Ethylene (C 2 H 4 ) is a unique plant-signaling molecule that regulates numerous developmental processes. The key enzyme in the two-step biosynthetic pathway of ethylene is 1-aminocyclopropane-1-carboxylate synthase (ACS), which catalyzes the conversion of S-adenosylmethionine (AdoMet) to ACC, the precursor of ethylene. To understand the function of this important enzyme, we analyzed the entire family of nine ACS isoforms (ACS1, ACS2, ACS4-9, and ACS11) encoded in the Arabidopsis genome. Our analysis reveals that members of this protein family share an essential function, because individual ACS genes are not essential for Arabidopsis viability, whereas elimination of the entire gene family results in embryonic lethality. Phenotypic characterization of single and multiple mutants unmasks unique but overlapping functions of the various ACS members in plant developmental events, including multiple growth characteristics, flowering time, response to gravity, disease resistance, and ethylene production. Ethylene acts as a repressor of flowering by regulating the transcription of the FLOWERING LOCUS C. Each single and high order mutant has a characteristic molecular phenotype with unique and overlapping gene expression patterns. The expression of several genes involved in light perception and signaling is altered in the high order mutants. These results, together with the in planta ACS interaction map, suggest that ethylene-mediated processes are orchestrated by a combinatorial interplay among ACS isoforms that determines the relative ratio of homo-and heterodimers (active or inactive) in a spatial and temporal manner. These subunit isoforms comprise a combinatorial code that is a central regulator of ethylene production during plant development. The lethality of the null ACS mutant contrasts with the viability of null mutations in key components of the ethylene signaling apparatus, strongly supporting the view that ACC, the precursor of ethylene, is a primary regulator of plant growth and development.

1-Aminocyclopropane-1-carboxylate synthase (ACS) catalyzes the rate-limiting step in the ethylene biosynthetic pathway in plants. The Arabidopsis genome encodes nine ACS polypeptides that form eight functional (ACS2, ACS4-9, and ACS11) homodimers and one nonfunctional (ACS1) homodimer. Transgenic Arabidopsis lines were constructed expressing the b-glucuronidase (GUS) and green fluorescence protein (GFP) reporter genes from the promoter of each of the gene family members to determine their patterns of expression during plant development. All genes, except ACS9, are expressed in 5-d-old etiolated or light-grown seedlings yielding distinct patterns of GUS staining. ACS9 expression is detected later in development. Unique and overlapping expression patterns were detected for all the family members in various organs of adult plants. ACS11 is uniquely expressed in the trichomes of sepals and ACS1 in the replum. Overlapping expression was observed in hypocotyl, roots, various parts of the flower (sepals, pedicle, style, etc.) and in the stigmatic and abscission zones of the silique. Exogenous indole-3-acetic acid (IAA) enhances the constitutive expression of ACS2, 4, 5, 6, 7, 8, and 11 in the root. Wounding of hypocotyl tissue inhibits the constitutive expression of ACS1 and ACS5 and induces the expression of ACS2, 4, 6, 7, 8, and 11. Inducers of ethylene production such as cold, heat, anaerobiosis, and Li 1 ions enhance or suppress the expression of various members of the gene family in the root of light-grown seedlings. Examination of GUS expression in transverse sections of cotyledons reveals that all ACS genes, except ACS9, are expressed in the epidermis cell layer, guard cells, and vascular tissue. Similar analysis with root tip tissue treated with IAA reveals unique and overlapping expression patterns in the various cell types of the lateral root cap, cell division, and cell expansion zones. IAA inducibility is gene-specific and cell type-dependent across the root tip zone. This limited comparative exploration of ACS gene family expression reveals constitutive spatial and temporal expression patterns of all gene family members throughout the growth period examined. The unique and overlapping gene activity pattern detected reveals a combinatorial code of spatio-temporal coexpression among the various gene family members during plant development. This raises the prospect that functional ACS heterodimers may be formed in planta.

1-Amino-cyclopropane-1-carboxylate synthase (ACS, EC 4.4.1.14) is the key enzyme in the ethylene biosynthetic pathway in plants. The completion of the Arabidopsis genome sequence revealed the presence of twelve putative ACS genes, ACS1-12, dispersed among five chromosomes. ACS1-5 have been previously characterized. However, ACS1 is enzymatically inactive whereas ACS3 is a pseudogene. Complementation analysis with the Escherichia coli aminotransferase mutant DL39 shows that ACS10 and 12 encode aminotransferases. The remaining eight genes are authentic ACS genes and together with ACS1 constitute the Arabidopsis ACS gene family. All genes, except ACS3, are transcriptionally active and differentially expressed during Arabidopsis growth and development. IAA induces all ACS genes, except ACS7 and ACS9; CHX enhances the expression of all functional ACS genes. The ACS genes were expressed in E. coli, purified to homogeneity by affinity chromatography, and biochemically characterized. The gas ethylene has been known since the beginning of the century to be used by plants as a signaling molecule for regulating a variety of developmental processes and stress responses (1). These include seed germination, leaf and flower senescence, fruit ripening, cell elongation, nodulation, wounding and pathogen responses. Ethylene production is induced by a variety of external factors, including wounding, viral infection, elicitors, auxin treatment, and Li ϩ ions (2-7). Ethylene is biosynthesized from methionine, which is converted to AdoMet 1 by the enzyme AdoMet synthetase. AdoMet is converted by the enzyme ACS to ACC, the precursor of ethylene (5, 7-9). ACC is oxidized to ethylene by ACO. ACS is a pyridoxal phosphate-containing enzyme (2, 5, 10) and its activity is regulated at the transcriptional (11-18) and posttranscriptional levels (19 -21). The ethylene biosynthetic enzymes, AdoMet synthetase, ACS, and ACO, are encoded by multigene families in various plant species (5,9,16,17,(22)(23)(24)(25). The crystal structures of apple and tomato Le-ACS2 isozymes were recently elucidated (26, 27). The structures show that the enzyme is a homodimer, and its overall fold and active sites are similar to those of aminotransferases even though the two enzymes have completely different catalytic activities. The tertiary structures together with available biochemical data explain the catalytic roles of the conserved and non-conserved active site residues (28 -31).The sequencing of the Arabidopsis genome revealed that ACS genes are putatively encoded by twelve genes (32). The question immediately arises as to why there are so many ACS isozymes for synthesizing ethylene in Arabidopsis. It has been postulated that the presence of ACS isozymes may reflect tissue-specific expression that satisfies the biochemical environment of the cells or tissues in which each isozyme is expressed (15). For example, a group of cells or tissues with low concentration of the ACS substrate, AdoMet, would express a high affinity (low K m ) ACS isozyme. Accordingly, the ...

PHYTOCHROME-INTERACTING FACTOR5 (PIF5), a basic helix-loop-helix transcription factor, interacts specifically with the photoactivated form of phytochrome B (phyB). Here, we report that dark-grown Arabidopsis thaliana seedlings overexpressing PIF5 (PIF5-OX) exhibit exaggerated apical hooks and short hypocotyls, reminiscent of the triple response induced by elevated ethylene levels, whereas pif5 mutants fail to maintain tight hooks like those of wild-type seedlings. Silver ions, an ethylene receptor blocker, rescued the triple-response phenotype, and we show that PIF5-OX seedlings express enhanced levels of key ethylene biosynthesis enzymes and produce elevated ethylene levels. Exposure of PIF5-OX seedlings to prolonged continuous red light (Rc) promotes hypocotyl elongation relative to dark controls, the reciprocal of the Rc-imposed hypocotyl inhibition displayed by wild-type seedlings. In contrast with this PIF5-OX hyposensitivity to Rc, pif5 mutant seedlings are hypersensitive relative to wild-type seedlings. We show that this contrast is due to reciprocal changes in phyB protein levels in prolonged Rc. Compared with wild-type seedlings, PIF5-OX seedlings have reduced, whereas pif5 mutants have increased, phyB (and phyC) levels in Rc. The phyB degradation in the overexpressors depends on a functional phyB binding motif in PIF5 and involves the 26S proteasome pathway. Our data thus indicate that overexpressed PIF5 causes altered ethylene levels, which promote the triple response in darkness, whereas in the light, the interaction of photoactivated phyB with PIF5 causes degradation of the photoreceptor protein. The evidence suggests that endogenous PIF5 negatively regulates phyB-imposed hypocotyl inhibition in prolonged Rc by reducing photoreceptor abundance, and thereby photosensory capacity, rather than functioning as a signaling intermediate.