María del Carmen Rodríguez-Gacio

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.

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.

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.

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.

An Update on the Role of NCED and CYP707A ABA Metabolism Genes in Seed Dormancy Induction and the Response to After-Ripening and Nitrate

The molecular mechanisms underlying the regulation of seed dormancy and sensu stricto germination once dormancy is removed are still largely unknown. The hormone abscisic acid (ABA), through a complex crosstalk with other plant hormones, transcription factors and cellular signals belonging to the development program, directly controls the induction and maintenance of primary seed dormancy. A working hypothesis is that loss of primary dormancy and induction of germination requires that the severe control exercised by ABA during seed maturation is down-modulated and is replaced by different signaling pathways. Therefore, it is reasonable to speculate that both ABA metabolism and/or a decrease in ABA sensitivity is operative. The ABA-metabolic NCED and CYP707A gene families are considered key for several developmental plant processes that are ABA-controlled, including seed development and sensu stricto germination. Hence, we propose that ABA metabolism and sensitivity are involved in the mechanisms of action of after-ripening (AR), a process which overcomes the seed primary dormancy status. This review summarizes the current knowledge in the last decade on the controlling role of the NCED and CYP707A gene families as well as changes in ABA sensitivity during the seed life cycle. The expression of ABA-metabolic genes and seed sensitivity to ABA during AR and in the presence of nitrate are also considered.

Nitrate-induced early transcriptional changes during imbibition in non-after-ripened Sisymbrium officinale seeds.

We have here demonstrated for the first time that nitrate not only accelerates testa rupture of non- AR seeds but also modifies expression pattern of the cell-wall remodeling proteins (mannanases; SoMAN6 and SoMAN7) and key genes belonging to metabolism and signaling of ABA (SoNCED6, SoNCED9, SoCYP707A2 and SoABI5) and GAs (SoGA3ox, SoGA20ox, SoGA2ox and SoRGL2). These results were obtained during Sisymbrium officinale seed imbibition in the absence of endosperm rupture. Exogenous ABA induced a notable inhibition of testa rupture in both absence and presence of nitrate being this effect sharply reversed by GA(4+7). However, nitrate was capable to provoke testa rupture in absence of ABA synthesis. The expression of SoMAN6 and SoMAN7 were positively altered by nitrate. Although ABA synthesis seems apparent at the start of non-AR seed imbibition, taken together the results of SoNCED6, SoNCED9 and SoCYP707A2 expression seem to suggest that nitrate leads to a strong net ABA decrease. Likewise, nitrate positively affected the SoABI5 expression when the SoNCED9 expression was also stimulated. By contrast, at the early and final of imbibition, nitrate clearly inhibited the SoABI5 expression. The expression of SoGA2ox6 and SoGA3ox2 are strongly inhibited by nitrate whereas of SoGA20ox6 was stimulated. On the other hand, SoRGL2 transcript level decreased in the presence of nitrate. Taken together, the results presented here suggest that the nitrate signaling is already operative during the non-AR S. officinale seeds imbibition. The nitrate, in cross-talk with the AR network likely increases the favorable molecular conditions that trigger germination.

Molecular analysis of endo-β-mannanase genes upon seed imbibition suggest a cross-talk between radicle and micropylar endosperm during germination of Arabidopsis thaliana

The endo-β-mannanase (MAN) family is represented in the Arabidopsis genome by eight members, all with canonical signal peptides and only half of them being expressed in germinating seeds. The transcripts of these genes were localized in the radicle and micropylar endosperm (ME) before radicle protrusion and this expression disappears as soon as the endosperm is broken by the emerging radicle tip. However, only three of these MAN genes, AtMAN5, AtMAN7 and especially AtMAN6 influence the germination time (t50) as assessed by the analysis of the corresponding knock-out lines. The data suggest a possible interaction between embryo and ME regarding the role of MAN during the Arabidopsis germination process.

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.

Non-symbiotic hemoglobins in the life of seeds.

Non-symbiotic hemoglobins (nsHbs), ancestors of symbiotic-Hbs, are hexacoordinated dimeric proteins, for which the crystal structure is well described. According to the extent of hexacoordination, nsHbs are classified as belonging to class-1 (nsHbs1) or class-2 (nsHbs2). The nsHbs1 show weak hexacoordination, moderate rates of O(2)-binding, very small rates of O(2) dissociation, and a remarkably high affinity for O(2), all suggesting a function involving O(2) scavenging. In contrast, the nsHbs2 exhibit strong hexacoordination, low rates of O(2)-binding and moderately low O(2) dissociation and affinity, suggesting a sensing role for sustained low (μM) levels of O(2). The existence of spatial and specific expression of nsHbs1 suggests that nsHbs play tissue-specific rather than housekeeping functions. The permeation of O(2) into seeds is usually prevented during the desiccation phase and early imbibition, generating an internal hypoxic environment that leads to ATP limitation. During evolution, the seed has acquired mechanisms to prevent or reduce this hypoxic stress. The nsHbs1/NO cycle appear to be involved in modulating the redox state in the seed and in maintaining an active metabolism. Under O(2) deficit, NADH and NO are synthesized in the seed and nsHbs1 scavenges O(2), which is used to transform NO into NO(3)(-) with concomitant formation of Fe(3+)-nsHbs1. Expression of nsHbs1 is not detectable in dry viable seeds. However, in the seeds cross-talk occurs between nsHbs1 and NO during germination. This review considers the current status of our knowledge of seed nsHbs and considers key issues of further work to better understand their role in seed physiology.

Cloning and analysis of a cDNA encoding an endo-polygalacturonase expressed during the desiccation period of the silique-valves of turnip- tops (Brassica rapa L. cv. Rapa)

During zygotic embryogenesis of turnip-tops (Brassica rapa L. cv. Rapa), the polygalacturonase activity (PG; EC 3.2.1.15), measured as a decrease in viscosity of polygalacturonic acid, reached a high when the desiccation process in the seeded silique was triggered and the valves had lost more than 70-75% of their moisture (45-50 DPA). The PG activity was not detected in any phases of developing seeds. This work also characterizes a cDNA with an open reading frame of 1303 bp and that codes for a putative PG called BrPG1. This falls into the category of clade-B, which includes PG related to shattering and abscission processes. The deduced BrPG1 sequence predicted a 434-residue-long precursor protein (46.7kDa) with a transit peptide sequence 23 amino acids long. A molecular mass of 44.3 kDa was calculated for the mature form of BrPG1, which showed high sequence similarity to PGA1 (97%) of B. napus (X98373) and ADPG1 (87%) of Arabidopsis thaliana (AJ002532). All conserved amino acids at the catalytic site of PGs belonging to clade-B were preserved on BrPG1. This BrPG1 gene was specifically expressed in the silique valves of turnip-tops and was temporally expressed at the beginning of its desiccation.