Angel J. Matilla

Ethylene in seed formation and germination

In seed formation the role of ethylene has received little attention. The data available on zygotic embryogenesis suggest an association of the ethylene biosynthetic pathway and seed maturation. Over the course of dicot embryogenesis, ACC-oxidase mRNA can be expressed in the cotyledons and embryonic axis. However, as maturation proceeds, cotyledonary ACC-oxidase expression disappears. In some seeds that develop primary dormancy, ethylene synthesis can be among the prerequisites for breaking dormancy. Moreover, the persistence of dormancy may be related to the difficulty of the embryonic axis to produce the necessary ethylene levels or to low tissue sensitivity. The use of inhibitors of ethylene biosynthesis or its action has provided data implicating an ethylene requirement for seed dormancy or germination in some species. However, the role of ethylene in germination remains controversial. Some authors hold that gas production is a consequence of the germination process, while others contend that ethylene production is a requirement for germination. Furthermore, among seeds that require ethylene, some are extremely sensitive to the gas, while others require relatively high levels to trigger germination. Recent studies with Xanthium pennsylvanicum seeds suggest that � cyanoalanine-synthase is involved in ethylene-dependent germination. In addition, regulation of the partitioning of S-adenosyl-L-methionine (AdoMet) between the ethylene vs polyamine biosynthetic pathways may be a way of controlling germination in some seeds. Such regulation may also apply to the reversal of seed thermoinhibition, which can occur when polyamine synthesis is inhibited, thereby strongly channelling AdoMet towards ethylene. The biological models and approaches that may shed additional light on the role of ethylene during seed germination are presented.

Seed dormancy and ABA signaling: The breakthrough goes on

The seed is an important organ of higher plants regarding plant survival and species dispersion. The transition between seed dormancy and germination represents a critical stage in the plant life cycle and it is an important ecological and commercial trait. A dynamic balance of synthesis and catabolism of two antagonistic hormones, abscisic acid (ABA) and giberellins (GAs), controls the equilibrium between seed dormancy and germination. Embryonic ABA plays a central role in induction and maintenance of seed dormancy, and also inhibits the transition from embryonic to germination growth. Therefore, the ABA metabolism must be highly regulated at both temporal and spatial levels during phase of dessication tolerance. On the other hand, the ABA levels do not depend exclusively on the seeds because sometimes it becomes a strong sink and imports it from the roots and rhizosphere through the xylem and/or phloem. All theses events are discussed in depth here. Likewise, the role of some recently characterized genes belonging to seeds of woody species and related to ABA signaling, are also included. Finally, although four possible ABA receptors have been reported, not much is known about how they mediate ABA signalling transduction. However, new publications seem to shown that almost all these receptors lack several properties to consider them as such.

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).

Softening-up mannan-rich cell walls

The softening and degradation of the cell wall (CW), often mannan enriched, is involved in several processes during development of higher plants, such as meristematic growth, fruit ripening, programmed cell death, and endosperm rupture upon germination. Mannans are also the predominant hemicellulosic CW polymers in many genera of green algae. The endosperm CWs of dry seeds often contain mannan polymers, sometimes in the form of galactomannans (Gal-mannans). The endo-β-mannanases (MANs) that catalyse the random hydrolysis of the β-linkage in the mannan backbone are one of the main hydrolytic enzymes involved in the loosening and remodelling of CWs. In germinating seeds, the softening of the endosperm seed CWs facilitates the emergence of the elongating radicle. Hydrolysis and mobilization of endosperm Gal-mannans by MANs also provides a source of nutrients for early seedling growth, since Gal-mannan, besides its structural role, serves as a storage polysaccharide. Therefore, the role of mannans and of their hydrolytic enzymes is decisive in the life cycle of seeds. This review updates and discusses the significance of mannans and MANs in seeds and explores the increasing biotechnological potential of MAN enzymes.

After-ripening alters the gene expression pattern of oxidases involved in the ethylene and gibberellin pathways during early imbibition of Sisymbrium officinale L. seeds

After-ripening (AR) in Sisymbrium officinale seeds altered SoACS7, SoACO2, SoGA20ox2, SoGA3ox2, and SoGA2ox6 gene expression. Except for SoGA20ox2 expression, which sharply diminished, the expression of the other genes rose during development, particularly that of SoACS7. In contrast, only the SoACO2 and SoGA2ox6 transcripts increased with seed desiccation; the others decreased. AR increased the SoGA3ox2 transcript in dry seed, but dramatically decreased the SoACS7 transcript. At the onset of imbibition, AR inhibited SoACS7 and SoACO2 expression and stimulated that of SoGA20ox2, SoGA3ox2, and SoGA2ox6, demonstrating that the participation of ethylene (ET) and gibberellins (GAs) differs in after-ripened and non-after-ripened seeds. The inhibition of SoACO2 expression in the presence of GA4+7, paclobutrazol (PB), inhibitors of ET synthesis and signalling (IESS), and notably ET+GA4+7 indicated ET–GA cross-talk in non-after-ripened seeds. A positive effect of AR in reversing this inhibition was found. The idea of ET–GA cross-talk is also supported by the effect of ET on SoGA3ox2 expression, notably induced by the AR process. In contrast, SoGA20ox2 expression did not appear to be susceptible to AR. SoGA2ox6 expression, poorly known in seeds, suggests that AR prompted an up-regulation under all treatments studied, whereas in non-after-ripened seeds expression was down-regulated. On the other hand, the β-mannanase (MAN) activity dramatically increased in dry after-ripened seed, being significantly boosted by ET. The absence of MAN inhibition by IESS suggests that although ET seems to be one of the factors controlling MAN, its presence did not appear to be essential. GA4+7 only increased MAN in seeds wich were after-ripened. Here, it is proposed that ET and GAs participate actively in establishing the AR process.

Progress in research on dry afterripening

The transition from the dormant to the non-dormant state of a viable and mature seed can take place at low hydration by exposure to air-dry storage conditions (dry afterripening; AR). The events occurring during this loss of dormancy are of considerable physiological, ecological and agricultural interest. AR may be attributable to increased sensitivity to germination-stimulating factors and a widening of the temperature window for germination. Genetic, –omics and physiological studies on this mode of dormancy breaking provide support for a key role of the balance between gibberellin (GA) and abscisic acid (ABA) metabolism and sensitivity. Recent evidence also supports a possible role for ethylene (ET) in this complex signalling network that is necessary for AR implementation. However, hormone-independent signals, such as reactive oxygen species (ROS), nitrate ( ) or nicotinamide adenine dinucleotide (NAD + ), also appear to be involved. The way in which hormone- and non-hormone-signalling pathways affects each other (cross-talk) is still under study. This review provides updated information on the programmes that overcome seed dormancy. Thus, we have reviewed: (1) the –omic status in dry seeds; (2) the relationship between temperature and nitrate signalling and AR; (3) alterations in ABA/GA synthesis and signalling; (4) the action of hormone molecules other than ABA and GA (i.e. ET, salicylic and jasmonic acids); and (5) participation of reactive oxygen species (ROS), NAD + and protein carbonylation. Taken together, the acquisition and implementation of dry AR involve a complex signalling network that is difficult to disentangle.

1-aminocyclopropane-1-carboxylate oxidase from embryonic axes of germinating chick-pea ( Cicer arietinum L.) seeds: cellular immunolocalization and alterations in its expression by indole-3-acetic acid, abscisic acid and spermine

A recombinant protein (approximately 38 kDa by SDS/PAGE), induced by expression in Escherichia coli of a cDNA encoding a 1-aminocyclopropane-1-carboxylate oxidase (ACO) isolated from embryonic axes of Cicer arietinum , was recognized by an antibody raised against an apple ACO. A monoclonal antibody, obtained from recombinant ACO of chick-peas, was used for immunolocalization experiments in the embryonic axes of chick-pea seeds. The results indicate that most of the ACO was present in the apoplast of the cell wall. No evidence of this protein in the vacuole was detected. During germination of C. arietinum seeds, the production of ethylene was induced in the embryonic axis; its highest value was reached when radicle emergence occurred. At this moment there was an accumulation of 1-aminocyclopropane-1-carboxylate (ACC), transcription of ACO mRNA, as well as maximal ACO activity. In the post-germinative period the activity of the last step of ethylene biosynthesis decreased. This decrease was eliminated by indole-3-acetic acid (IAA), which caused significant transcription of ACO mRNA. It is suggested that gene expression of ACO may be induced by auxins and spermine (Spm) and inhibited by abscisic acid (ABA).

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.

Organic Acids and Soluble Sugars in Edible and Nonedible Parts of Damson Plum (Prunus domestica L. subsp. insititia cv. Syriaca) Fruits During Development and Ripening

The contribution of the seed and pericarp to the content of malic, quinic, citric and fumaric acids, and sucrose, fructose and glucose was determined during development and ripening of damson plum fruits. In whole fruit, (i) malic and quinic acids were the principal organic acids (OA) and their levels varied significantly, the highest being found at the beginning of the late-green stage; and (ii) the content of citric and fumaric acids was scarce but fluctuated remarkably towards development and ripening. In the seed, the levels of malic, quinic and fumaric acid were lower in ripening than at the beginning of maturation, and a notable synthesis of citric was found from the middle of maturation onwards. In mesocarp, however, malic, quinic, and citric acids peaked in the middle of maturation, whereas fumaric acid notably increased towards ripening. In epicarp, the maximum for the quinic and malic was found at the beginning of ripening and maturation, respectively. In the seed, all soluble sugars (SS) studied peaked at the middle of maturation, and while fructose and glucose (the most abundant SS) tended to be stored during ripening, sucrose (the most abundant in the edible part of fruit) decreased. All the SS studied tend to increase in mesocarp and epicarp throughout maturation and ripening.