Daphne J. Osborne

Function of DNA synthesis and DNA repair in the survival of embryos during early germination and in dormancy

DNA synthesis following the addition of water to excised embryos of non-dormant rye and to embryos of non-dormant and dormant genetic lines of Avena fatua has been examined. All the samples exhibit an early unscheduled DNA synthesis, have similar responses to DNA polymerase inhibitors and a similar increase in DNA ligase function for the first 24 h of imbibition, irrespective of whether they germinate or remain dormant. A β-polymerase-mediated DNA repair activity is indicated immediately upon imbibition with the stable incorporation of 3 H-methyl-thymidine into high molecular weight DNA. Following γ-irradiation of dry or imbibed embryos, inhibitor results suggest the appearance of an additional α- or δ-polymerase activity in the ensuing repair. Abscisic acid permits the early unirradiated repair synthesis, but like aphidicolin, it inhibits replicative DNA synthesis and partly inhibits the post-irradiation incorporation of thymidine. DNA synthesis takes place continuously throughout dormancy of imbibed embryos: precursor incorporation into DNA cannot be chased in the short term and occurs in the absence of an S-phase or endoreduplication of nuclear DNA. It is proposed that dormant imbibed embryos maintain the integrity of the genome by a continuous but slow replacement of DNA steered by a non-amplifying and modified form of replicative DNA synthesis and that abscisic acid may play a determining role in this process.

DNA and desiccation tolerance

This article reviews mechanisms by which specialized cells of different life forms have overcome the lethal effects of dehydration and considers how the maintenance of genetic information is central to survival As a dynamic and hydrated molecule in vivo, DNA can assume different conformational structures depending upon the water activity, the base sequence and the presence of specific binding proteins The attainment of stable secondary structures that are resistant to degradation in vivo at low water potentials is proposed as a likely accompaniment to desiccation tolerance In addition, chemical modification of bases in DNA, the extent of methylation and conformational changes could determine the expression of different gene sequences as cells pass from desiccation-tolerant to desiccationintolerant states We monitored the integrity of extracted DNA in embryos of seeds and in winddispersed pollen during transition from their desiccation tolerance to desiccation intolerance on hydration and germination. We present evidence to show that the DNA of these two stages is different and that it is the DNA from desiccation-tolerant cells only that retains integrity when the cells are subjected to desiccation regimes We discuss these findings in relation to certain hydration-sensitive DNA structures and to other relevant biological systems

The requirement for DNA repair in desiccation tolerance of germinating embryos

It is proposed that desiccation tolerance in the embryo of seeds depends upon the capacity to repair damage to genomic DNA when the desiccated embryo is rehydrated. From a study of imbibed and hydrated embryos of rye (Secale cereale) and wild oat (Avena fatua) evidence is provided that it is neither the extent of water uptake by the cells, the ensuing stability of the DNA to desiccation, nor the onset of S-phase DNA synthesis in the first cell cycle of germination that determines whether the desiccated embryo will survive. It is shown that when aand p-polymerases of DNA repair are inhibited by aphidicolin and dideoxythymidine-5-triphosphate, respectively, a -y-irradiation-induced DNA fragmentation cannot be fully repaired. It is shown that in hydrated embryos, at a stage when desiccation tolerance is lost, embryo cells still repair irradiation-induced damage, but this repaired DNA is unstable to desiccation and cannot be rerepaired when water is again made available. The failure to re-repair on rehydration appears to be critical to embryo survival and successful germination.

Special publication Evidence for a non-ACC ethylene biosynthesis pathway in lower plants☆

Abstract A wide range of lower plants is shown to lack the ability to convert ACC into ethylene although they produce ethylene continuously at rates similar to those in higher plants. [ 14 C]-ACC has been fed to leaf material of a semi-aquatic fern ( Regnellidium diphyllum ) and to axenic cultures of a liverwort ( Riella helicophylla ). Products of metabolism have been compared with those of [ 14 C]-ACC fed to a higher land plant ( Phaseolus vulgaris ) and to the semi-aquatic ( Nymphoides peltata ). ACC is readily taken up by all and readily decarboxylated, but [ 14 C]-ethylene is not released by the lower plants. The higher plants readily convert ACC into an ACC conjugate from which [ 14 C]-ethylene can be released in vitro , but although the lower plants also convert a small proportion of ACC into a conjugate its conversion into ethylene is low in vitro and absent in vivo . Precursors of ethylene in other organisms and substances that regulate the pathway of ethylene production in higher plants have been tested. The results indicate that lower plants operate their ethylene production pathway differently from higher plants. We conclude that the regulatable ethylene synthesis route of higher plants arose late in plant evolution and speculate upon factors that may have determined its establishment.

Value of the mucilaginous pellicle to seeds of the sand-stabilizing desert woody shrub Artemisia sphaerocephala (Asteraceae)

Seeds (achenes) of certain desert species (Artemisia) deposit considerable amounts of fixed carbon into an external polysaccharide pellicle that has a high capacity for holding water and can be hydrated and dehydrated many times. One function is considered to be the adhesion of the seed to sand particles during dew which protects from predation by ants. We have questioned whether the germinating embryo could utilize this pellicle as a nutrient source in the poor desert soil. Embryo extracts from dry imbibing and germinating seeds were assayed for a number of specific endo-glycosylase and exo-glycosylase activities and for the ability of these enzymes to degrade preparations of isolated pellicle. No evidence was found for any enzymic cleavage or hydrolysis of the pellicle or for any products to be available for utilization during germination of the seeds. Of commercial enzymes tested only polygalacturonase released reducing sugars from pellicle preparations after long incubation times indicating the high level of resistance of pellicle to enzymic cleavage. Comparisons of the water holding capacity of seeds with or minus their pellicles showed that periods of hydration were extended after dew deposition if pellicle was present. We suggest that a value of pellicle at low water availability is the provision of an enhanced opportunity for metabolic events in the embryo including those of DNA repair, the maintenance of genomic integrity and sustained viability in the seed bank.

Desiccation and survival in the recalcitrant seeds of Avicennia marina: DNA replication, DNA repair and protein synthesis

An autoradiographic study was made of leucine and thymidine incorporation into the meristematic root primordia and hypocotyl tips of seeds of the recalcitrant mangrove species, Avicennia marina. The investigations show that although there is a temporary reduction of protein synthesis at shedding, root primordia and surrounding hypocotyl cells of the axis never wholly cease incorporation of [ 3 H]leucine and regain preshedding levels of activity within a day. Precursor studies using methyl-[ 3 H]thymidine show that, at shedding, there is a temporary cessation of incorporation into root meristem nuclei that lasts no longer than 48 h and, within a day, pre-shedding levels are regained in the meristem nuclei. Analysis of DNA fragmentation patterns in root tips at the time of shedding, and their ability to repair radiation-induced DNA damage, indicate that DNA repair processes are markedly compromised in these cells if water loss reaches 22%. Protein synthesis and DNA replication are reduced by more than half by a water loss of 18% and 16%, respectively. DNA replication does not fully recover on rehydration after only 8% water loss. DNA fragmentation to nucleosomes indicates a programme of cell death at a water loss of 10%. We suggest that the feature of continuous protein synthesis activity with only a temporary interruption in active cell cycling in A. marina root primordia helps to explain both the rapidity in seedling establishment and the extreme vulnerability to desiccation.

The study of a monocotyledon abscission zone using microscopic, chemical, enzymatic and solid state 13C CP/MAS NMR analyses.

We have investigated distinguishing features in cells of the abscission zone of a monocotyledon fruit, the oil palm Elaeis guineensis. The cell walls of the abscission zone and the subtending mesocarp and pedicel have been analysed by light and transmission electron microscopy, by chemical methods and by solid state 13C CP/MAS NMR spectroscopy. Results show that these abscission zone cells have specific characteristics which include high levels of unmethylated pectin in the walls and an inducible (x35) polygalacturonase enzyme expression. Together these findings help to explain the localised precision of cell separation events.

HAZARDS OF A GERMINATING SEED: AVAILABLE WATER AND THE MAINTENANCE OF GENOMIC INTEGRITY

Embryos of seeds die at different rates under different conditions. The amount of water they receive determines the extent of cell hydration, and this in turn determines the pattern of degradation of genomic DNA whether by DNase nucleosomecleavage or by random fragmentation. Progress of the first cell cycle to the first mitotic divisions is dictated by water availability in accelerated aged and primed seeds. The loss of desiccation tolerance at the border of G2M and cytokinesis is discussed in relation to nucleases, DNA conformation, DNA damage, and stages of arrest in the first cell cycle.

Telomeres and their relevance to the life and death of seeds

Referee: Professor A. Carl Leopold, Boyce Thompson Institute for Plant Research, Cornell University, Tower Road, Ithaca, NY 14853 The highly conserved multiple repeats of short oligonucleotide sequences that cap the ends of eukaryotic chromosomes have become of increasing interest and speculation as their relationship with cell cycling and cell life-spans has been revealed. Most new information has come from the study of animal cells, where the progressive loss of telomeres at each division in somatic cell cultures acts as a “mitotic clock” with cell crisis and death occurring after a prescribed number of cycles. The importance of these closely similar sequences in cell division and cell survival in different parts of plants is much less clear. For seeds and their mature embryos, where no cell division occurs once they are dry, the means for maintaining telomere multiples for viability and successful germination is an intriguing question. This review describes a number of aspects of telomeres in eukaryotes. First, their discovery in seeds of Zea mays, their structure and unusual replication by the enzyme telomerase, which, unlike other polymerases, possesses a reverse transcriptase function from an encoded telomere RNA. Then, evidence that loss of capping telomere multiples could be responsible for cell cycle arrest or programmed cell death. Finally, how restoration of telomere repeats can lead to the immortalization of cell lines in both plants and animals. Although there are differences between plants and animals and seeds seem a special case, a correlation between the maintenance of telomere repeats and the deferral of senescence and cell death appears to exist. It is clear from the remarkable commonality of these telomeric termini that they have an intrinsic part to play in retaining nuclear chromosome stability in growing plant cells, but they may also be markers of the maintenance of genomic integrity in the embryos of dry and germinating seeds.

Failed expression of an endo-β-1,4-glucanhydrolase (cellulase) in a non-abscinding mutant of Lupinus angustifolius cv Danja

Cellulase expressions in a normal shedding wild-type and a non-abscinding single gene mutant of Lupinus angustifolius have been studied during ethylene treatments of leaf abscission zone explants. Of the range of different glycohydrolases investigated only the abscission cell-specific beta-1,4-glucanhydrolase (cellulase) was not produced in the non-abscinding mutant. An endo-polygalacturonase was induced equally in both wild-type and mutant and other glycohydrolases were equally up-regulated. The abscission cell-specific cellulase induced at shedding of wild-type is antigenically similar to the Phaseolus vulgaris induced leaf abscission pI 9.5 cellulase but with a higher molecular mass (50 kD compared with 48 kD) and like the bean abscission-specific cellulase that of lupin is not glycosylated. Causes of the loss of function of cellulase expression in the non-shedding mutant are discussed.