The Plant Journal zyxwvutsrqpo (1 992) 2(3), 301-309 zyxwvutsrq Characterization of DNA sequences that mediate nuclear protein binding to the regulatory region of the z Pisum safiwum (pea) chlorophyl a/b binding protein gene A680: identification of a repeated heptamer motif Gerard0 Arguello, Enrique Garcia-Hernandez, Mireya Shnchez, Patricio Gariglio, Luis Herrera-Estrella and June Simpson* CINVESTAV, 1. P.N., U. lrapuato., Department of Genetic Engineering, Apdo. Postal zyxwvuts 629, Irapuato, Gto., 36500 Mexico Summary Two protein factors binding to the regulatory region of the pea chlorophyl a/b binding protein gene A680 have been identified. One of these factors is found only in green tissue but not in etiolated or root tissue. The second factor (denominated ABF-2) binds to a DNA sequence element that contains a direct heptamer repeat TCTCAAA. It was found that presence of both of the repeats is essentialfor binding. ABF-2 is present in both green and etiolated tissue and in roots and factors analogous to ABF-2 are present in several plant species. Computer analysis showed that the TCTCAAA motif is present in the regulatory region of several plant genes. Introduction In many eukaryotic genes cis-acting regulatory sequences carry out their functions by interacting with trans-acting protein factors that are present in the cell nucleus and which modulate in a positive or negative manner the transcription of the gene with which they interact (for recent reviews see Gilmartin et a/., 1990, Muller and Schaffner, 1990; Schaffner, 1989; Serfling eta/., 1985). In the case of light regulation in plants, one of the gene families most intensively studied to date is that encoding the chlorophyl a/b binding (Cab) proteins (Kuhlemeir et a/., 1987; Tobin and Silverthorne, 1985). The cab genes are found in the plant cell nucleus ( l i mko and Cashmore, 1983) and following translation of the mRNA on cytoplasmic ribosomes (Ape1 and Kloppstech, 1978), the cab pre- proteins are translocated to the chloroplasts where they are imported through the outer membrane and become part of the light harvesting complex found in the thylakoid membrane (Arntzen, 1978; Boardman et a/., 1978; Ellis, Received 26 August 1991: revised 3 December 1991 'For correspondence (fax +52 462 51282). 1981). Expression of these genes has been shown to be regulated by phytochrome (Kaufman eta/., 1984, 1985; Silverthorne and Tobin, 1984; Tobin et a/., 1984) and limited to green tissue (Lamppa et a/., 1985; Mayfield et a/., 1986; Mitra eta/., 1989; Muller et al., 1980) in several plant species. The 5' flanking sequences of these genes carry all the cis information to direct tissue-specific and light-inducible phytochrome-mediated expression (An, 1989; Nagy et a/., 1986a, 1986b, 1988; Simpson et a/., 1985, 1986a). In the case of the pea cab gene AB80(Cashmore, 1984), it has been shown that a 247 bp fragment (-347 to - 100) of the 5' flanking sequence, is capable of regulating a heterologous promoter in a light-regulated and tissue- specific manner (Simpson eta/., 1986b).This demonstrated that the 247 bp sequence could act as both an enhancer or upstream activating element to increaseexpression in the light and as a silencer or negatively acting element to prevent expression in root tissue. Since cis -acting regulatory elements are normally sites of interaction of trans -acting factors we were interested to investigate the nuclear factors binding to the pea A680 element and to attempt to determine, in more detail, the sequences to which they bind and the nature of these trans-acting factors. Here we report the identification of two protein factors which specifically interact with the A580 247 bp regulatory element (F247). One of these factors (ABF-2) is distinct from other factors previously described to be associated with light-inducible genes, since binding of ABF-2 is mediated by a previously un- reported short 7 bp direct repeat situated within the A680 enhancerkilencer element. Factors analogous to ABF-2 were found in both monocotyledons and dicotyledons, suggesting that ABF-2 is an evolutionarily conserved nuclear factor present in a wide range of plants. Results Factors that specifically bind to the AB80 regulatory element To determine whether the A680 F247 fragment interacts with nuclear transacting factors, gel retardation assays using this DNA fragment and pea leaf nuclear proteins were carried out. Lane 3 of Figure 1 a shows the migration 301