Isoenzymes of the Flavonoid and Phenylpropanoid Pathways Show Organ-Specific Regulation during Apple Fruit Development.

Elucidating the molecular mechanisms controlling fruit development is a primary target for the improvement of new apple (Malus × domestica Borkh.) cultivars. The first two weeks of development following pollination are crucial to determine fruit characteristics. During this period, a lot of changes take place in apple fruit, going from rapid cell division to the production of important metabolites. In this work, attention was focused on the phenylpropanoid and flavonoid pathways responsible for the production of numerous compounds contributing to fruit quality, such as flavonols, catechins, dihydrochalcones and anthocyanins. A total of 17 isoenzymes were identified, belonging to seven classes of the phenylpropanoid and flavonoid pathways that, despite showing more than 80% sequence identity, showed differential expression regulation during the first two weeks of apple fruit development. This feature seems to be quite common for most of the enzymes of both pathways. Differential regulation of isoenzymes was shown to be present in both 'Golden Delicious' and a wild relative (Malus mandshurica), even though differences were also present. Each isoenzyme showed a specific pattern of expression in the flower and fruit organs, suggesting that genes coding for enzymes with the same function may control different aspects of plant biology. Finally, promoter analysis was performed in order to highlight differences in the number and type of regulatory motifs. Overall, our results indicate that the control of the expression of genes involved in the phenylpropanoid and flavonoid pathways may be very complex as not only enzymes belonging to the same class, but even putative isoenzymes, can have different roles for the plant. Such genes may represent an important regulatory mechanism, as they would allow the plant to fine-tune the processing of metabolic intermediates towards different branches of the pathway, for example, in an organ-specific way.

Molecular Approaches for Low-Cost Point-of-Care Pathogen Detection in Agriculture and Forestry.

Early detection of plant diseases is a crucial factor to prevent or limit the spread of a rising infection that could cause significant economic loss. Detection test on plant diseases in the laboratory can be laborious, time consuming, expensive, and normally requires specific technical expertise. Moreover, in the developing countries, it is often difficult to find laboratories equipped for this kind of analysis. Therefore, in the past years, a high effort has been made for the development of fast, specific, sensitive, and cost-effective tests that can be successfully used in plant pathology directly in the field by low-specialized personnel using minimal equipment. Nucleic acid-based methods have proven to be a good choice for the development of detection tools in several fields, such as human/animal health, food safety, and water analysis, and their application in plant pathogen detection is becoming more and more common. In the present review, the more recent nucleic acid-based protocols for point-of-care (POC) plant pathogen detection and identification are described and analyzed. All these methods have a high potential for early detection of destructive diseases in agriculture and forestry, they should help make molecular detection for plant pathogens accessible to anyone, anywhere, and at any time. We do not suggest that on-site methods should replace lab testing completely, which remains crucial for more complex researches, such as identification and classification of new pathogens or the study of plant defense mechanisms. Instead, POC analysis can provide a useful, fast, and efficient preliminary on-site screening that is crucial in the struggle against plant pathogens.

Gene expression and metabolite accumulation during strawberry (Fragaria × ananassa) fruit development and ripening.

MAIN CONCLUSION: A coordinated regulation of different metabolic pathways was highlighted leading to the accumulation of important compounds that may contribute to the final quality of strawberry fruit. Strawberry fruit development and ripening involve complex physiological and biochemical changes, ranging from sugar accumulation to the production of important volatiles compounds that contribute to the final fruit flavor. To better understand the mechanisms controlling fruit growth and ripening in cultivated strawberry (Fragaria × ananassa), we applied a molecular approach combining suppression subtractive hybridization and next generation sequencing to identify genes regulating developmental stages going from fruit set to full ripening. The results clearly indicated coordinated regulation of several metabolic processes such as the biosynthesis of flavonoid, phenylpropanoid and branched-chain amino acids, together with glycerolipid metabolism and pentose and glucuronate interconversion. In particular, genes belonging to the flavonoid pathway were activated in two distinct phases, the first one at the very early stages of fruit development and the second during ripening. The combination of expression analysis with metabolomic data revealed that the functional meaning of these two inductions is different, as during the early stages gene activation of flavonoid pathway leads to the production of proanthocyanidins and ellagic acid-derived tannins, while during ripening anthocyanins are the main product of flavonoid pathway activation. Moreover, the subtractive approach allowed the identification of different members of the same gene family coding for the same or very similar enzymes that in some cases showed opposite regulation during strawberry fruit development. Such regulation is an important trait that can help to understand how plants specifically channel metabolic intermediates towards separate branches of a biosynthetic pathway or use different isoforms of the same enzyme in different organs or developmental stages.

Xylella fastidiosa: Host Range and Advance in Molecular Identification Techniques.

In the never ending struggle against plant pathogenic bacteria, a major goal is the early identification and classification of infecting microorganisms. Xylella fastidiosa, a Gram-negative bacterium belonging to the family Xanthmonadaceae, is no exception as this pathogen showed a broad range of vectors and host plants, many of which may carry the pathogen for a long time without showing any symptom. Till the last years, most of the diseases caused by X. fastidiosa have been reported from North and South America, but recently a widespread infection of olive quick decline syndrome caused by this fastidious pathogen appeared in Apulia (south-eastern Italy), and several cases of X. fastidiosa infection have been reported in other European Countries. At least five different subspecies of X. fastidiosa have been reported and classified: fastidiosa, multiplex, pauca, sandyi, and tashke. A sixth subspecies (morus) has been recently proposed. Therefore, it is vital to develop fast and reliable methods that allow the pathogen detection during the very early stages of infection, in order to prevent further spreading of this dangerous bacterium. To this purpose, the classical immunological methods such as ELISA and immunofluorescence are not always sensitive enough. However, PCR-based methods exploiting specific primers for the amplification of target regions of genomic DNA have been developed and are becoming a powerful tool for the detection and identification of many species of bacteria. The aim of this review is to illustrate the application of the most commonly used PCR approaches to X. fastidiosa study, ranging from classical PCR, to several PCR-based detection methods: random amplified polymorphic DNA (RAPD), quantitative real-time PCR (qRT-PCR), nested-PCR (N-PCR), immunocapture PCR (IC-PCR), short sequence repeats (SSRs, also called VNTR), single nucleotide polymorphisms (SNPs) and multilocus sequence typing (MLST). Amplification and sequence analysis of specific targets is also mentioned. The fast progresses achieved during the last years in the DNA-based classification of this pathogen are described and discussed and specific primers designed for the different methods are listed, in order to provide a concise and useful tool to all the researchers working in the field.

Fine-tuning of the flavonoid and monolignol pathways during apple early fruit development.

MAIN CONCLUSION: A coordinated regulation of different branches of the flavonoid pathway was highlighted that may contribute to elucidate the role of this important class of compounds during the early stages of apple fruit development. Apple (Malus × domestica Borkh.) is an economically important fruit appreciated for its organoleptic characteristics and its benefits for human health. The first stages after fruit set represent a very important and still poorly characterized developmental process. To enable the profiling of genes involved in apple early fruit development, we combined the suppression subtractive hybridization (SSH) protocol to next-generation sequencing. We identified and characterized genes induced and repressed during fruit development in the apple cultivar 'Golden Delicious'. Our results showed an opposite regulation of genes coding for enzymes belonging to flavonoid and monolignol pathways, with a strong induction of the former and a simultaneous repression of the latter. Two isoforms of phenylalanine ammonia-lyase and 4-coumarate:CoA ligase, key enzymes located at the branching point between flavonoid and monolignol pathways, showed opposite expression patterns during the period in analysis, suggesting a possible regulation mechanism. A targeted metabolomic analysis supported the SSH results and revealed an accumulation of the monomers catechin and epicatechin as well as several forms of procyanidin oligomers in apple fruitlets starting early after anthesis, together with a decreased production of other classes of flavonoids such as some flavonols and the dihydrochalcone phlorizin. Moreover, gene expression and metabolites accumulation of 'Golden Delicious' were compared to a wild apple genotype of Manchurian crabapple (Malus mandshurica (Maxim.) Kom.). Significant differences in both gene expression and metabolites accumulation were found between the two genotypes.

Suppression Substractive Hybridization and NGS Reveal Differential Transcriptome Expression Profiles in Wayfaring Tree (Viburnum lantana L.) Treated with Ozone.

Tropospheric ozone (O3) is a global air pollutant that causes high economic damages by decreasing plant productivity. It enters the leaves through the stomata, generates reactive oxygen species, which subsequent decrease in photosynthesis, plant growth, and biomass accumulation. In order to identify genes that are important for conferring O3 tolerance or sensitivity to plants, a suppression subtractive hybridization analysis was performed on the very sensitive woody shrub, Viburnum lantana, exposed to chronic O3 treatment (60 ppb, 5 h d(-1) for 45 consecutive days). Transcript profiling and relative expression assessment were carried out in asymptomatic leaves, after 15 days of O3 exposure. At the end of the experiment symptoms were observed on all treated leaves and plants, with an injured leaf area per plant accounting for 16.7% of the total surface. Cloned genes were sequenced by 454-pyrosequencing and transcript profiling and relative expression assessment were carried out on sequenced reads. A total of 38,800 and 12,495 high quality reads obtained in control and O3-treated libraries, respectively (average length of 319 ± 156.7 and 255 ± 107.4 bp). The Ensembl transcriptome yielded a total of 1241 unigenes with a total sequence length of 389,126 bp and an average length size of 389 bp (guanine-cytosine content = 49.9%). mRNA abundance was measured by reads per kilobase per million and 41 and 37 ensembl unigenes showed up- and down-regulation respectively. Unigenes functionally associated to photosynthesis and carbon utilization were repressed, demonstrating the deleterious effect of O3 exposure. Unigenes functionally associated to heat-shock proteins and glutathione were concurrently induced, suggesting the role of thylakoid-localized proteins and antioxidant-detoxification pathways as an effective strategy for responding to O3. Gene Ontology analysis documented a differential expression of co-regulated transcripts for several functional categories, including specific transcription factors (MYB and WRKY). This study demonstrates that a complex sequence of events takes place in the cells at intracellular and membrane level following O3 exposure and elucidates the effects of this oxidative stress on the transcriptional machinery of the non-model plant species V. lantana, with the final aim to provide the molecular supportive knowledge for the use of this plant as O3-bioindicator.

Identification of low temperature stress regulated transcript sequences and gene families in Italian cypress.

Cold acclimation is a complex transcriptionally controlled process regulated by many different genes and genic-interactions in plants. The northward spreading of woody species is mainly limited by winter harshness. To increase our knowledge about the biological processes underlying cold acclimation, plants evolved in warmer climates can serve as models. In this work, a Suppression Subtractive Hybridization approach using PCR-select was used to isolate Italian cypress (Cupressus sempervirens L.) transcript sequences putatively expressed under low temperature stress. After assessing the reliability of the subtractive step, a total of 388 clones were selected and sequenced. Following sequence assembly and removal of the redundant cDNAs, 156 unique transcripts were identified and annotated in order to assign them a putative functional class. Most of the identified transcripts were functionally classified pertaining to stress in cellular and chloroplast membranes, which are previously known to be severely damaged by cold treatment. Among the identified functional gene families, the extensively represented ones were dehydrins, early light-inducible proteins, senescence-associated genes and oleosins. The last three gene families were further selected for phylogenetic analysis, with the corresponding protein sequences across the complete genomes of the model plants Populus trichocarpa, Vitis vinifera, Physcomitrella patens, and Arabidopsis thaliana. The relationship with the ortholog sequences coming from these species and their further implications are discussed.

Evidence for regulation of columnar habit in apple by a putative 2OG-Fe(II) oxygenase.

Understanding the genetic mechanisms controlling columnar-type growth in the apple mutant 'Wijcik' will provide insights on how tree architecture and growth are regulated in fruit trees. In apple, columnar-type growth is controlled by a single major gene at the Columnar (Co) locus. By comparing the genomic sequence of the Co region of 'Wijcik' with its wild-type 'McIntosh', a novel non-coding DNA element of 1956 bp specific to Pyreae was found to be inserted in an intergenic region of 'Wijcik'. Expression analysis of selected genes located in the vicinity of the insertion revealed the upregulation of the MdCo31 gene encoding a putative 2OG-Fe(II) oxygenase in axillary buds of 'Wijcik'. Constitutive expression of MdCo31 in Arabidopsis thaliana resulted in compact plants with shortened floral internodes, a phenotype reminiscent of the one observed in columnar apple trees. We conclude that MdCo31 is a strong candidate gene for the control of columnar growth in 'Wijcik'.

Leaf plasticity to light intensity in Italian cypress (Cupressus sempervirens L.): adaptability of a Mediterranean conifer cultivated in the Alps.

Italian cypress (Cupressus sempervirens L.) is native to the eastern Mediterranean, an area characterised by hot, dry summers and mild winters. Over the centuries, however, the species has been introduced into more northerly regions, a long way from its native range. The current, generally warmer climatic conditions brought about by global warming have favoured its cultivation in even more northerly areas in the Alps and other European alpine regions. Given that not only temperature, but also light availability are limiting factors for the spread of cypress in these environments, it is important to ascertain how this species copes with low light conditions. The photosynthetic characteristics of cypress leaves collected from different portions of the crown with contrasting light availability were evaluated by several methods. Chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoid (Car) content was found to be higher in shade leaves than in sun leaves when measured on a fresh mass basis, although enzymatic activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) and nitrate reductase were lower in shade leaves. When the efficiency of PSII was measured by chlorophyll fluorescence, a marked reduction in F(m) was found in shade leaves, while F(o) remained unchanged. The use of exogenous electron donors diphenyl carbazide (DPC) and NH(2)OH actually improved the photosynthetic efficiency of shade leaves, and the same effect was found when PSII electron transport activity was measured as O(2) evolution. Altogether, these results seem to indicate lesser photosynthetic efficiency in shade leaves, probably an impairment on the donor side of the PSII. At the same time, analysis by SDS-PAGE revealed differences in the polypeptide composition of the thylakoid membranes of sun and shade leaves: the bands corresponding to 23 kDa, 28-25 kDa and 33 kDa polypeptides were less intense in the thylakoid membranes extracted from shade leaves. These results were further confirmed by an immunological study showing that the content of the 33 kDa protein, corresponding to the extrinsic PSII protein PsbO, was significantly diminished in shade leaves. The high plasticity of cypress leaves appears to be an advantageous trait in the plant's response to variations in environmental conditions, including global change. Implications for the management of this Mediterranean species at the northern edge of its distribution are discussed.

The genome of the domesticated apple (Malus × domestica Borkh.).

We report a high-quality draft genome sequence of the domesticated apple (Malus × domestica). We show that a relatively recent (>50 million years ago) genome-wide duplication (GWD) has resulted in the transition from nine ancestral chromosomes to 17 chromosomes in the Pyreae. Traces of older GWDs partly support the monophyly of the ancestral paleohexaploidy of eudicots. Phylogenetic reconstruction of Pyreae and the genus Malus, relative to major Rosaceae taxa, identified the progenitor of the cultivated apple as M. sieversii. Expansion of gene families reported to be involved in fruit development may explain formation of the pome, a Pyreae-specific false fruit that develops by proliferation of the basal part of the sepals, the receptacle. In apple, a subclade of MADS-box genes, normally involved in flower and fruit development, is expanded to include 15 members, as are other gene families involved in Rosaceae-specific metabolism, such as transport and assimilation of sorbitol.

Lévy processes and stochastic von Bertalanffy models of growth, with application to fish population analysis.

The study of animal growth is a longstanding crucial topic of theoretical biology. In this paper we introduce a new class of stochastic growth models that enjoy two crucial properties: the growth path of an individual is monotonically increasing and the mean length at time t follows the classic von Bertalanffy model. Besides the theoretical development, the models are also tested against a large set of length-at-age data collected on Atlantic herring (Clupea harengus): the mean lengths and variances of the cohorts were directly estimated by least squares. The results show that the use of subordinators can lead to models enjoying interesting properties, in particular able to catch some specific features often observed in fish growth data. The use of subordinators seems to allow for an increased fidelity in the description of fish growth, whilst still conforming to the general parameters of the traditional von Bertalanffy equation.

Genotype-specific regulation of cold-responsive genes in cypress (Cupressus sempervirens L.).

Cold acclimation in plants involves a very complex molecular response, with the regulation of many different genes and metabolic pathways. In this work fifteen cypress (Cupressus sempervirens) genes putatively regulated during cold exposure were isolated and their expression was studied in five cypress genotypes, along 15 days of treatment at 3 degrees C. Treated samples of shoots were collected from four year old cypress seedlings and a subtractive hybridization approach (PCR-Select) was performed after mRNA extraction. Fifteen genes were selected according to sequence similarities after a GenBank search and their expression was studied using Real-time PCR. Among these genes, five (ELIP, aquaporin, dehydrin and two cold-induced proteins) and four (oleosin, chlorophyll a/b-binding protein, oxidoreductase and rubisco activase) resulted respectively up- and down-regulated by the treatment in all tested genotypes. Finally, three genes (metal-binding protein, nodulin-like protein and beta-amylase) showed remarkable different pattern among genotypes. A consistent relationship was found between the cold regulation of the genes studied and their putative function, suggesting the existence of different cold response pathways in cypress. The possible roles of the low temperature-regulated sequences and of the individual expression differences during cypress cold acclimation are proposed and discussed.

cor Gene expression in barley mutants affected in chloroplast development and photosynthetic electron transport.

The expression of several barley (Hordeum vulgare) cold-regulated (cor) genes during cold acclimation was blocked in the albino mutant a(n), implying a chloroplast control on mRNAs accumulation. By using albino and xantha mutants ordered according to the step in chloroplast biogenesis affected, we show that the cold-dependent accumulation of cor14b, tmc-ap3, and blt14 mRNAs depends on plastid developmental stage. Plants acquire the ability to fully express cor genes only after the development of primary thylakoid membranes in their chloroplasts. To investigate the chloroplast-dependent mechanism involved in cor gene expression, the activity of a 643-bp cor14b promoter fragment was assayed in wild-type and albino mutant a(n) leaf explants using transient beta-glucuronidase reporter expression assay. Deletion analysis identified a 27-bp region between nucleotides -274 and -247 with respect to the transcription start point, encompassing a boundary of some element that contributes to the cold-induced expression of cor14b. However, cor14b promoter was equally active in green and in albino a(n) leaves, suggesting that chloroplast controls cor14b expression by posttranscriptional mechanisms. Barley mutants lacking either photosystem I or II reaction center complexes were then used to evaluate the effects of redox state of electron transport chain components on COR14b accumulation. In the mutants analyzed, the amount of COR14b protein, but not the steady-state level of the corresponding mRNA, was dependent on the redox state of the electron transport chain. Treatments of the vir-zb63 mutant with electron transport chain inhibitors showed that oxidized plastoquinone promotes COR14b accumulation, thus suggesting a molecular relationship between plastoquinone/plastoquinol pool and COR14b.

Chromosome regions and stress-related sequences involved in resistance to abiotic stress in Triticeae.

Drought, low temperature and salinity are the most important abiotic stress factors limiting crop productivity. A genomic map of major loci and QTLs affecting stress tolerance in Triticeae identified the crucial role of the group 5 chromosomes, where the highest concentration of QTLs and major loci controlling plant's adaptation to the environment (heading date, frost and salt tolerance) has been found. In addition, a conserved region with a major role in drought tolerance has been localized to the group 7 chromosomes. Extensive molecular biological studies have led to the cloning of many stress-related genes and responsive elements. The expression of some stress-related genes was shown to be linked to stress-tolerant QTLs, suggesting that these genes may represent the molecular basis of stress tolerance. The development of suitable genetic tools will allow the role of stress-related sequences and their relationship with stress-tolerant loci to be established in the near future.