Activation of acid growth in germinating horse chestnut seeds

The validity of the acid-growth hypothesis is proved for the case of cell elongation initiation in germinating seeds of horse chestnut (Aesculus hippocastanum L.), the embryo axes of which are known to extend during the first stages of germination only by cell elongation. During seed imbibition, H⁺-ion excretion was firstly low; it increased several times prior to radicle emergence and was maintained at a high level during growth initiation and further cell elongation. Cell wall acidification and radicle emergence were enhanced in the presence of 0.02 mM fusicoccin, thus indicating the involvement of the plasma membrane H⁺-ATPase in the execution of acid growth. The presence of this enzyme and its activator (14-3-3 protein) in microsomal fractions obtained from radicles and hypocotyls of the embryo axes during and after initiation of cell elongation was demonstrated immunochemically. It is supposed that the initiation of cell elongation at early germination occurs via the activation of the plasma membrane H⁺-ATPase and results in the acidification of cell walls, leading to their higher extensibility, in accordance with the hypothesis of acid growth.     

Acid vacuolar invertase in dormant and germinating seeds of the horse chestnut

A high water content is maintained in the tissues of the axial organs of horse chestnut seeds after the fruit is shed and down to the time the seeds germinate. The plant cell vacuoles, features of whose metabolism can influence the cells’ preparation to initiate growth in germination, are preserved. It was shown that the activity of acid invertase and its capacity to hydrolyze both sucrose and raffinose remain stable throughout the period of dormancy and the transition to germination, as do the molecular weight of its subunits (63 and 65 kDa) and multimer (500 to 550 kDa). The activity of the enzyme increases when the seeds swell under optimal conditions for germination; this is associated with the synthesis of new molecules of the enzyme in long-lived mRNA templates. The storability of the enzyme in the vacuoles of dormant seeds, together with the increase in its activity when seeds coming out of dormancy swell, ensures the rapid hydrolysis of sucrose issuing from the seeds’ cotyledons, thus leading to increased osmotic pressure and, as a result, the beginning of cell elongation, i.e., germination.     

Dynamics of carbohydrates in the embryo axes of horse chestnut seeds during their transition from dormancy to germination

It was shown that the content of carbohydrates and their composition in embryo axes of horse chestnut seeds changed as seeds acquired a capability of dormancy release and germination. Sucrose prevailed among carbohydrates, comprising to 150–160 mg/g dry wt. During the first half of the seed imbibition time, oligosaccharides, namely raffinose and stachyose, degraded, whereas the contents of glucose and fructose were very low. The second half of the imbibition period (until radicle protrusion) was characterized by a cessation of oligosaccharide breakdown and accumulation of monosaccharides. Carbohydrate balance showed that the contribution of oligosaccharide breakdown to sucrose and monosaccharide accumulation was rather small, and monosaccharides accumulated mostly at the expense of sucrose gradually coming from cotyledons during imbibition. The trend of carbohydrate metabolism in imbibing axial organs was similar during the entire period of a seed dormancy release in the course of stratification. A readiness for the commencement of these processes during the entire dormancy period implies that carbohydrate conversions in embryo axes are not a trigger for a dormancy release. Monosaccharide accumulation in embryo axes before radicle protrusion produces an increase in the osmotic pressure, as compared to that provided by sucrose, by approximately 20%. Recalcitrance of the horse chestnut seeds is discussed in relation to the role of carbohydrates and other endogenous osmotica in the establishment of osmotic properties.     

Water relations in germinating seeds

Life strategy of plants depends on successful seed germination in the available environment, and sufficient soil water is the most important external factor. Taking into account a broad spectrum of roles played by water in seed viability and its maintenance during germination, the review embraces early germination events in seeds different in their water status. Two seed types are compared, namely orthodox and recalcitrant seeds, in terms of water content in the embryonic axes, vacuole biogenesis, and participation of water channels in membrane water transport. Mature orthodox seeds desiccate to low water content and remain viable during storage, whereas mature recalcitrant seeds are shed while well hydrated but die during desiccation and cannot be stored. In orthodox Vicia faba minor air-dry seeds remaining viable at 8–10% water content in embryonic axes, the vacuoles in hypocotyl are preserved as protein storage vacuoles, then restored to vacuoles in imbibing seeds in the course of protein mobilization. However, in newly produced meristematic root cells, the vacuoles are formed de novo from provacuoles. In recalcitrant Aesculus hippocastanum seeds, embryonic axes have a water content of 63–64% at shedding and they lack protein storage vacuoles but preserve vacuoles preformed in maturing seeds. Independent of the vacuolar biogenetic patterns, their further trend is similar; they expand and fuse, thus producing an osmotic compartment, which precedes and becomes an obligatory step for the initiation of cell elongation. Prior to this, water moves in imbibing seeds through the membranes by diffusion, although the aquaporins forming water channels are present. In both seed types, water channels are opened and actively participate in water transport only after growth initiation. Aquaporin gene expression and their composition change in broad bean embryonic axes after growth initiation. This is the way how a mass water flow into growing seedling cells is achieved, independent of differences in seed water content and vacuole biogenesis patterns.