The completion of the Arabidopsis thaliana genome sequence allows a comparative analysis of transcriptional regulators across the three eukaryotic kingdoms. Arabidopsis dedicates over 5% of its genome to code for more than 1500 transcription factors, about 45% of which are from families specific to plants. Arabidopsis transcription factors that belong to families common to all eukaryotes do not share significant similarity with those of the other kingdoms beyond the conserved DNA binding domains, many of which have been arranged in combinations specific to each lineage. The genome-wide comparison reveals the evolutionary generation of diversity in the regulation of transcription.

Tables S1 to S3, S5, S6, and S9 as zipped Excel files

In plants, low temperature and dehydration activate a set of genes containing C-repeat/dehydration-responsive elements in their promoter. It has been shown previously that the Arabidopsis CBF/DREB1 transcription activators are critical regulators of gene expression in the signal transduction of cold acclimation. Here, we report the isolation of an apparent homolog of the CBF/DREB1 proteins (CBF4) that plays the equivalent role during drought adaptation. In contrast to the three already identified CBF/DREB1 homologs, which are induced under cold stress, CBF4 gene expression is up-regulated by drought stress, but not by low temperature. Overexpression of CBF4 in transgenic Arabidopsis plants results in the activation of C-repeat/dehydration-responsive element containing downstream genes that are involved in cold acclimation and drought adaptation. As a result, the transgenic plants are more tolerant to freezing and drought stress. Because of the physiological similarity between freezing and drought stress, and the sequence and structural similarity of the CBF/DREB1 and the CBF4 proteins, we propose that the plant's response to cold and drought evolved from a common CBF-like transcription factor, first through gene duplication and then through promoter evolution.Many plants increase their tolerance to freezing after exposure to low nonfreezing temperatures-a phenomenon known as cold acclimation (Hughes and Dunn, 1996;Thomashow, 1998). The major component of this acquired freezing tolerance is the tolerance to dehydration stress caused by extracellular ice formation during the freezing process. The presence of ice lowers the water potential extracellularly and causes water to flow out of cells (Pearce, 1999). Thus, a major cause of freezing damage is the freeze-induced dehydration (Steponkus and Webb, 1992;Thomashow, 1998). Because a plant's ability to survive freezeinduced dehydration is related to its adaptation to drought, it is not surprising that plants respond to low temperature and drought very similarly at the molecular level . Many genes, such as RD (responsive to dehydration), ERD (early responsive to dehydration), COR (cold regulated), LTI (low-temperature induced), and KIN (cold inducible), are induced by both low temperature and drought stress (Ingram and Bartels, 1996;Pearce, 1999; Thomashow, 1999; Shinozaki and YamaguchiShinozaki, 2000). The similarity of cold and drought stresses is further demonstrated by experiments showing that mild drought stress can result in increased freezing tolerance in plants (Clavitier and Siminovitch, 1982;Siminovitch and Cloutier, 1983; Guy et al., 1992).Recently, a major transcriptional regulatory system that controls abscisic acid (ABA) independent gene expression in response to low temperature has been identified (Stockinger et al., 1997;Liu et al., 1998). The system is based on the C-repeat (CRT)/ dehydration-responsive element (DRE) cis-acting element and the trans-acting DNA-binding protein CBF/DREB1 (CRT-binding factor or DRE-binding protein). There are three CB...

In many organisms, the circadian clock is composed of functionally coupled morning and evening oscillators. In Arabidopsis, oscillator coupling relies on a core loop in which the evening oscillator component TIMING OF CAB EXPRESSION 1 (TOC1) was proposed to activate a subset of morning-expressed oscillator genes. Here, we show that TOC1 does not function as an activator but rather as a general repressor of oscillator gene expression. Repression occurs through TOC1 rhythmic association to the promoters of the oscillator genes. Hormone-dependent induction of TOC1 and analysis of RNA interference plants show that TOC1 prevents the activation of morning-expressed genes at night. Our study overturns the prevailing model of the Arabidopsis circadian clock, showing that the morning and evening oscillator loops are connected through the repressing activity of TOC1.

The MADS domain homeotic proteins APETALA1 (API), APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) act in a combinatorial manner to specify the identity ofArabidopsis floral organs. The molecular basis for this combinatorial mode of action was investigated.Immunoprecipitation experiments indicate that all four proteins are capable of interacting with each other. However, these proteins exhibit "partner-specificity" for the formation of DNA-binding The study of homeotic mutants in Arabidopsis thaliana and Antirrhinum majus has led to the establishment of a genetic model (the ABC model) that explains how the fates of floral organ primordia are determined (1, 2). According to the ABC model, the identities of the organs of an Arabidopsis flower (four sepals, four petals, six stamens, and two carpels) are specified by the action of at least five organ identity genes, all of which have been cloned: APETALA1 (AP1), APETALA2 (AP2),APETALA3 (AP3), PISTILLATA (PI), andAGAMOUS (AG) (3-7). In situ hybridization and ectopic expression experiments (8-10) have provided strong evidence supporting the ABC model, showing that each whorl of a flower primordium has a unique combination of organ identity activities, which combinatorially specify organ identity. The specification of sepals is dependent on class A gene activities (AP1 and AP2), petals are specified by a combination of class A and class B (AP3 and PI) gene activities, stamens are specified by the combined activities of classes B and C (AG), and specification of the carpels is achieved by class C activity.AP1, AP3, PI, and AG are all MADS domain-containing proteins (Fig. 1), while AP2 bears similarity to a different class of DNA binding proteins (4, 13). The MADS domain is a conserved region of 56 amino acids present in a variety of dimeric transcription factors from different organisms. The MADS regions of SRF and MCM1 have been characterized as DNA binding and dimerization domains (11,14). Within the family of MADS box proteins, the plant proteins are unique in that they contain another conserved region, the K box, that may form amphipathic alpha helices (ref. 12; Fig. 1 Here, we show that AP1, AP3, PI, and AG are all capable of interacting with each other, but that only AP1/AP1, AP3/PI, and AG/AG dimers (and also heterodimers formed by truncated AG and AP1) bind to CArG-box containing sequences. This DNA-binding partner specificity is mediated to a large degree by the L region of these proteins, a 31-35 aa segment located between the MADS and K boxes. MATERIALS AND METHODSPlasmid Constructioh. AP1, AP3, PI, and AG cDNA coding sequences were cloned into the pSPUTK in vitro translation vector (Stratagene). Because the initiating ATG codon was not found in the AG cDNA clones (7), the wild-type sequence 5'-CATTTT ... at the beginning of theAG cDNA was changed to 5'-ATGGGG.... The in vivo functionality of such an altered AG protein has been shown in ectopic expression experiments (8). The construction of PPIFLAG, to make FLAG epitopetagged PI protein, has been ...

No abstract

Epicuticular wax forms a layer of hydrophobic material on plant aerial organs, which constitutes a protective barrier between the plant and its environment. We report here the identification of WIN1, an Arabidopsis thaliana ethylene response factor-type transcription factor, which can activate wax deposition in overexpressing plants. We constitutively expressed WIN1 in transgenic Arabidopsis plants, and found that leaf epidermal wax accumulation was up to 4.5-fold higher in these plants than in control plants. A significant increase was also found in stems. Interestingly, Ϸ50% of the additional wax could only be released by complete lipid extractions, suggesting that not all of the wax is superficial. Gene expression analysis indicated that a number of genes, such as CER1, KCS1, and CER2, which are known to be involved in wax biosynthesis, were induced in WIN1 overexpressors. This observation indicates that induction of wax accumulation in transgenic plants is probably mediated through an increase in the expression of genes encoding enzymes of the wax biosynthesis pathway.

The Arabidopsis FLOWERING LOCUS C ( FLC ) gene is a key floral repressor in the maintenance of a vernalization response. In vernalization-sensitive genetic backgrounds, FLC levels are high, and they decline after exposure to long cold periods. Four FLC paralogs ( MAF2 [ MADS AFFECTING FLOWERING2 ] to MAF5 ) are arranged in a tandem array on the bottom of Arabidopsis chromosome V. We used a reverse genetics approach to analyze their functions. Loss-of-function and gainof-function studies indicate that MAF2 acts as a floral repressor. In particular, maf2 mutant plants display a pronounced vernalization response when subjected to relatively short cold periods, which are insufficient to elicit a strong flowering response in the wild type, despite producing a large reduction in FLC levels. MAF2 expression is less sensitive to vernalization than that of FLC , and its repressor activity is exerted independently or downstream of FLC transcription. Thus, MAF2 can prevent premature vernalization in response to brief cold spells. Overexpression of MAF3 or MAF4 produces alterations in flowering time that suggest that these genes also act as floral repressors and might contribute to the maintenance of a vernalization requirement. However, the final gene in the cluster, MAF5 , is upregulated by vernalization. Therefore, MAF5 could play an opposite role to FLC in the vernalization response.