Essay 11.7

The Role of Respiration in Desiccation Tolerance

Folkert A. Hoekstra, Graduate School Experimental Plant Sciences, Wageningen University, The Netherlands

July, 2005

Respiration exerts a dual effect on the survival of plant cells undergoing water loss. Sufficient respiration is necessary to generate the chemical energy required for the production of molecules that protect against the adverse effects of water loss. Examples of such molecules are the compatible solutes, such as proline and glycinebetaine. These solutes are called "compatible" because they do not interfere with cellular activity, even at elevated concentrations in the cytoplasm. Other examples are dehydration-induced proteins, such as the late embryogenesis abundant (LEA) proteins and small heat shock proteins. On the other hand, high respiratory activity may lead to the production of potentially dangerous reactive oxygen species (ROS) (see Web Essay 11.5). Disturbance of the electron transport chain as a result of water loss might add to the production of ROS. Membranes are a primary target for ROS, which explains why drought- and desiccation-sensitive organisms often lose the integrity of their plasma membranes under conditions of water stress.

Down-Regulation of Metabolism

Typically, drought- and desiccation-tolerant cells suffer less oxidative damage than cells that are sensitive to these stresses. This might be the result of an effective decrease in the ROS produced, or an activation of effective antioxidant systems, or both. One hypothesis proposes that tolerant cells have a curtailed metabolism, so as to reduce the generation of ROS. Support for this hypothesis has come from the following observations. Mature seeds that are unable to survive air-drying have higher initial rates of respiration than desiccation-tolerant seeds. If their metabolic activity is curtailed during drying, for example by cooling-dehydration, injury is less (Leprince et al. 1995). This is in agreement with the finding that the rate of respiration before drying corresponds with the amount of free radicals that accumulate in the dried specimen. Another example of the link between respiration rate and survival of dehydration can be found in germinated seeds just after radicle protrusion. Desiccation tolerance in these seeds can be reinduced by slow dehydration for some days in a -1.5 MPa polyethylene glycol solution. Such an osmotic treatment considerably reduces the respiration rate of the radicles when compared with that of desiccation-sensitive radicles that are dried to a similar water content without osmotic adjustment. This difference in respiration rate is maintained during further drying. Evidence is growing that a number of genes linked with metabolism are down-regulated upon drought and desiccation stress.

Rapidly respiring systems sometimes tolerate desiccation to the air-dry state with the help of a superior antioxidant defense, but in those cases they tend to be more short-lived than less metabolically active systems (Hoekstra 2005). This is true for pollen, some of which are metabolically so advanced that they are capable of commencing rapid pollen tube growth (1–4 cm/hour) within a few minutes of rehydration. Dried pollen typically survive for a few weeks at room temperature as opposed to years or even decades in the case of seeds.

It is thought that the plant hormone abscisic acid (ABA), subsequent signal transduction sequences, and ensuing gene products play important roles in the down-regulation of metabolism. This view is supported by the observation that some ABA-insensitive mutant seeds of Arabidopsis lack quiescence and, with sufficient water available, may germinate right away in the pods before being shed. They are unable to acquire sufficient desiccation tolerance during maturation to survive the air-dry state. By contrast, wild-type seeds become quiescent and are able to survive drying to the air-dry state. The intrinsically desiccation-sensitive somatic embryos are a good example of ABA advancing desiccation tolerance. Upon preculture in the presence of ABA, somatic embryos suspend growth, assume low rates of respiration, and reduce the uptake and consumption of sugars. Subsequent slow-drying completes the acquisition of desiccation tolerance. Slow-drying without ABA addition is generally insufficient for somatic embryos to acquire tolerance because metabolism is insufficiently depressed.

Decrease of Respiration during Water Loss in Desiccation-Tolerant Systems

After some initial drying, the relatively low rate of respiration in desiccation-tolerant systems declines with a further loss of water. Respiration becomes immeasurably low when cytoplasmic bulk water disappears, which is just below 25% water content (on a fresh weight basis). Until about 40% water content is reached, the energy charge (EC) remains high, with values around 0.85. As explained below, this high EC value is evidence that the decline in respiration is the result of a reduced metabolic requirement for ATP rather than the inability of the respiratory system to produce ATP.

The EC, defined as ([ATP]+0.5 [ADP])/([ATP]+[ADP]+[AMP]), is a measure of the relative proportion of high-energy phosphate bonds among the adenylate phosphates in a cell. If only ATP is present, the EC reaches its maximum value of 1, whereas its minimum value of 0 is reached in the case of only AMP being present; a value of 0.5 is reached when only ADP is present. In normally metabolizing cells in which the ATP-generating reactions are in balance with the ATP-utilizing reactions, EC values are usually around 0.8–0.9, indicating that ATP is prevalent in such cells.

A possible decrease in EC value can be a valuable marker of the imbalance between ATP generation and utilization. In desiccation-tolerant seeds and pollen, the EC decreases when water content falls below 40%, illustrating that the respiratory system cannot fulfill the demand for ATP. At the same time, the viscosity of the cytoplasm increases exponentially and the mitochondrial redox carriers, cytochrome oxidase and cytochrome c, increasingly occur in the chemically reduced state. It is suggested that the behavior of the mitochondrial respiratory system at low-water content is the result of a decreased availability of O₂ caused by a slow diffusion of O₂ at high viscosity (Leprince and Hoekstra 1998). When water becomes available again, respiration increases, and high ATP levels are restored in desiccation-tolerant systems, but not in desiccation-sensitive systems because of the loss of membrane integrity.

Toxic By-Products of Unbalanced Metabolism in Desiccation-Sensitive Systems

Dehydration of desiccation-sensitive seed axes may lead to the emission of acetaldehyde and ethanol, at water contents as high as 60% to 70% (fresh weight basis) (Leprince et al. 2000). Such products of fermentation do not arise during drying of tolerant axes. The appearance of these compounds as a sign of unbalanced metabolism usually precedes the general loss of membrane integrity. It might be that acetaldehyde further aggravates desiccation-induced damage to membranes. Apparently, respiratory metabolism is a primary target of injury during water loss.

During dehydration of desiccation-sensitive seeds, the respiration rate may even increase, but it decreases upon the subsequent loss of membrane integrity (Leprince et al. 1999). Such an upsurge of respiration has been explained in light of the partitioning of amphiphilic substances from the cytoplasm into mitochondrial membranes during drying. This amphiphile partitioning is driven by the reduction in cytoplasmic volume (Hoekstra and Golovina 2002). One scenario could be that the mitochondrial membranes become leaky to protons as a result of the perturbing effects of these amphiphiles. Loss of the electrochemical proton gradient makes it increasingly difficult for mitochondria to produce sufficient ATP.

The question that emerges is why amphiphile partitioning into membranes would not lead to similar problems in desiccation-tolerant systems, as it does in desiccation-sensitive systems. The answer may be twofold. First, systems that respire at a low rate might maintain high ATP levels more easily upon a slight impairment of mitochondrial proton gradients. Second, there is evidence that membrane perturbance during drying is actively prevented in desiccation-tolerant systems.

Thus, it appears that a coordinated down-regulation of energy metabolism in desiccation-tolerant systems early during drying may play an important role in avoiding oxidative stress conditions and/or accumulation of by-products of metabolism to toxic levels (Figure 1).

Figure 1 Relationship between respiration rate in the hydrated state and survival of the plant when desiccated, to the air-dry state. Timely down-regulation of metabolism, thought to be regulated by the plant hormone ABA, reduces the generation of ROS and toxic by-products of metabolism during water loss. Thus, the integrity of membranes is maintained and the survival of desiccation is secured. (Click image to enlarge.)

References

Hoekstra, F. A. (2005) Differential longevities in desiccated anhydrobiotic plant systems. Integrat. Comp. Biol. A 45(5): 725–733.

Hoekstra, F. A., and Golovina, E.A. (2002) The role of amphiphiles. Comp. Biochem. Physiol. 131A: 527–533.

Leprince, O., and Hoekstra, F.A. (1998) The responses of cytochrome redox state and energy metabolism to dehydration support a role for cytoplasmic viscosity in desiccation tolerance. Plant Physiol. 118: 1253–1264.

Leprince, O., Buitink, J., and Hoekstra, F. A. (1999) Axes and cotyledons of recalcitrant seeds of Castanea sativa Mill. exhibit contrasting responses of respiration to drying in relation to desiccation sensitivity. J. Exp. Bot. 338: 1515–1524.

Leprince, O., Vertucci, C. W., Hendry, G. A. F., and Atherton, N. M. (1995) The expression of desiccation-induced damage in orthodox seeds is a function of oxygen and temperature. Physiol. Plant. 94: 233–240.

Leprince, O., Harren, F. J. M., Buitink, J., Alberda, M., and Hoekstra, F. A. (2000) Metabolic dysfunction and unabated respiration precede the loss of membrane integrity during dehydration of germinating radicles. Plant Physiol. 122: 597–608.