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Background and Aims Given the lack of specific studies on floral development in melon (Cucumis melo L.), we carried out an extensive study involving morphological and transcriptomic analyses to characterize floral development in this species, Methods Using an andromonoecious line, we analysed the development of floral buds in male and hermaphrodite flowers with both light microscopy and scanning electron microscopy. Based on flower lengths, we established a correlation between the developmental stages and four main episodes of floral development and conducted an extensive RNA sequencing analysis of these episodes, Key Results We identified 12 stages of floral development, from the appearance of the floral meristems to anthesis. The main structural differences between male and hermaphrodite flowers appeared between stages 6 and 7; later stages of development leading to the formation of organs and structures in both types of flowers were also described. We analysed the gene expression patterns of the four episodes in flower development to find the genes that were specific to each given episode. Among others, we identified genes that defined the passage from one episode to the next according to the ABCDE model of floral development, Conclusions This work combines a detailed morphological analysis and a comprehensive transcriptomic study to enable characterization of the structural and molecular mechanisms that determine the floral development of an andromonoecious genotype in melon. Taken together, our results provide a first insight into gene regulation networks in melon floral development that are crucial for flowering and pollen formation, highlighting potential targets for genetic manipulation to improve crop yield of melon in the future

Pepino mosaic virus (PepMV) is pandemic in tomato crops, causing important economic losses world-wide. No PepMV-resistant varieties have been developed yet. Identification of host factors interacting with PepMV proteins is a promising source of genetic targets to develop PepMV-resistant varieties. The interaction between the PepMV coat protein (CP) and the tomato glutathione S-transferase (GST) SlGSTU38 was identified in a yeast two-hybrid (Y2H) screening and validated by directed Y2H and co-immunoprecipitation assays. SlGSTU38-knocked-out Micro-Tom plants (gstu38) generated by the CRISPR/Cas9 technology together with live-cell imaging were used to understand the role of SlGSTU38 during infection. The transcriptomes of healthy and PepMV-infected wild-type (WT) and gstu38 plants were profiled by RNA-seq analysis. SlGSTU38 functions as a PepMV-specific susceptibility factor in a cell-autonomous manner and relocalizes to the virus replication complexes during infection. Besides, knocking out SlGSTU38 triggers reactive oxygen species accumulation in leaves and the deregulation of stress-responsive genes. SlGSTU38 may play a dual role: On the one hand, SlGSTU38 may exert a proviral function depending on its specific interaction with the PepMV CP; and on the other hand, SlGSTU38 may delay PepMV-infection sensing by participating in the redox intracellular homeostasis in a nonspecific manner

The cap-binding protein eIF4E, through its interaction with eIF4G, constitutes the core of the eIF4F complex, which plays a key role in the circularization of mRNAs and their subsequent cap-dependent translation. In addition to its fundamental role in mRNA translation initiation, other functions have been described or suggested for eIF4E, including acting as a proviral factor and participating in sexual development. We used CRISPR/Cas9 genome editing to generate melon eif4e knockout mutant lines. Editing worked efficiently in melon, as we obtained transformed plants with a single-nucleotide deletion in homozygosis in the first eIF4E exon already in a T0 generation. Edited and non-transgenic plants of a segregating F2 generation were inoculated with Moroccan watermelon mosaic virus (MWMV); homozygous mutant plants showed virus resistance, while heterozygous and non-mutant plants were infected, in agreement with our previous results with plants silenced in eIF4E. Interestingly, all homozygous edited plants of the T0 and F2 generations showed a male sterility phenotype, while crossing with wild-type plants restored fertility, displaying a perfect correlation between the segregation of the male sterility phenotype and the segregation of the eif4e mutation. Morphological comparative analysis of melon male flowers along consecutive developmental stages showed postmeiotic abnormal development for both microsporocytes and tapetum, with clear differences in the timing of tapetum degradation in the mutant versus wild-type. An RNA-Seq analysis identified critical genes in pollen development that were down-regulated in flowers of eif4e/eif4e plants, and suggested that eIF4E-specific mRNA translation initiation is a limiting factor for male gametes formation in melon

Plant viruses are obligate parasites that need to usurp plant cell metabolism in order to infect their hosts. Imaging techniques have been used for quite a long time to study plant virus–host interactions, making it possible to have major advances in the knowledge of plant virus infection cycles. The imaging techniques used to study plant–virus interactions have included light microscopy, confocal laser scanning microscopy, and scanning and transmission electron microscopies. Here, we review the use of these techniques in plant virology, illustrating recent advances in the area with examples from plant virus replication and virus plant-to-plant vertical transmission processes.

[Background] Vectors based on plant viruses are important tools for functional genomics, cellular biology, plant genome engineering and molecular farming. We previously reported on the construction of PepGFP2a, a viral vector based on pepino mosaic virus (PepMV) which expressed GFP efficiently and stably in plants of its experimental host Nicotiana benthamiana, but not in its natural host tomato. We have prepared a new set of PepMV-based vectors with improved stability that are able to express a wide range of reporter genes, useful for both N. benthamiana and tomato., [Results] We first tested PepGFPm1 and PepGFPm2, two variants of PepGFP2a in which we progressively reduced a duplication of nucleotides encoding the N-terminal region of the coat protein. The new vectors had improved GFP expression levels and stability in N. benthamiana but not in tomato plants. Next, we replaced GFP by DsRed or mCherry in the new vectors PepDsRed and PepmCherry, respectively; while PepmCherry behaved similarly to PepGFPm2, PepDsRed expressed the reporter gene efficiently also in tomato plants. We then used PepGFPm2 and PepDsRed to study the PepMV localization in both N. benthamiana and tomato cells. Using confocal laser scanning microscopy (CLSM), we observed characteristic fluorescent bodies in PepMV-infected cells; these bodies had a cytoplasmic localization and appeared in close proximity to the cell nucleus. Already at 3 days post-agroinoculation there were fluorescent bodies in almost every cell of agroinoculated tissues of both hosts, and always one body per cell. When markers for the endoplasmic reticulum or the Golgi apparatus were co-expressed with PepGFPm2 or PepDsRed, a reorganisation of these organelles was observed, with images suggesting that both are intimately related but not the main constituents of the PepMV bodies. Altogether, this set of data suggested that the PepMV bodies are similar to the potato virus X (PVX) “X-bodies”, which have been described as the PVX viral replication complexes (VRCs). To complete the set of PepMV-based vectors, we constructed a vector expressing the BAR herbicide resistance gene, useful for massive susceptibility screenings., [Conclusions] We have significantly expanded the PepMV tool box by producing a set of new vectors with improved stability and efficiency in both N. benthamiana and tomato plants. By using two of these vectors, we have described characteristic cellular bodies induced by PepMV infection; these bodies are likely the PepMV VRCs.

In situ hybridization (ISH) is an informative and relatively accessible technique for the localization of viral genomes in plant tissue and cells. However, simultaneous visualization of related plant viruses in mixed infections may be limited by the nucleotide similarity in the genomes and the single chromogenic detection over the same sample preparation. To address this issue, we used two Pepino mosaic virus isolates and performed ISH over consecutive serial cross-sections of paraffin-embedded leaf samples of single and mixed infected Nicotiana benthamiana plants. Moreover, the probe design was optimized to reduce cross-hybridisation, and co-localization was based on the overlapping of consecutive cross-sections from mixed infected leaves; thus, our results showed that both Pepino mosaic virus isolates co-localized in the same leaf tissue. In turn, both isolates were localized in the cytoplasm of the same cells. These results provide valuable information for studying mixed infections in plants by using a simple ISH procedure that is accessible to any pathology laboratory.

The emerging lettuce big-vein disease (LBVD) is causing losses in lettuce production ranging from 30 to 70% worldwide. Several studies have associated this disease with Mirafiori lettuce big-vein virus (MiLBVV) alone or in mixed infection with lettuce big-vein associated virus (LBVaV). We used Illumina small RNA sequencing (sRNA-seq) to identify viruses present in symptomatic lettuce plants from commercial fields in Southern Spain. Data analysis using the VirusDetect tool showed the consistent presence of MiLBVV and LBVaV in diseased plants. Populations of MiLBVV and LBVaV viral small RNAs (sRNAs) were characterized, showing features essentially similar to those of other viruses, with the peculiarity of an uneven asymmetric distribution of MiLBVV virus-derived small RNAs (vsRNAs) for the different polarities of genomic RNA4 vs. RNAs1 to 3. Sanger sequencing of coat protein genes was used to study MiLBVV and LBVaV phylogenetic relationships and population genetics. The Spanish MiLBVV population was composed of isolates from three well-differentiated lineages and reflected almost all of the diversity reported for the MiLBVV species, whereas the LBVaV population showed very little genetic differentiation at the regional scale but lineage differentiation at a global geographical scale. Universal primers were used to detect and quantify the accumulation of MiLBVV and LBVaV in field samples; both symptomatic and asymptomatic plants from affected fields carried equal viral loads, with LBVaV accumulating at higher levels than MiLBVV.

Potyviruses constitute the second largest genus of plant viruses and cause important economic losses in a large variety of crops; however, the atomic structure of their particles remains unknown. Infective potyvirus virions are long flexuous filaments where coat protein (CP) subunits assemble in helical mode bound to a monopartite positive-sense single-stranded RNA [(+)ssRNA] genome. We present the cryo-electron microscopy (cryoEM) structure of the potyvirus watermelon mosaic virus at a resolution of 4.0 Å. The atomic model shows a conserved fold for the CPs of flexible filamentous plant viruses, including a universally conserved RNA binding pocket, which is a potential target for antiviral compounds. This conserved fold of the CP is widely distributed in eukaryotic viruses and is also shared by nucleoproteins of enveloped viruses with segmented (−)ssRNA (negative-sense ssRNA) genomes, including influenza viruses.

Cucumber mosaic virus (CMV) has the broadest host range among plant viruses, causing enormous losses in agriculture. In melon, strains of subgroup II are unable to establish a systemic infection in the near-isogenic line SC12-1-99, which carries the recessive resistance gene cmv1 from the accession PI 161375, cultivar ‘Songwhan Charmi’. Strains of subgroup I overcome cmv1 resistance in a manner dependent on the movement protein. We characterized the resistance conferred by cmv1 and established that CMV-LS (subgroup II) can move from cell to cell up to the veins in the inoculated leaf, but cannot enter the phloem. Immunogold labelling at transmission electron microscopy level showed that CMV-LS remains restricted to the bundle sheath (BS) cells in the resistant line, and does not invade vascular parenchyma or intermediary cells, whereas, in the susceptible line ‘Piel de Sapo’ (PS), the virus invades all vein cell types. These observations indicate that the resistant allele of cmv1 restricts systemic infection in a virus strain- and cell type-specific manner by acting as an important gatekeeper for virus progression from BS cells to phloem cells. Graft inoculation experiments showed that CMV-LS cannot move from the infected PS stock into the resistant cmv1 scion, thus suggesting an additional role for cmv1 related to CMV transport within or exit from the phloem. The characterization of this new form of recessive resistance, based on a restriction of virus systemic movement, opens up the possibility to design alternative approaches for breeding strategies in melon.

The aim of this work is the mechanical properties evaluation of well-intergrowth free-standing c-oriented silicalite cantilevers. A laser-based optical vibrometer has been developed for the characterization of the nanoporous cantilevers resonant properties. The system has proven to be useful to determine the resonance frequencies and Q-factor of the zeolite-only cantilevers at room temperature and atmospheric pressure. Young modulus of the prepared silicalite polycrystalline cantilevers around 30.09 ± 5.29 GPa has been evaluated. We have also built a controlled gas circuit attached to the vibrometer with the aim to make a dynamic characterization of the nanoporous cantilevers in presence of ethanol vapours to assess about their capability as chemical sensors. © 2009 Elsevier B.V. All rights reserved.

The province of Murcia (Murcia Region) is the one of the most important apricot growing regions in Europe. In recent years a fruit disorder named by growers as “degeneración” has been detected in apricot commercial orchards of this region, mainly in the variety Velázquez Fino. Affected fruits are characterized by changes in their external appearance involving rugosity and a loss of organoleptic characteristics, which leads to an unmarketable product. In order to identify the causal agent of this disorder, the presence of the most important viruses affecting stone fruit trees and Hop stunt viroid (HSVd) was tested. While negative results were obtained for all viruses analysed, the viroid was detected in all the symptomatic trees. Within a single tree, the viroid was restricted to branches bearing fruits with the characteristic symptoms but was usually absent from the rest of the tree. Sequence analysis of several isolates of HSVd obtained from these affected trees revealed the presence of two variants previously detected in other apricot cultivars. Taken together, these results suggest that degeneration is associated with HSVd.

The combination of electron microscopy (EM) cytochemical with immunocytochemical methods is used to characterize the interchromatin region (IR) of the plant cell nucleus. Cryoprocessing of the sample provides a better ultrastructural preservation and allows the observation of some differences in the fine structure of the IR which shows a denser aspect resulting from the lower extraction of components with low-temperature methods. A complex network of fibrillar structures and isolated or clustered 30 to 50-nm granules are observed in the IR. Anti-DNA antibodies combined with the NAMA-Ur method for DNA or the EDTA staining, preferential for RNPs, allow the detection of chromatin fibers in the IR. Bismuth staining reveals the presence of highly phosphorylated proteins in some interchromatin structures. The spliceosomal snRNPs are immunolocalized on cryosections and Lowicryl sections of plant cells using monoclonal and polyclonal antibodies. They provide a homogeneous immunofluorescence pattern with no speckles. This is in correlation with the labeling at EM, immunogold particles decorate the EDTA-positive fibrillar structures of the IR but no labeling is found over the 30 to 50-nm granules. The presence of the spliceosomal snRNPs, DNA and phosphorylated proteins in the IR indicate that this nuclear domain plays a major role in pre-messenger RNA splicing and, possibly in transcription, in the plant cell nucleus.

The nuclei of the secretory tapetum of Scilla peruviana L. and Capsicum annuum L. are studied by light and transmission electron microscopy using cryoprocessed material. The NAMA-Ur method, specific for DNA, anti-DNA and anti-histones immunogold labelling show the condensed chromatin pattern and reveal the presence of chromatin fibres in the interchromatin region. Different antigens of the nucleoplasmic snRNPs, involved in the splicing of the pre-mRNAs, are immunolocalized over the interchromatin fibres. Nucleolar proteins related to different steps of rRNA processing are immunodetected on the nucleolar components. These approaches give new insights in the nuclear function of the tapetum during pollen development.

The Mr 34000 nucleolar protein B-36 was immunolocalized using a mouse monoclonal antibody in different plant cells by light and electron microscopy. Specific immunofluorescence was observed in nucleoli of cryofixed and cryosectioned material. Immunoelectron microscopy was performed on ultrathin cryosections and Lowicryl sections. Immunogold labelling revealed that B-36 is mainly localized in the nucleolar dense fibrillar component (DFC). The fibrillar centres and the granular component were practically devoid of gold particles. Nucleoli with different rates of transcriptional activity and, as a consequence, with different morphologies showed the same immunolocalization. This was true for both somatic and germinal tissues of the different plant species studied: Allium cepa root meristematic cells and Scilla peruviana and Capsicum annuum pollen grains. The presence of the B-36 antigen in the DFC of nucleoli with very different rates of transcriptional activity indicates that it is a constitutive protein. Moreover, the localization suggests that it could play a role in the transcription and/or early processing of rRNA. The presence of B-36 in the different plant tissues and species studied, confirms once more its high level of conservation throughout evolution. Another Mr 34000 protein called fibrillarin was also found in the DFC. Taking these and other published data together, we think that the antibodies used in the different studies are probably binding different epitopes of identical or very similar Mr 34000 nucleolar proteins.

We have developed a new, simple, and reproducible cytochemical method to specifically stain DNA at the electron microscopic level: the NAMA-Ur. It is based on the extraction of RNA and phosphate groups from phosphoproteins by a weak alkali hydrolysis (NA) which does not affect DNA, followed by blockage of the amino and carboxyl groups by methylation and acetylation (MA). Finally, sections are stained by uranyl (Ur), which can bind only to DNA. The efficiency of the pre-treatment (NA and MA) was demonstrated by X-ray microanalysis at the transmission electron microscopic level. The NAMA-Ur method has been successfully performed en bloc and on Lowicryl sections in mammalian and plant cells. A specific contrast is observed in the DNA-containing structures after this method, whose sensitivity allows visualization of electron-dense chromatin fibers of 10-12 nm composed of 3-nm DNA fibrils. This staining method has been combined with anti-DNA antibodies, providing complementary information to detect DNA in situ. We propose the NAMA-Ur as an easy method to investigate the chromatin organization in situ at the ultrastructural level.

The use of nucleolar silver staining in plant cells in conditions of impaired transcription, either natural (quiescent cells) or induced (actinomycin D, ethidium bromide and cordycepin treatments), shows that nucleoli with very low transcription rates, compared with controls, contain similar amounts of argyrophilic proteins, as well as the same structural location. This demonstrates the absence of correlation between silver staining and present transcriptional activity. The effects of conditions used on the structure of nucleolar chromatin support our previous suggestion on the involvement of “Ag-NOR” proteins in the process of decondensation of NOR chromatin.

The ultrastructure and morphometric features of nucleolar fibrillar centers have been studied in three different plant species over a long period of gametogenesis, extending from premeiosis to after the first postmeiotic mitosis, with respect to variations in nucleolar activity which occur throughout this process. Fibrillar centers represent the structural counterpart of the transcriptionally inactive portions of the nucleolar organizing region (NOR); in some gametogenic stages, no discrete fibrillar centers can be observed and, therefore, the structure of the whole NOR has been studied. Two clearly differentiated types of fibrillar center structure are described, with a close relationship to different stages of nucleolar activity. Stages with high activity show “homogeneous” fibrillar centers, whose chromatin is decondensed, and stages with low activity show “heterogeneous” fibrillar centers with both condensed and decondensed chromatin. These two types had previously been characterized cytochemically by us in somatic cells. Medium-active stages show fibrillar centers “intermediate” between the two main types. The morphometric behavior of fibrillar centers is also related to activity. The number of fibrillar centers per nucleolar section increases with the nucleolar activity, while their size decreases. However, the location of fibrillar centers in the nucleolus does not follow any special pattern, except in middle meiotic prophase I, in which the whole NOR occupies a peripheral position. This is due to a special NOR activity at these stages, whose products are stored as “nucleolar dense bodies.” All these data allow us to conclude that fibrillar centers are highly dynamic structures, extremely sensitive to changes in nucleolar activity.

We have made an ultrastructural study of the nuclear acid phosphatase localization in the different tissues of Allium cepa L. anthers, after meiosis. The enzyme is preferentially located in the fibrillar component, within the nucleolus,with a greater reaction density around the nucleolar organizing region (NOR). The specificity of the reaction was proved by contrastting the results with those obtained in two different series of anthers. One of these series was incubated in Gomori's medium with an acid phosphatasic inhibitor, and the other in Gomori's medium without substrate. In both cases the acid phosphatase reaction was negative. Some authors (Hardin and Spicer, 1970; Tandler et al., 1970; Rodríguez-García and Stockert, 1979) have observed an accumulation of inorganic cations (Ca++, Mg++. ... and Na+) within the nucleolar region where the phosphatase acid activity was detected by us. The coincidence of this localization is discussed, suggesting that these cations are probably involved in the phosphatasic activity of these nucleolar zones. Our results agree with the idea that this enzyme contributes to an increase in the amount of nuclear ortophosphate ions (Harris, 1963). On the other hand this enzyme could play a part in the successive triming of the different preribosomal RNA species that occurs during their maturation (Dalgarno and Shine, 1977).

The pattern of nucleolar silver staining was determined in the interphase and mitosis of plant cells using two cytological techniques, the selectivity of which was tested densitometrically. For complete accuracy, silver grains were removed and the sections were then observed under uranyl and lead staining. During interphase, the highest density of silver grains occurred in the portions of the dense fibrillar component nearest to the fibrillar centres; the latter were also stained except for the condensed chromatin inclusions typical of the heterogeneous type. During mitosis, not only the nucleolar organiser region (NOR) was stained, but also the prenucleolar material in anaphase and telophase. Two different proteins or sets of proteins are suggested to be responsible for this staining pattern; one of them is associated with NOR chromatin (which is not contained in the mitotic prenucleolar material) and the other with early ribosomal precursors (which have never been found in fibrillar centres). From these two proteins, the “Ag-NOR” reveals either transcriptionally active or potentially active chromatin, characterized by an uncoiled, extended structure, regardless of the state of transcription. This is the structural feature detected by Ag-NOR staining.

In an attempt to clarify the physiology of plant nuclear coiled bodies, three different cytochemical tests related to nuclear RNA metabolism were made: 1)localization of an acid phosphatasic activity; 2) presence of orthophosphate ions; and 3)presence of divalent cations. The three cytochemical tests gave positive results, which are discussed in view of the present knowledge on nuclear RNA synthesis and metabolism.

Using a high resolution nucleolar silver staining technique, we have studied the distribution of silver grains at interphase and mitosis. The nucleolar fibrillar centers and the fibrillar component, as well as the mitotic nucleolar organizing region (NOR), are argyrophilic. Of the NOR structures, the fibrillar component is transcriptionally active, but the fibrillar centers and the mitotic NORs are not. Treatment with cycloheximide (CHM), which indirectly impairs rRNA synthesis, does not affect the pattern of silver staining. The currently assumed correlation between activity and Ag-NOR staining appears questionable. On the other hand, all Ag-NOR-stained structures contain decondensed chromatin, whereas condensed portions of this chromatin are not stained by silver. In CHM-treated cells there is apparently no chromatin condensation accompanying inhibition of rRNA synthesis.