N. W. Pammenter

A review of recalcitrant seed physiology in relation to desiccation-tolerance mechanisms

A suite of mechanisms or processes that together have been implicated in the acquisition and maintenance of desiccation tolerance in orthodox seeds is discussed in the context of the behaviour of desiccation-sensitive seeds, and where appropriate, parallels are drawn with the situation in vegetative plant tissues that tolerate dehydration. Factors included are: physical characteristics of cells and intracellular constituents; insoluble reserve accumulation; intracellular de-differentiation; metabolic ‘switching off’; presence, and efficient operation, of antioxidant systems; accumulation of putatively protective substances including LEAs, sucrose and other oligosaccharides, as well as amphipathic molecules; the presence and role of oleosins; and the presence and operation of repair systems during rehydration. The variable response to dehydration shown by desiccation-sensitive seeds is considered in terms of the absence or incomplete expression of this suite of mechanisms or processes. Three categories of damage are envisaged: (i) reduction in cell volume which can lead to mechanical damage; (ii) aqueous-based degradative processes, probably consequent upon deranged metabolism at intermediate water contents. This is termed ‘metabolism-induced damage’ and its extent will depend upon the metabolic rate and the rate of dehydration; and (iii) the removal of water intimately associated with macromolecular surfaces leading to denaturation: this is referred to as desiccation damage sensu stricto . The effects of drying rate and the maturity status of seeds are considered in relation to the responses to dehydration, leading to the conclusion that the concept of critical water contents on a species basis is inappropriate. Viewing seed postharvest physiology in terms of a continuum of behaviour is considered to be more realistic than attempting precise categorization. Rapid dehydration of excised embryonic axes (or other explants) from desiccation-sensitive seeds permits retention of viability (in the short term) to water contents approaching the level of non-freezable water. This opens up the possibility of long-term conservation, by cryopreservation techniques, of the genetic resources of species producing non-orthodox seeds.

Effects of differential drying rates on viability retention of recalcitrant seeds of Ekebergia capensis

The drying rate of whole seeds of Ekebergia capensis (Meliaceae) was shown to influence the response to desiccation, with rapidly dried seeds surviving to lower water contents. Short-term rapid drying (to water contents higher than those leading to viability loss) actually increased the rate of germination. The form of the time course of decline of axis water content varied with drying rate; slow drying could be described by an exponential function, whereas with rapid drying initial water loss was faster than predicted by an exponential function. These observations suggest that slow drying brought about homogeneous dehydration and that the rapid drying was uneven across the tissue. This raised the possibility that the different responses to dehydration were a function of different distributions of water in the axis tissue under the two drying regimes. However, ultrastructural observations indicated that different deleterious processes may be occurring under the different drying treatments. It was tentatively concluded that a major cause of viability loss in slowly dried material was likely to be a consequence of aqueous-based processes leading to considerable membrane degradation. Uneven distribution of tissue water could not be rejected as a contributory cause of the survival of rapidly dried seeds to low bulk water contents. The differential response to dehydration at different drying rates implies that it is not possible to determine a ‘critical water content’ for viability loss by recalcitrant seeds.

Seed development in relation to desiccation tolerance: A comparison between desiccation-sensitive (recalcitrant) seeds of Avicennia marina and desiccation-tolerant types

Development of the highly desiccation-sensitive (recalcitrant) seeds of primarily one species, Avicennia marina , is reviewed and compared with the ontogeny of desiccation-tolerant (orthodox) seeds. A. marina seeds undergo no maturation drying and remain metabolically active throughout development, which grades almost imperceptibly into germination. While PGR control of histodifferentiation is essentially similar to that characterizing desiccation-tolerant seeds, the phase of growth and reserve deposition is characterized by exceedingly high cytokinin levels which, it is proposed, promote a sink for assimilate import. While some starch accumulation does occur, the predominant reserves are soluble sugars which are readily available for the immediate onset of seedling establishment upon shedding. ABA levels are negligible in the embryo tissues during seed maturation, but increase in the pericarp, which imposes a constraint upon germination until these outer coverings are sloughed or otherwise removed. The pattern of proteins synthesized remains qualitatively similar throughout seed development in A. marina , and no LEA proteins are produced. This suggests both that seedling establishment is independent of maturation proteins and that the absence of LEAs and desiccation sensitivity might be causally related. The study on A. marina reveals that for this recalcitrant seed-type, germination per se cannot be defined: rather, it is considered as the continuation of development temporarily constrained by the pericarp ABA levels. This leads to a reexamination of the role of rehydration as key event sensu stricto , in the germination processes in desiccation-tolerant (orthodox) seeds.

Homoiohydrous (recalcitrant) seeds: Developmental status, desiccation sensitivity and the state of water in axes of Landolphia kirkii Dyer

The desiccation sensitivity in relation to the stage of development was investigated in embryonic axes from the homoiohydrous (recalcitrant) seeds of Landolphia kirkii. Electrolyte leakage, used to assess membrane damage after flash (very rapid) drying, indicated that axes from immature (non-germinable) seeds were the most desiccation-tolerant, followed by those from mature seeds, while axes from seeds germinated for increasing times were progressively more desiccation-sensitive. Differential scanning calorimetry was used to study the relationship between desiccation sensitivity and the properties of water in the tissues. Axes from immature seeds had a lower content of non-freezable water than that of any other developmental stage and a higher enthalpy of melting of freezable water. For mature and immature axes electrolyte leakage increased at the point of loss of freezable water. At other developmental stages the water content at which electrolyte leakage increased markedly correlated with the other properties of the water, such as the change in the shape of the melting endotherm and the onset temperature. Ultrastructural studies of axes at the various developmental stages showed changes in the degree and pattern of vacuolation, the presence and quantities of lipid and starch, and the degree of endomembrane development. The results are discussed in relation to current hypotheses on the basis of desiccation tolerance.

Cryopreservation of desiccation-sensitive axes of Camellia sinensis in relation to dehydration, freezing rate and the thermal properties of tissue water

Summary The inter-relationships between water content, rate of freezing to −196 °C, thermal properties of water, and survival were studied in excised embryonic axes of tea seeds. Three freezing rates were used: 10°C min−1, about 200°C min−1 and rapid freezing (attained by plunging samples into nitrogen slush). Differential scanning calorimetry yielded three categories of melting endotherms: those without endothermic peaks (no freezable water), those with a broad endothermic peak with onset temperature varying with water content, and those with an additional sharp peak at about −2°C. When axes were cooled slowly, the sharp peak was present at high water contents (> 1.8 g H2O g−1 dw) and was diminished as axes were dried. The sharp peak was not apparent when axes were subjected to rapid freezing. Axes at moisture contents and subjected to freezing rates such that the sharp peak was present in the melting endotherms did not survive in tissue culture. Axes rapidly dried to water contents between 1.1 and 1.6 g H2O g−1 dry mass prior to rapid freezing showed 100 % survival in tissue culture. Ultrastructural studies of freeze fractured replicas showed considerable freezing damage at intermediate freezing rates, but good preservation of subcellular detail under rapid freezing conditions. It is suggested that the sharp peak observed on melting endotherms represents the melting of pure water arising from ice crystals formed during freezing. If axes with a sufficiently high water content are cooled at a freezing rate that prevents the formation of large ice crystals, damage is minimized and survival enhanced. If axes are dried to water content close to the level of non-freezable water, the additional stress of freezing is deleterious.

Effects of developmental status and dehydration rate on characteristics of water and desiccation-sensitivity in recalcitrant seeds of Camellia sinensis

The effect of rate of dehydration was assessed for embryonic axes from mature seeds of Camellia sinensis and the desiccation sensitivity of axes of different developmental stages was estimated using electrolyte leakage. Rapidly (flash) dried excised axes suffered desiccation damage at lower water contents (0.4 g H 2 O (g DW) −1 ) than axes dried more slowly in the whole seed (0.9 g H 2 O (g DW) −1 ). It is possible that flash drying of isolated axes imposes a stasis on deteriorative reactions that does not occur during slower dehydration. Differential scanning calorimetry (DSC) of the axes indicated that the enthalpy of the melting and the amount of non-freezable water were similar, irrespective of the drying rate. Very immature axes that had completed morphogenesis and histodifferentiation only were more sensitive to desiccation (damage at 0.7 g H 2 O (g DW) −1 ) than mature axes or axes that were in the growth and reserve accumulation phase (damage at 0.4 g H 2 O (g DW) −1 ). As axes developed from maturity to germination, their threshold desiccation sensitivity increased to a higher level (1.3−1.4 g H 2 O (g DW) −1 ). For the very immature axes, enthalpy of the melting of tissue water was much lower, and the level of non-freezable water considerably higher, than for any other developmental stage studied. There were no marked correlations between desiccation sensitivity and thermal properties of water. Desiccation sensitivity appears to be related more to the degree of metabolic activity evidenced by ultrastructural characteristics than to the physical properties of water.

The Basis of Recalcitrant Seed Behaviour

The term ‘recalcitrance’, defined as obstinate disobedience, refers to seeds that undergo no maturation drying as the final phase of development, tolerate very little post-shedding desiccation and are often chilling-sensitive. Such seeds are unstorable by any of the methods used for air-dry orthodox seeds. Since these terms were introduced by Roberts in 1973, much of the widely-disseminated literature has been systematically collated to afford an overview of recalcitrant seeds, particularly those of crop species (Chin and Roberts, 1980). Two major unresolved issues emerged from that overview: there was no explanation of the basis of recalcitrant seed behaviour, and no successful storage regimes had been established. The present contribution deals with progress that has been made towards an understanding of the responses of post-harvest, recalcitrant seeds in terms of their cell biology.

The effect of drying rate on viability retention of recalcitrant propagules of Avicennia marina

Recalcitrant propagules of Avicennia marina were stored under different relative humidities to achieve both rapid and slow drying. Irrespective of conditions, short-term (4–8 days) storage was accompanied by increased rates of protein synthesis and respiratory activity, the initiation of vacuolation and cell division and also by enhanced rates of germination. These data indicate that the germination process is initiated upon shedding. Storage for longer periods resulted in reduced rates of germination and ultimately, in loss of viability. However, propagules dried rapidly retained viability to a lower moisture content than those dried slowly. It is suggested that as germination changes occur during storage, the propagules become increasingly sensitive to desiccation, which might coincide with the degree of vacuolation. Rapidly dried propagules have not proceeded as far along the germination pathway and, at a given moisture content, are not as desiccation sensitive as those dried slowly. Thus viability loss is dependent upon rates of drying rather than on absolute moisture content or storage time, considered independently. S. Air. J. Bot. 1985, 51: 432–438 Weerspannige propagule van Avicennia marina is teen verskillende relatiewe vogtigheidswaardes geberg om vinnige sowel as stadige uitdroging teweeg te bring. Ongeag omstandighede, is korttermyn-berging (4–8 dae) gekenmerk deur ‘n verhoogde tempo van proteiensintese en respiratoriese aktiwiteit, aanvang van selholtevorming en seldeling, asook ‘n verhoogde ont-kiemingstempo. Hierdie gegewens dui daarop dat die proses van ontkieming ingelei word deur propaguulverlies. Berging vir langer tydperke het gelei tot verlaagde ontkiemingstempo, en uiteindelik tot verminderde kiemkrag. Propagule wat vinnig uitgedroog is, het egter hul kiemkrag tot op ‘n laer voggehalte behou as die wat stadig uitgedroog is. Daar word voorgestel dat die propagule toenemend sensitief word vir uitdroging, soos ontkiemingsveranderinge gedurende berging plaasvind, en dat dit moontlik saamval met die graad van selholtevorming. Vinnig-gedroogde propagule het nog nie so ver gevorder tot ontkieming nie, en by ‘n spesifieke voggehalte is hulle nie so droogte-sensitief soos die wat stadig uitgedroog het nie. Verlies aan kiemkragtigheid is gevolglik eerder afhanklik van uitdro-gingstempo as van absolute voggehalte of bergingstyd. S.-Afr. Tydskr. Plantk. 1985, 51: 432–438

Subcellular organization and metabolic activity during the development of seeds that attain different levels of desiccation tolerance

Water contents, desiccation tolerance, respiratory rates and subcellular characteristics of three contrasting seed types were studied during development. Avicennia marina (a tropical wetland species) and Aesculus hippocastanum (a temperate species) produce recalcitrant seeds and Phaseolus vulgaris produces orthodox seeds. During development, A. hippocastanum and P. vulgaris seeds showed a decline in water content and respiration rate with a concomitant increase in desiccation tolerance. These parameters did not change during the development of A. marina seeds once they had become germinable. There was a decrease in the degree of vacuolation and an increase in the deposition of insoluble reserves in A. hippocastanum and P. vulgaris seeds, while A. marina seeds remained highly vacuolated and did not accumulate insoluble reserves. Mitochondria and endomembranes degenerated during the development of A. hippocastanum and P. vulgaris seeds, but remained unchanged in A. marina seeds. The data are consistent with the hypothesis that extensive vacuolation and high metabolic rates contribute to desiccation sensitivity. However, the development of recalcitrant A. hippocastanum seeds is similar to that of orthodox P. vulgaris seeds. These data are in accord with the concept of seed recalcitrance being a consequence of truncated development. The results suggest that there may be three categories of seeds: orthodox seeds which develop desiccation tolerance, seeds which show similar development to orthodox seeds, but are shed before desiccation tolerance is well developed, and seeds which show no developmental trends giving rise to increased tolerance.

End product feedback effects on photosynthetic electron transport

The inhibition of photosynthetic electron transport when starch and sucrose synthesis limit the overall rate of photosynthesis was studied inPhaseolus vulgaris L. andXanthium strumarium L. The starch and sucrose limitation was established by reducing photorespiration by manipulation of the partial pressure of O2 and CO2. Chlorophylla fluorescence quenching, the redox state of Photosystem I (estimated by the redox status of NADP-dependent malate dehydrogenase), and the intermediates of the xanthophyll cycle were investigated. Non-photochemical fluorescence quenching increased, NADP-dependent malate dehydrogenase remained at 100% activity, and the amount of violaxanthin decreased when starch and sucrose synthesis limited photosynthesis. In addition, O2-induced feedback caused a decrease in photochemical quenching. These results are consistent with a downward regulation of photosynthetic electron transport during end product feedback on photosynthesis. When leaves were held in high CO2 for 4 hours, the efficiency of Photosystem II was reduced when subsequently measured under low light. The results indicate that the quantum efficiency of open Photosystem II centers was reduced by the 4 hour treatment. We interpret the results to indicate that feedback from starch and sucrose synthesis on photosynthetic electron transport stimulates mechanisms for dissipating excess light energy but that these mechanisms do not completely protect leaves from long-term inhibition of photosynthetic electron transport capacity.