Phytoene synthase catalyzes the dimerization of two molecules of geranylgeranyl pyrophosphate to phytoene and has been shown to be rate limiting for the synthesis of carotenoids. To elucidate if the capacity to produce phytoene is limiting also in the seed of Arabidopsis (Wassilewskija), a gene coding for an endogenous phytoene synthase was cloned and coupled to a seed-specific promoter, and the effects of the overexpression were examined. The resulting transgenic plants produced darker seeds, and extracts from the seed of five overexpressing plants had a 43-fold average increase of ␤-carotene and a total average amount of ␤-carotene of approximately 260 g g Ϫ1 fresh weight. Lutein, violaxanthin, and chlorophyll were significantly increased, whereas the levels of zeaxanthin only increased by a factor 1.1. In addition, substantial levels of lycopene and ␣-carotene were produced in the seeds, whereas only trace amounts were found in the control plants. Seeds from the transgenic plants exhibited delayed germination, and the degree of delay was positively correlated with the increased levels of carotenoids. The abscisic acid levels followed the increase of the carotenoids, and plants having the highest carotenoid levels also had the highest abscisic acid content. Addition of gibberellic acid to the growth medium only partly restored germination of the transgenic seeds.In higher plants, carotenoids are synthesized in the plastid via the 1-deoxy-d-xylulose-5-phosphate (DOXP) isoprenoid biosynthetic pathway (Lichtenthaler et al., 1997). These pigments can act as visual attractants, function as structural components in the photosystems, and trigger biochemical reactions. Phytoene synthases dimerize two geranylgeranyl pyrophosphate molecules to prephytoene diphosphate and the following conversion to phytoene, a noncolored hydrophobic C 40 carbon molecule (Dogbo et al., 1988). Consecutive desaturation, isomerization, cyclization, and oxygenation result in a number of different carotenoids (Fig. 1). Phytoene synthases are regulated both at the transcriptional and posttranscriptional levels and are a rate-limiting key enzyme in the biosynthetic pathway of carotenoid synthesis (Burkhardt et al., 1997;Hirschberg, 2001). In white mustard (Sinapis alba), the enzyme is inactive in darkgrown plantlets and localized to the prolamellar body of the plastid. White light results in relocalization, formation of thylakoids, and activation of the enzyme (Welsch et al., 2000). Purified phytoene synthase requires Mn 2ϩ and ATP for its activity, and ␤-carotene was shown to inhibit the reaction (Fraser et al., 2000). Constitutive expressions of phytoene synthases in tomato (Lycopersicon esculentum) and tobacco (Nicotiana tabacum) resulted in dwarfism, chlorosis, and differential coloring of the plants (Fray et al., 1995;Busch et al., 2002). In the tomato plants, the levels of GA were decreased, and in some instances, a small decrease of the levels of abscisic acid (ABA) was also found (Fray et al., 1995). By using tissue-specific promoters f...

SummaryThe cuticle plays a critical role in plant survival during extreme drought conditions. There are, however, surprisingly, many gaps in our understanding of cuticle biosynthesis.An Arabidopsis thaliana T-DNA mutant library was screened for mutants with enhanced transpiration using a simple condensation spot method. Five mutants, named cool breath (cb), were isolated.The cb5 mutant was found to be allelic to bodyguard (bdg), which is affected in an a/bhydrolase fold protein important for cuticle structure. The analysis of cuticle components in cb5 (renamed as bdg-6) and another T-DNA mutant allele (bdg-7) revealed no impairment in wax synthesis, but a strong decrease in total cutin monomer load in young leaves and flowers. Root suberin content was also reduced. Overexpression of BDG increased total leaf cutin monomer content nearly four times by affecting preferentially C18 polyunsaturated x-OH fatty acids and dicarboxylic acids. Whole-plant gas exchange analysis showed that bdg-6 had higher cuticular conductance and rate of transpiration; however, plant lines overexpressing BDG resembled the wild-type with regard to these characteristics. This study identifies BDG as an important component of the cutin biosynthesis machinery in Arabidopsis. We also show that, using BDG, cutin can be greatly modified without altering the cuticular water barrier properties and transpiration.