Mercedes Gallardo

Structural, physiological and molecular aspects of heterogeneity in seeds: a review

Higher plants have several strategies to perpetuate themselves under adequate ecophysiological conditions. The production of heterogeneous seeds is one such strategy. That is, to ensure the survival of the next generation, an individual plant might produce seeds that are heterogeneous with respect to the extent of dormancy, dispersion and persistence within the seed bank. Heterogeneity can affect not only certain physiological and molecular properties related to seed germination, but also such characteristics as colour, size and shape, parameters commonly used to differentiate morphs within a heterogeneous seed population. In heterogeneous seeds, the above features determine seed behaviour and alter their mechanism of germination. In this work, emphasis is placed on the existence of seed mutants having major alterations in characteristics of the testa and hormonal response. These mutants constitute a valuable tool for elucidating the mechanism of dormancy, germination and perpetuation of seeds. Finally, ontogeny and heterogeneity are reviewed, providing the first data related to the possible hormonal control of heterogeneity in seeds. These results raise the hypothesis that one of the factors triggering differences in germination among heterogeneous seeds may be an alteration in the signalling and action mechanism of ethylene and abscisic acid (ABA).

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.

The heterogeneity of turnip-tops (Brassica rapa) seeds inside the silique affects germination, the activity of the final step of the ethylene pathway, and abscisic acid and polyamine content

The mature silique of turnip-tops (Brassica rapa L. cv. Rapa) contains seeds that are heterogeneous in colour. From these seeds, we have selected three homogeneous lots: black (B), dark brown (DB) and light brown (LB). The dry seeds of these lots contained different levels of free and conjugated 1-aminocyclopropane-1-carboxylic acid (ACC), polyamines (PA) and ABA, the levels of the latter being inversely related to the germinative capacity. The water uptake (WU) rate was much faster in LB seeds than in B. This fact was probably related to the breaking of the seed coat, the speed of which was B >> DB > LB. The ABA, spermidine (Spd) and spermine (Spm) contents decreased in the seeds during germination, whereas the putrescine (Put) levels rose sharply (B > DB > LB). For the first time in seeds, heterogeneity is reported with respect to ethylene sensitivity and synthesis. Whereas exogenous ethylene did not alter the percentage of germination in lot B, germination was higher in DB and LB (LB>> DB) in the presence of ethylene. The final step of the ethylene pathway was altered concomitantly with this change in germinating capacity, affecting the levels of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), ACC, ACC-oxidase (ACO) and ethylene production. The gene BrACO1, recently characterised by us, is expressed differently in the three seed lots, particularly in the LB, where little transcription occurs. Finally, ethylene inhibits Put, Spd and Spm levels at different intensities in the three lots. The results point towards variation in the channelling of ACC towards synthesis of ethylene and / or PA, caused by the heterogeneity.

Involvement of calcium in ACC-oxidase activity from Cicer arietinum seed embryonic axes

Both in vivo and in vitro ACC-oxidase activities as well as ethylene production from embryonic axes of chickpea seeds were strongly inhibited by EGTA, a selective extracellular Ca2+ ion chelator, indicating that the influx of Ca2+ is important for enzymatic activity. EGTA inhibition was restored by exogenous Ca2+. Treatments of embryonic axes with either Verapamil and LaCl3 (both Ca2+ channel blockers) or TMB-8 (an intracellular Ca2+ antagonist) provoked an inhibition of both ACC-oxidase activity and ethylene production. These results suggest an involvement of calcium fluxes and intracellular calcium levels in the activity of the last step of the ethylene biosynthetic pathway, which is, in turn, intimately correlated with germination of Cicer arietinum seeds.

Polyamine contents, ethylene synthesis, and BrACO2 expression during turnip germination

Contents of total free [PA(S)] and conjugated polyamines [PA(SH), PA(PH)] were higher in turnip (Brassica rapa L. cv. Rapa) seeds during imbibition (0–36 h) and radicle protrusion (36–48 h) than during the further growth (10 d). Ethylene production was activated with the protrusion, reaching a maximum at the second day of germination and dropping afterwards. The application of ethrel accelerated radicle emergence but the direct intervention of ethylene in the breaking of the seed coat was not clear from the use of ethylene-biosynthesis inhibitors (CoCl2 and AVG). Finally, in this work the gene BrACO2 was characterized. Although its expression was not detected in seeds through zygotic embryogenesis, it increased concomitantly with the germination process.

Modulation of sphingolipid long-chain base composition and gene expression during early olive-fruit development, and putative role of brassinosteroid

Sphingolipids are abundant membrane components and signalling molecules in various aspects of plant development. However, the role of sphingolipids in early fleshy-fruit growth has rarely been investigated. In this study, we first investigated the temporal changes in sphingolipid long-chain base (LCB) content, composition, and gene expression that occurred during flower opening and early fruit development in olive (Olea europaea L. cv Picual). Moreover, the interaction between sphingolipid and the plant hormone, brassinosteroid (BR), during the early fruit development was also explored. For this, BR levels were manipulated through the application of exogenous BRs (24-epibrassinolide, EBR) or a BR biosynthesis inhibitor (brassinazole, Brz) and their effects on early fruit development, sphingolipid LCB content, and gene expression were examined in olive fruit at 14 days post-anthesis (DPA). We here show that sphingolipid with C-4 hydroxylation and Δ8 desaturation with a preference for (E)-isomer formation are quantitatively the most important sphingolipids in olive reproductive organs. In this work, the total LCB amount significantly decreased at the anthesis stage, but olive sphingosine-1-phosphate lyase (OeSPL) gene was expressed exclusively in flower and upregulated during the anthesis, revealing an association with the d18:1(8E) accumulation. However, the LCB content increased in parallel with the upregulation of the expression of genes for key sphingolipid biosynthetic and LCB modification enzymes during early fruit development in olive. Likewise, we found that EBR exogenously applied to olive trees significantly stimulated the fruit growth rate whereas Brz inhibited fruit growth rate after 7 and 14 days of treatment. In addition, this inhibitory effect could be counteracted by the application of EBR. The promotion of early fruit growth was accompanied by the down-regulation of sphingolipid LCB content and gene expression in olive fruit, whereas Brz application raised levels of sphingolipid LCB content and gene expression in olive fruit after 7 and 14 days of treatment. Thus, our data indicate that endogenous sphingolipid LCB and gene-expression levels are intricately controlled during early fruit development and also suggest a possible link between BR, the sphingolipid content/gene expression, and early fruit development in olive.

Sphingolipid Distribution, Content and Gene Expression during Olive-Fruit Development and Ripening

Plant sphingolipids are involved in the building of the matrix of cell membranes and in signaling pathways of physiological processes and environmental responses. However, information regarding their role in fruit development and ripening, a plant-specific process, is unknown. The present study seeks to determine whether and, if so, how sphingolipids are involved in fleshy-fruit development and ripening in an oil-crop species such as olive (Olea europaea L. cv. Picual). Here, in the plasma-membranes of live protoplasts, we used fluorescence to examine various specific lipophilic stains in sphingolipid-enriched regions and investigated the composition of the sphingolipid long-chain bases (LCBs) as well as the expression patterns of sphingolipid-related genes, OeSPT, OeSPHK, OeACER, and OeGlcCerase, during olive-fruit development and ripening. The results demonstrate increased sphingolipid content and vesicle trafficking in olive-fruit protoplasts at the onset of ripening. Moreover, the concentration of LCB [t18:1(8Z), t18:1 (8E), t18:0, d18:2 (4E/8Z), d18:2 (4E/8E), d18:1(4E), and 1,4-anhydro-t18:1(8E)] increases during fruit development to reach a maximum at the onset of ripening, although these molecular species decreased during fruit ripening. On the other hand, OeSPT, OeSPHK, and OeGlcCerase were expressed differentially during fruit development and ripening, whereas OeACER gene expression was detected only at the fully ripe stage. The results provide novel data about sphingolipid distribution, content, and biosynthesis/turnover gene transcripts during fleshy-fruit ripening, indicating that all are highly regulated in a developmental manner.

Melatonin and related bioactive compounds in commercialized date palm fruits (Phoenix dactylifera L.): correlation with some antioxidant parameters

The presence of melatonin in plant foodstuffs is a matter of growing interest since its antioxidant properties and the possible benefits for health of consumers. Fruits of date palm (Phoenix dactylifera, L.) are highly appreciated because of their nutritional value and healthy properties related to high content in antioxidant compounds present in them. In this study, we show that dates’ antioxidant properties persist in several commercialized varieties typical of the European markets, as compared to values referenced from studies in date fruit varieties collected in the origin countries. We also show for the first time that melatonin and one of its isomers are present in dates, being that the isomer content is several times higher than melatonin (in the range of 1xa0µg per 100xa0g of fresh weight). The isomer content was highly correlated with total phenolic compounds and total antioxidant capacity in the date varieties. Additionally, high levels of the indoleamines, serotonin and auxin accumulate in dates, which could be related to fruit ripening. All the indole derivatives analyzed in dates exhibited antioxidant activity as assayed by DPPH, with differences among the compounds and concentrations used. These results support that melatonin, its isomer, and related indoles present in dates could contribute to the reported health benefits of this fruit by increasing its natural antioxidant potential.