Absicisic acid synthesis and signaling in Arabidopsis seed development – ABSIG

To study the ABA signaling network, 'omics' approaches are used on ABA deficient mutants, whose seeds are harvested at different stages of development, before and after dormancy induction. Transcriptome analysis is performed at URGV-Evry by CATMAV6 microarray hydridization. The total soluble proteome is analyzed by two-dimensional gel electrophoresis and the differentially expressed proteins are identified by mass spectrometry. Finally metabolome analysis by mass spectrometry aims to determine the content of ABA and its catabolites, as well as the major metabolites accumulated in the seed. The differentially expressed genes and proteins will be studied further.
The search for regulators of ABA biosynthesis is performed by two approaches. The first aims at the identification of mutations that deregulate the expression of an ABA biosynthesis gene. Mutant selection is performed by binocular observation of the expression of a reporter gene placed downstream of the promoter of the gene studied. The mutated genes are identified by next generation sequencing. The other approach, called yeast one-hybrid, tests in yeast the binding ability of a number of Arabidopsis transcription factors to a promoter fragment.
Candidate genes derived from mutagenesis, one-hybrid and 'omics' experiments are studied by genetic, physiological, biochemical and molecular methods, using insertion mutants available in seed banks. These include germination assays, hormone measurements, gene expression and protein activity analysis.

After seed mutagenesis, we selected thirty mutants potentially deregulated for the expression of an ABA biosynthesis gene, called NCED6. We also identified a family of transcription factors that interact with the NCED9 promoter. Mutants and candidate genes are being studied.

The identification of genes involved in seed dormancy regulation has a potential interest for plant breeding. When dormancy is too low, pre-harvest sprouting can occur under certain climatic conditions, in wheat for instance, and cause high economic losses due to poor grain quality. In contrast some species exhibit a deep dormancy, preventing rapid and synchronous germination in the field.

An article published in Plant Journal describes the tissue specificity of expression of an ABA biosynthesis gene family in Arabidopsis developing seeds, and gene member contribution to dormancy induction.
Frey A, Effroy D, Lefebvre V, Seo M, Perreau F, Berger A, Sechet J, To A, North HM, Marion-Poll A (2012) Epoxycarotenoid cleavage by NCED5 fine-tunes ABA accumulation and affects seed dormancy and drought tolerance with other NCED family members. Plant J 70, 501-512

In seeds, ABA is synthesized in tissues which are genetically distinct, the testa is constituted of maternal tissues derived from ovule integuments, whereas the triploid endosperm and the diploid embryo both result from double fertilization and contain maternal and paternal genome equivalents. In addition, a large fraction of the ABA accumulated in developing seeds is transported from the mother plant tissues. Levels of seed ABA derived from maternal sources are maximal during the maturation phase and are required for the stimulation of reserve storage, while embryonic ABA is accumulated at late developmental stages and induces seed dormancy and desiccation tolerance.
Our previous study of the expression of NCED (9-cis epoxycarotenoid dioxygenase) genes, encoding key enzymes of the ABA biosynthesis pathway, has shown that both embryo and endosperm expression contribute to dormancy induction. An objective of this project is to understand how the different ABA pools are regulated in seeds and whether downstream signaling networks and targets differ depending on the site of ABA production. To achieve this goal, omics and reverse/forward genetic approaches will be employed to identify which elements of the ABA signaling pathway operate in developing seeds and their targets. We will take advantage of novel genetic material that has been generated recently in our group, in which ABA synthesis is restricted to specific seed tissues (maternal/embryo/endosperm). These mutants are devoid of the pleiotropic vegetative phenotypes characteristic of severe ABA-deficient or signaling mutants frequently used in previous studies. We shall determine which of the known ABA signaling elements are differentially regulated by ABA in different seed tissues and also identify new factors involved in dormancy induction by ABA.
The precise regulation of hormone levels has been shown to be crucial for the control of plant physiological responses by ABA. As yet, however, no gene has been proven to directly regulate ABA levels. Since NCED biosynthesis genes catalyze the key-step of ABA synthesis, our second objective will be the identification of factors that regulate NCED expression. We have already generated homozygous transformants carrying both pNCED6::GUS and pNCED6::GFP that will be used to identify mutants with altered NCED6 expression; in the wild type this is specific to the endosperm. Furthermore, in collaboration with our Japanese partner, the same material will be exploited in chemical genetic screens. Deletion series of NCED promoters have also been generated and will be used in one-hybrid screens to identify promoter-interacting factors.
A number of loci have been identified that are involved in ABA signaling in vegetative tissues to control stomatal aperture and stress responses. The integration of many of these into a comprehensive network has not been possible. A number of pieces are still missing from the puzzle, as illustrated by the recent discovery that a START protein family is involved in ABA perception. The ABA signaling network in seeds is even less well understood because seed is less amenable than stomata for the study of ABA signaling. In previous screens we have isolated two potentially new mutants with seed germination and ABA phenotypes. We propose in the third part of this project to identify the mutant loci and functionally characterize the corresponding genes, thereby adding two new genes to the ABA signaling network controlling seed dormancy and germination. Their relation to candidate genes identified in the first two parts of the project will also be investigated.