The unique carnation stunt-associated pararetroviroid.

The discovery and description of a pararetroviroid associated with carnation stunt was one of the most outstanding achievements gained by the research team directed by Ricardo Flores, as it opened up a whole new world in plant virology and quickly led to the identification of further viroid diseases. The carnation stunt-associated viroid-like RNA has been proved to exist in both RNA and DNA form, a discovery that pointed to new ways of studying the co-evolution of plants and viroid-like RNAs. This paper aims to summarise the scientific work of Ricardo Flores, a source of inspiration to both present and future generations of scientists.

Bioeconomy: Biomass and biomass-based energy supply and demand.

This paper addresses the challenges of the transition from a fossil fuel-dependent to a bio-based economy and implications related to the production of food, feed, bioenergy and other bio-based materials. The objective is to provide a comprehensive review of global biomass and biomass-based energy supplies and demand, with particular attention to the EU. Furthermore, factors related to setting priorities in the use of non-food biomass are discussed, as food security will remain the top priority. Finally, the changes in the bioenergy balance indicators in the Member States of the EU and new plant breeding technologies are analyzed. Overall, this study describes the complexity of the bio-based value chains in making decisions on how best to use biomass. The article presents a comprehensive review on global biomass and biomass based energy supplies and demand, discusses the European chemical industry perspective, analyzes the changes in the biomass based energy balance indicators in the Member States of the EU, and considers the challenges of the new plant breeding technologies.

Molecular Modeling for Better Understanding of Cucumovirus Pathology.

Cucumber mosaic virus (CMV) is a small RNA virus capable of infecting a wide variety of plant species. The high economic losses due to the CMV infection made this virus a relevant subject of scientific studies, which were further facilitated by the small size of the viral genome. Hence, CMV also became a model organism to investigate the molecular mechanism of pathogenesis. All viral functions are dependent on intra- and intermolecular interactions between nucleic acids and proteins of the virus and the host. This review summarizes the recent data on molecular determinants of such interactions. A particular emphasis is given to the results obtained by utilizing molecular-based planning and modeling techniques.

A single point mutation on the cucumber mosaic virus surface induces an unexpected and strong interaction with the F1 complex of the ATP synthase in Nicotiana clevelandii plants.

A previous study showed that a single amino acid difference in the cucumber mosaic virus (CMV) capsid protein (CP) elicits unusual symptoms. The wild-type strain (CMV-R) induces green mosaic symptoms and malformation while the mutant strain (CMV-R3E79R) causes chlorotic lesions on inoculated leaves and strong stunting with necrosis on systemic leaves. Virion preparations of CMV-R and CMV-R3E79R were partially purified from Nicotiana clevelandii A. Gray and analysed by two-dimensional gel electrophoresis. Their separated protein patterns showed remarkable differences at the 50-75 kDa range, both in numbers and intensity of spots, with more protein spots for the mutant CMV. Mass spectrometry analysis demonstrated that the virion preparations contained host proteins identified as ATP synthase alpha and beta subunits as well as small and large Rubisco subunits, respectively. Virus overlay protein binding assay (VOPBA), immunogold electron microscopy and modified ELISA experiments were used to prove the direct interaction between the virus particle and the N. clevelandii ATP synthase F1 motor complex. Protein-protein docking study revealed that the electrostatic change in the mutant CMV can introduce stronger interactions with ATP synthase F1 complex. Based on our findings we suggest that the mutation present in the CP can have a direct effect on the long-distance movement and systemic symptoms. In molecular view the mutant CMV virion can lethally block the rotation of the ATP synthase F1 motor complex which may lead to cell apoptosis, and finally to plant death.

Comprehensive Analysis of DWARF14-LIKE2 (DLK2) Reveals Its Functional Divergence from Strigolactone-Related Paralogs.

Strigolactones (SLs) and related butenolides, originally identified as active seed germination stimulants of parasitic weeds, play important roles in many aspects of plant development. Two members of the D14 α/ß hydrolase protein family, DWARF14 (D14) and KARRIKIN INSENSITIVE2 (KAI2) are essential for SL/butenolide signaling. The third member of the family in Arabidopsis, DWARF 14-LIKE2 (DLK2) is structurally very similar to D14 and KAI2, but its function is unknown. We demonstrated that DLK2 does not bind nor hydrolyze natural (+)5-deoxystrigol [(+)5DS], and weakly hydrolyzes non-natural strigolactone (-)5DS. A detailed genetic analysis revealed that DLK2 does not affect SL responses and can regulate seedling photomorphogenesis. DLK2 is upregulated in the dark dependent upon KAI2 and PHYTOCHROME INTERACTING FACTORS (PIFs), indicating that DLK2 might function in light signaling pathways. In addition, unlike its paralog proteins, DLK2 is not subject to rac-GR24-induced degradation, suggesting that DLK2 acts independently of MORE AXILLARY GROWTH2 (MAX2); however, regulation of DLK2 transcription is mostly accomplished through MAX2. In conclusion, these data suggest that DLK2 represents a divergent member of the DWARF14 family.

Salicylic Acid Induction of Flavonoid Biosynthesis Pathways in Wheat Varies by Treatment.

Salicylic acid is a promising compound for the reduction of stress sensitivity in plants. Although several biochemical and physiological changes have been described in plants treated with salicylic acid, the mode of action of the various treatments has not yet been clarified. The present work reports a detailed comparative study on the effects of different modes of salicylic acid application at the physiological, metabolomic, and transcriptomic levels. Seed soaking and hydroponic treatments were found to induce various changes in the protective mechanisms of wheat plants. The possible involvement of the flavonoid metabolism in salicylic acid-related stress signaling was also demonstrated. Different salicylic acid treatments were shown to induce different physiological and biochemical processes, with varying responses in the leaves and roots. Hydroponic treatment enhanced the level of oxidative stress, the expression of genes involved in the flavonoid metabolism and the amount of non-enzymatic antioxidant compounds, namely ortho-hydroxycinnamic acid and the flavonol quercetin in the leaves, while it decreased the ortho-hydroxycinnamic acid and flavonol contents and enhanced ascorbate peroxidase activity in the roots. In contrast, seed soaking only elevated the gene expression level of phenylalanine ammonia lyase in the roots and caused a slight increase in the amount of flavonols. These results draw attention to the fact that the effects of exogenous salicylic acid application cannot be generalized in different experimental systems and that the flavonoid metabolism may be an important part of the action mechanisms induced by salicylic acid.

Recombination analysis of Maize dwarf mosaic virus (MDMV) in the Sugarcane mosaic virus (SCMV) subgroup of potyviruses.

Recombination among RNA viruses is a natural phenomenon that appears to have played a significant role in the species development and the evolution of many strains. It also has particular significance for the risk assessment of plants which have been genetically modified for disease resistance by incorporating viral sequences into their genomes. However, the exact recombination events taking place in viral genomes are not investigated in detail for many virus groups. In this analysis, different single-stranded positive-sense RNA potyviruses were compared using various in silico recombination detection methods and new recombination events in the Sugarcane mosaic virus (SCMV) subgroup were detected. For an extended in silico recombination analysis, two of the analyzed Maize dwarf mosaic virus full-length genomes were sequenced additionally during this work. These results strengthen the evidence that recombination is a major driving force in virus evolution, and the emergence of new virus variants in the SCMV subgroup, paired with mutations, could generate viruses with altered biological properties. The intra- and interspecific homolog recombinations seem to be a general trait in this virus group, causing little or no changes to the amino acid of the progenies. However, we found a few breakpoints between the members of SCMV subgroup and the weed-infecting distant relatives, but only a few methods of the RDP3 package predicted these events with low significance level.

Alanine scanning of cucumber mosaic virus (CMV) 2b protein identifies different positions for cell-to-cell movement and gene silencing suppressor activity.

The multifunctional 2b protein of CMV has a role in the long distance and local movement of the virus, in symptom formation, in evasion of defense mediated by salicylic acid as well as in suppression of RNA silencing. The role of conserved amino acid sequence domains were analyzed previously in the protein function, but comprehensive analysis of this protein was not carried out until recently. We have analyzed all over the 2b protein by alanine scanning mutagenesis changing three consecutive amino acids (aa) to alanine. We have identified eight aa triplets as key determinants of the 2b protein function in virus infection. Four of them (KKQ/22-24/AAA, QNR/31-33/AAA, RER/34-36/AAA, SPS/40-42/AAA) overlap with previously determined regions indispensable in gene silencing suppressor function. We have identified two additional triplets necessary for the suppressor function of the 2b protein (LPF/55-57/AAA, NVE/10-12/AAA), and two other positions were required for cell-to-cell movement of the virus (MEL/1-3/AAA, RHV/70-72/AAA), which are not essential for suppressor activity.

Oral immunogenicity of a plant virus vector based porcine circovirus antigen - short communication.

A recombinant cucumber mosaic virus based expression system has been developed for the production of an immunogenic porcine circovirus epitope. The resulting nanoparticle was shown to elicit specific immune response in mice and pigs, when administered parenterally. To evaluate the oral applicability of this vaccine candidate, two experiments were performed. In the first one, the resistance of the vector itself to mucosal environment was tested in mice. Cucumber mosaic virus particles fed to mice were able to elicit specific mucosal and serum antibody production. In the second experiment, recombinant cucumber mosaic virus fed to piglets resulted in the appearance of porcine circovirus specific serum antibodies. The vector proved to be able to survive in the gastrointestinal tract, so that an epitope expressed on its surface could induce specific immune response. These results indicate that the developed plant virus based expression system offers an effective method for mucosal vaccine production.

The C-terminal domain of the 2b protein of Cucumber mosaic virus is stabilized by divalent metal ion coordination.

The main function of the 2b protein of Cucumber mosaic virus (CMV) is binding permanently the double stranded siRNA molecules in the suppression process of post-transcriptional gene silencing (PTGS). The crystal structure of the homologue Tomato aspermy virus (TAV) 2b protein is known, but without the C-terminal domain. The biologically active form is a tetramer: four 2b protein molecules and two siRNA duplexes. Regarding the complete 2b protein structure, we performed a molecular dynamics (MD) simulation of the whole siRNA-2b ribonucleoprotein complex. Unfortunately, the C-terminal domain is proved to be partially unstructured. Multiple sequence alignment showed a well conserved motif between residues 94 and 105. The negatively charged residues of the C-terminal domain are supposed to take part in coordination of a divalent metal ion and stabilize the three-dimensional structure of the C-terminal domain. MD simulations were performed on the detached C-terminal domains (aa 65-110). 0.15 M MgC2, CaCl2, FeCl2 and ZnCl2 salt concentrations were used in the screening simulations. Among the tested divalent metal ions Mg²âº proved to be very successful because Asp95, Asp96 and Asp98 forms a quasi-permanent Mg²âº binding site. However the control computations have resulted in any (at least) divalent metal ion remains in the binding site after replacement of the bound Mg²âº ion. A quadruple mutation (Rs2DDTD/95-98/AAAA) was introduced into the position of the putative divalent metal ion binding site to analyze the biological relevance of molecular modeling derived hypothesis. The plant inoculation experiments proved that the movement of the mutant virus is slower and the symptoms are milder comparing to the wild type virus. These results demonstrate that the quadruple mutation weakens the stability of the 2b protein tetramer-siRNA ribonucleoprotein complex.

The epitopes in wheat proteins for defining toxic units relevant to human health.

Wheat-related disorders are well-studied health problems. Knowledge of the composition and amounts of epitopes present in a single wheat sample represents a significant gap, and the detailed wheat proteome datasets now available can provide the necessary information to carry out an estimation of allergen prediction for a single cultivar. The combined use of genome sequence and allergen databases, prediction methodology, and cereal chemistry results in better understanding of the level of toxicity present in the end-products produced from wheat flour. The workflow presented in this review provides information about the number and distribution of epitopes at single protein, or protein fraction, levels. In addition, epitopes present in the highest frequency and harmful proteins expressed in the highest amount can be identified. The "epitope toxicity" value obtained in this way is a significant research output from the analysis of large datasets that can be applied to the food industry.

Molecular aspects of the antagonistic interaction of smoke-derived butenolides on the germination process of Grand Rapids lettuce (Lactuca sativa) achenes.

Smoke-derived compounds provide a strong chemical signal to seeds in the soil seed bank, allowing them to take advantage of the germination niche created by the occurrence of fire. The germination stimulatory activity of smoke can largely be attributed to karrikinolide (KAR(1) ), while a related compound, trimethylbutenolide (TMB), has been shown to have an inhibitory effect on germination. The aim of this study was to characterize the interaction of these potent fire-generated compounds. Dose-response analysis, leaching tests and a detailed transcriptome study were performed using highly KAR(1) -sensitive lettuce (Lactuca sativa cv 'Grand Rapids') achenes. Dose-response analysis demonstrated that the compounds are not competitors and TMB modulates germination in a concentration-dependent manner. The transcriptome analysis revealed a contrasting expression pattern induced by the compounds. KAR(1) suppressed, while TMB up-regulated ABA, seed maturation and dormancy-related transcripts. The effect of TMB was reversed by leaching the compound, while the KAR(1) effect was only reversible by leaching within the first 2 h of KAR(1) treatment. Our findings suggest that the compounds may act in concert for germination-related signaling. After the occurrence of fire, sufficient rainfall would contribute to post-germination seedling recruitment by reducing the concentration of the inhibitory compound.

Effect of light on the gene expression and hormonal status of winter and spring wheat plants during cold hardening.

The effect of light on gene expression and hormonal status during the development of freezing tolerance was studied in winter wheat (Triticum aestivum var. Mv Emese) and in the spring wheat variety Nadro. Ten-day-old plants (3-leaf stage) were cold hardened at 5°C for 12 days under either normal (250 µmol m(-2) s(-1) ) or low (20 µmol m(-2) s(-1) ) light conditions. Comprehensive analysis was carried out to explore the background of frost tolerance and the differences between these wheat varieties. Global genome analysis was performed, enquiring about the details of the cold signaling pathways. The expression level of a large number of genes is affected by light, and this effect may differ in different wheat genotypes. Photosynthesis-related processes probably play a key role in the enhancement of freezing tolerance; however, there are several other genes whose induction is light-dependent, so either there is cross-talk between signaling of chloroplast originating and other protective mechanisms or there are other light sensors that transduce signals to the components responsible for stress tolerance. Changes in the level of both plant hormones (indole-3-acetic acid, cytokinins, nitric oxide and ethylene precursor 1-aminocyclopropane-1-carboxylic acid) and other stress-related protective substances (proline, phenolics) were investigated during the phases of the hardening period. Hormonal levels were also affected by light and their dynamics indicate that wheat plants try to keep growing during the cold-hardening period. The data from this experiment may provide a new insight into the cross talk between cold and light signaling in wheat.

A cucumber mosaic virus based expression system for the production of porcine circovirus specific vaccines.

Potential porcine circovirus type 2 (PCV2) capsid protein epitopes, suitable for expression on the surface of cucumber mosaic virus (CMV) particles were determined by a thorough analysis of the predicted PCV capsid protein structure. The ab initio protein structure prediction was carried out with fold recognition and threading methods. The putative PCV epitopes were selected on the basis of PCV virion models and integrated into the plant virus coat protein, after amino acid position 131. The recombinants were tested for infectivity and stability on different Nicotiana species and stable recombinant virus particles were purified. The particles were tested for their ability to bind to PCV induced porcine antibodies and used for specific antibody induction in mice and pigs. The results showed that PCV epitopes expressed on the CMV surface were recognized by the porcine antibodies and they were also able to induce PCV specific antibody response. Challenge experiment with PCV2 carried out in immunized pigs showed partial protection against the infection. Based on these results it was concluded that specific antiviral vaccine production for the given pathogen was feasible, offering an inexpensive way for the mass production of such vaccines.

Identification a coat protein region of cucumber mosaic virus (CMV) essential for long-distance movement in cucumber.

To characterise the long-distance movement determinant of cucumoviral coat proteins (CPs), five mutants were engineered into the CMV CP bearing the corresponding tomato aspermy virus (TAV) loops exposed on the surface of the virion. Both viruses can move long-distance in Nicotiana clevelandii, but only CMV can move long-distance in cucumber. Investigation of the CMV chimeras identified three amino acids of the ßB-ßC loop that were essential for the CMV long-distance movement in cucumber. Introducing these mutations into the TAV CP was not sufficient for long-distance movement, indicating that this is not the sole region causing long-distance movement deficiency.

Salicylic acid treatment of pea seeds induces its de novo synthesis.

Salicylic acid (SA), which is known as a signal molecule in the induction of defense mechanisms in plants, could be a promising compound for the reduction of stress sensitivity. The aim of the present work was to investigate the distribution of SA in young pea (Pisum sativum L.) seedlings grown from seeds soaked in (3)H-labeled SA solution before sowing, and to study the physiological changes induced by this seed treatment. The most pronounced changes in SA levels occurred in the epicotyl and the seeds. Radioactivity was detected only in the bound form of SA, the majority of which was localized in the seeds, and only a very low level of radioactivity was detected in the epicotyl. SA pre-treatment increased the expression of the chorismate synthase and isochorismate synthase genes in the epicotyl. Pre-soaking the seeds in SA increased the activities of some antioxidant enzymes, namely ascorbate peroxidase (EC 1.11.1.11) and guaiacol peroxidase (EC 1.11.1.7) and the level of ortho-hydroxycinnamic acid, but decreased the level of polyamines. These results suggest that the increased level of free and bound SA detected in plants growing from seeds soaked in SA solution before sowing is the product of de novo synthesis, rather than having been taken up and mobilized by the plants.

Role of conserved non-coding regulatory elements in LMW glutenin gene expression.

Transcriptional regulation of LMW glutenin genes were investigated in-silico, using publicly available gene sequences and expression data. Genes were grouped into different LMW glutenin types and their promoter profiles were determined using cis-acting regulatory elements databases and published results. The various cis-acting elements belong to some conserved non-coding regulatory regions (CREs) and might act in two different ways. There are elements, such as GCN4 motifs found in the long endosperm box that could serve as key factors in tissue-specific expression. Some other elements, such as the AACA/TA motifs or the individual prolamin box variants, might modulate the level of expression. Based on the promoter sequences and expression characteristic LMW glutenin genes might be transcribed following two different mechanisms. Most of the s- and i-type genes show a continuously increasing expression pattern. The m-type genes, however, demonstrate normal distribution in their expression profiles. Differences observed in their expression could be related to the differences found in their promoter sequences. Polymorphisms in the number and combination of cis-acting elements in their promoter regions can be of crucial importance in the diverse levels of production of single LMW glutenin gene types.

Transcriptome analysis of germinating maize kernels exposed to smoke-water and the active compound KAR1.

BACKGROUND: Smoke released from burning vegetation functions as an important environmental signal promoting the germination of many plant species following a fire. It not only promotes the germination of species from fire-prone habitats, but several species from non-fire-prone areas also respond, including some crops. The germination stimulatory activity can largely be attributed to the presence of a highly active butenolide compound, 3-methyl-2H-furo[2,3-c]pyran-2-one (referred to as karrikin 1 or KAR1), that has previously been isolated from plant-derived smoke. Several hypotheses have arisen regarding the molecular background of smoke and KAR1 action. RESULTS: In this paper we demonstrate that although smoke-water and KAR1 treatment of maize kernels result in a similar physiological response, the gene expression and the protein ubiquitination patterns are quite different. Treatment with smoke-water enhanced the ubiquitination of proteins and activated protein-degradation-related genes. This effect was completely absent from KAR1-treated kernels, in which a specific aquaporin gene was distinctly upregulated. CONCLUSIONS: Our findings indicate that the array of bioactive compounds present in smoke-water form an environmental signal that may act together in germination stimulation. It is highly possible that the smoke/KAR1 'signal' is perceived by a receptor that is shared with the signal transduction system implied in perceiving environmental cues (especially stresses and light), or some kind of specialized receptor exists in fire-prone plant species which diverged from a more general one present in a common ancestor, and also found in non fire-prone plants allowing for a somewhat weaker but still significant response. Besides their obvious use in agricultural practices, smoke and KAR1 can be used in studies to gain further insight into the transcriptional changes during germination.

The solution structures of the cucumber mosaic virus and tomato aspermy virus coat proteins explored with molecular dynamics simulations.

The three-dimensional structures of two cucumovirus coat proteins (CP), namely Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV), were explored by molecular dynamics (MD) simulations. The N-terminal domain and the C-terminal tail of the CPs proved to be intrinsically unstructured protein regions in aqueous solution. The N-terminal alpha-helix had a partially unrolled conformation. The thermal factor analysis of the CP loop regions demonstrated that the CMV CP had more flexible loop regions than the TAV CP. The principal component analysis (PCA) of the MD trajectories showed that the first three eigenvectors represented the three main conformational motions in the CPs. The first motion components with the highest variance contribution described an opening movement between the hinge and the N-terminal domain of both CPs. The second eigenvector showed a closing motion, while the third eigenvector represented crosswise conformational fluctuations. These new findings, together with previous results, suggest that the hinge region of CPs plays a central role in the recognition and binding of viral RNA.

Genetic diversity of Hungarian Maize dwarf mosaic virus isolates.

The genetic diversity of the coat-protein (CP) region and the untranslated C-terminal region (3'UTR) of Maize dwarf mosaic virus (MDMV) was analyzed to evaluate the variability between isolates (inter-isolate sequence diversity). The results of inter-isolate sequence diversity analysis showed that the diversity of the MDMV CP gene is fairly high (p-distance: up to 0.136). During sequence analysis, a 13 amino-acid residue insertion and an 8 amino-acid residue deletion were found within the N-terminal region of the CP gene. The phylogenetic analysis showed that-unlike other potyvirus species in this subgroup-the MDMV isolates could not be distinguished on the basis of their host plants or geographic origins.