Transcriptional regulation of seed dormancy and seed longevity

The timing of seed germination is regulated by seed dormancy. There is ample natural variation for seed dormancy among as well as within plant species. In Arabidopsis several DELAY OF GERMINATION quantitative trait loci have been identified, of which DOG1 is best studied. Here we report the identification of DOG6, a quantitative trait locus with a similar strong effect on seed dormancy as DOG1. DOG6 affects the timing of germination both in laboratory as well as in field conditions. Complementation cloning revealed that DOG6 encodes the membrane bound transcription factor ANAC060. The absence of the ANAC060 protein or its sequestration outside the nucleus results in increased seed dormancy levels. The different natural variants of ANAC060 differ for the presence of the membrane binding domain, either due to the fact that this domain is absent in the genomic sequence or because the cDNA is alternatively spliced. Our data indicates that ANAC060, when present in the nucleus, inhibits seed dormancy by attenuating the expression of protein phosphatases 2C class A proteins including ABI 5 BINDING FACTOR 3, REDUCED DORMANCY5/DOG18 and ABA insensitive 4.The NAC family of transcription factors is involved in plant development and various biotic and abiotic stresses. The Arabidopsis thaliana DELAY OF GERMINATION 6 (ANAC060) protein is inhibiting seed dormancy and as such determines the timing of seed germination. ANAC040 and ANAC089 are the two closest homologs of ANAC060 based on protein and nucleotide sequence similarity. These three genes are predicted to be membrane bound transcription factors (MTFs) containing a conserved NAC domain but divergent C-terminal regions. Mutations in ANAC040 lead to higher seed germination under salt stress. A premature stop codon in the ANAC089 Cvi allele results in seeds exhibiting insensitivity to high concentrations of fructose. Thus, these three which genes are highly homologous at the protein/gene level confer distinct functions, although all germination related. In order to investigate whether the differences in function are caused by either a differential spatial or temporal regulation as a result of differences in the promoter regions or due to differences in their coding region (CDS) we have performed swapping experiments in which the promoter regions and the CDS of the three different NAC transcription factors have been exchanged. We examined seed dormancy and salt and fructose sensitivity of transgenic swapping lines in mutant backgrounds. We show that there is functional redundancy between ANAC060 and ANAC040, but not between ANAC060 and ANAC089.Seeds are usually stored in well controlled storehouses with restrained relative low temperature and humidity to conserve seed quality for a long period. Nevertheless, seeds will gradually lose vigor and eventually die. Oxygen has been identified as a main factor influencing seed viability during long term storage, however it plays a dual role during the seed lifespan. On the one hand, its product Reactive Oxygen Species (ROS) are beneficial for seed dormancy alleviation during after ripening. On the other hand, ROS progressively cause damage in the seed by altering the fluidity of cell membrane, influencing the transport of ions, enzyme deactivation, protein carbonylation and DNA and RNA damage, which all together ultimately result in seed death. Among these, the integrity of genetic materials largely impacts seed viability. RNA is vulnerable to ROS as it has single strand structure and an RNA repair mechanism is lacking at dry seed state. Oxidation of messenger (m) RNA results in the inhibition of translation or the production of abnormal proteins. Protein oxidation causes protein carbonylation, which irreversibly alters protein function and leads to detrimental protein cross-links. To uncover the oxidation pattern of seed stored RNAs and proteins along with seed ageing in a limited time span, an artificial ageing system elevated partial pressure of oxygen (EPPO), which mimics and accelerates seed ageing, has been applied. The integrity of total stored RNAs, the dynamic expression patterns of seed stored mRNAs and the oxidation profiles of seed storage proteins were explored. We have identified severe mRNA degradation during seed ageing and show that this degradation correlates with the germination capacity of seeds. Moreover, we have a strong indication that the degradation of monosome associated transcripts is delayed.