ABA crosstalk with ethylene and nitric oxide in seed dormancy and germination

1Survival of plant species mainly relies on the sexual reproduction to give birth to new individuals. In flowering plants, the seed is the main unit of dispersal and allows colonization of new geographic areas. As a consequence of the double fertilization process, a mature angiosperm seed contains a diploid embryo and protective layers comprising the triploid endosperm, a nourishing tissue for the embryo, and the seed coat of maternal origin. During development on the mother plant, after embryogenesis completion, reserve accumulation takes place and is followed, in so-called orthodox seeds, by an intense dehydration leading to low seed water content upon dispersal. In many species, a dormant state is also induced during the maturation phase, preventing pre-harvest germination and allowing seed survival until environmental conditions become suitable for germination and seedling establishment.

2Dormancy has been defined as a developmental state in which a viable seed fails to germinate under favorable environmental conditions, but different definitions and classifications have been proposed. summarized a classification proposed by, based on the fact that dormancy results from physiological and developmental (or morphological) properties of the seed. Dormancy is therefore divided in five classes: (1) physiological dormancy (PD) can be released by different stratification (moist chilling) treatments depending on its depth, (2) morphological dormancy (MD) is due to a delay of embryo development, (3) morphophysiological dormancy (MPD) is combining both PD and MD, (4) physical dormancy (PY) is correlated with seed coat impermeability to water and needs disruption of the seed coat (scarification) to be released, and finally (5) combinational dormancy combining PY and PD. Most species display a non-deep PD corresponding to a dormancy that can be released, depending on the species, by gibberellin (GA) treatment, stratification, scarification, or a period of dry storage (after-ripening). In this case, seeds generally combine a coat-imposed dormancy due to the covering layers of the seed (seed coat and endosperm) that prevent the radicle protrusion, and an embryo dormancy due to its incapacity to induce radicle growth.

3When dormancy is released, seeds can germinate under favorable conditions, specific to each species. The germination process, that begins with seed imbibition and finishes with a developed plantlet, is divided in three distinct phases of water uptake. Phase I starts with a fast water uptake and the activation of respiratory metabolism and transcriptional and translational activities. During phase II water uptake ceases, seed reserve mobilization begins and testa rupture occurs. Later, in the third phase water uptake resumes and endosperm rupture allows radicle protrusion; then starts the post-germination phase with high water uptake, mobilization of the major part of reserves and first cell divisions, until the complete seedling development. Germination sensu stricto ends with radicle protrusion. It is often described has the resulting consequence of the growth potential of the embryo and the resistance of the surrounding layers. Endosperm weakening is an essential part of the modification of seed envelopes for the progress of germination and involves the activation of cell-wall modifying enzymes. After dormancy release, storage/imbibition of non-dormant seeds in unfavorable conditions for germination can trigger a secondary dormancy. This is a way to protect seeds against germination too late in the year and induce a seasonal cycling of dormancy level in seeds.

4The regulation of seed dormancy and germination by the hormonal balance between abscisic acid (ABA) and GA, in response to environmental signals, is well documented in a number of recent reviews. The present review will describe recent knowledge about key players in the ABA metabolism and signaling pathways that control dormancy induction and maintenance and convergent evidences supporting the role of two other signaling compounds, nitric oxide (NO) and ethylene, in dormancy breakage and germination, and their interactions with ABA metabolism and signaling pathways.

5Abscisic acid is formed by cleavage of C40 oxygenated carotenoids, also called xanthophylls, which are produced in plastids from C5 precursors. Key genes encoding enzymes of the ABA biosynthesis pathway have been identified through mutant selection for altered germination phenotypes, giving further evidence of the major role of ABA in the regulation of seed dormancy and germination (Figure 1). For instance, the first ABA-deficient mutant, identified in Arabidopsis thaliana, was isolated in a GA biosynthesis mutant ga1 suppressor screen, on its ability to germinate in the absence of GA. It was shown to be defective in zeaxanthin epoxidase (ZEP) activity, like a Nicotiana plumbaginifolia mutant selected later on its early germination phenotype. ZEP catalyzes the epoxidation of zeaxanthin into violaxanthin and is encoded, in Arabidopsis, by the ABA1 gene. Violaxanthin is then converted into neoxanthin, by neoxanthin synthase (NSY), likely encoded by the Arabidopsis ABA4 gene. Despite impairment in ABA4 function completely prevents neoxanthin synthesis, the aba4 mutant exhibits no obvious dormancy phenotype, due the formation of cis-violaxanthin by an alternate pathway. Both cis-violaxanthin and cis-neoxanthin cleavage gives rise to xanthoxin, the C15 aldehyde precursor of ABA. Since cis-isomerization of violaxanthin and neoxanthin is required prior to cleavage, an unknown isomerase might be involved. The VIVIPAROUS14 (VP14) gene in maize (Zea mays) has been shown to encode a 9-cis-epoxycarotenoid dioxygenase (NCED), which catalyzes the oxidative cleavage of either 9′-cis-neoxanthin or 9-cis-violaxanthin. NCED genes have been then identified in a number of other plant species. In Arabidopsis, VP14-related gene family is composed of nine members, five of which (NCED2, NCED3, NCED5, NCED6, and NCED9) encode xanthoxin-producing enzymes.

6In Arabidopsis plastids, ZEP is associated mainly to envelope and slightly to thylakoid membranes (Figure 1). In contrast NSY/ABA4 is presumably tightly bound to the envelope since this protein is predicted to contain four transmembrane domains and is exclusively found in the envelope fraction. In contrast, NCED proteins have been detected either in stroma or thylakoid membrane-bound compartments, or both. In addition, recent VP14 structural analysis suggested that this enzyme might penetrate the surface of thylakoid membrane to access and transfer carotenoid substrates to its catalytic center. The scattered location of ZEP, NSY, and NCED suggests that the production of xanthoxin inside plastids may require transport mechanisms of lipid-soluble carotenoid molecules, which are not currently understood. Since the following enzymatic reactions take place in the cytosol, xanthoxin is also presumed to migrate from plastid to cytosol by a still unknown mechanism.

7Abscisic aldehyde is synthesized from xanthoxin, by an enzyme belonging to short-chain dehydrogenase/reductase family, which is named SDR1 and is encoded by the ABA2 gene in Arabidopsis. The oxidation of the ABA-aldehyde is the final step of ABA biosynthesis, and is catalyzed by an abscisic aldehyde oxidase. In Arabidopsis, four homologous aldehyde oxidase (AAO) genes have been characterized, but only one of them, AAO3, encodes a protein that has proven activity on abscisic aldehyde. Activity of this molybdoenzyme requires the activation of its molybdenum co-factor (Moco) by addition of a sulfur atom to the Mo center, which is catalyzed by a Moco sulfurase, which has been named ABA3 in Arabidopsis.

8Abscisic acid inactivation is a crucial mechanism to fine-tune ABA levels, which occurs by either oxidation or conjugation (Figure 1). The major catabolic route is the 8′-hydroxylation of ABA by the CYP707A subfamily of P450 monooxygenases. Spontaneous 8′-hydroxy-ABA isomerization gives rise to phaseic acid (PA), which is then converted to dihydrophaseic acid (DPA) by a still unknown reductase. ABA can also be hydroxylated at the C-7′ and C-9′ positions. As 8′-hydroxylation, 9′-hydroxylation is catalyzed by CYP707A as a side reaction, and neoPA is then formed by spontaneous isomerization. The conjugation of ABA with glucose to form the ABA-glucose ester (ABA-GE) is catalyzed by an ABA glucosyltransferase, and in Arabidopsis only UGT71B6 exhibits a selective glucosylation activity toward the natural enantiomer (+)-ABA. Subsequent hydrolysis of conjugates constitutes an alternative pathway for ABA synthesis in response to dehydration stress. Two glucosidases BG1 and BG2, localizing respectively in the endoplasmic reticulum or the vacuole, have been identified.

9Deficiency in either ABA synthesis or ABA inactivation by 8′-hydroxylation leads to strong dormancy phenotypes, respectively dormancy loss or strengthening. In contrast, reports on functional analysis of mutant or overexpressing lines in ABA conjugation or ABA-GE hydrolysis did not yet describe the implication of these processes in dormancy control. Nevertheless, ABA conjugation may contribute to ABA breakdown upon germination, as shown in lettuce (Lactuca sativa; ).

10Genetic analyses suggest that PYR/PYL/RCAR (pyrabactin resistance1/PYR1-like/regulatory components of ABA receptor) ABA receptors, clade A type 2C protein phosphatases (PP2C) and group III sucrose non-fermenting1-related protein kinase2 (SnRK2) subfamily are essential core components of the upstream signal transduction network that regulates ABA-responsive processes, including dormancy and germination (reviewed in ). PYR/PYL/RCAR proteins constitute a 14-member family, belonging to the START-domain superfamily, also called Bet v I-fold. ABA binding induces receptor conformation changes allowing the formation of a protein complex with PP2C and the inhibition of phosphatase activity (Figure 2). The clade A PP2C, including ABA INSENSITIVE1 (ABI1) and ABI2, also interact with three SnRK2 (SnRK2.2, SnRK2.3, and SnRK2.6) and, in the absence of ABA, dephosphorylate a serine residue whose phosphorylation is required for kinase activity. When ABA is present, PP2C binding to the receptor releases inhibition of SnRK2 activity, which can phosphorylate downstream targets.

11Other types of receptors and a large number of genes, whose mutations alter ABA germination sensitivity, have been reported to participate in ABA signaling. In particular, regulatory mechanisms such as RNA processing, RNA/protein stability or chromatin remodeling have an important role. However, they will not be detailed here, since their role in ABA crosstalk with ethylene and NO in seeds still requires further investigation. In Arabidopsis seeds, extensive evidence including mutant phenotypes strongly supports a central role of the PYR/PYL, PP2C, SnRK2 complex in ABA signaling (reviewed in; ). Germination of a pyr/pyl sextuple mutant is highly insensitive to ABA, as also observed for the snrk2.2 snrk2.3 snrk2.6 triple mutant. Moreover the snrk2 triple mutant exhibits loss of dormancy and even seed vivipary under high humidity conditions. Conversely, in accordance with PP2C being negative regulators of ABA signaling, germination in triple pp2c mutants was slower than in wild type and was inhibited by very low ABA concentrations. In contrast, the gain-of-function mutations abi1-1 and abi2-1, which prevent PP2C binding to PYL/PYR/RCAR, lead to ABA insensitivity and reduced dormancy.

12Basic leucine zipper transcription (bZIP) factors of the ABA-RESPONSIVE ELEMENTS (ABRE) BINDING FACTOR/ABA RESPONSE ELEMENT BINDING FACTOR/ABA INSENSITIVE5 (ABF/AREB/ABI5) clade have been shown in different species to constitute SnRK2 downstream targets and regulate ABRE containing genes. Several family members are expressed at different seed stages and exhibit partially redundant or antagonistic functions, and ABI5 appears to have a predominant role in the regulation of a subset of late embryogenesis abundant (LEA) proteins during late seed development. abi5 mutation confers ABA-insensitive germination, but it does not impair seed dormancy, suggesting that other factors might be involved in dormancy induction. Nevertheless, ABI5 has been clearly proven to act as a major inhibitor of germination processes in imbibed seeds, notably through its up-regulation by stress-induced ABA accumulation. ABI3/VIVIPAROUS1 (VP1) interacts with ABI5 for the regulation of a number of ABA-responsive genes during seed maturation and germination. However, in contrast to abi5, abi3 mutants do not only exhibit ABA-resistant germination, but also other phenotypes including desiccation intolerance and precocious germination. They share these maturation defects with fusca3 (fus3) and leafy cotyledon2 (lec2) mutants, which, like abi3, carry mutations in B3 transcription factor family genes. These factors form a complex network regulating the expression of reserve storage and LEA genes by their binding to RY motif, and it has been suggested that the lack of dormancy induction in mutants might indirectly result from early seed developmental defects. Nevertheless fus3 mutation has been shown to affect ABA levels in developing seeds. In addition, ABA-specific phenotypes of abi3/vp1 mutants strongly suggest an involvement in ABA-regulated dormancy induction, but downstream dormancy genes still remain elusive. Nevertheless, one of these might be the recently identified seed dormancy4 (Sdr4) gene in rice, which encodes a nuclear protein of unknown function.

13The Arabidopsis DELAY OF GERMINATION1 (DOG1) gene, whose precise function is still unknown, has been identified as a major regulator of seed dormancy. In accordance, protein accumulation in dry seeds well correlates with dormancy depth, and both transcript and protein levels are increased upon cool conditions of seed maturation, which increase seed dormancy. Despite dog1 dormancy phenotypes are similar to ABA synthesis and signaling mutants, current evidence suggests that DOG1 and ABA act in independent pathways. Nevertheless regulation of dormancy depth by DOG1 requires a functional ABA signaling pathway, and DOG1 has been reported to be implicated in the ABA-mediated sugar signaling pathway, together with ABI4, an APETALA2 transcription factor involved in reserve mobilization at germination. Another mutation, named despierto (dep), also causes dormancy loss. DEP gene encodes a C3HC4 RING (Really Interesting New Gene)-finger protein, whose targets are unknown. In addition to similarity in mutant phenotypes, expression of both DEP and DOG1 genes is maximal during late seed development and decreases during imbibition. Moreover dep mutation reduces DOG1 transcript levels in developing seeds and vice versa. It also down-regulates the expression of several ABA biosynthesis and signaling genes, including NCED6, NCED9, and ABI3, suggesting its action in dormancy induction may involve the ABA signaling pathway.

14Abscisic acid is produced in all seed tissues (testa, endosperm, embryo), as suggested by the spatiotemporal expression of ABA biosynthesis genes. However, ABA accumulated in seeds also originates from synthesis in vegetative tissues and transport to the seed. Several ABA transporters have been recently identified, which belong to either the ATP-binding cassette (ABC) or nitrate transporter 1 (NRT1)/peptide transporter (PTR) families. ABC transporter G family member 25 (ABCG25) functions as a plasma membrane ABA exporter, whereas both ABCG40 and AIT1 (ABA IMPORTER1) are plasma membrane uptake transporters. Despite mutations in these three genes induce alterations in germination sensitivity to ABA, suggesting a possible function in seeds, the precise contribution of any of them to either ABA supply from mother plant to seeds or its translocation between maternal and/or embryonic seed tissues needs further investigation. Another ABC transporter gene, ABCG22, has been reported to be involved in ABA-regulated water stress tolerance, but its function in ABA transport remains uncertain. ABA levels are maximal during mid-seed development, with a large fraction produced in maternal tissues. Maternal ABA has a major contribution to the regulation of many aspects of seed development, but only ABA produced by zygotic tissues at late maturation stages imposes dormancy.

15Carotenoid cleavage by NCED and ABA inactivation by CYP707A 8′-hydroxylase have been proven to constitute key regulatory steps for the control of ABA levels, which affect seed dormancy and germination in response to environmental cues. Among the five Arabidopsis NCED genes, NCED6 and NCED9 exhibit the highest expression levels in developing seeds and show distinctive expression patterns. NCED6 is specifically expressed in endosperm, whereas NCED9 expression is detected in testa and embryo. Furthermore mutant analysis indicated that ABA production in both embryo and endosperm contributes to dormancy induction. In barley (Hordeum vulgare), the two HvNCED genes also exhibit differential spatiotemporal patterns of expression. In contrast to HvNCED2, HvNCED1 transcript levels vary depending on environmental conditions during grain development and modulate ABA accumulation at late maturation stages. ABA inactivation by CYP707A during seed maturation also regulates dry seed ABA levels and dormancy depth, as deduced from cyp707a mutant analysis. Moreover, the seed dormancy increase under cold-maturation conditions is not only correlated with DOG1 up-regulation, as mentioned above, but also with CYP707A2 down-regulation.

16Upon imbibition, dormancy maintenance and germination are also regulated by both ABA catabolism and neo-synthesis. A decrease in ABA levels at imbibition has been observed in both dormant and non-dormant seeds in several species; nevertheless dormant seeds maintain higher ABA levels and in accordance exhibit lower CYP707A transcript levels, as shown in Arabidopsis and barley. Barley HvABA8’OH1 transcripts were detected in coleorhiza cells near the root apex and Arabidopsis CYP707A2 in endodermis and micropylar endosperm next to the radicle. Moreover, it is well documented in several species that unfavorable light or temperature conditions prevent germination by coordinated regulation of NCED and CYP707A gene expression. Furthermore, dormancy cycling by seasonal variation of soil temperature has been recently linked to the regulation of ABA metabolism and signaling genes. Deep dormancy in winter is correlated with increased ABA levels and NCED6 expression, together with that of DOG1 and MOTHER OF FLOWERING LOCUS T (MFT). MFT encodes a phosphatidylethanolamine-binding protein, which is regulated by ABI3 and ABI5, and feedback regulates ABA signaling by repressing ABI5. In contrast, shallow dormancy in summer is correlated with a reduction in ABA levels and an up-regulation of CYP707A2 and ABI2, which negatively regulates ABA signaling.

17In Arabidopsis, despite endosperm consists in a single cell layer in mature seeds, convergent evidence demonstrated its major role in ABA control of seed dormancy and germination. Firstly, whereas the removal of whole seed coat (endosperm and testa) releases mechanical constraints and allows development of embryos dissected from dormant seeds, the preservation of the endosperm after testa removal maintains dormancy. Secondly, using a “seed coat bedding assay,” showed that diffusion of endospermic ABA from dormant seed envelopes could prevent growth of non-dormant embryos, including those of ABA-deficient aba2 mutants. In isolated embryos, translocated ABA was able to induce ABI5 protein accumulation, whose level was correlated with dormancy maintenance. In addition, in a previous study, ABI5 transcript was detected in the embryo and the micropylar endosperm of imbibed seeds, suggesting a role in the inhibition of both embryo growth and endosperm rupture by ABA. The tissue-specificity of ABA sensitivity is also likely regulated by the spatiotemporal expression of upstream ABA signaling components, as suggested by the differential expression of PYR/PYL genes in embryo and/or endosperm of imbibed seeds.

18Ethylene biosynthesis pathway in germinating seeds is the same as that described in other plant organs (Figure 3), in which S-adenosyl-methionine (S-AdoMet) and 1-aminocyclopropane-1-carboxylic acid (ACC) are the main intermediates. The first step of ethylene biosynthesis is the conversion of S-AdoMet to ACC catalyzed by ACC synthase (S-adenosyl-L-methionine methylthioadenosine-lyase, ACS), the by-product being 5′-methylthioadenosine (MTA), which is recycled back to methionine through the Yang cycle. The second step corresponds to the oxidation of ACC by ACC oxidase (ACO) to form ethylene, CO2, and hydrogen cyanide (HCN). Cyanide produced during this final step of ethylene synthesis is detoxified to β-cyanoalanine by β-cyanoalanine synthase (β-CAS). Both ACS and ACO are encoded by a multigene family. In Arabidopsis, nine active ACS genes have been characterized. Most of them can be induced by cycloheximide (ACS2, ACS4, ACS6), wounding (ACS2, ACS4), and ethylene treatment (ACS2, ACS6; reviewed in ). In addition, ACS6 can also be induced by cyanide or ozone treatment. ACO activity controls in vivo ethylene production and has fundamental contribution during seed germination.

19In Arabidopsis, five membrane-localized receptors have been identified: ethylene resistant 1 (ETR1), ETR2, ethylene response sensor 1 (ERS1), ERS2, and ethylene insensitive 4 (EIN4; Figure 2). Among them, ETR1 and ERS1 contain three transmembrane domains in the N-terminus and a histidine kinase domain in the C-terminus. In contrast, ETR2, EIN4, and ERS2 have four transmembrane regions and a serine–threonine kinase domain in the C-terminus. Binding of C2H4 to the receptors occurs in the hydrophobic N-terminal part of the receptor dimer and requires a copper co-factor. The signaling pathway of C2H4 is controlled by CTR1 (constitutive triple response 1), a serine–threonine protein kinase that acts as a negative regulator, downstream of the receptor and upstream of EIN2. C2H4 binding results in the inactivation of the receptor–CTR1 complex, and in turn allows activation of a kinase cascade controlling EIN2 and its transcription factors in the nucleus such as EIN3, EIL1, ethylene-responsive element binding proteins (EREBPs)/ethylene-responsive factors (ERFs), which activate the transcription of ethylene-responsive genes. EIN2 works downstream of CTR1 and upstream of EIN3. Recently, demonstrated that EIN2 protein level is regulated through its degradation by the proteasome in the presence of the hormone via 2 F-Box proteins ETP1 and ETP2; in the presence of C2H4, ETP1 and ETP2 levels are low, thus increasing EIN2 protein level.

20The influence of ethylene on seed germination is well documented. Ethylene, ethephon (an ethylene-releasing compound), or ACC (the precursor of ethylene) stimulate seed germination in numerous species, among which several parasitic plants such as Orobanche ramosa and some Striga species. Application of ethylene promotes germination of either primary dormant or secondary dormant seeds (Table 1). It breaks seed coat-imposed dormancy in cocklebur (Xanthium pennsylvanicum;; ), subterranean clover (Trifolium subterraneum; ), Rumex crispus and Arabidopsis, and embryo dormancy in apple (Malus domestica;; ), sunflower (Helianthus annuus; ), and beechnut (Fagus sylvatica; ). It can also overcome thermodormancy in lettuce or secondary dormancy in sunflower, Amaranthus caudatus, and Amaranthus paniculatus. Likewise, it stimulates germination of non-dormant seeds placed in non-optimal conditions. For example, it can overcome the inhibition of germination imposed by high temperatures or osmotic agents, and alleviates the salinity effect in numerous halophytes.

21The stimulatory effect of exogenous ethylene increases with hormone concentration, and the efficient concentrations range from 0.1 to 200 μL L-1, depending on species and depth of their dormancy. Ethylene at 1.25 μL L-1 allows 100% germination of dormant Arabidopsis seeds incubated at 25°C in darkness, when dormant sunflower seeds required 12.5 μL L-1 to fully germinate at 15°C. Breaking of dormancy during chilling of apple seeds, or during dry storage of sunflower achenes, results in an increasing sensitivity to ethylene. In Stylosanthes humilis, non-dormant seeds are at least 50-fold more sensitive to ethylene than freshly harvested dormant ones. Improvement of dormant seed germination does not require a continuous application of ethylene; a short treatment in the presence of this compound is sufficient to improve germination of dormant seeds in various species. Seed responsiveness to ethylene decreases during prolonged pre-incubation under conditions favoring the maintenance of dormancy, probably due to an induction of a secondary dormancy.

22Ethylene production begins as the imbibition phase starts and increases with the germination progression. Its development differs among species (reviewed in;; ), however, the radicle protrusion through the seed coat is always associated with a peak of ethylene release. A close relationship between the ability to produce ethylene and seed vigor has been reported in numerous species, and ACC-dependent C2H4 production was proposed as a marker of seed quality.

23Ethylene production depends on both ACS activity that modulates ACC content, and the activity of ACO, the key enzyme that converts ACC into ethylene. Evolution of ethylene production during germination is associated with an increase in ACO activity, as well as a progressive accumulation of ACS and ACO transcripts, with generally a sharp increase during endosperm rupture or/and radicle protrusion. In Sisymbrium officinale, SoACS7 level is very low during seed imbibition, a more notable expression being detected when endosperm rupture reached 50–100%, whereas SoACO2 expression is detected at early stages during seed imbibition, and then rises during the germination process. Similarly, expression of PsACO1 in pea (Pisum sativum; ) and BrACO1 in turnip (Brassica rapa; ) is maximal at radicle emergence. In two Brassicaceae species, Arabidopsis and Lepidium sativum, ACO1 and ACO2 have been demonstrated to be the major ACOs involved in ethylene synthesis in seeds. In Lepidium sativum, the correlation between ACO1 and ACO2 transcript accumulation with in vivo ACO enzyme activity suggests that ACO is regulated at the transcriptional level during germination.

24Ethylene has been shown to regulate its own synthesis by inducing ACO transcription. It is required for the stimulation of ACO gene expression in pea, beechnut, and turnip. In contrast, expression of SoACS7 in Sisymbrium officinale and PsACS1 in pea is not affected.

25Induction of thermodormancy is often associated with a reduced ethylene production, which may result in chickpea (Cicer arietinum) from a greater ACC-malonyltransferase activity and an S-AdoMet channeling toward the polyamine pathway, thus reducing ethylene precursor availability, or also from ACO activity inhibition, as observed in chickpea and sunflower. Incubation at high temperature (35°C) of lettuce seeds induces a reduction in ethylene production, associated with a complete repression of LsACS1 and a reduced expression of ACO-A (homologous to AtACO4; ).

26In contrast, treatments (chilling, GA, HCN…) that break seed dormancy often lead to an increase in ethylene production (reviewed in; ). Cyanide treatment, which breaks embryo dormancy in apple and sunflower, stimulates ethylene production. In apple 5-day-old seedlings, it increases ACS and ACO activities, whereas in sunflower it reduces in vivo ACC-dependent ethylene production (i.e., in vivo ACO activity) and HaACS and HaACO expression. However, in Arabidopsis, cold stratification down-regulates the expression of ACOs, but results in transient expression of ACS.

27Studies using inhibitors of ACS activity (AVG: amino-ethoxyvinylglycine; AOA: amino-oxyacetic acid), ACO activity (CoCl2; α-AIB: α-aminoisobutyric acid), or ethylene action (2,5 NBD: 2,5-norbornadiene; STS: silver thiosulfate) demonstrated that ethylene evolved by seeds plays a promotive role in germination and dormancy breakage. Conversely, application of exogenous ACC stimulates germination of various ethylene-sensitive seeds such as lettuce, sunflower, cocklebur, Amaranthus caudatus and Amaranthus retroflexus, chickpea, sugar beet (Beta vulgaris; ). Thermodormancy in lettuce, Amaranthus caudatus and chickpea is also reversed by exogenous ACC. This stimulatory effect of ACC suggests that dormancy might be related to low C2H4 production due to insufficient levels of endogenous ACC, i.e., low ACS activity.

28Analysis of mutant lines altered in ethylene biosynthesis and signaling pathway demonstrated the involvement of ethylene in regulating seed germination. Mutations in ETHYLENE RESISTANT1 (ETR1) and ETHYLENE INSENSITIVE2 (EIN2) genes result in poor germination and deeper dormancy compared to wild type, in contrast constitutive triple response1 (ctr1) seeds germinate slightly faster. ERFs genes might also play a key (pivotal) role in ethylene responsiveness and germination regulation. In beechnut, demonstrated that the expression of FsERF1, a transcription factor involved in C2H4 signaling and sharing high homology with Arabidopsis ERFs, increases during dormancy release in the presence of ethephon or after chilling. In sunflower, ERF1 expression is fivefold higher in non-dormant than in dormant embryos, and also markedly stimulated by gaseous HCN, which breaks dormancy. Beechnut FsERF1 is almost undetectable in dormant seeds incubated under high temperature conditions that maintain dormancy, or in the presence of germination inhibitors, either ABA or AOA, an inhibitor of ethylene biosynthesis, but increases during moist chilling that progressively breaks dormancy. In tomato (Solanum lycopersicon), SlERF2 transcript accumulation is higher in germinating seeds than in non-germinating ones, and its overexpression in transgenic lines results in premature seed germination. Interestingly, in lettuce seeds, expression of genes involved in ethylene signaling (CTR1, EIN2, and ETR1) is less affected by high temperature than that of biosynthesis genes (ACS and ACO; ).

29The antagonistic interaction between ABA and C2H4 during germination was demonstrated in numerous species. In Arabidopsis and Lepidium sativum, ethylene counteracts the inhibitory effects of ABA on endosperm cap weakening and endosperm rupture. ABA also increases the ethylene requirement to release primary and secondary dormancies. Inhibition of germination by ABA is associated with a reduction in ethylene production. ABA clearly inhibits in vivo ACO activity, and this inhibition correlates with a decreased accumulation of ACO transcripts. In Arabidopsis, the accumulation of ACO1 transcripts in both the embryo and endosperm during germination is inhibited by ABA, and the high levels of ACO1 transcripts in ABA-insensitive mutants suggests the regulation of ACO expression by ABA. In the embryo, ACO2 transcript accumulation is also inhibited by ABA. In Lepidium sativum, inhibition of both ACO1 and ACO2 by ABA is restricted to the endosperm cap. In accordance, microarray analysis in Arabidopsis aba2 mutant detected an up-regulation of ACO transcript accumulation. Moreover, inhibition of shoot growth in tomato ABA-deficient mutants, flacca and notabilis, and in Arabidopsis aba2 results from increased ethylene production. In contrast to pea, chickpea, Lepidium sativum, and Arabidopsis, there is an ABA-mediated up-regulation of ACC accumulation and ACO expression in sugar beet seeds.

30Treatment with exogenous ethylene or ACC does not affect ABA content nor expression of genes involved in ABA biosynthesis in Lepidium sativum and sugar beet. Nevertheless, seeds of Arabidopsis ethylene-insensitive mutants, etr1 and ein2, exhibit higher ABA content than wild type and consistently germinate more slowly. ABA-GE levels are reduced in etr1–2 seeds; increased ABA accumulation might therefore be attributed to a decrease in ABA conjugation. However, ethylene may also regulate other enzymatic steps, since a microarray analysis reported NCED3 up-regulation in ein2 and CYP707A2 down-regulation in etr1-1. High ABA levels in ein2 were also associated with an up-regulation of ABA1, which was, however, not detected on microarrays.

31Several reports suggest that, during germination, ethylene not only acts on ABA metabolism to reduce ABA levels, but also negatively regulates ABA signaling. Indeed, mutations that reduce ethylene sensitivity (e.g., etr1, ein2, and ein6) result in an increase in ABA sensitivity, while increased ethylene sensitivity in ctr1 and eto1 reduces ABA sensitivity. Mutations in CTR1, for example, enhance the ABA insensitivity of abi1-1 seeds, when C2H4-insensitive mutants like ein2 reduce it. However, no significant difference in ABA sensitivity is observed in ein3, ein4, ein5, and ein7.

32In addition, overexpression in Arabidopsis seeds of a beechnut tyrosine phosphatase, FsPTP1, reduces dormancy, through both ABA signaling down-regulation and EIN2 up-regulation, suggesting that the negative role of FsPTP1 in ABA signaling might result from modulation of C2H4 signaling. This central role of EIN2 in mediating cross-links between hormonal response pathways has also been reported in plant response to abiotic and biotic stresses.

33Despite the existence of interactions between the ABA and ethylene signaling pathways, genetic evidence indicates that they may mainly act in parallel, since double mutants obtained by crossing ethylene mutants (ctr1, ein1, ein3, and ein6) with the aba2 mutant exhibit phenotypes resulting from both ABA deficiency and altered ethylene sensitivity.

34Nitric oxide is an inorganic, uncharged, gaseous free radical that can readily diffuse through cell membranes. Upon production, released NO can adjust to the cellular redox environment leading to the formation of diverse biologically active compounds referred to as reactive nitrogen species (RNS; ). Thus, its biological half-life is assumed to be in the order of seconds depending on the redox environment and the initial amount. While NO production can be beneficial at relatively low levels, uncontrolled accumulation, referred to as nitrosative stress, can result in detrimental consequences in plant cells. A strict control of NO levels is therefore required for cell survival. The regulation of NO biosynthesis, localization, and duration along with the control of NO removal (or storage) is therefore of paramount importance in determining the biological consequences of NO accumulation and thus for its role as secondary messenger. The chemical reactivity of NO makes it an unusual signal molecule that can readily act on a wide range of targets, especially proteins. The signal it mediates can also be modulated along the signal transduction pathways depending on the biological environment, thus adding to the complexity of NO signaling.

35Due to their importance as basis for NO-mediated signaling, the biosynthesis pathways of NO in plants have been the subjects of intense investigations during the last decade. The existence of several sources of NO associated with enzymatic or non-enzymatic reactions has been reported but only a few have been completely elucidated so far. Here we will mainly focus on the reactions proven or suggested to be relevant in the context of seed physiology (Figure 4), as NO synthesis was previously reviewed in and.

36In animals, NO biosynthesis is mainly catalyzed by three isoforms of NO synthase (NOS; ). These enzymes metabolize L-arginine (L-Arg) into L-citrulline and NO via the following reaction:

37To date, despite the identification of a green alga NOS, the search for a NOS homolog enzyme in higher plants only encountered failure, although biochemical assay highlighted the existence of a NOS-like activity in several plant tissues and organelles. Moreover, exogenous application of NOS inhibitors (structural analogs of L-Arg such as L-NAME, N-nitro-L-arginine methyl ester) significantly reduced NO release under diverse conditions in several plant species. Using these approaches, a NOS-like activity was detected in sorghum (Sorghum bicolor) and soybean (Glycine max) imbibed seeds. Nonetheless, the liability of such proofs is now debated in light of the discovery of other L-Arg-dependent NO synthesis pathways. Moreover, the recent finding that L-NAME can affect NO production by interfering with nitrate reductase (NR) activity discredits its use as a NOS inhibitor in plants. Thus, after more than a decade of intense research in the area and despite the proven occurrence of L-Arg-dependent NO biosynthesis, the mere existence of NOS is now questioned in plants.

38Apart from its well-known role in nitrate reduction and assimilation, the cytosolic NR has been shown to catalyze the reduction of nitrite to NO, using NAD(P)H as electron donor, both in vitro and in vivo, via the following reaction:

39In vivo, NR would be responsible at least in part for the basal level of NO production with a low reduction efficiency (in the order of 1% of the total NR activity). However, the nitrite reductase activity of NR (NR-NiR) can drastically increase under certain conditions such as oxygen deprivation. Overall, conditions leading to NR-mediated nitrite production exceeding the rate of nitrite removal can lead to a substantial increase in NO production by NR. Both the nitrate and nitrite reductase activities of NR are tightly controlled by post-translational modifications (PTM;;; ). In Arabidopsis, NR and NR-NiR activities are stimulated by sumoylation mediated by the E3 SUMO ligase AtSIZ1. Furthermore, the H2O2-induction of NO biosynthesis in Arabidopsis roots was recently proposed to depend on mitogen-activated protein kinase 6 (MPK6)-mediated phosphorylation of one of the NR isoforms (Ser 627 in Arabidopsis NIA2;, ). Moreover, NO was reported to inhibit NR activity in wheat leaves. In Arabidopsis seedlings, GA may also negatively regulate light-induced NR activity at post-translational level.

40Distinct studies reported an implication of NR in NO-mediated signal transduction pathways. In seeds, the NO-mediated positive effect of and on dormancy release supports an involvement of nitrite-dependent reductive pathways in NO biosynthesis, possibly via NR-NiR activity or at least depending on NR activity in the case of exogenous. Accordingly, NR activity was detected concomitantly with a NOS-like activity in soybean and sorghum embryonic axes, both enzymatic activities appeared to parallel the accumulation of NO upon seed imbibition.

41In Arabidopsis, NR is encoded by two homologous genes, NIA1 and NIA2. The relative contribution of these two isoforms to NO production was suggested to differ with a possible predominant involvement of NIA1 in NO production. Despite NO has been demonstrated to break seed dormancy, NR involvement in Arabidopsis seed germination remains unclear. Two distinct research groups assessed the germination characteristics of the nia1nia2 double mutant (also named G′4-3), obtained by. In the first study, G′4-3 seeds were found to be less dormant than wild type seeds, but more dormant in the second. Differences in culture environments of mother plants, germination conditions or duration of seed storage may explain these contrasted results.

42Upon exogenous application of the polyamines, spermine (spm) and spermidine (spd), a rapid NO production from Arabidopsis seedlings has been observed under aerobic conditions. In plants, the tri-amine Spd and tetra-amine Spm are formed by successive additions of aminopropyl groups [resulting from S-AdoMet decarboxylation] to the diamine putrescine (Put; reviewed in ). Put can be synthesized either from L-Arg (by L-Arg decarboxylase) or from L-ornithine (by ornithine decarboxylase). However, as Arabidopsis lacks ornithine decarboxylase activity, polyamines are exclusively produced from L-Arg. Thus, NO biosynthesis from polyamines can be considered as a L-Arg-dependent pathway in Arabidopsis.

43Plant cells are also able to produce NO through hydroxylamine oxidation and this reaction is promoted by reactive oxygen species (ROS) accumulation. Thus, NO might be responsible for the positive effect of exogenous hydroxylamines on seed germination. However, the relevance of such pathway to NO synthesis remains unclear.

44The existence of a root specific plasma membrane nitrite-NO reductase (Ni-NOR) was reported in tobacco (Nicotiana tabacum; ). This enzyme would catalyze the reduction of nitrite into NO in the apoplast and could act in tandem with a plasma membrane-bound NR (PM-NR; ). Its implication has been proposed in several physiological processes in roots, but has not been so far investigated in seeds.

45The non-enzymatic reduction of nitrite to NO can also occur under acidic pH and could be promoted by the presence of reductants:

46This non-enzymatic reaction may be of paramount importance in seeds as an intense NO production was observed during early Arabidopsis seed imbibition next to the aleurone layer. Sodium nitroprusside (SNP) releases dormancy by generating both NO and cyanide. In C24 dormant seeds, the cell impermeable NO scavenger, cPTIO (2-(4-carboxyphenyl)phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide), was demonstrated to efficiently impede SNP dormancy release, suggesting that the apoplast might be either an important pathway for NO movement or a site for NO production.

47Depending on the oxygen availability, several hemeproteins can either act as NO scavengers or NO producers. In hypoxic mitochondria, deoxyhemeproteins can catalyze a NR-independent nitrite reduction into NO using electrons from the electron transport chain. The re-oxidation of NO into nitrite can then occur either non-enzymatically inside the mitochondria, or in the cytosol, through the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent dioxygenase activity of class-1 non-symbiotic hemoglobin (nsHb1) that metabolizes NO into nitrate, which is subsequently reduced into nitrite by NR. These reactions constitute the so-called hemoglobin-NO cycle (displayed in red in Figure 4; ). nsHb1 proteins participate in NO scavenging, thereby playing an essential role in NO homeostasis. Accordingly, modulation of nsHb1 expression in plants was shown to directly impact NO levels at distinct developmental stages including in seeds and in diverse environmental conditions.

48The very active mitochondrial respiration upon seed imbibition may result in an oxygen consumption exceeding the atmospheric diffusion, thus leading to localized hypoxia in germinating seeds. In such conditions, nitrite-dependent NO production may occur in mitochondria and modulate respiration through reversible NO-mediated inhibition of cytochrome c oxidase (COX), thereby regulating oxygen consumption to avoid anoxia. Therefore, this nitrite-dependent NO biosynthesis in mitochondria may be of significant importance in germinating seeds. However, its possible role in NO-mediated dormancy release has not yet been established.

49Overall, current evidence supports the co-existence of several distinct NO biosynthesis pathways in seeds. Their relative contribution is probably highly dependent on both oxygen and ROS levels that may change along the time-course of imbibition. Further investigations will be required to elucidate the regulation of NO accumulation during seed imbibition.

50As for plant hormones, any mechanism directly influencing NO levels besides biosynthesis pathways may have a pivotal role in the regulation of NO signaling. In particular, since NO can react with reduced glutathione (GSH) to form S-nitrosoglutathione (GSNO), GSNO has been proposed to constitute a storage and transport form for NO in plants and seeds. Such modulation of NO storage pool would have a significant impact on NO levels. GSNO can further be metabolized by the GSNO reductase (GSNOR). Accordingly, gsnor mutants have multiple phenotypes suggesting GSNOR involvement in several growth and developmental processes including seed germination.

51Due to its chemical nature, NO is highly reactive and can interact with diverse molecules in plant cells. A number of NO-regulated genes have been identified in plants. These genes encode proteins involved in a wide range of functions from signal transduction to stress responses. However, the main challenge remains to pinpoint the direct molecular targets of NO, which are still poorly documented in plants. However, it is generally assumed that proteins constitute direct relevant NO targets. Besides its capacity to bind to transition metals of metalloproteins, NO can cause protein PTM, such as cysteine S-nitrosylation or tyrosine nitration. These modifications remain poorly characterized in plants and particularly in seeds. However, as discussed below, there is strong experimental evidence indicating that NO signaling in seeds could principally rely on PTM of specific proteins.

52Many S-nitrosylated proteins identified in plants are implicated in various metabolic processes. In dry Arabidopsis seeds, a β-subunit of the mitochondrial ATP synthase complex was found to be S-nitrosylated, suggesting that NO could participate in the regulation of the seed energy status. In wheat seeds, a parallel increase in NO and protein S-nitrosylation was reported during sensu stricto germination. At least 13 modified proteins were detected, but not identified. In recalcitrant Antiaris toxicaria seeds, desiccation impedes subsequent germination by enhancing H2O2 accumulation. This stress is associated with an increased carbonylation and a reduced S-nitrosylation of the antioxidant enzymes of the ascorbate-GSH pathway. Conversely, NO pre-treatments promote germination of desiccated seeds through PTM pattern reversion that enhances antioxidant enzyme activities. The balance between carbonylation and S-nitrosylation of these proteins was proposed to act as molecular switch tuning their activity according to the redox environment.

53In stomatal guard cells, ABA-induced stomatal closure is mediated by the successive accumulation of ROS and NO, acting as secondary messengers in ABA signaling. Even though similar actors are present in seeds, the picture is quite different, as both ROS and NO counteract ABA-inhibition of seed dormancy release and germination. This obvious discrepancy of NO action between seeds and stomata highlights the specificity of the seed signaling pathways (Figure 2).

54In imbibed seeds, the application of ABA biosynthesis inhibitors, fluridone or norflurazon, reduces ABA neo-synthesis and promotes dormancy release and germination. In tomato seeds, the NO scavenger, cPTIO, was shown to prevent germination stimulation by fluridone. Conversely, in dormant Arabidopsis C24 seeds, SNP enhances the positive effect of norflurazon on germination and also decreases seed sensitivity to exogenous ABA. Taken together, these results suggest that NO reduces both ABA accumulation and sensitivity. In agreement, pharmacological experiments demonstrated that NO enhances CYP707A2 gene expression in Arabidopsis seeds. Indeed, during the first stage of seed imbibition, a rapid accumulation of NO, possibly at the endosperm layer, was suggested as required for rapid ABA catabolism and dormancy breaking. A similar NO accumulation during imbibition was also observed in germinating seeds from other species. Recently, in Arabidopsis, NO was suggested to act upstream of GA in a signaling pathway leading to vacuolation of protein storage vacuoles in aleurone cells, a process inhibited by ABA. Since the growth of isolated embryos was unaffected by NO donors or scavengers, the endosperm layer might be the primary site of NO synthesis and action in seeds, and in accordance was shown to perceive and respond to NO. Besides its effect on the hormonal balance, it has been speculated that NO may accelerate flux through the pentose phosphate pathway by indirectly increasing the oxidation of NADPH. An increase in glucose catabolism via this pathway may in turn promote dormancy release.

55Several lines of evidence suggest that NO crosstalk with ABA and ethylene may involve protein modifications. Among the proteins recently identified as candidates for a regulation by tyrosine nitration in Arabidopsis seedlings, at least two may be involved in the interplay between ABA and NO in seeds. The first one is the Moco sulfurase ABA3 that catalyzes the conversion from the de-sulfo to the sulfo form of the Moco. The de-sulfo form of Moco (also call the “oxo” form) is the co-factor of NR, involved in nitrite and NO generation in plants while the sulfo form is the co-factor of the aldehyde oxydase required for the last step of ABA synthesis. If proven, modulation of ABA3 activity by nitration could affect the equilibrium between ABA and NO production in plants. The second protein is the E3 SUMO ligase AtSIZ1 recently demonstrated to stimulate NR and NR-NiR activities, and negatively regulate ABA signaling by ABI5 sumoylation. Thus, such modifications could have an important impact in seeds. Similarly, PTM contribution in the NO regulation of ethylene action has been also reported. In Arabidopsis, the up-accumulation of NO under hypoxia stimulates ethylene biosynthesis, possibly through PTM of key enzymes such as ACS and ACO by S-nitrosylation. In contrast, ethylene biosynthesis can be reversibly inhibited by NO through S-nitrosylation of methionine adenosyltransferase (MAT), leading to the reduction of the S-AdoMet pool.

56S-AdoMet is the precursor of ethylene and polyamines, thus a negative feedback regulation may exist between ethylene and the polyamine-dependent NO biosynthesis. Consistently, NO and ethylene accumulation are negatively correlated in ripe fruits. In addition, exogenous Spm was shown to reduce ethylene production in apple seeds. Accordingly, an antagonism may exist between a positive polyamine effect mediated by NO and a negative effect due to a competition with ethylene biosynthesis for S-AdoMet. Furthermore, a copper amine oxidase (CuAO1) involved in polyamine catabolism has also been shown to regulate NO biosynthesis and participate to ABA signaling. Indeed, seedlings of Arabidopsis cuao1 mutant are impaired in both polyamine and ABA-induced NO synthesis, and mutant seeds also display a reduced sensitivity to exogenous ABA during germination.

57As mentioned above, in Brassicaceae species, ethylene positively regulates seed germination by stimulating the weakening and rupture of seed testa and endosperm by counteracting the inhibitory action of ABA on radicle protrusion. In apple embryos, inhibition of ethylene biosynthesis prevents the promotion of dormancy release and germination by NO donors. Dormancy breaking of apple seeds by NO induces a transient production of ROS, stimulating ethylene accumulation thanks to an increase in both ACS and ACO activity. NO may also act on ethylene signaling since EREBPs were described as a class of transcription factors induced by NO. Moreover during tobacco seed germination, EREBP-3 that is transiently induced just before endosperm rupture is stimulated by ethylene and inhibited by ABA. Therefore, a synergic link seems to exist, at different levels, between NO and ethylene during seed germination, that counteracts ABA action.

58Significant advances have been recently obtained in the understanding of the ABA and ethylene metabolism and signaling pathways. In contrast, current knowledge on NO biosynthesis, signaling and action is far too incomplete, especially in seeds, and would require further investigation. Future research efforts should also lead to the identification of downstream target genes of signaling components, in order to fully understand how ABA is able to induce and maintain dormancy, or ethylene to stimulate germination. Moreover unraveling the role of post-translational mechanisms will be particularly crucial to developing a deeper understanding of hormonal pathways and deciphering NO regulatory network.

59Nitric oxide and ethylene crosstalk with ABA involves interactions at multiple levels in metabolism and signaling pathways. It would be important to discriminate the hierarchy among these signaling pathways, identify major regulatory nodes and determine whether the environmental factors, which regulate germination and dormancy, modulate this hierarchy.

60Moreover, control of seed dormancy and germination involves distinct physiological processes, in tissues of different origin, to achieve a coordinated regulation of embryo arrest or growth and surrounding structure maintenance or rupture. Although hormonal signaling networks in seeds and whole plants share common components, sets of specific regulatory factors, among which only few are known, are likely working in restricted seed territories. Current research combining genetic tools and recent technologies including microdissection, transcriptome profiling, high-throughput proteomics, metabolomics, and system biology, should help to identify missing regulatory components and unravel complex interactions between signal transduction pathways.

61The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.