Physiological aspects of seed recalcitrance: a case study on the tree Aesculus hippocastanum.

Recalcitrant seeds are typical of some tropical and subtropical trees. Their post-shedding life activity proceeds in humid air and wet litter. They are desiccation sensitive and, for this reason, have a short life span and need some special procedures for cryopreservation. This review is devoted to the post-shedding life strategy of recalcitrant seeds, which includes the maintenance of high hydration status, metabolic readiness and ability to rapidly germinate before desiccation-induced damage exerts a lethal effect. The main physiological aspects of recalcitrant seeds are considered starting from mature seeds, followed during dormancy if occurs and resulting in germination. The collected data embrace the metabolic processes in embryonic axes and whole seeds. The up-to-date results are integrated covering the main metabolic processes, namely water status and transport, protein and carbohydrate metabolism, antioxidant defense, axis-cotyledon relations, hormonal control and germination. Among the representatives of various taxa, the seeds of which exhibit recalcitrance, attention was given to horse chestnut seeds as one of most studied recalcitrants.

Deterioration mechanisms in air-dry pea seeds during early aging.

The deteriorative reactions underlying seed aging, namely, lipid peroxidation and non-enzymatic carbohydrate hydrolysis, were studied in pea seeds differing in quality. Aging air-dry seeds were subdivided to three fractions using the application to individual seeds of room temperature phosphorescence. These fractions were strong seeds (fraction I) producing normal seedlings, weak seeds (fraction II) producing mainly abnormal seedlings, and dead seeds (fraction III). Enzymatic processes cannot operate in dry seeds due to the absence of free water, and thus an analytical method was needed that does not require the addition of water. The content of lipid peroxidation products was similar in both strong and weak seeds; this excluded the possibility that lipid peroxidation induced the transition of strong to weak seeds during early aging. Lipid peroxidation was activated only in dying seeds. However, glucose content in weak seeds was much higher than in strong seeds, suggestive of non-enzymatic hydrolysis of carbohydrates, probably of oligosaccharides, which utilized bound water in air-dry seeds. This process resulted in lowered water content in weak seeds. Therefore, associated with deterioration of air-dry seeds during early aging is the non-enzymatic hydrolysis of carbohydrates, whereas lipid peroxidation is not the decisive event.

Vacuolar biogenesis and aquaporin expression at early germination of broad bean seeds.

A key event in seed germination is water uptake-mediated growth initiation in embryonic axes. Vicia faba var. minor (broad bean) seeds were used for studying cell growth, vacuolar biogenesis, expression and function of tonoplast water channel proteins (aquaporins) in embryonic axes during seed imbibition, radicle emergence and growth. Hypocotyl and radicle basal cells showed vacuole restoration from protein storage vacuoles, whereas de novo vacuole formation from provacuoles was observed in cells newly produced by root meristem. cDNA fragments of seven novel aquaporin isoforms including five Tonoplast Intrinsic Proteins (TIP) from three sub-types were amplified by PCR. The expression was probed using q-RT-PCR and when possible with isoform-specific antibodies. Decreased expression of TIP3s was associated to the transformation of protein storage vacuoles to vacuoles, whereas enhanced expression of a TIP2 homologue was closely linked to the fast cell elongation. Water channel functioning checked by inhibitory test with mercuric chloride showed closed water channels prior to growth initiation and active water transport into elongating cells. The data point to a crucial role of tonoplast aquaporins during germination, especially during growth of embryonic axes, due to accelerated water uptake and vacuole enlargement resulting in rapid cell elongation.

Vacuolar status and water relations in embryonic axes of recalcitrant Aesculus hippocastanum seeds during stratification and early germination.

BACKGROUNDS AND AIMS: In tropical recalcitrant seeds, their rapid transition from shedding to germination at high hydration level is of physiological interest but difficult to study because of the time constraint. In recalcitrant horse chestnut seeds produced in central Russia, this transition is much longer and extends through dormancy and dormancy release. This extended time period permits studies of the water relations in embryonic axes during the long recalcitrant period in terms of vacuolar status and water transport. METHODOLOGY: Horse chestnut (Aesculus hippocastanum) seeds sampled in Moscow were stratified in cold wet sand for 4 months. Vacuole presence and development in embryonic axes were examined by vital staining, light and electron microscopy. Aquaporins and vacuolar H(+)-ATPase were identified immunochemically. Water channel operation was tested by water inflow rate. Vacuolar acid invertase was estimated in terms of activity and electrophoretic properties. PRINCIPAL RESULTS: Throughout the long recalcitrant period after seed shedding, cells of embryonic axes maintained active vacuoles and a high water content. Preservation of enzyme machinery in vacuoles was evident from retention of invertase activity, substrate specificity, molecular mass and subunit composition. Plasmalemma and tonoplast aquaporins and the E subunit of vacuolar H(+)-ATPase were also present. In non-dormant seeds prior to growth initiation, vacuoles enlarged at first in hypocotyls, and then in radicles, with their biogenesis being similar. Vacuolation was accompanied by increasing invertase activity, leading to sugar accumulation and active osmotic functioning. After growth initiation, vacuole enlargement was favoured by enhanced water inflow through water channels formed by aquaporins. CONCLUSIONS: Maintenance of high water content and desiccation sensitivity, as well as preservation of active vacuoles in embryonic axes after shedding, can be considered a specific feature of recalcitrant seeds, overlooked when studying tropical recalcitrants due to the short duration. The retained physiological activity of vacuoles allows them to function rapidly as dormancy is lost and when external conditions permit. Cell vacuolation precedes cell elongation in both hypocotyl and radicle, and provides impetus for rapid germination.