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Germination ability is regulated by a combination of environmental and endogenous signals with both synergistic and antagonistic effects. Nitric oxide (NO) is a potent dormancy-releasing agent in many species, including
Survival of plant species mainly relies on the sexual reproduction which gives rise to new populations. During this process, the adult angiosperm plants produce flowers which upon fertilization give rise to seeds, the main unit of dispersal of flowering plants. In the plant life cycle, the seed and seedling stages are key developmental stages conditioning the final yield of crops. Indeed, seed dormancy, viability, and germination vigor are among the main concerns for agricultural productivity. High vigor seed lots display a low dormancy and lead to seedlings able to withstand extreme stress conditions. If not completely released, dormancy will negatively influence seed germination, which is detrimental to crop yield. However, from an agronomical point of view, lack of dormancy is not a desirable trait as it may lead to pre-harvest sprouting (
Numerous distinct nitrogen-containing compounds have been shown to positively influence seed germination especially by releasing seed dormancy and improving seed vigor in a wide range of species (
Under natural conditions, an appropriate timing of seed germination is determinant to ensure optimal growth conditions for the young seedlings and guarantee the survival of the species (
Abscisic acid (ABA) is considered as the pivotal hormone responsible for the induction and maintenance of seed dormancy (
Seed germination is temporally defined as the sequence of molecular and physiological events initiated upon imbibition of non-dormant seed and leading to the radicle protrusion through the seed external envelopes (testa and endosperm) that marks the end of germination
If the seed encounters suitable conditions for germination during its life, it may, if still viable, allow the young seedling establishment. But as a consequence of aging, the seed germination vigor can be severely affected. In other words, the capacity of a seed lot to germinate rapidly, uniformly and in a wide range of environmental conditions can be impaired or destroyed. As the seed germination process mainly relies on stored mRNA and proteins (
Nitrate (
Exogenous treatments with nitrates were shown to promote seed germination in
The reduced dormancy of NR deficient seeds, impaired in nitrate assimilation, along with the finding that glutamine, another nitrogen source did not affect seed germination suggest that the effect of nitrate is unrelated to plant nutrition (
Nitric oxide is a gaseous diatomic free radical detected at low levels in the atmosphere. It is also present in the soils at a concentration depending on the micro-biotic environment (
Recent data disclosed that the improvement of dormant-seeds germination provided by exogenous treatments with various nitrogenous molecules, including nitrate, and nitrite, most presumably occurs through NO production (
Indeed, NO is well known to release seed dormancy in numerous species (
In tomato seeds, the NO scavenger, carboxy-2-phenyl-4,4,5-tetramethylimidazole-1-oxyl 3-oxide (cPTIO), was shown to prevent germination stimulation by fluridone, an ABA synthesis inhibitor (
A rapid accumulation of NO, possibly in the endosperm layer, during the first stage of
As a conclusion, NO is a likely player of a signaling pathway that promotes loss of dormancy and has been suggested to behave as an endogenous regulator of this process. However, the direct targets of NO in seeds remain unclear. Nonetheless, some consequences of NO accumulation on seed metabolism have been highlighted and pinpoint an implication in the regulation of ABA metabolism.
In parallel to NO, ROS have emerged as key players in the control of seed dormancy and germination (
Recently, it has been proposed that ROS might coordinate the reduction of ABA-imposed dormancy with the onset of GA-stimulated germination (
In stomatal guard cells, one of the well-established signaling pathway for ABA-induced stomatal closure involve the successive accumulation of ROS and NO, acting as secondary messengers of ABA signal (
Nitric oxide is well known to play a dual role in stress responses in plants (
In seeds, pharmacological experiments highlighted that NO did not significantly influence the germination of non-dormant (fully after-ripened)
Nitric oxide (NO•) is an uncharged, gaseous and lipophilic free radical that can readily diffuses across biological membranes. Thus, NO can interact with numerous distinct molecules in plant cells and therefore acts as a signaling element. Free NO• is a transient compound displaying a high reactivity toward other free radicals (e.g., superoxide anion) and transition metal ions (e.g., iron;
Distinct pathways have been proposed to account for NO generation in plant cells (Reviewed in
Hemoglobins are well known in the animal kingdom for their role as oxygen carrier. In plants, non-symbiotic hemoglobins (nsHb) are divided into two main classes with distinct properties. Class 2 nsHb are the only proteins with an affinity for oxygen fitting with a direct role in oxygen storage and supply (
The over-expression of
Previous studies relying on modulation of nsHb1 expression in seeds mainly focused on seed maturation (
The investigations on the mode of action of NO in plant cells still suffer from several technical limitations. Indeed, the improvement of NO detection and quantification, pharmacological approaches and biochemical assay for the analysis of NO-induced PTMs are still required.
Most of the known implications of NO in plant physiology were first highlighted through pharmacological experiments employing NO donors and/or NO scavengers (
In animal cells, the absence of nitrate reduction pathways allows the use of assay based on nitrogen oxides, especially nitrite, quantification to evaluate NO production (nitrate and nitrite being considered as by-products of NO production and subsequent oxidation). In plant, such methodology is excluded due to the existence of an active nitrate assimilation pathway responsible for most of nitrite production. Consequently, distinct other methodologies have been applied including fluorescent probes based detection, EPR spectroscopy, electrochemistry, ozone based chemiluminescence, laser photoacoustic, mass spectrometry and the oxyhemoglobin assay. A short discussion on some of these techniques is provided below, for a complete review refer to (
Several distinct fluorescent probes can be used to investigate NO biosynthesis or release by a given tissue. The diaminofluoresceins (DAF; DAF-FM, 4-amino-5-methylamino-2′,7′-difluorofluorescein) or the diaminorhodamine 4M (DAR-4M) and their cell permeable forms DAF diacetate (DAF-2DA, DAF-FM DA) and DAR-4M acetoxymethyl ester (DAR-4M AM) are the most commonly used (
Electron paramagnetic resonance spectroscopy is a more specific method that can be applied to the direct detection of radical species including NO both
Another widely used approach is based on the chemiluminescent reaction between gaseous NO and ozone. This technique can allow the direct quantification of NO release from a tissue placed in a sealed compartment under a gaseous flux driving the gas released in the environment to an analyser. NO-specific electrodes are also available but are also difficult to apply to the study of the tiny
Overall, despites all the existing techniques, an accurate detection and quantification of NO generation in plant tissue remain difficult. In addition, most techniques require preparation steps or experimental conditions that can lead to undesirable signal. Thus, as for the pharmacological experiments, a cross validation with at least two distinct quantification methods is highly recommended (
The genetic resources available to investigate NO signaling remain restricted due to our limited actual knowledge of NO biosynthesis pathways in plants. Thus, most of the studies rely either on a pharmacological approach (as discussed previously) or on mutants affected in NO availability although their NO levels are not always explained. Some mutants somehow related to NO homeostasis in plants (e.g.,
Nitrate reductase, being the only identified enzyme proven to be directly involved in NO biosynthesis, NR-deficient mutants has been extensively used, especially the G′4–3 mutant in
Several other mutants known as affected in NO levels have also been used to investigate NO signaling in
Alternative strategies have been developed to get around the known limitations and pursue the investigations on NO signaling in plants. Promising examples include the use of transgenic lines with altered hemoglobin expression (
Aside from the long lasting question concerning the relevant NO sources in seeds, the re-constitution of NO signaling pathways require the identification of the NO biological targets. Yet, direct molecular targets of NO remain poorly documented in plants. NO could regulate physiological processes by affecting gene transcription. Indeed, several NO-regulated genes, involved in different functional and biological processes, have previously been described (
Nitric oxide-mediated
In wheat seeds, a parallel increase in NO and protein
Tyrosine nitration consists in the addition of a nitro group (–NO2) resulting in an alteration of diverse protein functions. The very fast reaction between NO and O2- gives rise to peroxynitrite (ONOO-) which is considered as a potent oxidizing and nitrating agent (
Most of the analysis published up to date pinpoint ABA content as a major determinant of dormancy release or maintenance. It appears that the decision to pursue the transition toward germination or maintain a dormant state can be taken during seed imbibition depending on environmental parameters. Thus, the control of ABA levels and sensitivity during early imbibition appears of paramount importance. During this phase, both NO and ROS accumulation has been reported. The intensity of the generation of these radicals could depend on both endogenous and environment cues. In turn, the interplay between ROS and RNS would determine both the extent of ABA catabolism (via the regulation of
However, despite a general consensus regarding NO importance in seed physiology, the pathways involved in its biosynthesis remain uncertain. This observation presumably reflects the complexity of the regulation of NO biosynthesis in plants. Indeed, multiple different endogenous sources all potentially depending on environmental and/or molecular parameters may contribute to NO accumulation in seeds. Moreover, the relevant reactions in seeds may be significantly different from those described at other physiological stages including the non-enzymatic reactions that may occur in the apoplast next to the aleurone layer (
In a similar way, we are firmly convinced that NO-related PTMs, namely tyrosine nitration and cysteine
The 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.
This work was supported by a doctoral and post-doctoral fellowship to Erwann Arc from the French Ministry of Higher Education and Research and from European Commission funding (EcoSeed project, KBBE), respectively. The post-doctoral fellowship to Marc Galland was funded by the French Ministry of Industry (FUI NUTRICE).