Organ detachment requires cell separation within abscission zones (AZs). Physiological studies have established that ethylene and auxin contribute to cell separation control. Genetic analyses of abscission mutants have defined ethylene-independent detachment regulators. Functional genomic strategies leading to global understandings of abscission have awaited methods for isolating AZ cells of low abundance and very small size. Here, we couple laser capture microdissection of Arabidopsis thaliana stamen AZs and GeneChip profiling to reveal the AZ transcriptome responding to a developmental shedding cue. Analyses focus on 551 AZ genes (AZ 551 ) regulated at the highest statistical significance (P # 0.0001) over five floral stages linking prepollination to stamen shed. AZ 551 includes mediators of ethylene and auxin signaling as well as receptor-like kinases and extracellular ligands thought to act independent of ethylene. We hypothesized that novel abscission regulators might reside in disproportionately represented Gene Ontology Consortium functional categories for cell wall modifying proteins, extracellular regulators, and nuclear-residing transcription factors. Promoter-b-glucuronidase expression of one transcription factor candidate, ZINC FINGER PROTEIN2 (AtZFP2), was elevated in stamen, petal, and sepal AZs. Flower parts of transgenic lines overexpressing AtZFP2 exhibited asynchronous and delayed abscission. Abscission defects were accompanied by altered floral morphology limiting pollination and fertility. Hand-pollination restored transgenic fruit development but not the rapid abscission seen in wild-type plants, demonstrating that pollination does not assure normal rates of detachment. In wild-type stamen AZs, AtZFP2 is significantly up-regulated postanthesis. Phenotype data from transgene overexpression studies suggest that AtZFP2 participates in processes that directly or indirectly influence organ shed.

SummaryEthylene perception in plants is co-ordinated by multiple hormone receptor candidates sharing sequence commonalties with prokaryotic environmental sensor proteins known as two-component regulators. Two tomato homologs of the Arabidopsis ethylene receptor ETR1 were cloned from a root cDNA library. Both cDNAs, termed LeETR1 and LeETR2, were highly homologous to ETR1, exhibiting~90% deduced amino acid sequence similarity and 80% deduced amino acid sequence identity. LeETR1 and LeETR2 contained all the major structural elements of two-component regulators, including the response regulator motif absent in LeETR3, the gene encoding tomato NEVER RIPE (NR). Using RNase protection analysis, the mRNAs of LeETR1, LeETR2 and NR were quantified in tissues engaged in key processes of the plant life cycle, including seed germination, shoot elongation, leaf and flower senescence, floral abscission, fruit set and fruit ripening. LeETR1 was expressed constitutively in all plant tissues examined. LeETR2 mRNA was expressed at low levels throughout the plant but was induced in imbibing tomato seeds prior to germination and was down-regulated in elongating seedlings and senescing leaf petioles. NR expression was developmentally regulated in floral ovaries and ripening fruit. Notably, hormonal regulation of NR was highly tissue-specific. Ethylene biosynthesis induced NR mRNA accumulation in ripening fruit but not in elongating seedlings or in senescing leaves or flowers. Furthermore, the abundance of mRNAs for all three LeETR genes remained uniform in multiple plant tissues experiencing marked changes in ethylene sensitivity, including the cell separation layer throughout tomato flower abscission.

Two structurally divergent endo-beta-1,4-glucanase (EGase) cDNAs were cloned from tomato. Although both cDNAs (Cel1 and Cel2) encode potentially glycosylated, basic proteins of 51 to 53 kD and possess multiple amino acid domains conserved in both plant and microbial EGases, Cel1 and Cel2 exhibit only 50% amino acid identity at the overall sequence level. Amino acid sequence comparisons to other plant EGases indicate that tomato Cel1 is most similar to bean abscission zone EGase (68%), whereas Cel2 exhibits greatest sequence identity to avocado fruit EGase (57%). Sequence comparisons suggest the presence of at least two structurally divergent EGase families in plants. Unlike ripening avocado fruit and bean abscission zones in which a single EGase mRNA predominates, EGase expression in tomato reflects the overlapping accumulation of both Cel1 and Cel2 transcripts in ripening fruit and in plant organs undergoing cell separation. Cel1 mRNA contributes significantly to total EGase mRNA accumulation within plant organs undergoing cell separation (abscission zones and mature anthers), whereas Cel2 mRNA is most abundant in ripening fruit. The overlapping expression of divergent EGase genes within a single species may suggest that multiple activities are required for the cooperative disassembly of cell wall components during fruit ripening, floral abscission, and anther dehiscence.

Endo-1,4-␤-D-glucanases (EGases, EC 3.2.1.4) are enzymes produced in bacteria, fungi, and plants that hydrolyze polysaccharides possessing a 1,4-␤-D-glucan backbone. All previously identified plant EGases are E-type endoglucanases that possess signal sequences for endoplasmic reticulum entry and are secreted to the cell wall. Here we report the characterization of a novel E-type plant EGase (tomato Cel3) with a hydrophobic transmembrane domain and structure typical of type II integral membrane proteins. The predicted protein is composed of 617 amino acids and possesses seven potential sites for N-glycosylation. Cel3 mRNA accumulates in young vegetative tissues with highest abundance during periods of rapid cell expansion, but is not hormonally regulated. Antibodies raised to a recombinant Cel3 protein specifically recognized three proteins, with apparent molecular masses of 93, 88, and 53 kDa, in tomato root microsomal membranes separated by sucrose density centrifugation. The 53-kDa protein comigrated in the gradient with plasma membrane markers, the 88-kDa protein with Golgi membrane markers, and the 93-kDa protein with markers for both Golgi and plasma membranes. EGase enzyme activity was also found in regions of the density gradient corresponding to both Golgi and plasma membranes, suggesting that Cel3 EGase resides in both membrane systems, the sites of cell wall polymer biosynthesis. The in vivo function of Cel3 is not known, but the only other known membrane-anchored EGase is present in Agrobacterium tumefaciens where it is required for cellulose biosynthesis.

We have previously described the construction and expression of a chimeric gene that allows developmentally regulated expression of tomato (Lycopersicon esculentum) polygalacturonase in ripening-impaired, mutant (rin) tomato fruit (JJ Giovannoni, D DellaPenna, AB Bennett, The Plant Cell 1: 53-63). We now show that expression of the chimeric polygalacturonase gene in rin tomato fruit resulted in the accumulation of all three polygalacturonase isozymes (PG1, PG2A, and PG2B). Polyuronide solubilization and polyuronide depolymerization both reached their maximal levels in transgenic rin fruit prior to the appearance of PG2 isozymes. These results demonstrate that PG1, PG2A, and PG2B all arise by differential processing of a single gene product and further suggest that the PG1 isozyme is sufficient to carry out both polyuronide solubilization and depolymerization in vivo.Tomato fruit ripening is characterized by a number of coordinated biochemical and physiological changes, which collectively alter fruit color, flavor, and texture. Biochemical and genetic analysis (6, 7) has indicated that many of the physiological processes associated with ripening are regulated at the level of gene expression, a view firmly supported by recent analysis ofgene expression during tomato fruit ripening (1,8,11,14,17,28).Among the ripening-associated enzymes studied in tomato, the cell wall-degrading enzyme polygalacturonase has been analyzed in the greatest detail (2,4,5,8,9,12,13,(24)(25)(26). The site of action of polygalacturonase is the cell wall, where it hydrolyzes polyuronides, or pectins. The increase in the level of chelator-soluble polyuronides during ripening and their corresponding decrease in molecular size have been well documented and are attributed to the action of polygalacturonase (15,22,23). Polygalacturonase activity isolated from ripe fruit is comprised of three closely related isozymes, PGl, PG2A, and PG2B (4,5,10,(18)(19)(20)