Manuel Rubio

Alteration in the chloroplastic metabolism leads to ROS accumulation in pea plants in response to plum pox virus

In this work, a recombinant plum pox virus (PPV, Sharka) encoding green fluorescent protein is used to study its effect on antioxidant enzymes and protein expression at the subcellular level in pea plants (cv. Alaska). PPV had produced chlorotic spots as well as necrotic spots in the oldest leaves at 13–15 d post-inoculation. At 15 d post-inoculation, PPV was present in the chlorotic and necrotic areas, as shown by the fluorescence signal produced by the presence of the green fluorescent protein. In the same areas, an accumulation of reactive oxygen species was noticed. Studies with laser confocal and electron microscopy demonstrated that PPV accumulated in the cytosol of infected cells. In addition, PPV infection produced an alteration in the chloroplast ultrastructure, giving rise to dilated thylakoids, an increase in the number of plastoglobuli, and a decreased amount of starch content. At 3 d post-inoculation, although no changes in the oxidative stress parameters were observed, an increase in the chloroplastic hydrogen peroxide levels was observed that correlated with a decrease in the enzymatic mechanisms involved in its elimination (ascorbate peroxidase and peroxidase) in this cell compartment. These results indicate that an alteration in the chloroplastic metabolism is produced in the early response to PPV. This oxidative stress is more pronounced during the development of the disease (15 d post-inoculation) judging from the increase in oxidative stress parameters as well as the imbalance in the antioxidative systems, mainly at the chloroplastic level. Finally, proteomic analyses showed that most of the changes produced by PPV infection with regard to protein expression at the subcellular level were related mainly to photosynthesis and carbohydrate metabolism. It seems that PPV infection has some effect on PSII, directly or indirectly, by decreasing the amount of Rubisco, oxygen-evolving enhancer, and PSII stability factor proteins. The results indicate that Sharka symptoms observed in pea leaves could be due to an imbalance in antioxidant systems as well as to an increased generation of reactive oxygen species in chloroplasts, induced probably by a disturbance of the electron transport chain, suggesting that chloroplasts can be a source of oxidative stress during viral disease development.

QTL analysis of resistance to sharka disease in the apricot ( Prunus armeniaca L.) ‘Polonais’ × ‘Stark Early Orange’ F1 progeny

Different hypotheses on the genetic control of the resistance to the plum pox virus (PPV) have been reported in apricot, but there was a lack of agreement about the number of loci involved. In recent years, apricot genetic maps have been constructed from progenies derived from ‘Stark Early Orange’ or ‘Goldrich’, two main sources of resistance, three of these including the mapping of the PPV resistance loci. As the location of the locus was not precisely established, we mapped the PPV resistance loci using interval mapping (IM), composite interval mapping (CIM), and the Kruskal–Wallis non-parametric test in the F1 progeny derived from a cross between the susceptible cv. ‘Polonais’ and ’Stark Early Orange’. Four genomic regions were identified as being involved in PPV resistance. One of these mapped to the upper region of linkage group 1 of ‘Stark Early Orange’, and accounted for 56% of the phenotypic variation. Its location was similar to the one previously identified in ‘Goldrich’ and Prunus davidiana. In addition, a gene strongly associated to these major quantitative trait loci (QTL) was found to be related to PPV infection. Two putative QTLs were detected on linkage groups 3 of ‘Polonais’ and 5 of both ‘Polonais’ and ‘Stark Early Orange’ with both parametric and non-parametric methods at logarithm of odds (LOD) scores slightly above the detection threshold. The last QTL was only detected in the early stage of the infection. PPV resistance is, thus, controlled by a major dominant factor located on linkage group 1. The hypothesis of recessive factors with lower effect is discussed.

New approaches to Prunus transcriptome analysis

The recent sequencing of the complete genome of the peach offers new opportunities for further transcriptomic studies in Prunus species in the called post-genomics era. First works on transcriptome analysis in Prunus species started in the early 2000s with the development of ESTs (expressed sequence tags) and the analysis of several candidate genes. Later, new strategies of massive analysis (high throughput) of transcriptomes have been applied, producing larger amounts of data in terms of expression of a large number of genes in a single experiment. One of these systems is massive transcriptome analysis using cDNA biochips (microarrays) to analyze thousands of genes by hybridization of mRNA labelled with fluorescence. However, the recent emergence of a massive sequencing methodology (“deep-sequencing”) of the transcriptome (RNA-Seq), based on lowering the costs of DNA (in this cases complementary, cDNA) sequencing, could be more suitable than the application of microarrays. Recent papers have described the tremendous power of this technology, both in terms of profiling coverage and quantitative accuracy in transcriptomic studies. Now this technology is being applied to plant species, including Prunus. In this work, we analyze the potential in using this RNA-Seq technology in the study of Prunus transcriptomes and the development of genomic tools. In addition, the strengths and limitations of RNA-Seq relative to microarray profiling have been discussed.

Quantitative Trait Loci (QTL) and Mendelian Trait Loci (MTL) Analysis in Prunus: a Breeding Perspective and Beyond

Trait loci analysis, a classic procedure in quantitative (quantitative trait loci, QTL) and qualitative (Mendelian trait loci, MTL) genetics, continues to be the most important approach in studies of gene labeling in Prunus species from the Rosaceae family. Since 2011, the number of published Prunus QTLs and MTLs has doubled. With increased genomic resources, such as whole genome sequences and high-density genotyping platforms, trait loci analysis can be more readily converted to markers that can be directly utilized in marker-assisted breeding. To provide this important resource to the community and to integrate it with other genomic, genetic, and breeding data, a global review of the QTLs and MTLs linked to agronomic traits in Prunus has been performed and the data made available in the Genome Database for Rosaceae. We describe detailed information on 760 main QTLs and MTLs linked to a total of 110 agronomic traits related to tree development, pest and disease resistance, flowering, ripening, and fruit and seed quality. Access to these trait loci enables the application of this information in the post-genomic era, characterized by the availability of a high-quality peach reference genome and new high-throughput DNA and RNA analysis technologies.

Clarifying omics concepts, challenges, and opportunities for Prunus breeding in the postgenomic era.

The recent sequencing of the complete genome of the peach, together with the availability of new high-throughput genome, transcriptome, proteome, and metabolome analysis technologies, offers new possibilities for Prunus breeders in what has been described as the postgenomic era. In this context, new biological challenges and opportunities for the application of these technologies in the development of efficient marker-assisted selection strategies in Prunus breeding include genome resequencing using DNA-Seq, the study of RNA regulation at transcriptional and posttranscriptional levels using tilling microarray and RNA-Seq, protein and metabolite identification and annotation, and standardization of phenotype evaluation. Additional biological opportunities include the high level of synteny among Prunus genomes. Finally, the existence of biases presents another important biological challenge in attaining knowledge from these new high-throughput omics disciplines. On the other hand, from the philosophical point of view, we are facing a revolution in the use of new high-throughput analysis techniques that may mean a scientific paradigm shift in Prunus genetics and genomics theories. The evaluation of scientific progress is another important question in this postgenomic context. Finally, the incommensurability of omics theories in the new high-throughput analysis context presents an additional philosophical challenge.

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.

Opportunities of marker-assisted selection for Plum pox virus resistance in apricot breeding programs

Evaluation of Plum pox virus (PPV) resistance is a laborious and expensive task, and the development of new accurate methods, including the use of molecular markers, would be very useful for breeding programs for resistance. In this work, the Plum pox virus resistance of 80 apricot genotypes of different genetic origins was evaluated in controlled greenhouse and natural field conditions. The genotypes for five simple sequence repeat (SSR) markers described as linked to PPV resistance were also determined. Depending on their behavior, cultivars were classified as resistant, susceptible, and uncertain, and the genotype was identified for each SSR linked to different phenotypes. Twenty genotypes were resistant and 37 susceptible in the greenhouse and in the field. However, 23 genotypes did not show clear behavior, probably due to the complex plant-virus interaction, so they were classified as uncertain. In general, results showed a narrow relationship between the SSRs PGS1.21 and PGS1.24, and resistance to PPV, although some genotypes did not show this relationship. Most of the susceptible genotypes did not show the alleles of resistance. Therefore, in most cases, marker-assisted selection (MAS) could be used as a means of screening new seedlings for early selection, making it possible to remove those that are susceptible. However, in certain cases, MAS using these markers has not proven to be completely effective. The origin of such discrepancies could be the presence of a second locus involved in PPV resistance. In addition, other factors affecting efficiency of MAS discussed in the work are the presence of null alleles and recombinant events. Resistant seedlings would have to be evaluated in greenhouse and natural conditions to confirm their actual behavior against PPV. From the breeding point of view, the use of homozygous resistant parents for the SSR resistance alleles, with good agronomic characteristics, would increase the efficiency of breeding programs, since all seedlings would be resistant regardless of the other parent. Finally, new molecular markers should be developed to accurately select resistant seedlings regardless of the resistant progenitors involved.

Variations in the cellular proliferation of prolactin cells from late pregnancy to lactation in rats

Lactation is a physiological process associated with hyperactivity of hypophyseal prolactin-producing cells. It is known that the percentage of these cells is increased during lactation, although there are discrepancies in the reports regarding the mechanisms responsible for increasing the number of prolactin cells. In order to analyse whether this increase is a result of previous proliferation, variations in the proliferation rate of prolactin-positive cells were determined from late pregnancy to lactation in adult female rats by means of observation of the immunohistochemical expression of PCNA as a marker of cellular proliferation. During late pregnancy, a very significant increase in the percentage of proliferating prolactin cells was observed in comparison to non-pregnant females in the proestrus phase (p < 0.01). Although the percentage of prolactin-positive cells after one week of lactation was higher than in non-lactating or in pregnant females (p < 0.01), the proliferation rate was lower than in the other groups studied. In sum, our results suggest that late pregnancy constitutes a preliminary proliferative phase preparatory to the ensuing lactation phase and that endocrine changes in late pregnancy involve the cellular proliferation of hypophyseal prolactin cells in order to prepare the gland for later demands and to prevent proliferative changes from occurring during lactation.

Gene Expression Analysis of Plum pox virus (Sharka) Susceptibility/Resistance in Apricot ( Prunus armeniaca L.)

RNA-Seq has proven to be a very powerful tool in the analysis of the Plum pox virus (PPV, sharka disease)/Prunus interaction. This technique is an important complementary tool to other means of studying genomics. In this work an analysis of gene expression of resistance/susceptibility to PPV in apricot is performed. RNA-Seq has been applied to analyse the gene expression changes induced by PPV infection in leaves from two full-sib apricot genotypes, “Rojo Pasión” and “Z506-7”, resistant and susceptible to PPV, respectively. Transcriptomic analyses revealed the existence of more than 2,000 genes related to the pathogen response and resistance to PPV in apricot. These results showed that the response to infection by the virus in the susceptible genotype is associated with an induction of genes involved in pathogen resistance such as the allene oxide synthase, S-adenosylmethionine synthetase 2 and the major MLP-like protein 423. Over-expression of the Dicer protein 2a may indicate the suppression of a gene silencing mechanism of the plant by PPV HCPro and P1 PPV proteins. On the other hand, there were 164 genes involved in resistance mechanisms that have been identified in apricot, 49 of which are located in the PPVres region (scaffold 1 positions from 8,050,804 to 8,244,925), which is responsible for PPV resistance in apricot. Among these genes in apricot there are several MATH domain-containing genes, although other genes inside (Pleiotropic drug resistance 9 gene) or outside (CAP, Cysteine-rich secretory proteins, Antigen 5 and Pathogenesis-related 1 protein; and LEA, Late embryogenesis abundant protein) PPVres region could also be involved in the resistance.

Prunus transcription factors: breeding perspectives.

Many plant processes depend on differential gene expression, which is generally controlled by complex proteins called transcription factors (TFs). In peach, 1533 TFs have been identified, accounting for about 5.5% of the 27,852 protein-coding genes. These TFs are the reference for the rest of the Prunus species. TF studies in Prunus have been performed on the gene expression analysis of different agronomic traits, including control of the flowering process, fruit quality, and biotic and abiotic stress resistance. These studies, using quantitative RT-PCR, have mainly been performed in peach, and to a lesser extent in other species, including almond, apricot, black cherry, Fuji cherry, Japanese apricot, plum, and sour and sweet cherry. Other tools have also been used in TF studies, including cDNA-AFLP, LC-ESI-MS, RNA, and DNA blotting or mapping. More recently, new tools assayed include microarray and high-throughput DNA sequencing (DNA-Seq) and RNA sequencing (RNA-Seq). New functional genomics opportunities include genome resequencing and the well-known synteny among Prunus genomes and transcriptomes. These new functional studies should be applied in breeding programs in the development of molecular markers. With the genome sequences available, some strategies that have been used in model systems (such as SNP genotyping assays and genotyping-by-sequencing) may be applicable in the functional analysis of Prunus TFs as well. In addition, the knowledge of the gene functions and position in the peach reference genome of the TFs represents an additional advantage. These facts could greatly facilitate the isolation of genes via QTL (quantitative trait loci) map-based cloning in the different Prunus species, following the association of these TFs with the identified QTLs using the peach reference genome.