Angelo Ramina

Signaling Pathways Mediating the Induction of Apple Fruitlet Abscission

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.

Cellulase and polygalacturonase involvement in the abscission of leaf and fruit explants of peach

Ethylene-induced abscission in leaf and fruit explants of peach involves different enzymes. In leaves abscission is accompanied by increased occurrence of cellulase forms differing in isoelectric point (pI 6.5 and 9.5). A polypeptide with a molecular mass of 51 kDa gives in a western blot a strong cross-reaction with an antibody raised against a maturation cellulase from avocado fruit. Cellulase activity is also found in abscising fruit explants but the amount is very low compared to that of the leaf explants. A northern analysis with a cellulase clone from avocado reveals the presence of two hybridizing mRNAs with a size of 2.2 kb and 1.8 kb, respectively. The steady-state level of the 2.2 kb mRNA is significantly increased by treatment with ethylene.Polygalacturonases are not detected in abscising leaves, but are strongly induced by ethylene in fruit explants. Of the three forms found, two are exopolygalacturonases while the third is an endoenzyme. Ethylene activates preferentially the endoenzyme and the basic exoenzyme but depresses the acid exopolygalacturonases. A northern analysis carried out with a cDNA coding for tomato endopolygalacturonase shows hybridization only with one endopolygalacturonase mRNA from in the fruit abscission zone. Treatment with ethylene causes an increase in the steady-state level of this mRNA. The differences in the enzyme patterns observed in fruit and leaf abscission zones and a differential enzyme induction suggest the feasibility to regulate fruit abscission in peach with the aid of antisense RNA genes.

Computational annotation of genes differentially expressed along olive fruit development

BackgroundOlea europaea L. is a traditional tree crop of the Mediterranean basin with a worldwide economical high impact. Differently from other fruit tree species, little is known about the physiological and molecular basis of the olive fruit development and a few sequences of genes and gene products are available for olive in public databases. This study deals with the identification of large sets of differentially expressed genes in developing olive fruits and the subsequent computational annotation by means of different software.ResultsmRNA from fruits of the cv. Leccino sampled at three different stages [i.e., initial fruit set (stage 1), completed pit hardening (stage 2) and veraison (stage 3)] was used for the identification of differentially expressed genes putatively involved in main processes along fruit development. Four subtractive hybridization libraries were constructed: forward and reverse between stage 1 and 2 (libraries A and B), and 2 and 3 (libraries C and D). All sequenced clones (1,132 in total) were analyzed through BlastX against non-redundant NCBI databases and about 60% of them showed similarity to known proteins. A total of 89 out of 642 differentially expressed unique sequences was further investigated by Real-Time PCR, showing a validation of the SSH results as high as 69%. Library-specific cDNA repertories were annotated according to the three main vocabularies of the gene ontology (GO): cellular component, biological process and molecular function. BlastX analysis, GO terms mapping and annotation analysis were performed using the Blast2GO software, a research tool designed with the main purpose of enabling GO based data mining on sequence sets for which no GO annotation is yet available. Bioinformatic analysis pointed out a significantly different distribution of the annotated sequences for each GO category, when comparing the three fruit developmental stages. The olive fruit-specific transcriptome dataset was used to query all known KEGG (Kyoto Encyclopaedia of Genes and Genomes) metabolic pathways for characterizing and positioning retrieved EST records. The integration of the olive sequence datasets within the MapMan platform for microarray analysis allowed the identification of specific biosynthetic pathways useful for the definition of key functional categories in time course analyses for gene groups.ConclusionThe bioinformatic annotation of all gene sequences was useful to shed light on metabolic pathways and transcriptional aspects related to carbohydrates, fatty acids, secondary metabolites, transcription factors and hormones as well as response to biotic and abiotic stresses throughout olive drupe development. These results represent a first step toward both functional genomics and systems biology research for understanding the gene functions and regulatory networks in olive fruit growth and ripening.

Peach fruit ripening and quality in relation to picking time, and hypoxic and high CO2 short-term postharvest treatments

Peach fruits (Prunus persica L. Batsch, cv Springcrest) were harvested at two ripening stages (flesh firmness of 60 N, first harvest, and 45 N, second harvest) and maintained at 20°C in air (control) or for 24 and 48 h in streams of ultra low (<1%) oxygen (ULO) or high (30%) CO2 concentration and then transferred to air for up to 8 days. The decline in flesh firmness was strongly reduced by ULO and CO2 treatments in fruits of both harvests, although the effect was stronger in fruits picked earlier in which ethylene biosynthesis remained at the basal level. In fruits of the second harvest, endo β-1,4-glucanase (EGase) activity was lower in ULO- and CO2-treated fruits than in control fruits at the end of the 24 h treatment and the following two days in air. Acetaldehyde (AA) gradually accumulated in control fruit and the highest concentrations were detected during late ripening. Both treatments induced a strong accumulation of AA but, with the exception of the 24 and 48 h CO2 treatments performed on fruits of the second harvest, a decrease in AA content was observed when the fruits were transferred to air. A slight increase in ethanol (EtOH) was found throughout the ripening process in control fruits; ULO and CO2 strongly stimulated EtOH production. When fruits were transferred to air, EtOH concentration declined rapidly. Alcohol dehydrogenase (ADH) activity significantly increased in control fruit only in the late stages of ripening. Greater ADH activity was found throughout the experimental period in fruits of the first harvest treated for 24 h in ULO and CO2, whereas, at day 8, control and treated fruits of the second harvest showed similar ADH activity values. Hypoxic and, to a lesser extent, CO2-enriched atmospheres stimulated Adh gene expression.

Genetic and environmental factors affecting allergen-related gene expression in apple fruit (Malus domestica L. Borkh)

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.

Cell enlargement and cell separation during peach fruit development

The increase in cell size and the activities of cell wall lytic enzymes during fruit growth supported by the mesocarp cell enlargement have been studied in peach of the freestone cultivar Redhaven. Four growth stages (S1-S4) in peach fruit were observed. Cell enlargement and formation of intercellular spaces in the mesocarp were directly correlated with the increase in fruit diameter. Cellulase and exopolygalacturonase showed the highest activity during the first stage of fast fruit growth (S1), while the endopolygalacturonase activity increased during the last growth stage (S4), when the fruit reached its final size and ripening started.

Cell wall hydrolases and amylase in kiwifruit softening

Abstract The activities of amylase, β-galactosidase (β-GAL), polygalacturonase (PG), and endo-1,4-β-glucanase (EG), and ethylene evolution, were measured during ripening in kiwifruit ( Actinidia deliciosa (A.Chev.) (C.F. Liang et A.R. Ferguson var deliciosa, cv. Hayward). Fruit, harvested at firmness values (FV) of 65 N and a soluble solid (SS) content of 9%, were maintained in air at 20 °C for 40 days, until they reached the edible stage. Fruit firmness decreased throughout the experimental period and the lowest FV (about 5 N) was reached after 38 days of storage. SS content increased rapidly to about 13% within 15 days, then the rate of accumulation slowed and the SS content of 14.1% was reached after 33 days. Ethylene climacteric was a late event occurring at a FV of 10 N. The highest amylase activity was measured at harvest. During storage it declined, although a slight rise was detected at 33 days. β-GAL activity was very low at the beginning of storage and increased throughout the experimental period, while PG activity was detected only after the fruit FV was below 10 N. EG activity decreased within the first three days of storage, then increased and peaked 15 days after harvest. Later, EG activity remained low but increased again at the end of the storage period. Application of propylene (500 ppm) to fruit that had softened to 30 N in air stimulated fruit softening and EG activity, and induced the accumulation of an EG-related peptide: the other enzymes appeared not to be affected by the gas.

A microarray approach to identify genes involved in seed-pericarp cross-talk and development in peach

BackgroundField 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.ResultsA 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.ConclusionsIn 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).

Structural and biochemical aspects of peach fruit abscission (Prunus persica L. Batsch.).

The physiological drop of immature fruits was studied in relation to the activation of the abscission zone located between the fruit and the receptacle. Light- and electron-microscopy observations demonstrated that this zone consisted of two types of parenchymatous cells: in the distal region, closer to the fruit, were groups of small thick-walled cells with few intercellular spaces; in the proximal region, closer to the pedicel, the stillgrouped cells were larger, polyphenolic-rich, and thick-walled but with many wide intercellular spaces. Separation of the fruit occurred by dissolution of the middle lamella of the cells of this zone followed by an increase in the size of the intercellular spaces. Lysis of the middle lamella began at the corners of the cells and spread from there across the entire wall surface. Structural changes were paralleled by an increase in soluble proteins, endo-cellulase and exo-polygalacturonase activity. Isoelectric focusing indicated that both enzymes were present as isoenzymes whose patterns were affected by embryoctomy and 2-chloroethylphosphonic acid treatments.

Fruitlet abscission: A cDNA-AFLP approach to study genes differentially expressed during shedding of immature fruits reveals the involvement of a putative auxin hydrogen symporter in apple (Malus domestica L. Borkh)

Apple Malus X domestica fruitlet abscission is preceded by a stimulation of ethylene biosynthesis and a gain in sensitivity to the hormone. This phase was studied by a differential screening carried out by cDNA-AFLP in abscising (AF) and non-abscising (NAF) fruitlet populations. Fifty-three primer combinations allowed for the isolation of 131, 66 and 30 differentially expressed bands from cortex, peduncle and seed, respectively. All sequences were then classified as up- or down-regulated by comparing the profile in AFs and NAFs. Almost all of these sequences showed significant homology to genes encoding proteins with known or putative function. The gene ontology analysis of the TDFs isolated indicated a deep change in metabolism, plastid and hormonal status, especially auxin. Furthermore, some common elements between abscission and senescence were identified. The isolation of the full length of one of these TDFs allowed for the identification of a gene encoding an auxin hydrogen symporter (MdAHS). Bioinformatic analysis indicated that the deduced protein shares some features with other auxin efflux carriers, which include PINs. Nevertheless the 3D structure pointed out substantial differences and a conformation largely dissimilar from canonical ion transporters. The expression analysis demonstrated that this gene is regulated by light and development but not affected by ethylene or auxin.