Maria C. Gomez-Jimenez

Molecular and Genetic Characterization of a Non-Climacteric Phenotype in Melon Reveals Two Loci Conferring Altered Ethylene Response in Fruit

Fruit ripening and abscission are associated with an ethylene burst in several melon (Cucumis melo) genotypes. In cantaloupe as in other climacteric fruit, exogenous ethylene can prematurely induce abscission, ethylene production, and ripening. Melon genotypes without fruit abscission or without ethylene burst also exist and are, therefore, non-climacteric. In the nonabscising melon fruit PI 161375, exogenous ethylene failed to stimulate abscission, loss of firmness, ethylene production, and expression of all target genes tested. However, the PI 161375 etiolated seedlings displayed the usual ethylene-induced triple response. Genetic analysis on a population of recombinant cantaloupe Charentais × PI 161375 inbred lines in segregation for fruit abscission and ethylene production indicated that both characters are controlled by two independent loci, abscission layer(Al)-3 and Al-4. The non-climacteric phenotype in fruit tissues is attributable to ethylene insensitivity conferred by the recessive allelic forms from PI 161375. Five candidate genes (two ACO, two ACS, and ERS) that were localized on the melon genetic map did not exhibit colocalization with Al-3 orAl-4.

Analysis of genes involved in L-ascorbic acid biosynthesis during growth and ripening of grape berries.

Recent data indicate the existence of at least three L-ascorbic acid (AsA) biosynthetic pathways in plant cells. Studying their occurrence in different plant organs and species may help to decipher the precise role(s) of AsA in plant cell physiology. In grape berries, AsA is of particular importance since it is known to be the precursor of tartaric acid, an essential component of the grape fruit. The concentration of AsA increases during development of the fruit to reach a maximum at the full ripe stage. We followed the expression of genes related to the various AsA biosynthetic pathways in this plant organ during fruit ontogeny by real time RT-PCR. Among them, a gene (VvGalUR), showing high homology to one from strawberry encoding a D-galacturonate reductase, was up-regulated during fruit ripening in parallel to the AsA content increase. Cloning of the corresponding full length cDNA showed highest similarity to the strawberry gene (FaGalUR). Moreover, VvGalUR gene expression in grape was also up-regulated by high light, a condition that increased AsA content in grape fruits, while none of the genes involved in the other possible biosynthetic pathways analyzed increased their transcript levels. The results are discussed in relation to the presence of several AsA biosynthetic pathways in grape fruits.

Polyamine-induced modulation of genes involved in ethylene biosynthesis and signalling pathways and nitric oxide production during olive mature fruit abscission

After fruit ripening, many fruit-tree species undergo massive natural fruit abscission. Olive (Olea europaea L.) is a stone-fruit with cultivars such as Picual (PIC) and Arbequina (ARB) which differ in mature fruit abscission potential. Ethylene (ET) is associated with abscission, but its role during mature fruit abscission remains largely uncharacterized. The present study investigates the possible roles of ET and polyamine (PA) during mature fruit abscission by modulating genes involved in the ET signalling and biosynthesis pathways in the abscission zone (AZ) of both cultivars. Five ET-related genes (OeACS2, OeACO2, OeCTR1, OeERS1, and OeEIL2) were isolated in the AZ and adjacent cells (AZ–AC), and their expression in various olive organs and during mature fruit abscission, in relation to interactions between ET and PA and the expression induction of these genes, was determined. OeACS2, OeACO2, and OeEIL2 were found to be the only genes that were up-regulated in association with mature fruit abscission. Using the inhibition of ET and PA biosynthesis, it is demonstrated that OeACS2 and OeEIL2 expression are under the negative control of PA while ET induces their expression in AZ–AC. Furthermore, mature fruit abscission depressed nitric oxide (NO) production present mainly in the epidermal cells and xylem of the AZ. Also, NO production was differentially responsive to ET, PA, and different inhibitors. Taken together, the results indicate that PA-dependent ET signalling and biosynthesis pathways participate, at least partially, during mature fruit abscission, and that endogenous NO and 1-aminocyclopropane-1-carboxylic acid maintain an inverse correlation, suggesting an antagonistic action of NO and ET in abscission signalling.

Transcriptomic Events Involved in Melon Mature-Fruit Abscission Comprise the Sequential Induction of Cell-Wall Degrading Genes Coupled to a Stimulation of Endo and Exocytosis

Background Mature-fruit abscission (MFA) in fleshy-fruit is a genetically controlled process with mechanisms that, contrary to immature-fruit abscission, has not been fully characterized. Here, we use pyrosequencing to characterize the transcriptomes of melon abscission zone (AZ) at three stages during AZ-cell separation in order to understand MFA control at an early stage of AZ-activation. Principal Findings The results show that by early induction of MFA, the melon AZ exhibits major gene induction, while by late induction of MFA, melon AZ shows major gene repression. Although some genes displayed similar regulation in both early and late induction of abscission, such as EXT1-EXT4, EGase1, IAA2, ERF1, AP2D15, FLC, MADS2, ERAF17, SAP5 and SCL13 genes, the majority had different expression patterns. This implies that time-specific events occur during MFA, and emphasizes the value of characterizing multiple time-specific abscission transcriptomes. Analysis of gene-expression from these AZs reveal that a sequential induction of cell-wall-degrading genes is associated with the upregulation of genes involved in endo and exocytosis, and a shift in plant-hormone metabolism and signaling genes during MFA. This is accompanied by transcriptional activity of small-GTPases and synthaxins together with tubulins, dynamins, V-type ATPases and kinesin-like proteins potentially involved in MFA signaling. Early events are potentially controlled by down-regulation of MADS-box, AP2/ERF and Aux/IAA transcription-factors, and up-regulation of homeobox, zinc finger, bZIP, and WRKY transcription-factors, while late events may be controlled by up-regulation of MYB transcription-factors. Significance Overall, the data provide a comprehensive view on MFA in fleshy-fruit, identifying candidate genes and pathways associated with early induction of MFA. Our comprehensive gene-expression profile will be very useful for elucidating gene regulatory networks of the MFA in fleshy-fruit.

Transcriptome Analysis of Mature Fruit Abscission Control in Olive

Mature fruit abscission (MFA) is a genetically controlled process, through poorly characterized mechanisms in fleshy fruit that include extensive transcriptional changes. While global transcriptome analyses have been used to investigate immature fruit abscission in fleshy fruit, no global gene expression changes specific to MFA have been described. Here we use pyrosequencing to characterize the transcriptomes of the olive abscission zone (AZ) during cell separation in order to understand MFA control at the stage of AZ activation. Analysis of gene expression from these AZs reveals that membrane microdomains involving sterols/sphingolipids and remorins together with signaling proteins are potentially involved in MFA. This is accompanied by gene activity related to sphingolipid turnover, suggesting potentially the involvement of long-chain base metabolism in regulating MFA. Activation of vesicle trafficking involving small GTPases is probably required for cell wall modifications during abscission. Analysis of transcription factors indicates that most members of the MYB and bZIP families are abundantly represented in the fruit AZ, and is consistent with a model by which most of the key transcription factors during abscission may regulate downstream processes mostly related to ABA. The data provide the first thorough analysis available for a comprehensive picture of the array of cellular responses controlled by gene expression that lead to MFA in fleshy fruit.

Regulation of polyamine metabolism and biosynthetic gene expression during olive mature-fruit abscission.

Exogenous ethylene and some inhibitors of polyamine biosynthesis can induce mature-fruit abscission in olive, which could be associated with decreased nitric oxide production as a signaling molecule. Whether H2O2 also plays a signaling role in mature-fruit abscission is unknown. The possible involvement of H2O2 and polyamine in ethylene-induced mature-fruit abscission was examined in the abscission zone and adjacent cells of two olive cultivars. Endogenous H2O2 showed an increase in the abscission zone during mature-fruit abscission, suggesting that accumulated H2O2 may participate in abscission signaling. On the other hand, we followed the expression of two genes involved in the polyamine biosynthesis pathway during mature-fruit abscission and in response to ethylene or inhibitors of ethylene and polyamine. OeSAMDC1 and OeSPDS1 were expressed differentially within and between the abscission zones of the two cultivars. OeSAMDC1 showed slightly lower expression in association with mature-fruit abscission. Furthermore, our data show that exogenous ethylene or inhibitors of polyamine encourage the free putrescine pool and decrease the soluble-conjugated spermidine, spermine, homospermidine, and cadaverine in the olive abscission zone, while ethylene inhibition by CoCl2 increases these soluble conjugates, but does not affect free putrescine. Although the impact of these treatments on polyamine metabolism depends on the cultivar, the results confirm that the mature-fruit abscission may be accompanied by an inhibition of S-adenosyl methionine decarboxylase activity, and the promotion of putrescine synthesis in olive abscission zone, suggesting that endogenous putrescine may play a complementary role to ethylene in the normal course of mature-fruit abscission.

Genetic diversity assessment in Portugal accessions of Olea europaea by RAPD markers

Eighty seven olive (Olea europaea ssp. sativa L.) cultivar accessions from Portugal were characterized by means of randomly amplified polymorphic DNA (RAPD) markers. Of the 11 arbitrary 10-mer primers tested a total of 92 polymorphic bands were obtained, representing 87.6 % of the total amplification products. Twenty nine different genotypes were clearly discriminated. Differences were not found among the amplification profiles from different individuals of the same cultivar. All the genotypes could be identified by the combination of three primers: OPR-1, OPK-14 and OPA-1, seven genotype-specific markers being detected. Genetic relationships were estimated by the unweighted pair-group method with arithmetic averaging (UPGMA). The genetic analysis of the results showed a gradual distance between the various cultivars, making it difficult to identify well-differentiated phylogenetic groups, although two clusters were distinguishable with 35 % similarity, in addition to three independent branches with lower similarity: Galega, Tentilheira and Redondal. The dendrogram reflect some relationships for most of the cultivars according to the use of the fruit and ecological adaptation.

Mature fruit abscission is associated with up-regulation of polyamine metabolism in the olive abscission zone.

This study investigates whether, and how, polyamines (PAs) are involved in mature fruit abscission of olive (Olea europaea L.). Physiological abscission was studied in relation to the activation of the abscission zone (AZ), located between fruit and peduncle, from two olive cultivars where the breakstrength profiles and the scanning electron micrographs illustrated differences in the abscission program, under natural conditions, of mature fruit. The localization and activities of diamine oxidase (DAO), polyamine oxidase (PAO) and PA biosynthetic enzymes, together with PA content were investigated in the fruit AZ during development and abscission. The activities of arginine decarboxylase and S-adenosyl-l-methionine decarboxylase in the fruit AZ were significantly increased and decreased, respectively, by mature fruit abscission, in good agreement with the rise in free putrescine (Put), and content in uncommon PAs there, such as homospermidine and cadaverine, while no significant differences in free spermidine (Spd) and spermine (Spm) contents were detected. By contrast, an abscission-induced decrease was noted in the contents of insoluble conjugated Put, Spd and Spm. The maximum activity of PAO coincided with the maximum content of Spd and Spm, and it was localized mainly in parenchyma cells of pith, while DAO was present mainly in parenchyma cells of pith and cortex as well as at the base of the vascular tissue. These results suggest a clear correlation between the PA distribution and mature fruit abscission. The regulation of PA metabolism is discussed in relation to mature fruit abscission.

Tissue-specific expression of olive S-adenosyl methionine decarboxylase and spermidine synthase genes and polyamine metabolism during flower opening and early fruit development.

Polyamines (PAs) are required for cell growth and cell division in eukaryotic and prokaryotic organisms. The present study is aimed at understanding the developmental regulation of PA biosynthesis and catabolism during flower opening and early fruit development in relation to fruit size and shape. Two full-length cDNA clones coding for S-adenosyl methionine decarboxylase (SAMDC) and spermidine synthase (SPDS) homologs, key steps in the PA biosynthesis pathway, in the stone-fruit of olive (Olea europaea L.) were identified and the spatial and temporal organization of these genes were described. In olive flowers, OeSAMDC gene transcripts were highly expressed in ovary wall, placenta and ovules, while OeSPDS transcript was confined to the ovules of ovary at anthesis stage. A correlation was detected between the SAMDC enzyme activity/accumulation transcript and spermidine (Spd) and spermine (Spm) levels during flower opening, implying that the synthesis of decarboxylated SAM might be a rate-limiting step in Spd and Spm biosynthesis. OeSAMDC and OeSPDS transcripts were co-expressed in fruit mesocarp and exocarp at all developmental stages analyzed as well as in nucellus, integuments and inner epidermis tissues of fertilized ovules. In contrast, the OeSAMDC and OeSPDS genes had different expression patterns during early fruit development. The results provide novel data about localization of PA biosynthesis gene transcripts, indicating that transcript levels of PA biosynthesis genes are all highly regulated in a developmental and tissue-specific manner. The differences between the two olive cultivars in the fruit size in relation to the differences in the accumulation patterns of PAs are discussed.

Comparative transcriptional profiling analysis of olive ripe-fruit pericarp and abscission zone tissues shows expression differences and distinct patterns of transcriptional regulation

BackgroundIn fleshy fruit, abscission of fully ripe fruit is a process intimately linked to the ripening process. In many fruit-tree species, such as olive (Olea europaea L. cv. Picual), there is a coupling of the full ripening and the activation of the abscission-zone (AZ). Although fully ripe fruit have marked physiological differences with respect to their AZs, dissimilarities in gene expression have not been thoroughly investigated. The present study examines the transcriptome of olive fruit and their AZ tissues at the last stage of ripening, monitored using mRNA-Seq.ResultsRoche-454 massive parallel pyrosequencing enabled us to generate 397,457 high-quality EST sequences, among which 199,075 were from ripe-fruit pericarp and 198,382 from AZ tissues. We assembled these sequences into 19,062 contigs, grouped as 17,048 isotigs. Using the read amounts for each annotated isotig (from a total of 15,671), we identified 7,756 transcripts. A comparative analysis of the transcription profiles conducted in ripe-fruit pericarp and AZ evidenced that 4,391 genes were differentially expressed genes (DEGs) in fruit and AZ. Functional categorization of the DEGs revealed that AZ tissue has an apparently higher response to external stimuli than does that of ripe fruit, revealing a higher expression of auxin-signaling genes, as well as lignin catabolic and biosynthetic pathway, aromatic amino acid biosynthetic pathway, isoprenoid biosynthetic pathway, protein amino acid dephosphorylation, amino acid transport, and photosynthesis. By contrast, fruit-enriched transcripts are involved in ATP synthesis coupled proton transport, glycolysis, and cell-wall organization. Furthermore, over 150 transcripts encoding putative transcription-factors (TFs) were identified (37 fruit TFs and 113 AZ TFs), of which we randomly selected eight genes and we confirmed their expression patterns using quantitative RT-PCR.ConclusionWe generated a set of EST sequences from olive fruit at full ripening, and DEGs between two different olive tissues, ripe fruit and their AZ, were also identified. Regarding the cross-talk between fruit and AZ, using qRT-PCR, we confirmed a set of TF genes that were differentially expressed, revealing profiles of expression that have not previously been reported, this offering a promising beginning for studies on the different transcription regulation in such tissues.