Functional stress responses in Glaucophyta: Evidence of ethylene and abscisic acid functions in Cyanophora paradoxa.

Glaucophytes, an enigmatic group of freshwater algae, occupy a pivotal position within the Archaeplastida, providing insights into the early evolutionary history of plastids and their host cells. These algae possess unique plastids, known as cyanelles that retain certain ancestral features, enabling a better understanding of the plastid transition from cyanobacteria. In this study, we investigated the role of ethylene, a potent hormone used by land plants to coordinate stress responses, in the glaucophyte alga Cyanophora paradoxa. We demonstrate that C. paradoxa produces gaseous ethylene when supplied with exogenous 1-aminocyclopropane-1-carboxylic acid (ACC), the ethylene precursor in land plants. In addition, we show that cells produce ethylene natively in response to abiotic stress, and that another plant hormone, abscisic acid (ABA), interferes with ethylene synthesis from exogenously supplied ACC, while positively regulating reactive oxygen species (ROS) accumulation. ROS synthesis also occurred following abiotic stress and ACC treatment, possibly acting as a second messenger in stress responses. A physiological response of C. paradoxa to ACC treatment is growth inhibition. Using transcriptomics, we reveal that ACC treatment induces the upregulation of senescence-associated proteases, consistent with the observation of growth inhibition. This is the first report of hormone usage in a glaucophyte alga, extending our understanding of hormone-mediated stress response coordination into the Glaucophyta, with implications for the evolution of signaling modalities across Archaeplastida.

On '1-Aminocyclopropanecarboxylate synthase, a key enzyme in ethylene biosynthesis' by Yeong-Biau Yu, Douglas O.Adams and Shang Fa Yang.

The significance of the paper by Yu et al. (1979) is discussed in the context of the long history of ethylene as a plant growth regulator. By launching the era of molecular analysis and biotechnological exploitation, this research made a vital contribution to crop production and quality.

Multigenic regulation in the ethylene biosynthesis pathway during coffee flowering.

Ethylene regulates different aspects of the plant's life cycle, such as flowering, and acts as a defense signal in response to environmental stresses. Changes induced by water deficit (WD) in gene expression of the main enzymes involved in ethylene biosynthesis, 1-aminocyclopropane-1-carboxylic acid synthase (ACS) and oxidase (ACO), are frequently reported in plants. In this study, coffee (Coffea arabica) ACS and ACO family genes were characterized and their expression profiles were analyzed in leaves, roots, flower buds, and open flowers from plants under well-watered (WW) and water deficit (WD) conditions. Three new ACS genes were identified. Water deficit did not affect ACS expression in roots, however soil drying strongly downregulated ACO expression, indicating a transcriptional constraint in the biosynthesis pathway during the drought that can suppress ethylene production in roots. In floral buds, ACO expression is water-independent, suggesting a higher mechanism of control in reproductive organs during the final flowering stages. Leaves may be the main sites for ethylene precursor (1-aminocyclopropane-1-carboxylic acid, ACC) production in the shoot under well-watered conditions, contributing to an increase in the ethylene levels required for anthesis. Given these results, we suggest a possible regulatory mechanism for the ethylene biosynthesis pathway associated with coffee flowering with gene regulation in leaves being a key point in ethylene production and ACO genes play a major regulatory role in roots and the shoots. This mechanism may constitute a regulatory model for flowering in other woody species. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01235-y.

Stablization of ACOs by NatB mediated N-terminal acetylation is required for ethylene homeostasis.

N-terminal acetylation (NTA) is a highly abundant protein modification catalyzed by N-terminal acetyltransferases (NATs) in eukaryotes. However, the plant NATs and their biological functions have been poorly explored. Here we reveal that loss of function of CKRC3 and NBC-1, the auxiliary subunit (Naa25) and catalytic subunit (Naa20) of Arabidopsis NatB, respectively, led to defects in skotomorphogenesis and triple responses of ethylene. Proteome profiling and WB test revealed that the 1-amincyclopropane-1-carboxylate oxidase (ACO, catalyzing the last step of ethylene biosynthesis pathway) activity was significantly down-regulated in natb mutants, leading to reduced endogenous ethylene content. The defective phenotypes could be fully rescued by application of exogenous ethylene, but less by its precursor ACC. The present results reveal a previously unknown regulation mechanism at the co-translational protein level for ethylene homeostasis, in which the NatB-mediated NTA of ACOs render them an intracellular stability to maintain ethylene homeostasis for normal growth and responses.

Low storage temperature for tree ripe mangoes under controlled atmospheres with elevated CO2 concentrations.

BACKGROUND: Tree-ripe mangoes are of a better quality than the more commonly marketed mature-green fruit. However, the postharvest life of tree-ripe mangoes at the chilling threshold temperature for mature-green fruit of 12 °C is insufficient to allow long distance transport for international marketing. Because the chilling sensitivity often decreases as fruit ripen, lower temperatures (5 and 8 °C) in combination with a controlled atmosphere of 5 kPa O2 plus 10 or 25 kPa CO2 were tested to determine whether the quality of tree-ripe mangoes could be maintained longer without chilling injury (CI). RESULTS: Tree-ripe 'Tommy Atkins' and 'Keitt' mangoes were stored for 14 or 21 days, respectively, in air or controlled atmosphere (CA) at 5 or 8 °C. Respiration rates were below 10 mL kg-1 h-1 during CA storage and increased three-fold during a 3-day shelf life period at 20 °C. Ethanol synthesis of fruit stored in 25 kPa CO2 , but not 10 kPa CO2 , increased during storage and remained high during shelf life, indicating physiological stress. Elevated electrolyte leakage and 1-aminocyclopropane-1-carboxylic acid concentrations in both cultivars stored in 25 kPa CO2 also indicated that mesocarp tissues were injured by the higher CO2 level. No CI symptoms were observed in air or CA at either 5 or 8 °C. CONCLUSION: Storage of tree-ripe mangoes in 5 kPa O2 plus 10 kPa CO2 at either 5 or 8 °C best maintained the quality of Tommy Atkins and Keitt fruit for 14 or 21 days, respectively, without evidence of either atmosphere injury or CI. © 2020 Society of Chemical Industry.

Overexpression of an ethylene-forming ACC oxidase (ACO) gene precedes the Minute Hilum seed coat phenotype in Glycine max.

BACKGROUND: To elucidate features of seed development, we investigated the transcriptome of a soybean isoline from the germplasm collection that contained an introgressed allele known as minute hilum (mi) which confers a smaller hilum region where the seed attaches to the pod and also results in seed coat cracking surrounding the hilum region. RESULTS: RNAs were extracted from immature seed from an extended hilum region (i.e., the hilum and a small ring of tissue surrounding the hilum in which the cracks form) at three different developmental stages:10-25, 25-50 and 50-100 mg seed fresh weight in two independent replicates for each stage. The transcriptomes of these samples from both the Clark isoline containing the mi allele (PI 547628, UC413, ii R t mi G), and its recurrent Clark 63 parent isoline (PI 548532, UC7, ii R T Mi g), which was used for six generations of backcrossing, were compared for differential expression of 88,648 Glyma models of the soybean genome Wm82.a2. The RNA sequence data obtained from the 12 cDNA libraries were subjected to padj value < 0.05 and at least two-fold expression differences to select with confidence genes differentially expressed in the hilum-containing tissue of the seed coat between the two lines. Glyma.09G008400 annotated as encoding an ethylene forming enzyme, ACC oxidase (ACO), was found to be highly overexpressed in the mi hilum region at 165 RPKMs (reads per kilobase per million mapped reads) compared to the standard line at just 0.03 RPKMs. Evidence of changes in expression of genes downstream of the ethylene pathway included those involved in auxin and gibberellin hormone action and extensive differences in expression of cell wall protein genes. These changes are postulated to determine the restricted hilum size and cracking phenotypes. CONCLUSIONS: We present transcriptome and phenotypic evidence that substantially higher expression of an ethylene-forming ACO gene likely shifts hormone balance and sets in motion downstream changes resulting in a smaller hilum phenotype and the cracks observed in the minute hilum (mi) isoline as compared to its recurrent parent.

The TuMYB46L-TuACO3 module regulates ethylene biosynthesis in einkorn wheat defense to powdery mildew.

Powdery mildew disease, elicited by the obligate fungal pathogen Blumeria graminis f.sp. tritici (Bgt), causes widespread yield losses in global wheat crop. However, the molecular mechanisms governing wheat defense to Bgt are still not well understood. Here we found that TuACO3, encoding the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase functioning in ethylene (ET) biosynthesis, was induced by Bgt infection of the einkorn wheat Triticum urartu, which was accompanied by increased ET content. Silencing TuACO3 decreased ET production and compromised wheat defense to Bgt, whereas both processes were enhanced in the transgenic wheat overexpressing TuACO3. TuMYB46L, phylogenetically related to Arabidopsis MYB transcription factor AtMYB46, was found to bind to the TuACO3 promoter region in yeast-one-hybrid and EMSA experiments. TuMYB46L expression decreased rapidly following Bgt infection. Silencing TuMYB46L promoted ET content and Bgt defense, but the reverse was observed when TuMYB46L was overexpressed. Hence, decreased expression of TuMYB46L permits elevated function of TuACO3 in ET biosynthesis in Bgt-infected wheat. The TuMYB46L-TuACO3 module regulates ET biosynthesis to promote einkorn wheat defense against Bgt. Furthermore, we found four chitinase genes acting downstream of the TuMYB46L-TuACO3 module. Collectively, our data shed a new light on the molecular mechanisms underlying wheat defense to Bgt.

Dendrobium orchids carrying antisense ACC oxidase: small changes in flower morphology and a delay of bud abortion, flower senescence, and abscission of flowers.

Four Dendrobium Sonia 'Earsakul' lines were generated by insertion of one, two or three antisense copies of a Carica papaya gene encoding 1-aminocyclopropane-1-carboxylic acid oxidase (CpACO). Whole vegetative plants of the transgenic lines showed about 50% of the basal ethylene production rate, while the increase in ethylene production in floral buds during opening and open flowers prior to visible senescence was delayed. Detailed analysis of more than 100 parameters in flowering plants showed no effect of antisense ACO on plant morphology and coloration, except for shorter length and width of some of the sepals and petals. In intact plants the water-soaking of floral buds as well as bud abscission were delayed by ACO antisense, as was the time to senescence of open flowers. Pollen viability and pollen tube growth were not affected in the transgenic lines. In cut inflorescences placed in water, bud yellowing, bud water soaking, and bud abscission were considerably delayed by the antisense construct, while the life span of open flowers were increased and abscission of open flowers were delayed. It is concluded that the reduction of ACO activity affected the shape of some petals/sepals and delayed the abortion in floral buds, and the senescence and abscission of open flowers.

Ethylene Biosynthesis Inhibition Combined with Cyanide Degradation Confer Resistance to Quinclorac in Echinochloa crus-galli var. mitis.

Echinochloa crus-galli var. mitis has rarely been reported for herbicide resistance, and no case of quinclorac resistance has been reported so far. Synthetic auxin-type herbicide quinclorac is used extensively to control rice weeds worldwide. A long history of using quinclorac in Chinese rice fields escalated the resistance in E. crus-galli var. mitis against this herbicide. Bioassays in Petri plates and pots exhibited four biotypes that evolved into resistance to quinclorac ranking as JS01-R > AH01-R > JS02-R > JX01-R from three provinces of China. Ethylene production in these biotypes was negatively correlated with resistance level and positively correlated with growth inhibition. Determination of the related ethylene response pathway exhibited resistance in biotypes that recorded a decline in 1-aminocyclopropane-1-carboxylic acid (ACC) content, ACC synthase oxidase activities, and less inducible ACS and ACO genes expressions than the susceptible biotype, suggesting that there was a positive correlation between quinclorac resistance and ethylene biosynthesis inhibition. Cyanides produced during the ethylene biosynthesis pathway mainly degraded by the activity of ß-cyanoalanine synthase (ß-CAS). Resistant biotypes exhibited higher ß-CAS activity than the susceptible ones. Nucleotide changes were found in the EcCAS gene of resistant biotypes as compared to sensitive ones that caused three amino acid substitutions (Asn-105-Lys, Gln-195-Glu, and Gly-298-Val), resulting in alteration of enzyme structure, increased binding residues in the active site with its cofactor, and decreased binding free energy; hence, its activity was higher in resistant biotypes. Moreover, these mutations increased the structural stability of the enzyme. In view of the positive correlation between ethylene biosynthesis inhibition and cyanide degradation with resistance level, it is concluded that the alteration in ethylene response pathway or at least variation in ACC synthase and ACC oxidase enzyme activities-due to less relative expression of ACS and ACO genes and enhanced ß-CAS activity, as well as mutation and increased relative expression of EcCAS gene-can be considered as a probable mechanism of quinclorac resistance in E. crus-galli var. mitis.

Methylobacterium oryzae CBMB20 influences photosynthetic traits, volatile emission and ethylene metabolism in Oryza sativa genotypes grown in salt stress conditions.

MAIN CONCLUSION: Inoculation of endophytic Methylobacterium oryzae CBMB20 in salt-stressed rice plants improves photosynthesis and reduces stress volatile emissions due to mellowing of ethylene-dependent responses and activating vacuolar H+-ATPase. The objective of this study was to analyze the impact of ACC (1-aminocyclopropane-1-carboxylate) deaminase-producing Methylobacterium oryzae CBMB20 in acclimation of plant to salt stress by controlling photosynthetic characteristics and volatile emission in salt-sensitive (IR29) and moderately salt-resistant (FL478) rice (Oryza sativa L.) cultivars. Saline levels of 50 mM and 100 mM NaCl with and without bacteria inoculation were applied, and the temporal changes in stress response and salinity resistance were assessed by monitoring photosynthetic characteristics, ACC accumulation, ACC oxidase activity (ACO), vacuolar H+ ATPase activity, and volatile organic compound (VOC) emissions. Salt stress considerably reduced photosynthetic rate, stomatal conductance, PSII efficiency and vacuolar H+ ATPase activity, but it increased ACC accumulation, ACO activity, green leaf volatiles, mono- and sesquiterpenes, and other stress volatiles. These responses were enhanced with increasing salt stress and time. However, rice cultivars treated with CBMB20 showed improved plant vacuolar H+ ATPase activity, photosynthetic characteristics and decreased ACC accumulation, ACO activity and VOC emission. The bacteria-dependent changes were greater in the IR29 cultivar. These results indicate that decreasing photosynthesis and vacuolar H+ ATPase activity rates and increasing VOC emission rates in response to high-salinity stress were effectively mitigated by M. oryzae CBMB20 inoculation.

Transient induction of a subset of ethylene biosynthesis genes is potentially involved in regulation of grapevine bud dormancy release.

KEY MESSAGE: Transient increases in ethylene biosynthesis, achieved by tight regulation of transcription of specific ACC oxidase and ACC synthase genes, play a role in activation of grapevine bud dormancy release. The molecular mechanisms regulating dormancy release in grapevine buds are as yet unclear. It has been hypothesized that its core involves perturbation of respiration which induces an interplay between ethylene and ABA metabolism that removes repression and allows regrowth. Roles for hypoxia and ABA metabolism in this process have been previously supported. The potential involvement of ethylene biosynthesis in regulation of dormancy release, which has received little attention so far, is now explored. Our results indicate that (1) ethylene biosynthesis is induced by hydrogen cyanamide (HC) and azide (AZ), known artificial stimuli of dormancy release, (2) inhibitors of ethylene biosynthesis and signalling antagonize dormancy release by HC/AZ treatments, (3) ethylene application induces dormancy release, (4) there are two sets of bud-expressed ethylene biosynthesis genes which are differentially regulated, (5) only one set is transiently upregulated by HC/AZ and during the natural dormancy cycle, concomitant with changes in ethylene levels, and (6) levels of ACC oxidase transcripts and ethylene sharply decrease during natural dormancy release, whereas ACC accumulates. Given these results, we propose that transient increases in ethylene biosynthesis prior to dormancy release, achieved primarily by regulation of transcription of specific ACC oxidase genes, play a role in activation of dormancy release.

Functional characterization of 1-aminocyclopropane-1-carboxylic acid oxidase gene in Arabidopsis thaliana and its potential in providing flood tolerance.

Ethylene is a phytohormone that has gained importance through its role in stress tolerance and fruit ripening. In our study we evaluated the functional potential of the enzyme involved in ethylene biosynthesis of plants called ACC (aminocyclopropane-1-carboxylic acid) oxidase which converts precursor ACC to ethylene. Studies on ethylene have proven that it is effective in improving the flood tolerance in plants. Thus our goal was to understand the potential of ACC oxidase gene overexpression in providing flood tolerance in transgenic plants. ACC oxidase gene was PCR amplified and inserted into the pBINmgfp5-er vector, under the control of a constitutive Cauliflower Mosaic Virus promoter. GV101 strain of Agrobacterium tumefaciens containing recombinant pBINmgfp5-er vector (referred herein as pBIN-ACC) was used for plant transformation by the 'floral dip' method. The transformants were identified through kanamycin selection and grown till T3 (third transgenic) generation. The flood tolerance was assessed by placing both control and transgenic plants on deep plastic trays filled with tap water that covered the soil surface. Our result shows that wild-type Arabidopsis could not survive more than 20 days under flooding while the transgenic lines survived 35 days, suggesting development of flood tolerance with overexpression of ACC oxidase. Further molecular studies should be done to elucidate the role and pathways of ACC oxidase and other phytohormones involved in the development of flood adaptation.

Regulation of ethylene-related gene expression by indole-3-acetic acid and 4-chloroindole-3-acetic acid in relation to pea fruit and seed development.

In pea, the auxins 4-chloroindole-3-acetic acid (4-Cl-IAA) and indole-3-acetic acid (IAA) occur naturally; however, only 4-Cl-IAA stimulates pericarp growth and gibberellin (GA) biosynthesis, and inhibits the ethylene response in deseeded ovaries (pericarps), mimicking the presence of seeds. Expression of ovary ethylene biosynthesis genes was regulated similarly in most cases by the presence of 4-Cl-IAA or seeds. PsACS1 [which encodes an enzyme that synthesizes 1-aminocyclopropane-1-carboxylic acid (ACC)] transcript abundance was high in pericarp tissue adjacent to developing seeds following pollination. ACC accumulation in 4-Cl-IAA-treated deseeded pericarps was driven by high PsASC1 expression (1800-fold). 4-Cl-IAA, but not IAA, also suppressed the pericarp transcript levels of PsACS4. 4-Cl-IAA increased PsACO1 and decreased PsACO2 and PsACO3 expression (enzymes that convert ACC to ethylene) but did not change ACO enzyme activity. Increased ethylene was countered by a 4-Cl-IAA-specific decrease in ethylene responsiveness potentially via modulation of pericarp ethylene receptor and signaling gene expression. This pattern did not occur in IAA-treated pericarps. Overall, the effect of 4-Cl-IAA and IAA on ethylene biosynthesis gene expression generally explains the ethylene evolution patterns, and their effects on GA biosynthesis and ethylene signaling gene expression explain the tissue response patterns in young pea ovaries.

Characterization of Cu(II)-reconstituted ACC Oxidase using experimental and theoretical approaches.

1-Aminocyclopropane-1-carboxylic acid oxidase (ACCO) is a non heme iron(II) containing enzyme that catalyzes the final step of the ethylene biosynthesis in plants. The iron(II) ion is bound in a facial triad composed of two histidines and one aspartate (H177, D179 and H234). Several active site variants were generated to provide alternate binding motifs and the enzymes were reconstituted with copper(II). Continuous wave (cw) and pulsed Electron Paramagnetic Resonance (EPR) spectroscopies as well as Density Functional Theory (DFT) calculations were performed and models for the copper(II) binding sites were deduced. In all investigated enzymes, the copper ion is equatorially coordinated by the two histidine residues (H177 and H234) and probably two water molecules. The copper-containing enzymes are inactive, even when hydrogen peroxide is used in peroxide shunt approach. EPR experiments and DFT calculations were undertaken to investigate substrate's (ACC) binding on the copper ion and the results were used to rationalize the lack of copper-mediated activity.

Gene expression analyses in tomato near isogenic lines provide evidence for ethylene and abscisic acid biosynthesis fine-tuning during arbuscular mycorrhiza development.

Plant responses to the environment and microorganisms, including arbuscular mycorrhizal fungi, involve complex hormonal interactions. It is known that abscisic acid (ABA) and ethylene may be involved in the regulation of arbuscular mycorrhiza (AM) and that part of the detrimental effects of ABA deficiency in plants is due to ethylene overproduction. In this study, we aimed to determine whether the low susceptibility to mycorrhizal colonization in ABA-deficient mutants is due to high levels of ethylene and whether AM development is associated with changes in the steady-state levels of transcripts of genes involved in the biosynthesis of ethylene and ABA. For that, tomato (Solanum lycopersicum) ethylene overproducer epinastic (epi) mutant and the ABA-deficient notabilis (not) and sitiens (sit) mutants, in the same Micro-Tom (MT) genetic background, were inoculated with Rhizophagus clarus, and treated with the ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG). The development of AM, as well as the steady-state levels of transcripts involved in ethylene (LeACS2, LeACO1 and LeACO4) and ABA (LeNCED) biosynthesis, was determined. The intraradical colonization in epi, not and sit mutants was significantly reduced compared to MT. The epi mutant completely restored the mycorrhizal colonization to the levels of MT with the application of 10 µM of AVG, probably due to the inhibition of the ACC synthase gene expression. The steady-state levels of LeACS2 and LeACO4 transcripts were induced in mycorrhizal roots of MT, whereas the steady-state levels of LeACO1 and LeACO4 transcripts were significantly induced in sit, and the steady-state levels of LeNCED transcripts were significantly induced in all genotypes and in mycorrhizal roots of epi mutants treated with AVG. The reduced mycorrhizal colonization in sit mutants seems not to be limited by ethylene production via ACC oxidase regulation. Both ethylene overproduction and ABA deficiency impaired AM fungal colonization in tomato roots, indicating that, besides hormonal interactions, a fine-tuning of each hormone level is required for AM development.

ACC oxidase and miRNA 159a, and their involvement in fresh fruit bunch yield (FFB) via sex ratio determination in oil palm.

Oil palm (Elaeis guineesis Jacq.) is the most productive oil-bearing crop, yielding more oil per area than any other oil-bearing crops. However, there are still efforts to improve oil palm yield, in order to serve consumer and manufacturer demand. Oil palm produces female and male inflorescences in an alternating cycle. So, high sex ratio (SR), the ratio of female inflorescences to the total inflorescences, is a favorable trait in term of increasing yields in oil palm. This study aims to understand the genetic control for SR related traits, such as fresh fruit bunch yield (FFB), by characterizing genes at FFB quantitative trait loci (QTLs) on linkage 10 (chromosome 6) and linkage 15 (chromosome 10). Published oil palm sequences at the FFB QTLs were used to develop gene-based and simple sequence repeat (SSR) markers. We used the multiple QTL analysis model (MQM) to characterize the relationship of new markers with the SR traits in the oil palm population. The RNA expression of the most linked QTL genes was also evaluated in various tissues of oil palm. We identified EgACCO1 (encoding aminocyclopropane carboxylate (ACC) oxidase) at chromosome 10 and EgmiR159a (microRNA 159a) at chromosome 6 to be the most linked QTL genes or determinants for FFB yield and/or female inflorescence number with a phenotype variance explained (PVE) from 10.4 to 15 % and suggest that these play the important roles in sex determination and differentiation in oil palm. The strongest expression of EgACCO1 and the predicted precursor of EgmiR159a was found in ovaries and, to a lesser extent, fruit development. In addition, highly normalized expression of EgmiR159a was found in female flowers. In summary, the QTL analysis and the RNA expression reveal that EgACCO1 and EgmiR159a are the potential genetic factors involved in female flower determination and hence would affect yield in oil palm. However, to clarify how these genetic factors regulate female flower determination, more investigation of their down regulation or target may be essential. Additionally, if more sex determination genes controlled by plant hormones are identified, it may possible to reveal a crosstalk of sex determination genes with hormones and environment factors.

Identification and active site analysis of the 1-aminocyclopropane-1-carboxylic acid oxidase catalysing the synthesis of ethylene in Agaricus bisporus.

BACKGROUND: 1-Aminocyclopropane-1-carboxylate oxidase (ACO) is a key enzyme that catalyses the final step in the biosynthesis of the plant hormone ethylene. Recently, the first ACO homologue gene was isolated in Agaricus bisporus, whereas information concerning the nature of the ethylene-forming activity of this mushroom ACO is currently lacking. METHODS: Recombinant ACO from A. bisporus (Ab-ACO) was purified and characterised for the first time. Molecular modelling combined with site-directed mutagenesis and kinetic and spectral analysis were used to investigate the property of Ab-ACO. RESULTS: Ab-ACO has eight amino acid residues that are conserved in the Fe (II) ascorbate family of dioxygenases, including four catalytic residues in the active site, but Ab-ACO lacks a key residue, S289. In comparison to plant ACOs, Ab-ACO requires ACC and Fe (II) but does not require ascorbate. In addition, Ab-ACO had relatively low activity and was completely dependent on bicarbonate, which could be ascribed to the replacement of S289 by G289. Moreover, the ferrous ion could induce a change in the tertiary, but not the secondary, structure of Ab-ACO. CONCLUSIONS: These results provide crucial experimental support for the ability of Ab-ACO to catalyse ethylene formation in a similar manner to that of plant ACOs, but there are differences between the biochemical and catalytic characteristics of Ab-ACO and plant ACOs. GENERAL SIGNIFICANCE: This work enhances the understanding of the ethylene biosynthesis pathways in fungi and could promote profound physiological research of the role of ethylene in the regulation of mushroom growth and development.

Cytokinins regulate spatially-specific ethylene production to control root growth in Arabidopsis.

The two principal growth regulators cytokinins and ethylene are known to interact in the regulation of plant growth. However, information about underlying molecular mechanism and positional specificity of the cytokinin/ethylene crosstalk in root growth control is scarce. We have identified spatial specificity of cytokinin-regulated root elongation and root apical meristem (RAM) size, both of which we demonstrate to be dependent on ethylene biosynthesis. Upregulation of the cytokinin biosynthetic gene ISOPENTENYLTRANSFERASE (IPT) in proximal and peripheral tissues leads to both root and RAM shortening. In contrast, IPT activation in distal and inner tissues reduces RAM size while leaving the root length comparable to mock-treated controls. We show that cytokinins regulate two steps specific to ethylene biosynthesis, the production of ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) by ACC SYNTHASEs (ACSs), and its conversion to ethylene by ACC OXIDASEs (ACOs). We describe cytokinin- and ethylene-specific regulation controlling the activity of ACSs and ACOs that are spatially discrete along both proximo/distal and radial root axes. Using direct ethylene measurements, we identify ACO2, ACO3 and ACO4 as being responsible for ethylene biosynthesis and the ethylene-regulated root and RAM shortening in cytokinin-treated Arabidopsis. Direct interaction between ARABIDOPSIS RESPONSE REGULATOR 2 (ARR2), a member of the multistep phosphorelay cascade and the C-terminal portion of ETHYLENE INSENSITIVE 2 (EIN2-C), a key regulator of canonical ethylene signaling is involved in the cytokinin-induced, ethylene-mediated control of ACO4. We propose tight cooperation between cytokinin and ethylene signaling in the spatial-specific regulation of ethylene biosynthesis as a key aspect of hormonal control over root growth.

Functional mechanism study of the allelochemical myrigalone A identifies a group of ultrapotent inhibitors of ethylene biosynthesis in plants.

Allelochemicals represent a class of natural products released by plants as root, leaf, and fruit exudates that interfere with the growth and survival of neighboring plants. Understanding how allelochemicals function to regulate plant responses may provide valuable new approaches to better control plant function. One such allelochemical, Myrigalone A (MyA) produced by Myrica gale, inhibits seed germination and seedling growth through an unknown mechanism. Here, we investigate MyA using the tractable model Dictyostelium discoideum and reveal that its activity depends on the conserved homolog of the plant ethylene synthesis protein 1-aminocyclopropane-1-carboxylic acid oxidase (ACO). Furthermore, in silico modeling predicts the direct binding of MyA to ACO within the catalytic pocket. In D. discoideum, ablation of ACO mimics the MyA-dependent developmental delay, which is partially restored by exogenous ethylene, and MyA reduces ethylene production. In Arabidopsis thaliana, MyA treatment delays seed germination, and this effect is rescued by exogenous ethylene. It also mimics the effect of established ACO inhibitors on root and hypocotyl extension, blocks ethylene-dependent root hair production, and reduces ethylene production. Finally, in silico binding analyses identify a range of highly potent ethylene inhibitors that block ethylene-dependent response and reduce ethylene production in Arabidopsis. Thus, we demonstrate a molecular mechanism by which the allelochemical MyA reduces ethylene biosynthesis and identify a range of ultrapotent inhibitors of ethylene-regulated responses.

[Effect of ACC oxidase gene AhACOs on salt tolerance of peanut].

ACC oxidase (ACO) is one of the key enzymes that catalyze the synthesis of ethylene. Ethylene is involved in salt stress response in plants, and salt stress seriously affects the yield of peanut. In this study, AhACO genes were cloned and their functions were investigated with the aim to explore the biological function of AhACOs in salt stress response, and to provide genetic resources for the breeding of salt-tolerant varieties of peanut. AhACO1 and AhACO2 were amplified from the cDNA of salt-tolerant peanut mutant M29, respectively, and cloned into the plant expression vector pCAMBIA super1300. The recombinant plasmid was transformed into Huayu22 by pollen tube injection mediated by Agrobacterium tumefaciens. After harvest, the small slice cotyledon was separated from the kernel, and the positive seeds were screened by PCR. The expression of AhACO genes was analyzed by qRT-PCR, and the ethylene release was detected by capillary column gas chromatography. Transgenic seeds were sowed and then irrigated with NaCl solution, and the phenotypic changes of 21-day-seedings were recorded. The results showed that the growth of transgenic plants were better than that of the control group Huayu 22 upon salt stress, and the relative content of chlorophyll SPAD value and net photosynthetic rate (Pn) of transgenic peanuts were higher than those of the control group. In addition, the ethylene production of AhACO1 and AhACO2 transgenic plants were 2.79 and 1.87 times higher than that of control peanut, respectively. These results showed that AhACO1 and AhACO2 could significantly improve the salt stress tolerance of transgenic peanut.