The Seed and the Metabolism Regulation

The Seed and the Metabolism Regulation: History

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Subjects: Plant Sciences

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Seeds are the reproductive units of higher plants. They have a significant place in agriculture and plant diversity maintenance. Because they are dehydrated, they can remain viable in the environment for centuries. The dry seed is a metabolically inactive organism, but well organized to protect its components and enter intensive repair to restore metabolic activities upon imbibition for the completion of germination.

seed
hydration force
energy
germination
dormancy
ageing
PM H+ ATPase

1. Dry Seed: Well-Organized to Resist

1.1. The Seed, a Special New Individual

The seed is composed of an embryo surrounded by reserve material and covering layers. It represents the plant dispersion organ formed by sexual reproduction as well as the new individual. The seed therefore occupies a critical position in the life cycle of the higher plant. The success of the establishment of the new individual is determined by physiological and biochemical features of the seeds in response to their environment.

The angiosperm seed generally consists of the embryo, the result of the fertilization of the egg cell and one of the male pollen nuclei, the endosperm, which is the result of the fusion of the two polar nuclei with the second pollen nuclei, and the perisperm, corresponding to the nucellus and the testa or seed coat formed from the integument around the ovule. The extent to which the endosperm or perisperm persists varies between species. For example, the Arabidopsis embryo is surrounded by an endosperm layer while the sunflower embryo is not (Figure 1). When the testa is underdeveloped, the outer structure being the pericarp or fruit coat, the dispersal unit is not a seed but a fruit, as in the case of sunflower and wheat. The embryo, which represents the new individual, is comprised of the embryonic axis and one or two cotyledons. The axis includes the embryonic root (radicle), the hypocotyl, and the shoot apex (plumule). Thus, as the seed corresponds to a diverse composition of such complex tissues that have distinct developmental programs [6], studying seeds implies the study of these programs and their coordination in time and space to achieve germination [7].

Figure 1. Seed morphology scheme presenting longitudinal section of sunflower fruit as a non-endospermic seed surrounded by the pericarp and Arabidopsis thaliana as an endospermic seed.

Desiccation represents the last phase of seed development and corresponds to a huge loss of water content that decreases the seed water percentage in orthodox seeds to less than 10% of the dry weight (DW), depending on species (e.g., mature sunflower seeds contain 4% g H₂O/g DW [1]). Such a low water content changes the cytoplasm from a fluid to glassy state, which severely reduces molecular diffusion and mobility, preventing chemical reactions [8].

Seed germination starts with water uptake and ends with radicle protrusion. The seed water absorption rate corresponds to three phases during which controlled physiological processes take place. As shown in the Figure 2.

Figure 2. Seed imbibition curve showing the three characteristic phases with their main biological processes.

However, germination is not generally possible for mature seeds because they are generally dormant. They need to undergo a post-maturation phase, called after-ripening, a period that allows them to acquire the capacity to germinate.

2.2. Water, “Matrix of Life”

If water is the matrix of life [20], dry seeds can hardly be considered as alive and yet they bear life in the form of the embryo. Water is an essential participant in the chemistry of life by sustaining the biochemistry of the cell. It acts as a liquid and solvent for biochemical reactions, but also influences macromolecule structures [21]. Water participates in the catalytic function of proteins and nucleic acids and physically in hydrophobic associated protein folding and complex formation through the hydrogen bond [22]. It was shown that in desiccated Arabidopsis seeds the chromatin is highly condensed and can be de-condensated after hydration [29]. The property of the seed to undergo a reversible chromatin condensation/de-condensation enables to withstand desiccation and the entry in active metabolism during imbibition.

Thus, high water binding forces in dry seeds are responsible for the lack of stability and activity of biomolecules causing low metabolism and energy. As membrane reorganization is one of the first events in the initiation of cell energization, more water (>24%) is needed to activate protein reorganization and activity for full plasma membrane and mitochondrial energy restoration (Figure 3).

Figure 3. Early cellular events during imbibition as related to water binding forces.

2.3. Respiration Resumption

Holding respiration may represent the major process allowing seed longevity. This is achieved with the support of several seed features. The seed structure itself may contribute to holding respiration by O₂ uptake limitation due to the space occupation by reserve molecules and the presence of the seed coat (for review [13,30]). More importantly, mitochondria in dry seeds are called promitochondria as their internal membranes are underdeveloped with a low number of cristae and low protein content [31,32]. Several studies using different biochemical approaches, such as adenylate pool or adenylate energy charge (AEC) ratio ((ATP + 0.5 ADP)/(AMP + ADP + ATP)), oxygen uptake, tricarboxylic cycle enzyme activity measurements, or cytological investigations, converge to state that respiration is reduced to a very low level in dry seeds and that the hydration induces an increase in mitochondria components and activity [33,34,35,36,37,38,39]. The proliferation and differentiation of mitochondria, called ‘mitochondrial biogenesis’, occur progressively upon imbibition [10,37,38]. These are considered as the prerequisite for the full reactivation of mitochondria and subsequent energy supply for germination [31,32]. However, isolated promitochondria were shown to be able to generate ATP and a membrane potential by oxidizing supplied succinate and/or NADH [31,40]. Such metabolic activity may be decisive at the onset of imbibition to help the biogenesis process to take place. Moreover, promitochondria seem to have an import apparatus ready for mitochondrial biogenesis [32]. In fact, the electron transport system is activated immediately after the initiation of imbibition and is dependent on AMP, ADP, cytochrome C oxidase, and ATPase that were recovered from dry seeds [36,41].

2.4. Plasma Membrane Potential

One of the fundamental properties of living cells is the establishment of an electrical potential difference across the plasma membrane. In dry seeds, the transport of ions is not possible due to the absence of water as a conductive fluid, but also due to the loss of the integrity of membranes and their protein components. Membrane deterioration has been highlighted by the high electrolyte leakage rate measured in dry seeds and many studies have reported that seed hydration induced a membrane leakage decrease, confirming that the cell membrane was repaired upon imbibition. It was shown that the leakage decrease depends on the moisture content of the seeds, being undetectable above 24% [9]. In fact, 20% water corresponds to the minimum amount of water needed to create a hydrophilic layer that stabilizes the organization of lipids in a bilayer [43]. Such a water content corresponds to phase I of germination (Figure 3), while lipid metabolism and repair were shown to take place during phase II [44,45], suggesting the need for an intact membrane before any repair event can be started. It also corresponds to the start point of the full hydration of proteins that should include membrane ion channels and transporters.

In plants, plasma membrane potential is driven by two major components, K⁺ gradient and H⁺ ATPase activity. The plasma membrane (PM) H⁺ ATPase is responsible for membrane energization by extruding H⁺ protons, which is necessary for the activity of nutrient transporters associated to electrochemical H⁺ gradient [46]. It was demonstrated that PM H⁺ ATPase is essential for growth since the knockout of the two major PM H⁺-ATPase genes, AHA1 and AHA2, is lethal in Arabidopsis embryos [47]. The role of PM H⁺ ATPase in physiological processes is regulated by post-translational modifications which correspond to the phosphorylation of C terminus residues [48,49]. It was shown that PM H⁺-ATPase presents two activity states, auto-inhibited and upregulated, depending on the coupling ratio between ATP hydrolysis and H⁺ pumping [48,50,51]. The basal state has a low coupling ratio, while the activated state has a high ratio [51]. Several signals, such as sugar or light, activate the phosphorylation of C terminus, allowing the activation state corresponding to high affinity for ATP [48]. Although H⁺-ATPase has not been actively studied in seeds, recent work has shown that high H⁺-ATPase activity was associated with germination capacity while dormant state was associated with low activity in sunflower seeds [52]. Considering that the imbibition of dry seeds is driven by the physical properties of water in the reorganization and remodeling of PM, including the proper folding of H⁺-ATPase as a protein component, and given the central role of mitochondria and reserve mobilization, the ATP/ADP ratio of the cell may be the major parameter affecting PM H⁺-ATPase activity in the seed germination process. Further investigations are needed to discover the pathways by which this protein is phosphorylated and dephosphorylated in the regulation of dormancy and germination.

3. Seed Dormancy: Higher Level of Resistance

After-ripening has fascinated researchers because dormancy is alleviated in dry conditions, suggesting that some processes operate in the dry seed. Biological reactions have been investigated and transcriptional programs have been proposed to be involved in the regulation of after-ripening-mediated seed dormancy alleviation in several seeds [53,54,55,56,57]. Given the restricted molecular mobility due to the glassy state in dry seed cells, the existence of a hydrated pocket within the cell enabling gene transcription has been hypothesized [53]. To address this issue, Meimoun et al. [1] investigated transcriptomic changes after the after-ripening period in sunflower seeds using two protocols, one allowing dormancy alleviation but not the other, in order to differentiate between changes in gene expression associated with dormancy alleviation and those associated with storage only. They showed that there is no significant variation between conditions, suggesting that gene expression did not take place during after-ripening, in agreement with the absence of metabolic activity in dry seeds [1].

Non-enzymatic oxidations are possible in low hydrated seeds and represent the most plausible lead to explain the observed molecular changes reported during after-ripening [60]. Indeed, mRNA oxidation was shown to be associated with dormancy release during after-ripening in sunflower and wheat [61,62], which alters the stability of stored mRNAs, being finally degraded or translated into non-functional proteins [63]. However, if a fraction of stored mRNA is inactivated, the one involved in germination has to be protected from oxidation.

3.2. Internal Determinants of Dormancy

It is well established that dormancy is regulated by the hormonal balance between the main positive regulator abscisic acid (ABA) and negative ones, such as hormones like gibberellic acid (GA), ethylene (ET), auxins, or brassinosteroids, as well as some other molecules, like ROS or nitric oxide (NO). The involvement of each of them and their interactions in the whole process of germination depend on the structure of the seed and the environment. Nevertheless, ABA represents the highly conserved component of the process across species and the unique dormancy determinant as opposed to the multiple stimulants of germination. Thus, to illustrate the regulation and function of hormones in the physiology of germination, without elaborating on all the hormones and their complex signaling, the case of the ABA is the most appropriate.

Mitochondria play a central role in energy supply and they are also associated with ABA sensitivity based on works showing that several mutants of RNA processing for subunits of the electron chain display reduced ABA sensitivity. This regulation involves retrograde, anterograde, and inter-organelle signals in the transcription control of the ABA biosynthesis gene, NCED [75]. On the other hand, Paszkiewicz et al. [10] have shown that mitochondrial dynamics associated with germination condition was slightly affected by ABA treatment, arguing that mitochondrion reactivation depends only on the physical conditions of hydration and temperature. Based on these works, the optimal differentiation and functioning of mitochondrion are associated with an ABA sensitivity decrease. Accordingly, it is easy to consider that in dormant seeds, the impairment of mitochondrial activity occurs. However, it has long been established that inhibitors of oxidative phosphorylation such as cyanid can break dormancy. This paradox has still not been elucidated. The activation of the pentose phosphate pathway, the metabolic pathway that supplies reducing energy to cells, has been the most plausible hypothesis proposed [76]. Indeed, in reduced mitochondrial activity, glycolysis is activated to obtain ATP, a phenomenon known the “Pasteur effect”, leading to pyruvate production and the accumulation of fermentation by-products. Thus, anaerobic metabolism facilitates reserve breakdown and it might operate, in normal conditions, at the onset of germination when the mitochondria are not yet fully reactivated. All these data point to the importance of cell metabolism and energy regulation for successful germination.

On the other hand, it was proposed that the ABA inhibition of growth in germinating Arabidopsis seeds is driven by its inhibitory action on PM H⁺-ATPase activity [77]. ABA inhibition was less effective in Arabidopsis mutants with increased capacity for H⁺ efflux, suggesting that cytosolic acidification due to reduced H⁺-ATPase activity was the main mechanism driving growth inhibition [77]. Similarly, in sunflower, ABA induced the inhibition of PM H⁺-ATPase in non-dormant seeds, which display hyperpolarization and subsequent membrane energization. Meanwhile, in dormant seeds, PM H⁺-ATPase activity was reduced even if the corresponding proteins were present and the levels of ATP were comparable to that on ND [52]. PM H⁺-ATPase activity is also regulated by ROS and ethylene in the opposite way [52], as proposed in the model presented Figure 4.

Figure 4. A model for seed cell polarization regulation in the control of dormancy in sunflower [52]. ABA, abscisic acid; PrxII, cell wall peroxidase III; POX, cytosolic peroxidase; ET, ethylene; Vm, PM potential.

At the scale of the whole organ, specialized tissues play a critical role upon imbibition. The most striking events are the transport of hormones from/to the different tissues of the seed and consequently their specific contribution in each tissue to regulating seed germination. For example, in endospermic seeds, ABA is produced in the endosperm and transported to the embryo, while GA goes in the opposite way [78].

3.3. Environmental Impact on Dormancy

Environmental factors are of high importance in the awakening of the seed. Their effect on seed performance was shown to surpass genetic impact [80]. Temperature and soil moisture oscillations are the major players under natural conditions. Indeed, alternating temperatures more than constant ones can promote germination via the interplay between ROS signaling and hormones [81]. On the other hand, light and nitrate also play important roles. Their effects are associated in dormancy cycling [85,86,87]. Seed sensitivity to both of them depends on the season and depth of dormancy. A low concentration of nitrate (around 0.1 mM) is able to promote seed germination in several species [85]. Several evidences converge towards nitrate induction of CYP707A2 leading to ABA decrease more than GA biosynthesis in dormancy breakdown [85]. However, GA biosynthesis gene involvement has also been reported in response to environmental cues in Arabidopsis seeds from lab but also soil seed bank experiences [88,89]. Moreover, the analysis of the whole transcriptome change by nitrate treatment during seed imbibition showed the upregulation of genes involved in nitrate assimilation and transport, hormone metabolism, and energy, such as Glucose-6-phosphate dehydrogenase2, highlighting the importance of the pentose phosphate pathway [90]. In fact, dormancy is tightly regulated in natural conditions as in the soil, when seeds experience several scenarios of temperature, light, nitrate, and moisture, as well as microbial environment. The latter corresponds to a wide range of microbes, as pathogenic ones can induce decrease in seed longevity due to infection, and others can influence seed dormancy by breaking down the seed coat [12,91].

4. Seeds: The Ability to Recover from Ageing

4.1. Seed Ageing

Seed ageing was defined as the loss of seed quality and viability over time [93]. Aged seeds germinate poorly giving abnormal seedlings or ultimately are unable to germinate. Orthodox seeds are resistant to ageing for very long time because they have a very low water content, resulting in reduced cell metabolism especially respiration which is responsible for the major production of ROS [94]. Indeed, as for the dry after-ripening process described above, enzymatic reactions and respiration are restricted by the lack of free available water preventing cellular damage. It was shown that long term storage resulting in seed loss of viability is associated with the impairment of mitochondrial activity and protein synthesis machinery [95,96]. Mitochondrion membrane integrity was identified as the primary target for ageing leading to the deregulation of its oxidative properties [97]. ROS are considered as the major cause of seed deterioration due to the oxidation of its components [98].

4.2. Seed Priming

Seeds possess effective repair machinery to cope with ageing-associated oxidative damage. Several non-enzymatic antioxidants have been proposed to be determinant in seed longevity, e.g., glutathione or ascorbic acid [107,108]. The antioxidant enzymes are also of importance in ROS detoxification, such as catalase, superoxide dismutase, ascorbate peroxidase, or glutathione reductase [109,110]. Other enzymes acting on specific macromolecules are also activated, such as DNA or protein repair enzymes [101,111,112]. Such machinery operates when seed hydration occurred, and its efficiency depends on the plant species and the extent of ageing damage. Based on this feature, a priming technique has been developed to improve seed quality from alterations caused by several stresses. Priming treatment consists of seed pre-hydration with a controlled amount of water which does not allow radicle elongation, i.e., a water amount corresponding phase II of the germination sensu stricto, which is sufficient to trigger the reparation processes. The addition of beneficial molecules, such as antioxidants or hormones, during priming treatment can further increase seed reparation and subsequent quality. Several priming techniques have been developed depending on the plant species and subsequent use. They all lead to an improvement of seed performance under variable environmental conditions [113]. It was shown that repair mechanisms and oxidative management in primed seeds represent the main processes associated with priming induced germination improvement, but DNA replication, cell cycle advancement, the modification of the membrane structure, and restoration of mitochondrial integrity were also proposed to explain the priming effect in germination improvement [114]. In fact, the seed engages growth preparation processes during imbibition while maintaining the desiccation tolerance machinery which allowed a successful dehydration after the priming treatment. The dried primed seed is ready to grow better, even under stress conditions.

5. Conclusion

Understanding the successful entry and exit from desiccation is fundamental for the improvement of seed germination in the coming challenging conditions due to global warming. The application in plant germplasm conservation in seed banks is of high importance in the maintenance of genetic resources for food and environment security. In this review, several layers of regulations of seed performance were shown, from the organization and physical protection of cell components to the regulation of several signaling processes in a coordinated crosstalk. However, their implementation and coordination of these mechanisms during seed development deserve more investigations.

This entry is adapted from the peer-reviewed paper 10.3390/biology11020168

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