Genetic and molecular interactions between BELL1 and MADS box factors support ovule development in Arabidopsis

In Arabidopsis thaliana and many other plant species, ovules arise from carpel tissue as new meristematic formations. Cell fate in proliferating ovule primordia is specified by particular ovule identity factors, such as the homeodomain factor BELL1 (BEL1) and MADS box family members SEEDSTICK (STK), SHATTERPROOF1 (SHP1), SHP2, and AGAMOUS. Both in the bel1 mutant and the stk shp1 shp2 triple mutant, integuments are transformed into carpelloid structures. Combining these mutants in a bel1 stk shp1 shp2 quadruple mutant, we showed that the bel1 phenotype is significantly enhanced. We also demonstrate that ovule differentiation requires the regulation of the stem cell maintenance gene WUSCHEL, repression of which is predominantly maintained by BEL1 during ovule development. Based on yeast three-hybrid assays and genetic data, we show that BEL1 interacts with the ovule identity MADS box factors when they dimerize with SEPALLATA proteins. We propose a model for ovule development that explains how the balance between carpel identity activity and ovule identity activity is established by a MADS box homeodomain protein complex.     

Genetic analysis of seed coat development in Arabidopsis

In the angiosperms, fertilization initiates the formation of the seed from the ovule, including the differentiation of the seed coat from the ovule integuments. Seed coat differentiation includes some of the most dramatic cellular changes of seed development and culminates in the death of the seed coat cells. Recently, genetic analyses in Arabidopsis have contributed substantially to our understanding of many aspects of seed coat biology and it might not be long before the entire differentiation pathway is understood. Such an advance would contribute substantially to our understanding of many important cellular events, including secondary cell wall synthesis, cell morphogenesis, vacuolar targeting and cell death, and would provide tools for the manipulation of seed dormancy and germination.     

BANYULS, a novel negative regulator of flavonoid biosynthesis in the Arabidopsis seed coat

A mutant of Arabidopsis that accumulates a high level of red pigments within the seed coat has been isolated from a population of T-DNA-transformed plants. Genetic analysis revealed that the mutation is recessive and affects maternal seed tissues only. Due to the color of the immature seeds, this mutation was named banyuls ( ban ). Pigments accumulated continuously from early seed development to the desiccation stage in the seed coat of the mutant. The phenotype of the double mutant banyuls/transparent testa confirmed the flavonoid nature of the pigments and enabled assignment of the regulatory TT ( Transparent Testa ) genes to two groups according to their epistatic relationship to ban . The flavonoid content of germinated ban and wild-type seedlings was similar. Plants harbouring the ban mutation had a normal formation of trichomes and root hairs and were not affected in their responses to light. The seeds of ban plants exhibited reduced germination compared to wild-type which may be a direct consequence of the high level of pigments. These results suggest that BANYULS functions as a negative regulator of flavonoid biosynthesis that prevents accumulation of pigments in the seed coat during early embryogenesis in Arabidopsis .     

The female gametophyte and the endosperm control cell proliferation and differentiation of the seed coat in Arabidopsis

Double fertilization of the female gametophyte produces the endosperm and the embryo enclosed in the maternal seed coat. Proper seed communication necessitates exchanges of signals between the zygotic and maternal components of the seed. However, the nature of these interactions remains largely unknown. We show that double fertilization of the Arabidopsis thaliana female gametophyte rapidly triggers sustained cell proliferation in the seed coat. Cell proliferation and differentiation of the seed coat occur in autonomous seeds produced in the absence of fertilization of the multicopy suppressor of ira1 (msi1) mutant. As msi1 autonomous seeds mostly contain autonomous endosperm, our results indicate that the developing endosperm is sufficient to enhance cell proliferation and differentiation in the seed coat. We analyze the effect of autonomous proliferation in the retinoblastoma-related1 (rbr1) female gametophyte on seed coat development. In contrast with msi1, supernumerary nuclei in rbr1 female gametophytes originate mainly from the endosperm precursor lineage but do not express an endosperm fate marker. In addition, defects of the rbr1 female gametophyte also reduce cell proliferation in the ovule integuments before fertilization and prevent further differentiation of the seed coat. Our data suggest that coordinated development of the seed components relies on interactions before fertilization between the female gametophyte and the surrounding maternal ovule integuments and after fertilization between the endosperm and the seed coat.     

Differentiation of mucilage secretory cells of the Arabidopsis seed coat

In some plant species, including Arabidopsis, fertilization induces the epidermal cells of the outer ovule integument to differentiate into a specialized seed coat cell type with a unique morphology and containing large quantities of polysaccharide mucilage (pectin). Such seed coat mucilage cells are necessary for neither viability nor germination under normal laboratory conditions. Thus, the Arabidopsis seed coat offers a unique system with which to use genetics to identify genes controlling cell morphogenesis and complex polysaccharide biosynthesis and secretion. As a first step in the application of this system, we have used microscopy to investigate the structure and differentiation of Arabidopsis seed coat mucilage cells, including cell morphogenesis and the synthesis, secretion, and extrusion of mucilage. During seed coat development in Arabidopsis, the epidermal cells of the outer ovule integument grow and differentiate into cells that produce large quantities of mucilage between the primary cell wall and plasma membrane. Concurrent with mucilage production, the cytoplasm is shaped into a column in the center of the cell. Following mucilage secretion the cytoplasmic column is surrounded by a secondary cell wall to form a structure known as the columella. Thus, differentiation of the seed coat mucilage cells involves a highly regulated series of events including growth, morphogenesis, mucilage biosynthesis and secretion, and secondary cell wall synthesis.     

TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana

Genetic 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 tt19-4 mutant differentially accumulates proanthocyanidin and anthocyanin through a 3' amino acid substitution in glutathione S-transferase

The Arabidopsis transparent testa (tt) mutant tt19-4 shows reduced seed coat colour, but stains darkly with DMACA and accumulates anthocyanins in aerial tissues. Positional cloning showed that tt19-4 was allelic to tt19-1 and has a G-to-T mutation in a conserved 3'-domain in the TT19-4 gene. Soluble and unextractable seed proanthocyanidins and hydrolysis of unextractable proanthocyanidin differ between wild-type Col-4 and both mutants. However, seed quercetins, unextractable proanthocyanidin hydrolysis, and seedling anthocyanin content, and flavonoid gene expression differ between tt19-1 and tt19-4. Transformation of tt19-1 with a TT19-4 cDNA results in vegetative anthocyanins, whereas TT19-4 cDNA cannot complement the proanthocyanidin and pale seed coat phenotype of tt19-1. Both recombinant TT19 and TT19-4 enzymes are functional GSTs and are localized in the cytosol, but TT19 did not function with wide range of flavonoids and natural products to produce conjugation products. We suggest that the dark seed coat of Arabidopsis is related to soluble proanthocyanidin content and that quercetin holds the key to the function of TT19. In addition, TT19 appears to have a 5' GSH-binding domain influencing both anthocyanin and proanthocyanidin accumulation and a 3' domain affecting proanthocyanidin accumulation by a single amino acid substitution.