Summary The CBF cold response pathway has a prominent role in cold acclimation. The pathway includes action of three transcription factors, CBF1, 2 and 3 (also known as DREB1b, c and a, respectively), that are rapidly induced in response to low temperature followed by expression of the CBF-targeted genes (the CBF regulon) that act in concert to increase plant-freezing tolerance. The results of transcriptome profiling and mutagenesis experiments, however, indicate that additional cold response pathways exist and may have important roles in life at low temperature. To further understand the roles that the CBF proteins play in configuring the low temperature transcriptome and to identify additional transcription factors with roles in cold acclimation, we used the Affymetrix GeneChip containing probe sets for approximately 24,000 Arabidopsis genes to define a core set of cold-responsive genes and to determine which genes were targets of CBF2 and 6 other transcription factors that appeared to be coordinately regulated with CBF2. A total of 514 genes were placed in the core set of cold-responsive genes, 302 of which were upregulated and 212 downregulated. Hierarchical clustering and bioinformatic analysis indicated that the 514 cold-responsive transcripts could be assigned to one of seven distinct expression classes and identified multiple potential novel cis-acting cold-regulatory elements. Eighty-five cold-induced genes and eight cold-repressed genes were assigned to the CBF2 regulon. An additional nine cold-induced genes and 15 cold-repressed genes were assigned to a regulon controlled by ZAT12. Of the 25 core cold-induced genes that were most highly upregulated (induced over 15-fold), 19 genes (84%) were induced by CBF2 and another two genes (8%) were regulated by both CBF2 and ZAT12. Thus, the large majority (92%) of the most highly induced genes belong to the CBF and ZAT12 regulons. Constitutive expression of ZAT12 in Arabidopsis caused a small, but reproducible, increase in freezing tolerance, indicating a role for the ZAT12 regulon in cold acclimation. In addition, ZAT12 downregulated the expression of the CBF genes indicating a role for ZAT12 in a negative regulatory circuit that dampens expression of the CBF cold response pathway.

The Arabidopsis CBF1, 2, and 3 genes (also known as DREB1b, c, and a, respectively) encode transcriptional activators that have a central role in cold tolerance. CBF1-3 are rapidly induced upon exposing plants to low temperature, followed by expression of CBF-targeted genes, the CBF regulon, resulting in an increase in plant freezing tolerance. At present, little is known about the cold-sensing mechanism that controls CBF expression. Results presented here indicate that this mechanism does not require a cold shock to bring about the accumulation of CBF transcripts, but instead, absolute temperature is monitored with a greater degree of input, i.e. lower temperature, resulting in a greater output, i.e. higher levels of CBF transcripts. Temperature-shift experiments also indicate that the cold-sensing mechanism becomes desensitized to a given low temperature, such as 4°C, and that resensitization to that temperature requires between 8 and 24 h at warm temperature. Gene fusion experiments identified a 125-bp section of the CBF2 promoter that is sufficient to impart cold-responsive gene expression. Mutational analysis of this cold-responsive region identified two promoter segments that work in concert to impart robust cold-regulated gene expression. These sequences, designated ICEr1 and ICEr2 (induction of CBF expression region 1 or 2), were also shown to stimulate transcription in response to mechanical agitation and the protein synthesis inhibitor, cycloheximide.Many plants increase in freezing tolerance in response to low nonfreezing temperatures, a phenomenon known as cold acclimation (Guy, 1990;Thomashow, 1999). In Arabidopsis, cold acclimation involves action of the CBF cold-response pathway (Thomashow, 2001). Within 15 min of exposing plants to low temperatures, transcripts accumulate for a family of genes designated CBF1, CBF2, and CBF3 Jaglo-Ottosen et al., 1998; Medina et al., 1999), or DREB1b, DREB1c, and DREB1a (Liu et al., 1998), respectively, which encode transcriptional activators that are members of the AP2/EREBP family of DNA-binding proteins (Riechmann and Meyerowitz, 1998). These transcription factors bind the cold-and dehydration-responsive DNA regulatory element designated the CRT (C-repeat)/ DRE (dehydration response element); (Baker et al., 1994;Yamaguchi-Shinozaki and Shinozaki, 1994;Stockinger et al., 1997) that is present in the promoters of COR and many other cold-responsive genes and stimulate their transcription. Expression of the CBF regulon of target genes then leads to an increase in freezing tolerance Jaglo-Ottosen et al., 1998;Liu et al., 1998;Kasuga et al., 1999). Multiple mechanisms appear to contribute to the enhancement of freezing tolerance, including the synthesis of cryoprotective polypeptides, such as COR15a (Artus et al., 1996;Steponkus et al., 1998), and the accumulation of compatible solutes that have cryoprotective properties, including Suc, raffinose, and Pro (Nanjo et al., 1999;Gilmour et al., 2000;Taji et al., 2002).Currently, little is known about how the CBF gen...

SummaryArabidopsis thaliana has nine genes that constitute a family of putative carotenoid cleavage dioxygenases (CCDs). While five members of the family are believed to be involved in synthesis of the phytohormone abscisic acid, the functions of the other four enzymes are less clear. Recently two of the enzymes, CCD7/MAX3 and CCD8/MAX4, have been implicated in synthesis of a novel apocarotenoid hormone that controls lateral shoot growth. Here, we report on the molecular and genetic interactions between CCD1, CCD7/MAX3 and CCD8/ MAX4. CCD1 distinguishes itself from other reported CCDs as being the only member not targeted to the plastid. Unlike ccd7/max3 and ccd8/max4, both characterized as having highly branched phenotypes, ccd1 loss-of-function mutants are indistinguishable from wild-type plants. Thus, even though CCD1 has similar enzymatic activity to CCD7/MAX3, it does not have a role in synthesis of the lateral shoot growth inhibitor. Rather, it may have a role in synthesis of apocarotenoid flavor and aroma volatiles, especially in maturing seeds where loss of function leads to significantly higher carotenoid levels.

In many organisms, various enzymes mediate site-specific carotenoid cleavage to generate biologically active apocarotenoids. These carotenoid-derived products include provitamin A, hormones, and flavor and fragrance molecules. In plants, the CCD1 enzyme cleaves carotenoids at 9,10 (9,10) bonds to generate multiple apocarotenoid products. Here we systematically analyzed volatile apocarotenoids generated by maize CCD1 (ZmCCD1) from multiple carotenoid substrates. ZmCCD1 did not cleave geranylgeranyl diphosphate or phytoene but did cleave other linear and cyclic carotenoids, producing volatiles derived from 9,10 (9,10) bond cleavage. Additionally the Arabidopsis, maize, and tomato CCD1 enzymes all cleaved lycopene to generate 6-methyl-5-hepten-2-one. 6-Methyl-5-hepten-2-one, an important flavor volatile in tomato, was produced by cleavage of the 5,6 or 5,6 bond positions of lycopene but not geranylgeranyl diphosphate, -carotene, or phytoene. In vitro, ZmCCD1 cleaved linear and cyclic carotenoids with equal efficiency. Based on the pattern of apocarotenoid volatiles produced, we propose that CCD1 recognizes its cleavage site based on the saturation status between carbons 7 and 8 (7 and 8) and carbons 11 and 12 (11 and 12) as well as the methyl groups on carbons 5, 9, and 13 (5, 9, and 13).Apocarotenoids are terpenoid compounds derived from the oxidative cleavage of carotenoids (1). They are generated when double bonds in a carotenoid are cleaved by molecular oxygen, forming an aldehyde or ketone in each product at the site of cleavage. Carotenoids can be cleaved at any of their conjugated double bonds, resulting in a diverse set of apocarotenoids. This structural diversity is the consequence of the large number of carotenoid precursors (more than 600) and subsequent modifications such as oxidation, reduction, and conjugation. Although apocarotenoid formation can also occur via nonspecific oxidation, biologically active forms with regulatory functions are expected to be generated via site-specific cleavage.Apocarotenoids are widely distributed in nature and serve important biological functions. Examples of biologically active apocarotenoids include retinoids in animals (2), trisporic acid in fungi (3), and abscisic acid in higher plants (4). A variety of other biologically important compounds are believed to be derived by oxidative cleavage of carotenoids. Among these compounds are those associated with mycorrhizal colonization, including mycorradicin (5), blumenin (6), and strigolactone (7).Various enzymes mediate the site-specific carotenoid cleavage needed to generate biologically active apocarotenoids. The founding member of the carotenoid-cleaving enzymes is maize VIVIPAROUS14 (VP14) (8, 9). This 9-cis-epoxycarotenoid dioxygenase (NCED) generates abscisic acid via asymmetrical cleavage at the 11,12 double bond of neoxanthin and/or violaxanthin. NCED genes have been identified in a variety of plant species (9 -13). Using sequence similarity to VP14, the 15,15Ј-dioxygenases responsible for vitamin A biosynthesis w...