Genetic analysis of seed development in Arabidopsis thaliana = [Genetische analyse van de zaadontwikkeling in Arabidopsis thaliana]

This thesis deals with the genetic aspects of seed development in Arabidopsisthaliana. Mutants affected in several aspects of seed development and, more specifically, in seed maturation have been isolated by various selection procedures. The mutants have been analyzed genetically, physiologically, and morphologically. Some of the mutants are impaired in the biosynthesis or sensitivity to the plant hormone, abscisic acid (ABA). All ABA-related mutants show reduced seed dormancy, indicating the important role of this hormone in the establishment of dormancy. In a direct screen for reduced dormancy, two mutants (rdo) with reduced dormancy were found. These were not ABA-deficient and showed the same sensitivity to ABA, ethylene, auxin, and cytokinin as the wild-type. In contrast to this embryo-determined reduced dormancy, reduced dormancy can also originate in an altered seed coat (testa), like in the altered testa shape ( ats ) mutant. Here, the altered testa shape is caused by a defect in the development of the integuments. Extreme ABA-insensitive mutants ( abi3 ) have green seeds that fail to complete many other aspects of seed maturation, including the induction of dormancy and desiccation tolerance, and the accumulation of seed storage proteins and lipids. In addition to abi3 mutants, lec and fus mutants exhibit such a severely disturbed seed maturation as well, with dark purple seeds due to anthocyanin accumulation. The fus3 mutant shows normal ABA-sensitivity. These various seed maturation mutants indicate that specific genes, some acting dependently and some acting independently from ABA, are responsible for seed maturation programs. The seed maturation mutants were subjected to a physiological and biochemical analysis. A GA-deficient mutant was combined with these mutants. Analysis of these double mutants indicated that seeds of the abi3 and lec mutants did not require GA for germination, in contrast to fus3 seeds. This correlates with ABA-sensitivity for germination. The composition of storage proteins and carbohydrates in abi3 , lec, and fus3 mutant seeds has been compared. The abi3 , lec, and fus3 mutants all showed severely reduced storage proteins. The desiccation intolerance of these seed maturation mutants was not correlated with the lack of specific carbohydrates. Furthermore, the mutants had a higher total content of carbohydrates. This is probably a consequence of the lower levels of storage lipids and proteins

A Permeable Cuticle Is Associated with the Release of Reactive Oxygen Species and Induction of Innate Immunity

Wounded leaves of Arabidopsis thaliana show transient immunity to Botrytis cinerea, the causal agent of grey mould. Using a fluorescent probe, histological staining and a luminol assay, we now show that reactive oxygen species (ROS), including H2O2 and O2−, are produced within minutes after wounding. ROS are formed in the absence of the enzymes Atrboh D and F and can be prevented by diphenylene iodonium (DPI) or catalase. H2O2 was shown to protect plants upon exogenous application. ROS accumulation and resistance to B. cinerea were abolished when wounded leaves were incubated under dry conditions, an effect that was found to depend on abscisic acid (ABA). Accordingly, ABA biosynthesis mutants (aba2 and aba3) were still fully resistant under dry conditions even without wounding. Under dry conditions, wounded plants contained higher ABA levels and displayed enhanced expression of ABA-dependent and ABA-reporter genes. Mutants impaired in cutin synthesis such as bdg and lacs2.3 are already known to display a high level of resistance to B. cinerea and were found to produce ROS even when leaves were not wounded. An increased permeability of the cuticle and enhanced ROS production were detected in aba2 and aba3 mutants as described for bdg and lacs2.3. Moreover, leaf surfaces treated with cutinase produced ROS and became more protected to B. cinerea. Thus, increased permeability of the cuticle is strongly linked with ROS formation and resistance to B. cinerea. The amount of oxalic acid, an inhibitor of ROS secreted by B. cinerea could be reduced using plants over expressing a fungal oxalate decarboxylase of Trametes versicolor. Infection of such plants resulted in a faster ROS accumulation and resistance to B. cinerea than that observed in untransformed controls, demonstrating the importance of fungal suppression of ROS formation by oxalic acid. Thus, changes in the diffusive properties of the cuticle are linked with the induction ROS and attending innate defenses

A Seed Shape Mutant of Arabidopsis That Is Affected in Integument Development.

A seed shape mutant of Arabidopsis was isolated from an ethyl methanesulfonate-treated population. Genetic analysis revealed that the heart-shaped phenotype was maternally inherited, showing that this is a testa mutant. This indicated the importance of the testa for the determination of the seed shape. This recessive aberrant testa shape (ats) gene was located at position 59.0 on chromosome 5. A comparison was made between ovules and developing and mature seeds of the wild type and of the mutant using light and scanning electron microscopy. We showed that the mutant seed shape is determined during the first few days after fertilization, when the embryo occupies only a very small part of the seed. The integuments of ats ovules consisted of only three rather than five cell layers. In double mutants, the effect of ats was additive to other testa mutations, such as transparent testa, glabra (ttg), glabrous2 (gl2), and apetala2 (ap2). The ats mutation resulted in a reduced dormancy, which was maternally inherited. This effect of a testa mutation on germination was also seen in ttg seeds, in which the outer layer of the testa was disturbed. This indicated the importance of the testa as a factor in determining dormancy in Arabidopsis

Acquisition of Desiccation Tolerance and Longevity in Seeds of Arabidopsis thaliana (A Comparative Study Using Abscisic Acid-Insensitive abi3 Mutants).

Two new abscisic acid (ABA)-insensitive mutants of Arabidopsis thaliana affected in the abi3 locus are described. These new mutants are severely ABA insensitive. Like the earlier described abi3-1 and the ABA-deficient and -insensitive double mutant aba,abi3, these new mutants vary in the extent of ABA-correlated physiological responses. Mutant seeds fail to degrade chlorophyll during maturation and show no dormancy, and desiccation tolerance and longevity are poorly developed. Carbohydrate accumulation as well as synthesis of LEA or RAB proteins are often suggested to be essential for acquisition of desiccation tolerance. In this work two points are demonstrated. (a) Accumulation of carbohydrates as such does not correlate with acquisition of desiccation tolerance or longevity. It is suggested that a low ratio of mono- to oligosac-charides rather than the absolute amount of carbohydrates controls seed longevity or stability to desiccation tolerance. (b) Synthesis of a few assorted proteins, which is responsive to ABA in the later part of seed maturation, is not correlated with desiccation tolerance or longevity

Angiosperm ovules: diversity, development, evolution

In angiosperms the presence of an outer integument appears to be instrumental for ovule curvature, as indicated from studies on ovule diversity through the major clades of angiosperms, molecular developmental genetics in model species, abnormal ovules in a broad range of angiosperms, and comparison with gymnosperms with curved ovules. Lobation of integuments is not an atavism indicating evolution from telomes, but simply a morphogenetic constraint from the necessity of closure of the micropyle. Ovule shape is partly dependent on locule architecture, which is especially indicated by the occurrence of orthotropous ovules. Some ovule features are even more conservative than earlier assumed and thus of special interest in angiosperm macrosystematics