The Arabidopsis LEAFY COTYLEDON1 (LEC1) gene is required for the specification of cotyledon identity and the completion of embryo maturation. We isolated the LEC1 gene and showed that it functions at an early developmental stage to maintain embryonic cell fate. The LEC1 gene encodes a transcription factor homolog, the CCAAT box-binding factor HAP3 subunit. LEC1 RNA accumulates only during seed development in embryo cell types and in endosperm tissue. Ectopic postembryonic expression of the LEC1 gene in vegetative cells induces the expression of embryo-specific genes and initiates formation of embryo-like structures. Our results suggest that LEC1 is an important regulator of embryo development that activates the transcription of genes required for both embryo morphogenesis and cellular differentiation.

The hormonal action of abscisic acid (ABA) in plants is controlled by the precise balance between its biosynthesis and catabolism. In plants, ABA 8 0 -hydroxylation is thought to play a predominant role in ABA catabolism. ABA 8 0 -hydroxylase was shown to be a cytochrome P450 (P450); however, its corresponding gene had not been identified. Through phylogenetic and DNA microarray analyses during seed imbibition, the candidate genes for this enzyme were narrowed down from 272 Arabidopsis P450 genes. These candidate genes were functionally expressed in yeast to reveal that members of the CYP707A family, CYP707A1-CYP707A4, encode ABA 8 0 -hydroxylases. Expression analyses revealed that CYP707A2 is responsible for the rapid decrease in ABA level during seed imbibition. During drought stress conditions, all CYP707A genes were upregulated, and upon rehydration a significant increase in mRNA level was observed. Consistent with the expression analyses, cyp707a2 mutants exhibited hyperdormancy in seeds and accumulated six-fold greater ABA content than wild type. These results demonstrate that CYP707A family genes play a major regulatory role in controlling the level of ABA in plants.

Screening for brassinosteroid (BR) biosynthesis inhibitors was performed to find chemicals that induce dwarfism in Arabidopsis, mutants that resembled BR biosynthesis mutants that can be rescued by BR. Through this screening experiment, the compound brassinazole was selected as the most potent chemical. In dark-grown Arabidopsis, brassinazoleinduced morphological changes were nearly restored to those of wild type by treatment with brassinolide. The structure of brassinazole is similar to pacrobutrazol, a gibberellin biosynthesis inhibitor. However, in assays with cress (Lepidium sativum) plants, brassinazole-treated plants did not show recovery after the addition of gibberellin but showed good recovery after the addition of brassinolide. These data demonstrate that brassinazole is a specific BR biosynthesis inhibitor. Brassinazole-treated cress also showed dwarfism, with altered leaf morphology, including the downward curling and dark green color typical of Arabidopsis BR-deficient mutants, and this dwarfism was reversed by the application of 10 nm brassinolide. This result suggests that BRs are essential for plant growth, and that brassinazole can be used to clarify the function of BRs in plants as a complement to BR-deficient mutants. The brassinazole action site was also investigated by feeding BR biosynthesis intermediates to cress grown in the light.

A secreted luciferase from the marine ostracod, Vargula hilgendorfii, is a useful tool for gene expression assays in living mammalian cells. We have cloned the cDNA of a new secreted luciferase from the ostracod Cypridina noctiluca, which inhabits the coast of Japan. C. noctiluca luciferase consists of 553 amino acid residues with a molecular mass of 61,415 Da, as deduced from the nucleotide sequence. The homologies of nucleotide and amino acid sequences with V. hilgendorfii luciferase are 79.2% and 83.1%, respectively. C. noctiluca luciferase can expressed in and secreted from cultured mammalian cells. The characteristic properties of expressed C. noctiluca luciferase are similar to those of V. hilgendorfii luciferase. However, the activity of C. noctiluca luciferase in culture medium is much higher than that of V. hilgendorfii luciferase, suggesting that C. noctiluca luciferase is a highly potent reporter enzyme for real-time and continuous monitoring of gene expression in living cells.

In the chloroplast psbD light-responsive promoter (LRP), a highly conserved sequence exists upstream from the bacterial Ϫ10/Ϫ35 elements. Multiple sequence-specific DNA binding proteins are predicted to bind to the conserved sequence as transcription factors. Using yeast one-hybrid screening of an Arabidopsis cDNA library, a possible DNA binding protein of the psbD LRP upstream sequence was identified. The protein, designated PTF1, is a novel protein of 355 amino acids (estimated molecular weight of 39.6) that contains a basic helix-loop-helix DNA binding motif in the predicted N-terminal region of the mature protein. Transient expression assay of PTF1-GFP fusion protein showed that PTF1 was localized in chloroplasts. Using the modified DNA sequence in the one-hybrid system, the ACC repeat was shown to be essential for PTF1 binding. The rate of psbD LRP mRNA accumulation was reduced in a T-DNA-inserted Arabidopsis ptf1 mutant. Compared with wild-type plants, the mutant had pale green cotyledons and its growth was inhibited under short-day conditions. These results suggest that PTF1 is a trans-acting factor of the psbD LRP.In the early stage of light-induced chloroplast development, the transcription activity of chloroplast genes increases, leading to increased mRNA accumulation (Klein and Mullet, 1990;Rapp et al., 1992; DuBell and Mullet, 1995). Transcription rates vary among promoters, and in most cases, they reflect the amount of transcripts and the stoichiometric composition of proteins (Rapp et al., 1992). Although mRNA stability is an important regulatory factor in mature chloroplasts (Kawaguchi et al., 1992; Kim et al., 1993;Staub and Maliga, 1993;Shiina et al., 1998), the transcription rate plays a primary role in controlling gene expression in developing chloroplasts (for review, see Mullet, 1993; Mayfield et al., 1995;Stern et al., 1997).Recent studies have clarified many of the molecular mechanisms of transcriptional regulation in plastids by successive cloning of RNA polymerase core and accessory subunits for plastid transcription (for review, see Maliga, 1998; Hess and Bö rner, 1999). One is nuclear-encoded bacteriophage-type RNA polymerase (Hedtke et al., 1997), which functions in transcription from the nuclear-encoded bacteriophage-type RNA polymerase promoters that exist in most nonphotosynthetic genes (Hajdukiewicz et al., 1997; Kapoor et al., 1997). On the other hand, many plastid genes have eubacterial 70 -type promoters, which are preceded by "Ϫ10" and "Ϫ35" elements (consensus TATAAT and TTGACA, respectively; Hanley- Bowdoin and Chua, 1987; Igloi and Kö ssel, 1992). These promoters are recognized by plastid-encoded RNA polymerase (PEP), which is composed of plastid-encoded catalytic core subunits associated with nuclear-encoded subunits (for review, see Link, 1996;Maliga, 1998; and Hess and Bö rner, 1999). Many plastid-factors have been cloned in higher plants (Isono et al., 1997;Tanaka et al., 1997; Kestermann et al., 1998;Tozawa et al., 1998). As in bacterial RNA polymerase, plastid-fac...