The ripening-impaired tomato mutant Never-ripe (Nr) is insensitive to the plant hormone ethylene. The gene that cosegregates with the Nr locus encodes a protein with homology to the Arabidopsis ethylene receptor ETR1 but is lacking the response regulator domain found in ETR1 and related prokaryotic two-component signal transducers. A single amino acid change in the sensor domain confers ethylene insensitivity when expressed in transgenic tomato plants. Modulation of NR gene expression during fruit ripening controls response to the hormone ethylene.

A structure-based model describing the interaction of the two-domain PI-SceI endonuclease with its 31-base pair DNA substrate suggests that the endonuclease domain (domain II) contacts the cleavage site region of the substrate, while the protein splicing domain (domain I) interacts with a distal region that is sufficient for high affinity binding. To support this model, alanine-scanning mutagenesis was used to assemble a set of 49 PISceI mutant proteins that were purified and assayed for their DNA binding and cleavage properties. Fourteen mutant proteins were 4-to >500-fold less active than wild-type PI-SceI in cleavage assays, and one mutant (T225A) was 3-fold more active. The yeast PI-SceI endonuclease catalyzes the hydrolysis of two specific phosphodiester bonds within an asymmetrical recognition site (1). This enzyme is a homing endonuclease (for a review, see Ref. 2) that occurs as an intein situated within an H ϩ -ATPase protein subunit. Like other homing endonucleases, PI-SceI recognizes an extremely long sequence (31 bp) 1 and cuts DNA to yield 5Ј-phosphate and 3Ј-hydroxyl ends (3, 4). Mutagenesis and biochemical studies indicate that the PI-SceI recognition sequence can be divided into two regions (4, 5).Region I contains the cleavage site that is cut by the enzyme to generate a 4-base pair overhang, and region II includes an adjacent 17-bp sequence (the minimal binding sequence) that is sufficient for high affinity binding. Mutagenesis of the substrate reveals that PI-SceI tolerates substitutions at numerous positions, since substitutions at only nine positions in the substrate lead to severely reduced activity (4). Like the other homing endonucleases that have been studied, PI-SceI requires Mg 2ϩ as a cofactor. The metal ion is likely to be required for the hydrolytic reaction, since it is required for catalysis but not for specific binding. Mn 2ϩ can substitute for Mg 2ϩ , and it stimulates more efficient cleavage by the enzyme at cognate and noncognate sites (1, 5).The three-dimensional structure of PI-SceI has been recently determined by x-ray crystallography and reveals a bipartite domain structure (6). Domain I contains the protein splicing active site, which is composed of the N-and C-terminal amino acids and two other His residues that have been shown to be required for activity or have been implicated in the reaction (7,8). The residues that compose the putative endonucleolytic active site, a lysine (Lys 301 ) and two aspartic acid residues (Asp 218 and Asp 326 ), are present in domain II and form a catalytic triad that displays structural similarity to charged clusters found in restriction enzymes (6, 9). By using the PISceI structural information and the knowledge that the enzyme contacts two discrete regions of the recognition sequence, a model for the docking of PI-SceI with its substrate was constructed where domains I and II of the protein contact regions II and I, respectively, of the substrate (Fig. 1). In this model, both domains are proposed to contact the substrate, since the bindi...

Fruit ripening represents a complex system of genetic and hormona1 regulation of eukaryotic development unique to plants. We are using tomato ripening mutants as tools to elucidate genetic components of ripening regulation and have recently demonstrated that the Never-ripe (Nr) Ripening of fleshy fruits represents a tightly synchronized system of development unique to plant species. Numerous biochemical and physiological processes are coordinated during ripening to effect changes in color, texture, flavor, and aroma, thereby making the fruit organ desirable to seed-dispersing organisms. Nuclear genes encoding enzymes contributing to many aspects of this process have been isolated and characterized in terms of expression patterns and function (for reviews, see Speirs and Brady, 1991;Gray et al., 1992; Giovannoni, 1993). In climacteric fruit such as tomato, banana, apple, peach, and many others, ripening is coordinated by the biosynthesis of the gaseous plant growth regulator ethylene (Abeles et al., 1992). Fruit-ripening-related ethylene biosynthesis is governed by the induction of genes encoding enzymes of the ethylene biosynthetic pathway that are themselves influ-

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Ethylene can alter plant morphology due to its effect on cell expansion. The most widely documented example of ethylene-mediated cell expansion is promotion of the "triple response" of seedlings grown in the dark in ethylene. Roots and hypocotyls become shorter and thickened compared with controls due to a reorientation of cell expansion, and curvature of the apical hook is more pronounced. The epinastic (epi) mutant of tomato (Lycopersicon esculentum) has a dark-grown seedling phenotype similar to the triple response even in the absence of ethylene. In addition, in adult plants both the leaves and the petioles display epinastic curvature and there is constitutive expression of an ethylene-inducible chitinase gene. However, petal senescence and abscission and fruit ripening are all normal in epi. A double mutant (epi/epi;Nr/Nr) homozygous for both the recessive epi and dominant ethylene-insensitive Never-ripe loci has the same dark-grown seedling and vegetative phenotypes as epi but possesses the senescence and ripening characteristics of Never-ripe. These data suggest that a subset of ethylene responses controlling vegetative growth and development may be constitutively activated in epi. In addition, the epi locus has been placed on the tomato RFLP map on the long arm of chromosome 4 and does not demonstrate linkage to reported tomato CTR1 homologs.