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Content archived on 2024-04-30

Genetic and molecular markers for seed quality

Deliverables

Extensive expression studies using a battery of Arabidopsis cell cycle genes were performed in cabbage (Brassica oleracea f. oleracea L.) and Arabidopsis seeds as target organs. By carrying out these studies we were able to precisely define the spatial and temporal expression profiles of different cell cycle genes during imbibition and early seed germination. From our studies we could show that most cell cycle genes are not expressed in dry seeds and are only activated during or following radicle protrusion. These results lead us to conclude that cell division activity per se does not seem to be fundamental for the initiation of the seed germination process but might be essential to overpass a particular developmental stage during post-germination. Moreover, we have shown that it was not possible to distinguish between cabbage lines with different germination performances based on the expression of the cell cycle genes. These lines seem to present only a temporal disparity on cell cycle gene expression, meaning that cell cycle gene expression is indeed affected (delayed) in poor germinating seeds. However, this might be due to a delay in the activation of the general transcription machinery rather than being directly associated to the cell cycle. Based on these data, we conclude that the studied cell cycle genes may not be appropriate markers for seed quality.
In collaboration with partner 2 (INRA) and partner 4 (Wageningen University) we proceeded to the mRNA expression analysis of several genes (DD1-1, DD1-2, DD1-5, DD1-5, DD1-10, CR11 and WGT157), which have been isolated during the course of this project due to their particular involvement in germination. The majority of these genes were expressed in the radicle and radicle tip, during and following protrusion. These studies suggest that during seed imbibition, transcript accumulation in the embryonic radicle tip might be essential not only for proper radicle growth and consequent protrusion, but also for further seedling development.
The pre-germination process (priming) has been studied with sugarbeet and tomato seeds particularly with regard to the solubilisation of seed storage proteins during the process. In our previous work on sugar beet seeds, we showed that priming induces the mobilisation of 11S globulin, resulting in the solubilisation in water of part of the globulin (its B subunit). In this work, specific antibodies have been obtained for the 11S-globulin B-subunit of tomato seeds. Furthermore, a comparative study between this 11S-globulin solubilisation and the induction of cell cycle activity (as measured by flow cytometry of seed nuclei) during priming of tomato seeds, shows that the two phenomena are induced at different timing during priming, the globulin being first mobilised, followed by cell cycle reactivation. Based upon use of antibodies against 11S-globulin B-subunit from tomato and sugarbeet, specific ELISAs have been designed and/or optimised. Such assays now allow readily following and optimising the priming treatments, without solely relying on germination assays. The ELISAs can also been used in single seed assays, allowing to characterise biochemical heterogeneity of commercial seed lots, in relation with their physiological heterogeneity (seed germination). Finally, the ELISAs can be used to assess the initial quality of commercial seed lots (certification analyses), i.e. to decipher whether they have been primed either intentionally or naturally (during seed development on the mother plant). Such assays can be used commercially.
Proteomics has been developed to globally investigate the germination and priming processes using Arabidopsis seeds as a model system. An optimised experimental protocol is provided. Reference maps of seed proteins, including more than 100 proteins at different stages of germination and priming have been obtained in this way.
Flow cytometry offers the possibility of high throughput analysis of individual cells or cell organelles. This technique is employed for analyses of nuclei isolated from seeds, during seed development, maturation, germination and priming. It enables to analyse the onset of DNA replication, which occurs in most species prior to radicle protrusion. Imbibition of seeds is accompanied by modification of DNA protein structure and can be followed using this technique. Different seed species may require different nuclear extraction buffers. Protocols for a multitude of seed species have been developed.

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