Abscisic acid and assimilate partitioning during seed development

This thesis describes the influence of abscisic acid (ABA) on the transport of assimilates to seeds and the deposition of reserves in seeds. It is well-known from literature that ABA accumulates in seeds during development, and that ABA concentrations in seeds correlate rather well with seed size and seed growth rates. However, since ABA is at least partly synthesized in the leaves and transported to the seeds via the phloem, a correlation between ABA levels and growth rate can easily be explained as the result of the combined transport of ABA and assimilates. Reports about the effect of applied ABA on transport of assimilates to seeds are contradictory (Table 1.I). Moreover, application of ABA has several disadvantages: the application technique itself may cause artefacts, and the results are difficult to interpret since the endogenous ABA level after application depends on penetration, transport and metabolism in the tissue. For these reasons, we have chosen for a different approach, viz . the use of hormone mutants. Two species were used: Pisum sativum and Arabidopsis thaliana .Growth and development of the ABA-deficient ' wilty ' mutant of pea is described in detail (Chapter 2). A non-wilty isogenic line was obtained after six successive backcrosses of the mutant with a closely approximating line. The plants were grown at conditions of high relative humidity and cultured on hydroponics, since leaves of ABA-deficient plants fail to accumulate ABA at drought stress and consequently do not close their stomata- For the same reason, mutant leaves have a higher dry matter content than wild-type leaves. The mutant grew slower and especially root growth was reduced; this resulted in a considerably larger shoot/root ratio. Similar effects have been found in ABA-deficient mutants of several other species. This root-growth promotive effect of ABA can be explained as a measure to prevent an undesirable water status of the leaves by increasing the volume of soil explored under dry conditions.ABA-deficient plants had fewer and smaller seeds than wild-type plants, but since the mutants plants themselves were also smaller, the weight ratio of reproductive to vegetative parts was similar in both lines. The seeds of mutant plants contained about five times less ABA than wild-type seeds. It was concluded that the lower growth rate of both vegetative and reproductive parts was not directly caused by the lower ABA content of these organs, but by disturbed water relations.One of the reasons to choose the pea mutant was that transport of assimilates to legume seeds can be studied by the empty-seed-coat technique. After removal of a part of the pod wall and the seed coat, the embryo is replaced by a buffer, while leaving most of the maternal tissue intact. This buffer receives assimilates from the seed-coat and is regularly analysed for the presence of sucrose. The rate of sucrose efflux calculated from the seed-coat into the medium is assumed to be a measure for phloem import, especially during the period of near-constant sucrose release (4-10 hours after the start of the experiment). The effect of ABA on sucrose release was studied by applying various ABA concentrations to the buffer (Figure 3. 1) and expressing the amount of sucrose released into these buffers relative to the amount present in a control seed-coat (a surgically modified seed-coat containing buffer without ABA). It was shown that hardly any ABA leaked from one seed-coat to another. The experiments were performed with both wildtype and ABA-deficient plants, either or not at source-limited conditions, since it was assumed that a possible effect of ABA might be more pronounced in ABA-deficient plants and at source-limited conditions. Source-limiting indeed caused a reduction of the sucrose release- rate. However, no effect of ABA on sucrose release could be discerned, irrespective of the experimental conditions.Another advantage of the use of mutants is the possibility to study competition between genetically different seeds, for the same source of assimilates (Figure 1.3). In pea, this was achieved by crossing an ABA-deficient mother plant with pollen from plants that were heterozygous for this trait. Chapter 4 describes experiments on ABA-deficient pea plants bearing pods with both ABA-deficient and ABA-containing seeds in the same pod. Seeds in the same pod usually have the same growth rate. In these pods, the growth rate of the seeds was determined by measuring the diameter of the seeds with a pair of callipers. In a control experiment it was shown that these manipulations (opening of the pod and measuring the seeds) did not disturb the normal growth pattern of the seeds. No effect of the genotype on the growth rate of the seeds was detected.Similar studies were performed with Arabidopsis mutants (Chapter 5). In one series of experiments, successive flowers of a recombinant of an ABA-deficient and an ABA-insensitive mutant (aba,abi3) were alternatingly pollinated with pollen from either wildtype or double-mutant plants. In another series of experiments, a double-mutant that was both ABA-deficient and starchless was used as a mother plant; the amount of available assimilates in these plants was reduced by decreasing the light intensity. The growth rate of the seeds was determined by exposing the mother plants to radiolabelled CO 2 and detecting the amount of radioactivity in the seeds. The weight of the seeds of these crosses was determined on a high-precision balance. In these experiments, again no significant influence of the genotype on either the import of radioactivity or the weight of the seeds could be detected.The possible effect of ABA on the deposition of reserve material in seeds was studied with some Arabidopsis mutants. Arabidopsis is a crucifer and its seeds initially accumulate starch which is degraded and converted to lipids during seed maturation. Seeds of the ABA-deficient (aba) and the ABA-insensitive (abi3) mutant and their recombinant (aba,abi3) were collected during development and their lipid and carbohydrate composition was analysed and compared with wild-type seeds. The maximum dry and fresh weight of the seeds was not influenced by the genotype. All mutants had considerably reduced levels of eicosenoic acid (20: 1) in the triacylglycerol fraction as compared to wild- type seeds; it is concluded that ABA is involved in the regulation of elongation of fatty acids. The total amount of neutral lipids in seeds of the single mutants was similar to that in wild-type seeds (about 30-35 % on a dry weight basis), but doublemutant seeds contained only half this amount. On the other hand, double-mutant seeds had elevated levels of starch and soluble sugars. Apparently, the blockade in lipid synthesis in these mutants is so strong that it results in starch accumulation and finally in accumulation of soluble sugars. It is concluded that both the presence of ABA and the sensitivity to ABA are required for normal acyl-chain elongation and lipid accumulation; the absence of both factors results in a higher proportion of the imported assimilates being stored as carbohydrates.From the above-mentioned experiments, it was concluded that ABA has no major influence on the long-distance transport of assimilates, at least not in the species Pisum sativum and Arabidopsis thaliana. However, ABA appears to be involved in the distribution of assimilates over the various types of storage material during seed development.