense deposits were observed in the vacuoles.

The effect of glufosinate on nitrogen assimilation at the physiological, biochemical and molecular levels in Phaeodactylum tricornutum.

This study investigated the effects of glufosinate, a widely used herbicide, on the marine diatom Phaeodactylum tricornutum through short-term toxicity tests at the physiological and gene transcriptional levels. Glufosinate (4 mg L(-1)) decreased the amount of pigments but increased reactive oxygen species (ROS) and malondialdehyde levels. As a glutamine synthetase (GS) inhibitor, glufosinate affected the transcripts and activities of key enzymes related to nitrogen assimilation. Transcript levels of GS and nitrate reductase (NR) in P. tricornutum decreased to only 57 and 26 % of the control. However, transcript levels of nitrate transporter (NRT) and the small subunit of glutamate synthase (GltD) were 1.79 and 1.76 times higher than that of the control. The activities of NRT, GS and GOGAT were consistent with gene expression except for NR, which was regulated mainly by post-translational modification. Furthermore, the results of electron microscopy showed that chloroplast structure was disrupted in response to glufosinate exposure. These results demonstrated that glufosinate first disturbed nitrogen metabolism and caused a ROS burst, which disrupted chloroplast ultrastructure. Ultimately, the growth of P. tricornutum was greatly inhibited by glufosinate.

NbTMP14 Is Involved in Tomato Spotted Wilt Virus Infection and Symptom Development by Interaction with the Viral NSm Protein.

Tomato spotted wilt virus (TSWV) is one of the most destructive plant viruses, causing severe losses in many important crops worldwide. The non-structural protein NSm of TSWV is a viral movement protein that induces viral symptoms. However, the molecular mechanisms by which NSm contributes to symptom development are unclear. Here, we present evidence that NSm directly interacts with Nicotiana benthamiana chloroplast thylakoid membrane protein TMP14 (NbTMP14) by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. The interaction between NSm and NbTMP14 led to the translocation of the NbTMP14 protein from the chloroplast to the cytoplasm in TSWV-infected plants, and overexpressing NSm decreased NbTMP14 mRNA accumulation. In addition, abnormal chloroplasts and starch accumulation were observed in TSWV-infected plants. Silencing of NbTMP14 by TRV VIGS also showed similar results to those of TSWV-infected plants. Overexpressing NbTMP14 in transgenic N. benthamiana plants impeded TSWV infection, and silencing NbTMP14 in N. benthamiana plants increased disease symptom severity and virus accumulation. To our knowledge, this is the first report showing that the plant chloroplast TMP14 protein is involved in viral infection. Knowledge of the interaction between NSm and NbTMP14 advances our understanding of the molecular mechanisms underlying TSWV symptom development and infection.

Organized Disassembly of Photosynthesis During Programmed Cell Death Mediated By Long Chain Bases.

In plants, pathogen triggered programmed cell death (PCD) is frequently mediated by polar lipid molecules referred as long chain bases (LCBs) or ceramides. PCD interceded by LCBs is a well-organized process where several cell organelles play important roles. In fact, light-dependent reactions in the chloroplast have been proposed as major players during PCD, however, the functional aspects of the chloroplast during PCD are largely unknown. For this reason, we investigated events that lead to disassembly of the chloroplast during PCD mediated by LCBs. To do so, LCB elevation was induced with Pseudomonas syringae pv. tomato (a non-host pathogen) or Fumonisin B1 in Phaseolus vulgaris. Then, we performed biochemical tests to detect PCD triggering events (phytosphingosine rises, MPK activation and H2O2 generation) followed by chloroplast structural and functional tests. Observations of the chloroplast, via optical phenotyping methods combined with microscopy, indicated that the loss of photosynthetic linear electron transport coincides with the organized ultrastructure disassembly. In addition, structural changes occurred in parallel with accumulation of H2O2 inside the chloroplast. These features revealed the collapse of chloroplast integrity and function as a mechanism leading to the irreversible execution of the PCD promoted by LCBs.

Primary Endosymbiosis: Emergence of the Primary Chloroplast and the Chromatophore, Two Independent Events.

The emergence of semiautonomous organelles, such as the mitochondrion, the chloroplast, and more recently, the chromatophore, are critical steps in the evolution of eukaryotes. They resulted from primary endosymbiotic events that seem to share general features, i.e., an acquisition of a bacterium/cyanobacteria likely via a phagocytic membrane, a genome reduction coinciding with an escape of genes from the organelle to the nucleus, and finally the appearance of an active system translocating nuclear-encoded proteins back to the organelles. An intense mobilization of foreign genes of bacterial origin, via horizontal gene transfers, plays a critical role. Some third partners, like Chlamydia, might have facilitated the transition from cyanobacteria to the early chloroplast. This chapter describes our current understanding of primary endosymbiosis, with a specific focus on primary chloroplasts considered to have emerged more than one billion years ago, and on the chromatophore, having emerged about one hundred million years ago.

Rice TSV3 Encoding Obg-Like GTPase Protein Is Essential for Chloroplast Development During the Early Leaf Stage Under Cold Stress.

The Spo0B-associated GTP-binding (Obg) proteins are essential for the viability of nearly all bacteria. However, the detailed roles of Obg proteins in higher plants have not yet been elucidated. In this study, we identified a novel rice (Oryza sativa L.) thermo-sensitive virescent mutant (tsv3) that displayed an albino phenotype at 20° before the three-leaf stage while being a normal green at 32° or even at 20° after the four-leaf stage. The mutant phenotype was consistent with altered chlorophyll content and chloroplast structure in leaves. Map-based cloning and complementation experiments showed that TSV3 encoded a small GTP-binding protein. Subcellular localization studies revealed that TSV3 was localized to the chloroplasts. Expression of TSV3 was high in leaves and weak or undetectable in other tissues, suggesting a tissue-specific expression of TSV3 In the tsv3 mutant, expression levels of genes associated with the biogenesis of the chloroplast ribosome 50S subunit were severely decreased at the three-leaf stage under cold stress (20°), but could be recovered to normal levels at a higher temperature (32°). These observations suggest that the rice nuclear-encoded TSV3 plays important roles in chloroplast development at the early leaf stage under cold stress.

Passive anion transport through the chloroplast inner envelope membrane measured by osmotic swelling of intact chloroplasts.

It has been shown that chloride channels are located in the envelope membranes of chloroplasts [5,11]. In this report, we use the light-scattering technique to measure quantitatively the rate of anion transport through the inner envelope membrane of isolated intact chloroplasts. Our results permit to assign the anion transport to the inner envelope of chloroplasts. The anionic selectivity determined from the kinetics of light scattering indicates that the chloride pathway is also highly permeable for NO-2 and NO-3. The sulfate and phosphate anions are impermeant. The chloride flux is not inhibited by DIDS or NEM and is temperature-dependent. The activation energy of the transport process suggests that the Cl- flux occurs through a channel.

Thermostability of photosynthesis in two new chlorophyll b-less rice mutants.

Leaves of the two new chlorophyll b-less rice mutants VG28-1, VG30-5 and the wild type rice cv. Zhonghua 11 were subjected to temperatures 28, 36, 40, 44 and 48 degrees C in the dark for 30 min or gradually elevated temperature from 30 degrees C to 80 degrees C at 0.5 degrees C/min. The thermostability of photosynthetic apparatus was estimated by the changes in chlorophyll fluorescence parameters, photosynthetic rate and pigment content, chloroplast ultrastructure and tissue location of H2O2 accumulation. There were different patterns of F(o)-temperature curves between the Chl b-less mutants and the wild type plant, and the temperature of F(o) rising threshold was shifted 3 degrees C lower in the Chl b-less mutants (48 degrees C) than in the wild type (51 degrees C). At temperature up to about 45 degrees C, chloroplasts were swollen and thylakoid grana became misty accompanied with the complete loss of photosynthetic oxygen evolution in the two Chl b-less mutants, but chloroplast ultrastructure in the wild type showed no obvious alteration. After 55 degrees C exposure, the disordered thylakoid and significant H2O2 accumulation in leaves were found in the two Chl b-less mutants, whereas in the wild type plant, less H2O2 was accumulated and the swollen thylakoid still maintained a certain extent of stacking. A large extent of the changes in qP, NPQ and Fv/Fm was consistent with the Pn decreasing rate in the Chl b-less mutants during high temperature treatment as compared with the wild type. The results indicated that the Chl b-less mutants showed a tendency for higher thermosensitivity, and loss of Chl b in LHC II could lead to less thermostability of PSII structure and function. Heat damage to photosynthetic apparatus might be partially attributed to the internal oxidative stress produced at severely high temperature.

[Identification and localization of virus RNA in pepper (Capsicum anuum L.) chloroplasts by means of the PCR method]. / Identifikatsiia i opredelenie lokalizatsii virusnoi RNK v khloroplastakh pertsa (Capsicum annuum L.) metodom polimeraznoi tsennoi reaktsii.

Localization of virus RNA in stroma of Capsicum anuum L. chloroplasts was determined by the PCR method. Accumulation of virus protein in the membranes and stroma of infected pepper chloroplasts has been studied. It is concluded that the virus protein synthesis takes place in the pepper chloroplasts.

[Effect of tobacco mosaic virus on the ultrastructure of leaf mesophyll cells of the pepper Capsicuum anuum L]. / Vliianie virusa tabachnoi mozaiki na ul'trastrukturu kletok mezofilla lista pertsa Capsicuum anuum L.

Chlorotic areas of Capsicuum anuum L. leaves infected with tobacco mosaic virus (TMV) have been investigated. Pathological changes of chloroplasts were found out. Swelling, more osmophilic plastoglobuli, loosened thylakoid structure were observed. It was shown that chloroplasts did not take part in the process of virus replication. Some structural changes of mitochondria were discovered too. They were most tolerant to the virus infection in comparison with other organoids.

Proteomic analysis of chloroplast biogenesis (clb) mutants uncovers novel proteins potentially involved in the development of Arabidopsis thaliana chloroplasts.

Plant cells outstand for their ability to generate biomass from inorganic sources, this phenomenon takes place within the chloroplasts. The enzymatic machinery and developmental processes of chloroplasts have been subject of research for several decades, and this has resulted in the identification of a plethora of proteins that are essential for their development and function. Mutant lines for the genes that code for those proteins, often display pigment-accumulation defects (e.g., albino phenotypes). Here, we present a comparative proteomic analysis of four chloroplast-biogenesis affected mutants (cla1-1, clb2, clb5, clb19) aiming to identify novel proteins involved in the regulation of chloroplast development in Arabidopsis thaliana. We performed 2D-PAGE separation of the protein samples. These samples were then analyzed by computational processing of gel images in order to select protein spots with abundance shifts of at least twofold, statistically significant according to Student's t-test (P<0.01). These spots were subjected to MALDI-TOF mass-spectrometry for protein identification. This process resulted in the discovery of three novel proteins potentially involved in the development of A. thaliana chloroplasts, as their associated mutant lines segregate pigment-deficient plants with abnormal chloroplasts, and altered mRNA accumulation of chloroplast-development marker genes. BIOLOGICAL SIGNIFICANCE: This report highlights the potential of using a comparative proteomics strategy for the study of biological processes. Particularly, we compared the proteomes of wild-type seedlings and four mutant lines of A. thaliana affected in chloroplast biogenesis. From this proteomic analysis it was possible to detect common mechanisms in the mutants to respond to stress and cope with heterotrophy. Notably, it was possible to identify three novel proteins potentially involved in the development or functioning of chloroplasts, also it was demonstrated that plants annotated to carry T-DNA insertions in the cognate genes display pigment-deficient phenotypes, aberrant and underdeveloped chloroplasts, as well as altered mRNA accumulation of chloroplast biogenesis marker genes.

Coat protein mutations in an attenuated Cucumber mosaic virus encoding mutant 2b protein that lacks RNA silencing suppressor activity induces chlorosis with photosynthesis gene repression and chloroplast abnormalities in infected tobacco plants.

In tobacco plants, the Cucumber mosaic virus (CMV) pepo strain induces mosaic symptoms, including pale green chlorosis and malformed tissues. Here, we characterized the involvement of 2b protein and coat protein (CP) in the development of mosaic symptoms. A 2b mutant (R46C) that lacks viral suppressor of RNA silencing (VSR) activity showed an asymptomatic phenotype with low levels of virus accumulation. Tomato spotted wilt virus NSs protein did not complement the virulence of the R46C, although it did restore high-level virus accumulation. However, R46C mutants expressing mutated CP in which the amino acid P129 was mutated to A, E, C, Q, or S induced chlorosis that was associated with reduced expression of chloroplast and photosynthesis related genes (CPRGs) and abnormal chloroplasts with fewer thylakoid membranes. These results suggest that the CP of the CMV pepo strain acquires virulence by amino acid mutations, which causes CPRG repression and chloroplast abnormalities.

Expression of Potato virus Y cytoplasmic inclusion protein in tobacco results in disorganization of parenchyma cells, distortion of epidermal cells, and induces mitochondrial and chloroplast abnormalities, formation of membrane whorls and atypical lipid accumulation.

Infection of plant cells by potyviruses induces the formation of cytoplasmic inclusions ranging in size from 200 to 1000 nm. To determine if the ability to form these ordered, insoluble structures is intrinsic to the potyviral cytoplasmic inclusion protein, we have expressed the cytoplasmic inclusion protein from Potato virus Y in tobacco under the control of the chrysanthemum ribulose-1,5-bisphosphate carboxylase small subunit promoter, a highly active, green tissue promoter. No cytoplasmic inclusions were observed in the leaves of transgenic tobacco using transmission electron microscopy, despite being able to clearly visualize these inclusions in Potato virus Y infected tobacco leaves under the same conditions. However, we did observe a wide range of tissue and sub-cellular abnormalities associated with the expression of the Potato virus Y cytoplasmic inclusion protein. These changes included the disruption of normal cell morphology and organization in leaves, mitochondrial and chloroplast internal reorganization, and the formation of atypical lipid accumulations. Despite these significant structural changes, however, transgenic tobacco plants were viable and the results are discussed in the context of potyviral cytoplasmic inclusion protein function.

Targeted overexpression of the Escherichia coli MinC protein in higher plants results in abnormal chloroplasts.

Higher plant chloroplast division involves some of the same types of proteins that are required in prokaryotic cell division. These include two of the three Min proteins, MinD and MinE, encoded by the min operon in bacteria. Noticeably absent from annotated sequences from higher plants is a MinC homologue. A higher plant functional MinC homologue that would interfere with FtsZ polymerization, has yet to be identified. We sought to determine whether expression of the bacterial MinC in higher plants could affect chloroplast division. The Escherichia coli minC (EcMinC) gene was isolated and inserted behind the Arabidopsis thaliana RbcS transit peptide sequence for chloroplast targeting. This TP-EcMinC gene driven by the CaMV 35S(2) constitutive promoter was then transformed into tobacco (Nicotiana tabacum L.). Abnormally large chloroplasts were observed in the transgenic plants suggesting that overexpression of the E. coli MinC perturbed higher plant chloroplast division.

Cytological modifications in zucchini yellow mosaic virus (ZYMV)-infected Styrian pumpkin plants.

The present research demonstrates severe ultrastructural changes induced by zucchini yellow mosaic virus (ZYMV) within the cells of older and younger leaves of Styrian pumpkin plants (Cucurbita pepo L. subsp. pepo var. styriaca GREB.). Cylindrical inclusions (pinwheels), proliferated endoplasmatic reticulum and filamentous viral particles were found throughout the cytoplasm of ZYMV-infected cells and within sieve elements. ZYMV-infection also induced severe modifications in the number and ultrastructure of chloroplasts, whereas mitochondria, nuclei and peroxisomes remained unaffected. A significantly lower number of chloroplasts was observed in all tissues of both ZYMV-infected leaf types when compared to control plants. Statistical quantification revealed that in chloroplasts of ZYMV-infected older and younger leaves the amount of plastoglobuli and starch increased significantly, whereas the amount of thylakoids significantly decreased. The present research gives a more precise insight in ZYMV-induced modifications within single cells and organelles, and provides statistical data of the most affected chloroplasts.