The testa of higher plant seeds protects the embryo against adverse environmental conditions. Its role is assumed mainly by controlling germination through dormancy imposition and by limiting the detrimental activity of physical and biological agents during seed storage. To analyze the function of the testa in the model plant Arabidopsis, we compared mutants affected in testa pigmentation and/or structure for dormancy, germination, and storability. The seeds of most mutants exhibited reduced dormancy. Moreover, unlike wild-type testas, mutant testas were permeable to tetrazolium salts. These altered dormancy and tetrazolium uptake properties were related to defects in the pigmentation of the endothelium and its neighboring crushed parenchymatic layers, as determined by vanillin staining and microscopic observations. Structural aberrations such as missing layers or a modified epidermal layer in specific mutants also affected dormancy levels and permeability to tetrazolium. Both structural and pigmentation mutants deteriorated faster than the wild types during natural aging at room temperature, with structural mutants being the most strongly affected.

Novel Arabidopsis mutants with lowered levels of endogenous abscisic acid (ABA) were isolated. These were selected in a screen for germination in the presence of the gibberellin biosynthesis inhibitor paclobutrazol. Another mutant was isolated in a screen for NaCl tolerance. The ABA-deficiency was caused by two monogenic, recessive mutations, aba2 and aba3, that were both located on chromosome 1. The mutants showed a phenotype that is known to be characteristic for ABA-deficiency: a reduced seed dormancy and excessive water loss, leading to a wilty phenotype. Double mutant analysis, combining different aba mutations, indicated the leaky nature of the mutations.

Phenolic compounds that are present in the testa interfere with the physiology of seed dormancy and germination. We isolated a recessive Arabidopsis mutant with pale brown seeds, transparent testa12 ( tt12 ), from a reduced seed dormancy screen. Microscopic analysis of tt12 developing and mature testas revealed a strong reduction of proanthocyanidin deposition in vacuoles of endothelial cells. Double mutants with tt12 and other testa pigmentation mutants were constructed, and their phenotypes confirmed that tt12 was affected at the level of the flavonoid biosynthetic pathway. The TT12 gene was cloned and found to encode a protein with similarity to prokaryotic and eukaryotic secondary transporters with 12 transmembrane segments, belonging to the MATE (multidrug and toxic compound extrusion) family. TT12 is expressed specifically in ovules and developing seeds. In situ hybridization localized its transcript in the endothelium layer, as expected from the effect of the tt12 mutation on testa flavonoid pigmentation. The phenotype of the mutant and the nature of the gene suggest that TT12 may control the vacuolar sequestration of flavonoids in the seed coat endothelium. INTRODUCTIONFlavonoids represent a highly diverse group of plant aromatic secondary metabolites. The major forms, which are anthocyanins (red to purple pigments), flavonols (colorless to pale yellow pigments), and proanthocyanidins (PAs), also known as condensed tannins (colorless pigments that brown with oxidation), are present in proportions and amounts that vary according to the plant species, the organ, the stage of development, and environmental growth conditions. Arabidopsis contains flavonoid pigments in vegetative tissues and in seeds (Shirley et al., 1995). In the mature testa, flavonoids were detected in both the endothelium and the three crushed parenchymal layers just above the endothelium . PAs have been shown to accumulate exclusively in the endothelium layer (Devic et al., 1999). Wild-type seed color depends on the stage of development: as long as the testa is transparent, until ‫ف‬ 10 days after pollination, the seed has the color of the underlying endosperm and embryo. At maturity, the testa becomes opaque after the oxidation of flavonoid pigments, which gives the seed its characteristic brown color. Therefore, the color of mutants with seed flavonoid defects ranges from pale yellow (complete lack of visible pigments in the testa) to pale brown, depending on the accumulated intermediate flavonoids .In Arabidopsis, mutants at 21 loci have been isolated on the basis of altered testa pigmentation (Figure 1). The genes disrupted in the transparent testa ( tt ) mutants tt3 , tt4 , tt5 , tt6 , and tt7 (Shirley et al., 1995) and the banyuls ( ban ) mutant (Albert et al., 1997) code for the enzymes dihydroflavonol-4-reductase (Shirley et al., 1992), chalcone synthase (Feinbaum and Ausubel, 1988), chalcone isomerase (Shirley et al., 1992), flavonol 3-hydroxylase (Pelletier and Shirley, 1996; Wisman et al., 1998), flavonol 3 Ј -hydroxylase (Ko...

Two new abscisic acid (ABA)-insensitive mutants of

Summary In order to identify new factors involved in Em (a class I Late Embryogenesis Abundant protein) gene expression, Arabidopsis mutants with an altered expression of an Em promoter GUS fusion construct and a modified accumulation of Em transcripts and proteins were isolated. Germination tests on ABA showed that the most affected mutant had a weak abi phenotype. Complementation tests further revealed this mutant to be a new abi5 allele, consequently named abi5–5. In addition to reducing the final level of Em transcripts in the dry seed, the abi5–5 mutation causes a delay in the accumulation of AtEm1 during seed development. An additional characteristic of the abi5–5 mutant, is the ability of its seeds to germinate at high concentrations of salt and mannitol. The abi5–5 mutation was characterized at the molecular level and was shown to result from a two base pair deletion in the coding sequence of the ABI 5 gene. The wild type and mutant recombinant proteins were produced in E. coli and were assayed for DNA‐binding activity on their target promoters by electrophoretic mobility shift assay (EMSA). The ABI5 recombinant protein binds the ABRE sequence in the AtEm6 promoter as shown by Dnase footprinting. Among the ABRE‐type sequences selected on both Em promoters, the G‐box type AGACACGTGGCATGT element of the AtEm6 promoter shows the strongest binding by EMSA quantification.

Abscisic acid (ABA)-deficient mutants in a variety of species have been identified by screening for precocious germination and a wilty phenotype. Mutants at two new loci, aba2 and aba3, have recently been isolated i n Arabidopsis fhaliana (L.) Heynh. (

Phenolic compounds that are present in the testa interfere with the physiology of seed dormancy and germination. We isolated a recessive Arabidopsis mutant with pale brown seeds, transparent testa12 (tt12), from a reduced seed dormancy screen. Microscopic analysis of tt12 developing and mature testas revealed a strong reduction of proanthocyanidin deposition in vacuoles of endothelial cells. Double mutants with tt12 and other testa pigmentation mutants were constructed, and their phenotypes confirmed that tt12 was affected at the level of the flavonoid biosynthetic pathway. The TT12 gene was cloned and found to encode a protein with similarity to prokaryotic and eukaryotic secondary transporters with 12 transmembrane segments, belonging to the MATE (multidrug and toxic compound extrusion) family. TT12 is expressed specifically in ovules and developing seeds. In situ hybridization localized its transcript in the endothelium layer, as expected from the effect of the tt12 mutation on testa flavonoid pigmentation. The phenotype of the mutant and the nature of the gene suggest that TT12 may control the vacuolar sequestration of flavonoids in the seed coat endothelium.

No abstract