Isabel María Sánchez-Calle

The relationships between ethylene production and germination ofCicer arietinum seeds

The germination percentage of chick-pea (Cicer arietinum) Seeds was greatly reduced by temperatures of 30°C and 35°C. This thermoinhibition was overcome by ethylene (ethrel). Both ABA and PEG diminished ethylene production and germination percentage in a parallel way. FC, MGBG and CHA stimulated both ethylene production and germination. AVG reduced ethylene production to some extent but did not inhibit germination. CoCl2 and PG completely prevented both ethylene production and germination; this effect was reversed by ethylene but not by its immediate precursor ACC. NBE prevented both germination and ethylene production. Our results suggest that high ethylene production rates are not essential for germination of chick-pea seeds but that certain quantities of ethylene may be required.

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.

Germination of chick-pea seeds in relation to manipulation of the ethylene pathway and polyamine biosynthesis by inhibitors

Abstract Cyclohexylamine and methylglioxalbis(guanylhydrazone), inhibitors of polyamine synthesis, when added to the germination medium of Cicer arietinum L. seeds, increase the germination percentage and ethylene level in both control (25°C) and thermo-inhibited seeds (30°C). This stimulation is inhibited at higher temperatures (35°C), ethylene is not produced and putrescine and spermine are accumulated, both in free and bound forms. The inhibition of the biosynthetic pathway or action of ethylene by norbornadiene and CoCl2 not only causes an important accumulation of free and bound polyamines, but also prevents germination. In the seed response to aminoethoxyvinylglycine, both biosynthetic pathways are altered. The physiological implications of the distribution of S-adenosylmethionine is discussed in relation to the importance of ethylene synthesis in Cicer arietinum seed germination.

Alterations of the ethylene pathway in germinating thermoinhibited chick-pea seeds caused by the inhibition of polyamine biosynthesis

Abstract The alterations in the ethylene biosynthetic pathway provoked by cyclohexylamine (CHA) and/or methylglyoxal-bis(guanylhydrazone) (MGBG), inhibitors of the polyamine synthesis, have been studied in thermoinhibited seeds (delayed germination by supraoptimal temperatures) of the chick-pea (Cicer arietinum). CHA and/or MGBG stimulated ethylene synthesis both at 25°C (control) and at 30°C (temperature at which thermoinhibition is induced, and reversed by both inhibitors). Moreover, both inhibitors caused: (1) an increase in ACC-synthase (EC 4.4.1.14) and free ACC; (2) a stimulation of in vivo ACC-oxidase; and (3) a stimulation of (malonyl)-1-aminocyclopropane-1-carboxylic acid synthesis at 25°C (since the supraoptimal temperatures had no effect). Compared with the control, both supraoptimal temperatures lead to a decrease the AdoMet-decarboxylase (AdoMetDC) (EC 4.1.1.50) activity. This was not affected by CHA. However, MGBG induced an increase of this activity at 25°C and at 30°C, which diminished strongly up to 35°C. The fact that the inhibition of spermidine and spermine synthesis by CHA and/or MGBG provoked an induction of the enzyme of ethylene biosynthesis and a concomitant increase in ethylene production and a reversion of thermoinhibition in chick-pea seeds suggests that the ethylene and polyamine pathways compete during germination, placing polyamines in the possible role of an endogenous germination regulator possibly by modulating ethylene biosynthesis.

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.

Content and Distribution of Free and Bound Polyamines in Embryonic Axes of Chick-Pea Seeds

Summary Changes in free (S) and bound polyamine contents were investigated in both the embryonic axes attached to the cotyledons and the embryonic axes excised from the whole chick-pea seeds before germination (isolated embryonic axes) during the first 24 hours at 25 °C, 30 °C or 35 °C. We also studied the distribution of these polyamines (PAs) along the embryonic axis of seeds germinated for 65 hours at 25 °C and 30 °C. The content in (S) polyamines was in general higher in embryonic axes separated from whole seeds than in isolated axes, suggesting a transport of these PAs from the cotyledons to the axis. At supraoptimal temperatures (S) polyamines decreased as germination proceeded, reaching higher levels than under the optimum temperature and the highest Spd and Spm accumulations were at 30 °C during the first 12 hours. The absence of cotyledons induced an important linkage of putrescine (Put) that strongly decrease with temperature. Methylglyoxalbis-(guanyl hydrazone) (MGBG) did not modify the total values of (S) and conjugated acid soluble (SH) polyamines, although it d change their distribution and increased conjugated acid insoluble (PH) polyamines.

Thermoinhibition alters the polyamine levels in cotyledons and embryonic axes during germination of stratified chick-pea seeds

Abstract The aim of the present work is to know how much the levels of both free and bound polyamines are altered in the cotyledons of Cicer arietinum by the presence or absence of embryonic axes during the first 24 h of germination under thermoinhibitory conditions (30°C). As an approach, embryonic axes and cotyledons were separated from dry seeds and also from germinated whole seeds. All classes of polyamines decreased both in cotyledons and embryonic axes from dry seeds during the stratification (0–4°C) period (12 month). At 25°C (control) all the polyamine types studied decreased in the cotyledons from germinated whole seeds, and accumulated in the isolated ones (embryonectomized). In cotyledons from germinated whole seeds, a supraoptimal temperature of 30°C provoked a rise in free spermine, free and bound spermidine, and putrescine bound to macromolecules, as compared to the control. However, all polyamines, except putrescine and spermine bound to small substances which remained unaffected, rose at 30°C in the embryonectomized cotyledons. In the embryonic axes separated from whole seeds, the free polyamine content was higher than in isolated ones, and vice versa for polyamines bound to low and high molecular compounds; this last effect being decreased by supraoptimal temperatures.

Effects of polyamines on the ion content and solutes excretion of isolated embryonic axes and the germination rate of seeds of chickpea

Abstract The effect that three polyamines, spermine, spermidine and cadaverine, have on the growth rate and the endogenous ion content of isolated, chickpea (Cicer arietinum, L.) embryonic axes and the germination of whole seeds has been studied. Both the growth rate and ion content are in the same direction, but depending upon each individual polyamine and the concentration. In moderate quantities, spermine increases growth rate, spermidine less so and cadaverine not at all; while at high quantities, spermine greatly reduces growth rate, spermidine less so and cadaverine has no effect. Cadaverine has more effect on the germination of whole seeds. The excretion of aminoacids and sugars into the medium in response to these polyamines was significantly augmented.

Modifications to polyamines provoked by short-chain saturated fatty acids in Cicer arietinum seeds

Short-chain saturated fatty acids (SCSFA), ranging from pentanoic (C5) to decanoic acid (C~0), when added at micromolar concentrations to the germination medium of chick-pea seeds, did not significantly affect radicle emergence, although fresh weight diminished. Only heptanoic acid (C7) and octanoic acid (Cs) stimulated ethylene production and free spermidine and spermine content, whereas putrescine reached a maximum with nonanoic acid (C9). All the SCSFA tested increased the polyamines (PAs) bound to substances of low Mr; however, only hexanoic acid (C6) and C9 acid induced a strong appearance of PAs bound to substances of high Mr. Proline synthesis was stimulated in the presence of C7 acid and C9 acid, but values lower than control were obtained with the other SCSFA studied. (C 1997 Elsevier Science Ltd. All rights reserved