David Macherel and Marie-Hélène Avelange-Macherel, UMR 1191 Physiologie Moléculaire des Semences, Université d’Angers, France

November, 2012

Seeds Are Anhydrobiotes

The evolutionary success of angiosperms is largely due to the invention of the seed, a development that offered a major advantage for survival and dispersal of species. In most species, seeds undergo almost total desiccation in the late stage of their development. Seeds are thus anhydrobiotes (i.e., organisms capable of surviving desiccation). Although anhydrobiosis can be found occasionally in other eukaryote lineages, it is a widespread property in most plant seeds, the so-called orthodox seeds. (Desiccation-sensitive seeds, such as walnut, coffee, and many tropical seeds, are called recalcitrant.) In the dry state, seeds are metabolically inactive, highly tolerant to environmental conditions, and can remain viable for long periods of time (2000-year-old date seeds actually produced healthy plants; Sallon et al. 2008). Such properties are key factors for the dispersion of angiosperm generations over space and time. When appropriate conditions (water, temperature, oxygen) are met, and in the absence of dormancy, seeds rapidly resume metabolism upon imbibition and shift to a new developmental program leading to germination and seedling establishment. It is noteworthy that agriculture, and thus civilizations, emerged because of, and still rely on, the capacity of seeds to tolerate desiccation. Since water is essential for the continuation of life, and even for the maintenance of biological structures, surviving desiccation is not trivial and requires sophisticated cellular adaptations, notably at the molecular level. The mechanisms underlying desiccation tolerance in seeds and other anhydrobiotes are thus multifaceted, and involve a concerted array of protective and repair mechanisms, including the accumulation of membrane and macromolecule protectants (sugars, stress proteins) and the control of metabolism and oxidative load (see reviews by Hoekstra et al. 2001; França et al. 2007). Anhydrobiosis is thus a vital property of seeds, with a profound impact on cellular physiology.

The Importance of Respiration in Seeds

While many seeds remain green and photosynthetically active during their development (see below), chlorophyll is usually lost during late seed maturation. Therefore, seed germination (defined as radicle protrusion), as well as early seedling growth, is entirely heterotrophic, relying on respiratory metabolism. The oxidation of carbon reserves (starch, lipids, proteins) accumulated during seed maturation provides the energy required for germination, emergence, and development of the seedling until transition to autotrophy. In nature, these events usually take days or weeks depending on species and environmental conditions, highlighting the importance of respiratory metabolism for seedling establishment (i.e., reaching autotrophic status). Accordingly, while oxygen sensitivity of seeds can differ among species, oxygen is absolutely required for germination (Al-Ani et al. 1985). Such energy-demanding processes cannot rely on fermentative metabolism, whose energy yield is too low. In addition to the dependence of germination upon respiration, the fact that seeds are anhydrobiotes gives rise to an unusual situation for mitochondria, which is sketched in Figure 1.

Figure 1  Mitochondria are exposed to water stress, desiccation, and hypoxic conditions in the course of seed development and germination. The scheme shows a typical timeline of seed development, storage, and germination, with changes in development of dry weight, water content, and respiration. Mitochondria are exposed to severe water stress during late seed maturation, desiccation, and imbibition. They also need to be functional at the onset of imbibition, and thus to retain sufficient integrity during storage in the dry state, which can last for long periods of time. In many species, mitochondria operating during seed maturation until late desiccation, as well as during seed imbibition and germination, are likely to face internal hypoxic conditions, requiring a control of respiration to prevent anoxia. 

Mitochondrial energy transduction must operate in fluctuating water contents, (i.e., during dehydration and imbibition phases), and it appears crucial to preserve the structure and function of the organelle throughout the dry state. In summary, respiration plays a major role in the physiology of seeds, which are heterotrophic organisms capable of anhydrobiosis.

Properties of Seed Mitochondria

Little is known about the precise structure and functional state of mitochondria within a dry seed, although electron microscopy images reveal mitochondria with few cristae and a light matrix, suggesting an undifferentiated state (pro-mitochondria). However, since resumption of respiratory oxygen consumption is recognized as one of the earliest events during imbibition (see Bewley 1997; Benamar et al. 2003), mitochondria are expected to become rapidly competent in oxidative phosphorylation once seed tissues are hydrated. Studies with organelles isolated from imbibing seeds suggest that mitochondria rely initially on the oxidation of exogenous NADH and succinate to provide cellular ATP, a mechanism that uses a simple but efficient energy transduction pathway, since complex I and a full TCA cycle are not required (Logan et al. 2001; Benamar et al. 2003). Interestingly, experiments with imbibing pea embryos subjected to dehydration and re-imbibition revealed that the capacity of mitochondria to recover their integrity and function was well linked to desiccation tolerance (Wang et al. 2012).

Mitochondrial repair and biogenesis mechanisms, such as protein import, become rapidly operational, leading to the progressive restoration of mitochondria with more cristae and a full TCA cycle (Howell et al. 2006). Large-scale gene expression analysis in Arabidopsis indicated that transcription of genes encoding biogenesis functions (RNA metabolism and import components) preceded a later cascade of gene expression encoding the bioenergetic and metabolic functions of mitochondria (Law et al. 2012). Because mitochondria expressing a GFP reporter could not be detected during stratification and early germination, it was concluded that the organelle could not become functional in energy transduction until after several hours of imbibition, during which extensive biogenesis was required (Law et al. 2012). These observations contrast with the fact that mitochondrial respiration resumes very rapidly during imbibition, arguing for the maintenance of functional mitochondria in dry seeds (Macherel et al. 2007). Further investigation is thus required to decipher the exact nature of mitochondrial function and biogenesis during germination.

Pea seed mitochondria have been shown to harbor a small heat-shock protein (HSP22) and a late embryogenesis abundant protein (LEAM) likely related to desiccation tolerance (Bardel et al. 2002). Small heat shock proteins are ubiquitous stress-induced proteins that are especially abundant in plants, with representatives in the cytosol and most organelles. In vitro evidence indicates that small HSPs play a major role as passive chaperones, reducing aggregation of misfolded proteins (Sharma et al. 2009), and/or as membrane stabilizers (Horvath et al. 2008). However, the function of the mitochondrial sHSP, which is highly expressed upon heat or oxidative stress in vegetative tissues or during seed development, has not been elucidated. Late embryogenesis abundant (LEA) proteins are highly hydrophilic proteins that accumulate to high concentrations during late seed maturation, and which are also found in most anhydrobiotes, suggesting a prominent role in desiccation tolerance (for a comprehensive review see (Tunnacliffe and Wise 2007). Biophysical and biochemical analysis of LEAM revealed that the protein was intrinsically disordered (a frequent feature of LEA proteins) and was located in the matrix space of mitochondria, but was able to fold into amphipathic alpha-helices during drying (Tolleter et al. 2007). Molecular modeling and biophysical analyses suggest that, at low hydration, LEAM inserts into the inner leaflet of the mitochondrial membrane, parallel to the plane, exposing the hydrophobic face of the helix to the core of the membrane, and being stabilized by electrostatic interactions between the charged residues of the protein and of the polar moiety of phospholipids (Figure 2). Interestingly, putative orthologs of LEAM can be found in other plant species, and a related mitochondrial LEA protein was characterized in the brine shrimp (Artemia franciscana) embryo, an anhydrobiotic invertebrate (Menze et al. 2009). Considering their role in bioenergetics, preserving mitochondrial function is likely to be of primary importance for eukaryotic anhydrobiotes.

Figure 2  Topological model of LEAM within the inner mitochondrial membrane at low hydration. Model showing how LEAM in its alpha-helical form could insert within the inner leaflet of the inner mitochondrial membrane at low hydration, providing reinforcement of the membrane in the dry state. The helix would be stabilized by hydrophobic interactions with the fatty acid core of the membrane, and ionic interactions between positively charged residues (red) and negative charges of phospholipids. The negatively charged residues on the protein (blue) form a crest facing the matrix space. The scheme is based on the work of Tolleter et al. (2007).

Interestingly, pea seed mitochondria were found to display an unusual capacity to operate oxidative phosphorylation at temperatures as high as 40°C or as low as –3.5°C (Figure 3; Stupnikova et al. 2006). This suggests that the adaptations of seed mitochondria, which are essential in the context of desiccation tolerance (e.g., LEAM and HSP22 accumulation), contribute to an increased tolerance to other environmental factors, such as extreme temperatures. In higher plants, it can therefore be expected that seed mitochondria are the most robust form of the organelle (Stupnikova et al. 2006; Macherel et al. 2007).

Figure 3  Oxidative phosphorylation by pea seed mitochondria at freezing temperature. The graph shows the oxygen consumption of isolated pea seed mitochondria at –3.5°C. The reaction medium remained liquid at –3.5°C because of the presence of osmoticum (0.6 M mannitol). Although the rates of oxygen consumption are low (nmol O₂ min–1mg protein–1), respiration is well coupled, with a respiratory control (RC) over 3 and a strong stimulating effect of the uncoupler FCCP. This indicates that seed mitochondria can efficiently oxidize exogenous NADH and produce ATP at negative temperatures (Stupnikova et al. 2006).

The Oxygen Challenge in Seeds

It is well known that oxygen, the terminal acceptor of electrons in mitochondrial respiration, is mandatory for germination. Oxygen deprivation is thus exploited in some mechanisms of coat-imposed dormancy, where the tissues surrounding the embryo form a chemical barrier to oxygen, thus preventing or constraining efficient energy metabolism in the imbibed seed. In developing and germinating seeds that display a very active metabolism, and hence oxygen consumption, the internal tissues are likely to face hypoxic conditions because of limitation in oxygen diffusion. To cope with oxygen intake and diffusion, animals have sophisticated oxygen transport systems that fuel their metabolically active tissues with an adequate supply of oxygen. Since plants generally display a high surface-to-volume ratio in their organs (e.g., leaves, fine roots), and also produce oxygen in their chlorophyllous parts in the light, tissue hypoxia is not considered a major issue, except in flooded soils where fermentative pathways in roots act as temporary energy suppliers.

The use of oxygen microsensors has revealed that developing legume seed tissues are strongly hypoxic because their high rate of respiratory oxygen consumption is not matched by oxygen diffusion from outside (Rolletschek et al. 2002). A similar situation was found with cereals, and further work suggested that photosynthesis played a major role within developing seeds in supplying a supplement of oxygen for respiration during the day (Borisjuk and Rolletschek 2009). An intriguing question is how respiratory oxygen consumption is regulated under the hypoxic conditions that prevail in developing seeds in the dark, or in germinating seeds, in which carbon metabolism is heterotrophic. Indeed, most seeds lose chlorophyll during late development and are expected to germinate in the soil, hidden from light. In mammals, the gaseous radical nitric oxide (NO) is a major regulator of oxygen homeostasis through various mechanisms, and in recent years it has also emerged as an important signaling molecule in plants (Besson-Bard et al. 2008). NO is a potent reversible inhibitor of cytochrome oxidase, and experiments using microsensors inserted into developing legume seeds showed that respiratory oxygen consumption was balanced under hypoxia by NO production derived from nitrite (Borisjuk et al. 2007). At low O₂ concentration, mitochondria reduce nitrite to NO, which diffuses to the active site of cytochrome oxidase, where its binding inhibits oxygen consumption, preserving oxygen levels (Benamar et al. 2008). Bound NO can be recycled back to nitrite chemically or by cytochrome oxidase when oxygen levels increase (Liu et al. 1998; Cooper 2002), thus maintaining steady state levels of NO that finely tune mitochondrial oxygen consumption rate. The system allows mitochondria to operate oxidative phosphorylation at the highest possible rate without leading to anoxia (Figure 4).

Figure 4  NO–nitrite control of mitochondrial respiration under hypoxic conditions. A high oxygen consumption rate by mitochondria combined with a limited diffusion of oxygen within seed tissues can lead to anoxia, a condition where oxidative phosphorylation cannot be maintained. At low oxygen concentration, nitrite (NO₂–) can be reduced to nitric oxide (NO) by the electron transfer chain (ETC). NO reversibly inhibits complex IV, decreasing oxygen consumption, thus allowing the oxygen level to increase. NO has a short half-life and can be recycled to nitrite. The system, which can be demonstrated with isolated organelles, would allow mitochondria to finely tune their oxygen consumption under hypoxic conditions to prevent anoxia while maximizing energy transduction (see Benamar et al. 2008). 

The control of oxygen homeostasis has recently emerged as an important issue for developing and germinating seeds that exhibit high respiratory rates. In the light, green seeds benefit from photosynthetic oxygen evolution to sustain respiration. In the dark, or in germinating seeds, the regulation of mitochondrial respiration by NO appears as an efficient way to maximize energy production while maintaining oxygen levels at a minimum, thus preventing anoxia.

In conclusion, seed mitochondria appear exquisitely adapted to tolerate and operate within unusual cellular environments imposed by natural desiccation, and also by internal hypoxic conditions reigning in actively respiring seeds. Seed mitochondria might therefore be one of the most robust forms of the organelle in eukaryotes.

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