[Show abstract][Hide abstract] ABSTRACT: Abscission is the regulated process of detachment of an organ from a plant. In apple the abscission of fruits occurs during their early development to control the fruit load depending on the nutritional state of the plant. In order to control production and obtain fruits with optimal market qualities, the horticultural procedure of thinning is performed to further reduce the number of fruitlets. In this study we have conducted a transcriptomic profiling of seeds from two different types of fruitlets, according to size and position in the fruit cluster. Transcriptomic profiles of central and lateral fruit seeds were obtained by RNAseq. Comparative analysis was performed by the functional categorization of differentially expressed genes by means of Gene Ontology (GO) annotation of the apple genome. Our results revealed the overexpression of genes involved in responses to stress, hormone biosynthesis and also the response and/or transport of auxin and ethylene. A smaller set of genes, mainly related to
PLoS ONE 03/2015; 10(3-3):e0120503. DOI:10.1371/journal.pone.0120503 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Apple research has undergone great improvements in the last years, in both quantitative and qualitative terms. Huge amount of data are now available, especially as far as the early development and the ripening phase are concerned. Moreover, the recent release of the apple genome sequence is significantly speeding up research, allowing on one hand to shed light on the most critical aspects of fruit development with almost immediate practical implications and, on the other hand, to identify new molecular markers that will improve the future breeding programs. In this context, apple is being increasingly considered as a model for fruit development studies, although many gaps still exist in apple research. These gaps are being filled by coupling the next generation high-throughput technologies with new physiological approaches, aimed at achieving both new basic knowledge and innovative tools to improve the final quality of the fruit. In this review, the available information on the regulatory aspects of apple fruit development will be reported and discussed in the light of the future perspectives of apple research.
[Show abstract][Hide abstract] ABSTRACT: A ripening specific bZIP gene of peach was studied by ectopically expressing it in tomato. Two lines, with either a mild or a strong phenotype, respectively, were analyzed in detail. Transgenic fruit morphology was normal, yet the time spent to proceed through the various ripening stages was longer compared to wild type. In agreement with this finding the transgenic berries produced less ethylene, and also had a modified expression of some ripening-related genes that was particularly evident in berries with a strong phenotype. In particular, in the latter fruits polygalacturonase and lipoxygenase genes, but also genes coding for transcription factors (TFs) important for tomato ripening (i.e. TAGL1, CNR, APETALA2a, NOR) did not show the expected decreased expression in the red berries. As regards the RIN gene, its expression continued to increase in both mild and strong lines, and this is in agreement with the dilated ripening times. Interestingly, a metabolomic analysis of berries at various stages of ripening showed that the longer time spent by the transgenic berries to proceed from a stage to another was not due to a slackened metabolism. In fact, the differences in amount of stage-specific marker metabolites indicated that the transgenic berries had a very active metabolism. Therefore, the dilated ripening and the enhanced metabolism of the berries over-expressing the bZIP gene suggest that such gene might regulate ripening by acting as a pacemaker for some of the ripening metabolic pathways.
[Show abstract][Hide abstract] ABSTRACT: Apple (Malus domestica L. Borkh) fruitlet abscission represents an interesting model system to study the early phases of the shedding process, during which major transcriptomic changes and metabolic rearrangements occur within the fruit. In apple, the drop of fruits at different positions within the cluster can be selectively magnified through chemical thinners, such as benzyladenine and metamitron, acting as abscission enhancers. In the present research, different abscission potentials were obtained within the apple fruitlet population by means of the above cited thinners. A metabolomic study was conducted on the volatile organic compounds (VOCs) emitted by abscising fruitlets, allowing to identify isoprene as an early marker of abscission induction. A strong correlation was also observed between isoprene production and abscisic acid (ABA) levels in the fruit cortex, which were shown to increase in abscising fruitlets with respect to non-abscising ones. Transcriptomic evidence indicated that abscission-related ABA is biologically active and its increased biosynthesis is associated with the induction of a specific ABA-responsive NCED (9-cis-epoxycarotenoid dioxygenase) gene. According to a hypothetical model, ABA may transiently cooperate with other hormones and secondary messengers in the generation of an intra-fruit signal leading to the downstream activation of the abscission zone. The shedding process therefore appears to be triggered by multiple interdependent pathways, whose fine regulation, exerted within a very short temporal window by both endogenous and exogenous factors, determines the final destiny of the fruitlets.
[Show abstract][Hide abstract] ABSTRACT: Specific bioinformatic analyses were carried out in peach and olive, starting with the ab initio identification of genes responsible for flower differentiation (‘ABCDE’ genes). The peach MADS-box genes involved in this process, already identified and partially characterized, were used to validate the bioinformatic approach herein adopted using the recent release of the genome. A double experimental approach including Hidden Markov Models and Blast was set up by exploiting all available genes with function validated not only in model plants but also in crop species. Since the genome sequence of olive is not available, a 454 collection recently generated from flower buds at different developmental stages was used as a target database. In peach, the list of candidates was implemented with further members, such as four AP2- and two AP3-like genes, with a putative ‘A’ and ‘B’ function, respectively. In olive, a higher number of candidates was identified compared to peach, probably due both to the larger size and the polyploid origin of its genome, as well as to the presence of different alleles of the same gene (being most loci heterozygous in olive and homozygous in peach). Phylogenetic analyses pointed out homogeneous clusters in which candidates of the same class group together with proteins already characterized in model species (i.e. Arabidopsis and Antirrhinum). Expression analyses of candidates are currently in progress in order to assess the organ specificity and the timing of expression.
[Show abstract][Hide abstract] ABSTRACT: Background
Auxins act as repressors of ripening inception in grape (véraison), while ethylene and abscisic acid (ABA) play a positive role as inducers of the syndrome. Despite the increasing amount of information made available on this topic, the complex network of interactions among these hormones remains elusive. In order to shed light on these aspects, a holistic approach was adopted to evaluate, at the transcriptomic level, the crosstalk between hormones in grape berries, whose ripening progression was delayed by applying naphtalenacetic acid (NAA) one week before véraison.
The NAA treatment caused significant changes in the transcription rate of about 1,500 genes, indicating that auxin delayed grape berry ripening also at the transcriptional level, along with the recovery of a steady state of its intracellular concentration. Hormone indices analysis carried out with the HORMONOMETER tool suggests that biologically active concentrations of auxins were achieved throughout a homeostatic recovery. This occurred within 7 days after the treatment, during which the physiological response was mainly unspecific and due to a likely pharmacological effect of NAA. This hypothesis is strongly supported by the up-regulation of genes involved in auxin conjugation (GH3-like) and action (IAA4- and IAA31-like). A strong antagonistic effect between auxin and ethylene was also observed, along with a substantial ‘synergism’ between auxins and ABA, although to a lesser extent.
This study suggests that, in presence of altered levels of auxins, the crosstalk between hormones involves diverse mechanisms, acting at both the hormone response and biosynthesis levels, creating a complex response network.
[Show abstract][Hide abstract] ABSTRACT: Humic substances (HS), as component of soil organic matter, have been widely studied. Their positive effects on plant physiology have been further demonstrated, but there are no clear evidences about the direct as well as indirect mechanisms regulating these processes.In this paper a transcriptomic approach based on the detection of cDNA-AFLP markers was used to identify candidate genes potentially involved in the regulation of the response to HS in Arabidopsis thaliana.Using 160 primer combinations, the cDNA-AFLP enabled to identify 133 genes putatively involved in plant–HS interaction. Sequence analysis and Gene Ontology classification indicated that a large number of genes involved in developmental and metabolic processes, as well as in transcription regulation or RNA metabolism were identified as HS-regulated. Real-Time PCR analyses confirmed transcription levels of 32 HS-regulated genes.This study demonstrates that HS exert their effects on plant physiology by means of complex transcriptional networks. From the overall transcriptomic results we may hypothesize that HS exert their function through a multifaceted mechanism of action, partially connected to their well demonstrated auxin activity, but involving also IAA-independent signalling pathways.
[Show abstract][Hide abstract] ABSTRACT: Field observations and a few physiological studies have demonstrated that peach embryogenesis and fruit development are tightly coupled. In fact, attempts to stimulate parthenocarpic fruit development by means of external tools have failed. Moreover, physiological disturbances during early embryo development lead to seed abortion and fruitlet abscission. Later in embryo development, the interactions between seed and fruit development become less strict. As there is limited genetic and molecular information about seed-pericarp cross-talk and development in peach, a massive gene approach based on the use of the μPEACH 1.0 array platform and quantitative real time RT-PCR (qRT-PCR) was used to study this process.
A comparative analysis of the transcription profiles conducted in seed and mesocarp (cv Fantasia) throughout different developmental stages (S1, S2, S3 and S4) evidenced that 455 genes are differentially expressed in seed and fruit. Among differentially expressed genes some were validated as markers in two subsequent years and in three different genotypes. Seed markers were a LTP1 (lipid transfer protein), a PR (pathogenesis-related) protein, a prunin and LEA (Late Embryogenesis Abundant) protein, for S1, S2, S3 and S4, respectively. Mesocarp markers were a RD22-like protein, a serin-carboxypeptidase, a senescence related protein and an Aux/IAA, for S1, S2, S3 and S4, respectively.The microarray data, analyzed by using the HORMONOMETER platform, allowed the identification of hormone-responsive genes, some of them putatively involved in seed-pericarp crosstalk. Results indicated that auxin, cytokinins, and gibberellins are good candidates, acting either directly (auxin) or indirectly as signals during early development, when the cross-talk is more active and vital for fruit set, whereas abscisic acid and ethylene may be involved later on.
In this research, genes were identified marking different phases of seed and mesocarp development. The selected genes behaved as good seed markers, while for mesocarp their reliability appeared to be dependent upon developmental and ripening traits. Regarding the cross-talk between seed and pericarp, possible candidate signals were identified among hormones.Further investigations relying upon the availability of whole genome platforms will allow the enrichment of a marker genes repertoire and the elucidation of players other than hormones that are involved in seed-pericarp cross-talk (i.e. hormone peptides and microRNAs).
[Show abstract][Hide abstract] ABSTRACT: Mob1 genes are primarily involved in the cell cycle progression and mitosis exit in yeasts and animals. The function of a Mob1-like gene (At5g45550) from Arabidopsis thaliana was investigated using RNAi and immunological staining. AtMob1-like RNAi silenced lines showed a reduced radial expansion of the inflorescence stem and a reduced elongation zone of the primary root. Morphological features of plant organs were accompanied by a reduction in cell size. The fertility of AtMob1-like RNAi silenced lines was very low as seed production was strongly reduced. About 2% of the progeny of AtMob1-like RNAi silenced plants were tetraploid. The female and male sporogenesis was affected differentially. The ovules developed irregularly and one third of the megaspores and embryo sacs degenerated prematurely. Up to 20% of the ovules produced binucleated megaspores that failed to develop further, being their degeneration likely accompanied with a delayed programmed cell death. The anthers produced about 30% of aborted pollen grains, showing also a strong variation in their size. Together, the results show that Arabidopsis MOB1-like is required to regulate cell expansion and cell division, presumably by affecting the mitotic as well as the meiotic cell cycle.
[Show abstract][Hide abstract] ABSTRACT: Apple (Malus × domestica) represents an interesting model tree crop for studying fruit abscission. The physiological fruitlet drop occurring in this species can be easily magnified by using thinning chemicals, such as benzyladenine (BA), to obtain fruits with improved quality and marketability. Despite the economic importance of this process, the molecular determinants of apple fruitlet abscission are still unknown. In this research, BA was used to obtain fruitlet populations with different abscission potentials to be analyzed by means of a newly released 30K oligonucleotide microarray. RNAs were extracted from cortex and seed of apple fruitlets sampled over a 4-d time course, during which BA triggers fruit drop, and used for microarray hybridization. Transcriptomic profiles of persisting and abscising fruitlets were tested for statistical association with abscission potential, allowing us to identify molecular signatures strictly related to fruit destiny. A hypothetical model for apple fruitlet abscission was obtained by putting together available transcriptomic and metabolomic data. According to this model, BA treatment would establish a nutritional stress within the tree that is primarily perceived by the fruitlet cortex whose growth is blocked by resembling the ovary growth inhibition found in other species. In weaker fruits, this stress is soon visible also at the seed level, likely transduced via reactive oxygen species/sugar and hormones signaling cross talk, and followed by a block of embryogenesis and the consequent activation of the abscission zone.
[Show abstract][Hide abstract] ABSTRACT: The fruits of Rosaceae species may frequently induce allergic reactions in both adults and children, especially in the Mediterranean area. In peach, true allergens and cross-reactive proteins may cause hypersensitive reactions involving a wide diversity of symptoms. Three known classes of allergenic proteins, namely, Pru p 1, Pru p 3, and Pru p 4, have been reported to be mostly involved, but an exhaustive survey of the proteins determining the overall allergenic potential, their biological functions, and the factors affecting the expression of the related genes is still missing. In the present study, the expression profiles of some selected genes encoding peach allergen isoforms were studied during fruit growth and development and upon different fruit load and light radiation regimens. The results indicate that the majority of allergen-encoding genes are expressed at their maximum during the ripening stage, therefore representing a potential risk for peach consumers. Nevertheless, enhancing the light radiation and decreasing the fruit load achieved a reduction of the transcription rate of most genes and a possible decrease of the overall allergenic potential at harvest. According to these data, new growing practices could be set up to obtain hypoallergenic peach fruits and eventually combined with the cultivation of hypoallergenic genotypes to obtain a significant reduction of the allergenic potential.
Journal of Agricultural and Food Chemistry 01/2009; 57(2):724-34. DOI:10.1021/jf802709k · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: SUMMARY Apples can cause allergic reactions in the worldwide population because of the presence of four classes of allergens (Mal d 1, Mal d 2, Mal d 3, and Mal d 4), and their cross-reactivity with sensitising allergens in other species. Knowledge of the factors that affect the allergenic potential of apples would provide important information to apple growers and consumers for the adoption of agronomic practices to decrease the allergenic potential, and for the consumption of fruits with reduced levels of allergens. Expression studies on apple genes that encode allergens were performed by means of real-time PCR. Samples were collected from fruit in different trials set up to assess the effects of shading, elevation, storage, and water stress on the expression of apple allergen genes. Shading, elevation, and storage significantly affected the transcription of all genes encoding allergens, whereas water stress had only a slight influence on the expression of the Mal d 4 family of genes. The implications of these results for growers and consumers are critically discussed.
Journal of Horticultural Science and Biotechnology 01/2009; 84. · 0.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: l-Aminoethoxyvinylglycine (AVG) and 1-naphthylacetic acid (NAA) are known to affect preharvest fruit drop, fruit quality and
fruit maturation in Golden Delicious apples (Malus×domestica Borkh). Experiments were carried out on GD/M9 trees treated at three different developmental stages (41, 28 and 17days before
the beginning of the commercial harvest) with AVG and NAA. Both chemicals significantly reduced fruit drop without significantly
affecting the fruit weight. Background colour development and ripening were both delayed by AVG, whereas NAA significantly
enhanced yellowing without affecting the evolution of ripening. Ethylene evolution and transcription profiles of genes involved
in ethylene biosynthesis (MdACS1 and MdACO1) and action (MdETR1, MdERS1 and MdCTR1) were monitored in cortex from the date of the first treatment until the beginning of fruit drop in the control trees (21days
after the beginning of commercial harvest). AVG blocked or efficiently reduced the ethylene evolution. This effect was paralleled
by a down-regulation of MdACS1, MdACO1, MdETR1 and MdERS1. NAA at the second and third date of application enhanced the onset of ethylene evolution, although, at the end of the experiment,
no difference was found between control and treated fruits. The chemical applied in the first date significantly down-regulated
the transcription of the genes at the end of the experiment. MdCTR1 expression, basically unaffected by AVG and NAA, appeared to be transiently down-regulated. The initial down-regulation is
under developmental control, whereas the late regain of transcript accumulation paralleled the ethylene evolution.
[Show abstract][Hide abstract] ABSTRACT: Aspergillus carbonarius is responsible for the majority of mycotoxin contaminations in grapes and its derivatives. Most of A. carbonarius strains are ochratoxin A (OTA) producers, even though at very different levels. This broad variability was used to identify genes whose expression is linked with the ability of producing OTA. A cDNA-AFLP differential display screening was performed in two strains of A. carbonarius, antagonists for the ability of producing OTA, allowing the identification of 119 differentially expressed sequences putatively involved in the regulation of OTA biosynthesis. A likely connection was pointed out between the biosynthesis of the toxin, vegetative growth and sexual/asexual developmental progression, along with common signalling pathways involving G protein and Ca2+/calmodulin dependent phosphorylation and dephoshorylation cascades.
International Journal of Food Microbiology 10/2008; 127(1-2):105-15. DOI:10.1016/j.ijfoodmicro.2008.06.037 · 3.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Freshly consumed apples can cause allergic reactions because of the presence of four classes of allergens, namely, Mal d 1, Mal d 2, Mal d 3, and Mal d 4, and their cross-reactivity with sensitizing allergens of other species. Knowledge of environmental and endogenous factors affecting the allergenic potential of apples would provide important information to apple breeders, growers, and consumers for the selection of hypoallergenic genotypes, the adoption of agronomical practices decreasing the allergenic potential, and the consumption of fruits with reduced amount of allergens. In the present research, expression studies were performed by means of real-time PCR for all the known allergen-encoding genes in apple. Fruit samples were collected from 15 apple varieties and from fruits of three different trials, set up to assess the effect of shadowing, elevation, storage, and water stress on the expression of allergen genes. Principal components analysis (PCA) was performed for the classification of varieties according to gene expression values, pointing out that the cultivars Fuji and Brina were two good hypoallergenic candidates. Shadowing, elevation, and storage significantly affected the transcription of the allergen-encoding genes, whereas water stress slightly influenced the expression of only two genes, in spite of the dramatic effect on both fruit size and vegetative growth of the trees. In particular, shadowing may represent an important cultural practice aimed at reducing apple cortex allergenicity. Moreover, elevation and storage may be combined to reduce the allergenic potential of apple fruits. The possible implications of the results for breeders, growers, and consumers are discussed critically.
Journal of Agricultural and Food Chemistry 08/2008; 56(15):6707-16. DOI:10.1021/jf800813d · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A full-length cDNA encoding a putative high-affinity nitrate transporter (ZmNrt2.2) from maize was isolated and characterised, together with another previously identified transporter (ZmNrt2.1), in terms of phylogenesis, protein structure prediction and regulation of transcript accumulation in response to nitrate and sugar availability. The expression of both genes was evaluated by quantitative and semi-quantitative RT-PCR in response to nitrate and sugar supply and the in planta localisation of mRNA was studied by in situ hybridisation. Data obtained suggested similar genetic evolution and identical transmembrane structure prediction between the two deduced proteins, and differences in both regulation of their expression and mRNA localisation in response to nitrate, leading us to hypothesise a principal role for ZmNRT2.1 in the influx activity and the major involvement of ZmNRT2.2 in the xylem loading process. Our data suggest opposing sugar regulation by ZmNrt2.1 and ZmNrt2.2 transcription in the presence or absence of nitrate and the existence of both hexokinase-dependent and hexokinase-independent transduction mechanisms for the regulation of ZmNrt2.1 and ZmNrt2.2 expression by sugars.
[Show abstract][Hide abstract] ABSTRACT: After 10-year-use of AFLP (Amplified Fragment Length Polymorphism) technology for DNA fingerprinting and mRNA profiling, large repertories of genome- and transcriptome-derived sequences are available in public databases for model, crop and tree species. AFLP marker systems have been and are being extensively exploited for genome scanning and gene mapping, as well as cDNA-AFLP for transcriptome profiling and differentially expressed gene cloning. The evaluation, annotation and classification of genomic markers and expressed transcripts would be of great utility for both functional genomics and systems biology research in plants. This may be achieved by means of the Gene Ontology (GO), consisting in three structured vocabularies (i.e. ontologies) describing genes, transcripts and proteins of any organism in terms of their associated cellular component, biological process and molecular function in a species-independent manner. In this paper, the functional annotation of about 8,000 AFLP-derived ESTs retrieved in the NCBI databases was carried out by using GO terminology.
Descriptive statistics on the type, size and nature of gene sequences obtained by means of AFLP technology were calculated. The gene products associated with mRNA transcripts were then classified according to the three main GO vocabularies. A comparison of the functional content of cDNA-AFLP records was also performed by splitting the sequence dataset into monocots and dicots and by comparing them to all annotated ESTs of Arabidopsis and rice, respectively. On the whole, the statistical parameters adopted for the in silico AFLP-derived transcriptome-anchored sequence analysis proved to be critical for obtaining reliable GO results. Such an exhaustive annotation may offer a suitable platform for functional genomics, particularly useful in non-model species.
Reliable GO annotations of AFLP-derived sequences can be gathered through the optimization of the experimental steps and the statistical parameters adopted. The Blast2GO software was shown to represent a comprehensive bioinformatics solution for an annotation-based functional analysis. According to the whole set of GO annotations, the AFLP technology generates thorough information for angiosperm gene products and shares common features across angiosperm species and families. The utility of this technology for structural and functional genomics in plants can be implemented by serial annotation analyses of genome-anchored fragments and organ/tissue-specific repertories of transcriptome-derived fragments.