Step In Ethylene Biosynthesis Research Articles

Chemical Modification of 1-Aminocyclopropane Carboxylic Acid (ACC) Oxidase: Cysteine Mutational Analysis, Characterization, and Bioconjugation with a Nitroxide Spin Label.

1-Aminocyclopropane carboxylic acid oxidase (ACCO) catalyzes the last step of ethylene biosynthesis in plants. Although some sets of structures have been described, there are remaining questions on the active conformation of ACCO and in particular, on the conformation and potential flexibility of the C-terminal part of the enzyme. Several techniques based on the introduction of a probe through chemical modification of amino acid residues have been developed for determining the conformation and dynamics of proteins. Cysteine residues are recognized as convenient targets for selective chemical modification of proteins, thanks to their relatively low abundance in protein sequences and to their well-mastered chemical reactivity. ACCOs have generally 3 or 4 cysteine residues in their sequences. By a combination of approaches including directed mutagenesis, activity screening on cell extracts, biophysical and biochemical characterization of purified enzymes, we evaluated the effect of native cysteine replacement and that of insertion of cysteines on the C-terminal part in tomato ACCO. Moreover, we have chosen to use paramagnetic labels targeting cysteine residues to monitor potential conformational changes by electron paramagnetic resonance (EPR). Given the level of conservation of the cysteines in ACCO from different plants, this work provides an essential basis for the use of cysteine as probe-anchoring residues.

Arabidopsis ACC Oxidase 1 Coordinated by Multiple Signals Mediates Ethylene Biosynthesis and Is Involved in Root Development.

Ethylene regulates numerous aspects of plant growth and development. Multiple external and internal factors coordinate ethylene production in plant tissues. Transcriptional and post-translational regulations of ACC synthases (ACSs), which are key enzymes mediating a rate-limiting step in ethylene biosynthesis have been well characterized. However, the regulation and physiological roles of ACC oxidases (ACOs) that catalyze the final step of ethylene biosynthesis are largely unknown in Arabidopsis. Here, we show that Arabidopsis ACO1 exhibits a tissue-specific expression pattern that is regulated by multiple signals, and plays roles in the lateral root development in Arabidopsis. Histochemical analysis of the ACO1 promoter indicated that ACO1 expression was largely modulated by light and plant hormones in a tissue-specific manner. We demonstrated that point mutations in two E-box motifs on the ACO1 promoter reduce the light-regulated expression patterns of ACO1. The aco1-1 mutant showed reduced ethylene production in root tips compared to wild-type. In addition, aco1-1 displayed altered lateral root formation. Our results suggest that Arabidopsis ACO1 integrates various signals into the ethylene biosynthesis that is required for ACO1’s intrinsic roles in root physiology.

The pattern of 1-aminocyclopropane-1-carboxylate oxidase induction in the tomato leaf petiole abscission zone is independent of expression of the ribonuclease-LX-encoding LeLX gene.

The abscission of tomato leaves occurs in the petiole abscission zone, and its late stage includes two spatially divided processes: cell separation and programmed cell death (PCD). Both of these processes are regulated by ethylene. The last step in ethylene biosynthesis is conversion of 1‐aminocyclopropane‐1‐carboxylic acid to ethylene, which is catalysed by the enzyme 1‐aminocyclopropane‐1‐carboxylate oxidase (ACO); however, the location of ACO in the leaf petiole abscission zone is not known. The tomato gene LeLX encodes ribonuclease LX, which is a marker for PCD and is induced by ethylene during abscission, but its association with ACO has not been explored.In a tomato transgenic line 1‐7 with inhibited expression of LeLX showing delayed leaf abscission, the morphology and ultrastructure of the leaf petiole abscission zone was examined. In this zone of the cv.’VF36’ and of a transgenic line 1‐7, spatiotemporal differences in expression of LeACO1 and LeACO4 were analysed and ACO protein was detected immunohistochemically.In comparison to wild‐type plants, there were no obvious morphological and ultrastructural features in the abscission zone of plants of a transgenic line 1‐7 before and after abscission induction. LeACO1 expression was low before abscission induction, and increased 24 h after induction, although with no apparent spatial pattern. In contrast, LeACO4 was expressed before abscission induction, and its transcript level declined 24 h after induction on the distal side of the abscission zone fracture. In the LeLX‐inhibited transgenic line, there were no significant differences in LeACO1 and LeACO4 expression in the petiole abscission zone, in comparison to wild‐type plants. In addition, the ACO protein was immunolocalised to the vascular tissues that traverse the petiole abscission zone in plants of wild type and of a transgenic line 1‐7; and additionally in the plane of future abscission zone fracture of transgenic‐line plants.The results suggest temporal differential expression of the LeACO genes in tomato leaf petioles and vascular localisation of ACO1 protein. Additionally, the results indicate that expression of LeACO genes is not affected by suppression of the LeLX expression.

Reduced nitric oxide levels during drought stress promote drought tolerance in barley and is associated with elevated polyamine biosynthesis

Nitric oxide (NO) is a key messenger in plant stress responses but its exact role in drought response remains unclear. To investigate the role of NO in drought response we employed transgenic barley plants (UHb) overexpressing the barley non-symbiotic hemoglobin gene HvHb1 that oxidizes NO to NO3−. Reduced NO production under drought conditions in UHb plants was associated with increased drought tolerance. Since NO biosynthesis has been related to polyamine metabolism, we investigated whether the observed drought-related NO changes could involve polyamine pathway. UHb plants showed increases in total polyamines and in particular polyamines such as spermidine. These increases correlated with the accumulation of the amino acid precursors of polyamines and with the expression of specific polyamine biosynthesis genes. This suggests a potential interplay between NO and polyamine biosynthesis during drought response. Since ethylene has been linked to NO signaling and it is also related to polyamine metabolism, we explored this connection. In vivo ethylene measurement showed that UHb plants significantly decrease ethylene production and expression of aminocyclopropane-1-carboxylic acid synthase gene, the first committed step in ethylene biosynthesis compared with wild type. These data suggest a NO-ethylene influenced regulatory node in polyamine biosynthesis linked to drought tolerance/susceptibility in barley.

Regulation of the turnover of ACC synthases by phytohormones and heterodimerization in Arabidopsis.

Ethylene influences many aspects of plant growth and development. The biosynthesis of ethylene is highly regulated by a variety of internal and external cues. A key target of this regulation is 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACS), generally the rate-limiting step in ethylene biosynthesis, which is regulated both transcriptionally and post-transcriptionally. Prior studies have demonstrated that cytokinin and brassinosteroid (BR) act as regulatory inputs to elevate ethylene biosynthesis by increasing the stability of ACS proteins. Here, we demonstrate that several additional phytohormones also regulate ACS protein turnover. Abscisic acid, auxin, gibberellic acid, methyl jasmonic acid, and salicylic acid differentially regulate the stability of ACS proteins, with distinct effects on various isoforms. In addition, we demonstrate that heterodimerization influences the stability of ACS proteins. Heterodimerization between ACS isoforms from distinct subclades results in increased stability of the shorter-lived partner. Together, our study provides a comprehensive understanding of the roles of various phytohormones on ACS protein stability, which brings new insights into crosstalk between ethylene and other phytohormones, and a novel regulatory mechanism that controls ACS protein stability through a heterodimerization of ACS isoforms.

Assay Methods for ACS Activity and ACS Phosphorylation by MAP Kinases In Vitro and In Vivo.

Ethylene, a gaseous phytohormone, has profound effects on plant growth, development, and adaptation to the environment. Ethylene-regulated processes begin with the induction of ethylene biosynthesis. There are two key steps in ethylene biosynthesis. The first is the biosynthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) from S-Adenosyl-Methionine (SAM), a common precursor in many metabolic pathways, which is catalyzed by ACC synthase (ACS). The second is the oxidative cleavage of ACC to form ethylene under the action of ACC oxidase (ACO). ACC biosynthesis is the committing and generally the rate-limiting step in ethylene biosynthesis. As a result, characterizing the cellular ACS activity and understanding its regulation are important. In this chapter, we detail the methods used to measure, (1) the enzymatic activity of both recombinant and native ACS proteins, and (2) the phosphorylation of ACS protein by mitogen-activated protein kinases (MAPKs) in vivo and in vitro.

An ACC Oxidase Gene Essential for Cucumber Carpel Development

Sex determination in plants gives rise to unisexual flowers that facilitate outcrossing and enhance genetic diversity. In cucumber and melon, ethylene promotes carpel development and arrests stamen development. Five sex-determination genes have been identified, including four encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase that catalyzes the rate-limiting step in ethylene biosynthesis, and a transcription factor gene CmWIP1 that corresponds to the Mendelian locus gynoecious in melon and is a negative regulator of femaleness. ACC oxidase (ACO) converts ACC into ethylene; however, it remains elusive which ACO gene in the cucumber genome is critical for sex determination and how CmWIP1 represses development of female flowers. In this study, we discovered that mutation in an ACO gene, CsACO2, confers androecy in cucumber that bears only male flowers. The mutation disrupts the enzymatic activity of CsACO2, resulting in 50% less ethylene emission from shoot tips. CsACO2 was expressed in the carpel primordia and its expression overlapped with that of CsACS11 in female flowers at key stages for sex determination, presumably providing sufficient ethylene required for proper CsACS2 expression. CmACO3, the ortholog of CsACO2, showed a similar expression pattern in the carpel region, suggesting a conserved function of CsACO2/CmACO3. We demonstrated that CsWIP1, the ortholog of CmWIP1, could directly bind the promoter of CsACO2 and repress its expression. Taken together, we propose a presumably conserved regulatory module consisting of WIP1 transcription factor and ACO controls unisexual flower development in cucumber and melon.

Cloning and expression of the 1-aminocyclopropane-1-carboxylic oxidase gene from Agrostis stolonifera.

A gene encoding 1-aminocyclopropane-1-carboxylic oxidase (ACO), which catalyzes the terminal step in ethylene biosynthesis, was isolated from Agrostis stolonifera. The AsACO gene is composed of 975 bp, encoding 324 amino acids. Three exons interspersed by two introns form AsACO gDNA. A BLAST search of the nucleotide sequence revealed a high level of similarity (79-91%) between AsACO and ACO genes of other plants. A phylogenetic tree was constructed via BLAST in the NCBI, and revealed the highest homology with wheat TaACO. The calculated molecular mass and predicted isoelectric point of AsACO were 36.25 and 4.89 kDa, respectively. Analysis of subcellular localization revealed that AsACO is located in the nucleus and cytoplasm. The Fe(II)-binding cofactors and cosubstrate were identified, pertaining to the ACO family. The expression patterns of AsACO were determined by quantitative real time PCR. AsACO expression was highest in the stem, and was strongly up-regulated in response to ethephon, methyl jasmonate, salicylic acid, and cold temperature, but down-regulated in response to drought and NaCl treatment. The protein encoded by AsACO exhibited ACC oxidase activity in vitro. Taken together, these findings suggest that AsACO contains domains common to the ACO family, and is induced in response to exogenous hormones. Conversely, some abiotic stress conditions can inhibit AsACO expression.

Cadmium-induced ethylene production and responses in Arabidopsis thaliana rely on ACS2 and ACS6 gene expression.

BackgroundAnthropogenic activities cause metal pollution worldwide. Plants can absorb and accumulate these metals through their root system, inducing stress as a result of excess metal concentrations inside the plant. Ethylene is a regulator of multiple plant processes, and is affected by many biotic and abiotic stresses. Increased ethylene levels have been observed after exposure to excess metals but it remains unclear how the increased ethylene levels are achieved at the molecular level. In this study, the effects of cadmium (Cd) exposure on the production of ethylene and its precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and on the expression of the ACC Synthase (ACS) and ACC Oxidase (ACO) multigene families were investigated in Arabidopsis thaliana.ResultsIncreased ethylene release after Cd exposure was directly measurable in a system using rockwool-cultivated plants; enhanced levels of the ethylene precursor ACC together with higher mRNA levels of ethylene responsive genes: ACO2, ETR2 and ERF1 also indicated increased ethylene production in hydroponic culture. Regarding underlying mechanisms, it was found that the transcript levels of ACO2 and ACO4, the most abundantly expressed members of the ACO multigene family, were increased upon Cd exposure. ACC synthesis is the rate-limiting step in ethylene biosynthesis, and transcript levels of both ACS2 and ACS6 showed the highest increase and became the most abundant isoforms after Cd exposure, suggesting their importance in the Cd-induced increase of ethylene production.ConclusionsCadmium induced the biosynthesis of ACC and ethylene in Arabidopsis thaliana plants mainly via the increased expression of ACS2 and ACS6. This was confirmed in the acs2-1acs6-1 double knockout mutants, which showed a decreased ethylene production, positively affecting leaf biomass and resulting in a delayed induction of ethylene responsive gene expressions without significant differences in Cd contents between wild-type and mutant plants.

A wheat aminocyclopropane-1-carboxylate oxidase gene, TaACO1, negatively regulates salinity stress in Arabidopsis thaliana.

TaACO1 could catalyze ACC into ethylene in vitro. Constitutive expression of TaACO1 in Arabidopsis conferred salt sensitivity, and TaACO1 regulates salt stress mainly via the DREB1/CBF signal transduction pathway. Ethylene signaling plays essential roles in mediating plant responses to biotic and abiotic stresses, besides regulating plant growth and development. The roles of ethylene biosynthesis in abiotic stress, however, remain elusive. In this study, an aminocyclopropane-1-carboxylate oxidase gene, TaACO1, affecting the terminal step in ethylene biosynthesis, was isolated from a salt-tolerant bread wheat introgression line Shanrong No. 3 (SR3) and its effect on salt-stress response was examined. Purified recombinant protein of TaACO1 heterogenously expressed in Escherchia coli could catalyze ACC into ethylene in vitro. TaACO1 transcripts were down-regulated by salt, drought, oxidative stress and ABA. TaACO1-transgenic plants conferred salt sensitivity as judged from the seed germination, cotyledon greening and the relative root growth under salt stress. Constitutive expression of TaACO1 in Arabidopsis increased AtMYB15 expression and suppressed the expression of stress-responsive genes AtRAB18, AtCBF1 and AtCBF3. These findings are helpful in understanding the roles of ethylene biosynthesis in plant salt-stress response.

Transgenic alteration of ethylene biosynthesis increases grain yield in maize under field drought‐stress conditions

A transgenic gene-silencing approach was used to modulate the levels of ethylene biosynthesis in maize (Zea mays L.) and determine its effect on grain yield under drought stress in a comprehensive set of field trials. Commercially relevant transgenic events were created with down-regulated ACC synthases (ACSs), enzymes that catalyse the rate-limiting step in ethylene biosynthesis. These events had ethylene emission levels reduced approximately 50% compared with nontransgenic nulls. Multiple, independent transgenic hybrids and controls were tested in field trials at managed drought-stress and rain-fed locations throughout the US. Analysis of yield data indicated that transgenic events had significantly increased grain yield over the null comparators, with the best event having a 0.58Mg/ha (9.3bushel/acre) increase after a flowering period drought stress. A (genotype×transgene)×environment interaction existed among the events, highlighting the need to better understand the context in which the down-regulation of ACSs functions in maize. Analysis of secondary traits showed that there was a consistent decrease in the anthesis-silking interval and a concomitant increase in kernel number/ear in transgene-positive events versus nulls. Selected events were also field tested under a low-nitrogen treatment, and the best event was found to have a significant 0.44Mg/ha (7.1bushel/acre) yield increase. This set of extensive field evaluations demonstrated that down-regulating the ethylene biosynthetic pathway can improve the grain yield of maize under abiotic stress conditions.

1-Aminocyclopropane-1-carboxylic acid as a signalling molecule in plants

Ethylene influences many aspects of plant growth and development including germination, leaf and floral senescence and abscission, fruit ripening, and the response to abiotic and biotic stress. The pathways involved in the biosynthesis of and response to ethylene have been elucidated. The first committed and generally rate-limit- ing step in ethylene biosynthesis is the conversion of S-adenosyl-methionine to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase (ACS). This enzyme is encoded by a gene family in most plants and is subject to both transcriptional and post-transcriptional control in response to numerous endogenous and environmental cues. In addition to its well-described function as the immediate precursor of ethylene, recent studies suggest an alternative, non-canonical role for ACC. These studies found that in some contexts, chemical inhibitors of ethylene biosynthesis caused effects on root cell expansion that were not observed when ethylene perception or signalling was disrupted, suggesting that ACC, but not ethylene, played a role in these specific processes. Furthermore, muta- tion of all eight ACS genes in Arabidopsis was found to result in embryo lethality, in contrast to the relatively modest developmental effects of null mutants in the ethylene signalling components. This divergence suggests that ACC may play a role as a signalling molecule in plants. Here, we first review the ethylene biosynthesis pathway and then discuss these studies suggesting an independent role of ACC as a signalling molecule.

Cloning and analysis of cross-intron genomic gene encoding ACO in Paeonia suffruticosa Andr.

The last step of ethylene biosynthesis in higher plants is to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by ACC oxidase (ACO). In our investigation, a cross-introns genomic DNA gene encoding ACC oxidase, PsgACO, was isolated from Paeonia suffruticosa. It revealed that PsgACO (FJ855434) has 1281 bases, containing four exons and three introns, encoding 312 amino acids. The four exons stretched from 1 to 105, 217 to 434, 592 to 925 and 1000 to 1281 bases. A splicing junction sequence of all the exon-introns conformed to the GT-AG rule in the cross-intron genomic DNA sequence of the ACO gene. PsgACO showed high homology to many characterized ACC oxidases both at the nucleic acid and amino acid levels. As well, twelve amino acid residues were conserved among many ACOs from other species. A phylogenetic tree analysis indicated that the amino acid of ACOs is quite conserved among the different eudicots. The phylogenetic tree showed that both the tree peony and herbaceous peony are quite isolated taxa. Bioinformatic analysis showed that the molecular weight of ACO is 35.29 kD, with a theoretical pI of 5.25. It is a non-secrete, stable hydrophilic protein, located in the cytoplasm.

MiRNA regulation in the early development of barley seed

BackgroundDuring the early stages of seed development many genes are under dynamic regulation to ensure the proper differentiation and establishment of the tissue that will constitute the mature grain. To investigate how miRNA regulation contributes to this process in barley, a combination of small RNA and mRNA degradome analyses were used to identify miRNAs and their targets.ResultsOur analysis identified 84 known miRNAs and 7 new miRNAs together with 96 putative miRNA target genes regulated through a slicing mechanism in grain tissues during the first 15 days post anthesis. We also identified many potential miRNAs including several belonging to known miRNA families. Our data gave us evidence for an increase in miRNA-mediated regulation during the transition between pre-storage and storage phases. Potential miRNA targets were found in various signalling pathways including components of four phytohormone pathways (ABA, GA, auxin, ethylene) and the defence response to powdery mildew infection. Among the putative miRNA targets we identified were two essential genes controlling the GA response, a GA3oxidase1 and a homolog of the receptor GID1, and a homolog of the ACC oxidase which catalyses the last step of ethylene biosynthesis. We found that two MLA genes are potentially miRNA regulated, establishing a direct link between miRNAs and the R gene response.ConclusionOur dataset provides a useful source of information on miRNA regulation during the early development of cereal grains and our analysis suggests that miRNAs contribute to the control of development of the cereal grain, notably through the regulation of phytohormone response pathways.

ACC-OXIDASE IS THE RATE LIMITING STEP IN ETHYLENE BIOSYNTHESIS DURING POSTHARVEST STORAGE OF TOMATO

It has been well documented in literature that ACC-synthase is the rate limiting step in ethylene biosynthesis during climacteric ripening of tomato. This manuscript investigates how the ethylene metabolism further evolves during post harvest storage of tomato. The current data show how ethylene production declines after tomato fruit reaches the red stage. Metabolite screening revealed that ACC and MACC are highly abundant in red ripe and stored fruits. Surprisingly, although ACC-oxidase in-vitro activity was declining, ACC-synthase in vitro activity was increasing again during postharvest storage. These data suggest that the decline in ethylene production during storage must be regulated at the level of ACC-oxidase. The exact reason why ACC-synthase is still highly active during these stages remains peculiar and needs further investigation.

A Ser/Thr protein kinase phosphorylates MA-ACS1 (Musa acuminata 1-aminocyclopropane-1-carboxylic acid synthase 1) during banana fruit ripening

1-Aminocyclopropane-1-carboxylic acid synthase (ACS) catalyzes the rate-limiting step in ethylene biosynthesis during ripening. ACS isozymes are regulated both transcriptionally and post-translationally. However, in banana, an important climacteric fruit, little is known about post-translational regulation of ACS. Here, we report the post-translational modification of MA-ACS1 (Musa acuminata ACS1), a ripening inducible isozyme in the ACS family, which plays a key role in ethylene biosynthesis during banana fruit ripening. Immunoprecipitation analyses of phospholabeled protein extracts from banana fruit using affinity-purified anti-MA-ACS1 antibody have revealed phosphorylation of MA-ACS1, particularly in ripe fruit tissue. We have identified the induction of a 41-kDa protein kinase activity in pulp at the onset of ripening. The 41-kDa protein kinase has been identified as a putative protein kinase by MALDI-TOF/MS analysis. Biochemical analyses using partially purified protein kinase fraction from banana fruit have identified the protein kinase as a Ser/Thr family of protein kinase and its possible involvement in MA-ACS1 phosphorylation during ripening. In vitro phosphorylation analyses using synthetic peptides and site-directed mutagenized recombinant MA-ACS1 have revealed that serine 476 and 479 residues at the C-terminal region of MA-ACS1 are phosphorylated. Overall, this study provides important novel evidence for in vivo phosphorylation of MA-ACS1 at the molecular level as a possible mechanism of post-translational regulation of this key regulatory protein in ethylene signaling pathway in banana fruit during ripening.

1-Aminocyclopropane-1-carboxylic acid synthase 2 is phosphorylated by calcium-dependent protein kinase 1 during cotton fiber elongation

The reaction catalyzed by 1-aminocyclopropane-1-carboxylic acid synthase (ACS) is proposed to be the rate-limiting step in ethylene biosynthesis, which has been found as one of the most up-regulated metabolic pathways during cotton fiber development. However, the transcripts of the identified ACS genes did not increase in a similar manner as those of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) genes, implicating a possible post-transcriptional modification or regulatory mechanism. In this work, cotton ACS2 was shown to interact with Ca(2+)-dependent protein kinase 1 (CPK1). Bacterially expressed and purified recombinant ACS2 was phosphorylated by CPK1 in vitro and site-directed mutagenesis studies suggest that ACS2 S460 is a possible phosphorylation site for CPK1. Phosphorylated ACS2 significantly increased ACS activity, leading to elevated ethylene production. We thus speculated that CPK1 is involved in cotton fiber growth regulation by phosphorylating ACS2, which results in enhanced ethylene production in vitro.

Methyl jasmonate-induced defense responses are associated with elevation of 1-aminocyclopropane-1-carboxylate oxidase in Lycopersicon esculentum fruit

It has been known that methyl jasmonate (MeJA) interacts with ethylene to elicit resistance. In green mature tomato fruits (Lycopersicon esculentum cv. Lichun), 0.02mM MeJA increased the activity of 1-aminocyclopropane-1-carboxylate oxidase (ACO), and consequently influenced the last step of ethylene biosynthesis. Fruits treated with a combination of 0.02 MeJA and 0.02 α-aminoisobutyric acid (AIB, a competitive inhibitor of ACO) exhibited a lower ethylene production comparing to that by 0.02mM MeJA alone. The increased activities of defense enzymes and subsequent control of disease incidence caused by Botrytis cinerea with 0.2mM MeJA treatment was impaired by AIB as well. A close relationship (P<0.05) was found between the activity alterations of ACO and that of chitinase (CHI) and β-1,3-glucanase (GLU). In addition, this study further detected the changes of gene expressions and enzyme kinetics of ACO to different concentrations of MeJA. LeACO1 was found the principal member from the ACO gene family to respond to MeJA. Accumulation of LeACO1/3/4 transcripts followed the concentration pattern of MeJA treatments, where the largest elevations were reached by 0.2mM. For kinetic analysis, K(m) values of ACO stepped up during the experiment and reached the maximums at 0.2mM MeJA with ascending concentrations of treatments. V(max) exhibited a gradual increase from 3h to 24h, and the largest induction appeared with 1.0mM MeJA. The results suggested that ACO is involved in MeJA-induced resistance in tomato, and the concentration influence of MeJA on ACO was attributable to the variation of gene transcripts and enzymatic properties.

Dissecting the role of climacteric ethylene in kiwifruit (Actinidia chinensis) ripening using a 1-aminocyclopropane-1-carboxylic acid oxidase knockdown line

During climacteric fruit ripening, autocatalytic (Type II) ethylene production initiates a transcriptional cascade that controls the production of many important fruit quality traits including flavour production and softening. The last step in ethylene biosynthesis is the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene by the enzyme ACC oxidase (ACO). Ten independent kiwifruit (Actinidia chinensis) lines were generated targeting suppression of fruit ripening-related ACO genes and the fruit from one of these lines (TK2) did not produce detectable levels of climacteric ethylene. Ripening behaviour in a population of kiwifruit at harvest is asynchronous, so a short burst of exogenous ethylene was used to synchronize ripening in TK2 and control fruit. Following such a treatment, TK2 and control fruit softened to an 'eating-ripe' firmness. Control fruit produced climacteric ethylene and softened beyond eating-ripe by 5 d. In contrast, TK2 fruit maintained an eating-ripe firmness for >25 d and total volatile production was dramatically reduced. Application of continuous exogenous ethylene to the ripening-arrested TK2 fruit re-initiated fruit softening and typical ripe fruit volatiles were detected. A 17 500 gene microarray identified 401 genes that changed after ethylene treatment, including a polygalacturonase and a pectate lyase involved in cell wall breakdown, and a quinone oxidoreductase potentially involved in volatile production. Many of the gene changes were consistent with the softening and flavour changes observed after ethylene treatment. However, a surprisingly large number of genes of unknown function were also observed, which could account for the unique flavour and textural properties of ripe kiwifruit.

An insight into the sequential, structural and phylogenetic properties of banana 1-aminocyclopropane-1-carboxylate synthase 1 and study of its interaction with pyridoxal-5′-phosphate and aminoethoxyvinylglycine

In banana, ethylene production for ripening is accompanied by a dramatic increase in 1-aminocyclopropane-1-carboxylate (ACC) content, transcript level of Musa acuminata ACC synthase 1 (MA-ACS1) and the enzymatic activity of ACC synthase 1 at the onset of the climacteric period. MA-ACS1 catalyses the conversion of S-adenosyl-L-methionine (SAM) to ACC, the key regulatory step in ethylene biosynthesis. Multiple sequence alignments of 1-aminocyclopropane-1-carboxylate synthase (ACS) amino acid sequences based on database searches have indicated that MA-ACS1 is a highly conserved protein across the plant kingdom. This report describes an in silico analysis to provide the first important insightful information about the sequential, structural and phylogenetic characteristics of MA-ACS1. The three-dimensional structure of MA-ACS1, constructed based on homology modelling, in combination with the available data enabled a comparative mechanistic analysis of MA-ACS1 to explain the catalytic roles of the conserved and non-conserved active site residues. We have further demonstrated that, as in apple and tomato, banana- ACS1 (MA-ACS1) forms a homodimer and a complex with cofactor pyridoxal-5'-phosphate (PLP) and inhibitor aminoethoxyvinylglycine (AVG). We have also predicted that the residues from the PLP-binding pocket, essential for ligand binding, are mostly conserved across the MA-ACS1 structure and the competitive inhibitor AVG binds at a location adjacent to PLP.