A Combinatorial Interplay Among the 1-Aminocyclopropane-1-Carboxylate Isoforms Regulates Ethylene Biosynthesis in Arabidopsis thaliana

Atsunari Tsuchisaka; Guixia Yu; Hailing Jin; Jose M Alonso; Joseph R Ecker; Xiaoming Zhang; Shang Gao; Athanasios Theologis

doi:10.1534/genetics.109.107102

. 2009 Nov;183(3):979–1003. doi: 10.1534/genetics.109.107102

A Combinatorial Interplay Among the 1-Aminocyclopropane-1-Carboxylate Isoforms Regulates Ethylene Biosynthesis in Arabidopsis thaliana

Atsunari Tsuchisaka ^*, Guixia Yu ^*, Hailing Jin ^†, Jose M Alonso ^‡,¹, Joseph R Ecker ^‡, Xiaoming Zhang ^†, Shang Gao ^†, Athanasios Theologis ^*,²

PMCID: PMC2778992 PMID: 19752216

Abstract

Ethylene (C₂H₄) 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.

THE gas ethylene (C₂H₄) has long been known to be a signaling molecule that regulates a variety of developmental processes and stress responses in plants (Abeles et al. 1992). These include seed germination, leaf and flower senescence, fruit ripening, cell elongation, nodulation, and pathogen responses. Ethylene production is enhanced by a variety of external factors, including wounding, viral infection, elicitors, hormone treatment, chilling injury, drought, Cd²⁺ and Li⁺ ions, O₃, SO₂, and other pollutants (Yang and Hoffman 1984; Abeles et al. 1992; Bleecker and Kende 2000; Thomma et al. 2001). Enhancement of ethylene production serves as a signaling mechanism with profound physiological consequences (Guo and Ecker 2004).

Ethylene is synthesized from methionine by its conversion to S-adenosylmethionine (AdoMet), which is converted by the enzyme 1-aminocyclopropane-1-carboxylate synthase (ACS, EC 4.4.1.14) into methylthioadenosine (MTA) and 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene (Bleecker and Kende 2000). ACC is oxidized to C₂H₄, CO₂, and HCN by ACC oxidase (ACO) (Dong et al. 1992). Alternatively, ACC can be diverted from conversion to ethylene by forming the conjugate N-malonyl-ACC (Yang and Hoffman 1984). The activity of ACS is regulated at the transcriptional level (Bleecker and Kende 2000; Guo and Ecker 2004) and posttranscriptional level (Argueso et al. 2007).

ACS is a cytosolic enzyme with a short half-life and requires pyridoxal phosphate (PLP) as a cofactor (Yang and Hoffman 1984; Yip et al. 1990). The enzyme functions as a homodimer whose active site is formed from the interaction of residues from the monomeric subunits, similar to AspAT (Tarun and Theologis 1998). In particular, the Y⁹² residue, which helps anchor the PLP cofactor to the ACS apoenzyme, interacts with active-site residue K²⁷⁸, which forms a covalent Schiff base with the PLP cofactor from the adjacent subunit. The three-dimensional structure of ACS has confirmed this model (Capitani et al. 1999), and together with available biochemical data explains the catalytic roles of the conserved and nonconserved active site residues (Tarun and Theologis 1998).

ACS is encoded by a multigene family in every plant species examined (Bleecker and Kende 2000). The Arabidopsis genome contains 12 genes annotated as ACS (ACS1–12), dispersed among the five chromosomes (Arabidopsis Genome Initiative 2000). However, ACS3 is a pseudogene whereas ACS10 and 12 encode aminotransferases (Yamagami et al. 2003). The remaining nine genes (ACS1, ACS2, ACS4–9 and ACS11) are authentic ACSs and constitute the Arabidopsis ACS gene family (Yamagami et al. 2003). Among the nine ACS polypeptides, eight of them (ACS2, ACS4–9, and ACS11) form functional homodimers and one (ACS1) forms a nonfunctional homodimer (Tsuchisaka and Theologis 2004a). The highly variable carboxylic end of the proteins serves as a regulatory domain responsible for post-translational regulation of the enzyme whereas the nonvariable amino terminus harbors the catalytic domain. The ACS proteins comprise three phylogenetic branches, depending on their C terminus heterogeneity (Wang et al. 2004; Argueso et al. 2007; Hansen et al. 2008).

The biological significance of multigene families in general and of the ACS gene family in particular is unknown. Biochemical characterization of the ACSs reveals that all active isoforms are biochemically distinct (Yamagami et al. 2003). This has been viewed to reflect that each isoform may have a distinct biological function defined by its biochemical properties, which in turn define its tissue-specific expression (Yamagami et al. 2003). For example, if a group of cells or tissues has low concentrations of the ACS substrate, AdoMet, then these cells express a high affinity (low K_m) ACS isozyme. Such a concept underscores the physiological fine-tuning of the cell and demands that the enzymatic properties of each isozyme be distinct. In addition, the subunits of all isozymes have the capacity to form active and inactive heterodimers in Escherichia coli. The ACS polypeptides can potentially form 45 homo- and heterodimers of which 25 are functional (Tsuchisaka and Theologis 2004a). Functional heterodimerization may further enhance the isozyme diversity of the family and provides physiological versatility by being able to operate in a broad gradient of AdoMet concentration in various cells/tissues during plant growth and development. The formation of heterodimers in planta is possible since the expression of the ACS gene family members is overlapping during plant development (Tsuchisaka and Theologis 2004b). However, it is not known whether ACS heterodimers are formed in planta.

To understand the function and regulatory roles of each ACS gene in ethylene production during plant development, we analyzed the family of nine ACS isoforms encoded by the Arabidopsis genome. We used a combination of approaches, including T-DNA insertions/amiRNA technology, genome expression profiling, and in planta interactome mapping to analyze the essential and nonessential roles of the Arabidopsis ACS genes. We found that disruption of any single ACS gene causes no overt phenotype but had unique effects on gene expression profiles, indicating that the ACSs perform distinct nonessential roles. But they must have at least one essential function in common since elimination of all ACS genes resulted in embryo lethality. Phenotypic characterization of single, double, and high order mutants revealed specific and overlapping functions among the various ACS gene family members on plant developmental events such as differential growth, flowering time, gravitostimulation, and disease resistance. These results, coupled with the findings of an in planta ACS “interactome” map suggest that ethylene-mediated processes are regulated by a combinatorial interplay among the nine ACS subunits, which can form 45 different dimeric isoforms. This interplay provides a combinatorial code that determines the relative ratio of homo- and heterodimers (active or inactive) in a spatiotemporal manner and is the central regulator of ethylene production during plant development. The lethality of the ACS null mutant, in contrast to the viability of null mutations in key components of the ethylene signaling apparatus, strongly support the idea that ACC, the precursor of ethylene, is a primary regulator of plant growth and development.

MATERIALS AND METHODS

Materials, strains, and transformation vectors:

Restriction enzymes were obtained from New England BioLabs. All chemicals used for this study were of analytical grade and purchased from Aldrich and Sigma. Oligonucleotides were purchased from Operon Technologies (Alameda, CA). See File S1 in the supporting information for details regarding transformation and transgenic line selection protocols.

Plant material and growth conditions:

Arabidopsis thaliana ecotype Columbia (Col) was used throughout this study. Growth conditions and characterization of mutant phenotypes are described in File S1.

Identification and characterization of T-DNA insertion alleles:

We used a PCR-based approach to identify T-DNA insertion mutations in ACS gene family members. See File S1 for technical details and primer information and Figure S8 for the integration pattern of T-DNA.

Construction of high order mutants:

We used the strategy outlined in File S1.

Inactivation of the ACS8 and ACS11 genes with an amiR:

An artificial microRNA (amiR) containing transgene that specifically inhibit both ACS8 and ACS11 gene expression was constructed by overlapping PCR using the pRS300 plasmid as template containing the MIR319a (Schwab et al. 2006). See File S1 for technical details and primer information.

Complementation of the octuple (amiR) line:

The amiR target sequences of the ACS8 and ACS11 ORFs were mutated by site-directed mutagenesis giving rise to ACS8m and ACS11m ORFs that encode functional proteins. The pQE80-ACS8 and pQE801-ACS11 plasmids (Tsuchisaka and Theologis 2004a) were used as templates. See File S1 for technical details and primer information.

Mapping the insertion sites of the amiR, amiR complementation and BiFC transgenes:

We used thermal asymmetric interlaced (TAIL) PCR for mapping the integration sites of the amiR, amiR complementation and BiFC transgenic lines following the procedure described in File S1.

Complementation of the acs6-1 and acs9-1 mutants:

Both mutants were complemented by expressing the ACS6 and ACS9 ORFs from their own promoters (2.5 kb). The 3′-UTRs of each gene (1 kb) were also included in the constructs to ensure appropriate tissue-specific expression. See File S1 for technical details and primer information.

Ethylene production:

Ethylene production was determined in 5- or 10-day-old light-grown seedlings and 30- or 40-day-old light-grown plants (only the aerial parts were used) as described in File S1.

Histochemical GUS Assay:

The ACSpromoter-GUS constructs reported by Tsuchisaka and Theologis (2004b) were used to determine the ACS gene expression in the region of the shoot apical meristem (SAM) of young light-grown Arabidopsis seedlings. See File S1 for technical details.

RT–PCR analysis for ACS and flowering gene expression:

Total RNA was isolated using RNeasy (QIAGEN, Valencia, CA) and polyA⁺ RNA was purified from total RNA using Oligotex (QIAGEN). See File S1 for technical details and primer information.

Bimolecular fluorescence complementation (BiFC) in planta:

The homo- and heterodimeric interactions among the various subunits of the ACS gene family members were determined in planta by BiFC (Hu et al. 2002). See File S1 for technical details regarding construct design and imaging of yellow fluorescence in planta.

Pathogen infection assay:

Arabidopsis plants were grown at 24° under a 12 hr light/12 hr dark cycle for 4 weeks before the pathogen inoculation. See File S1 for pathogen strains used and other experimental details.

Global gene expression analysis:

Total RNA was prepared using RNAqueous RNA isolation kit with Plant RNA isolation aid (Ambion, Austin, TX). After LiCl precipitation, RNA was purified using RNeasy columns (QIAGEN, Valencia, CA) and reprecipitated with LiCl. RNA pellets were washed with 70% EtOH (three times), and resuspended in DEPC-treated water. Affymetrix ATH1 array was used. Ten micrograms of total RNA was used for biotin-labeled cRNA probe synthesis. cRNA probe synthesis were performed according to the manufacturer's protocols (Affymetrix, Santa Clara, CA) Scanned arrays were analyzed with Affymetrix MAS 5.0 software and then normalized with gcRMA obtained from bioconductor (http://bioconductor.org). See File S1 for details regarding data analysis.

RESULTS

Characterization of ACS T-DNA insertion mutants:

We used a PCR-based screening approach to identify T-DNA insertion mutations in ACS gene family members (see Experimental Procedures in File S1). Inactivation of the ACS genes by the T-DNA insertions was confirmed by RT–PCR analysis of RNA isolated from CHX-treated seedlings (to increase the very low basal level of ACS mRNA expression) using the “black” set of primers shown in Figure S1A. All T-DNA insertions inhibit the expression of wild-type (wt) full-length ACS mRNAs [Figure S1Ba; compare lane 3 (T/T) with lanes 1 (wt) and 2 (t/t)]. A small amount of ACS2 transcript is detected in the acs2-2 mutant, which may be due to a low level splicing event (Figure S1Ba; lane 3). Similar analysis using primers upstream (“red” set) and downstream of the insertions (“green” set of primers shown in Figure S1A) was also carried out and the results are shown in Figure S1Bb (red set) and Figure S1Bc (green set), respectively. ACS transcripts are detected in all mutants upstream of the insertion except acs5-2 [Figure S1Bb; compare lane 3 (T/T) with lanes 1 (wt) and 2 (t/t)]. Accumulation of a larger truncated transcript in acs5-1 is attributed to incomplete splicing (Figure S1Bb; lane 3). ACS transcripts are also detected in all mutants downstream of the insertion except acs4-1 and acs7-2 [Figure S1Bc; compare lane 3 (T/T) with lanes 1 (wt) and 2 (t/t)]. The transcripts detected with the “red set” or “green set” of primers may derive from full-length T-DNA insertion products that could have failed to be amplified for technical reasons using the “black set” of primers. However, the transcripts detected downstream of the insertions may also be generated by a promoter activity inside the T-DNA insertion. The potential proteins derived from these various transcripts are almost certainly enzymatically inactive. In addition all the transcripts detected downstream of the insertions cannot be translated because of the presence of early translational stop signals. The activity of the ACS4-1 protein truncated at Q³³¹ (includes the key catalytic residue K²⁷³) must be nil because deletion analysis reveals that the C terminus of Le-ACS2 beyond the conserved R⁵¹² is necessary for enzyme activity (A. S. Tarun and A. Theologis, unpublished results). All lines were backcrossed twice.

Phenotypic characterization:

We observed six major phenotypic alterations in the mutants in: (1) plant growth, (2) flowering time (bolting), (3) response to shoot gravitostimulation, (4) resistance to pathogens, (5) developmental defects, and (6) ethylene production. We examined single, double, pentuple hexuple, heptuple, and octuple mutants.

Single and double mutants:

Seedling growth:

The acs1-1 and acs9-1 mutations enhance the hypocotyl length in etiolated seedlings, while acs4-1 has an inhibitory effect (Figure 1A). The effect of acs2-1, acs5-2, and acs6-1 on hypocotyl growth is nil. However, light-grown seedlings of these six single mutants have greatly enhanced hypocotyl length (Figure 1B). The same mutations also enhance cotyledon size, with acs1-1 having the most pronounced effect (Figure 1C). Etiolated seedlings of all single and double mutants examined have normal hook formation, and their response to gravitostimulation is normal (data not shown).

Growth of adult plants:

All single mutants except acs2-2 have the same height as wt plants during the early stages of growth (20 day old). The acs2-2 plants are much shorter than the wt plants, but another allele of ACS2, acs2-1, has no effect on plant height (Figure 1F/ 20 day old). However, after 30 days of growth all single mutants exhibit an obvious and statistically significant increase in height (Figure 1F). A visual phenotypic comparison of 40-day-old wt plants with those of single mutants is shown in Figure 1G.

The most prominent effect of inactivating multiple ACS genes is enhancement of plant height. Some of the double mutants examined, such as acs2-1acs4-1 and acs2-2acs4-1, are taller than the single mutants during the early stages of plant growth (Figure 1F; compare the height of single with that of the double mutants after 20 days of growth), and all double mutants are taller than the single mutants after 30 days of growth (Figure 1F). However, as growth progresses there is no significant difference in shoot length between single and double mutants (Figure 1F; 50 days of growth). Overall, the single mutants examined have phenotypic characteristics similar to wt plants. For example, their rosette leaf number, silique length, and seed number/silique are normal (Figure S2, A, C, and D). However, the diameter of the inflorescence stem of acs1-1 is thinner than that of the wt (Figure S2B).

Ethylene production:

Ethylene production is unaffected by inactivation of the ACS2, ACS5, and ACS6 isozymes (Figure 1D). However, inactivation of ACS1 (inactive isozyme) inhibits ethylene production by ∼30% (Figure 1D). An enhancement in ethylene evolution is observed in the acs4-1 (40%) and acs9-1 (15%) mutants (Figure 1D). While single acs mutations have broad effects on ethylene production, ranging from inhibition to stimulation, this effect is not apparent in double acs mutants (Figure 1E).

Flowering time:

The acs1-1, acs6-1, acs7-1, and acs9-1 single mutants start to form bolts earlier than wt (Figure 2A); acs2-1, acs4-1, and acs5-2 start to bolt at the same time as wt (Figure 2A). A strong antagonistic interaction between the two early flowering mutants, acs6-1 and acs7-1, was noticed (Figure 2B). While both single mutants flower earlier than wt, the acs6-1acs7-1 double mutant flowers later than wt (Figure 2, B and C). The double mutant has a higher rosette leaf number than the single mutants, consistent with its late flowering phenotype. Ethylene production is slightly inhibited by loss of ACS7, and is unaffected by loss of ACS6 (Figure 2D). However, the inactivation of both genes enhances the loss of ethylene production detected in the acs7-1 mutant (Figure 2D). These results reveal that total ethylene production by a seedling is not a good predictor for the flowering behavior of the mutants. While loss of ethylene production in the double mutant results in late flowering, slight (acs7-1) or no loss (acs6-1) of ethylene production in the single mutants results in early flowering. It appears that the amount of ethylene produced at the site(s) responsible for flowering initiation (leaf primordia in shoot apical meristem region; SAM) is likely the key determinant of flowering time.

The inhibitory effect of the acs6-1acs7-1 double mutant on flowering time prompted us to investigate the expression of key regulators of flowering in young seedlings. The most dramatic effect of the double mutant is on the gene expression of the FLOWERING REPRESSOR C (FLC; Figure 2E). The FLC expression is enhanced in the double mutant compared to the wt control and the two single, acs6-1 and acs7-1 mutants in both 3- and 7-day-old seedlings (Figure 2E). The expression of FLC is inhibited in the acs7-1 but is expressed in similar levels to the wt control in the acs6-1 mutant (Figure 2E). Concomitantly with the increase in FLC gene expression, a decrease in the expression of the positive flowering regulator FLOWERING LOCUS T (FT) is detected in the double mutant compared to the wt and the two single mutants (Figure 2E). However, the FT expression is enhanced in the two early flowering single mutants compared to the wt in 7-day-old seedlings, which is consistent with their early flowering phenotype (Figure 2E). FT expression is undetectable in 3-day-old seedlings (Figure 2E). We noticed less dramatic changes in the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), a positive flowering regulator, in all three mutants, compared to the wt control in 7-day-old seedlings (Figure 2E). However, an enhancement in SOC1 expression is detected in acs7-1 in 3-day-old seedlings (Figure 2E). Finally, the expression of CONSTANS (CO) does not show any dramatic changes in any mutant in both 3- and 7-day-old seedlings, although its expression is slightly enhanced in the double mutant (Figure 2E).

Complementation of acs6-1 and acs9-1 mutants:

To determine whether differences in flowering time of some single mutants is indeed due to the T-DNA insertions rather than to mutations in other chromosomal loci, we complemented the acs6-1 and acs9-1 mutations by transforming them with the corresponding full-length cDNAs expressed from their own promoters. A wide range of flowering time was observed among the transformants. Lines with flowering time similar to wt were among the transformants, indicating complementation and suggesting that the early flowering phenotype is linked to the T-DNA insertion in the corresponding gene. Surprisingly, some lines flowered later than wt plants. While the majority of the acs9-1 transformants have a late flowering phenotype, only one acs6-1 transformant (line no. 29) flowers later (compare the results shown in Figure S3A with those shown in Figure S3B). We were curious to know why complementation of two loss-of-function mutations, acs6-1 and acs9-1 (early flowering) with their corresponding cDNAs, gives rise to plants with a gain-of-function phenotype (late flowering). We investigated further some lines of both mutants that flower like wt, and some lines that flower early, like the mutants (early flowering), and some lines that flower late [Figure 2, F (acs6-1) and I (acs9-1)]. The expression profiles of the various ACS gene family members are altered and are different among the three types of transformants. Their ACS expression profiles are also different when compared to those of the wt and to the untransformed mutant lines (see Figure 2, G and J1, which show a quantitation of the RT–PCR data). For example, the RNA expression pattern of the line that flowers like wt is different from that of the wt control and both mutants (see Figure 2, G and J1). The same is true for the early- and late-flowering lines and the original mutants (Figure 2, G and J1). We noticed that the late flowering mutant acs6-1 (line no. 29) overexpresses the ACS6 transcript (Figure 2G). The absence of ACS1 and ACS9 transcripts in the data of Figure 2 (G and J1) is due to their low abundance in total RNA. However, we were able to detect overexpression of the ACS9 transcript in the late flowering line no.18 of acs9-1 using polyA⁺ RNA for the RT–PCR analysis (Figure 2J2). The overexpression of ACS6 and ACS9 transcripts in the late flowering mutants acs6-1 (line no. 29) and acs9-1 (line no. 18) may be due to high copy number of transgenes or to higher promoter activity due to a chromosomal position effect.

The different ACS gene expression patterns observed in the three types of transformant lines are reflected in the different amounts of ethylene produced in their 5-day-old light-grown seedlings. The early-flowering transformant of acs6-1 (line no. 1) produces the same amount of ethylene as the acs6-1 mutant and the wt control. The wt-like line no. 3 also produces the same amount of ethylene as the wt control, but the late-flowering line no. 29 is an ethylene overproducer (Figure 2H). Transformation of acs6-1 created two new mutants; one that produces the same amount of ethylene as acs6-1 but that flowers as the wt, and another that overproduces ethylene and flowers later than wt. The results of the acs9-1 lines are different. The early-flowering line no. 15 produces more ethylene than the wt and the original acs9-1 mutant, while the wt-like transformant line produces the same amount of ethylene as wt. Interestingly, production of ethylene in the late-flowering line of acs9-1 is inhibited 10% (Figure 2K). These observations suggest that total ethylene production of an intact seedling is not a good predictor of flowering time. While the two late flowering lines of acs6-1 and acs9-1 mutants produce different amounts of ethylene compared to the wt control, they both have the same flowering time phenotype (compare the results of Figure 2H with those of K). Furthermore, we were surprised to observe that the transformant lines are also defective in their response to gravitostimulation and growth characteristics. The response to gravity was reduced in the late-flowering line of acs6-1, and all three lines of acs9-1 have a reduced response to gravitostimulation (see Figure S4, A–C). On the other hand the hypocotyl growth of etiolated seedlings is enhanced in all three lines of both acs6-1 and acs9-1 transformants (Figure S4, A–C).

These results reveal a communication network among the ACS proteins. We imagine that introduction of an exogenous ACS gene disturbs the balance of ACS isoforms. Since ethylene is known to stimulate its own synthesis, the possibility exists that the exogenous ACS gene causes local changes in ethylene production (e.g., leaf primordial in the SAM region) that alter the expression of various ACS gene family members. Also the striking phenotypes described earlier with the inactivation of ACS1 alone (acs1-1), which forms an inactive homodimer, are attributed to the loss of ACS1 heterodimers with other ACS isoforms.

Construction of high order mutants:

We generated double, triple, quadruple, and pentuple mutants with the various insertion lines shown in Figure S1A. We did not characterize the double, triple, and quadruple mutants phenotypically in a great detail because of the large number of them and the absence of dramatic phenotypes. The most prominent effect of inactivating multiple ACS genes is the enhancement of plant height.

pentuple mutants:

Plant growth:

All four pentuple mutants characterized are taller than the wt during the entire period of plant growth and development (Figure 3, A and C). Hypocotyl length of 10-day-old light-grown seedlings is longer in all four pentuple mutants compared to wt (Figure 3B). We subsequently compared the growth characteristics between the pentuple2 with two ethylene perception mutants, ein2-5 (Alonso et al. 1999) and etr1-1 (Chang et al. 1993), respectively. The hypocotyl length of the pentuple2, ein2-5, and etr1-1 etiolated (3 day old) and light-grown (10 day old) seedlings is longer than that of wt (Figure 3D). The pentuple2 and ein2-5 mutants are taller than wt throughout the plant life cycle; etr1-1 plants become taller than the wt after 2 months of growth (Figure 3E). The pentuple2 mutant, however, has a wt-like hook, in contrast to the hookless phenotype of ein2-5 and etr1-1 plants (Figure 3F). Since hypocotyl lengths are longer than the wt, the cortical cell length of 10-day-old light-grown pentuple2, ein2-5, and etr1-1 mutant seedlings is longer than in wt plants (Figure 3G), suggesting that their longer hypocotyl size is due to longitudinal cell expansion rather than an increase in cell number.

Flowering time:

All the pentuple mutants start to flower earlier than do wt plants (Figure 3H). Rosette leaf number is the same in all four mutants and is similar to the rosette leaf number of wt plants, even though the mutants start to bolt earlier than wt (see inserted table in Figure 3H).

Ethylene production:

Ethylene production is inhibited ∼40% in young 5-day-old light-grown seedlings of the pentuple1, 3, and 4 mutants; the inhibition is slightly smaller (30%) in the pentuple2 mutant (Figure 3I). The inhibition of ethylene production is greater (almost 80%) in 1-month-old intact plants in all four pentuple mutants (Figure 3J). These results are consistent with the view that loss of ethylene biosynthetic capacity results in early flowering. Additional phenotypic parameters were studied and quantified during the course of the characterization of the pentuple2 mutant. The findings are summarized in Table S1 and show that 80% loss in ACS biosynthetic capacity in the pentuple2 mutant causes mild phenotypic abnormalities.

hexuple, heptuple, and octuple mutants:

Our ultimate goal was to construct a null ACS mutant in Arabidopsis. When the project initiated 9 years ago, we were able to identify insertions of five genes (ACS2, ACS4, ACS5, ACS6, and ACS9) leading to the construction and characterization of the pentuple mutants. However, 3 years ago the identification of insertion mutations in ACS1 and ACS7 enabled the construction of a hexuple mutant; acs2-1acs4-1acs5-2acs6-1acs7-1acs9-1 and a heptuple mutant; acs1-1acs2-1acs4-1acs5-2acs6-1acs7-1acs9-1. Mutations in the two remaining genes, ACS8 and ACS11, are not available, so we used artificial micro RNA (amiR) technology to inhibit their activity. An amiR sequence was designed to specifically inhibit both genes according to the rules described by Schwab et al. (2006). This amiR was introduced by transformation into the hexuple mutant, giving rise to the octuple mutant; acs2-1acs4-1acs5-2acs6-1acs7-1acs9-1amiRacs8acs11. The hexuple background was used for the construction of the octuple mutant to expedite the process since the ACS1 gene, which is not inactivated in the octuple mutant, encodes an inactive homodimer, and its heterodimers ACS1/ACS8 and ACS1/ACS11 are also enzymatically inactive (Tsuchisaka and Theologis 2004a).

Growth of mature plants:

The growth phenotypes of high order mutant plants during various stages of plant development are shown in Figure 4, A and B. The height of the pentuple2, hexuple, and heptuple mutants is greater than that of the wt after 40 days of growth, but the height of the octuple mutant is similar to that of the wt control (Figure 4A). However, the height of the octuple becomes greater than that of wt plants and very similar to that of the other high order mutants after 50 days of growth (Figure 4B). Eventually the octuple plants are the tallest among all the high order mutants (Figure 4C). Growth of the octuple mutant is delayed during the initial stages of plant development. The octuple plants are less bushy due to the reduced branching (Figure 4B). One of the most prominent characteristic of the octuple plants is their delayed senescence. While the wt, pentuple2, hexuple, and heptuple mutant plants start to senesce after 60 days of growth the octuple mutant plants are quite green and healthy.

Growth of seedlings:

The hypocotyl length of etiolated seedlings is greatly enhanced in all four high order mutants compared to the wt control. There is a progressive increase in hypocotyl length among the pentuple2, hexuple, and heptuple mutants, but a reduction in the octuple mutant (Figure 4E). Two prominent phenotypic changes were observed in the hexuple, heptuple, and octuple mutants that are not seen in the pentuple2 mutant. First, there is progressive loss of hook formation among these three mutants; the octuple mutant is hookless (Figure 4E). Second, their response to gravitostimulation is also greatly reduced compared to the wt control and pentuple2 mutant (Figure 4G).

The enhancement of hypocotyl length in light-grown seedlings observed in the pentuple2 mutant is inhibited in hexuple, heptuple, and octuple mutants (Figure 4F). Their hypocotyl lengths are similar to the wt control (Figure 4F). Two prominent phenotypic characteristics of light-grown octuple seedlings are as follows:

(1) reduced size of cotyledons compared to the wt control and to the other three high order mutants (Figure 4F).
(2) the size and shape of its leaves: the leaf blade is smaller and has a downward curling tip (Figure 4D), reminiscent of the ifl1/rev mutation of the INTERFASCICULAR FIBRLESS/REVOLUTA gene (Talbert et al. 1995).

The overall plant architecture of the octuple mutant plant is similar to the ifl1/rev with its cauline paraclades highly elongated (Talbert et al. 1995). This mutation is defective in auxin transport/signaling, resulting in abnormal fiber and vascular differentiation (Zhong and Ye 2001). All other high order mutants have normal leaves (data not shown).

Flowering time:

The early flowering phenotype of the pentuple2 mutant is greatly enhanced in the hexuple and heptuple mutants (Figure 5A). This phenotype is absent from the octuple mutant, but it still flowers somewhat earlier than do wt plants (Figure 5A). The number of rosette leaves is reduced in all mutants, but most dramatically in the hexuple and heptuple mutants (see inserted table in Figure 5A), a phenotypic characteristic that is in agreement with the observed early flowering phenotype.

The strong effect of the high order mutations on flowering time prompted us to investigate the expression of key regulators of flowering in young seedlings. The most dramatic effect of the high order mutations is their inhibitory effect on expression of the flowering repressor FLC in 3- and 9-day-old seedlings (Figure 5B). Concomitantly with the decrease in FLC gene expression, an increase in FT gene expression is detected in the hexuple, heptuple, and octuple mutants in 3-day-old seedlings (Figure 5B). The FT expression is greatly enhanced in 9-day-old seedlings and is higher than in wt plants in all mutants (Figure 5B). The SOC1 activator is expressed at low levels in 3-day-old seedlings and its expression is greatly enhanced in older seedlings. All mutants express SOC1 at a level similar to that in the wt control in both 3- and 9-day-old seedlings (Figure 5B). CO gene expression does not show any dramatic changes in any mutant in both 3- and 9-day-old seedlings (Figure 5B). The early flowering phenotype of the high order mutants raises the question of whether all ACS gene family members are expressed in the leaf primordial where FLC exerts its negative role on FT (Wigge et al. 2005; Searle et al. 2006). Figure 5C shows longitudinal sections of 5-day-old light-grown seedlings of ACSpromoter-GUS lines, showing that all ACS genes except ACS9 are expressed in the shoot apical meristems (SAM) and in the neighboring tissues such as leaf primordial, stipules and the vasculature at that stage of development.

Ethylene production:

Ethylene production is greatly inhibited in all high order mutants, with the octuple mutant having the lowest ethylene evolution in both 10-day-old seedlings and 35-day-old plants (Figure 5D). Ethylene production is inhibited by 92 and 86% in the octuple seedlings and mature plants, respectively (Figure 5D). Both hexuple and heptuple mutants produce approximately the same amount of ethylene in both tissues, which corresponds to ∼25% of the ethylene produced by wt plants (Figure 5D). RT–PCR analysis with RNA from 5-day-old seedlings indicates that the low but detectable amount of ethylene produced by the octuple mutant is due to the incomplete inhibition of ACS8 and ACS11 gene expression by the amiR (Figure 5E). Inactivation of multiple ACS genes does not cause an enhancement of gene expression of the noninactivated genes (Figure 5E). For example, ACS7, 8, and 11 gene expression in the pentuple2 mutant is the same as in the wt control (Figure 5E). The same is true for the hexuple and heptuple mutants, where the expression of ACS8 and 11 is not altered compared to the pentuple2 mutant (Figure 5E). ACS1 mRNA is of very low abundance and cannot be detected in isolated total or polyA⁺ RNA.

Embryo lethality in the octuple/amiR lines:

Attempts to isolate additional homozygous octuple mutant lines that produce less ethylene were unsuccesful. We screened 62 independent amiR transformants (out of 192 total) that were determined to have the amiR construct at a single locus and were unable to isolate another homozygous octuple line that produced less ethylene and expressed lower amounts of the ACS8 and 11 mRNAs than the octuple line described above. These results suggested to us that such an octuple mutant line may not exist because it is not viable. Indeed, that was the case because the siliques of T1 plants from 24 independent amiR lines show signs of embryo lethality (Figure 6A). We see three types of siliques: some are like the ones of line no. 3; others are like those of line no. 20; others are like those of no. 24 (Figure 6A). Closer examination of the siliques of the viable octuple line show that they are shorter than those of the control, contain half of the seeds present in siliques of wt plants, and show signs of embryo lethality because of empty seed spaces (Figure 6B). It is interesting that heterozygous octuple plants have normal silique length but half the number of seeds than do wt plants and also show signs of embryo lethality (Figure 6B). The other high order mutants have normal size siliques and the same amount of seeds/silique as the wt control (Figure 6C). Furthermore, the embryonic lethality associated with the octuple mutant lines was recently confirmed by the isolation of a second octuple line that exists only as a heterozygous plant. No homozygous plants can be recovered from its progeny. Segregation analysis shows that seeds from this heterozygous line segregate in a ratio of 1 hexuple to 1 heterozygous for the amiR insertion. This ratio suggests male or female gametophytic defect in this second octuple line. A phenotypic comparison between these two octuple lines is presented in Figure S5. The results show that both lines have quite similar phenotypes but the silique phenotype is attenuated in the heterozygous line. All the experiments reported here have been carried out with the homozygous octuple line.

Figure 6.— — Embryo lethality of the *octuple/amiR* lines (A–C). The siliques from 24 T1 independent *amiR* plants are shown in A, indicating embryo lethality. The three magnified siliques at the bottom show prezygotic lethality marked with an asterisk and embryonic lethality marked with a box. The silique morphology and seed content of the heterozygous and homozygous *octuple* (*amiR*) plants are shown in B. The silique length and seed content in the high order mutants are shown in C (N = 10). Bars represent the standard deviation (SD). The asterisk (*) has been defined in the legend of Figure 1. Rescue of the *octuple* mutant phenotype by backcrossing to wt (D–G). The photos in D compare the phenotypes of 20-day-old light-grown plants among wt, *hexuple*, *octuple*, and five backcrossed lines. Bar = 1 cm. The silique morphology of the backcrossed lines expressing different *ACS* genes is shown in E. Quantitative comparison of the silique length and seed content of the backcrossed lines among the wt, *hexuple*, and *octuple* mutants is shown in F (N = 10). The genotyping of the backcrossed lines is shown in G. The genotypes of the backcrossed lines are shown at the bottom. Bars represent the standard deviation (SD). The asterisk (*) has been defined in the legend of Figure 1.

Specificity of the amiR:

Is the embryonic lethality and the various seedling phenotypes observed in the octuple mutant lines due to the inhibition of ACS8 and 11 gene expression by the amiR or is due to inhibition of other genes necessary for these various phenotypes? We carried out three experiments to address this question.

(1) We rescued the octuple light-grown and etiolated seedling phenotypes by germinating mutant seeds on plates containing various amounts of the ethylene precursor, ACC (Figure S5). The phenotypes of both types of octuple mutant seedlings reverted back to the wt phenotype (Figure S5). The octuple mutant leaves were wt-like after ACC treatment (Figure S5, A and B). However, the octuple is hypersensitive to ACC compared to the wt: the roots of the octuple are shorter and have a proliferation of root hairs, unlike the wt (Figure S5, A and B). The growth of octuple etiolated seedlings is inhibited to the same degree as in wt with exogenous ACC and their hookless phenotype is reverted to wt-like (Figure S5, C and D). They also show a hypersensitivity to ACC because of root hair proliferation unlike the wt (Figure S5, C and D). For both types of seedlings, the basis of this observed ACC hypersensitivity is unknown.
(2) We rescued the octuple phenotype by backcrossing the mutant to wt and selecting segregants that are homozygous for the amiR insertion but have lost the T-DNA insertion from various ACS genes. We isolated and analyzed five such segregant lines (Figure 6G). A visual comparison among the octuple mutant and the backcrossed lines in 20-day light-grown plants shows that the octuple phenotype has been reverted back to the wt- or hexuple-like phenotype. The small size and abnormal morphology of the rosette leaves, a prominent characteristic of the octuple mutant is reverted back to almost the wt- or hexuple-like rosette leaves in all lines (Figure 6D). Furthermore the small silique size and low seed number of the octuple mutant reverted to the wt-like phenotype (Figure 6, E and F). These data clearly show that the ACS8 and ACS11 genes are not essential for Arabidopsis viability and that the embryonic lethality of the octuple lines is not due to the nonspecific effect of the amiR. While the octuple silique phenotype is revered back to wt-like by activating endogenous ACS genes the phenotype of octuple siliques from ethylene-treated plants fail to revert back to wt-like (Figure S7, A, B, and C). This suggests that the embryonic lethality associated with the octuple phenotype maybe due to the low availabilty of endogenous ACC.
(3) We complemented the octuple mutant with a transgene that expresses ACS8 and ACS11 cDNAs with altered amiR target sequences but that encode enzymatically active isozymes. Three lines (nos. 8, 10, and 14) homozygous for the introduced transgene were isolated and compared to the hexuple mutant. A visual comparison among the mutant and complemented lines in both etiolated and light-grown seedlings shows that the octuple phenotype has been reverted to the hexuple-like phenotype (Figure 7, A and B) The hookless phenotype of the etiolated octuple mutant seedlings is reverted to the hexuple-like hook in all three complemented lines (Figure 7A). The hypocotyl length of all three lines is longer than that of the octuple mutant, very similar to the hexuple mutant (Figure 7A; etiolated). The small cotyledon size, a prominent characteristic of the octuple mutant, is reverted back to almost the hexuple-like cotyledon size in all three complementation lines (Figure 7B; light grown). Ethylene production dramatically increased in all three lines and it is higher than that of the hexuple mutant (Figure 7C). The higher ethylene production of the complementation lines is due to the overexpression of the ACS8 and ACS11 transcripts by 35S promoter (Figure 7D). We noticed that the three complementation lines overexpress one of the two ACS genes introduced into them, but not both: lines no. 8 and no. 14 overexpress only ACS8; line no. 10 overexpresses ACS11 (Figure 7D). We think that this may be due to a recombination event during transformation resulting in the deletion of one of the genes in the 35S double gene construct used for this experiment (see materials and methods). Further examination of mature plants shows that while the small size of the rosette leaves has been corrected in the complemented lines, their revoluta-like morphology remains octuple-like (Figure 7E). The complementation lines also flower earlier than the octuple but not as early as the hexuple mutant (Figure 7F), indicating a partial phenotypic reversal. The same was observed with the reversal of the octuple silique phenotype. While silique length was partially corrected, the seed number per silique remained octuple-like in the complementation lines (Figure 7, G and H). We think that the partial reversal of some octuple phenotypes may be due to expression of only one of the two genes, but perhaps both ACS8 and ACS11 are required for correcting certain phenotypes. Alternatively, the 35S promoter used for this experiment may not be active in some cells and tissues responsible for the phenotypic defects.

Figure 7.— — Complementation of the *octuple* mutant. The photos in A compare the phenotypes of 3-day-old etiolated seedlings among wt, *hexuple*, *octuple*, and three complementation line nos. 8, 10, and 14. Bar = 1 cm. The graph on the right of A compares the hypocotyl length and hook curvature among wt, *hexuple*, *octuple*, and line nos. 8, 10, and 14 (N = 10). The photos in B compare the phenotypes of 5-day-old light-grown seedlings between wt, *hexuple*, *octuple*, and line nos. 8, 10, and 14. Bar = 1 cm. The graph on the right of B compares the hypocotyl length and cotyledon area among wt, *octuple*, and line nos. 8, 10, and 14. (N = 10). The comparison of the ethylene production among wt, *hexuple*, *octuple*, and line nos. 8, 10, and 14 in 5-day-old light-grown seedlings is shown in C (N = 3). The expression of the *ACS8* and 11 in wt, *hexuple*, *octuple*, and line nos. 8, 10, and 14 in 5-day-old light-grown seedlings is shown in D. The *ACT8* gene was used as a nondifferential expressed gene. The graph on the right of D shows the quantitation of the RT–PCR data. The photos in E compare the phenotypes of 20- (first column) and 40-day (second column)-old light-grown plants among wt, *hexuple*, *octuple*, and three transformation lines. Their rosette leaves are also compared (third column). The flowering time of wt, *hexuple*, *octuple*, and line nos. 8, 10, and 14 is shown in F. The silique morphology of wt, *hexuple*, *octuple*, and line nos. 8, 10, and 14 is shown in G. Quantitative comparison of the silique length and seed content of wt, *hexuple*, *octuple*, and line nos. 8, 10, and 14 is shown in H (N = 10). Bars represent the standard deviation (SD). The asterisk (*) has been defined in the legend of Figure 1.

Response to pathogens:

A vast amount of literature suggests that ethylene is involved in various pathogen responses (Broekaert et al. 2006). Here, we characterized the responses of the high order mutants to necrotrophic pathogens Botrytis cinerea and Alternaria brassicicola. The pentuple2 mutant showed slightly enhanced susceptibility to B. cinerea, whereas hexuple, heptuple, and octuple mutants displayed much stronger disease symptoms compared with wt and pentuple2 mutants (Figure 8). The disease symptoms progressed and led to complete decay of the octuple mutant at 7 days postinoculation (dpi) (Figure 8). However, we observed no significant difference between the mutants and wt after A. brassicicola infection (data not shown).

Figure 8.— — Response of the high order mutants to pathogen infection. The *pentuple2*, *hexuple*, *heptuple*, and *octuple* mutants show increased disease symptoms to *Botrytis cinerea.* Plants were inoculated by spraying spore suspension of the *B. cinerea* (strain BO5-10) at a concentration of 2 × 10⁵ spores/ml. The pictures were taken at 7 dpi. The experiments were repeated twice and similar results were observed.

The ACS interactome map:

The nine ACS proteins form active and inactive heterodimers in E. coli (Tsuchisaka and Theologis 2004a), but it is not known whether ACS heterodimers form in planta. Furthermore, our results point to the operation of a combinatorial control mechanism among the nine ACS isoforms, so we determined the homo- and heterodimeric interaction among the various ACS subunits in planta using BiFC (Hu et al. 2002). All possible active and inactive homo- and heterodimers of the ACS gene family members can be detected in auxin-treated Arabidopsis etiolated seedlings (Figure 9A). Auxin treatment was necessary for detecting the various homo- and heterodimeric interactions because the hormone enhances transcription of all the ACS gene family members except ACS1 (Yamagami et al. 2003). The root tip was used for the assay because our previous studies with ACSpromoter-GUS fusions have shown that most of the genes (but not ACS1 and ACS9) are expressed in various root cell types after auxin treatment (Tsuchisaka and Theologis 2004b). The heterodimeric interaction between bJun and bFos in the root tip of Arabidopsis served as a positive control (see left bottom of Figure 9A).

The data presented in Figure 9A provide the foundation for a meaningful estimation of the effect of inactivating various ACS genes on the composition and diversity of the ACS family in various cells and tissues. A diagrammatic presentation of the number of active and inactive isozymes in the various mutants is shown in Figure 9B. The data show the effect of deleting single or multiple genes on the relative ratio between active and inactive isozymes, and provide a framework for understanding the complexity of ethylene biosynthesis. This complexity is greatly magnified if one considers the heterogeneity of the C terminus among the ACS polypeptides, which is responsible for their stability (Argueso et al. 2007). The ACS proteins can be classified into three types on the basis of their C terminus: type 1 have the longest C terminus with a single putative calcium-dependent protein kinase (CDPK) phosphorylation site and three mitogen-activated protein kinase (MAPK) phosphorylation sites; type 2 have a medium size C terminus containing a single CDPK site; type 3 have a short C terminus with no predicted protein kinase phosphorylation sites. A diagrammatic presentation of the heterogeneity among the active and inactive isoform based on their C terminus is presented in Figure 9C. The relative ratio of all the different ACS isoforms shown in Figure 9C is greatly altered in the mutants presented in this analysis (Figure 9D). The homo- and heterodimerization capacity of ACS family coupled with its heterogeneity at the C terminus provides a framework for understanding the complexity of ethylene biosynthesis as well as for interpreting the phenotypic defects of the various mutants.

Global gene expression analysis:

To determine how the single and high order mutations affect the molecular phenotype of young Arabidopsis seedlings, we profiled their global gene expression. The results of the analysis are presented in Figure S9, Figure S10, Figure S11, Table S2, Table S3, Table S4, Table S5, Table S6, Table S7, Table S8, Table S9, and Table S10.

DISCUSSION

Lessons from the single mutants:

Each ACS performs a specific and unique function:

All the null mutants of any single ACS gene are viable. Mutations inactivating specific ACS isozymes affect three developmental processes: hypocotyl growth, flower time, and cotyledon size. Four single mutants, acs1-1, acs6-1, acs7-1, and acs9-1, flower earlier than does wt, suggesting that ACS1, ACS6, ACS7, and ACS9 are negative regulators of the flowering process that are involved in proper regulation of flowering time. Remarkably, some interactions between ACSs appear to be antagonistic, because the early flowering phenotype of acs6-1 and acs7-1 is suppressed in the double mutant, which has a late flowering phenotype.

Some of the mutations affect the hypocotyl growth of etiolated seedlings positively or negatively. For example, acs1-1 and acs9-1 enhance hypocotyl length whereas acs4-1 inhibits it, suggesting that ACS1 and ACS9 are negative regulators, whereas ACS4 is a positive regulator of growth in the dark. A unique phenotypic alteration associated with the acs1-1 mutation is inhibition of inflorescence stem diameter. All single mutants enhance hypocotyl length and the size of cotyledons in light-grown seedlings, with the acs1-1 having the strongest effect. The enhancement in plant height in light-grown plants is evident throughout the life cycle. These data suggest that the ethylene produced by ACS1, ACS2, ACS4, ACS5, ACS6, and ACS9 isozymes acts as a negative regulator of plant growth in the light.

These developmental abnormalities do not correlate well with the total amount of ethylene produced in young seedlings. Loss of ACS4 and ACS9 function results in moderate ethylene overproduction, but loss of ACS1, which is the inactive isozyme, results in inhibition of ethylene evolution. The rest of the single mutants produce the same amount of ethylene as the wt. These results reveal an interaction among the ACS isoforms that regulate the overall output/activity of the ACS family. Again some of the interactions between ACSs are antagonistic, because ethylene overproduction caused by acs4-1 and acs9-1 is suppressed in the double mutant. Total ethylene production by a seedling appears not to be a good indicator/predictor for evaluating developmental phenotypic responses mediated by the hormone. We believe that the localized cellular sites of ethylene production, rather than the absolute amount of ethylene produced by an intact seedling or plant, determine the developmental response and outcome. Even though it is volatile and therefore highly diffusible, ethylene can induce different local responses depending on its site(s) of production and/or perception (Stepanova et al. 2008; Thomann et al. 2009). This agrees with the findings that the ACS genes have distinct patterns of expression during Arabidopsis development (Tsuchisaka and Theologis 2004b). Local hormone biosynthesis and deactivation have emerged as key regulatory factor in various developmental processes (Zhao 2008; Chandler 2009).

A second, independent evaluation of the relationship among the ACS genes is the global gene expression profiles of their mutants. Distinct differences between all single acs mutants and wt are apparent, suggesting that each ACS has a specific function. The differences in gene expression profiles between two alleles of the same gene may reflect dominant negative interactions of the truncated polypeptides with the rest of the family members resulting in alterations of ethylene production in specific cellular sites. When individual members of a gene family are disrupted and have no obvious phenotypic consequences, functional redundancy is offered as a possible explanation. Our data establish that every family member executes a unique function in each cell by regulating the cellular ethylene production via its interactions with the rest of the ACS family members.

Lessons from the high order mutants:

The ACS family members each perform a common essential function:

The phenotypes of the high order acs mutants led us to some simple conclusions and revealed a wealth of phenotypic complexity. To emphasize the most salient result, the inactivation of all nine genes caused embryonic lethality, indicating that the ethylene biosynthetic pathway is required for Arabidopsis viability. Our inability to recover a truly null octuple mutant that produces no ethylene was not due to a nonspecific effect of the amiR used to inactivate the ACS8 and ACS11 genes. The octuple mutant provides an excellent resource for constructing lines of plants expressing individual ACS genes. This strategy has the potential to assign specific biological function(s) to each ACS member.

Phenotypic resistance to the loss of ethylene: the pentuple paradox:

We were quite surprised that inactivation of the ACS biosynthetic capacity by 80% in the pentuple mutant resulted in mild phenotypic changes. While flowering time and growth of etiolated and light-grown seedlings/plants were more pronounced in the pentuple mutants compared to the single mutants, the majority of its phenotypic characteristics/responses were similar to those of wt plants (Table S1). Its global gene expression profile was also quite similar to wt (see File S1, Results and Discussion). This may be due to the low sensitivity of the microarray analysis to detect changes in gene expression in specific cells and tissues responsible for the phenotypic changes. A pentuple mutant cell that expresses all four remaining noninactivated genes, ACS1, ACS7, ACS8, and ACS11, potentially has five active (ACS7/ACS7, ACS8/ACS8, ACS11/ACS11, ACS8/ACS11, and ACS7/ACS11) and five inactive homo- and heterodimeric isoforms (ACS1/ACS1, ACS1/ACS7, ACS1/ACS8, ACS1/ACS11, and ACS7/ACS11), and the in planta interactome map supports such a proposition. The difference in ethylene production between young seedlings and mature plants may reflect differences in the ratio of active and inactive isoforms. This may be due to differences in expression of the various genes and/or to preferential protein stabilization that compensates for the loss of ethylene biosynthetic capacity. The hexuple and heptuple mutants exhibited more marked phenotypic changes associated with reduced ethylene production. Inactivation of the ACS biosynthetic capacity by 88%, resulting in approximately 70–75% inhibition in total ethylene, revealed phenotypic changes associated with differential growth and response to pathogens. Response of these mutants to gravitostimulation is greatly diminished, and their hook structure is less prominent than in the pentuple mutant. Both mutations also enhanced phenotypes previously detected in the pentuple mutant such as enhanced plant growth, cotyledon size, and early flowering, and pathogen susceptibility. However, while their hypocotyl length of etiolated seedlings is further enhanced in both mutants compared to the pentuple mutant, their light-grown hypocotyl length is like that of wt, in sharp contrast to the pentuple mutant, which has longer hypocotyls than does wt. This may reflect a lower rate of cell growth at this low level of ethylene production because the mature hexuple and heptuple plants are taller than the pentuple plants.

The octuple Arabidopsis plant:

The octuple mutant produces ∼10% of the wt level ethylene because is not a truly null for ACS8 and ACS11. It is a handsome plant, the tallest among the high order mutants, lives longer than all the other mutants, senesces 3 weeks later than the wt, and produces fertile seeds, albeit at 50% yield compared to the wt and the other high order mutants. However, it is highly vulnerable because of its compromised defense to pathogens and probably to herbivores because of its defects in the glucosinolate biosynthetic pathway (see File S1, Results and Discussion). The delayed senescence of the octuple mutant reminds us that inhibition of plant senescence requires very low ethylene production. This is in agreement with the observation that inhibition of tomato fruit ripening by antisense RNA requires almost complete inhibition of ethylene production (Oeller et al. 1991). Many of the phenotypic characteristics of the octuple mutant reflect enhancement or reversal of those seen in the hexuple and heptuple mutations, but additional phenotypes were also observed. For example, an octuple etiolated seedling is indeed hookless and its hypocotyl length is longer than that of wt but shorter than that of the other high order mutants. Its response to gravity is diminished and is quite variable compared to the hexuple and heptuple mutants. A light-grown octuple seedling has the same hypocotyl length as the wt, but the size of its cotyledons is greatly inhibited. It appears that the rate of growth is greatly diminished in the octuple seedling and young plant, but its overall size as a mature plant is the tallest among all the other high order mutants. Furthermore, its susceptibility to pathogen is greatly enhanced, and it flowers later than the other two high order mutants. In addition we see changes in leaf size (smaller) and morphology, siliques size, and seed content/silique compared to the hexuple and heptuple, which have wt-like leaves and siliques. If all possible combinations and permutations of the nine ACS family members were constructed and analyzed (N = 2⁹ − 1 = 511), the phenotypic complexity can be massive. The order of unmasking various phenotypic changes depends on the extent of the inactivation of the ACS biosynthetic capacity. Growth and flowering time are the most sensitive parameters to alterations in ethylene biosynthetic capacity. The two differential growth parameters, hook formation, response to gravity, resistance to pathogens, and inhibition of senescence are resilient to changes in ethylene biosynthetic capacity. It should be pointed out, however, that this order may be quite different if different combinations and permutations of gene mutations were analyzed. We constructed 91 mutants (∼18% of all possible) and analyzed 26 mutants (∼5% of total). It should be noted that the ACS6 and ACS9 overexpression lines shown in Figure 2 are defective to their response to gravity and etiolated hypocotyl growth (Figure S4), indicating that local disturbances in the ACS family have major phenotypic consequences. This observation reveals the operation of a communication network among the ACS members that controls local ethylene production.

Ethylene functions as a transcriptional rheostat:

It is of a great interest that Arabidopsis genes respond differentially to different ethylene concentrations (De Paepe et al. 2004). The global gene expression profiles of the single and high order mutants shown in File S1 (Results and Discussion) resemble an ethylene dose response curve of the transcriptional output of all Arabidopsis cells at a given developmental stage. The pentuple mutant behaves like its ACS activity is balanced, as if the positive and negative influences on its ACS activity are equal. Results of our analysis of the mutants reinforce the concept that ethylene acts as a rheostat to regulate transcription of key genes involved in numerous developmental and physiological processes. We believe this is a consequence of a combinatorial interplay among the various ACS isoforms that regulate ethylene production in a temporal and spatial manner.

Highlights of the genome expression profiling analysis:

A discussion on the most highly induced and repressed genes in the high order mutants listed in Table S10, A and B is presented in File S1 (Results and Discussion). Among them are genes involved in glucosinolate metabolism and light perception/signaling.

Ethylene and plant growth:

Ethylene is the simplest of the six small molecule hormones plants use to integrate a myriad of extrinsic (e.g., light) and intrinsic signals to regulate cell expansion and ensure optimal growth and development (Davies 2004; Nemhauser et al. 2006; Michael et al. 2008). Comparative global gene expression analysis has shown that each hormone regulates unique and nonoverlapping transcriptional networks (Nemhauser et al. 2006), suggesting that cell expansion driven by each specific hormone is qualitatively distinct. Recently, the DELLA repressor proteins whose activity is regulated by gibberelic acid (GA) have emerged as key integrators of plant growth by light and high order hormonal signals including ethylene (Fu and Harberd 2003; Achard et al. 2006; Schwechheimer 2008; Schwechheimer and Willige 2009). The enlarged cotyledon size of the acs mutants can be attributed to the loss of DELLA function. The same phenotypic abnormality has been seen in the spt10 mutant of the SPATULA (SPT) gene which is a repressor of the GA biosynthetic gene GA3 oxidase (Penfield et al. 2006). Since ethylene is known to positively regulate DELLA repressing function by inhibiting GA biosynthesis and enhancing DELLA function (Achard et al. 2003, 2007; Schwechheimer 2008) the prospect arises that the enlarged cotyledon phenotype of the acs mutants may be due to the loss of DELLA function. Alternatively, loss of DELLA function may be due to the inhibition of ethylene-regulated auxin production (Swarup et al. 2007; Stepanova et al. 2008; Tao et al. 2008). Auxin is known to enhance DELLA repressing function (Fu and Harberd 2003).

Ethylene generally inhibits growth of plants (Vandenbussche et al. 2005; Dugardeyn and Van Der Straeten 2008), but in a few cases it stimulates cell expansion (Smalle et al. 1997). Results of our analysis of acs mutants clearly demonstrate that ethylene is a repressor of cell growth in dark- or light-grown plants, because progressive inhibition of the ethylene biosynthetic capacity causes a progressive enhancement of plant size. The stimulatory effect of the loss of ethylene biosynthetic capacity on plant growth is in sharp contrast to the loss of auxin, GA, and BR biosynthetic capacity, which causes dwarfism (Sun et al. 1992; Li et al. 1996; Zhao 2008). Some of the phenotypes of the high order acs mutants, such as long hypocotyl and internodal length, early flowering, and decreased seed yield seen in the octuple mutant are reminiscent of those observed in shade avoidance syndrome (SAS) (Smith and Whitelam 1997; Jiao et al. 2007; Tao et al. 2008). More broadly, the repressing activity of ethylene on plant growth is reminiscent of the similar activity of light on plant expansion (Chen et al. 2004; Jiao et al. 2007). The long hypocotyls of light-grown acs mutants is reminiscent of the reduced capacity of the light-mediated inhibition of hypocotyl elongation in the hy mutants of Arabidopsis (Chory et al. 1989). Our microarray data also indicate the operation of an extensive communication network between light signaling and ethylene biosynthesis, since expression of many light-related genes is altered by the loss of ethylene production (Vandenbussche et al. 2005; Michael et al. 2008; Alabadí and Blázquez 2009). The possibility exists that the acs mutants are also defective in clock-regulated growth because the expression of key clock components, such as LHY and FKF, has been altered in the mutants (Thain et al. 2004; Michael et al. 2008). Since ethylene interacts with all the known hormone biosynthetic and signaling pathways it is reasonable to suggest that all these pathways have been uncoupled and the growth of the various mutants is an expression of the growth capacity present in Arabidopsis cells under various levels of ethylene production.

Ethylene and differential growth:

Hook formation:

Recognition of ethylene's involvement in localized differential growth in response to light (phototropism), gravity (gravitropism), and formation of an apical hook to ensure early seedling establishment is as old as the discovery of the hormone (Neljubow 1901). It is generally accepted that localized differential growth is due to auxin gradients that are established by local auxin biosynthesis and directional intercellular auxin transport (Tanaka et al. 2006; Vanneste and Friml 2009). The development of an apical hook involves a complex interplay of at least three hormone-responsive pathways: ethylene, auxin, GA and light (Achard et al. 2003; Li et al. 2004). It is yet not clear whether asymmetric auxin biosynthesis is due to asymmetric expression of ACS isoforms or asymmetric expression of the auxin biosynthetic enzymes (Stepanova et al. 2008). Alternatively, the ethylene-regulated HOOKLESS (HLS1) gene may be responsible for auxin asymmetry by regulating auxin signaling via ARF2 (Li et al. 2004). Our data indicate that the loss of hook formation is quite resistant to the loss of ethylene biosynthetic capacity. It was not until the partial loss of the ACS8 and ACS11 genes in the octuple mutant that a hookless phenotype resulted.

Gravity sensing:

It is widely accepted that asymmetric auxin distribution is responsible for the differential cell elongation on opposite sides of cells that leads to gravitotropic curvature upon gravitostimulation (Terao-Morita and Tasaka 2004). Loss-of-function mutations in the auxin-regulated transcriptional activator NPH4/ARF7 disrupts the gravitotropic response in Arabidopsis (Harper et al. 2000). Ethylene is involved in gravity sensing by suppressing the NPH4/ARF7 loss-of-function phenotype and this may explain to diminished response of the acs mutants to gravitostimulation. However, it is quite intriguing that while the high order acs mutants have lost their ability to respond to gravity, they are not agravitropic. This suggests that a component of the gravity sensing apparatus responsible for gravitostimulation maybe defective in the mutants (Terao-Morita and Tasaka 2004). It also suggests that the ethylene produced by the octuple mutant is sufficient for maintaining an auxin biosynthesis/signaling apparatus for proper gravity sensing. Our gene expression profiling data raise the possibility that blue-light mediated processes, such as tropisms, may be defective in the high order acs mutants. Expression of two homologs of NON PHOTOTROPIC HYPOCOTYL 3 (NPH3), a BTB/POZ-containing protein involved in phototropism signaling, is induced in the hexuple and heptuple mutants (Table S10; Cheng et al. 2007; Pedmale and Liscum 2007).

Ethylene and flowering time:

Ethylene is known to be involved in the flowering process (Abeles et al. 1992; Boss et al. 2004; Achard et al. 2006). One of the highlights of our analysis is that ethylene represses flowering in Arabidopsis. Inactivation of specific ACS gene products enhances flowering time, and this enhancement is further potentiated by the progressive loss of ACS biosynthetic capacity in the high order mutants. We attribute the diminished early flowering phenotype of the octuple mutant to major alterations in the flowering machinery caused by the severe inhibition in ethylene production.

Several lines of evidence indicate that ethylene controls flowering time through a rheostat in which the level of ethylene production in the leaf primordial/SAM is proportional to the lateness to flower. For example, complementation of transgenic acs6-1 and acs9-1 mutants by their corresponding ORFs results in a broad range of flowering times, presumably due to variation in ACS6 or ACS9 expression caused by variation in transgene copy number and/or chromosomal position effects. Our data indicate that ethylene exerts its effect on flowering by regulating the expression of the FLOWERING LOCUS C (FLC) (Michaels and Amasino 2001). FLC also acts as a rheostat to repress flowering (Michaels and Amasino 1999) through repression of the floral pathway integrators FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC1) (Blázquez and Weigel 2000; Samach et al. 2000; Wigge et al. 2005). The early flowering phenotype of the high order acs mutants is associated with a progressive loss of FLC mRNA in light-grown seedlings and a concomitant increase of the positive flowering activator FT mRNA. The opposite is observed in the late flowering double mutant acs6-1acs7-1. The increase in SOC1 mRNA is moderate. The early flowering phenotype appears not to be due to a disruption in the photoperiodic-regulated flowering process because the mRNA of CONSTANS (CO), a master regulator of this process, is slightly repressed in the acs mutants. In addition two key components of the photoperiodic-regulated flowering machinery, LHY, a component of the clock and FKF, a novel blue light photoreceptor that controls CO transcript pattern (Bäurle and Dean 2006; Imaizumi and Kay 2006), are repressed in the acs mutants. We do not know whether ethylene regulates FLC transcription directly or indirectly. All known hormonal networks interfere with the flowering process (Boss et al. 2004), and since ethylene communicates with all of them (Nemhauser et al. 2006; Alabadí and Blázquez 2009) the possibility exists that the effect of ethylene on early flowering is indirect. Alternatively, ethylene may regulate FLC expression by regulating the chromatin state, which has emerged as an important mechanism in the control of FLC expression (Bäurle and Dean 2006; Domagalska et al. 2007; Pien et al. 2008).

It has been recently shown that ethylene delays flowering by modulating DELLA activity. Ethylene-enhanced DELLA accumulation in turn delays flowering via repression of the floral meristem-identity genes LEAFY (LFY) and SOC1 (Achard et al. 2007). These findings are based on the observation that constitutively active ethylene signaling in the ctr1 mutant reduces GA levels, and the late flowering phenotype of the ctr1 mutant is partially rescued by loss-of-function mutations in DELLA genes (Achard et al. 2007). A possible explanation for this difference between those results and ours may be that a different flowering pathway operates in the ctr1 mutant and in our acs mutants.

The antagonistic effect of the acs6-1 and acs7-1 mutations on flowering, together with the flowering time of plants overexpressing ACS6 and ACS9 reveal the operation of a communication network among ACS proteins that may cause local changes in ethylene production. We think that the late-flowering phenotype of the acs6-1acs7-1 double mutant may be due to ethylene overproduction in the leaf primordial/SAM region brought about by alteration in the relative ratio of various active and inactive ACS isoforms.

Ethylene and plant pathogens:

Ethylene plays an important role in plant pathogen responses associated with disease resistance or disease susceptibility, depending on the type of pathogen and plant species. Ethylene has been proposed to be more effective against necrotrophic pathogens, such as B. cinerea than against biotrophic pathogens. Ethylene-insensitive mutants ein2, ein3, and etr1 show enhanced susceptibility to B. cinerea (Thomma et al. 1999; Ferarri et al. 2003). Plants that overexpress transcription factors involved in the ethylene and JA pathways exhibit an increased resistance to several necrotrophs (Berrocal-Lobo et al. 2002; Berrocal-Lobo and Molina 2004; Pré et al. 2008). Overexpression of AP2C1 in Arabidopsis, which encodes a Ser/Thr protein type 2C phosphatase, reduces ethylene production and compromises resistance to the necrotrophic pathogen B. cinerea (Schweighofer et al. 2007). We observed that the high order acs mutants have enhanced susceptibility to necrotrophic pathogen B. cinerea, which suggests that ethylene is important for nonhost resistance to B. cinerea in Arabidopsis, and our gene expression profiling results support this proposition. We have also examined the responses of these mutants to bacterial pathogens, Xanthomonas campestris pv. vesicatoria (Xcv), and X. campestris pv. campestris (Xcc). No significant changes of bacterial growth were observed in these mutants as compared with that in the wt (data not shown), suggesting that ethylene production has no obvious effect in biotrophs or hemibiotrophs. Several B. cinerea-induced genes [AtGenExpress; response to B. cinerea infection, provided by Ferrari et al. (2007)], including genes encoding a putative NADP-dependent oxidoreductase (At5g16980), a serine carboxypeptidase S28 family protein (At5g22860), two APS reductases (APR1 and APR3), and a cytochrome P450 gene CYP71A12, were suppressed in at least one of the high order acs mutants (Table S5 and Table S9). By contrast, a group of B. cinerea-repressed genes was induced in the mutants. Among them, a glutaredoxin family gene (At3g62950) was induced in all four mutants (Table S4 and Table S8). Two B. cinerea-repressed beta-ketoacyl-CoA synthase genes, KCS12 and KCS16, were also induced in at least two of the mutants (Table S4 and Table S8). Thus, inhibition of ethylene synthesis alters expression of many B. cinerea-responsive genes and represses defense related genes, which leads to the enhanced susceptibility to B. cinerea infection.

Is ACC a primary plant growth regulator?

The most obvious interpretation of our inability to recover a truly null ACS mutant is that the ethylene biosynthetic pathway is required for Arabidopsis embryo and/or gametophytic development. We know ethylene regulates key genes involved in auxin biosynthesis and transport (Stepanova et al. 2007, 2008; Swarup et al. 2007; Tao et al. 2008; Chandler 2009), processes known to have a central role in embryo patterning and development (Tanaka et al. 2006; Vanneste and Friml 2009). It is also possible that ethylene may regulate DNA methylation, which is critical for Arabidopsis embryogenesis and seed viability (Xiao et al. 2006). However, there is a potential conflict with such an interpretation. While the octuple acs mutant causes embryonic lethality, single or double ein2 and ctr1 mutants, which are missing key components of the signaling apparatus are viable (Kieber et al. 1993; Roman et al. 1995; Alonso et al. 1999) This suggests that ethylene is not required for Arabidopsis viability and embryo development. Since inactivation of all ACS genes eliminates the production of ethylene and inhibits the biosynthesis of its precursor, ACC, the prospect arises that ACC is a primary plant growth regulator responsible for embryo development, as it may be for cell expansion mediated by the FEI/SOS pathway (Xu et al. 2008). Perhaps the alternative MKK9-MPK3/6 dependent pathway of ACC synthesis is responsible for embryo development (Yoo et al. 2008). However, since the ctr1ein2 double mutant is not embryonic lethal, this alterative pathway cannot be responsible for embryo development. If there is an alternative signaling pathway responsible for embryo development, it should branch from the linear pathway at the receptor level, above the CTR1-catalyzed step. Construction of a heptuple mutant, etr1ers1etr2ein4ers2ctr1ein2 that inactivates all five receptors plus CTR1 and EIN2 has the potential to offer a definitive answer to the question of whether ACC is a primary growth regulator. Furthermore, the construction of a null ACO mutant has the potential to shine light to this intriguing possibility. There are 17 annotated ACO genes in the Arabidopsis genome (Arabidopsis Genome Initiative 2000), but whether all of them encode genuine ACC oxidase remains to be determined.

While we have presented above the rationale for ACC being a signaling molecule in its own right, another interpretation of the data is possible. Specifically, the elimination of the ACS-mediated pathway may affect the flux through the AdoMet pathway leading to increased levels of other metabolites that may cause embryo lethality.

“The Arabidopsis ACS symphony orchestra”:

Our results can be interpreted to indicate that ACC production, and, by extension, ethylene evolution, is a product of the collective action of the members of the ACS protein family. We view the ACS protein family as a “symphony orchestra” (45-member when all nine genes are expressed in a cell) that regulates ethylene-mediated processes by generating appropriate amounts of ACC in the proper spatial and temporal manner through their harmonious interplay. At any given moment, the orchestra is tuned by various inducers to produce ACC sufficient to mediate myriad ethylene responses (Figure 10). Each ACS dimeric isoform can be viewed as a particular instrument that interacts with its colleagues to produce the “melody” that coordinates plant growth development together with those of other hormonal and light regulatory networks. Disturbances in the tuning of the orchestra caused by mutations in the acs genes result in phenotypic cacophonies. Such disturbances can also be caused by expressing exogenous family members in a tissue-specific manner, as we saw in the complementation of the acs6-1 and acs9-1 single mutants. The number of players in each cell will depend on the number of genes expressed there. There are potentially 511 (N = 2⁹ − 1) harmonies in the ACS orchestra in the 5 × 10⁵ cells of a 10-day-old light-grown Arabidopsis seedling (assuming that the seedling volume is 4 μl because its average weight is ∼4 mg and the density ρ = 1 g/ml; the cell size is 20 μm × 20 μm × 20 μm; the cell volume 8 × 10⁻¹⁵ m³). The relative abundance of the players in the orchestras will depend on mRNA abundance, protein stability of various homo- and heterodimeric isoforms as well as on the K_d's of the various ACS polypeptides. The capacity of the various isozymes to form active heterodimers together with their C terminus heterogeneity provides vast biochemical diversity among the 511 harmonies capable of operating under a very broad spectrum of AdoMet concentration during the plant cycle. This diversity provides the molecular basis for explaining the pleiotropic effects of ethylene. The ACS family is a paradigm for the concept that gene redundancy provides biochemical and metabolic flexibility (Graur and Li 2000). The combinatorial complexity among the ACS family members is reminiscent of the auxin signaling apparatus, in which the multiple Aux/IAA, ARF, and TIR/AFB gene family members collaborate in the pleiotropic effects of auxin (Mockaitis and Estelle 2008).

The view presented in Figure 10 also indicates that the final biological outcome depends not only on the ACS family. ACC produced by the ACS family is processed by at least a 10-member ACO family, whose members act as monomers (Dong et al. 1992). Very little is known about its biochemical diversity and expression patterns during plant growth. Finally, ethylene is sensed by five different receptors that have the capacity to heterodimerize (Grefen et al. 2008) and form 5 homo- and 15 heterodimeric isoforms. The combinatorial interplay among the receptor isoforms in a spatial and temporal manner coupled with potential differences in their affinity for ethylene in vivo may provide the molecular basis for cell- and tissue-specific ethylene responses (Hall et al. 2007; Stepanova et al. 2008; Tao et al. 2008). Furthermore, differences in spatial and temporal expression of different receptor isoforms with different affinities for ethylene may act as a shield during its diffusion through neighboring cells to prevent undesired biological responses.

Conclusions and future directions:

Our results provide strong support for the hypothesis that the “Yang” ethylene biosynthetic pathway is the only route of ethylene production in Arabidopsis. The ethylene precursor ACC, may be a signaling molecule, regulating a variety of yet unidentified processes. Ethylene evolution is regulated by combinatorial interplay of the ACS polypeptides that serves as a rheostat controlling unique sets of genes that mediate the myriad ethylene-mediated processes during plant development. We still need to know and understand how the ACS symphony orchestra of each cell is coordinated with those in all the other Arabidopsis cells. Understanding how Arabidopsis coordinates the activity (output) of each ACS orchestra in each cell with those in the rest of the cells is a task for the future. More important, how the ethylene biosynthetic and signaling machineries are coordinated with the other hormonal and light networks to regulate plant growth is a major challenge for the future.

Knowledge of the temporal and spatial concentrations of substrates and intermediates of the ethylene biosynthetic pathway, together with the biochemical parameters of its enzymes promises to greatly advance our understanding of the regulation of the ACS gene family. The development of single cell biosensors using riboswitches (Lai 2003) and Biophotonics (West and Halas 2003) for detecting AdoMet, ACC, or even ethylene may provide some of the tools for achieving this goal. Furthermore, single cell protein profiling will advance our understanding of the ACS family. Determining the protein stability and the biochemical properties of the various homo- and heterodimeric forms will require new technological innovations. The mutants generated for this study may be valuable for analysis to elucidate the post-translational regulation of the various ACS polypeptides. Finally, establishing whether ACC is a primary plant growth regulator should be a major goal for the future because it will augment the repertoire of plant growth regulators used by plants to control their growth.

Acknowledgments

Note: Any questions regarding the mutants and transgenic lines that have been deposited to ABRC should be addressed to Atsunari Tsuchisaka at atsunari@nature.berkeley.edu.

We thank Rebecca Schwab and Detlef Weigel for designing the specific amiRNA sequences to inhibit ACS8 and ACS11 gene expression and for their generous gift of pRS300 plasmid containing the amiRNA backbone; Leor Eshed Williams for her advice and discussions regarding the amiRNA experiments; and Jennifer Fletcher and Pablo Leivar for useful discussions during the course of this work. This research was supported by the U.S. Department of Agriculture-Agricultural Research Service (CRIS 5335-21430-005-00D) (to A.T.).

Supporting information is available online at http://www.genetics.org/cgi/content/full/genetics.109.107102/DC1.

Microarray sequence data have been submitted to the NCBI gene expression data repository under the accession no. GSE14496.

All mutants and transgenic lines have been deposited to the Arabidopsis Biological Resource Center (ABRC).

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PERMALINK

A Combinatorial Interplay Among the 1-Aminocyclopropane-1-Carboxylate Isoforms Regulates Ethylene Biosynthesis in Arabidopsis thaliana

Atsunari Tsuchisaka

Guixia Yu

Hailing Jin

Jose M Alonso

Joseph R Ecker

Xiaoming Zhang

Shang Gao

Athanasios Theologis

Abstract

MATERIALS AND METHODS

Materials, strains, and transformation vectors:

Plant material and growth conditions:

Identification and characterization of T-DNA insertion alleles:

Construction of high order mutants:

Inactivation of the ACS8 and ACS11 genes with an amiR:

Complementation of the octuple (amiR) line:

Mapping the insertion sites of the amiR, amiR complementation and BiFC transgenes:

Complementation of the acs6-1 and acs9-1 mutants:

Ethylene production:

Histochemical GUS Assay:

RT–PCR analysis for ACS and flowering gene expression:

Bimolecular fluorescence complementation (BiFC) in planta:

Pathogen infection assay:

Global gene expression analysis:

RESULTS

Characterization of ACS T-DNA insertion mutants:

Phenotypic characterization:

Single and double mutants:

Seedling growth:

Figure 1.—

Growth of adult plants:

Ethylene production:

Flowering time:

Figure 2.—

Complementation of acs6-1 and acs9-1 mutants:

Construction of high order mutants:

pentuple mutants:

Plant growth:

Figure 3.—

Flowering time:

Ethylene production:

hexuple, heptuple, and octuple mutants:

Growth of mature plants:

Figure 4.—

Growth of seedlings:

Flowering time:

Figure 5.—

Ethylene production:

Embryo lethality in the octuple/amiR lines:

Figure 6.—

Specificity of the amiR:

Figure 7.—

Response to pathogens:

Figure 8.—

The ACS interactome map:

Figure 9.—

Global gene expression analysis:

DISCUSSION

Lessons from the single mutants:

Each ACS performs a specific and unique function:

Lessons from the high order mutants:

The ACS family members each perform a common essential function:

Phenotypic resistance to the loss of ethylene: the pentuple paradox:

The octuple Arabidopsis plant:

Ethylene functions as a transcriptional rheostat:

Highlights of the genome expression profiling analysis:

Ethylene and plant growth:

Ethylene and differential growth:

Hook formation:

Gravity sensing:

Ethylene and flowering time:

Ethylene and plant pathogens:

Is ACC a primary plant growth regulator?

“The Arabidopsis ACS symphony orchestra”:

Figure 10.—

Conclusions and future directions:

Acknowledgments

References