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Flavonoids are secondary metabolites that accumulate in most plant seeds and are involved in physiological functions such as dormancy or viability. This review presents a current view of the genetic and biochemical control of flavonoid metabolism during seed development. It focuses mainly on proanthocyanidin accumulation in Arabidopsis, with comparisons to other related metabolic and regulatory pathways. These intricate networks and their fine-tuned regulation, once they are determined, should contribute to a better understanding of seed coat development and the control of PA and flavonol metabolism. In addition, flavonoids provide an interesting model to study various biological processes and metabolic and regulatory networks.

SummaryGenetic analyses have demonstrated that together with TTG1, a WD-repeat (WDR) protein, TT2 (MYB), and TT8 (bHLH) are necessary for the correct expression of BANYULS (BAN). This gene codes for the core enzyme of proanthocyanidin biosynthesis in Arabidopsis thaliana seed coat. The interplays of TT2, TT8, and their closest MYB/bHLH relatives, with TTG1 and the BAN promoter have been investigated using a combination of genetic and molecular approaches, both in yeast and in planta. The results obtained using glucocorticoid receptor fusion proteins in planta strongly suggest that TT2, TT8, and TTG1 can directly activate BAN expression. Experiments using yeast two-and three-hybrid clearly demonstrated that TT2, TT8, and TTG1 can form a stable ternary complex. Furthermore, although TT2 and TT8 were able to bind to the BAN promoter when simultaneously expressed in yeast, the activity of the complex correlated with the level of TTG1 expression in A. thaliana protoplasts. In addition, transient expression experiments revealed that TTG1 acts mainly through the bHLH partner (i.e. TT8 or related proteins) and that TT2 cannot be replaced by any other related A. thaliana MYB proteins to activate BAN. Finally and consistent with these results, the ectopic expression of TT2 was sufficient to trigger BAN activation in vegetative parts, but only where TTG1 was expressed. Taken together, these results indicate that TT2, TT8, and TTG1 can form a ternary complex directly regulating BAN expression in planta.

The Arabidopsis LEAFY COTYLEDON2 (LEC2) gene is a central embryonic regulator that serves critical roles both early and late during embryo development. LEC2 is required for the maintenance of suspensor morphology, specification of cotyledon identity, progression through the maturation phase, and suppression of premature germination. We cloned the LEC2 gene on the basis of its chromosomal position and showed that the predicted polypeptide contains a B3 domain, a DNA-binding motif unique to plants that is characteristic of several transcription factors. We showed that LEC2 RNA accumulates primarily during seed development, consistent with our finding that LEC2 shares greatest similarity with the B3 domain transcription factors that act primarily in developing seeds, VIVIPAROUS1/ABA INSENSITIVE3 and FUSCA3. Ectopic, postembryonic expression of LEC2 in transgenic plants induces the formation of somatic embryos and other organ-like structures and often confers embryonic characteristics to seedlings. Together, these results suggest that LEC2 is a transcriptional regulator that establishes a cellular environment sufficient to initiate embryo development.

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SummaryThe WRINKLED1 (WRI1) transcription factor has been shown to play a role of the utmost importance during oil accumulation in maturing seeds of Arabidopsis thaliana. However, little is known about the regulatory processes involved. In this paper, comprehensive functional analyses of three new mutants corresponding to null alleles of wri1 confirm that the induction of WRI1 is a prerequisite for fatty acid synthesis and is important for normal embryo development. The strong expression of WRI1 specifically detected at the onset of the maturation phase in oil-accumulating tissues of A. thaliana seeds is fully consistent with this function. Complementation experiments carried out with various seed-specific promoters emphasized the importance of a tight regulation of WRI1 expression for proper oil accumulation, raising the question of the factors controlling WRI1 transcription. Interestingly, molecular and genetic analyses using an inducible system demonstrated that WRI1 is a target of LEAFY COTYLEDON2 and is necessary for the regulatory action of LEC2 towards fatty acid metabolism. In addition to this, quantitative RT-PCR experiments suggested that several genes encoding enzymes of late glycolysis, the fatty acid synthesis pathway, and the biotin and lipoic acid biosynthetic pathways are targets of WRI1. Taken together, these results indicate new relationships in the regulatory model for the control of oil synthesis in maturing A. thaliana seeds. In addition, they exemplify how metabolic and developmental processes affecting the developing embryo can be coordinated at the molecular level.

Anthocyanidin reductase encoded by the BANYULS ( BAN ) gene is the core enzyme in proanthocyanidin (PA) biosynthesis. Here, we analyzed the developmental mechanisms that regulate the spatiotemporal expression of BAN in the developing Arabidopsis seed coat. PA-accumulating cells were localized histochemically in the inner integument (seed body and micropyle) and pigment strand (chalaza). BAN promoter activity was detected specifically in these cells. Gain-of-function experiments showed that an 86-bp promoter fragment functioned as an enhancer specific for PA-accumulating cells. Mutations in regulatory genes of PA biosynthesis abolished BAN promoter activity ( transparent testa2 [ tt2 ], tt8 , and transparent testa glabra1 [ ttg1 ]), modified its spatial pattern ( tt1 and tt16 ), or had no influence ( ttg2 ), thus revealing complex regulatory interactions at several developmental levels. Genetic ablation of PA-accumulating cells targeted by the BAN promoter fused to BARNASE led to the formation of normal plants that produced viable yellow seeds. Importantly, these seeds had no obvious defects in endosperm and embryo development.